Somatostatin-14 Research Articles

Cholecystokinin Does not Stimulate Prosomatostatin Derived Peptides in Man*

In man, plasma cholecystokinin (CCK) and somatostatin-28 (S-28) levels increase after ingestion of a mixed meal. Both peptides originate from the gastrointestinal tract. In supra- and periphysiological doses, CCK stimulates the release of somatostatin-14 from in vitro pancreatic islets and gastric cells and increases circulating somatostatin-like immunoreactivity in dogs, leading to the conjecture that CCK regulates somatostatin-like immunoreactivity secretion. Nonetheless, whether CCK is responsible in part for the meal-induced rise in S-28 in man has not been established. Therefore, the present study was designed to determine if CCK, at both physiological and supraphysiological concentrations, increases the circulating levels of prosomatostatin (proS)-derived peptides in humans. On 3 separate days, five healthy men ate a mixed liquid meal or received iv infusions of CCK at rates of 18 or 38 pmol/kg.h. Plasma levels of pro-S-derived peptides, including pro-S, S-14, S-13, S-28, and CCK, were measured. Basal CCK levels averaged 0.9 +/- 0.1 pmol/L and increased after the meal to a peak level of 5.4 +/- 1.5 pmol/L and averaged 3.1 +/- 1.2 pmol/L over 90 min. The mean basal levels of pro-S, S-14, and S-13, measured collectively, was 6.1 +/- 0.4 pmol/L eq S14 and was unaltered by food intake. The S-28 level was 6.7 +/- 0.6 pmol/L and rose to a zenith of 13.1 +/- 3.3 pmol/L by 90 min. Infusion of CCK at 18 and 38 pmol/kg.h produced steady state plasma CCK levels of 4.1 +/- 1.1 and 9.9 +/- 1.5 pmol/L, respectively. Basal levels of pro-S-derived peptides were unaltered during the infusion of either the low or high dose of CCK. We conclude that CCK by itself is not a physiological signal to the release of pro-S-derived peptides in man.

Depolarizing influences regulate somatostatin synthesis and processing in cultured cerebral cortical cells

There is increasing evidence that persistent depolarization plays a critical role not only in excitation-secretion coupling, but also in the mechanisms linking excitation of neuronal cells to long-term adaptative changes in biosynthesis of neuropeptides. Somatostatin (SRIF) release and synthesis are affected by numerous agents, such as high concentrations of potassium that cause depolarization of cellular membrane. In the present work, we tried to determine whether prolonged exposure to veratridine (VTD) regulates SRIF synthesis. We found that exposure to VTD (100 μM) resulted in the stimulation of total (cell content + media) immunoreactive SRIF (IR-SRIF). This effect was calcium- and sodium-dependent, since it was prevented when verapamil (VPM) 20 μM or tetrodotoxin (TTX) 1 μM were added simultaneously with VTD. Cerebral cortical cells were exposed to high potassium concentrations, and the nature of the IR-SRIF was characterized by high-pressure liquid chromatography (HPLC) or gel filtration. It was evident that chronic exposure to high potassium concentrations modified the elution profile of medium IR-SRIF on HPLC and gel filtration, causing an increase in somatostatin-28 (S-28) and a decrease in somatostatin-14 (S-14). The results indicate that chronic exposure to VTD or high potassium concentration increases immunoreactive somatostatin and augments synthesis of its high-molecular-weight forms. This suggests that chronic membrane depolarization activating sodium and calcium channels initiates the entry of calcium ions, which triggers somatostatin release and causes a depletion of its intracellular stores. The stimulation of somatostatin secretion could be coupled to synthesis of the peptide.

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.

