Search

cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation., (© 2015 Wiley Periodicals, Inc.)

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)-α and interleukin (IL)-1β on cyclic AMP-phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF-α or IL-1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL-1β but remained suppressed with TNF-α through 24 h. Cyclic AMP was also reduced in TNF-α-stimulated primary microglia, albeit to a lesser extent. Accompanying TNF-α-induced cyclic AMP reductions, but not IL-1β, was increased cyclic AMP-PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF-α; IL-1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF-α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF-α but not IL-1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX-42(+) microglia; PDE4B was absent in OX-42(+) cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up-regulation from 24 h to 1 wk post-SCI, the peak of microglia activation. These studies show that TNF-α and IL-1β differentially affect cyclic AMP-PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases., (Copyright © 2012 Wiley Periodicals, Inc.)

During maturation, oocytes must undergo a process of nuclear disassembly, or "germinal vesicle breakdown" (GVBD), that is regulated by signaling pathways involving cyclic AMP (cAMP). In vertebrate and starfish oocytes, cAMP elevation typically prevents GVBD. Alternatively, increased concentrations of intra-oocytic cAMP trigger, rather than inhibit, GVBD in several groups of marine invertebrates. To integrate what is known about the stimulation of GVBD by intra-oocytic cAMP, this article reviews published data for ascidian, bivalve, brittle star, jellyfish, and nemertean oocytes. The bulk of the review concentrates on the three most intensively analyzed groups known to display cAMP-induced GVBD-nemerteans, ascidians, and jellyfish. In addition, this synopsis also presents some previously unpublished findings regarding the stimulatory effects of intra-oocytic cAMP on GVBD in jellyfish and the annelid worm Pseudopotamilla occelata. Finally, factors that may account for the currently known distribution of cAMP-induced GVBD across animal groups are discussed., (Copyright © 2011 Wiley Periodicals, Inc.)

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis., (Copyright © 2011 Wiley Periodicals, Inc.)

Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures., (Copyright © 2011 Wiley Periodicals, Inc.)

Adenosine is an important mediator of inhibition of platelet activation. This metabolite is released from various cells, as well as generated via activity of ecto-enzymes on the cell surface. Binding of adenosine to A(2) subtypes (A(2A) or A(2B)), G-protein coupled adenosine receptors, results in increased levels of intracellular cyclic adenosine monophosphate (cAMP), a strong inhibitor of platelet activation. The role and importance of adenosine and its receptors in platelet physiology are addressed in this review, including recently identified roles for the A(2B) adenosine receptor as a modulator of platelet activation through its newly described role in the control of expression of adenosine diphosphate (ADP) receptors.

Secretin plays a key role in the regulation of normal cholangiocyte physiology via secretin receptor (SCTR). SCTR expression is upregulated during extrahepatic cholestasis induced by bile duct ligation and closely associated with cholangiocyte proliferative responses. Although well studied in normal cholangiocytes, the role of secretin and the expression of SCTR in the regulation of cholangiocarcinoma proliferation are unknown. In vitro, secretin (10(-7) M) displayed differential effects on normal cholangiocyte [H-69 and human intrahepatic biliary epithelial cell line (HIBEpiC)] and cholangiocarcinoma (Mz-ChA-1, HuH-28, TFK-1, SG231, CCLP1 and HuCC-T1) cell lines as such secretin is mitogenic for normal cholangiocytes and antiproliferative for cholangiocarcinoma. As expected in normal cholangiocytes (HIBEpiC), secretin increased intracellular cyclic adenosine monophosphate (cAMP) levels. However, the effect of secretin on intracellular cAMP levels was suppressed in Mz-ChA-1 cells. Secretin-stimulated intracellular cAMP levels in Mz-ChA-1 were restored by pretreatment with pertussis toxin, suggesting that the receptor coupled to Galpha(i) rather than Galpha(s). SCTR expression was found to be downregulated in 4 of the 6 cholangiocarcinoma cell lines evaluated and in human cholangiocarcinoma biopsy samples. In vivo, secretin significantly inhibited the tumor size and more than doubled tumor latency, which was associated with a decrease in proliferating cell nuclear antigen and an increase in cleaved-caspase 3 expression levels. Our results demonstrate that secretin and/or the modulation of SCTR expression might have potential as a therapeutic tool in the treatment of cholangiocarcinoma.

The aim of the current study was to investigate the cAMP-dependent regulation of arginase-1 (ARG1) expression in RAW-macrophages. Basal ARG1 mRNA expression was low and increased upon incubation with the cAMP analogue Br-cAMP. We used selective agonists of protein kinase A type I (PKAI), type II (PKAII) and exchange protein directly activated by cAMP (EPAC) to determine the pathway responsible for ARG1 expression. Activation of PKAI led to a significant up-regulation of ARG1 mRNA expression and arginase enzyme activity. In contrast, neither activation of PKAII nor activation of EPAC affected ARG1 expression. In addition, it has been shown that histone deacetylase (HDAC) activity plays a critical role in cAMP-dependent transcriptional regulation. Incubation with Br-cAMP and the HDAC inhibitor trichostatin A (TSA) led to a concentration-dependent suppression of ARG1 expression. These data indicate that cAMP-dependent activation of ARG1 expression is mediated by PKAI and requires histone deacetylation., ((c) 2007 Wiley-Liss, Inc.)

The factors regulating astroglial resting membrane potential (RMP) are unresolved. Here, we have examined developmental, morphological, and intracellular factors that may regulate the RMP in astrocytes of isolated intact optic nerves of rats and mice aged postnatal day (P3) to adult. The astroglial RMP ranged from -25 to -85 mV, independent of age and morphological phenotype. There was a developmental negative shift in the astroglial RMP from a non-Gaussian distribution in perinatal nerves, to a bimodal distribution of RMPs after P15, with peaks at -52 and -74 mV in adults. Blockade of Kir with 100 microM BaCl(2) significantly depolarized the RMP to -30 mV; the RMP was unaffected by TEA or agents that modulated ATP-sensitive potassium channels. Raising intracellular cyclic AMP (cAMP) with dbcAMP or forskolin induced a significant hyperpolarization by approximately 15 mV, whereas inhibition of cAMP-dependent protein kinase (PKA) depolarized the astroglial RMP to -40 mV. The hyperpolarizing action of dbcAMP was blocked by 100 microM BaCl(2). The effects of BaCl(2) indicate that the developmental negative shift in the RMP and the cAMP-mediated hyperpolarization were dependent on Kir. This study provides evidence that the heterogeneous RMP of mature astrocytes is regulated by cAMP and PKA signaling.

