325 results on '"Tashjian AH Jr"'
Search Results
2. Teriparatide [human PTH(1-34)]: 2.5 years of experience on the use and safety of the drug for the treatment of osteoporosis.
- Author
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Tashjian AH Jr and Gagel RF
- Subjects
- Bone Density Conservation Agents administration & dosage, Bone Density Conservation Agents adverse effects, Humans, Teriparatide administration & dosage, Teriparatide adverse effects, Bone Density Conservation Agents therapeutic use, Osteoporosis drug therapy, Teriparatide therapeutic use
- Published
- 2006
- Full Text
- View/download PDF
3. p21(Waf1/Cip1) and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells.
- Author
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Perletti G, Marras E, Dondi D, Osti D, Congiu T, Ferrarese R, de Eguileor M, and Tashjian AH Jr
- Subjects
- Blotting, Western, Cell Differentiation, Colonic Neoplasms enzymology, Colonic Neoplasms pathology, Cyclin-Dependent Kinase Inhibitor p21, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Neoplastic, Humans, Microscopy, Electron, Scanning, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Protein Kinase C-delta, Tumor Suppressor Proteins metabolism, Up-Regulation, Colonic Neoplasms metabolism, Cyclins metabolism, Enzyme Inhibitors metabolism, Protein Kinase C metabolism, Tumor Suppressor Protein p53 metabolism
- Abstract
We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.
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- 2005
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4. Commentary on clinical safety of recombinant human parathyroid hormone 1-34 in the treatment of osteoporosis in men and postmenopausal women.
- Author
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Tashjian AH Jr and Chabner BA
- Subjects
- Animals, Biological Assay, Consumer Product Safety, Disease Models, Animal, Female, Humans, Male, Recombinant Proteins therapeutic use, Risk Assessment, Osteoporosis drug therapy, Osteoporosis, Postmenopausal drug therapy, Parathyroid Hormone therapeutic use, Peptide Fragments therapeutic use
- Published
- 2002
- Full Text
- View/download PDF
5. Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides. Appendix: thermodynamic interpretation of the activation by concentrated salts.
- Author
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Huang X, Knoell CT, Frey G, Hazegh-Azam M, Tashjian AH Jr, Hedstrom L, Abeles RH, and Timasheff SN
- Subjects
- Base Sequence, DNA Primers, Escherichia coli genetics, Humans, Mass Spectrometry, Nuclear Magnetic Resonance, Biomolecular, Prostate-Specific Antigen chemistry, Prostate-Specific Antigen genetics, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Prostate-Specific Antigen metabolism
- Abstract
Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.
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- 2001
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6. Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.
- Author
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Chen F, Selinger Z, Marks P, Belinsky G, and Tashjian AH Jr
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- Animals, Binding, Competitive immunology, Blotting, Western, Cell Line, Cell Membrane metabolism, DNA, Complementary genetics, DNA, Complementary metabolism, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Humans, Immune Sera metabolism, Immunohistochemistry, Immunosorbent Techniques, Microscopy, Confocal, Pituitary Gland cytology, Pituitary Gland metabolism, Rabbits, Rats, Receptors, Thyrotropin-Releasing Hormone genetics, Receptors, Thyrotropin-Releasing Hormone metabolism, Transfection, Antibody Specificity immunology, Immune Sera chemistry, Immune Sera isolation & purification, Receptors, Thyrotropin-Releasing Hormone immunology
- Abstract
Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy., (Copyright 2001 Academic Press.)
- Published
- 2001
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7. Novel action of pituitary adenylate cyclase-activating polypeptide. Stimulation of extracellular acidification in rat pituitary GH4C1 cells.
- Author
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Chen X, Tang S, and Tashjian AH Jr
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- Animals, Calcium metabolism, Cell Line, Colforsin pharmacology, Cyclic AMP-Dependent Protein Kinases metabolism, Enzyme Activation, Enzyme Inhibitors pharmacology, Extracellular Space physiology, Flavonoids pharmacology, Hydrogen-Ion Concentration, Kinetics, Mitogen-Activated Protein Kinases metabolism, Naphthalenes pharmacology, Pituitary Adenylate Cyclase-Activating Polypeptide, Pituitary Gland cytology, Protein Kinase C metabolism, Rats, Signal Transduction drug effects, Tetradecanoylphorbol Acetate pharmacology, Thyrotropin-Releasing Hormone pharmacology, Neuropeptides pharmacology, Pituitary Gland physiology, Signal Transduction physiology
- Abstract
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide/secretin family. Using microphysiometry, we have found that PACAP acutely (1 min) increased the extracellular acidification rate (ECAR) in GH4C1 cells approximately 40% above basal in a concentration-dependent manner. ECAR, maximally induced by PACAP, can be increased further by thyrotropin-releasing hormone (TRH), indicating that the signalling pathways for these two neuropeptides are not identical. In studies on the mechanism of PACAP-enhanced ECAR, we found that maximum stimulation of the cAMP/PKA pathway by treatment with FSK, or the PKC pathway with PMA, did not inhibit the ECAR response to PACAP. The PKC inhibitor calphostin C and the MAP kinase inhibitor PD98059 had no effect on the ECAR response to PACAP. Furthermore, PACAP induced little or no change in cytosolic Ca(2+) ([Ca(2+)](i)), while TRH induced a large increase in [Ca(2+)](i). However, the tyrosine kinase inhibitor genistein completely blocked PACAP-induced ECAR, suggesting involvement of tyrosine kinase(s). We conclude that PACAP causes an increase in ECAR in GH4C1 rat pituitary cells, which is not dependent on the PKA, PKC, MAP kinase or Ca(2+) signalling pathways, but does require tyrosine kinase activity.
- Published
- 2000
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8. Direct measurement of hormone-induced acidification in intact bone.
- Author
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Belinsky GS and Tashjian AH Jr
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, Adenylyl Cyclases physiology, Animals, Animals, Newborn, Bone Neoplasms pathology, Bone and Bones chemistry, Bucladesine pharmacology, Calcium Signaling drug effects, Carbonic Anhydrases physiology, Colforsin pharmacology, Cyclic AMP-Dependent Protein Kinases physiology, Enzyme Activation drug effects, Extracellular Space chemistry, Extracellular Space drug effects, Humans, Mice, Organ Culture Techniques, Organ Specificity, Osteoblasts metabolism, Osteosarcoma pathology, Protein Kinase C antagonists & inhibitors, Protein Kinase C physiology, Second Messenger Systems drug effects, Skull chemistry, Skull drug effects, Tetradecanoylphorbol Acetate pharmacology, Thapsigargin pharmacology, Tumor Cells, Cultured chemistry, Tumor Cells, Cultured drug effects, Bone and Bones drug effects, Hydrogen-Ion Concentration drug effects, Osteoblasts drug effects, Parathyroid Hormone pharmacology
- Abstract
Previous findings have shown that osteoblasts respond to parathyroid hormone (PTH) with an increase in extracellular acidification rate (ECAR) in addition to the known effect of PTH to increase local acidification by osteoclasts. We, therefore, investigated use of the Cytosensor to measure the ECAR response of whole intact bone to PTH employing microphysiometry. The Cytosensor measures a generic metabolic increase of cells to various agents. Using neonatal mouse calvaria, we found that the area surrounding the sagittal suture was particularly responsive to PTH. In this bone, the increase in ECAR was slower to develop (6 minutes) and more persistent than in cultured human osteoblast-like SaOS-2 cells and was preceded by a brief decrease in ECAR. Salmon calcitonin also produced an increase in ECAR in this tissue but with a different pattern than that elicited by PTH. Because PTH stimulates osteoclastic bone resorption in mouse calvaria via a cyclic adenosine monophosphate (cAMP)-mediated mechanism, we showed that the adenylyl cyclase activator forskolin also stimulated ECAR in this tissue. When the protein kinase A (PKA) pathway was activated by maintaining a high intracellular concentration of cAMP using N6-2'-0-dibutyryladenosine-cAMP (db-cAMP), there was a reduction of PTH-induced acidification, while isobutylmethylxanthine pretreatment potentiated the PTH-induced acidification, consistent with a PKA-mediated pathway. Thapsigargin and the protein kinase C (PKC) activator phorbol myristate acetate had no effect on the PTH-induced increase in ECAR in calvaria, indicating that PKC does not play a major role in the ECAR response in intact bone. These results indicate the utility of using microphysiometry to study ECAR responses in intact tissue and should enable elucidation of the relative importance of extracellular acidification by osteoblasts and osteoclasts to the anabolic and catabolic activities of PTH, respectively.
- Published
- 2000
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9. Ca(2+) and extracellular acidification rate responses to parathyroid hormone fragments in rat ROS 17/2 and human SaOS-2 cells.
- Author
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Belinsky GS, Morley P, Whitfield JF, and Tashjian AH Jr
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, 8-Bromo Cyclic Adenosine Monophosphate pharmacology, Alkaloids, Animals, Benzophenanthridines, Cell Line, Colforsin pharmacology, Cyclic AMP-Dependent Protein Kinases metabolism, Fura-2, Humans, Hydrogen-Ion Concentration, Kinetics, Peptide Fragments pharmacology, Phenanthridines pharmacology, Protein Kinase C metabolism, Rats, Signal Transduction, Spectrometry, Fluorescence, Tetradecanoylphorbol Acetate pharmacology, Calcium metabolism, Parathyroid Hormone pharmacology
- Abstract
To examine the importance of the N- or C-termini of PTH(1-34) the effects of truncated fragments of PTH on human receptors in osteoblast-like SaOS-2 cells and rat receptors in rats ROS 17/2 cells were examined. Fura-2-loaded cells were used to monitor cytosolic free Ca(2+) concentration ([Ca2+]i), and the Cytosensor microphysiometer was used to monitor extracellular acidification rate (ECAR). C-terminally truncated fragments (1-31) and (1-28) of hPTH(1-34)NH(2) stimulated an increase in [Ca(2+)](i) and ECAR in both cell lines. hPTH(3-34)NH(2) and other N-terminally truncated fragments did not stimulate [Ca(2+)](i) or ECAR in either cell type. The signal transduction pathway of PTH-induced ECAR in ROS 17/2 cells was investigated to compare with previous results in SaOS-2 cells. Potentiation by IBMX, attenuation by 8Br-cAMP and lack of effect of the PKC inhibitor chelerythrine chloride support a cAMP/PKA-mediated signal transduction pathway in ROS 17/2, while the protein kinase C pathway was predominant in SaOS-2 cells. We conclude that the intact N-terminus of PTH is essential in PTH signaling mediated via either the cAMP/PKA or inositol lipid/Ca(2+)/PKC pathways in osteoblast-like cells., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
10. Identification of distinct signalling pathways for somatostatin receptors SSTR1 and SSTR2 as revealed by microphysiometry.