Evidence for Multiple Protein Constituents of the Somatostatin Receptor in Pituitary Tumor Cells: Affinity Cross-Linking and Molecular Characterization*

Previous studies have shown that somatostatin receptors on AtT-20 and GH3 pituitary tumor cells show relative preference for binding somatostatin-28 (S-28) and somatostatin-14 (S-14), respectively. Here we have attempted to determine whether this selectivity can be explained by molecular heterogeneity of the receptor. Cells were incubated with [125I-Tyr11]S-14, [125I-Leu8-D-Trp22,Tyr25]S-28, and [125I-Tyr3]SMS, and the bound ligand was chemically cross-linked with bis-[2-succinimido-oxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, or dithiobis (succinimidyl propionate). The solubilized cross-linked material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. [125I-Tyr11]S-14 labeled three specific receptor proteins of 57K, 42K, and 27K mol wt in AtT-20 cells. The relative proportions of the protein bands were unaltered by the use of whole cells or cell membranes or by the inclusion of dithiothreitol or antiproteolytic agents. With both [125I-Tyr11]S-14 and [125I-LTT]S-28, the 57K protein constituted the major labeled component, representing 70-75% of the total cross-linked proteins. Labeling of the three protein species by [125I-Tyr11]S-14 and [125I-LTT]S-28 was inhibited by both S-14 and S-28 in a dose-dependent manner. S-28 was 10-20 times more potent than S-14 for inhibiting the labeling by both ligands of the principal receptor species of 57K. By contrast, when a radioiodinated derivative of the octapeptide analog octreotide ([125I-Tyr3]SMS) was used as ligand, the 27K protein was preferentially labeled, whereas the 57K and 42K bands were detected only as minor components. Labeling of GH3 cells with [125I-Tyr11]S-14 and [125I-LTT]S-28 revealed three cross-linked proteins of 57K, 42K, and 27K mol wt similar to those observed in AtT-20 cells. However, in this cell line the 27K protein, not the 57K species, was the dominant component identified with these two ligands, comprising 40-50% of the total cross-linked proteins. These results suggest that there are three somatostatin receptor proteins of 57K, 42K, and 27K in pituitary cells. In AtT-20 cells, the 57K protein constitutes the major receptor protein labeled by [125I-Tyr11]S-14 and [125I-LTT]S-28, whereas the 27K protein is the major species labeled by [125I-Tyr3]SMS. The 27K, not the 57K, moiety is the principal receptor form in GH3 cells. Such ligand- and tissue-selective binding by the somatostatin receptor provides strong evidence for receptor molecular heterogeneity.

Binding and internalization of somatostatin, insulin, and glucagon by cultured rat islet cells.

The pathways by which islet B, A, and D cells bind and internalize homologous (self) and heterologous (other) islet hormones were compared. [125I-Tyr]Somatostatin-14 (S-14), 125I-insulin, and 125I-glucagon were incubated with monolayer cultures of neonatal rat islet cells. Tissues were processed for quantitative electron microscopic autoradiography by the probability circle method coupled to morphometry. For all three radioligands and all three cell types surface labeling was rapidly followed by internalization of the radioligands into endocytotic vesicles. The further intracellular movement of the ligand occurred in a time- and temperature-related manner and depended on whether it was homologous or heterologous for the cell in question. Thus [125I-Tyr]S-14 in B and A cells, 125I-insulin in A and D cells, and 125I-glucagon in B and D cells were rapidly transferred from endocytotic vesicles to lysosomal structures. By contrast, [125I-Tyr]S-14 in D cells, 125I-insulin in B cells, and 125I-glucagon in A cells showed poor progression from endocytotic vesicles to downstream vesicular structures. We conclude that (a) each of the three radioligands is internalized by islet cells in a time- and temperature-dependent manner; (b) after initial internalization the further intracellular progression of the endocytosed radioligand occurs freely in cells heterologous for the radioligand but poorly in cells homologous for the radioligand; and (c) binding and endocytosis can be uncoupled from lysosomal degradation of ligand.

Circulating prosomatostatin-derived peptides. Differential responses to food ingestion.

