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The intense interest in SST as a model to study neuropeptide synthesis has provided a framework for understanding the biosynthetic pathways, the putative role of PCs, and the secretory pathways required for PSST maturation to its active products. Kinetic studies have demonstrated that PSST is rapidly and independently processed to SST-14, SST-28, and PSST[1-10]. Both PC1 and PC2 are capable of processing PSST to SST-14. Furin and PACE 4 effect monobasic cleavage and are candidate SST-28 convertases. Contrary to previous belief, cleavage at the NH2-terminus has been shown not require the monobasic Lys13 residue with SKI-1 acting as the most likely convertase. Secretory granules are not a requirement for PSST maturation. Additionally, the conserved amino terminal segment of PSST serves as a sorting for the regulated secretory pathway. Beyond the pure biochemical and basic research interest in defining the specific intracellular events implicated in processing and sorting of PSST, combining the knowledge gained from the processing and targeting aspects to treatments of various diseases would be the ultimate satisfaction of any scientist. Such an approach was elegantly attempted by Rivera and colleagues who used the ER as the storage compartment for genetically engineered secretory proteins such as insulin and growth hormone (53).

In kidney, nitric oxide (NO) synthesized by nitric oxide synthase (NOS) regulates sodium and water excretion, and renal medullary blood flow. The expression of constitutive NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS), were assessed in kidney of the spontaneously hypertensive rat (SHR) and the normotensive Wistar Kyoto (WKY) rat by Western blot analysis and immunocytochemistry. Neuronal NOS expression was observed in the cortex and eNOS was detected only in theinner medulla of both WKY and SHR. In SHR, expression of eNOS was attenuated to 35.1 +/- 10.8%, while expression of nNOS was only 57.5 +/- 5.7% of the levels seen in WKY rat. Immunocytochemical studies revealed decreased staining of nNOS in the macula densa, collecting ducts and in the glomerulus of SHR compared to WKY rat. Endothelial NOS immunoreactivity was restricted to vascular structures of the inner intima cells and smooth muscle cells, and was markedly reduced in the vasculature of SHR. The decreased renal blood flow observed in SHR may be linked to a diminished expression of eNOS and nNOS, underscoring the importance of these enzymes in the pathophysiology and maintenance of genetic hypertension.

The existence of dimers and higher oligomers of G-protein-coupled receptors (GPCRs) has been frequently reported using strategies based on coimmunoprecipitation or Western blot assays. These methods rely on highly artificial systems with overexpressed receptors, resulting in conflicting observations on the question of whether GPCR dimers are preformed or are formed in response to agonist treatment. Fluorescence resonance energy transfer (FRET) microscopy is a superior and less perturbing technique which can be performed on selected cell regions, e.g., plasma membrane of intact cells with a sensitivity high enough to allow study under physiological levels of receptor expression. Here we describe the application of photobleaching (pb) FRET microscopy for investigating ligand-dependent oligomerization of somatostatin receptors. Procedures for the introduction of suitable donor-acceptor fluorophores in a given GPCR are described. The competitive nature of FRET and photobleaching is exploited to enable the indirect measurement of FRET via its effect on donor photobleaching lifetimes on a pixel-by-pixel basis. The method allows enhanced resolution between 10 and 100A and represents a sensitive and specific biophysical tool for characterizing the assembly and regulation of GPCR oligomers on the cell surface.

The peptide somatostatin (SST) is widely synthesized in the brain and periphery and acts through a family of five receptors (SSTR1-5) to exert numerous effects. A gene product related to SST, cortistatin (CST), also interacts with SSTR1-5. Here we have investigated the regulation of SSTR1-5 and of CST in SST knockout (SSTKO) mice. The five SSTRs were quantitated individually by subtype-selective binding analysis, by immunocytochemistry, and by mRNA measurement and showed, in the brain of SSTKO mice, up-regulation of subtypes 1, 2, 4, and 5, and down-regulation of SSTR3. Peripheral tissues displayed both subtype- and tissue-specific changes in SSTR1-5 mRNA levels of expression. Lack of SST did not up-regulate normal CST expression in brain nor did it induce its expression in the periphery. SST-like immunoreactivity, however, was induced in the proximal midgut in SSTKO animals, suggesting intestinal expression of a novel SST-like gene. Fil: Ramírez, José L.. McGill University; Canadá Fil: Mouchantaf, Rania. McGill University; Canadá Fil: Kumar, Ujendra. McGill University; Canadá Fil: Otero Corchon, Veronica. Oregon Health and Science University; Estados Unidos Fil: Rubinstein, Marcelo. Oregon Health and Science University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Low, Malcolm J.. Oregon Health and Science University; Estados Unidos Fil: Patel, Yogesh C.. McGill University; Canadá

The somatostatin analogs octreotide and lanreotide, selective to receptor subtypes 2 and 5, failed clinical efficacy for the prevention of restenosis after percutaneous transluminal angioplasty. These findings might have been the result of targeting a wrong subset of receptors. In rat arteries, subtypes 1 and 4 are expressed 3-4 times more prominently than 2 and 5, and subtype 1 is the nearly exclusive subtype in atherosclerotic human vessels. Here, we demonstrate that daily s.c. injections (50-500 microg/kg/d) of CH275 (DesAA1,2,5(D-W8,IAmp9)Somatostatine-14), selective to subtypes 1 and 4, dose-dependently inhibited intimal hyperplasia 14 days after rat carotid denudation injury (for intimal area P=0.0002 across the dose range). CH275 was more effective than somatostatin-14 (equal affinity to all five subtypes, P=0.03), or octreotide (selective to subtypes 2 and 5, P=0.098). When rats were given the peptides for 14 days with end-point at 28 days, CH275 still significantly inhibited intimal area expansion. Both CH275 and octreotide inhibited the outgrowth of cells from postinjury aortic tissue punch-explants and the distance migrated in vitro, but not cell replication, which indicated that the effects of somatostatin analogs were directed on the migration of intimal cell progenitors rather than on their proliferation.

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo- and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family.