- Author
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Chen L and Tashjian AH Jr
- Subjects
- Animals, Cyclic AMP metabolism, GTP-Binding Proteins metabolism, Hormone Antagonists pharmacology, Peptides, Cyclic pharmacology, Pertussis Toxin, Rats, Sodium-Hydrogen Exchangers physiology, Somatostatin analogs & derivatives, Somatostatin pharmacology, Tumor Cells, Cultured, Virulence Factors, Bordetella pharmacology, Receptors, Somatostatin metabolism, Signal Transduction
- Abstract
Somatostatin receptors (SSTRs) are known to mediate diverse cellular responses. Most target cell express more than one SSTR isoform, making it difficult to define the signalling pathway used by individual receptor subtypes. Thus, we have expressed SSTR1 or SSTR2 in rat pituitary F4C1 cells which lack endogenous SSTRs. Using a silicon-based biosensor system, the Cytosensor microphysiometer, which measures the extracellular acidification rate (ECAR) in real time, we have studied the responses to SS mediated by either SSTR1 or SSTR2. In control F4C1 cells, SS had no effect on the basal ECAR. In transfected cells expressing only SSTR1, SS caused a unique decrease in ECAR in a concentration-dependent manner. Receptor-mediated decreases in ECAR have not been reported previously. In F4C1 cells expressing only SSTR2, SS induced a bidirectional ECAR response, a rapid increase followed by a decrease below basal. Two SS analogues, MK678 and CH275, induced characteristic ECAR responses with the expected receptor selectivities for SSTR1 or SSTR2. Pretreatment of F4C1 cells with pertussis toxin abolished the decreases in ECAR mediated by both SSTR1 and SSTR2, but only partially reduced the increase in ECAR mediated by SSTR2. The decrease in ECAR did not depend on a decrease in intracellular cAMP. The ECAR responses to SS were modestly attenuated by methylisobutylamiloride (MIA), an inhibitor of the ubiquitous Na(+)-H+ exchanger NHE1. Removal of extracellular Na+ greatly inhibited the ECAR responses to SS, demonstrating a role for both amiloride-sensitive and -insensitive Na(+)-dependent acid transport mechanisms in SS-induced extracellular acidification. In conclusion, we have identified and characterized different signalling pathways for SSTR1 and SSTR2 in pituitary cells as measured by microphysiometry.
- Published
- 1999
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- View/download PDF
11. Structural basis for the binding specificity of a SSTR1-selective analog of somatostatin.
- Author
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Chen L, Hoeger C, Rivier J, Fitzpatrick VD, Vandlen RL, and Tashjian AH Jr
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- Animals, Aspartic Acid metabolism, Cell Line, Protein Binding, Protein Conformation, Rats, Somatostatin chemistry, Somatostatin metabolism, Receptors, Somatostatin metabolism, Somatostatin analogs & derivatives
- Abstract
The availability of subtype-specific agonists and antagonists for somatostatin (SS) receptors (SSTRs) will be important for elucidation of the function of each receptor isoform in vivo. A SS analog, des-AA1,2,5-[D-Trp8, IAmp9]SS (CH275), has been shown previously to bind preferentially to SSTR1. In this report, we identify structural determinants in the ligand and receptor responsible for the selective binding of CH275 to SSTR1 by modifying both the ligand and the receptor. We propose that IAmp9 in CH275, like Lys9 in SS, interacts with Asp137 in the middle of the third transmembrane domain of SSTR1 to form an ion pair, while other residues unique to SSTR1 conbribute to binding selectivity of CH275 for SSTR1. Replacement of Asp137 with Asn resulted in loss of binding of radiolabeled SS and decreased potencies of both SS and CH275 to induce a change in the extracellular acidification rate measured by microphysiometry. The structural determinants for specific binding to SSTR1 were mapped in chimeric SSTR1/SSTR2 receptors. One chimera, 2beta, with the N-terminus to second transmembrane domain (TM2) from SSTR2 and the remainder of the receptor from SSTR1, had low affinity for CH275. Furthermore, when a single residue, Leu107, in TM2 of SSTR1 was replaced with Phe, the corresponding residue in SSTR2, a 20-fold decrease in affinity for CH275 with no significant change in affinity for SS was observed. A reciprocal change from Phe to Leu in the chimeric receptor 2beta resulted in a 10-fold increase in affinity for CH275. Thus, Leu107 is an important determinant for CH275 binding to SSTR1. To identify the moiety in CH275 which could interact with Leu107, a new analog des-AA1,2,5-[D-Trp8, Amp9]SS was prepared. This analog bound to both SSTR1 and SSTR2 with similar affinities; thus, subtype selectivity was lost. Collectively, these data support a binding model for CH275 in which the positively charged IAmp interacts with the negatively charged Asp137 in TM3 of SSTR1 and the isopropyl group of IAmp forms a hydrophobic interaction with Leu107 in TM2., (Copyright 1999 Academic Press.)
- Published
- 1999
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12. PKCdelta acts as a growth and tumor suppressor in rat colonic epithelial cells.
- Author
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Perletti GP, Marras E, Concari P, Piccinini F, and Tashjian AH Jr
- Subjects
- Animals, Calcium metabolism, Colonic Neoplasms genetics, Epithelial Cells enzymology, Epithelial Cells pathology, Growth Inhibitors genetics, Intestinal Mucosa enzymology, Intestinal Mucosa pathology, Isoenzymes genetics, Protein Kinase C genetics, Protein Kinase C-delta, Rats, Recombinant Proteins biosynthesis, Cell Transformation, Neoplastic, Colonic Neoplasms enzymology, Genes, src, Growth Inhibitors biosynthesis, Isoenzymes biosynthesis, Protein Kinase C biosynthesis
- Abstract
We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCdelta, PKCepsilon and PKCzeta, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCzeta and PKCepsilon showed similar protein levels, PKCdelta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCdelta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCdelta. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCdelta was involved in growth control and transformation in this model, we overexpressed PKCdelta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCdelta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.
- Published
- 1999
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13. Comparison of recombinant human PTH(1-34) (LY333334) with a C-terminally substituted analog of human PTH-related protein(1-34) (RS-66271): In vitro activity and in vivo pharmacological effects in rats.
- Author
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Frolik CA, Cain RL, Sato M, Harvey AK, Chandrasekhar S, Black EC, Tashjian AH Jr, and Hock JM
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- Animals, Binding, Competitive, Bone Resorption chemically induced, Calcium metabolism, Cattle, Cell Line, Humans, Male, Mice, Organ Culture Techniques, Rats, Rats, Inbred F344, Receptor, Parathyroid Hormone, Type 1, Receptors, Parathyroid Hormone metabolism, Signal Transduction, Teriparatide metabolism, Bone Density drug effects, Teriparatide analogs & derivatives, Teriparatide pharmacology
- Abstract
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are believed to exert their biological actions through binding and activation of a common cell surface receptor. Recently, an analog of PTHrP (RS-66271), was described that demonstrated reduced binding affinity for the PTH/PTHrP receptor compared with bovine PTH(1-34) but retained equal biological activity. The present study investigated the receptor binding affinities of synthetic RS-66271 and recombinant human PTH(1-34) (LY333334) and compared their in vitro and in vivo pharmacological effects. RS-66271 had one hundredth the activity of PTH(1-34) in competing for the binding of [125I] [Nle8,18, Tyr34]human PTH(1-34) to the human PTH/PTHrP receptor stably expressed in a human kidney cell line. Despite this reduced binding affinity, RS-66271 had equivalent activity in increasing both cAMP production in osteoblast-like cells and bone resorption in neonatal mouse calvariae. However, RS-66271 was 7. 6-fold less active in stimulating inositol phosphate production. For in vivo studies, young, male Fisher rats received a daily subcutaneous dose of either 10 or 40 microg/kg of peptide for 1, 2, or 4 weeks. Volumetric bone mineral density and total bone mineral content of the proximal tibia were determined by peripheral quantitative computerized tomography. Trabecular and cortical bone of the distal femur were analyzed for calcium and dry weight. Lumbar vertebrae (L4-L6) were analyzed by histomorphometry. Trabecular and cortical bone mass showed a dose- and time-dependent increase in the treated animals compared with the controls. These increases were evident as early as 1 week after initiation of dosing. There were no consistent significant differences in the comparative effects of PTH(1-34) and RS-66271 on the measured bone parameters. In conclusion, despite the reduced binding affinity of RS-66271 for the PTH/PTHrP receptor compared with human PTH(1-34), both peptides displayed similar in vitro and in vivo pharmacological effects.
- Published
- 1999
- Full Text
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14. The thyrotropin-releasing hormone-receptor complex and G11alpha are both internalised into clathrin-coated vesicles.
- Author
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Petrou C and Tashjian AH Jr
- Subjects
- Animals, Biological Transport, Active, Cell Line, Kinetics, Macromolecular Substances, Rats, Signal Transduction, Clathrin metabolism, Coated Vesicles metabolism, GTP-Binding Proteins metabolism, Receptors, Thyrotropin-Releasing Hormone metabolism, Thyrotropin-Releasing Hormone metabolism
- Abstract
It has been proposed that, after agonist binding, the thyrotropin-releasing hormone receptor (TRHR) becomes internalised associated with Gq, as part of a TRH-TRHR-Gq ternary complex [13]. We tested this hypothesis directly by examining the intracellular distribution of the TRHR and Gq/11 after agonist binding. The localisation of the TRH-TRHR complex and Gq/11alpha was studied by the biochemical isolation of clathrin-coated vesicles (CCVs). The internalised TRH-TRHR complex was localised in CCVs. The CCVs, which had internalised [3H]MeTRH, contained 4-fold higher levels of radiolabelled ligand than did CCVs from cells incubated with [3H]MeTRH at 4 degrees C. Like the receptor-ligand (RL) complex, G11alpha also translocated to these endocytic vesicles. For example, CCVs from cells with internalised TRH-TRHR complexes contained G11alpha, whereas CCVs from cells without internalised RL complexes lacked G11alpha. We conclude that, after agonist-induced TRHR-G11alpha coupling, both the TRH-TRHR complex and G11alpha are internalised in CCVs.
- Published
- 1998
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15. User-friendly and versatile software for analysis of protein hydrophobicity.
- Author
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Han B and Tashjian AH Jr
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Mathematical Computing, Molecular Sequence Data, Rats, Receptors, Thyrotropin-Releasing Hormone chemistry, Proteins chemistry, Software
- Abstract
We have developed a simple and flexible program to analyze regional hydrophobicity of a protein from its amino acid (aa) sequence. This program runs as a Microsoft Excel document into which aa sequence can be copied from any Windows-compatible or Macintosh word processor. The program returns the hydrophobicity index of each aa residue and other analyses that can be used to predict transmembrane domains, amphiphilic alpha helices and putative antigenic epitopes in a protein using established algorithms. The program can also be easily modified to test user-defined algorithms and to accommodate non-conventional aa residues or ambiguities in the aa sequence. Simple modification of the program allows direct use of nucleic acid sequence information for various analyses. Graphic visualization of the results is readily achieved using the graphics function of Microsoft Excel. Alternatively, the data can be imported into other graphics software for preparation of publication-quality figures. By running as a document in Microsoft Excel, which can be found in virtually all personal computers, this program provides easy access even to the computer novice.
- Published
- 1998
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16. Protein kinase Cepsilon is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway.
- Author
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Perletti GP, Concari P, Brusaferri S, Marras E, Piccinini F, and Tashjian AH Jr
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- Animals, Cell Division genetics, Cell Division physiology, Cell Line, Cell Line, Transformed, Cell Transformation, Neoplastic genetics, Colon metabolism, Epithelial Cells cytology, Genes, ras genetics, Genes, ras physiology, Isoenzymes genetics, Mutation genetics, Mutation physiology, Phosphorylation, Protein Kinase C genetics, Protein Kinase C-epsilon, Proto-Oncogene Proteins c-raf genetics, Proto-Oncogene Proteins c-raf metabolism, Signal Transduction genetics, Signal Transduction physiology, ras Proteins genetics, Colon cytology, Epithelial Cells metabolism, Isoenzymes physiology, Protein Kinase C physiology, ras Proteins physiology
- Abstract
We have shown previously that overexpression of the epsilon isoform of protein kinase C (PKCepsilon) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/epsilon cells (overexpressing PKCepsilon), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCepsilon-transformed D/epsilon cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCepsilon-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCepsilon. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCepsilon cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCepsilon, and that overexpression of PKCepsilon circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCepsilon exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.
- Published
- 1998
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17. New insights into interactions between the human PTH/PTHrP receptor and agonist/antagonist binding.
