Separate cell types that express two different forms of somatostatin in anglerfish islets can be immunohistochemically differentiated

The somatostatin-related peptides somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are synthesized at the C-terminal end of two separate pre-pro-somatostatins in anglerfish pancreatic islets. The purpose of this study was to determine whether these peptides are expressed in the same or different cell types. Antisera R141 and R293, which recognize the central region of SS-14 and the C-terminal region of aSS-28 ([Tyr7,Gly10] SS-14), respectively, were used in an immunohistochemical examination of anglerfish islets. The R293 antiserum-labeled cells were distributed individually or in small clusters. These same cells, as well as a separate set of cells arranged in large clusters, were stained by the R141 antiserum. Pre-absorption of the R141 antiserum with [Tyr7,Gly10] SS-14 eliminated staining by R141 of only those cells also labeled by R293, whereas pre-absorption of R141 with SS-14 prevented all staining. Pre-absorption of R293 with [Tyr7,Gly10] SS-14 eliminated all staining, whereas pre-absorption with SS-14 had no effect on aSS-28-like immunoreactivity. These results suggest the existence of two separate cell types which express either SS-14 or aSS-28. The cells that contained the somatostatin-related peptides were found to be distinct from those cells that contained insulin, glucagon, or anglerfish peptide Y. However, the cells stained by the R293 antiserum were distributed in close association with glucagon-containing cells. The implications of the existence of separate cell types which express SS-14 or aSS-28 are discussed with regard to processing of the biosynthetic precursors to these peptides.     

A comparison of the inhibitory effects of somatostatin-14, -25, and -28 on motility of the guinea pig ileum

A number of studies have suggested that somatostatin-14 (SS-14) and somatostatin-28 (SS-28) exhibit a similar spectrum of biological activities but have different potencies. In the present study the effects of SS-14, SS-28, and somatostatin-25 on electrically induced contractions of the guinea pig ileum have been compared. All three peptides exhibited equipotent inhibitory effects. Inhibition was obtained at a threshold concentration less than 10(-10) M, with maximal inhibition at 10(-7) M and IC50 values of 6.0-6.5 X 10(-10) M. The N-terminal 14 amino acid fragment of SS-28 had no effect either on motility, when added alone, or on the actions of SS-28, suggesting that this region of the molecule is not critical for biological activity.     

Isolation and characterization of S. cerevisiae mutants defective in somatostatin expression: cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites.

The peptide somatostatin exists as two different molecular species. In addition to the most common form, somatostatin-14, there is also a fourteen amino acid N-terminally extended form of the tetradecapeptide, somatostatin-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which upon cleavage generate either somatostatin-14 or -28, respectively. In some species of fish two distinct, but homologous, precursors (prosomatostatin-I and -II) give rise to somatostatin-14 and -28, respectively. Whereas anglerfish prosomatostatin-II was previously shown to release exclusively somatostatin-28, the yeast Saccharomyces cerevisiae proteolytically matures the homologous prosomatostatin-I precursor to somatostatin-28 and -14 as well as to a lysine-extended form of somatostatin-14. The Kex2 endoprotease appears to be essential for the formation of lysine somatostatin-14 and is involved either directly or indirectly in the release of mature somatostatin-14. The isolation of yeast mutants defective in somatostatin-28 expression (sex mutant) allowed the cloning of a non-essential gene, which encodes an aspartyl protease, whose disruption severely affects the cleavage of mature somatostatin-28 from both somatostatin precursors. We conclude that two distinct endoproteases, which demonstrate some cross specificity in vivo, are involved in the proteolytic maturation of prosomatostatin at mono- and dibasic processing sites in yeast.     