Prosomatostatin (pro-S) and its bioactive posttranslational products, somatostatin-14 (S-14), somatostatin-13 (S-13), and somatostatin-28 (S-28), were measured in human plasma by the use of immunoglobulins to the NH2-terminus of S-28 conjugated with agarose to separate them and, thereafter, by RIA with an antiserum recognizing the COOH-terminus of pro-S, and by specific RIA for the NH2-terminus of S-14 and pro-S. In healthy men, mean basal levels of pro-S were 4 pg equivalent S-14/ml; S-14/S-13 combined were 9 pg equivalent S-14/ml; and S-28 levels were 16 pg/ml. After a 700-kcal meal, pro-S, S-14, and S-14/S-13 did not change, whereas S-28 levels doubled by 120 min and remained elevated for 240 min. To evaluate the origins of these peptides, their levels were compared in peripheral, portal, gastric, and mesenteric veins of anesthetized patients and in patients with total resection of stomach and pancreas before and after nutrient intake. The stomach and small intestine were sources of both peptides; however, most S-28 originated in the small intestine. These findings suggest that, in contrast to S-14, S-28 is a hormone and may modulate postprandial nutrient absorption and use.

Sensitivity of rat pancreatic A and B cells to somatostatin

Islet A and B cells were purified from the rat pancreas and examined for their respective sensitivity to somatostatin. Both somatostatin-14 (S14) and -28 (S28) inhibited glucagon and insulin release through direct interactions with the corresponding cell types. A dose-dependent suppression of the secretory activities was paralleled by a reduction in cellular cyclic AMP formation with similar ED50 values for both actions. The somatostatin effects on pancreatic hormone release may thus be mediated via an inhibition of adenylate cyclase activity. In pancreatic A cells, S14 and S28 were equally potent inhibitors with ED50 values ranging from 2 x 10(-12) to 2 x 10(-11) mol/l. Pancreatic B cells exhibited a similar sensitivity to S28 as the A cells (ED50 of 2 to 5 x 10(-11) mol/l), but not to S14 (ED50 of 2 x 10(-9) mol/l). Extrapolation of these in vitro sensitivities of islet A and B cells to the in vivo situation suggests that both cell types can respond to circulating S28 levels and that A cells are sensitive to both locally and distally released S14. Islet B cells appear insensitive to the normal peripheral S14 levels but could respond to locally released somatostatin. The marked difference in the sensitivities of islet A and B cells to S14 suggest that these cell types are equipped with different somatostatin receptors. This notion was further supported by the cell-selective actions of the synthetic S14 analogues [D-Trp8, D-Cys14]S14 and desAsn5[D-Trp8, D-Ser13]S14.

Distribution of somatostatin receptors in the brain of the frog Rana ridibunda: Correlation with the localization of somatostatin‐containing neurons

The biochemical characterization and anatomical distribution of somatostatin binding sites were examined in the brain of the frog Rana ridibunda, and the distribution of the receptors was compared with the location of somatostatin immunoreactive neurons. The pharmacological profile of somatostatin receptors was determined in the frog brain by means of an iodinated superagonist of somatostatin, [125I-Tyr0,DTrp8]S-14. Membrane-enriched preparations from frog brain homogenates were shown to contain high-affinity receptors (KD = 0.78 +/- 0.34 nM; Bmax = 103 + 12.7 fmoles/mg protein) with pharmacological specificity for [DTrp] substituted S14 and S28 analogs. The distribution of somatostatin-binding sites was studied by autoradiography on coronal sections of frog brain. Various densities of somatostatin receptors were detected in discrete areas of the brain. The highest concentration of binding sites was observed in the olfactory bulb, in the pallium, and in the superficial tectum. Moderate binding was observed in the striatum, amygdaloid complex, preoptic area, and cerebellum. Immunocytochemical studies of the distribution of somatostatin-28 (S28) related peptides were also conducted in the frog brain. Two antisera that recognize distinct epitopes of the somatostatin molecule have been used for immunohistochemical mapping of the peptide. Antiserum SS9 recognizes both S28 and somatostatin-14 (S14) and allowed the labelling of perikarya. Antiserum S320 recognizes the N-terminal fragment (1-12) resulting from enzymatic cleavage of S28. This latter antiserum, which does not cross-react with S28, stained mainly neuronal processes. At the infundibular level, however, both antisera stained cell bodies and fibers. Immunoreactive somatostatin-related peptides were detected in many areas of the frog brain. In the diencephalon, a heavy accumulation of perikarya and fibers was seen in the preoptic nucleus, the dorsal and ventral infundibular nuclei, and the median eminence. Immunoreactive perikarya were also observed in the telencephalon, especially in the pallium and in thalamic nuclei. Immunostained processes were detected in many telencephalic areas and in the tectum. There was good correlation between the distribution of somatostatin-immunoreactive elements and the location of somatostatin-binding sites in several areas of the brain, in particular in the median pallium, the tectum, and the interpeduncular nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatostatin blocks a calcium current in rat sympathetic ganglion neurones.