The removal by phagocytosis of degenerated myelin is central for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). We tested for roles played by the cAMP cascade in the regulation of myelin phagocytosis mediated by complement receptor-3 (CR3/MAC-1) and scavenger receptor-AI/II (SRAI/II) separately and combined in mouse microglia and macrophages. Components of the cAMP cascade tested are cAMP, adenylyl cyclase (AC), Gi, protein kinase A (PKA), exchange protein directly activated by cAMP (Epac), and phosphodiesterases (PDE). PKA inhibitors H-89 and PKI(14-22) amide inhibited phagocytosis at normal operating cAMP levels (i.e., those occurring in the absence of reagents that alter cAMP levels), suggesting activation of phagocytosis through PKA at normal cAMP levels. Phagocytosis was inhibited by reagents that elevate endogenous cAMP levels to above normal: Gi-inhibitor Pertussis toxin (PTX), AC activator Forskolin, and PDE inhibitors IBMX and Rolipram. Phagocytosis was inhibited also by cAMP analogues whose addition mimics abnormal elevations in endogenous cAMP levels: nonselective 8-bromo-cAMP, PKA-specific 6-Benz-cAMP, and Epac-specific 8-CPT-2'-O-Me-cAMP, suggesting that abnormal high cAMP levels inhibit phagocytosis through PKA and Epac. Altogether, observations suggest a dual role for cAMP and PKA in phagocytosis: activation at normal cAMP levels and inhibition at higher. Furthermore, a balance between Gi-controlled cAMP production by AC and cAMP degradation by PDE maintains normal operating cAMP levels that enable efficient phagocytosis.

Previous studies have indicated that digoxin (DG) inhibits testosterone production by rat testicular interstitial cells through both in vivo and in vitro experiments. DG and digitoxin (DT), but not ouabain, inhibit the progesterone, pregnenolone, and corticosterone secretion by rat granulosa cells, luteal cells, and zona fasciculata-reticularis (ZFR) cells, respectively. However, the effect of DG and DT on the enzyme kinetics of cytochrome P450 side chain cleavage enzyme (P450scc), the protein expression of P450scc and steroidogenic acute regulatory protein (StAR), and mRNA expression of StAR are unclear. ZFR cells were prepared from adrenocortical tissues of ovariectomized rats, and then challenged with adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, A23187, cyclopiazonic acid (CPA), nicotinic acid adenine dinucleotide phosphate (NAADP), trilostane, 25-OH-Cholesterol, progesterone, or deoxycorticosterone in the presence of DG, DT, or ouabain for 1 h. Enzyme kinetics of P450scc, protein expression of acute regulatory protein (StAR) and P450scc, and mRNA expression of StAR were investigated. DG and DT but not ouabain suppressed basal and other evoked-corticosterone release significantly. DG and DT also inhibited pregnenolone production. The Vmax of the DG and DT group was the same as the control group, but the Km was higher in DG- and DT-treated group than in control group. DT and ouabain significant suppressed mRNA expression of StAR. DG and DT had no effect on the P450scc and StAR protein expression at basal state, but diminished ACTH-induced StAR protein expression to basal level. These results indicated that DG and DT have an inhibitory effect on corticosterone production via a Na+, K+-ATPase-independent mechanism by diminishing actions on cAMP-, Ca2+-pathway, competitive inhibition of P450scc enzyme and reduction of StAR mRNA expression., (Copyright 2005 Wiley-Liss, Inc.)

The role of cyclic adenosine monophosphate (cAMP) is poorly understood in the regulation of normal and abnormal hepatic cell growth. In this study, we examined the regulation of intracellular cAMP levels and its effect on nuclear cAMP responsive elements (CREs) in a rat model of hepatocellular carcinoma (HCC). Tumorigenic liver cells were cultured from an in vivo model of HCC and the role of cAMP in cell mitogenesis determined. These data demonstrated agents that elevate intracellular cAMP ([cAMP]i) levels caused significant dose-dependent inhibition of serum-stimulated mitogenesis in HCC cells. Cells were next analyzed for transcription factor expression and activity following increased [cAMP]i. These data demonstrated time- and dose-dependent increases in CRE binding protein (pCREB) activity, a maximal response occurring after 10-20 min before returning to basal levels within 60 min. In contrast, increased [cAMP]i levels led to sustained inducible cAMP early repressor (ICER) II/IIgamma mRNA and protein induction. To understand these data in relation to the in vivo setting, HCC tumors were analyzed and compared to pair-matched normal liver (NL) samples. These studies demonstrated significantly elevated Gsalpha-protein expression in HCC versus NL in the absence of significant changes in basal cAMP levels. Analysis of total and active CREB demonstrated significantly increased total CREB/pCREB in HCC versus NL. Further analysis of CRE expression demonstrated significantly increased expression of ICER mRNA and protein in HCC versus sham operated (Sh). These data demonstrate cAMP, while capable of stimulating promitogenic CREB activation inhibits cell mitogenesis in HCC possibly via ICER induction., (Copyright (c) 2005 Wiley-Liss, Inc.)

Cyclic AMP (cAMP) is a pleiotropic second messenger that regulates numerous cellular processes. In vascular smooth muscle cells (VSMCs), these include cell proliferation, migration, and contractility. Here we show that cAMP-elevating agents induce dramatic morphological changes in VSMCs, characterized by cell rounding and formation of long branching processes. The stellate morphology is associated with disassembly of actin stress fibers and lamellipodia, loss of focal adhesions, and the formation of small F-actin rings. Because of the importance of Rho family GTPases in regulating actin dynamics, we analyzed their individual roles in the cAMP phenotype. We found that pharmacological or genetic inhibition of Rac mimics cAMP effect in inducing a stellate morphology of VSMCs. Expression of activated Rac1 prevents forskolin-induced cAMP stellation, suggesting that cAMP affects cell morphology by inhibiting Rac function. Consistent with this, treatment with forskolin inhibits agonist-stimulated Rac activation in VSMCs. We further show that activated Rac1 containing the F37A effector loop substitution fails to rescue the cAMP phenotype. Our results suggest that cAMP modulates the morphology of VSMCs by inhibiting a Rac-dependent signaling pathway., ((c) 2005 Wiley-Liss, Inc.)