Hepatic stellate cells (HSCs) are considered therapeutic targets to decrease portal hypertension. To elucidate some of the hemodynamic effects of somatostatin (SST) on portal pressure, the presence and function of SST receptors (SSTRs) on HSCs were investigated.SSTR messenger RNA expression, and SSTR presence was investigated using reverse-transcription polymerase chain reaction, real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. The function of SSTRs was studied by examining the effects of SST and specific SSTR agonists on endothelin-1(ET-1)-induced HSC contraction.Specific amplicons for SSTR subtypes 1, 2, and 3 were demonstrated in rat liver and in activated HSCs. The presence of SSTR subtypes 1, 2, and 3 was confirmed by Western blotting. With immunohistochemistry, a strong staining of HSCs was obtained for SSTR subtypes 1, 2, and 3 in CCl4-treated rats, but not in normal rat liver. Incubation of HSCs on collagen gels with buffer, 10(-8) mol/L SST, and 2 x 10(-8) mol/L ET-1 resulted in collagen surface area decreases of 5.5% +/- 3.3%, 6.8% +/- 4.4%, and 49.8% +/- 8.3%, respectively. Relative contraction of gels preincubated with 10(-8) mol/L SST followed by 2 x 10(-8) mol/L ET-1 or vice versa as compared with maximal contraction (100%) with 2 x 10(-8) mol/L ET-1 were 72.6% +/- 17.9% and 76.2% +/- 12.6%, respectively (P0.05). SSTR agonist 1, but not SSTR agonist 2 or 3, was able to counteract the contractile effect of ET-1. CONCLUSIONA: Activated rat HSCs bear SSTR subtypes 1, 2, and 3. SST causes significant partial inhibition of ET-1-induced contraction of activated HSCs, mainly by stimulation of SSTR subtype 1.

Somatostatin and dopamine are two major neurotransmitter systems that share a number of structural and functional characteristics. Somatostatin receptors and dopamine receptors are colocalized in neuronal subgroups, and somatostatin is involved in modulating dopamine-mediated control of motor activity. However, the molecular basis for such interaction between the two systems is unclear. Here, we show that dopamine receptor D2R and somatostatin receptor SSTR5 interact physically through hetero-oligomerization to create a novel receptor with enhanced functional activity. Our results provide evidence that receptors from different G protein (heterotrimeric guanine nucleotide binding protein)–coupled receptor families interact through oligomerization. Such direct intramembrane association defines a new level of molecular crosstalk between related G protein–coupled receptor subfamilies.

Mammalian prosomatostatin (PSST) contains the bioactive peptides SST-14 and SST-28 at the COOH-terminal end of the molecule and a putative sorting signal in the propeptide segment for targeting the precursor to the regulated secretory pathway. The NH(2)-terminal segment of PSST consists of an amphipathic alpha-helix, which has been totally conserved throughout vertebrate evolution. We have analyzed the PSST-(3--15) region for sorting function by alanine scanning and deletional mutagenesis. Mutants created were stably expressed in AtT-20 cells. Regulated secretion was studied by analyzing basal and stimulated release of SST-14 LI and by immunocytochemistry for staining of SST-14 LI in punctate granules. Deletion of the PSST-(3--15) segment blocked regulated secretion and rerouted PSST for constitutive secretion as unprocessed precursor. Alanine scanning mutagenesis identified the region Pro(5)--Gln(12) as being important in precursor targeting, with Leu(7) and Leu(11) being critical. Molecular modeling demonstrated that these two residues are located in close proximity on a hydrophobic surface of the alpha-helix. Disruption of the alpha-helix did not impair the ability of PSST to be processed at the COOH terminus to SST-14 and SST-28. Processing, however, was shifted to the early compartments of the secretory pathway rather than storage granules and was relatively inefficient.

The twelve tasks comprising this four year project were completed and a' full report submitted in September 2000. However, in the course of these studies three new leads were pursued as three separate tasks (Tasks 13, 14, 15) which were not part of the original contract but because of their importance were nonetheless developed substantially and also reported upon in September 2000. We then requested and received approval for a no-cost extension to allow us to round off these three new tasks up to the period ending September 2001. We made reasonable progress in each of these three areas as outlined in this report despite the time and budgetary constraints and personnel departures. As emphasized in our Final Report, however, each of these three new tasks represented substantial new projects which we intend to pursue fully with renewed new longterm funding.

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.

Preprocortistatin (PPCST) has been recently identified as a novel somatostatin (SST)-related gene expressed only in brain. PPCST shares 11 of 14 residues with SST-14 at its C-terminal segment, where it features Lys-Lys and Lys-Arg basic sites for cleavage to putative cortistatin (CST)-14 and CST-29 peptides, respectively. Although synthetic replicates of the two putative CST peptides interact with SST receptors, they also display novel effects suggesting independent biological functions. Nothing is currently known about the naturally occurring mature cleavage products of PPCST posttranslational processing. Here we have cloned rat PPCST cDNA, stably expressed it in AtT-20 pituitary cells, and characterized the cellular and releasable products of PPCST processing by HPLC and radioimmunoassay using a SST-14 antibody that recognizes synthetic CST-14 and CST-29. Transfected cells released 120 +/- 21 pg of total CST-LI per plate basally, with an increase to 204 +/- 33 pg per plate with forskolin stimulation (p < 0.05). HPLC chromatograms of cell extracts revealed three peaks corresponding to CST-14, CST-29, and unprocessed PPCST (ratio, 41:55:4.5). CST was released preferentially as CST-14 (63-70%) compared with CST-29 (30-37%) under basal and forskolin-stimulated conditions. These studies demonstrate efficient processing of PPCST to both CST-14 and CST-29 through putative cleavage at both C-terminal dibasic sites of PPCST. Although the two peptides are synthesized approximately equally, CST-14 is released preferentially via the regulated secretory pathway.

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.