1. The effects of somatostatin and somatostatin analogues on a Ca2+ current from acutely isolated and short-term (24-48 h) cultured adult rat superior cervical ganglion (SCG) neurones were studied using the whole-cell variant of the patch-clamp technique. 2. [D-Trp8]Somatostatin (SOM) produced a rapid, reversible and concentration-dependent reduction of the Ca2+ current. Ca2+ current amplitude was reduced over the voltage range -15 to +40 mV with the greatest reduction occurring where the amplitude was maximal (ca +10 mV). In the presence of SOM, the Ca2+ current rising phase was slower and biphasic at potentials between 0 and +40 mV. 3. Application of 0.1 microM-SOM for greater than 10 s resulted in a desensitization of the response. During a 4 min application of 0.1 microM-SOM, Ca2+ current amplitude returned to about 90% of control. A second application of 0.1 microM-SOM produced less block than the initial application. 4. Concentration-response curves for SOM, somatostatin-14 (SOM-14) and somatostatin-28 (SOM-28) were fitted to a single-site binding isotherm. The concentrations producing half-maximal block and the maximal attainable blocks of the Ca2+ current for SOM, SOM-14 and SOM-28 were 3.3, 5.4 and 35 nM, respectively and 55, 51 and 54%, respectively. SOM-14 and SOM-28 slowed the Ca2+ current rising phase in a manner similar to that of SOM. Somatostatin-28 had no effect on the Ca2+ current at 1 microM. 5. The magnitude of the Ca2+ current block produced by 0.1 microM-SOM was not significantly altered in the presence of 1 microM-idazoxan, atropine, naloxone or the somatostatin antagonist aminoheptanoyl-Phe-D-Trp-Lys-O-benzyl-Thr. 6. Internal dialysis with solutions containing 500 microM-guanylyl-imidodiphosphate (Gpp(NH)p) or guanosine-5'-O-(3-thiotriphosphate)(GTP-gamma-S) decreased the Ca2+ current amplitude by 36 and 41%, respectively, and induced a biphasic rising phase in the Ca2+ current. Under these conditions, application of 0.1 microM-SOM produced significantly less block of Ca2+ current amplitude (7.1 and 14.7%, respectively) when compared with controls. 7. Internal dialysis with solutions containing 500 microM-guanosine-5'-O-(2-thiodiphosphate)(GDP-beta-S) had no significant effect on either the Ca2+ current amplitude or block produced by 0.1 microM-SOM. 8. Internal dialysis with solutions containing 500 microM-cyclic adenosine 3',5'-monophosphate (cyclic AMP) and 3-isobutyl-1-methylxanthine had no significant effect on either the Ca2+ current block produced by 0.1 microM-SOM or the Ca2+ current amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)

Further studies of the effects of somatostatin and related peptides in area CA1 of rabbit hippocampus.

1. In slice studies of mature and immature CA1 hippocampal pyramidal cells from rabbit, somatostatin 14 (SS14), the related peptide somatostatin 28(1-12) [SS(1-12)], and the synthetic analogue of somatostatin 14, SMS-201995 (SMS), had similar effects. When pressure-ejected onto cell somata, these peptides elicited depolarizations, often accompanied by action potential discharge. When applied to dendrites, the peptides produced depolarizations or hyperpolarizations. 2. When a large amount of one of the three somatostatin-related (SS) peptides was applied to the slice at some distance from the impaled cell, hyperpolarizations were observed that were not always blocked by tetrodotoxin (TTX) or low Ca2+. Since SS peptides were also found to depolarize interneurons in area CA1, it seems likely that the hyperpolarizations that were blocked by TTX or low Ca2+ were mediated via excitation of interneurons that in turn hyperpolarized pyramidal cells. 3. All SS peptides also had long-lasting effects on CA1 pyramidal cells that led to spontaneous firing of action potentials and an increase in the number of action potentials discharged in response to a given depolarizing current pulse; the spontaneous discharge effect was blocked by TTX or low Ca2+ plus Mn2+ and, thus, appeared to have a presynaptic mechanism. However, the increase in discharge in response to a constant depolarizing current pulse was not dependent on intact synaptic transmission and, therefore, was attributable to a direct postsynaptic effect of the SS peptides.