5-Hydroxytryptamine (5-HT), a neurotransmitter and neuromodulator in the central nervous system of the leech Hirudo medicinalis hyperpolarizes the giant glial cell in the neuropil of segmental ganglia at micromolar concentrations. The 5-HT-evoked glial response (EC(50) approximately 2.5 microM) is mediated by a non-desensitizing, G-protein-coupled receptor and due to activation of a Ca(2+)-independent K(+) conductance. The adenylyl cyclase inhibitor SQ22,536 blocks the response to 5-HT; in the presence of 1 mM db-cAMP, but not of 1 mM db-cGMP, the glial response is suppressed. The 5-HT-evoked response is reduced by Ba(2+) with half-maximal inhibition at 50 microM Ba(2+). The results suggest that release of 5-HT from serotonergic neurons, or the maintenance of micromolar levels of extracellular 5-HT in the ganglion, may help to set the glial membrane potential close to the K(+) equilibrium potential., (Copyright 2004 Wiley-Liss, Inc.)

In morphogenetic dynamics of neurons, and in adaptive physiology of brain function, transcription factors of the cyclicAMP signaling pathway, such as activator cyclicAMP responsive element binding protein (CREB) and inhibitor inducible cyclicAMP early repressor (ICER), play an important role. In particular, the presence of the transcription factor ICER in neurons or neuroendocrine cells suggests the need for the gating of an up-regulated gene expression. Little is known, however, about the natural distribution of the inhibitory transcription factor ICER. We, therefore, mapped the rodent brain and pituitary for an ICER immunoreaction and found a nuclear staining for this transcription factor. ICER-positive glial cells were found throughout the brain. ICER-positive neurons were found in sensory input centers, like the olfactory bulb, or sensory brain stem nuclei, and in hypothalamic nuclei involved in central neuroendocrine control. In addition, neuroendocrine/endocrine transducers, like the pituitary and the pineal gland showed a high basal presence of ICER. Our data show that a basic ICER level is required by many cell systems and can be seen as an anticipatory and/or a protective measure in systems with superior reactive dynamics., (2004 Wiley-Liss, Inc.)

Background: Mitochondrial aconitase (mACON), an iron-requiring enzyme, is a major target of nitric oxide (NO) in cells, which causes the oxidant-mediated disruption of the [4Fe-4S] prosthetic group of the enzyme. In this study, the effect of NO on mACON enzymatic activity and gene expression were investigated., Methods: Three NO generators, sodium nitroprusside (SNP), S-nitoso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN) were used to determine the regulation of mACON enzymatic activity by NO. The effect of SNP on mACON, which modulates citrate secretion and cellular bioenergetics in PC-3 cells, was investigated by determining the effect of SNP on mACON gene expression using Western blot and transient gene expression assays., Results: SNP upregulated mACON enzymatic activity and gene expression in PC-3 cells. However, treating cells with other NO generators, SNAP and SIN, resulted in decreased mACON enzymatic activity. The addition of ascorbic acid to the SNP treatment resulted in a decrease in mACON enzymatic activity and gene expression. Our results showed that both SNP and dibutyryl-cAMP increased the mACON promoter activity 2-fold while the effect was blocked by adding H-89. Mutation of the cAMP response element (CRE) to the AGAGCT abolished the activating effects of SNP and dibutyryl-cAMP on mACON promoter activity., Conclusions: These results establish the function of nitroprusside as a signaling molecule for mACON gene expression through the cAMP signal transduction pathway in human prostatic carcinoma cells., (Copyright 2004 Wiley-Liss, Inc.)

The adenylate cyclase (AC)/cAMP/cAMP-dependent protein kinase pathway controls many biological phenomena. The molecular mechanisms by which cAMP induces alternative commitment towards differentiation or proliferation are not still completely known. The differentiation of myoblast cell lines into myocytes/myotubes represents a well-established model of skeletal muscle differentiation. We analyzed the AC/cAMP pathway during terminal differentiation of H9c2 myoblasts. When cultured in low-serum containing medium, H9c2 myoblasts exit the cell cycle and differentiate into myocytes/myotubes. A key step of this process is the expression of myogenin, an essential transcription factor for the terminal differentiation into myocytes. During this phenomenon we observed a decrease in both cAMP levels and AC activity, which suggests a functional negative role of cAMP on the differentiation process of H9c2 cells. 8-Br-cAMP and other cAMP-elevating agents, such as forskolin, IBMX, and isoproterenol, negatively affected skeletal muscle differentiation of H9c2 myoblasts. Both AC activity down-regulation and intracellular cAMP reduction were accompanied by significant variations in the levels of membrane proteins belonging to the AC system (AC catalytic subunit, G(alphai-1), G(alphas)). The functional relationship between intracellular cAMP content and protein levels of AC system is discussed., (Copyright 2003 Wiley-Liss, Inc.)

Steroidogenic acute regulatory protein (StAR) plays a key role in the availability of cholesterol to the inner mitochondrial membrane, where the first step of steroidogenesis, its conversion to pregnenolone, takes place. Here, we demonstrate for the first time that the StAR gene is also expressed in the rat sciatic nerve and in cultured Schwann cells. The addition to the culture medium of the cAMP-elevating agent forskolin or of the cAMP analogue 8Br-cAMP produced a time-course extinction of StAR gene expression. An inverse relationship was demonstrated between StAR gene expression and the intracellular cAMP content. Accordingly, pharmacological inhibition of the activities of Schwann cell adenylyl cyclase or of phosphodiesterase IV resulted in modifications of StAR gene expression. Since StAR gene expression is stimulated by cAMP in classical steroidogenic cells, our work is the first demonstration of a negative regulation of StAR gene by cAMP., (Copyright 2003 Wiley-Liss, Inc.)