- Author
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Fukayama S, Royo M, Sugita M, Imrich A, Chorev M, Suva LJ, Rosenblatt M, and Tashjian AH Jr
- Subjects
- Animals, Cell Line, Epitope Mapping, Goats, Humans, Immune Sera, Parathyroid Hormone immunology, Parathyroid Hormone metabolism, Peptide Fragments immunology, Peptide Fragments metabolism, Protein Binding, Receptor, Parathyroid Hormone, Type 1, Receptors, Parathyroid Hormone immunology, Teriparatide analogs & derivatives, Teriparatide immunology, Teriparatide metabolism, Receptors, Parathyroid Hormone metabolism
- Abstract
We prepared a polyclonal antiserum [Ab-(88-97)] against residues 8-97 of the NH2-terminal tail of the human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor. Ab-(88-97) bound specifically to the receptor, as assessed by fluorescence-activated cell sorter analysis of HEK C21 cells, which stably express approximately 400,000 hPTH/PTHrP receptors per cell. Unlike PTH, Ab-(88-97) binding did not elicit either adenosine 3',5'-cyclic monophosphate or intracellular calcium concentration signaling responses in these cells. Incubation of C21 cells for 90 min at 4 degrees C with hPTH-(1-34) plus antiserum reduced the Ab-(88-97) binding to the cells by up to 40-50% of control values in a PTH concentration-dependent fashion with a half-maximal effective concentration of approximately 5 nM. The decrease in Ab-(88-97) binding caused by hPTH-(1-34) was completely reversed by coincubation with hPTHrP-(7-34). We conclude that residues 88-97 of the hPTH/PTHrPR are involved, either directly or indirectly, in agonist but not antagonist binding to the receptor.
- Published
- 1998
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18. A new action of parathyroid hormone. receptor-mediated stimulation of extracellular acidification in human osteoblast-like SaOS-2 cells.
- Author
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Barrett MG, Belinsky GS, and Tashjian AH Jr
- Subjects
- Cell Line, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Extracellular Space metabolism, Humans, Hydrogen-Ion Concentration, Osteoblasts metabolism, Peptides metabolism, Signal Transduction, Sodium-Hydrogen Exchangers metabolism, Osteoblasts drug effects, Parathyroid Hormone pharmacology, Receptors, Parathyroid Hormone metabolism
- Abstract
The major physiological function of parathyroid hormone (PTH) is the maintenance of Ca2+/Pi homeostasis via the parathyroid hormone/parathyroid hormone-related protein receptor (PTHR) in kidney and bone. An important consequence of PTHR activation in bone is enhanced local acidification of the extracellular space. Agonist activation of some seven transmembrane-domain receptors increases the extracellular acidification rate (ECAR). We utilized microphysiometry to investigate PTH-stimulated, receptor-mediated increases in ECAR in human osteoblast-like SaOS-2 cells. PTH-(1-34) elicited a large, acute, dose-dependent increase in ECAR with an EC50 of about 2 nM. The PTH-induced increase in ECAR was specific to cells expressing the PTHR and was inhibited by PTHR antagonists. Rapid, partial, homologous desensitization of the PTH-induced increase in ECAR was observed. Incubation of SaOS-2 cells with 8-bromo-cyclic AMP neither mimicked nor abrogated the PTH effect, and PTH stimulated an acute increase in ECAR in cAMP-resistant SaOS-2 Ca#4A cells. Stimulation of ECAR by PTH was independent of transient increases in cytosolic free calcium. Both inhibition and down-regulation of PKC reduced the PTH-induced increase in ECAR. Inhibition of Na+/H+ exchange did not affect the PTH-induced ECAR response. We conclude that PTH caused a receptor-mediated, concentration-dependent, increase in ECAR, which was not dependent on the cAMP/PKA signaling pathway or the Na+/H+ exchanger but involved the action of PKC. Thus, acid production in bone, a physiologically important action of PTH, is not confined to osteoclasts as previously considered but is also mediated by osteoblasts.
- Published
- 1997
- Full Text
- View/download PDF
19. Beta-adrenergic receptor kinase-1 acutely regulates PTH/PTHrP receptor signalling in human osteoblastlike cells.
- Author
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Fukayama S, Kong G, Benovic JL, Meurer E, and Tashjian AH Jr
- Subjects
- Cell Line, Colforsin pharmacology, Cyclic AMP pharmacology, Cyclic AMP-Dependent Protein Kinases genetics, Enzyme Activation, Humans, Mutation physiology, Osteoblasts cytology, Proto-Oncogene Proteins, Receptor, Parathyroid Hormone, Type 1, Sensitivity and Specificity, Signal Transduction drug effects, Transfection, beta-Adrenergic Receptor Kinases, Axl Receptor Tyrosine Kinase, Cyclic AMP-Dependent Protein Kinases metabolism, Oncogene Proteins, Osteoblasts enzymology, Receptor Protein-Tyrosine Kinases metabolism, Receptors, Parathyroid Hormone physiology, Signal Transduction physiology
- Abstract
To investigate whether G protein-coupled receptor kinases (GRKs) are involved in the regulation of the PTH/PTHrPR, we have established mutant SaOS-2 cells which stably overexpress (> 10-20-fold) a dominant negative form of the beta-adrenergic receptor kinase-1 (beta ARK-1). Acute (< or = 2 h) incubation with hPTH (1-34) induced significantly less (by up to 50%) downregulation of the PTH/PTHrPR in beta ARK-1 mutant SaOS-2 cells than observed in wild-type cells. Pretreatment of wild-type cells with PTH for 2 h induced homologous cAMP desensitisation to a second challenge with PTH, while the effect was blunted by up to 60% in beta ARK-1 mutant cells. We conclude that activation of beta ARK-1 (or a closely related GRK) is a critical component of the acute phase (< or = 2 h) of PTH-induced receptor downregulation and homologous cAMP desensitisation of the PTH/PTHrPR.
- Published
- 1997
- Full Text
- View/download PDF
20. Both overlapping and distinct signaling pathways for somatostatin receptor subtypes SSTR1 and SSTR2 in pituitary cells.
- Author
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Chen L, Fitzpatrick VD, Vandlen RL, and Tashjian AH Jr
- Subjects
- Adenylate Cyclase Toxin, Adenylyl Cyclase Inhibitors, Animals, CHO Cells, COS Cells, Calcium metabolism, Colforsin pharmacology, Cricetinae, Cyclic AMP metabolism, Inositol Phosphates metabolism, Membrane Potentials, Pertussis Toxin, Rats, Recombinant Fusion Proteins metabolism, Tumor Cells, Cultured, Type C Phospholipases metabolism, Virulence Factors, Bordetella pharmacology, Pituitary Gland metabolism, Receptors, Somatostatin metabolism, Signal Transduction
- Abstract
To elucidate the signaling events mediated by specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and SSTR2 individually in rat pituitary GH12C1 and F4C1 cells, which lack endogenous somatostatin receptors. In transfected GH12C1 cells, both SSTR1 and SSTR2 coupled to inhibition of Ca2+ influx and hyperpolarization of membrane potential via a pertussis toxin (PTx)-sensitive mechanism. These effects reflected modulation of ion channel activities which are important for regulation of hormone secretion. Somatostatin analogs MK678 and CH275 acted as subtype selective agonists as expected. In transfected F4C1 cells, both SSTR1 and SSTR2 mediated somatostatin-induced inhibition of adenylyl cyclase via a PTx-sensitive pathway. In addition, activation of SSTR2 in F4C1 cells, but not SSTR1, stimulated phospholipase C (PLC) activity and an increase in [Ca2+]i due to release of Ca2+ from intracellular stores. Unlike adenylyl cyclase inhibition, the PLC-mediated response was only partially sensitive to PTx. To determine the structural determinants in SSTR2 necessary for activation of PLC, we constructed chimeric receptors in which domains of SSTR2 were introduced into SSTR1. Chimeric receptors containing only the third intracellular loop, or all three intracellular loops from SSTR2, mediated inhibition of adenylyl cyclase, but failed to stimulate PLC activity as did wild-type SSTR2. Furthermore, the C-terminal tail of SSTR2 was not required for coupling to PLC. Thus, by expressing individual somatostatin receptor subtypes in pituitary cells, we have identified both overlapping and distinct signaling pathways for SSTR1 and SSTR2, and have shown that sequences other than simply the intracellular domains are required for SSTR2 to couple to the PLC signaling pathway.
- Published
- 1997
- Full Text
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21. A receptor-G protein coupling-independent step in the internalization of the thyrotropin-releasing hormone receptor.
- Author
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Petrou C, Chen L, and Tashjian AH Jr
- Subjects
- Animals, Antibodies, Monoclonal metabolism, Cells, Cultured, Fluorescent Antibody Technique, Indirect, Mutagenesis, Site-Directed, Pyrrolidonecarboxylic Acid analogs & derivatives, Rats, Receptors, Thyrotropin-Releasing Hormone genetics, Structure-Activity Relationship, Thyrotropin-Releasing Hormone analogs & derivatives, Thyrotropin-Releasing Hormone metabolism, Transferrin metabolism, GTP-Binding Proteins metabolism, Models, Biological, Receptors, Thyrotropin-Releasing Hormone metabolism, Signal Transduction
- Abstract
To determine whether functional receptor-G protein coupling or signaling are required for internalization of the thyrotropin-releasing hormone receptor (TRHR), we compared the endocytosis of Gq-coupled and uncoupled receptors. A hemagglutinin epitope-tagged TRHR (HA-TRHR) was in the Gq-coupled state when bound to the agonist, MeTRH, and in a nonsignaling state when bound to the HA antibody (12CA5). 12CA5 did not induce an increase in [Ca2+]i or inositol phosphates and did not inhibit [3H]MeTRH binding or MeTRH-induced production of second messengers. Both agonist- and antibody-bound HA-TRHRs were rapidly internalized via the same pathway; internalization was sensitive to hypertonic shock, and both types of internalized receptors were sorted into lysosomes. In addition, the amino acid sequence CNC (positions 335-337) in the C-terminal tail of the TRHR, which is important in ligand-induced receptor internalization as determined by deletion mutagenesis (Nussenzveig, D. R., Heinflink, M., and Gershengorn, M. C. (1993) J. Biol. Chem. 268, 2389-2392), was also important for 12CA5-induced internalization. We expressed two truncated receptors, HA-K338STOP and HA-C335STOP, in GH12C1 pituitary cells. Both HA-TRHR and HA-K338STOP were localized at the plasma membrane of untreated cells and were translocated to intracellular vesicles after MeTRH or 12CA5 binding; however, HA-C335STOP was internalized and recycled constitutively. The intracellular localization of HA-C335STOP was not altered by MeTRH; however, 12CA5 binding induced the disappearance of internalized HA-C335STOP and caused its localization at the plasma membrane, indicating that constitutively cycling HA-C335STOP cannot be reinternalized after antibody binding. Thus, amino acids 335-337, which are important for the internalization of Gq-coupled TRHRs, are also required for the sequestration of functionally uncoupled TRHRs, and in addition, they act as an inhibitory signal that prevents constitutive receptor internalization. Specifically, the Cys residues at positions 335 and 337 are important for preventing constitutive TRHR internalization, because a mutant HA-C335S/C337S receptor was sequestered constitutively. We conclude that release from a negative regulatory internalization sequence or domain is important for HA-TRHR internalization and that the role of the CNC sequence in internalization is independent of functional TRHR-Gq coupling.
- Published
- 1997
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22. Inhibition of capacitative Ca2+ entry into cells by farnesylcysteine analogs.