Somatostatin (SST), a regulatory peptide, is produced by neuroendocrine, inflammatory, and immune cells in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide is released in large amounts from storage pools of secretory cells, or in small amounts from activated immune and inflammatory cells, and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven transmembrane (TM) domain G-protein-coupled receptors that comprise five distinct subtypes (termed SSTR1-5) that are endoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. Short synthetic octapeptide and hexapeptide analogs bind well to only three of the subtypes, 2, 3, and 5. Selective nonpeptide agonists with nanomolar affinity have been developed for four of the subtypes (SSTR1, 2, 3, and 4) and putative peptide antagonists for SSTR2 and SSTR5 have been identified. The ligand binding domain for SST ligands is made up of residues in TMs III-VII with a potential contribution by the second extracellular loop. SSTRs are widely expressed in many tissues, frequently as multiple subtypes that coexist in the same cell. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated protein kinase (MAPK) through G-protein-dependent mechanisms. Some of the subtypes are also coupled to inward rectifying K(+) channels (SSTR2, 3, 4, 5), to voltage-dependent Ca(2+) channels (SSTR1, 2), a Na(+)/H(+) exchanger (SSTR1), AMPA/kainate glutamate channels (SSTR1, 2), phospholipase C (SSTR2, 5), and phospholipase A(2) (SSTR4). SSTRs block cell secretion by inhibiting intracellular cAMP and Ca(2+) and by a receptor-linked distal effect on exocytosis. Four of the receptors (SSTR1, 2, 4, and 5) induce cell cycle arrest via PTP-dependent modulation of MAPK, associated with induction of the retinoblastoma tumor suppressor protein and p21. In contrast, SSTR3 uniquely triggers PTP-dependent apoptosis accompanied by activation of p53 and the pro-apoptotic protein Bax. SSTR1, 2, 3, and 5 display acute desensitization of adenylyl cyclase coupling. Four of the subtypes (SSTR2, 3, 4, and 5) undergo rapid agonist-dependent endocytosis. SSTR1 fails to be internalized but is instead upregulated at the membrane in response to continued agonist exposure. Among the wide spectrum of SST effects, several biological responses have been identified that display absolute or relative subtype selectivity. These include GH secretion (SSTR2 and 5), insulin secretion (SSTR5), glucagon secretion (SSTR2), and immune responses (SSTR2).

Ligand-activated somatostatin receptors (SSTRs) initiate cytotoxic or cytostatic antiproliferative signals. We have previously shown that cytotoxicity leading to apoptosis was signaled solely via human (h) SSTR subtype 3, whereas the other four hSSTR subtypes initiated a cytostatic response that led to growth inhibition. In the present study we characterized the antiproliferative signaling mediated by hSSTR subtypes 1, 2, 4, and 5 in CHO-K1 cells. We report here that cytostatic signaling via these subtypes results in induction of the retinoblastoma protein Rb and G1 cell cycle arrest. Immunoblot analysis revealed an increase in hypophosphorylated form of Rb in agonist-treated cells. The relative efficacy of these receptors to initiate cytostatic signaling was hSSTR5hSSTR2hSSTR4 approximately = hSSTR1. Cytostatic signaling via hSSTR5 also induced a marginal increase in cyclin-dependent kinase inhibitor p21. hSSTR5-initiated cytostatic signaling was G protein dependent and protein tyrosine phosphatase (PTP) mediated. Octreotide treatment induced a translocation of cytosolic PTP to the membrane, whereas it did not stimulate PTP activity when added directly to the cell membranes. C-tail truncation mutants of hSSTR5 displayed progressive loss of antiproliferative signaling proportional to the length of deletion, as reflected by the marked decrease in the effects of octreotide on membrane translocation of cytosolic PTP, and induction of Rb and G1 arrest. These data demonstrate that the C-terminal domain of hSSTR5 is required for cytostatic signaling that is PTP dependent and leads to induction of hypophosphorylated Rb and G1 arrest.

The neuropeptide somatostatin was discovered in Guillemin's laboratory at the Salk Institute 18 years ago.1 From a relatively humble beginning as a hypothalamic growth hormone inhibitor, it has eme...

The subset of striatal neurons which colocalize SS/NPY/NADPH-d are selectively resistant to neurodegeneration in Huntington's Disease (HD) and to excitotoxic cell death induced experimentally with NMDA receptor (NMDAR) agonists. Here we have analyzed the expression of immunoreactive NMDAR-1 (NR1) subunit (as an index of NMDAR protein) and of huntingtin (the normal product of the HD gene) in primary cultures of rat striatum to see if differential expression of the two antigens in the subset of SS/NPY/NADPH-d and other striatal neurons can explain their selective resistance or vulnerability. Double-label histochemical and immunocytochemical studies were carried out using conventional and confocal laser scanning microscopy to characterize the cellular and subcellular expression of NR1 and SS, or NPY or bNOS, together with NADPH-d histochemistry. The percentages of cultured striatal neurons that were positive for NADPH-d, SS, NPY, bNOS, and NRI were, respectively, 3.8, 8.4, 10.2, 5.1, and 80%. The majority of striatal NADPH-d neurons coexpressed SS and NPY; 17% of SS-producing neurons were strongly positive for NR1; the remaining cells (approximately 80%) exhibited only weak NR1 expression. Comparable data were obtained for NPY-positive neurons, 15% of which colocalized NR1 strongly and 70-80% weakly. By double-label immunofluorescence, huntingtin was nonselectively expressed in virtually all striatal neurons including SS/NPY/NADPH-d neurons. These results show that the majority of striatal SS/NPY/NADPH-d neurons express NR1. The relative abundance of NR1 in SS/NPY/NADPH-d neurons, however, varies between a small subset of neurons that are receptor rich and the remainder that express low levels only and may determine susceptibility to NMDAR-mediated neurotoxicity. Huntingtin is nonselectively expressed in virtually all striatal neurons and does not appear to be a determinant of the selective resistance of normal striatal SS/NPY/NADPH-d neurons to NMDA toxicity.

This paper examines the role of medical expertise in clinical reasoning, using a complete workup of a patient with an endocrine disorder. Endocrinologists, housestaff, and final year medical students were asked to develop the case. This consisted of history-taking, interpreting physical examination results, request for tests and their interpretations, and providing therapeutic and patient management plans, and an explanation of the pathophysiology underlying the problem. The subjects were also asked to provide explanations supporting their decisions during the patient workup. A variety of techniques deriving from cognitive psychology were used to analyze the data. The main concern was how expertise affected the building of relationship between the components of the workup. Experts formed integrated knowledge-rich structures that were generated during the history-taking and used consistently throughout the workup. Housestaff formed more tentative and less integrated representations which were modified during the patient encounter. Students representations superficially resembled those of experts, but were knowledge-lean. The results are interpreted in terms of clinical performance of endocrinologists, housestaff, and students during the workup, and its relationship to the explanation task as an indicator of clinical competence of the patient problem provided by the subjects. It is proposed that the explanation given after the problem solving process could be used as an indicator of clinical competence.