Adenosine is an immunosuppressive molecule that is associated with the microenvironment of solid tumors. Mouse T cells activated with anti-CD3 antibody in the presence of adenosine with or without coformycin (to prevent adenosine breakdown by adenosine deaminase) exhibited decreased tyrosine phosphorylation of some intracellular proteins and were inhibited in their ability to proliferate and synthesize interleukin (IL)-2. In addition, adenosine interfered with activation-induced expression of the co-stimulatory molecules CD2 and CD28. Activation-induced CD2 and CD28 expression was also diminished when T cells were activated in the presence of anti-IL-2 and anti-CD25 antibodies to neutralize IL-2 bioactivity. Collectively, these data suggest that CD2 and CD28 up-regulation following T cell activation is IL-2-dependent; and that adenosine inhibits activation-induced T cell expression of CD2 and CD28 by interfering with IL-2-dependent signaling. The inhibitory effect of adenosine on activation-induced CD2 and CD28 expression could not be attributed to cyclic AMP (cAMP) accumulation resulting from the stimulation of adenylyl cyclase-coupled adenosine receptors, even though cAMP at concentrations much higher than those generated following adenosine stimulation was inhibitory for both CD2 and CD28 expression. We conclude that adenosine interferes with IL-2-dependent T cell expression of co-stimulatory molecules via a mechanism that does not involve the accumulation of intracellular cAMP., (Copyright 2003 Wiley-Liss, Inc.)

Bovine oocytes are arrested at the prophase of first meiotic cell cycle. Meiosis resumes in oocytes of pre-ovulatory follicles upon LH surge. However, oocytes from secondary follicles spontaneously resume meiosis in the absence of hormones if removed from the follicle and cultured in vitro. The nature of meiotic arrestor in bovine follicles is poorly understood. In this study we investigated the role of cell-cell interactions between granulosa and cumulus cells and the oocyte in mediating maintenance of meiotic arrest by cAMP. We sorted oocytes as granulosa-cumulus oocyte complexes (GCOC) if surrounded with cumulus cells attached to a large granulosa investment or cumulus oocytes complexes (COC) if surrounded with cumulus cells only and investigated the role cAMP in maintenance of meiotic arrest in these oocytes under various conditions. In hormone- and serum-free medium both GCOC and COC enclosed oocytes resumed meiosis. When [cAMP](i) was elevated with addition of invasive adenylate cyclase (iAC) GCOC enclosed oocytes were maintained in the prophase with intact germinal vesicle (GV) while COC enclosed oocytes underwent GV breakdown (GVBD). iAC elevated [cAMP](i) in both types of oocytes to the same level. If oocytes were liberated from the cumulus and granulosa cells, they re-initiated meiosis in serum and hormone free medium, but remained in the GV stage if iAC was added to the medium. Untreated GCOC and COC enclosed oocytes extruded first polar body at the same frequency in hormone-supplemented media. GCOC and COC enclosed oocytes but not denuded oocytes (DO) cultured without somatic cells acquired developmental competence if cultured in hormone-containing medium. It is concluded that maintenance of meiotic arrest is regulated by the interplay of [cAMP](i), and cumulus and granulosa cells., (Copyright 2003 Wiley-Liss, Inc.)

Steroid hormone biosynthesis in the adrenal cortex and gonads involves the coordinated transcription of the genes encoding the steroid hydroxylases, 3beta-hydroxysteroid dehydrogenase (3betaHSD), the steroidogenic acute regulatory protein (StAR), and adrenodoxin (Adx). Transcriptional regulation of steroidogenic genes is multifactorial, entailing developmental, tissue-specific, constitutive, and cAMP-dependent mechanisms. Optimal steroidogenic capacity is achieved by the actions of ACTH which exerts transcriptional pressure on all steroidogenic genes. The actions of ACTH in the adrenal cortex have been studied in great detail and is mediated by cAMP and protein kinase A (PKA) via two temporally distinct pathways. The acute response leads to mobilization of cholesterol, the initial substrate for all steroidogenic pathways, from cellular stores to the inner mitochondrial membrane where cholesterol sidechain cleavage cytochrome P450 (P45011A1) resides. The slower, chronic response of ACTH in the adrenal cortex directs transcription of the genes encoding the steroidogenic enzymes. Although steroidogenic gene transcription in response to ACTH is cAMP-dependent, the consensus cAMP response pathway (CRE/CREB) is not involved. Instead, each steroidogenic gene utilizes unique cAMP-responsive sequences (CRS) found in the promoters of each gene, which bind a diverse array of transcription factors. Moreover, once specific transcription factors are bound to the promoters of the steroidogenic genes, increased gene expression requires posttranslational modification (phosphorylation/dephosphorylation) of the transcription factors and binding of coactivator proteins. This review provides a general view (with emphasis on the human) of the important factors involved in regulating steroidogenic gene expression and ultimately steroid hormone biosynthesis., (Copyright 2003 Wiley-Liss, Inc.)

ACTH is the major regulator of adrenal cortex function, having acute and chronic effects on steroid synthesis and secretion. The precise molecular mechanisms by which ACTH stimulates steroid synthesis and secretion, as well as cell hypertrophy, survival, and migration are still poorly understood. Several studies have shown that ACTH action is mediated not only by cyclic adenosine monophosphate (cAMP), but also by calcium (Ca(2+)), both interacting closely through positive feedback loops to enhance steroid secretion. However, in spite of the evidence that ACTH could stimulate other signaling pathways, such as inositol phosphates and diacylglycerol or mitogenic-activated protein kinase pathway (MAPK), none is as potent as cAMP. Recent data indicate that duration and potency of the cAMP production could be modulated by several isoforms of adenylyl cyclases and phosphodiesterases. In addition, calcium is probably not a first second messenger per se; rather, there are several arguments indicating that its increase occurs following cAMP production. Finally, in addition to steroid secretion, ACTH, through cAMP, is a survival factor, protecting cells against apoptosis. All of the effects of ACTH are dependent on cytoskeleton integrity. In summary, after 30 years of intensive research in this field, cAMP remains the first obligatory second messenger of ACTH action. However, recent work emphasizes that cell environment (matrix and cytoskeleton) probably interacts with cAMP to coordinate functions other than steroid secretion., (Copyright 2003 Wiley-Liss, Inc.)

The importance of chloride channels for the cell is demonstrated by a number of serious human diseases that are due to mutations in chloride channels. The most well-known of these diseases is cystic fibrosis. Investigations into the mechanisms of the disease and possible treatments require the study of chloride fluxes at the level of individual cells. The present study compares two methods for studies of chloride transport: X-ray microanalysis and MQAE fluorescence with image analysis. As an experimental system, the cAMP-activated chloride channel in cultured respiratory epithelial cells was chosen. Both methods showed that stimulation with the cAMP-elevating agents forskolin and IBMX decreased the chloride content of the cells by about 20-27%. Inducing a driving force for chloride by replacing extracellular chloride by nitrate resulted in a chloride efflux that was significantly increased in the presence of forskolin and IBMX. This study shows that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride transport in individual cells., (Copyright 2002 Wiley-Liss, Inc.)