- Author
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Xu Y, Gilbert BA, Rando RR, Chen L, and Tashjian AH Jr
- Subjects
- Affinity Labels, Cell Line, Cysteine analogs & derivatives, Guanosine Triphosphate metabolism, Humans, Manganese metabolism, Protein Binding, Thapsigargin pharmacology, Calcium metabolism, Cysteine pharmacology
- Abstract
Capacitative Ca2+ influx, which occurs in response to mobilization of intracellular Ca2+ stores, is a general feature of many cell types. Although the mechanism of capacitative Ca2+ entry is not known, evidence suggests the involvement of small G proteins that are prenylated on a cysteine residue near their carboxyl termini. We have investigated the actions of farnesyl-cysteine analogs on capacitative Ca2+ influx. Using human embryonic kidney 293 cells, we found that S-farnesylthioacetic acid, N-acetyl-S-farnesyl-L-cysteine, N-pivaloyl-S-farnesyl-L-cysteine, and N-acetyl-S-gernylgernyl-L-cysteine blocked the activation of capacitative Ca2+ influx, whereas N-benzoyl-S-farnesyl-S-cysteine had no effect on capacitative Ca2+ entry. Inhibition by S-farnesylthioacetic acid was concentration dependent (5-20 microM) and specific for Ca2+ influx through non-voltage-gated Ca2+ channels. A single protein band of 26-28 kDa was labeled specifically with a photoaffinity analog of farnesylcysteine. GTP binding to the photoaffinity-labeled band was demonstrated. These findings suggest, but do not prove, that a prenylated substrate, possibly a small G protein, is linked functionally to capacitative Ca2+ entry in human embryonic kidney 293 cells.
- Published
- 1996
23. Pretreatment with 17 beta-estradiol attenuates basal- and PTH-stimulated membrane adenylyl cyclase activity in human osteoblast-like SAOS-2 cells.
- Author
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Monroe JJ and Tashjian AH Jr
- Subjects
- Adenosine Triphosphate metabolism, Cell Line, Cell Membrane enzymology, Colforsin pharmacology, Cyclic AMP metabolism, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, Humans, Kinetics, Manganese pharmacology, Peptide Fragments pharmacology, Phosphorus Radioisotopes, Teriparatide, Time Factors, Adenylyl Cyclases metabolism, Estradiol pharmacology, Osteoblasts enzymology, Parathyroid Hormone pharmacology
- Abstract
Parathyroid hormone (PTH)-stimulated production of cAMP by intact human osteoblast-like SaOS-2 cells is diminished by pretreatment with 17 beta-estradiol (E2). The goal of the present study was to determine whether E2, affected adenylyl cyclase activity in cell membranes. Cells were deprived of steroid hormones for 24 h and then incubated with E2 (1 nM) or vehicle for 12 h. Cell membranes were prepared and incubated with [alpha-32P]-ATP in the absence or presence of agonist, and the amount of [32P]-cAMP produced was measured to quantify adenylyl cyclase activity. There was less cAMP produced by membranes from E2-treated cells in response to PTH, GTP gamma S, forskolin, and Mn2+. E2 had no effect on the amounts of Gs or Gi. We propose that pretreatment of SaOS-2 cells with E2 reduced membrane cyclase activity, at least in part, by actions on the PTH-sensitive adenylyl cyclase and/or the G protein-adenylyl cyclase complex.
- Published
- 1996
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24. Enhanced expression of vascular endothelial growth factor in human SaOS-2 osteoblast-like cells and murine osteoblasts induced by insulin-like growth factor I.
- Author
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Goad DL, Rubin J, Wang H, Tashjian AH Jr, and Patterson C
- Subjects
- Animals, Blotting, Northern, Cell Line, Dactinomycin pharmacology, Drug Stability, Humans, Mice, Protein Synthesis Inhibitors pharmacology, RNA, Messenger biosynthesis, Vascular Endothelial Growth Factor A, Vascular Endothelial Growth Factors, Endothelial Growth Factors genetics, Gene Expression drug effects, Insulin-Like Growth Factor I pharmacology, Lymphokines genetics, Osteoblasts metabolism
- Abstract
Formation of new capillaries, a critical component of tissue growth and repair, is a recognized process in the development, formation, and remodeling of bone. Vascular endothelial growth factor (VEGF), a potent angiogenic factor with specific mitogenic actions on endothelial cells, is produced in a regulated manner by many cell types, including osteoblasts. The aim of the present investigation was to test the hypothesis that insulin-like growth factor I (IGF-I), a known osteogenic factor, modulates VEGF expression in osteoblasts. In human SaOS-2 osteoblast-like cells, 10 nM IGF-I increased the abundance of VEGF messenger RNA (mRNA) by 4-fold above the control value at 2h, and the elevated levels of mRNA returned to near basal by 8 h. IGF-I stimulated VEGF mRNA levels at IGF-I concentrations as low as 1-2 nM. The stability of VEGF mRNA was not increased after IGF-I treatment, and actinomycin D abrogated the enhanced expression of VEGF mRNA by IGF-I, indicating that the action of IGF-I was probably mediated by a transcriptional mechanism. The induction of VEGF mRNA by IGF-I in SaOS-2 cells was associated with an increase in immunoreactive VEGF protein, as detected by immunoblot analysis. IGF-I also increased the expression of VEGF mRNA in primary murine osteoblasts, which confirmed that the actions of IGF-I were not unique to SaOS-2 cells. We conclude that IGF-I enhances osteoblast synthesis of VEGF, which may then act locally on endothelium to stimulate angiogenesis, an essential component of bone growth and remodeling.
- Published
- 1996
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25. Palytoxin modulates cytosolic pH in human osteoblast-like Saos-2 cells via an interaction with Na(+)-K(+)-ATPase.
- Author
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Monroe JJ and Tashjian AH Jr
- Subjects
- Cell Line, Cnidarian Venoms pharmacology, Cytosol drug effects, Drug Resistance, Humans, Hydrogen-Ion Concentration drug effects, Ouabain pharmacology, Rubidium pharmacokinetics, Acrylamides pharmacology, Cytosol metabolism, Osteoblasts drug effects, Osteoblasts metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Palytoxin (PTx) at nanomolar concentrations enhances the permeability of mammalian cell membranes to both Na+ and Ca2+. In basal human osteoblast-like Saos-2 cells, PTx (8 nM) caused a persistent decrease in cytosolic pH (pHi) of about 0.2 units, which required the presence of extracellular Ca2+ (Cae2+) and Na+ (Nae+). We acidified Saos-2 cells by incubation with nigericin to examine the action of PTx in cells with an activated Na+/H+ antiporter. Under these conditions, PTx increased the pHi without requiring Cae2+ or Nae+, and the alkalinization was unaffected by hexamethylene amiloride. We conclude that the PTx-induced rise in pHi did not involve the Na+/H+ antiporter. PTx increased the rate of 86Rb+ efflux. We propose that PTx induced alkalinization in nigericin-acidified cells by collapsing the K+ gradient. Exposure to ouabain had no effect on pHi, but it prevented the actions of PTx on PHi in both basal and nigericin-acidified cells. Ouabain-resistant mutant cells were less sensitive to PTx in extruding 86Rb+ than their ouabain-sensitive parents. We conclude that PTx interacts with the Na(+)-K(+)-adenosinetriphosphatase to regulate pHi in both basal and nigericin-acidified Saos-2 cells.
- Published
- 1996
- Full Text
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26. Inositol 1-,4-,5-trisphosphate-dependent Ca2+ signaling by the recombinant human PTH/PTHrP receptor stably expressed in a human kidney cell line.
- Author
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Pines M, Fukayama S, Costas K, Meurer E, Goldsmith PK, Xu X, Muallem S, Behar V, Chorev M, Rosenblatt M, Tashjian AH Jr, and Suva LJ
- Subjects
- Blotting, Western, Cloning, Molecular, Cyclic AMP metabolism, Cytosol drug effects, Cytosol metabolism, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, GTP-Binding Proteins genetics, GTP-Binding Proteins metabolism, Humans, Kidney cytology, Kidney metabolism, Neoplasm Proteins pharmacology, Parathyroid Hormone pharmacology, Peptide Fragments pharmacology, Proteins pharmacology, Receptor, Parathyroid Hormone, Type 1, Receptors, Parathyroid Hormone drug effects, Recombinant Proteins metabolism, Recombinant Proteins pharmacology, Signal Transduction drug effects, Transfection, Tumor Cells, Cultured, Calcium metabolism, Inositol 1,4,5-Trisphosphate pharmacology, Parathyroid Hormone metabolism, Parathyroid Hormone-Related Protein, Receptors, Parathyroid Hormone metabolism, Signal Transduction physiology
- Abstract
We previously reported the preparation and partial characterization of a series of human embryonic kidney cell lines (HEK-293) stably expressing various numbers of the recombinant human (h) parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (Rc). Using this expression system we examined ligand (PTH or PTHrP) binding characteristics and cyclic AMP responsiveness. We have now extended these studies to investigate the calcium signal transduction pathways activated by the hPTH/PTHrP Rc. In parental HEK-293 cells, which lack endogenous PTH/PTHrP Rc, incubation with hPTH(1-34) had no effect on cytosolic free Ca2+ concentration [Ca2+]i. In HEK-293 clone C-21, stably expressing approximately 400,000 Rc/cell, PTH stimulated an increase in [Ca2+]i by Ca2+ release from intracellular stores; PTH released Ca2+ exclusively from the IP3 sensitive Ca2+ pool. Unlike previous studies, the ability of PTH to elicit both cAMP responses and [Ca2+]i transients occurred over a wide range of Rc numbers (between 400,000 and 3000 Rc/cell); both responses were always observed at PTH concentrations in the same dose range although the magnitude of the responses decrease with Rc number. Pretreatment of C-21 cells with pertussis toxin for 24 h, which significantly enhanced PTH-stimulated cAMP accumulation, did not modulate PTH-stimulated [Ca2+]i transients. At each PTH concentration tested which resulted in increased cAMP levels, there was also an increase in [Ca2+]i transients. Treatment of C-21 cells with a battery of midregion and C-terminal PTH or PTHrP peptides showed no effect on either [Ca2+]i transients or cAMP accumulation, indicating a lack of functional interactions between these peptides and the form of the hPTH/PTHrP Rc stably expressed in these cells. Immunological analysis of G-protein expression demonstrated the presence of Gs, Gi, and Gq in all parental and transfected cells lines examined. Taken together, these data demonstrate that the hPTH/PTHrP Rc, stably expressed in HEK-293 cells, elicits responses in both the cAMP and IP3-dependent [Ca2+]i pathways and is responsive only to N-terminal PTH/PTHrP peptides.
- Published
- 1996
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27. Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells.
- Author
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Perletti GP, Folini M, Lin HC, Mischak H, Piccinini F, and Tashjian AH Jr
- Subjects
- Animals, Blotting, Western, Cell Adhesion, Cell Division drug effects, Cell Line, Colonic Neoplasms pathology, Epithelial Cells, Epithelium enzymology, Epithelium pathology, Gene Expression, Isoenzymes biosynthesis, Mice, Mice, Nude, Rats, Recombinant Proteins biosynthesis, Tetradecanoylphorbol Acetate pharmacology, Transfection, Transplantation, Heterologous, Cell Transformation, Neoplastic, Colon enzymology, Colonic Neoplasms genetics, Genes, ras, Protein Kinase C biosynthesis
- Abstract
We have analysed the expression of five protein kinase C [PKC] isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D [D/WT] and in the related tumorigenic Ha-ras-transformed FRC/TEX CL D/H-ras line [D/ras]. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium.
- Published
- 1996
28. Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor.
- Author
-
Han B and Tashjian AH Jr
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cell Line, Gene Deletion, Glycosylation, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Pituitary Gland metabolism, Protein Structure, Secondary, Rats, Receptors, Thyrotropin-Releasing Hormone genetics, Structure-Activity Relationship, Thyrotropin-Releasing Hormone metabolism, Extracellular Space metabolism, Receptors, Thyrotropin-Releasing Hormone chemistry, Receptors, Thyrotropin-Releasing Hormone metabolism
- Abstract
The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.
- Published
- 1995
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29. Signaling by N- and C-terminal sequences of parathyroid hormone-related protein in hippocampal neurons.