Somatostatin (SS) and neuropeptide Y (NPY) are coproduced in a subpopulation of neurons that are selectively resistant to NMDA neurotoxicity. We have previously reported that quinolinic acid (QUIN), an NMDA receptor agonist, augments SS mRNA in cultured fetal rat cortical neurons. This study examines coregulation of SS and NPY by QUIN and NMDA in cultured cortical neurons and compares the effects of these agents with those of forskolin and phorbol 12-myristate 13-acetate (PMA), known to activate SS and NPY gene transcription by protein kinase A- and protein kinase C-dependent mechanisms. In addition, transcriptional regulation of the SS gene was investigated by acute transfection of cortical cultures with an SS promoter-chloramphenicol acetyltransferase (CAT) construct. QUIN and NMDA displayed dose-dependent fourfold augmentation of levels of mRNA for SS but not for NPY. In contrast, forskolin and PMA increased both SS and NPY mRNA levels. QUIN- and NMDA-mediated induction of SS mRNA was blocked by the NMDA receptor antagonist (−)-2-amino-5-phosphonovaleric acid and displayed regional brain specificity because it was not observed in fetal hypothalamic cell cultures. In time course studies, the effects of QUIN/NMDA on SS mRNA occurred after a latency of 8 h, indicating a delayed effect. Cortical cells transfected with pSS-750 CAT showed three- to fourfold stimulation of CAT activity with forskolin but not by QUIN or NMDA. These data reveal a dose-dependent, tissue-specific, NMDA receptor-mediated stimulation of SS but not NPY mRNA. Such stimulation of SS gene expression does not require activation of the protein kinase A or protein kinase C signaling pathways, is not transcriptionally mediated, and is likely posttranscriptional, as also suggested by the delay in SS mRNA induction.

Using a cDNA probe, genomic clones were obtained for the 5′ flanking promoter sequence of the human somatostatin receptor 2-encoding gene (SSTR2). A 3.8-kb sequence directly upstream from the start codon was analyzed. This sequence shares a number of characteristics with the promoters of other G-protein-coupled receptor (GPCR)-encoding genes including a number of G + C-rich regions, binding sites for several transcriptional factors, and the absence of coupled TATAA and CAAT sequences.

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR 4 ). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst 1 , sst 2 , sst 3 , sst 4 and sst 5 ). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF 1 ) appears to comprise sst 2 , sst 3 and sst 5 receptor subtypes. The other class (SRIF 2 ) appears to comprise the other two recombinant receptor subtypes (sst 1 and sst 4 ). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression, and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR1-4 display weak selectivity for SST-14 binding whereas hSSTR5 is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3, and 5 belong to a similar SSTR subclass. hSSTR1 and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtype-selective, and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3-7 which is lined by hydrophobic and charged amino acid residues.

25-hydroxycholesterol (25-OHC), an oxysterol that potently regulates cellular cholesterol metabolism, induced formation of novel fibrillar structures in normal mouse astrocytes as observed by fluorescence microscopy with the cholesterol probe, filipin. These fibrils were identified as lysosomes by their immunoreactivity for the lysosome associated membrane glycoprotein (LAMP). In contrast, astrocytes derived from the Niemann-Pick disease type C (NPC) mutant mouse were resistant to this oxysterol-induced lysosomal reorganization. NPC astrocytes have abnormal intracellular cholesterol storage as observed by brightly positive filipin staining of their lysosomes. These results show that lysosomal cholesterol storage in NPC astrocytes is associated with a block in oxysterol-mediated fibrillar reorganization of lysosomes.

The somatostatin (SS) gene is transcriptionally regulated via the cyclic AMP (cAMP) response element (CRE), located in the proximal promoter (-41 to -48 bp). We have previously reported that glucocorticoids induce dose-dependent cell-specific alterations in the steady-state SS mRNA level. Here we have investigated direct transcriptional control of the SS gene by glucocorticoids. We have examined transcriptional interaction between glucocorticoids and the cAMP signalling pathway and mapped the 5′ upstream regulatory region of the SS gene involved in glucocorticoid transactivation. Transcriptional regulation was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in PC12 rat pheochromocytoma cells and A126-1B2 (protein kinase A-deficient mutant PC12) cells, by acute transfection of 5′ flanking SS DNA (- 750, -250 and -71 bp) ligated to the reporter (CAT) gene. Dexamethasone (DEX) induced a dose-dependent 2.2-fold stimulation of SS gene transcription in PC12 cells, but not in A126-1B2 cells. Other steroid and thyroid hormones tested, and retinoic acid, were ineffective, while cAMP and forskolin stimulated gene transcription 4-5-fold in PC12 cells but not in A126-1B2 cells. DEX exerted an additive effect on cAMP-induced gene transcription. Deletion of the promoter from -750 to -71 bp (but not from -750 to -250 bp) abolished all stimulatory effects of DEX without affecting cAMP responsiveness. Mutation of the CRE abrogated both DEX- and cAMP-dependent gene enhancement. Gel electrophoretic mobility shift assays confirmed that the -250 to -71 bp region of the SS promoter (but not the -71 to +55 bp domain) binds specifically to a glucocorticoid response element-sensitive nuclear protein(s) from PC12 cells, suggesting a putative glucocorticoid receptor interaction with SS promoter DNA. We conclude that glucocorticoids regulate SS gene transcription positively. Glucocorticoid-induced transactivation shows dependence on protein kinase. A activity, and may be mediated via protein-protein interaction between the glucocorticoid receptor and the CRE binding protein. DNA sequences upstream from the CRE between -250 and -71 bp in the SS promoter appear to be the target of glucocorticoid action.

This study investigates hypothesis generation and evaluation in clinical problem solving by medical trainees. The study focuses on 1) directionality of reasoning and 2) use of confir mation and disconfirmation strategies in generating and evaluating hypotheses. Two clinical problems were divided into segments of information containing presenting complaint, past history, and physical examination. The initial information indicated a typical myocardial infarct but subsequent information contradicted it. The results showed that the participating students predominantly used forward reasoning and confirmation strategies. When faced with con tradictory evidence: 1) second-year students ignored cues in the problem or reinterpreted them to fit the hypothesis; 2) third-year students generated concurrent hypotheses to account for different sets of data; and 3) fourth-year students generated several initial hypotheses and subsequently narrowed the hypothesis space by generating a single coherent diagnostic explanation. The results are discussed in terms of coordination of clinical evidence and its relationship to scientific reasoning. Key words: hypothesis generation; diagnostic reasoning; novice strategies; medical problem solving. (Med Decis Making 1993;13:198-211)