Peptide immunocytochemistry in afferent neurons from lower gut in rats.

Several peptides were detected in primary sensory neurons located in nodose and dorsal root ganglia and projecting from rat cecum and rectosigmoid, through a combination of retrograde staining by the fluorescent tracer DY-2HCl and of the immunofluorescent procedure of Coons. The three larger cell populations thus identified stored immunoreactive components respectively similar to calcitonin gene-related peptide (CGRP), substance P (SP), and a peptide related to peptide histidine methionine (PHM). The later immunoreactivity consisted of a single molecular form with an apparent molecular weight smaller than PHM itself. Fewer cells contained components immunologically similar to somatostatin 14 (ST14), to the 1-14 N-terminal sequence of somatostatin 28 (1-14 S28), and to neuropeptide Y (NPY). Neonatal treatment with capsaicin resulted in a drastic reduction of immunoreactivity for SP, PHM, ST14, 1-14 S28, and in a partial reduction of CGRP-like positive perikarya. These results demonstrate that several peptides are potentially involved in the sensory innervation of the lower gut in rat.

Biosynthesis of somatostatin in canine fundic D cells.

The observation that virtually all of the somatostatin-like immunoreactivity in the stomach consists of somatostatin-14 (S14), to the exclusion of somatostatin-28 (S28), suggests a unique pattern of prosomatostatin posttranslational processing. In order to examine the mechanisms by which S14 is produced from its precursor in the stomach, we investigated the biosynthesis of somatostatin in isolated canine fundic D cells. D cells pulse-labeled with [35S]cysteine revealed a cycloheximide inhibitable time-dependent incorporation of radioactivity into S14. A small fraction of radioactivity was incorporated into S28 but not into larger precursors. However, when the cells were incubated with monensin (1 microM), incorporation of radioactivity into a presumed somatostatin precursor was noted. Upon transfer of [35S]cysteine prelabeled cells to radioactivity-free medium, no conversion of S28 to S14 could be detected and the decrease of labeled S14 in cells correlated with a complimentary increase in the culture medium. Exogenous somatostatin inhibited somatostatin biosynthesis in a fashion that could be blocked by pertussis toxin pretreatment. Stimulation of prelabeled D cells with tetradecanoyl phorbol 13-acetate (10(-7) M) or forskolin (10(-4) M) for 2 h resulted in release of 41 and 33% of the newly synthesized radioactive S14, respectively, while only 9 and 6% of the total cell content of radioimmunoassayable somatostatin was secreted. These data suggest that: (a) somatostatin is synthesized in fundic D cells primarily as S14, (b) S14 is produced by rapid processing of a larger precursor but there is little, if any, conversion of S28 to S14, (c) somatostatin biosynthesis is autoregulated, and (d) newly synthesized S14 is preferentially released from D cells in response to stimulation.

Effects of somatostatin and pancreatic polypeptide on exocrine and endocrine pancreas in the rats.

The effects of somatostatin-14 (SS) and rat pancreatic polypeptide (rPP) on endocrine and exocrine functions of the pancreas, stimulated by 40 ng/kg cholecystokinin octapeptide during continuous infusion of 1 U/kg/h secretin, were investigated in the rat, in vivo. Protein output in the pancreatic juice and integrated insulin (IRI) secretion were inhibited, in a dose related fashion, by continuous infusion of SS, in doses of 0.5, 5 and 50 micrograms/100g/h. On the other hand, bicarbonate and volume output were not inhibited by SS. The rPP significantly inhibited not only the protein output but also the bicarbonate and volume output of the pancreatic juice in doses of 0.1 and 1 microgram/100g/h. Integrated IRI secretion was inhibited significantly by rPP, but the inhibition of IRI secretion was much greater in the portal than in the jugular vein. Plasma SS and rPP concentrations showed physiological ranges during infusion of doses of 0.5 and 0.1 microgram/100g/h of SS and rPP, respectively. Therefore, there was a marked difference between the inhibition by rPP and that by SS on the exocrine and endocrine pancreas. These observations suggest that SS and PP may play a physiological role in the pancreas and that their effects on the pancreas relate to different mechanisms.