- Author
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Fukayama S, Tashjian AH Jr, Davis JN, and Chisholm JC
- Subjects
- Animals, Animals, Newborn, Calcium metabolism, Cells, Cultured, Cyclic AMP metabolism, Humans, Kinetics, Neurons drug effects, Rats, Receptor, Parathyroid Hormone, Type 1, Receptors, Parathyroid Hormone drug effects, Signal Transduction, Hippocampus physiology, Neurons physiology, Parathyroid Hormone-Related Protein, Peptide Fragments pharmacology, Proteins pharmacology, Receptors, Parathyroid Hormone physiology
- Abstract
Parathyroid hormone-related protein (PTHrP) is synthesized in the brain, and a single type of cloned receptor for the N-terminal portion of PTHrP and PTH is present in the central nervous system. Nothing is known about the physiological actions or signaling pathways used by PTHrP in the brain. Using cultured rat hippocampal neurons, we demonstrate that N-terminal PTHrP[1-34] and PTH[1-34] signal via cAMP and cytosolic calcium transients. The cAMP response showed strong acute (< or = 6 h) homologous and heterologous desensitization after preincubation with PTHrP or PTH. In contrast, the acute calcium response did not desensitize after preincubation with PTHrP; in fact, preincubation dramatically recruited additional responsive neurons. Unexpectedly, C-terminal PTHrP[107-139], which does not bind or activate the cloned PTH/PTHrP receptor, signaled in neurons via cytosolic calcium but not cAMP. Although some neurons responded to both PTHrP[1-34] and PTHrP[107-139], others responded only to PTHrP[1-34]. We conclude that certain hippocampal neurons exhibit dual signaling in response to PTHrP[1-34] and that some neurons have a receptor for C-terminal PTHrP that signals only via cytosolic calcium.
- Published
- 1995
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30. Identification of Asn289 as a ligand binding site in the rat thyrotropin-releasing hormone (THR) receptor as determined by complementary modifications in the ligand and receptor: a new model for THR binding.
- Author
-
Han B and Tashjian AH Jr
- Subjects
- Amino Acid Sequence, Animals, Aspartic Acid, Binding Sites, Calcium pharmacology, Egtazic Acid pharmacology, Female, Hydrogen Bonding, Kinetics, Ligands, Mutagenesis, Site-Directed, Oocytes physiology, Point Mutation, Rats, Receptors, Thyrotropin-Releasing Hormone biosynthesis, Recombinant Proteins biosynthesis, Recombinant Proteins metabolism, Restriction Mapping, Xenopus laevis, Asparagine, Receptors, Thyrotropin-Releasing Hormone metabolism, Thyrotropin-Releasing Hormone metabolism
- Abstract
To test the hypothesis that pGlu of the thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2) binds to Asn289 in the third extracellular loop (EL3) of its receptor through a hydrogen bonding interaction, we converted Asn289 to Asp (N289D mutant) and measured the potencies of TRH and Pro1TRH for the wild-type and mutant receptors. TRH was 100 times less potent for the N289D receptor than for the wild-type. In contrast, Pro1TRH, which has a protonated proline in place of the pGlu of TRH, was 10 times more potent for the N289D receptor than for the wild-type. A similar result was obtained when Asn289 was converted to Glu, while the potency of Pro1TRH did not change when Asn289 was converted to Ala, confirming that the increased potency of Pro1TRH for the N289D receptor was due to a charge interaction between Pro1TRH and the mutant receptor. These findings are inconsistent with a previous model indicating a direct interaction of the pGlu of TRH with Asn110 in the third transmembrane helix of the receptor (Perlman et al. (1994) J. Biol. Chem. 269, 23383-23386). When Asn110 was converted to Asp (N110D mutant), unlike the N289D receptor, the potency of Pro1TRH for the N110D receptor was decreased by > 10-fold rather than increased. Therefore, a direct interaction of Asn110 with the pGlu of TRH could not be supported by our experiments. We propose a new model in which the pGlu of TRH binds to Asn289 in EL3 and conclude that, unlike catecholamines which bind completely within the transmembrane domain of their receptors, this tripeptide binds, at least in part, to the extracellular domain of its receptor.
- Published
- 1995
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31. Actions of palytoxin on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells.
- Author
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Monroe JJ and Tashjian AH Jr
- Subjects
- Cell Line, Cnidarian Venoms pharmacology, Cytosol metabolism, Humans, Intracellular Membranes metabolism, Osmolar Concentration, Sodium-Potassium-Exchanging ATPase physiology, Acrylamides pharmacology, Calcium metabolism, Homeostasis drug effects, Osteoblasts metabolism, Sodium metabolism
- Abstract
Palytoxin (PTx) is a potent membrane-active agent produced by marine coelenterates that acts to stimulate bone resorption in organ culture at nanomolar concentrations. We report here the actions of PTx on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells. PTx induced a rise in the cytosolic free Na+ concentration ([Na]i) by causing entry of extracellular Na+ (Na(e)+). PTx also caused a concentration-dependent biphasic rise in the cytosolic free Ca2+ concentration ([Ca2+]i) by enhancing entry of extracellular Ca2+ (Ca(e)2+). Entry of Na+ was dependent on the presence of Ca(e)2+ and was prevented by the Na+/Ca2+ exchange antagonist 3,4-dichlorobenzamil (DCB). Entry of Ca2+ was dependent on the presence of Na(e)+ but was not prevented by DCB. The actions of PTx on [Na+]i and [Ca2+]i were completely inhibited by pretreatment of the cells with ouabain. Ouabain alone had no acute effect on [Na+]i or [Ca2+]i in Saos-2 cells. We propose that interaction of PTx with the Na+ pump created a channel that allowed influx of Na(e)+ and Ca(e)2+. The rise in [Ca2+]i then stimulated the activity of the plasma membrane Na+/Ca2+ exchanger, which further enhanced Na(e)+ entry.
- Published
- 1995
- Full Text
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32. Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.
- Author
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Tanaka Y and Tashjian AH Jr
- Subjects
- Adenosine Diphosphate Ribose pharmacology, Animals, Caffeine pharmacology, Calmodulin antagonists & inhibitors, Cyclic ADP-Ribose, Dose-Response Relationship, Drug, Drug Interactions, Models, Biological, Ovum metabolism, Ryanodine Receptor Calcium Release Channel, Sea Urchins, Subcellular Fractions metabolism, Sulfonamides pharmacology, Adenosine Diphosphate Ribose analogs & derivatives, Calcium metabolism, Calcium Channel Agonists pharmacology, Calcium Channels drug effects, Calmodulin pharmacology, Muscle Proteins drug effects
- Abstract
The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.
- Published
- 1995
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33. Agonist-independent, muscle-type-specific signal transduction pathways in cat esophageal and lower esophageal sphincter circular smooth muscle.
- Author
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Sohn UD, Han B, Tashjian AH Jr, Behar J, and Biancani P
- Subjects
- Animals, Cats, Female, GTP-Binding Proteins physiology, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, In Vitro Techniques, Male, Muscle Contraction drug effects, Type C Phospholipases physiology, Bombesin pharmacology, Esophagogastric Junction metabolism, Esophagus metabolism, Muscle, Smooth metabolism, Signal Transduction, Substance P pharmacology
- Abstract
Smooth muscle cells isolated from the circular muscle layer of cat esophagus and lower esophageal sphincter (LES) exhibit distinct contractile intracellular signal transduction pathways in response to acetylcholine. To determine whether these contractile pathways are muscle type dependent, the authors examined the signal transduction pathways utilized by substance P and bombesin, which in other tissues, use different signal transduction pathways, and by the GTP analog, guanosine 5'-O-3-thiotriphosphate (GTP gamma S), which activates all available G proteins. Western blot analysis of esophageal and LES circular muscle revealed the presence of Gq-G11 (42 kD), Gi1-Gi2 (40 kD) and Go-Gi3 (40 kD) types of G proteins. The responses of esophageal cells to bombesin and substance P were blocked by 1) a Gi3 protein antibody, 2) the inhibitor of specific phosphatidylcholine-phospholipase C (PLC) D609 potassium tricyclo-[5.2.1.0(2.6)]-decyl-(9[8])-xanthogenate, 3) inhibition of phosphatidic acid phosphohydrolase by propranolol, 4) the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and 5) incubation in Ca(++)-free medium. Conversely, the responses of LES muscle cells to bombesin and substance P were blocked by 1) a Gq-G11 antibody, 2) a phosphatidylinositol-specific PLC antagonist U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), 3) the calmodulin inhibitor CGS9343B (1,3-Dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin++ +-4 - yl)methyl-4-piperindinyl]-2H-benzimidazol-2-one maleate) and 4) incubation in Sr++. After permeabilization by saponin, inositol 1,4,5-trisphosphate contracted LES but not esophageal cells. The inositol 1,4,5-trisphosphate receptor antagonist heparin and depletion of intracellular Ca++ stores by thapsigargin or A23187 4-Benzoxazolecarboxylic acid, 5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol- 2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-, [6s-[6.alpha. (2S*,3S*),8.beta. (R*), 9.beta., 11. alpha.]]-(9Cl), blocked bombesin- and substance P-induced contraction of LES but not of esophageal muscle. In addition, contraction in response to GTP gamma S, which activates all G proteins, was blocked in esophageal cells by a Gi3-protein antibody, propranolol, D609 and H7. In LES muscle cells, the response to GTP gamma S was blocked by a Gq protein antibody, U-73122 and CGS934B. These data demonstrate that, in esophageal muscle, different agonists activate the same Gi3 protein, phosphatidylcholine-specific phospholipases and protein kinase C-dependent pathway.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1995
34. Cyclic ADP-ribose-induced calcium release in sea urchin egg homogenates is a cooperative process.
- Author
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Xu Y and Tashjian AH Jr
- Subjects
- Adenosine Diphosphate Ribose administration & dosage, Adenosine Diphosphate Ribose metabolism, Adenosine Diphosphate Ribose pharmacology, Animals, Caffeine administration & dosage, Caffeine pharmacology, Calcium Channels drug effects, Calcium Channels metabolism, Cyclic ADP-Ribose, Drug Synergism, Female, In Vitro Techniques, Kinetics, Sea Urchins, Second Messenger Systems, Adenosine Diphosphate Ribose analogs & derivatives, Calcium metabolism, Ovum drug effects, Ovum metabolism
- Abstract
Cyclic ADP-ribose (cADPR), an enzymatic product formed from NAD+ in sea urchin eggs and in several mammalian cell types, has been proposed as a second messenger for the regulation of cytosolic free Ca2+ concentration. Although cADPR induces Ca2+ release from an intracellular Ca2+ pool by a mechanism that differs from that used by inositol 1,4,5-trisphosphate, the precise action is unknown. We have analyzed the kinetics of cADPR-induced Ca2+ release at 15 degrees C in sea urchin egg homogenates using the calcium indicator fluo-3. In this system, Ca2+ release induced by cADPR was a positively cooperative process with a Hill coefficient of 1.8 +/- 0.32 (mean +/- SE, n = 15). We also examined the effect of caffeine, which potentiates the action of cADPR, on the kinetics of cADPR-induced Ca2+ release. Caffeine (3 mM) did not affect the degree of cooperativity induced by cADPR, but it did cause an increase in the apparent affinity of cADPR for its cellular target; the apparent Kd, derived from the kinetic analysis, decreased from 176 +/- 50 to 68 +/- 13 nM. We conclude that cADPR-induced Ca2+ release in sea urchin eggs is a cooperative process. A straightforward interpretation of the analysis is that opening of the Ca(2+)-release channel requires the binding of two molecules of cADPR (Hill coefficient of about 2). However, it is not yet known whether cADPR binds directly to the release channel. It is possible that potentiation of the action of cADPR by newly released Ca2+ could also contribute to the observed cooperative effect.