We report here on the cloning of a human intronless gene encoding a member of the G-protein linked somatostatin (SST) receptor subfamily, termed SSTR3. Based on the deduced amino acid sequence, this gene encodes a 418 amino acid protein displaying sequence similarity, particularly within putative transmembrane domains, with the recently cloned human SSTR1 (62%), SSTR2 (64%) and SSTR4 (58%) receptors. Membranes prepared from COS-7 cells transiently expressing the human SSTR3 gene bound [125I]Leu8,D-Trp22,Tyr25 SST-28 in a saturable manner with high affinity (approximately 200 pM) and with rank order of potency (D-Trp8 SST-14SST-14SMS-201-995SST-28) indicative of a somatostatin-14 selective receptor. The pharmacological profile of the expressed human SSTR3 receptor is similar but not identical to that reported for the rat homolog [(1992) J. Biol. Chem. 267, 20422] where the peptide selectivity is SST-28or = SST-14SMS-201-995. Northern blot analysis reveals the presence of an SSTR3 mRNA species of approximately 5 kb in various regions of the monkey brain, including the frontal cortex, cerebellum, medulla, amygdala, with little or no SSTR3 mRNA detectable in brain regions such as the striatum, hippocampus, and olfactory tubercle. The SSTR3 receptor gene maps to human chromosome 22. The existence of at least four distinct human genes encoding somatostatin-14 selective receptors with diverse pharmacological specificities may help to account for some of the multiple biological actions of somatostatin under normal and pathological conditions.

Biological activity now recognizable as that of somatostatin (SS) was first encountered in 1968 by Krulich et al. [33] during attempts to screen hypothalamic extracts for growth hormone (GH) releasing activity. The workers identified a GH-inhibitory substance, characterized it as a low-molecular-weight basic peptide, and were able to localize the biological activity to the median eminence and anterior hypothalamic area. A year later, Hellman and Lernmark [23] reported the presence of a potent insulin-inhibitory factor in extracts of pigeon pancreatic islets. These two apparently unrelated observations were to come into sharp focus in 1973 with the discovery by Brazeau et al. [10] of the tetradecapeptide SS-14 as the hypothalamic GH-inhibitory substance, a signal achievement duly recognized by the award of the Nobel Prize to Guillemin in 1977. Subsequent studies revealed that SS is not produced only in the hypothalamus but also occurs throughout the central nervous system (CNS), in peripheral neurons, in the gastrointestinal (GI) tract and in the pancreatic islets of Langerhans. Furthermore, SS-like immunoreactivity is heterogeneous and is distributed in many tissues in both vertebrate and invertebrate species of the animal kingdom and in the plant kingdom. Extensive investigation of SS has shown that its wide anatomical distribution is paralleled by an equally broad spectrum of biological effects.

To determine whether somatostatin receptor subtypes arise from molecular heterogeneity of the receptor protein, we have cross-linked the putative receptor in normal rat tissues and in AtT-20 and GH3 cells, both chemically with SS-14, SS-28 and Tyr3 SMS ligands, as well as by photoaffinity labeling with an azido derivative of Tyr3 SMS (EE 581). Three prominent somatostatin receptor proteins of 58-kDa, 32-kDa, and 27-kDa size have been identified. These proteins exhibit a tissue-specific distribution, ligand selectivity, and relative preference for SS-14 and SS-28 binding, and thus qualify as somatostatin receptor subtypes. Using EE 581 as a photoaffinity probe, the 58-kDa and 32-kDa proteins have been purified to homogeneity from brain and AtT-20 cells by successive SDS-PAGE. The 58-kDa form has been trypsinized and amino acid sequence data obtained from four tryptic fragments. With the help of synthetic oligonucleotides derived from these sequences, work is currently in progress to clone the 58-kDa protein to elucidate its complete sequence, its expression, and its functional relationship to the somatostatin receptor and its pharmacological subtypes.

This study assessed the pharmacokinetic and pharmacodynamic behaviour of alfentanil during and after coronary artery bypass grafting (CABG). Twenty-eight patients with good ventricular function having CABG were divided into three groups and premedicated with morphine 0.1 mg · kg−1 IM, scopolamine 0.005 mg · kg−1 IM and diazepam 0.1 mg · kg−1 PO. Group I patients received an infusion of 250 μg · kg−1 of alfentanil over one hour coincidental with a second infusion at 2.5 μg · kg−1 · min−1 which was continued to the end of surgery. Patients in group II received 300 μg · kg−1 and 3.0 μg · kg−1 · min−1 and patients in group III 350 μg · kg−1 and 3.5 μg · kg−1 · min−1. The tracheas of all patients were intubated after receiving alfentanil 96 μg-kg−1 and pancuronium 0.15 μg · kg−1. Haemodynamic responses to intubation and surgical stimuli (≥ 20 per cent increase in heart rate and/or systolic blood pressure from control) were treated with isoflurane, one to two per cent inspired, until abolished. Blood samples were taken during and after surgery for plasma alfentanil concentrations which were determined by radioimmunoassay. After surgery the times to awakening and extubation, and alfentanil elimination half-life (t1/2B = 0.6931−k) were determined for each patient. Haemodynamic responses occurred in 20 patients. There were no significant differences in any variable among the groups. The times to awakening and extubation for all patients were 3.2 ± 0.6 and 8.8 ± 1.2 hr (mean ± SEM) respectively. The elimination half-life for all patients was 5.1 ± 1.0 hr(mean ± SEM). We conclude that alfentanil given at these rates of infusion provides inadequate adrenergic suppression for CABG yet does not allow early postoperative tracheal extubation. The elimination half-time appears prolonged after cardiopulmonary bypass compared with values obtained from volunteers or from patients undergoing non-cardiac surgery.

Adrenocortical somatostatin receptors have been shown to interact with somatostatin-14 (S-14) and somatostatin-28 (S-28). To determine whether these peptides interact with the same or different receptor proteins, we chemically cross-linked these receptors using disuccinimidyl suberate to radioligands prepared from tyrosinated S-14 and S-28 analogs. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and subsequent autoradiography of [125I-Tyr11] S-14 and [Leu8, D-Trp22, 125I-Tyr25] S-28 cross-linked to their binding sites following solubilization in the presence of 50 mM DTT revealed the presence of a single labelled protein of Mr = 200,000. When the cross-linked material was treated under non-reducing conditions, this band was not observed. Furthermore, addition of excess S-14 and S-28 at the time of binding inhibited the incorporation of both radioligands into the receptor protein. These results demonstrate that adrenocortical membrane receptors for somatostatin contain a single receptor protein sub-unit or sub-units of identical size which interact with both S-14 and S-28.