Peptides derived from cleavage of prosomatostatin at carboxyl- and amino-terminal segments. Characterization of tissue and secreted forms in the rat.

Tissue and secreted forms of rat prosomatostatin and its cleavage products were characterized immunochemically using high performance liquid chromatography and sequence-specific radioimmunoassays directed against somatostatin-14 (S-14), S-28-(1-12), and S-28-(1-14). Acetic or hydrochloric acid extracts of hypothalamus, pancreas, stomach, and jejunum contained seven molecular forms of Mr = 10,400 (corresponding to prosomatostatin (pro-S], Mr = 6,800 (7-kDa peptide, consisting of an NH2-terminally truncated form of pro-S), Mr = 7,600 (8-kDa peptide, corresponding to pro-S-(1-76), i.e. pro-S minus the COOH-terminal -Arg-Lys-S-14), Mr = 5,600 (5-kDa peptide, corresponding to pro-S-(33-76)) and three peptides co-chromatographing with synthetic S-14, S-28, and S-28-(1-12). Acid/ethanol extracts of these tissues contained pro-S, 8-kDa peptide, S-28, S-14, and S-28-(1-12) forms, but not the 7- and 5-kDa species. Pepstatin inhibited 7- and 5-kDa peptide formation in acetic acid extracts of tissues. The secreted forms consisted of the same five forms present in acid/ethanol or acetic acid plus pepstatin tissue extracts. The 7- and 5-kDa peptides were not secreted and appeared to be derived artifactually, presumably through the action of renin- and cathepsin D-like acid proteases. Accurate quantitation of the 8-kDa peptide by acid/ethanol extraction revealed a variable tissue distribution. Since the presence of the 8-kDa form provides evidence for direct processing of pro-S----S-14 + 8-kDa peptide, the present data suggest that pro-S----S-14 conversion is important for S-14 synthesis in the hypothalamus and pancreas, tissues rich in the 8-kDa form, but not in the stomach and jejunal mucosa, which contain low concentrations of this peptide.

Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex.

The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme.

Anagelsic effect of intracerebroventricular (ICV) somatostatin-14 (SOM-14), arginine vasopressin (AVP) and oxytocin (OT) in a patient with terminal cancer pain

Anagelsic effect of intracerebroventricular (ICV) somatostatin-14 (SOM-14), arginine vasopressin (AVP) and oxytocin (OT) in a patient with terminal cancer pain

Internalization and subcellular distribution of radiolabeled somatostatin-28 in mouse anterior pituitary tumor cells.

The ACTH-secreting mouse AtT-20/D16-16 (AtT-20) tumor corticotroph possesses receptors for the tetradecapeptide somatostatin (S-14) to which the 14-amino acid N-terminal extension somatostatin-28 (S-28) also binds. When AtT-20 cells are exposed to either S-14 or S-28 for extended periods of time, a marked decrease in S-14 receptor density is observed. Since receptor down-regulation is frequently associated with internalization of ligand and/or receptor, the present study was designed to establish whether AtT-20 cells could in fact internalize S-28 and to determine the subcellular localization of internalized peptide. Cells were incubated in the presence of [Leu8, D-Trp22,125I-Tyr25] S-28 for 1, 4, and 18 h; washed with PBS; and harvested. Cell pellets were fixed, sectioned, and analyzed by light and electron microscopic autoradiography. Uptake of radiolabeled S-28 (90% of all cells) was inhibited by 80% when unlabeled S-14 was coincubated with [125I]S-28. Of the cell compartments examined, plasma membrane, secretory granules, lysosomes, Golgi apparatus, and nuclear membrane all had distinct time-dependent labeling patterns. Plasma membranes were maximally labeled 1 h after exposure to [125 I] S-28. Secretory granule and lysosomal labeling was observed within 1 h, but was maximal after 18 h of exposure. Labeling of the granule compartment preceded that of the Golgi apparatus. The nuclear compartment (membranes plus nuclei) was also labeled significantly after 18 h of incubation. However, the nuclear membrane itself was labeled after only 1 h of exposure to the ligand. The data suggest that radiolabel is transferred from the plasma membrane to the intracellular organelles as a function of exposure time. Labeling of the secretory compartment suggests that granules may bind and protect internalized peptide from lysosomal degradation. Appearance of label in the nuclear compartment suggests that S-28 (and S-14) may have effects on transcriptional activity in AtT-20 cells.