- Published
- 1995
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35. Alterations in the frequency and shape of Ca2+ fluctuations in GH4C1 cells induced by thyrotropin-releasing hormone and Bay K 8644.
- Author
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Brady KD, Wagner KA, Tashjian AH Jr, and Golan DE
- Subjects
- Action Potentials drug effects, Aniline Compounds, Animals, Cell Line, Fluorescence, Fluorescent Dyes, Kinetics, Pituitary Gland drug effects, Rats, Xanthenes, 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester pharmacology, Calcium metabolism, Pituitary Gland physiology, Thyrotropin-Releasing Hormone pharmacology
- Abstract
We have examined statistically the actions of thyrotropin-releasing hormone (TRH) and Bay K 8644, an L-type Ca(2+)-channel agonist, on the frequency and shape of cytosolic Ca2+ spikes in individual GH4C1 rat pituitary cells. TRH induced a brief (0-40 s) suppression of Ca2+ spikes followed by a period (40-200 s) of increased spike frequency. TRH treatment reduced the rate of rise and amplitude of Ca2+ spikes, and increased the rate of fall, relative to spontaneous spikes before treatment. TRH had no significant effect on the correlation between spike amplitude and the spike decay time constant tau, suggesting that the increased rate of fall was due to enhanced Ca2+ extrusion and not to decreased Ca(2+)-induced Ca2+ release. Bay K rapidly (t1/2 = 9-13 s) induced a 2-fold increase in the rate of rise of spikes with no change in the total rise time, leading to an increase in spike amplitude. It increased by 2-fold the fall time of spikes, as predicted solely by the previously observed relationship between spike amplitude and fall time. Bay K therefore appeared to increase the number of Ca2+ channels participating in each spike event without altering the kinetics of channel activation or deactivation, and without influencing Ca2+ extrusion. After addition of Bay K, the interval between spikes gradually (t1/2 approximately 100 s) increased, whereas the rate of rise remained constant and maximal. To explain these actions of TRH and Bay K, we postulate that a fraction of L-type Ca2+ channels are inactivated during each spike and must be re-activated in order to participate in a subsequent spike. We conclude further that the changes in spike frequency and profiles induced by these secretagogues are most consistent with a model in which TRH induces increases in both Ca2+ influx and efflux while Bay K induces a large increase in Ca2+ influx but has little effect on efflux.
- Published
- 1995
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36. Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other.
- Author
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Petrou CP and Tashjian AH Jr
- Subjects
- Animals, Anti-Bacterial Agents pharmacology, Cell Line, Cell Membrane metabolism, Cycloheximide pharmacology, Hydrogen-Ion Concentration, Kinetics, Methylation, Pituitary Gland metabolism, Rats, Macrolides, Receptors, Thyrotropin-Releasing Hormone metabolism, Thyrotropin-Releasing Hormone metabolism
- Abstract
We have examined the trafficking of the thyrotropin-releasing hormone receptor (TRHR) and its ligand, after TRHR-TRH internalization in rat pituitary GH4C1 cells. After rapid ligand-induced receptor sequestration, the cell surface receptor pool was replenished. Replenishment was insensitive to inhibition of protein synthesis and was dependent on the duration of internalization; therefore, the replenished receptors were not newly synthesized but recycled. The total amount of recycled receptors decreased with increasing internalization time, resulting in only partial replenishment of the cell-surface receptor pool after prolonged incubation with ligand. Thus, in addition to a receptor recycling pathway, a non-cycling route exists for TRHR sorting; this route became dominant with increasing internalization periods. TRHR entry into these pathways was not determined by the affinity of the receptor-ligand interaction, because the extent of receptor recycling was similar after TRH- and methyl-TRH (MeTRH)-induced internalization. Unlike results with the TRHR, the TRH recycling pool was not depleted by the noncycling pathway. After multiple rounds of [3H]MeTRH internalization, the amount of cell-associated radioactivity increased with increasing internalization time due to accumulation of the ligand or its metabolites in a non-cycling pathway, but the absolute amount of recycled ligand remained constant after short or long internalization times. The difference in the proportion of TRHR and MeTRH that were diverted into a noncycling pathway indicated intracellular dissociation of the internalized TRHR-TRH complex. Dissociation of the internalized TRHR-TRH complex was dependent on the acidic pH in an intracellular compartment. Although extracellular acidic pH did not enhance cell-surface receptor-ligand (RL) dissociation, bafilomycin A1 inhibited both receptor and ligand recycling. We conclude that the TRHR-TRH system is unique among recycling receptors because, after RL sequestration, the TRHR-TRH complex becomes dissociated intracellularly via a bafilomycin A1-sensitive, acidic pH-dependent mechanism, and both the unoccupied TRHR and TRH recycle disassociated from each other.
- Published
- 1995
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37. Kinetics and reversibility of thyrotropin-releasing hormone-stimulated guanine nucleotide exchange in membranes from GH4C1 cells.
- Author
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Brady KD, Han B, and Tashjian AH Jr
- Subjects
- Amino Acid Sequence, Binding Sites, Cell Line, Cell Membrane drug effects, GTP-Binding Proteins immunology, GTP-Binding Proteins metabolism, Immune Sera, Kinetics, Molecular Sequence Data, Pertussis Toxin, Virulence Factors, Bordetella pharmacology, Cell Membrane metabolism, Guanosine 5'-O-(3-Thiotriphosphate) metabolism, Thyrotropin-Releasing Hormone physiology
- Abstract
To evaluate the role of thyrotropin-releasing hormone (TRH)-stimulated guanine nucleotide exchange in the biphasic cellular responses to TRH, we have examined the kinetics, reversibility, and inhibition by QC120 (an antiserum recognizing the carboxyl terminus of alpha q/11) of TRH-stimulated guanosine-5'-(alpha-[35S] thio)triphosphate ([35S]GTP alpha S) binding in membranes from GH4C1 cells. Enhanced binding of [35S]GTP alpha S stimulated by TRH was dose dependent and readily detectable within 8 sec of TRH treatment. Binding measured within the first 20 sec was largely inhibited by QC120, whereas additional binding that accumulated during incubations of 3-6 min was not inhibited by even high concentrations of the antiserum. TRH-stimulated binding was reversible, in that, after membranes were incubated with TRH and [35S]GTP alpha S, subsequent addition of excess GTP caused exchange of 70-100% of the prebound radioligand. Exchange of TRH-stimulated [35S]GTP alpha S binding occurred in fast and slow phases, with half-times of < 5 sec and 187 sec, respectively. Addition of QC120 before the GTP chase inhibited the fast phase of exchange, whereas reduction of the TRH concentration in the preincubation selectively reduced the magnitude of the slow phase. Neither phase of exchange was affected by prior treatment of cells with pertussis toxin. Our observations indicate that Gq/11 is rapidly activated by the TRH receptor and that a second, unidentified, G protein is slowly activated by the TRH receptor.
- Published
- 1994
38. Structure-activity studies of the s-echistatin inhibition of bone resorption.
- Author
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Sato M, Garsky V, Majeska RJ, Einhorn TA, Murray J, Tashjian AH Jr, and Gould RJ
- Subjects
- Amino Acid Sequence, Animals, Bone Neoplasms pathology, Cell Adhesion drug effects, Cells, Cultured, Dose-Response Relationship, Drug, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Female, Giant Cell Tumor of Bone pathology, Humans, Intercellular Signaling Peptides and Proteins, Mice, Microscopy, Confocal, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Molecular Sequence Data, Organ Culture Techniques, Osteoclasts cytology, Osteoclasts ultrastructure, Platelet Aggregation drug effects, Platelet Aggregation Inhibitors pharmacology, Rats, Rats, Sprague-Dawley, Structure-Activity Relationship, Viper Venoms chemistry, Viper Venoms pharmacology, Bone Resorption drug therapy, Oligopeptides chemistry, Osteoclasts drug effects, Peptides, Platelet Aggregation Inhibitors therapeutic use, Viper Venoms therapeutic use
- Abstract
Synthetic Arg-Gly-Asp (RGD)-containing peptides were examined in bone resorption or attachment and detachment assays with isolated mammalian osteoclasts in an effort to elucidate the mechanistic and structural basis for the inhibition of bone resorption by s-echistatin. Bone resorption was the process most sensitive to inhibition by s-echistatin, with IC50 = 0.3 nM; inhibition of attachment to bone or detachment (lamellipodial retraction) was 30- to 70-fold less sensitive, with IC50 = 10 or 20 nM, respectively. Single amino acid substitutions within the 49-residue sequence of s-echistatin showed that although the efficacy of s-echistatin is dependent on the Arg24-Gly25-Asp26 sequence, additional residues, including Asp27, Met28, and Cys39, are also critical for potent inhibition of the resorbing activity of isolated rat osteoclasts. Because of the identification of the av beta 3 as the primary integrin on rat osteoclasts interacting the RGD peptides (Helfrich et al.), we examined the possibility of modeling bone resorption with other beta 3-mediated processes. Specifically, av beta 3 endothelial cell (human or rat) attachment to vitronectin and aIIb beta 3 platelet aggregation were compared with bone resorption for sensitivity to s-echistatin analogs, linear RGD peptides, and cyclic RGD peptides. Essentially no similarity in sensitivity to RGD peptides were observed between bone resorption, platelet aggregation, or endothelial cell attachment. Because rat osteoclasts and human giant cell tumors (osteoclastomas) shared similar sensitivity to s-echistatin and rat and human endothelial cells showed a similar sensitivity profile to RGD peptides, the dissimilarity of bone resorption to other beta 3-mediated processes cannot be explained in terms of species differences.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
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39. Relationships between amplitudes and kinetics of rapid cytosolic free calcium fluctuations in GH4C1 rat pituitary cells: roles for diffusion and calcium-induced calcium release.
- Author
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Brady KD, Wagner KA, Tashjian AH Jr, and Golan DE
- Subjects
- Aniline Compounds, Animals, Biophysical Phenomena, Biophysics, Cell Line, Cytosol metabolism, Diffusion, Fluorescent Dyes, Ion Transport physiology, Kinetics, Models, Biological, Rats, Xanthenes, Calcium metabolism, Pituitary Gland metabolism
- Abstract
We have examined statistical relationships between the amplitudes and the kinetics (rise times, fall times, and decay constants) of cytosolic free calcium fluctuations (spikes) in a population of 353 individual GH4C1 rat pituitary cells. The fast falling phase was approximated by a single exponential decay, and the decay time constant, tau, increased linearly with spike amplitude in 80% of the cells studied. The slope of the tau versus amplitude plot for each cell was inversely related to the cell's mean spike amplitude. Thus, some process responsible for prolonging the decay phase of spikes appeared to operate strongly in cells with spikes of low amplitude, but to become less prominent in cells with high amplitude spikes. Mean tau correlated more strongly with mean rise and fall times than with mean spike amplitude, indicating that the kinetic properties of spikes were not tightly coupled to spike amplitude. These findings are consistent with a model wherein the rise phase corresponds to entry of extracellular calcium via L-type calcium channels into localized sub-plasmalemmal domains, followed by diffusion of subplasmalemmal calcium into the cell interior; and the falling phase corresponds to further calcium diffusion combined with activation of cytoplasmic calcium-induced calcium release, which prolongs the falling phase.
- Published
- 1994
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40. Thimerosal potentiates Ca2+ release mediated by both the inositol 1,4,5-trisphosphate and the ryanodine receptors in sea urchin eggs. Implications for mechanistic studies on Ca2+ signaling.