We examined three issues: (1) properties of the intraclass correlation coefficient (r 1); (2) normality of its z transform; and (3) Fisher's formula for the variance of z. To do this we simulated 1,000 samples of r 1 and of z for several values of p, both for equal and for unequal family sizes. Those samples permitted us to evaluate some of Fisher's remarks on the bias, variance, and normally of r 1 Furthermore, they showed us that all simulated z transforms were normally distributed with variances that agreed with the values calculated from a modification of Fisher's formula. Those last two results affirm the applicability of the z transform and of Fisher's modified formula to studies in human genetics involving unequal family sizes.

Synthetic somatostatin-28 (S-28) as well as a related endogenous rat hypothalamic somatostatin-like compound (3K SLI) were incubated with hypothalamic extracts from which endogenous somatostatin-like immunoreactivity (SLI) had been removed by immunoabsorption. The reaction products were analyzed by gel chromatography, HPLC as well as two different radioimmunoassay for tetradecapeptide somatostatin (S-14) in which S-28 crossreacted either 100% (RIA R149) or less than 0.001% (RIA S39). The results indicate that incubation of S-28 with SLI free hypothalamic extracts results in a rapid decrease of total immunoreactivity measured with RIA R149 (t 1/2 = 14 min). By contrast, with RIA S39 a rise from zero to a peak value at 8 min was measured suggesting the formation of S-14. This was confirmed by subsequent analysis by gel chromatography and HPLC. Using endogenous 3K SLI a decrease of total R149-immunoreactivity with a similar time course (t 1/2 = 17 min) was observed simultaneously with the emergence of material that corresponded to S-14. This converting activity seems to be specific for SLI-containing tissues since similar rates of conversion were observed with extracts from cerebral cortex and cerebellum but not with lung and liver extracts. It is concluded that (1) S-28 is converted to S-14 by hypothalamic enzymes; (2) the processing of 3K SLI is similar, suggesting the two molecules are closely related, if not identical, and (3) the regulation of S-28 to S-14 conversion could represent an important mechanism for controlling the functional activity of somatostatinergic cells.

Flow cytometry has been examined as a method to separate islet cells into homogeneous subpopulations. Collagenase-isolated rat islets were dissociated into single cells and these were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. Light scatter histograms showed two peaks of viable cells. Radioimmunoassay of hormone content in cell fractions collected across the two peaks showed that glucagon- containing cells were concentrated towards the left side of the left peak and somatostatin-containing cells were concentrated towards the right side of the left peak, whereas insulin-containing cells were clearly enriched in the right peak. The B-cell-enriched fraction (90% B cells, 3% A cells, 2% D cells) exhibited significant insulin secretory responses to glucose (16.7 mM), and 3-isobutyl-1-methylxanthine (0.1 mM), during a 24-h culture period, and these responses were slightly greater than those observed in the original mixed islet cell preparation (66% B cells, 14% A cells, and 4% D cells). These results indicate that flow cytometry can be applied to sort pancreatic islet cells into populations enriched in specific endocrine cell types for further study of the functions of individual cell types.

The application of immunofluorescence technique with anti-insulin, anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide (PP) antisera to sections of precisely sampled regions of the human pancreas allowed the quantitative evaluation of the total content of these four endocrine cell populations in 13 nondiabetics, in 2 insulin-dependent diabetics (IDDM), and in 2 non-insulin-dependent diabetic subjects (NIDDM) of various age and sex. In nondiabetic subjects, PP-cells appear sex-related. Male individuals have a significantly greater volume of PP-cells than female. In diabetic subjects, the only marked difference as compared with nondiabetics is the reduction of insulin cell volume in IDDM. Other small differences between individual endocrine cell volumes are detectable in both IDDM and NIDDM as compared with nondiabetics, but their significance is at present unclear. The qualitative changes of islet structure accompanying insulin cell reduction in IDDM were not considered in the present Study.

Timed urine collections were obtained from normal men and women for a study of somatostatin in urine. Somatostatin-14-like immunoreactivity (S-14 LI) was extracted from urine by adsorption to C-18 silica cartridges, and the extracts were analyzed by RIA directly or after gel and high pressure liquid chromatography (HPLC). S-14 LI was consistently detected in all urine samples. The mean S-14 LI content in 24-h collections in men [10.96 +/- 0.91 (+/- SE) pmol/24 h; n = 10; total volume, 1.96 +/- 0.09 L] was not significantly different from that in women (9.09 +/- 0.85 pmol/24 h; n = 10; total volume, 1.87 +/- 0.09 L). Gel chromatography of 24-h urine collections revealed three major peaks of S-14 LI coeluting with S-14, S-28, and a 12,000 mol wt species corresponding to prosomatostatin. HPLC analysis confirmed the presence of S-14, S-28, and the 12,000 mol wt form and additionally revealed a major fourth peak of 3,000 mol wt, closely related to S-28. Immunoreactivity corresponding to [Des,Ala1]S-14 (S-13) was identified by HPLC coelution with synthetic S-13 and reactivity in a centrally, but not N-terminally directed, S-14 RIA. The relative proportions of the different HPLC peaks varied considerably during the 24-h period. S-14 and S-28 were excreted preferentially during the day, whereas the 12,000 and 3,000 mol wt forms were excreted preferentially at night. The ingestion of a mixed meal evoked parallel increases in plasma S-14 LI and urinary S-14 LI excretion in six normal subjects. We conclude that the principal circulating forms of S-14 LI (S-14, S-28, S-13, and pro-S) are present in urine and that the measurement of urinary S-14 LI could provide a reliable index of integrated plasma S-14 LI concentrations.

Quantitative in vivo autoradiography was used to identify and compare the regional distribution of specific binding sites for blood-borne [125I-Tyr11]somatostatin-14 (S-14§) and [Leu8,D-Trp22-125I-Tyr25]somatostatin-28 (S-28§) in the rat brain. Rats were given intracardiac injections of 17 pmol S-14§ (with or without unlabeled S-14) or 17 pmol S-28§ (with or without unlabeled S-28). After whole body perfusion and fixation, brains were processed for light microscopic autoradiography of S-14§- and S-28§-binding sites. Of the peripheral tissues, the adrenal glands showed the highest uptake of S-14§ and S-28§ (determined by counting) and were subsequently processed for autoradiography for comparison with brain. Specific autoradiographic grains (ARG) associated with both radioligands were identified only in the circumventricular organs (CVOs). The highest ARG density associated with S-14§ was found in the area postrema, followed in decreasing order by the subfornical organ and the organum vasculosum lamina ter...