Background: Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. ICER is a transcriptional repressor that negatively regulates cAMP-mediated gene expression. Here, we report the effect of ectopically increasing the expression of ICER on in vitro and in vivo proliferation of the highly metastatic and androgen-insensitive AT6.3 rat prostate cells., Methods: The proliferative potential of stable AT6.3 cell clones expressing ICER was studied by cell counts, thymidine incorporation, flow cytometry, colony formation in soft agar, and growth in immunodeficient nude mice., Results: cAMP inhibits the growth of AT6.3 cells. ICER mRNA and protein levels were markedly induced by cAMP in AT6.3 cells. Forced expression of ICER in AT6.3 cells did not affect cell growth, thymidine incorporation, or the cell cycle. However, these ICER-bearing AT6.3 cells were rendered unable to grow in soft agar or to form tumors in nude mice., Conclusion: These results show that ICER specifically affects the tumorigenicity of prostate cancer cell without affecting their growth. Therefore, the manipulation of ICER expression could be used for the treatment of androgen-insensitive prostate tumors without causing undesirable toxicity to the cells., (Copyright 2002 Wiley-Liss, Inc.)

Background: The endogenous substance lysophosphatidic acid (LPA) has been found to generate proliferation of cultured smooth muscle cells (SMC). Therefore, the effect of LPA on human benign prostate hyperplasia (BPH) could be of interest., Methods: The proliferative effect of LPA on cultured human prostatic SMC from specimens obtained at trans-urethral resection of the prostate (TURP) because of BPH, was analyzed by [3H]-thymidine and [35S]-methionine incorporation. In addition, LPA stimulated BPH SMC were treated with papaverin, forskolin, sildenafil or zaprinast, well known to increase the intracellular level of cAMP or cGMP., Results: LPA produced a dose-dependent increase in BPH SMC, both regarding DNA- and protein-synthesis with EC50 values of 3 and 10 microM, respectively. Furthermore, both papaverin, a general phosphodiesterase inhibitor regarding cAMP hydrolyzes, and forskolin, an adenylyl cyclase stimulating agent, inhibited the LPA-stimulated DNA replication in a dose dependent manner with IC50 = 2.5, and 0.35 microM, respectively. cGMP increasing agents, such as the NO-donors SIN-1 and SNAP, produced a weak anti-proliferative response. However, both phosphodiesterase 5 inhibitors sildenafil (Viagra) and zaprinast efficiently blocked DNA replication. In addition, when the protein synthesis was examined, we found that the LPA response was significantly inhibited by forskolin and papaverin., Conclusions: The major conclusion of this investigation is that the endogenous serum component LPA, is able to promote human BPH SMC growth. In addition, our study indicates that cyclic nucleotides can inhibit this effect. Future clinical studies will be needed to determine if different specific phosphodiesterase inhibitors per se or in combination could represent a new therapeutic possibility for the treatment of BPH., (Copyright 2002 Wiley-Liss, Inc.)

The LNCaP cell line is a versatile and useful model suitable for the study of human prostate cancer in vitro. It has been determined that the elevation of LNCaP intracellular cyclic adenosine monophosphate (cAMP) levels through the addition of membrane-permeable cAMP analogs, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal-transduction pathway can induce reversible neuroendocrine (NE) differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights into the molecular mechanisms governing NE differentiation, early detection of prostate cancer, and potential targets for gene therapy. In this study, differential display polymerase chain reaction (PCR) was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and were cloned. DNA sequencing and database comparisons were performed. To our knowledge, this is the first report of the use of differential gene expression techniques to analyze gene expression during cAMP-induced NE differentiation in LNCaP cells. Confirmation of NE differentiation reversibility also was accomplished., (Copyright 2002 Wiley-Liss, Inc.)

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic species). Third, the phosphoacceptor-serine 128 of CYP2B1 in the consensus sequence for interaction with the protein kinase A represents an on/off switch for the activation of CPA depending on the phosphorylation conditions in the cell. Fourth, evidence is presented that the above-described events also occur in vivo. In conclusion, a successful therapy with CPA, helped by forced expression of CYP2B1 in tumor cells (as recently proposed) will, in addition, be profoundly modified by its phosphorylation status., (Copyright 2001 Wiley-Liss, Inc.)

Effects of caffeine and adenosine on the function and in vitro penetration of frozen-thawed boar spermatozoa were examined. First, the effect on sperm function was determined by the chlortetracycline fluorescence assessment. Both caffeine and adenosine stimulated capacitation of spermatozoa. However, adenosine, but not caffeine, inhibited spontaneous acrosome loss. Second, sperm penetration into in vitro matured oocytes was compared among spermatozoa cultured in the absence or presence of caffeine or adenosine. Both caffeine and adenosine increased the penetration rate (99.1 +/- 0.9% in caffeine, 72.4 +/- 2.0% in adenosine vs. 54.8 +/- 5.1% in controls) but only caffeine decreased drastically the monospermic penetration rate (8.0 +/- 2.3% in caffeine vs. 75.4 +/- 4.8% in adenosine and 78.6 +/- 4.8% in controls). When oocytes were cocultured in various sperm concentrations, the proportion of monospermy changed in inverse proportion to sperm concentration in the presence of caffeine, but did not change in the presence of adenosine. A relatively high number of spermatozoa at the early stage of spontaneous acrosome reaction in the presence of caffeine may be one of the main causes of polyspermic penetration in porcine IVF system. These results indicate that replacement of caffeine with adenosine in fertilization medium improves monospermic penetration by frozen-thawed boar spermatozoa.