- Author
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Tanaka Y and Tashjian AH Jr
- Subjects
- Adenosine Diphosphate Ribose analogs & derivatives, Adenosine Diphosphate Ribose pharmacology, Aniline Compounds, Animals, Caffeine pharmacology, Calcium Channels drug effects, Cyclic ADP-Ribose, Dithiothreitol pharmacology, Female, Fluorescent Dyes, Inositol 1,4,5-Trisphosphate metabolism, Inositol 1,4,5-Trisphosphate Receptors, Ionomycin pharmacology, Kinetics, Microsomes drug effects, Microsomes metabolism, Muscle Proteins drug effects, Ovum drug effects, Receptors, Cytoplasmic and Nuclear drug effects, Ryanodine metabolism, Ryanodine Receptor Calcium Release Channel, Sea Urchins, Signal Transduction drug effects, Xanthenes, Calcium metabolism, Calcium Channels metabolism, Inositol 1,4,5-Trisphosphate pharmacology, Muscle Proteins metabolism, Ovum metabolism, Receptors, Cytoplasmic and Nuclear metabolism, Ryanodine pharmacology, Signal Transduction physiology, Thimerosal pharmacology
- Abstract
Two intracellular Ca2+ release pathways are modulated by Ca2+ itself, namely Ca(2+)-induced Ca2+ release (classical CICR), and Ca(2+)-sensitized inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release (CSIICR). Both of these pathways are thought to be important in generating cytosolic Ca2+ oscillations. Caffeine and the sulfhydryl reagent thimerosal (TMS) are frequently used as selective modulators of CICR and CSIICR, respectively. We have studied the modes of action of caffeine and TMS in sea urchin egg homogenates using the Ca2+ indicator fluo-3. Cyclic ADP-ribose (cADPR), the only known endogenous compound to cause Ca2+ release via the ryanodine receptor, was used as an agonist for the classical CICR pathway. cADPR and InsP3 each alone caused release of sequestered Ca2+. Coaddition of caffeine dose-dependently and dramatically enhanced Ca2+ release induced by cADPR. InsP3-induced Ca2+ release was not affected by caffeine, but it was amplified by TMS, indicating the existence of the CSIICR pathway in this preparation. Surprisingly, cADPR-induced Ca2+ release was also markedly potentiated by TMS. TMS alone caused a slow increase in Ca2+ release which was not inhibited by heparin and/or procaine, indicating that TMS-induced Ca2+ release was not due to the actions of endogenous agonists (InsP3 or cADPR) on their receptors. All observed actions of TMS were abolished by dithiothreitol. We conclude: 1) both the InsP3- and cADPR-controlled Ca2+ release pathways are potentiated by TMS, while caffeine is specific for the cADPR pathway, indicating that TMS cannot be used as a selective reagent for probing the CSIICR mechanism; and 2) the action of TMS on intracellular Ca2+ release is independent of endogenous Ca2+ channel agonists, suggesting a novel action of TMS possibly on intravesicular Ca2+ handling.
- Published
- 1994
41. Involvement of alkaline phosphatase in the modulation of receptor signaling in osteoblasts: evidence for a difference between human parathyroid hormone-related protein and human parathyroid hormone.
- Author
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Fukayama S and Tashjian AH Jr
- Subjects
- Cell Line, Cyclic AMP metabolism, Humans, Intestines enzymology, Kidney enzymology, Osteoblasts ultrastructure, Parathyroid Hormone metabolism, Parathyroid Hormone pharmacology, Parathyroid Hormone-Related Protein, Placenta enzymology, Proteins metabolism, Proteins pharmacology, Receptor, Parathyroid Hormone, Type 1, Receptors, Parathyroid Hormone metabolism, Vasoactive Intestinal Peptide pharmacology, Alkaline Phosphatase physiology, Osteoblasts enzymology, Osteoblasts physiology, Receptors, Parathyroid Hormone analysis, Receptors, Parathyroid Hormone physiology, Signal Transduction physiology
- Abstract
We have previously reported that alkaline phosphatase (ALPase) is functionally involved in calcium uptake by several osteoblast-like cell lines. We have extended these studies to investigate the actions of ALPase on the cAMP response to and the receptor binding of human parathyroid hormone (hPTH) and human parathyroid hormone-related protein (hPTHrP). Pretreatment of human osteoblast-like SaOS-2 cells with human placental ALPase (hpALPase) inhibited the cAMP response to hPTH(1-34) but had no effect on the actions of hPTHrP(1-34) or vasoactive intestinal peptide. The inhibitory effect was reversed by L-Phe-Gly-Gly, an inhibitor of hpALPase. Treatment of SaOS-2 cells with hpALPase modestly reduced the binding of hPTH to 70% of control values, with little or no effect on the binding of hPTHrP. Bovine kidney and calf intestine ALPases were without effect on either the cAMP response or binding of hPTH or hPTHrP in SaOS-2 cells. In rat osteoblast-like ROS 17/2.8 cells, hpALPase had no effect on cAMP production stimulated by hPTH(1-34) or hPTHrP(1-34), arguing against a nonspecific effect of hpALPase. We suggest that, in SaOS-2 cells, the common PTH/PTHrP receptor can differentiate between the agonist activities of hPTH and hPTHrP by a mechanism that is sensitive to hpALPase.
- Published
- 1994
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42. Characterization of beta-adrenergic receptors on rat and human osteoblast-like cells and demonstration that beta-receptor agonists can stimulate bone resorption in organ culture.
- Author
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Moore RE, Smith CK 2nd, Bailey CS, Voelkel EF, and Tashjian AH Jr
- Subjects
- Animals, Base Sequence, Binding Sites, Bone and Bones drug effects, Bone and Bones metabolism, Cyclic AMP metabolism, DNA, Complementary metabolism, Humans, Mice, Molecular Sequence Data, Norepinephrine pharmacology, Oligonucleotides, Antisense, Organ Culture Techniques, Osteoblasts cytology, Osteoblasts metabolism, Phosphodiesterase Inhibitors pharmacology, Polymerase Chain Reaction, RNA, Messenger metabolism, Rats, Tumor Cells, Cultured, Adrenergic beta-Agonists pharmacology, Bone Resorption chemically induced, Osteoblasts drug effects, Receptors, Adrenergic, beta metabolism
- Abstract
We have shown by receptor-binding analyses that the beta-2 adrenergic receptor is present on rat ROS 17/2.8 osteoblast-like cells. This was confirmed by PCR amplification of cDNA copied from the mRNA. The beta-1 adrenoreceptor subtype was absent and its mRNA was not detectable, even at the level of sensitivity afforded by PCR analysis. The beta-adrenergic receptors present on ROS 17/2.8 cells were functional as measured by ligand-induced enhancement of cAMP production. We investigated whether adrenergic agonists could mimic the action of PTH to stimulate bone resorption in neonatal mouse calvariae in organ culture. PTH induced a large increase in cAMP while norepinephrine and isoproterenol induced a small but significant increase. In the presence of a phosphodiesterase inhibitor and an antioxidant, norepinephrine consistently stimulated bone resorption. In order to determine whether functional beta-adrenergic receptors were unique to ROS 17/2.8 cells, human SaOS-2 osteoblast-like cells were also examined for enhancement of cAMP production by norepinephrine, and essentially the same results were obtained. Thus, adrenergic agonists efficiently activate beta-receptors on two osteoblast-like cells and can stimulate bone resorption in intact mouse calvariae.
- Published
- 1993
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43. Functional identification and quantitation of three intracellular calcium pools in GH4C1 cells: evidence that the caffeine-responsive pool is coupled to a thapsigargin-resistant, ATP-dependent process.
- Author
-
Tanaka Y and Tashjian AH Jr
- Subjects
- Adenosine Triphosphatases antagonists & inhibitors, Animals, Cells, Cultured, Drug Resistance, Inositol 1,4,5-Trisphosphate pharmacology, Microsomes drug effects, Microsomes metabolism, Rats, Thapsigargin, Adenosine Triphosphate metabolism, Caffeine pharmacology, Calcium metabolism, Terpenes pharmacology
- Abstract
We have recently reported that basal oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) in intact GH4C1 cells are dependent on a Ca(2+)-induced Ca2+ release (CICR) mechanism. The purpose of the present study was to characterize the uptake and release pathways for intracellular Ca2+ in GH4C1 cells. We have used both permeabilized cells and microsome preparations, and we have monitored the change in ambient [Ca2+] using the dye, fluo 3. We find that there are three functionally distinct nonmitochondrial, ATP-dependent Ca2+ pools in these cells: Pool 1 is an inositol 1,4,5-trisphosphate (InsP3) responsive pool which is filled by a thapsigargin (Tg) sensitive Ca(2+)-ATPase; pool 2 is a second Tg-sensitive pool which is InsP3-unresponsive; and pool 3 is a Tg-resistant pool, at least a part of which has the characteristics of a CICR mechanism. These pools were established as follows. Tg caused additional Ca2+ release after maximum release was induced by prior addition of InsP3. In contrast, the InsP3 response was abolished in a time-dependent manner after pretreatment with Tg. Ambient Ca2+, added after maximum blockade by Tg, was still able to be sequestered. Ionomycin released Ca2+ even after maximum depletion by Tg. The ionomycin-releasable pool remaining after Tg treatment was also ATP-dependent, because this pool was completely discharged by ATP-depletion. Two additional inhibitors of intracellular Ca(2+)-ATPases, 2,4-di(tert-butyl)hydroquinone and cyclopiazonic acid, which are structurally unrelated to Tg, acted on the same targets as Tg. To estimate accurately the distribution of Ca2+ among compartments, we developed a new approach based on the analysis of two equilibrium states of Ca2+ distribution. Using this method, the size of the Tg-sensitive pools (pools 1 + 2) was estimated to be 63 +/- 2.5% of total non-mitochondrial Ca2+ in our preparation. Caffeine induced Ca2+ release, and this action was observed even after complete depletion of the Tg-sensitive pool, indicating that pool 3 had the characteristics of a CICR compartment. Because caffeine pretreatment caused an increase in the size of pools 1 + 2, the CICR-like mechanism operated primarily on pool 3. These new results strengthen our model, in which a distinct CICR-like pool is responsible for Ca2+ oscillations in GH4C1 cells, and also support the concept that different types of Ca2+ efflux pathways occur in Ca(2+)-storing nonmitochondrial organelles containing different types of Ca(2+)-ATPases.
- Published
- 1993
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44. Insulin-like growth factor-I inhibits parathyroid hormone-stimulated and enhances prostaglandin E2-stimulated adenosine 3',5'-monophosphate production by human osteoblast-like SaOS-2 cells.
- Author
-
Goad DL and Tashjian AH Jr
- Subjects
- Cell Line, Dose-Response Relationship, Drug, Humans, Insulin pharmacology, Insulin-Like Growth Factor II pharmacology, Vasoactive Intestinal Peptide pharmacology, Cyclic AMP antagonists & inhibitors, Cyclic AMP biosynthesis, Dinoprostone pharmacology, Insulin-Like Growth Factor I pharmacology, Osteoblasts metabolism, Parathyroid Hormone pharmacology
- Abstract
Insulin-like growth factor-1 (IGF-I), an endocrine and autocrine/paracrine factor that enhances collagen synthesis and bone matrix formation by osteoblasts, has been implicated in the coupling of bone formation with bone resorption. We have found, using SaOS-2 osteoblastic cells, that IGF-I inhibits PTH-stimulated cAMP production. Pretreatment of SaOS-2 cells with IGF-I for 24 h inhibited cAMP production stimulated by PTH with an IC50 of 1 nM and maximal inhibition to 10-20% of control values at an IGF-I concentration of 10 nM. Pretreatment with IGF-I had no effect on vasoactive intestinal peptide-stimulated cAMP production, but it enhanced cAMP production stimulated by prostaglandin E2 (PGE2) by as much as 5-fold at a concentration of 10 nM and with an EC50 of 1 nM. Pretreatment of SaOS-2 cells with IGF-I did not affect cholera toxin- or forskolin-stimulated cAMP accumulation. Taken together, these findings indicated that IGF-I did not affect Gs alpha, coupling of Gs alpha to adenylate cyclase, or adenylate cyclase itself. Binding experiments using [125I] chicken PTH-related peptide (PTHrP)-(1-36)-[Tyr36]NH2 demonstrated that IGF-I reduced PTH/PTHrP receptor number to 25% of the control value without affecting receptor affinity. IGF-I and the related growth factors, insulin and IGF-II, inhibited PTH-stimulated cAMP production with a rank order of potency of IGF-I > or = IGF-II > insulin, indicating that the actions of IGF-I on SaOS-2 cells were probably mediated by the IGF-I receptor. We conclude that physiologically relevant concentrations of IGF-I specifically inhibited PTH-stimulated and enhanced PGE2-stimulated production of cAMP by an action at the level of PTH and PGE2 receptors and/or coupling of the receptors to Gs alpha.