Inhibitory effect of somatostatin-28 on pancreatic polypeptide, glucagon and insulin secretion in normal man

We have compared the effects of equimolar doses of intravenous somatostatin-28 (SS-28) and somatostatin-14 (SS-14) (250 micrograms and 125 micrograms, respectively) on the secretion of pancreatic polypeptide (PP), glucagon and insulin evoked by a protein-rich meal in normal subjects. Both peptides reduced the fasting plasma levels of these hormones and completely abolished their responses to the alimentary stimulus; in addition, they caused an early decrease of plasma glucose followed by a hyperglycemic phase. As compared to SS-14, SS-28 elicited a longer-lasting inhibition of PP and insulin secretion and displayed greater hypo- and hyperglycemic effects. A somatostatin-like component, similar to SS-28, has been identified in pancreatic extracts as well as in peripheral plasma. Thus, it might be hypothesized that this peptide plays a role in the control of pancreatic hormone release.     

Comparison of the gastric exocrine inhibitory activities and plasma kinetics of somatostatin-28 and somatostatin-14 in cats

The gastric exocrine inhibitory activities of somatostatin-28 (SS-28) and somatostatin-14 (SS-14) were determined in conscious cats prepared with gastric fistulae. Gastric acid and pepsin secretions were stimulated with pentagastrin. Expressed in terms of exogenous doses, SS-14 (ID50: 1.49 nmol . kg-1 . h-1) was 3.4 times more potent than SS-28 (ID50: 5.12 nmol . kg-1 . h-1) as an inhibitor of gastric acid secretion. Similarly SS-14 (ID50: 0.25 nmol . kg-1 . h-1) was 3.8 times more potent than SS-28 (ID50: 0.96 nmol . kg-1 . h-1) as an inhibitor of pepsin secretion. Expressed in terms of circulating plasma concentration measured by radioimmunoassay, SS-14 (ID50: H+, 232 and pepsin 73 pM) was 8-9 times more potent than SS-28 (ID50: H+, 2112 and pepsin, 611 pM) as an inhibitor of gastric exocrine secretions. The plasma immunoreactive half-life of SS-28 (6.1 min) was double that for SS-14 (2.4 min) possibly due to a slower theoretical metabolic clearance rate of the larger peptide (30 and 87 ml . kg-1 . min-1, respectively). Both peptides had similar apparent distribution volumes (SS-14, 306 and SS-28, 263 ml . kg-1). As judged by gel chromatography of plasma samples, there was no evidence for the conversion of SS-28 to SS-14 in vivo. The reduced activity of SS-28, compared with SS-14, against gastric exocrine secretions contrasts with its more potent effects in the pituitary and pancreas.     

The antidiabetic action of somatostatin-28 as assessed by the artificial endocrine pancreas: greater potency than somatostatin-14

In order to investigate the action of somatostatin-28 (SS-28) on the metabolic homeostasis of insulin-dependent diabetics, we compared its effects to those of somatostatin-14 (SS-14) in terms of insulin sparing, changes in dextrose demands, glucose fluctuations and behavior of growth hormone and glucagon secretion. Eight insulin-dependent subjects were connected to Artificial Endocrine Pancreas (Biostator) for 84 hours during which they received intravenous infusions of either SS-14, SS-28 or isotonic saline in a randomized order, after a steady state of metabolism had been achieved. Five of the patients received SS-28 100 micrograms/h and SS-14 250 micrograms/h for 10 hours and three of them SS-28, 50 micrograms/h and SS-14 250 micrograms/h for 12 hours. Identical doses of both peptides were administered as bolus infusions prior to the continuous ones. Under SS-28 100 micrograms/h and SS-14 250 micrograms/h patients required 13.5 +/- 2.3 and 14.5 +/- 1.9 U of insulin respectively vs 40 +/- 5.6 U under isotonic saline infusion (mean +/- SEM, P less than 0.005 and P less than 0.01). At the same period the apparatus delivered 15 times more dextrose under SS-28 and 20 times more under SS-14. The magnitude of glucose fluctuations diminished from 64.6 +/- 2.47 mg% without to 41.4 +/- 2 mg% under SS-14 (P less than 0.01) and 46 +/- 3.8 mg% under SS-28 (P less than 0.02). Similar changes were observed in the remaining three patients who received SS-28 in the dose of 50 micrograms/h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue

Preprosomatostatin-I (PPSS-I) is processed in anglerfish islets to release a 14-residue somatostatin (SS-14). However, very little is known regarding other processing events that affect PPSS-I. This is the first study to identify and quantify the levels of nonsomatostatin products generated as a result of processing of this somatostatin precursor in living islet tissue. The products of PPSS-I processing in anglerfish islet tissue were identified in radiolabeling studies using a number of criteria. These criteria included immunoreactivity, specific radiolabeling by selected amino acids, radiolabel sequencing, and chromatographic comparison to isolated, structurally characterized fragments of anglerfish PPSS-I using reverse-phase high performance liquid chromatography. Intact prosomatostatin-I (aPSS-I) was isolated from tissue incubated with [3H]tryptophan and [14C]leucine. Significant 14C radioactivity was observed in the products of 11 of the first 44 sequencer cycles in positions consistent with the generation of a 96- residue prosomatostatin. These results indicate that signal cleavage occurs after the cysteine located 25 residues from the initiator Met of PPSS-I, resulting in a signal peptide 25 amino acids in length. Nonsomatostatin-containing fragments of the precursor were also found in tissue incubated with a mixture of 3H-amino acids. Only a small quantity of the dodecapeptide representing residues 69-80 in the prohormone was found (10 nmol/g tissue). Two other fragments of aPSS-I, also observed to be present in low abundance, were found to correspond to residues 1-27 (16 nmol/g tissue) and to residues 1-67 (7 nmol/g tissue) of aPSS-I. No evidence for the presence of the fragment corresponding to residues 29-67 was found. However, large quantities of SS-14 were observed (287 nmol/g tissue), indicating that the major site of aPSS-I cleavage is at the basic dipeptide immediately preceding SS- 14. Recovery of much lower levels of the nonsomatostatin fragments of aPSS-I suggests that prohormone processing at the secondary sites identified in this study occurs at a low rate relative to release of SS- 14 from aPSS-I.     

Gastric secretion and gastrin under progressive doses of somatostatin-14 and -28 administered intraperitoneally to the rat

The actions of progressive doses of intraperitoneally (IP) administered somatostatin-14 (SS-14) and -28 (SS-28) on gastric secretion (acid, pepsin) and mucosal blood flow (MBF) were studied in conscious gastric fistula rats both under basal conditions and under additional administration of pentagastrin. Also, somatostatin-like immunoreactivity was measured in aortal blood in all groups as well as aortal gastrin levels under basal conditions. IP infusion of equimolar doses of SS-14 and SS-28 resulted in an equal and dose-dependent inhibition of basal as well as pentagastrin-stimulated gastric acid secretion. MBF was reduced by either peptide both in the basal and pentagastrin experiments. Under basal conditions pepsin secretion was significantly increased by infusion of SS-14 at the higher doses, by infusion of SS-28 only at the intermediate dose (3.1 nmole kg-1.hr-1). In the pentagastrin experiments, low and intermediate doses of SS-14 tended to lower pepsin outputs but the highest dose of SS-14 stimulated pepsin secretion, whereas SS-28 had no effect on pepsin. Administration of SS-28 inhibited gastrin only at the highest dose (12.3 nmole kg-1.hr-1), and SS-14 had no influence at all on gastrin. After IP infusion of both peptides, plasma SLI rose dose-dependently under basal and stimulated conditions. Gel chromatography indicated an in-vivo conversion of SS-28 to SS-14 or intermediate fragments. It is concluded that SS-14 and SS-28 delivered by IP infusion, inhibit basal and stimulated gastric acid equally in the rat without suppressing gastrin. The mechanism underlying SS-mediated pepsin stimulation is unknown.     