In the circulation, most of IGFs are bound to a high molecular mass complex of 150 kDa that consists of IGF-I (or IGF-II), IGFBP-3 and the acid-labile subunit (ALS). Within rat liver, biosynthesis of these components has been localized to different cell populations with hepatocytes as source of ALS and nonparenchymal cells (endothelial and Kupffer cells (KC)) as source of IGFBP-3. In the present study, the regulatory effects of the cAMP analogs dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP (8-br-cAMP) on IGF-I, ALS, and IGFBP expression were evaluated in primary cultures of rat hepatocytes, KC as well as in cocultures of hepatocytes and KC. In cocultures, biosynthesis of IGFBP-3 and ALS was inhibited dose-dependently by db-cAMP and 8-br-cAMP while that of IGF-I, IGFBP-1, and -4 was stimulated as demonstrated by ligand and Northern blotting. IGFBP-3 expression in primary cultures of pure KC did not respond to cAMP treatment indicating the importance of a cellular interaction between KC and hepatocytes for the decreased IGFBP-3 synthesis. The inhibition of IGFBP-3 in db-cAMP-treated cocultures was due to a decrease of IGFBP-3 mRNA level accompanied by a reduced cellular degradation of IGFBP-3. We conclude that cAMP stimulate the biosynthesis of IGF-I, IGFBP-1, and -4 in cocultures of hepatocytes and KC thereby enabling the formation of binary IGF/IGFBP complexes while the formation of the 150 kDa complex is impaired through downregulation of IGFBP-3 and ALS. This complex regulation may be a prerequisite for the effects of cAMP-dependent hormones on the transfer of IGFs from circulation to peripheral tissues., (Copyright 2001 Wiley-Liss, Inc.)

The epithelial cells of the choroid plexus are the structural basis of the blood-cerebrospinal fluid (CSF)-barrier. Here we summarise our recent efforts to culture those cells mainly on permeable supports in vitro. Isolated from porcine brains, we report a simple protocol for the primary culture using cytosine arabinoside as an additive that is cytotoxic for other cells except the plexus epithelial cells. Enhanced barrier properties are obtained by withdrawal of serum from the culture medium after confluency is reached. Cells improve their polarity, permeability for hydrophilic substrates is lowered, electrical resistance is increased tenfold, and a pH-gradient is built up across the cell monolayer. Polarised secretion of proteins and most importantly fluid secretion into the apical filter compartment was attained and proven to be dependent on the Na(+),K(+)-ATPase activity. Active transport processes (penicillin G, riboflavin, myo-inositol, ascorbic acid) were studied and clearly showed the involvement of the organic anion transporter. The permeability of the barrier was found to be regulated by cyclic adenosine monophosphate (cAMP). Moreover, we report that cell proliferation and differentiation is controlled by components of the extracellular matrix. The present culture model could now be used as an in vitro system to quantify drug transport across the blood-CSF-barrier., (Copyright 2001 Wiley-Liss, Inc.)

It has been well established that the key role of noradrenaline is the induction of uncoupling-protein-1 (UCP-1) expression, the unique marker of brown adipocytes. However, its implication on proliferation and the pathways involved are not as well characterized. By using rat fetal brown adipocytes as a model, we show that, although noradrenaline activates extracellular regulated kinases (ERKs) through beta-, alpha1-, and alpha2-receptors, only beta-receptors mediate cell growth by a mechanism that requires ERKs activation but is independent of cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA). Conversely, the cAMP/PKA cascade mediates noradrenaline-induced UCP-1 expression, whereas ERKs pathway attenuates thermogenic differentiation. On the other hand, alpha1- and alpha2-receptors have an antiproliferative effect that is enhanced by ERK inhibition., (Copyright 2000 Wiley-Liss, Inc.)

Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium., (Copyright 2000 Wiley-Liss, Inc.)

Background: Androgen-independent growth leads to progressive prostate cancer after androgen-ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand-independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells., Methods: We established and characterized the androgen-independent FGC-DCC from the androgen-dependent LNCaP fast growing colony (FGC) cell line. The androgen-independent DU-145, FGC-DCC, and PC-3, and the androgen-dependent LNCaP and PC-346C cell lines were used to study growth modulation of gastrin-releasing peptide (GRP), calcitonin (CT), serotonin (5-HT), and vasoactive intestinal peptide (VIP) by (3)H-thymidine incorporation. Specificity of the growth-modulating effects was tested with the anti-GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides., Results: Androgen-independent growth stimulation by neuropeptides was shown in DU-145 and PC-346C. 2A11 inhibited GRP-induced (3)H-thymidine incorporation in DU-145 and PC-346C and inhibited proliferation of the FGC-DCC and PC-3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP-induced growth effect in DU-145 and PC-346C, whereas oxadiazoloquinoxaline-1-one (ODQ) had no effect on (3)H-thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC-DCC, or PC-3., Conclusions: GRP-induced growth of DU-145 and PC-346C was specific and cAMP-mediated. Androgen-independent growth of FGC-DCC cells was mainly due to an induction of Bcl-2 expression and possibly through the activation of an autocrine and NE-like pathway, as has been shown also for the PC-3 cell line. Growth induction of non-NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer., (Copyright 2000 Wiley-Liss, Inc.)

Freshly isolated human mucosal T lymphocytes in vitro can markedly diminish an important property of intestinal epithelium-its barrier function. On the other hand, cytokines and their cellular receptors, which maintain homeostasis of epithelia, limit epithelial permeability, and preserve barrier function, are not well characterized. Using a described human colonic epithelial cell monolayer system, we found that transforming growth factor-beta1 (TGF-beta1) preserved 75% or more of epithelial barrier function, quantitated electrophysiologically, even in the presence of cytokines generated by a high density of barrier-disruptive mucosa-derived mononuclear cells. In opposing the TGF-beta1 effect, cytokines able to reduce barrier function were spontaneously secreted by mucosal T cells and were increased in their barrier effect after T-lymphocyte activation. Further, neutralization of individual cytokines with specific monoclonal antibodies abrogated the lymphocyte-induced reduction in epithelial barrier function, and identified interferon gamma (IFN-gamma), interleukin (IL)-4, and IL-10, but not IL-6, as the primary cytokines whose barrier effects were curtailed by TGF-beta1. Receptors (RI and RII) for TGF-beta1 were found to be localized primarily to the apical and basal membranes of surface epithelium in colonic crypts. These findings provide the scientific basis for new strategies to pharmacologically enhance the barrier function of epithelia in mucosal organs regularly exposed to environmental antigens and to T-lymphocyte products., (Copyright 1999 Wiley-Liss, Inc.)