To determine directly whether somatostatin-28 (S-28) physiologically regulates GH secretion, and what its contribution is relative to somatostatin-14 (S-14), we have compared the effects of immunoneutralization with a specific S-28 antibody with those of an anti S-14/S-28 serum on GH secretory dynamics in the rat. Plasma samples were obtained every 15 min for 7 h (1000-1700 h) from conscious, chronically cannulated rats after iv administration of 1 ml of one of the following sera: 1) rabbit anti S-28 (reacts with S-28, but not with S-14; maximum binding, 297 pmol/ml), 2) nonimmune rabbit serum, 3) sheep anti-S-14/S-28 serum (maximum binding, 9.4 nmol S-14 or S-28/ml), and 4) nonimmune sheep serum. A comparison of the mean integrated plasma GH levels during peak and trough periods showed significantly higher trough GH levels in both antibody-treated groups compared to those in the corresponding controls. In the anti-S-14/S-28-treated group, the elevation of trough period GH levels (40.5 +/- 3.5 ng/ml) represented a 3.25-fold increase (P less than 0.01) compared to the control value (12.5 +/- 1.5 ng/ml). In the anti-S-28-treated group, trough period GH levels (14 +/- 1.6 ng/ml) showed a 2.3-fold increase (P less than 0.01) compared to the control value (6.1 +/- 0.9 ng/ml). Mean peak period GH levels were 1.35-fold higher (P less than 0.05) than control values in the anti-S-14/S-28-treated group; anti-S-28 serum did not change mean peak GH levels. These data provide strong evidence that circulating S-28 (like S-14) physiologically regulates trough GH secretion and that the contribution of S-28 to GH inhibition is as important as that of S-14.

We examined the effects of intracerebroventricular (icv) administration of the somatostatin peptides, S-14 and S-28, and an analog, [D-Trp22]S-28, on spontaneous GH and glucose secretion in freely moving rats bearing chronic icv and intraatrial cannulae. Normal saline icv-treated control rats exhibited the typical pulsatile pattern of GH secretion. Central injection of S-14, S-28, and [D-Trp22]S-28 (each at two different doses, 5 and 10 micrograms) caused an early significant suppression of plasma GH levels which was of longer duration after S-28 or [D-Trp22]S-28 than after S-14 injection. The icv administration of S-28 and [D-Trp22]S-28 (but not S-14) also resulted in significant hyperglycemia, which persisted for periods up to 1 h. In a second study designed to determine whether somatostatin administered via the brain ventricle can reach the peripheral circulation, we measured plasma levels of S-14-like immunoreactivity (S-14 LI) and S-28-(15-28) LI at frequent time intervals after icv injection of S-14 (5 micrograms) and S-28 (10 micrograms). Plasma S-14 LI rose from a basal value of 0.16 +/- 0.02 (+/- SE) ng/ml to a peak of 3.0 +/- 1.0 ng/ml at 1 min, and S-28 icv resulted in a 150-fold increase in plasma S-28-(15-28) LI 1 min after injection. Sephadex G-50 gel chromatography revealed that the plasma immunoreactivity consisted of a single molecular species (either S-14 or S-28) corresponding to the centrally administered counterpart. These results demonstrate that after icv administration at high doses, both forms of somatostatin cause an acute inhibition of spontaneous GH secretion and that the larger form also causes hyperglycemia. We correlate these events with massive hypersomatostatinemia resulting from leakage of the somatostatin peptides from the cerebrospinal fluid into the systemic circulation. The findings indicate that icv injected hypothalamic hormones can reach the peripheral circulation and exert significant biological actions on distant target organs; thus, they have important implications for the design and interpretation of experiments in which peptides are administered via the cerebral ventricles.

Changes in immunoreactive somatostatin were examined in islets, whole pancreas, stomach, and hypothalamus of streptozotocin-diabetic rats. There was no change in islet somatostatin content at 2 days after the administration of streptozotocin, but thereafter, somatostatin progressively increased in the diabetic animals by 45% at 2 weeks, 230% at 6 weeks, and 500% by 6 months. By contrast, islet glucagon rose acutely and maintained a constant 2-fold elevation irrespective of the duration of the diabetes. Morphometric analysis of the somatostatin- and glucagon-producing cells in the islets revealed an apparent augmentation of both cell types. The concentration of somatostatin per total pancreas was also increased in the diabetic animals, suggesting that the islet increase was part of a true increase in pancreatic somatostatin. Pancreatic glucagon was unchanged despite the islet increase. The increase in pancreatic somatostatin was paralleled by an elevation in gastric somatostatin concentration, implying a common mechanism in response to streptozotocin for the somatostatin cells in these two sites. There was no change in hypothalamic somatostatin concentration. Islet somatostatin was also increased in alloxan-diabetic rats. suggesting that streptozotocin does not stimulate the D cells directly.

Hypothalamic somatostatin concentration is increased in normal rats treated with growth hormone. Somatostatin is reduced in the median eminence of hypophysectomized rats but restored to normal following growth hormone administration. These results suggest that growth hormone exerts a positive feedback effect on hypothalamic somatostatin mechanism by which it may regulate its own secretion.

We studied the pancreatic and enteric hormone profile of a 46-year-old woman who had hyperglycemia and a pancreatic tumor. Before operation, there was no evidence of overproduction of glucagon or insulin. The tumor's ultrastructure had a distinctive endocrine morphology, resembling D cells. Prompted by the recent demonstration of somatostatin in D cells of pancreatic islets, we analyzed the tumor and found a large quantity of immunoreactive somatostatin (301 ng per milligram of tissue). Insulin, glucagon, gastrin, vasoactive intestinal polypeptide and human pancreatic polypeptide were present in only trace quantities. The tumor cells were cultured in monolayers, which remained viable up to 51 days and released somatostatin into the culture medium. In seven insulinomas and two glucagonomas, we found the somatostatin content either much lower (less than 0.6 ng per milligram of tissue) or undetectable. After complete resection of the tumor, our patient became euglycemic and has remained so for the past 20 months.

In the present study we characterized and compared the relative amounts of the different molecular forms of somatostatin-14 like immunoreactivity (S-14 LI) and of somato-statin-28(1–2) like immunoreactivity (S-28(1–2) LI) in extracts of tumor and peripheral plasma of a patient with a pancreatic somatostatinoma. Tissue and plasma were chromatographed on Sephadex G-50 columns equilibrated with 6 m urea. Immuno-reactivity in the eluting fractions was assayed with two separate, region specific RIAs using antibodies R149 (S-14 LI) and S309 (S-28(1–2)LI). RIA R149 recognizes the 6-8 and 14 regions of the S-14 sequence and detects S-14, S-28, and prosomatostatin, an approximately 14,000 mol wt precursor for the two peptides. RIA S309 recognizes the 2–11 segment of S-28 and reacts with S-28, S-28(1–2), and higher mol wt S-28(1–2) LI but not S-14. Total tumor S-14 LI was 190 pmol/mg protein and consisted of three peaks of immunoreactivity of apparent 14,000 mol wt (14K S-14 LI), 3,200 mol wt (3.2K corresponding to...