Chemical cross-linking of somatostatin receptors in rat adrenal cortex

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.

Effect of somatostatin on the vagal motor neuron in the rat

Somatostatin-14 (SOM) effects on electrical properties of membrane in rat brainstem slice preparations were studied in vitro by intracellular recording. Vagal motoneurons in the nucleus dorsalis motoris nervi vagi (DMV) were hyperpolarized by SOM. SOM increased both negativity of membrane potential and input membrane conductance, and decreased synaptic noise. The effects persisted during synaptic blockade by tetrodotoxin (TTX) or [Ca] 6-free-high-[Mg] 0 perfusion. The reversal potential of the hyperpolarization induced by SOM depended on [K] 0 concentration. Hill's coefficient calculated from the dose-response curve was 2. The results suggest that SOM may inhibit visceral organ functions through the DMV.

Somatostatin receptors on rat pancreatic acinar cells. Pharmacological and structural characterization and demonstration of down-regulation in streptozotocin diabetes.

The binding of somatostatin-14 (S-14) to rat pancreatic acinar cell membranes was characterized using [125I-Tyr11]S-14 as the radioligand. Maximum binding was observed at pH 7.4 and was Ca2+-dependent. Such Ca2+ dependence of S-14 receptor binding was not observed in other tissues. Scatchard analysis of the competitive inhibition by S-14 of [125I-Tyr11]S-14 binding revealed a single class of high affinity sites (Kd = 0.5 +/- 0.07 nM) with a binding capacity (Bmax) of 266 +/- 22 fmol/mg of protein. [D-Trp8]S-14 and structural analogs with halogenated Trp moiety exhibited 2-32-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 was equipotent with S-14. The affinity of somatostatin-28 for binding to these receptors was 50% of that of S-14. Cholecystokinin octapeptide (CCK-8) inhibited the binding of [125I-Tyr11]S-14, but its inhibition curve was not parallel to that of S-14. In the presence of 1 nM CCK-8, the Bmax of S-14 receptors was reduced to 150 +/- 17 fmol/mg of protein. Dibutyryl cyclic GMP, a CCK receptor antagonist, partially reversed the inhibitory action of CCK-8, suggesting that CCK receptors mediate the inhibition of S-14 receptor binding. GDP, GTP, and guanyl-5'-yl imidodiphosphate inhibit S-14 receptor binding in this tissue. The inhibition was shown to be due to decrease in binding capacity and not due to change in affinity. Specifically bound [125I-Tyr11]S-14 cross-linked to the S-14 receptors was found associated with three proteins of approximate Mr = 200,000, 80,000, and 70,000 which could be detected under both reducing and nonreducing conditions. Finally, pancreatic acinar cell S-14 receptors were shown to be down-regulated by persistent hypersomatostatinemia 1 week after streptozotocin-induced diabetes characterized by decreased Bmax (105 +/- 13 fmol/mg of protein) without any change in affinity. We conclude that pancreatic acinar cell membrane S-14 receptors require Ca2+ for maximal binding and thus differ from S-14 receptors in other tissues, S-14 receptors in this tissue also exhibit selective ligand specificities, these receptors are regulated by CCK-8 and guanine nucleotides, three receptor proteins of apparent Mr = 200,000, 80,000, and 70,000 specifically bind S-14, and (v) these receptors are regulated by S-14 in vivo as evidenced by decreased binding in streptozotocin diabetic rats characterized by hypersomatostatinemia.