We have studied the interaction between P1 and P2 purinoceptors in purified type-1 astrocyte cultures from postnatal days 7-8 rat cerebella using single cell microfluorimetry with fura-2. The stimulation of astrocytes with ATP elicits rapid [Ca2+]i transients showing an EC50 value of 7.9 +/- 0.3 microM. Costimulation of type-1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 microM) evoked [Ca2+]i transients that correspond to 60% of the maximal ATP response. NECA (5'-N-ethylcarboxamidoadenosine) was the only agonist that mimicked the adenosine effect and showed an EC50 value of 0.17 +/- 0.01 microM. This value was identical to that obtained for the cAMP production stimulation, indicating that A2B receptors coupled to adenylate cyclase activation were involved. The presence of A2B adenosine receptors was also confirmed by immocytochemistry experiments. When astrocytes were costimulated with isoproterenol and ineffective ATP concentrations similar [Ca2+]i transients were observed. The treatment of astrocytes with cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 +/- 0.2 microM. However, the pretreatment of cells with forskolin or a permeable cAMP analogue had no effect on ATP calcium responses. These results indicated that the potentiation mechanism was elicited before the adenylate cyclase activation. We could conclude that in type-1 astrocytes, the activation of A2B adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic calcium responses to ATP. The potentiation was parallel but independent on cAMP accumulation suggesting the involvement of beta gamma subunits released after Gs stimulation.

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

Thyroid hormones are important for neurogenesis and gliogenesis during brain development. We have previously demonstrated that triiodothyronine (T3) treatment induced proliferation in primary culture astrocytes derived from the cerebellum of neonatal rats. Conditioned medium obtained from those T3-treated astrocytes (T3CM) mimicked the effect of hormonal treatment on these cells. Because neuron-glia interaction plays an important role in brain development, we tested the ability of such T3-glial CM to influence neuronal physiology. With that aim, neurons from 19-day embryonic cerebella were cultivated for 24 h in the presence of CM obtained from T3-treated cerebellar astrocytes. Interestingly, the cerebellar neuronal population increased by 60-80% in T3CM. Addition of 5 microM forskolin enhanced the responsiveness of cerebellar neurons to astrocytes T3CM, but it did not interfere with neuronal survival in control medium. Conversely, inhibition of adenylate cyclase by its specific inhibitor, SQ22536, reversed the T3CM effect on neurons. These data strongly suggest that cAMP signal transduction pathways might be implicated in such an event. Analysis of bromodeoxyuridil incorporation revealed that the increase in neuron number in T3CM was partially due to neuron proliferation, because the proliferation index was three times higher in T3CM than in control medium. Neutralizing antibody assays demonstrated that T3CM effects on neurons are due, at least in part, to the presence of tumor necrosis factor-beta and epidermal growth factor. Thus, we report here a novel molecular mechanism of action of thyroid hormone on cerebellar neuronal cells: Thyroid hormone induces astrocytes to secrete growth factors that can interfere with neuronal proliferation via a paracrine pathway.

Acute effects and action mechanisms of prolactin (PRL) on aldosterone secretion in zona glomerulosa (ZG) cells were investigated in ovariectomized rats. Administration of ovine PRL (oPRL) increased aldosterone secretion in a dose-dependent manner. Incubation of [3H]-pregnenolone combined with oPRL increased the production of [3H]-aldosterone and [3H]-deoxycorticosterone but decreased the accumulation of [3H]-corticosterone. Administration of oPRL produced a marked increase of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in ZG cells. The stimulatory effect of oPRL on aldosterone secretion was attenuated by the administration of angiotensin II (Ang II) and high potassium. The Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 10(-2) M), inhibited the basal release of aldosterone and completely suppressed the stimulatory effects of oPRL on aldosterone secretion. The stimulatory effects of oPRL on aldosterone secretion were attenuated by the administration of nifedipine (L-type Ca2+ channel blocker) and tetrandrine (T-type Ca2+ channel blocker). These data suggest that the increase of aldosterone secretion by oPRL is in part due to (1) the increase of cAMP production, (2) the activation of both L- and T-type Ca2+ channels, and (3) the activation of 21-hydroxylase and aldosterone synthase in rat ZG cells.

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide in the mammalian nervous system. NAAG meets the traditional criteria of a neurotransmitter, including localization in synaptic vesicles, depolarization-induced release, low potency activation of some N-methyl-D-aspartate receptors, and highly selective activation of a cAMP-coupled metabotropic glutamate receptor (mGluR) with potency approaching that of glutamate. The peptide is present in cultured cortical glia in high concentration and is hydrolyzed by cell surface peptidase activity. In the present work, we tested the hypothesis that NAAG selectively activates a subclass of metabotropic receptors on cultured rat cerebellar glia, primarily astrocytes. These glial cells express mRNA for mGluR subtypes 1, 3, 4, 5, 6, 7, and 8. We were not able to detect message for mGluR2 in these cells using the reverse transcriptase-polymerase chain reaction. Cerebellar glia responded to NAAG, glutamate, and trans-ACPD with a decrease in forskolin-stimulated cAMP formation. AP4, an agonist of the group III receptors mGluR4, mGluR6, mGluR7, and mGluR8, had little or no effect on stimulated cAMP levels. Treatment with low micromolar NAAG significantly increased uptake of radioactive thymidine by cultured astrocytes through activation of metabotropic glutamate receptors. Antagonists of group II mGluRs prevented the decrease in cAMP and the increase in uptake of thymidine by NAAG. Cultured cerebellar astrocytes expressed 20 pmol NAAG per mg protein, a value that is at least 30-fold lower than that expressed by mixed glial cultures prepared from mouse cortex. We conclude that cerebellar astrocytes respond to NAAG via the mGluR3 receptor and that the peptide may selectively activate this receptor subtype in astrocytes following release from neurons or glia.

Fluorescence videomicroscopy allows the temporal behavior of biological specimens to be studied at the cellular level. We describe two types of methods that can be used for extracting quantitative information from image sequences: the modeling approach, which is mainly local, and multivariate statistical analysis, which provides a global approach. The potentials for use of these two methods are illustrated through a simulation example and actual examples dealing with the study of chloride secretion by airway epithelial cells. We define some guidelines for making a choice between these two approaches, bearing in mind that a blend of these two methodologies is also possible.

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC3H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE1]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC3H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC3H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC3H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC3H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation.

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-3. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed beta-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation.