- Published
- 1993
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45. Regulation of endogenous thyrotropin-releasing hormone receptor messenger RNA in GH4C1 cells: roles of protein and RNA synthesis.
- Author
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Yang J and Tashjian AH Jr
- Subjects
- Animals, Base Sequence, Blotting, Northern, Cell Line, Cell Nucleus metabolism, Cycloheximide pharmacology, Dactinomycin pharmacology, Dexamethasone pharmacology, Dichlororibofuranosylbenzimidazole pharmacology, Gene Expression drug effects, Kinetics, Molecular Sequence Data, Oligodeoxyribonucleotides, Pituitary Neoplasms, Protein Biosynthesis drug effects, RNA, Messenger analysis, RNA, Messenger biosynthesis, RNA, Ribosomal, 18S analysis, RNA, Ribosomal, 18S biosynthesis, Rats, Receptors, Thyrotropin-Releasing Hormone metabolism, Transcription, Genetic drug effects, Tumor Cells, Cultured, RNA, Messenger metabolism, Receptors, Thyrotropin-Releasing Hormone biosynthesis
- Abstract
In previous studies on the regulation of the endogenous TRH receptor (TRHR) in GH4C1 cells, we have shown that the synthetic glucocorticoid dexamethasone (Dex) increased TRHR abundance on the plasma membrane of these cells as well as increasing the concentration of TRHR mRNA. In the present investigation, we determined whether the action of Dex on TRHR mRNA was direct or whether it involved the synthesis of intermediary protein(s). We found that the protein synthesis inhibitors cycloheximide or anisomycin alone enhanced TRHR mRNA accumulation and, in the presence of Dex, caused a 10- to 20-fold superinduction of TRHR mRNA. The superinduction required inhibition of protein synthesis. Results of nuclear run-on assays showed that superinduction was accompanied by a large increase in transcription of TRHR mRNA. Experiments designed to examine the stability of TRHR mRNA transcripts revealed that inhibitors of RNA synthesis (actinomycin D and dichlorobenzimidazole riboside) caused an increase in endogenous TRHR mRNA concentrations by themselves, complicating their use to estimate TRHR mRNA half-life. We conclude that Dex stimulates transcription of endogenous TRHR mRNA by a mechanism that does not require enhanced synthesis of new proteins. In fact, ongoing protein synthesis reduces steady state TRHR mRNA concentrations consistent with a role for a labile protein(s) in suppressing TRHR gene transcription; an additional action of a labile protein to enhance TRHR mRNA degradation is also possible.
- Published
- 1993
- Full Text
- View/download PDF
46. Transcriptional regulation by dexamethasone of endogenous thyrotropin-releasing hormone receptor messenger ribonucleic acid in rat pituitary GH4C1 cells.
- Author
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Yang J and Tashjian AH Jr
- Subjects
- Animals, Blotting, Northern, Cell Line, Kinetics, Rats, Receptors, Thyrotropin-Releasing Hormone, Dexamethasone pharmacology, Pituitary Gland metabolism, RNA, Messenger metabolism, Receptors, Neurotransmitter genetics, Transcription, Genetic drug effects
- Abstract
Previous molecular studies on the mechanism of regulation of TRH receptor (TRHR) mRNA used transfected cells and concluded that the major mode of regulation is via mRNA degradation. Glucocorticoid hormones induce an increase in the number of TRHRs in rat pituitary GH cells, the model system used most extensively to characterize these receptors. We prepared a probe from the recently cloned rat lactotroph TRHR cDNA and investigated the mechanism of regulation by dexamethasone of endogenous TRHR mRNA in GH4C1 cells. Incubation with dexamethasone (100 nM for 6-48 h) induced a 3-to 4-fold increase in the steady state TRHR mRNA concentration (maximum at 24 h), as determined by Northern blot analysis. The stimulation was specific for glucocorticoids and dose dependent over the range 1-500 nM (maximum effect reached at 10 nM). The rate of transcription of the TRHR gene, measured by a nuclear run-on assay, was increased 3.0 +/- 0.57-fold (mean +/- SE) in cells treated for 12 h with 100 nM dexamethasone. We conclude that glucocorticoids regulate endogenous TRHR mRNA expression in GH4C1 cells in large part by controlling the rate of transcription of the TRHR gene.
- Published
- 1993
- Full Text
- View/download PDF
47. Regulation of endogenous thyrotropin-releasing hormone (TRH) receptor messenger RNA by TRH in GH4C1 cells.
- Author
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Yang J and Tashjian AH Jr
- Subjects
- Animals, Calcium metabolism, Cell Line, Enzyme Activation drug effects, Ionomycin pharmacology, Kinetics, Pituitary Gland drug effects, Pituitary Gland metabolism, Protein Biosynthesis, Protein Kinase C metabolism, Rats, Receptors, Thyrotropin-Releasing Hormone, Second Messenger Systems, Tetradecanoylphorbol Acetate pharmacology, RNA, Messenger metabolism, Receptors, Neurotransmitter genetics, Thyrotropin-Releasing Hormone pharmacology, Transcription, Genetic drug effects
- Abstract
Incubation with TRH causes a decrease (over 2-24 h) in the number of TRH receptors (TRHRs) in GH4C1 rat pituitary cells. Using a homologous cDNA probe prepared from the TRHR cloned from these cells, we demonstrate here that TRH modulates the rate of transcription of TRHR mRNA. Incubation of GH4C1 cells for 4 h with TRH caused an 80% reduction in TRHR mRNA concentration. The TRHR mRNA level remained low for 48 h in the continued presence of TRH. Run-on transcription assays demonstrated that TRH caused a decrease in TRHR mRNA transcription of about 60% at 4 h. The rate of transcription remained low at 12 h. Inhibition of protein synthesis did not prevent the TRH-induced decrease in TRHR mRNA. Activation of protein kinase C with the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate decreased TRHR mRNA by about 40% in 4 h. Elevation of [Ca2+]i with ionomycin decreased TRHR mRNA by about 25%. The two agents together decreased TRHR mRNA by approximately 70% in 4 h, an effect comparable to that elicited by TRH. We conclude that regulation of endogenous TRHR mRNA by TRH is modulated, at least in part, at the level of transcription by a mechanism that probably involves both activation of protein kinase C and an increase in [Ca2+]i.
- Published
- 1993
- Full Text
- View/download PDF
48. Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells.
- Author
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Zhao D, Yang J, Jones KE, Gerald C, Suzuki Y, Hogan P, Chin WW, and Tashjian AH Jr
- Subjects
- Amino Acid Sequence, Animals, Molecular Sequence Data, Receptors, Thyrotropin-Releasing Hormone, DNA genetics, Rats genetics, Receptors, Neurotransmitter genetics
- Published
- 1993
- Full Text
- View/download PDF
49. Mechanism of spontaneous intracellular calcium fluctuations in single GH4C1 rat pituitary cells.
- Author
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Wagner KA, Yacono PW, Golan DE, and Tashjian AH Jr
- Subjects
- 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester pharmacology, Animals, Cells, Cultured, Membrane Potentials, Pituitary Gland cytology, Pituitary Gland drug effects, Rats, Thyrotropin-Releasing Hormone pharmacology, Calcium metabolism, Pituitary Gland metabolism
- Abstract
Individual unstimulated GH4C1 cells exhibited spontaneous dynamic fluctuations in cytosolic free Ca2+ concentration ([Ca2+]i). Either chelation of extracellular Ca2+ with EGTA or treatment with nifedipine inhibited spontaneous [Ca2+]i fluctuations, indicating that the [Ca2+]i profile was dependent on the entry of extracellular Ca2+ via voltage-operated Ca2+ channels (VOCC). Spontaneous [Ca2+]i fluctuations did not resume immediately after exposure of EGTA-pretreated cells to extracellular Ca2+, supporting the hypothesis that the complex [Ca2+]i profiles observed in unstimulated cells required filling of an intracellular Ca2+ pool. BAY K 8644 elicited large rapid oscillations in [Ca2+]i. After chelation of extracellular Ca2+, however, re-addition of Ca2+ plus BAY K 8644 did not result in [Ca2+]i oscillations. The intracellular Ca2+ pool necessary for BAY K-induced oscillations was not the same Ins(1,4,5)P3-sensitive pool stimulated by thyrotropin-releasing hormone (TRH), because the TRH-stimulated Ins(1,4,5)P3-induced [Ca2+]i spike and the BAY K 8644-induced oscillations were differentially sensitive to chelation of extracellular Ca2+ and thapsigargin. Caffeine caused an increase in [Ca2+]i fluctuations in quiescent cells, supporting a role for Ca(2+)-induced Ca2+ release (CICR) in the generation of spontaneous [Ca2+]i fluctuations. In conclusion, the complex spontaneous changes in [Ca2+]i observed in single GH4C1 cells depend on both the influx of extracellular Ca2+ through VOCC and the action of an intracellular Ca2+ pool that increases [Ca2+]i through a CICR-like mechanism.
- Published
- 1993
- Full Text
- View/download PDF
50. Production of prolactin by smooth muscle cells cultured from human uterine fibroid tumors.
- Author
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Nowak RA, Rein MS, Heffner LJ, Friedman AJ, and Tashjian AH Jr
- Subjects
- Base Sequence, Cells, Cultured, Female, Humans, Leiomyoma pathology, Molecular Probes genetics, Molecular Sequence Data, Muscle, Smooth pathology, Polymerase Chain Reaction, Prolactin genetics, RNA, Messenger metabolism, Tumor Cells, Cultured, Uterine Neoplasms pathology, Leiomyoma metabolism, Muscle, Smooth metabolism, Prolactin biosynthesis, Uterine Neoplasms metabolism
- Abstract
Uterine leiomyomas, which are myometrial smooth muscle tumors, secrete PRL. We investigated the actions of several hormones known to stimulate PRL secretion by the pituitary gland or decidua on PRL secretion by leiomyoma-derived smooth muscle cells (SMC) in monolayer culture. Cultures were verified to be SMC by immunostaining for smooth muscle alpha-action and desmin. Hormone treatments were performed in serum-free medium for 72 h. Medium was harvested every 24 h and assayed for PRL. 17 beta-Estradiol, progesterone, TRH, insulin-like growth factor-I, epidermal growth factor, and the GnRH agonist leuprolide did not affect PRL secretion by these SMC. Insulin caused a significant suppression of PRL secretion by 72 h, and this was accompanied by a 64% increase in total cell protein per well, which represented an increase in cell number. Cells were also plated at various densities to determine the effects of cell number on PRL secretion. The amount of PRL secreted per 1000 cells decreased significantly as cell number per well increased. Northern blot analysis identified PRL mRNA in fresh leiomyoma tissue. PRL mRNA in three independent cultures of SMC was then detected by reverse transcription and the polymerase chain reaction. Hybridization occurred only with the expected band of approximately 423 basepairs in size. We conclude that leiomyomas express PRL mRNA in vivo and that leiomyoma-derived SMC in culture continue to express the PRL mRNA and secrete PRL in the absence of ovarian steroids. PRL secretion by SMC in culture appears to be modulated primarily by changes in cell density.
- Published
- 1993
- Full Text
- View/download PDF
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