We have identified and characterized specific receptors for tetradecapeptide somatostatin (SRIF; somatotropin release-inhibiting factor) in rat brain using [125I]Tyr11]SRIF as the radioligand. These receptors are present in membranes obtained from a subfraction of synaptosomes. Membranes derived from cerebral cortex bind SRIF with high affinity (Ka = 1.25 X 10(10) M-1) and have a maximum binding capacity (Bmax) of 0.155 X 10(-12) mol/mg. Neither opiates nor other neuropeptides appear to influence the binding of SRIF to brain membranes. Synthetic analogs with greater biological potency than SRIF--[D-Trp8]SRIF, [D-Cys14]SRIF, and [D-Trp8, D-Cys14]SRIF--bind to the receptors with greater avidity than SRIF, whereas inactive analogs [(2H)Ala3]SRIF and [Ala6]SRIF exhibit low binding. The ratio of receptor density to endogenous somatostatin is high in the cortex, thalamus, and striatum, low in the hypothalamus, and extremely low in the brain stem and cerebellum. Thus, SRIF receptors in the brain appear to be a distinct, new class of receptors with a regional distribution different from that of endogenous somatostatin.

Spontaneously diabetic C57BL/6J obob and C57BL/Ks dbdb mice have been shown to have significantly decreased immunoassayable pancreatic somatostatin concentrations compared to lean littermate controls at 11-12 weeks: obob 1.06+/-0.15 pg/mug protein (n=10) vs control 1.94+/-0.-6 pg/mug protein (n=10) (mean +/- SE; p less than 0.005); dbdb 0.7+/-0.2 pg/mug protein (n=8) vs control 1.5+/-0.2 pg/mug protein (n=8) (mean +/- SE; p less than 0.005). An inverse relationship between circulating insulin levels and pancreatic SRIF concentration is suggested.

No information is at present available on the mode of SRIF biosynthesis. Since anglerfish pancreatic islet tissue is comprised of approximately 30% D cells, we have examined this tissue for SRIF synthesis . The following known differences in amino acid composition of islet peptides were used advantageously in this study: anglerfish proinsulin: Trp-0, Ile-2, Cys-6; anglerfish glucagon: Trp-1, Ile-0, Cys-0; mammalian SRIF: Trp-1, Ile-0, Cys-2. After incubating islet tissue with [3H]tryptophan and [14C]isoleucine or [35S]cystine for various time periods, proteins were extracted in 2 M acetic acid and desalted by Bio-Gel P-2 gel filtration. P-2 void volume proteins were then subjected to P-10 gel filtration and isolated by polyacrylamide gel electrophoresis (PAGE) at alkaline pH. The predominant amount of the immumoreactive SRIF in the extracts appeared in a peak eluting just before the salt volume on P-10 filtration and migrated slowly toward the cathode during PAGE. The behavior of synthetic SRIF was identical. The anglerfish SRIF immunoreactive peptide could be labeled with Trp and Cys but not Ile during incubations longer than 1 h. The Trp- and Cys-labeled peptide could be bound on columns to which the immunoglobulin fraction of antisera to SRIF had been complexed. Cycloheximide inhibited isotope incorporation into all islet proteins. These results indicate that islet SRIF is synthesized in situ. Moreover, the immunological activity, size, and charge characteristics of anglerfish islet SRIF appear to be similar to those of mammalian hypothalamic SRIF. When islets were subjected to short pulse incubations with labeled Trp and Cys, only peptides eluting in the 7,000-13,000 dalton portion of the filtration eluate became labeled. No appreciable isotope incorporation into SRIF was observed. However, when pulse incubations were followed by incubation in the presence of cycloheximide or excess unlabeled amino acids in isotope-free medium (chase), the incorporation of Trp and Cys into SRIF increased with the length of chase, suggesting the participation of a larger precursor in SRIF synthesis.

Quantitative electron microscopic autoradiography was used for comparing the binding of labeled somatostatin-14 (S-14) and somatostatin-28 (S-28 section) to islet cells. Monolayer cultures of rat islet cells were incubated with [125I-Tyr11]S-14 (S-14 section) or [125I-Leu8, D-Trp22, Tyr25]S-28 (S-28 section) in the presence or absence of excess unlabeled peptides. Autoradiographic grains (ARG) associated with individual islet cells were identified and expressed as the mean number per B, A, and D cells. Specific ARG associated with S-14 were found over B and A cells. S-28 section-related specific ARG were concentrated over B, A, as well as D cells. The highest density of S-14 section labeling occurred over A cells, which under conditions of maximum labeling (37 degrees C for 60 min) contained five times as many ARG as did B cells. By contrast, under the same incubation conditions, the labeling density with S-28 section was maximal over B cells, which contained four and five times as many grains as A and D cells, respectively. These observations show preferential association of S-14 section with the A cell and S-28 section with the B cel provide strong evidence for the existence of separate binding sites for S-14 section and S-28 section on A and B cells, respectively, which presumably mediate the previously reported glucagon selective inhibitory effect of S-14 and the insulin-selective action of S-28.

The action of enzymes extracted from rat hypothalamus on the previously characterized high molecular weight forms of hypothalamic somatostatin-like immunoreactivity (4 K SLI and 25 K SLI) has been investigated in vitro in order to further define the role of these molecules as possible precursors for tetradecapeptide somatostatin (SRIF). Studies of the degradation of endogenous SLI and of synthetic SRIF by hypothalamic enzymes showed that the time course of breakdown of endogenous SLI is markedly slower than that of synthetic SRIF due to the relative stability of 25 K SLI as well as the generation of at least two new immunoreactive molecules. Incubation of purified 25 K SLI with SLI-free hypothalamic extract showed after 10 to 30 min newly formed immunoreactive material of an intermediate size between 25 K SLI and 4 K SLI and after 60 min the emergence of material coeluting with SRIF. These data show that the hypothalamus contains the enzymes necessary for degrading endogenous SLI and for processing the 25 K SLI molecule to SRIF providing further evidence that 25 K SLI might be a biosynthetic precursor for SRIF.