The growth-promoting properties of the retroviral v-erbA oncogene, a highly mutated version of the chicken thyroid hormone receptor (TR) alpha, have so far exclusively been linked to dominant repression of the antimitogenic roles of TR and retinoic acid receptors. Here we show that when expressed in Xenopus oocytes v-ErbA induced ultrastructural changes characteristic of early and intermediate events of meiotic maturation by activating gene transcription. v-ErbA-induced maturation events occurred without activation of the cAMP/maturation-promoting factor signal pathway and were arrested prior to meiotic spindle formation. The effects of v-ErbA were not mimicked by a dominant negative in vitro-generated mutant of human TR, suggesting that v-ErbA can contribute to cell cycle reentry by interference with regulatory pathways distinct from those involving TR. Interestingly, a portion of v-ErbA expressed in oocytes was present at the cytoplasmic fibrils of the nuclear pore complexes, suggesting that in addition to its intranuclear function v-ErbA may modulate nucleocytoplasmic transport.

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism whose action is mediated by high affinity cell surface receptors and bioactivity and bioavailability regulated, in part, by IGF-1 binding proteins (IGFBPs). Prostaglandin E2 (PGE2) stimulates collagen and proteoglycan synthesis in cartilage via an autocrine feedback loop involving IGF-1. We determined whether the eicosanoid could regulate IGFBP-4, a major form expressed by chondrocytes and, as such, act as a modifier of IGF-1 action at another level. Using human articular chondrocytes in high-density primary culture, Western and Western ligand blotting to measure secreted IGFBP-4 protein, and Northern analysis to monitor IGFBP-4 mRNA levels, we demonstrated that PGE2 provoked a 2.7 +/- 0.3- and 3.8 +/- 0.5- (n = 3) fold increase in IGFBP-4 mRNA and protein, respectively. This effect was reversed by the Ca(++) channel blocker, verapamil, and the Ca(++)/calmodulin inhibitor, W-7. The Ca(++)ionophore, ionomycin, mimicked the effects of PGE2. The phorbol ester, PMA, which activated phospholipid-dependent protein kinase C (PKC) in chondrocytes, had no effect on IGFBP-4 production. Cyclic AMP mimetics and PKA activators, IBMX, and Sp-cAMP, inhibited the expression of the binding protein as did the PGE2 secretagogue, interleukin-1 beta (IL-beta). The inhibitory effect of the latter cytokine was mediated by a erbstatin/genistein (tyrosine) sensitive kinase. Dexamethasone, an inhibitor of cyclooxygenase (COX-2) expression and PGE2 synthesis, down-regulated control, constitutive levels of IGFBP-4 mRNA and protein, eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE2-receptor signalling pathways. The results suggest that extracellular signals control IGFBP-4 production by a number of different transducing networks with changes in Ca(++) and calmodulin activity exerting a strong positive influence, possibly maintaining the constitutivity of IGFBP-4 synthesis under basal conditions. PGE2 activation of the IGF-1/IGFBP axis may play a pivotal role in the metabolism of cartilage and possibly connective tissues in general. Eicosanoid biosynthesis may be a rate-limiting step in cartilage repair processes.

A novel developmental gene, yelA, has been found that plays as essential role in regulating terminal differentiation of Dictyostelium discoideum. Strains in which yelA is disrupted by plasmid insertion are arrested at the tight mound stage but accumulate the bright yellow pigment characteristic of mature sori. Although these mutant strains do not form fruiting bodies, many of the cells encapsulate within the mounds. Sporulation occurs about 6 hours earlier in yelA- cells than in wild-type cells, accompanied by precocious expression of a prespore gene, spiA. However, the spores are defective and lose viability over a period of several hours. Unencapsulated cells also die unless they are dissociated from the mounds and shaken in suspension. The yelA gene was isolated by plasmid rescue and found to encode a protein of 102 kDa in which the N-terminal sequence shows significant similarity to domains found in the eIF-4G subunits of the translational initiation complex eIF-4F. In wild-type cells yelA mRNA first accumulates at 8 hours of development and is maintained in both prespore and prestalk cells until culmination when it is found only is stalk cells. Mutations in yelA can partially suppress the block to sporulation in mutant strains in which either of the prestalk genes tagB or tagC is disrupted such that an encapsulation signal is not produced. It appears that premature encapsulation is normally inhibited by YelA until a signal is received from prestalk cells during culmination.

The aim of the present study was to investigate the mechanisms regulating endothelin-1 (ET-1) secretion in rat thyroid FRTL-5 cells. ET-1 was found to be secreted after stimulation with adenosine and ATP. The release of ET-1 was sensitive to pertussis toxin, indicating a role of G-proteins in the stimulus-secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine- and ATP-mediated ET-1 secretion was abolished. These evidences suggest a role of a P1-adenosine receptor in the secretion of ET-1. Increasing cyclic AMP with forskolin decreased the adenosine-mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni2+ prevented the response. Protein kinase C (PKC) is also partly involved in ET-1 secretion in FRTL-5 cells. Activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated the secretion of ET-1 in a time- and dose-dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET-1 stimulated by adenosine. In conclusion, ET-1 secretion in FRTL-5 cells is stimulated via a pertussis toxin-sensitive P1-receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca2+, and PKC.

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17 beta-estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0-1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose-dependent manner (P < 0.01). Generation of inositol-phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by 3H-thymidine incorporation in serum-free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose-dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF2 alpha production by epithelial cells (P < 0.0001) but had no effect on PGE2 production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro.

Forskolin concentration-dependently increased the short-circuit current (Isc) across the isolated mucosa of rat colon, which was carried mainly by Cl- secretion from the mucosal membrane. The sulfonylureas such as glibenclamide, tolbutamide, glipizide and the ATP-sensitive K+ channel opener cromakalim inhibited the forskolin (1 microM)-induced increase of short-circuit current (delta Isc) when these drugs were applied to the basolateral side. The rank order of potency for inhibition of delta Isc was: glibenclamide > cromakalim > tolbutamide > glipizide. Glibenclamide (100 microM) and cromakalim (100 microM) caused transient or small reduction of the A23187-induced delta Isc when applied to the basolateral side. Glibenclamide, tolbutamide and cromakalim decreased the forskolin-induced delta Isc when applied to the mucosal side; however, the responses produced by basolateral application were greater and faster than those elicited by mucosal application. None of these four agents affected the basal transepithelial current. The results indicate that the cAMP-dependent Cl- secretion in the rat colon could be modulated by ATP-sensitive K+ channel regulators.

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKC alpha had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKC alpha in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1.