Effects of somatostatin-25 on lipid mobilization from rainbow trout,Oncorhynchus mykiss,liver and adipose tissue incubated in vitro. Comparison with somatostatin-14

Summary The physiological effects of the pancreatic peptides somatostatin-14 and somatostatin-25 on lipid metabolism in rainbow trout were evaluated by in vitro culture of liver and adipose tissue. The culture medium was subsequently analyzed for glycerol and fatty acid content and triacylglycerol lipase activity was measured within the tissues. Both somatostatin-14 and somatostatin-25 stimulated hepatic fatty acid and glycerol release within 3 h after treatment. Liver triacylglycerol lipase activity was elevated following treatment with somatostatin-14 (76% above control) or somatostatin-25 (94% above control). Somatostatin-14 and somatostatin-25 also significantly stimulated the release of fatty acid and glycerol from adipose tissue. Triacylglycerol lipase activity in adipose tissue also was enhanced by both somatostatins. These results indicate that somatostatin-14 and somatostatin-25 directly stimulate the mobilization of triacylglycerol from liver and adipose tissue, suggesting that these peptides are important systemic modulators of lipid metabolism in fish.Abbreviations bw body weight - cAMP cyclic adenosine monophosphate - FA ratty acids - fw fresh weight - GLU glucagon - INS insulin - MS-222 tricaine-methane sulphonate - SS-14 somatostatin-14 - SS-25 somatostatin-25 - TG triacylglycerol     

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity

Rat preprosomatostatin (rPPSS) is processed to two bioactive peptides, somatostatin-14 and somatostatin-28. In anglerfish islets, the two peptides are synthesized by distinct cell types and are derived from different precursors, anglerfish preprosomatostatin-1 (a(I)PPSS) and anglerfish preprosomatostatin-2 (a(II)PPSS). To determine the basis of the differential processing, we introduced a(I)PPSS or a(II)PPSS expression vectors into mammalian endocrine cell lines that can accomplish both patterns of processing. Both precursors were processed identically, indicating that cellular factors must determine the processing pattern. Although similar processing sites are present in both precursors, high levels of unprocessed anglerfish prosomatostatin-2 were secreted constitutively from the transfected cells. A hybrid protein containing the leader sequence and a portion of the pro-region of rPPSS fused to the carboxy-terminal third of a(II)PPSS was processed and secreted via a regulated pathway. We conclude that the amino-terminal 78 residues of rPPSS contain sufficient information to correct the targeting deficiency of a(II)PPSS in mammalian endocrine cell lines.     

Prosomatostatin is processed in the Golgi apparatus of rat neural cells.

Proteolytic processing of somatostatin precursor produces several peptides including somatostatin-14 (S-14), somatostatin-28 (S-28), and somatostatin-28 (1-12) (S-28(1-12)). The subcellular sites at which these cleavages occur were identified by quantitative evaluation of these products in enriched fractions of the biosynthetic secretory apparatus of rat cortical or hypothalamic cells. Each of the major cellular compartments was obtained by discontinuous gradient centrifugation and was characterized both by specific enzyme markers and electron microscopy. The prosomatostatin-derived fragments were measured by radioimmunoassay after chromatographic separation. Two specific antibodies were used, allowing the identification of either S-28(1-12) or S-14 which results from peptide bond hydrolysis at a monobasic (arginine) and a dibasic (Arg-Lys) cleavage site, respectively. These antibodies also revealed prosomatostatin-derived forms containing at their COOH terminus the corresponding dodeca- and tetradecapeptide sequences. Whereas the reticulum-enriched fractions contained the highest levels of prosomatostatin, the proportion of precursor was significantly lower in the Golgi apparatus. In the latter fraction, other processed forms were also present, i.e. S-14 and S-28(1-12) together with the NH2-terminal domain (1-76) of prosomatostatin (pro-S(1-76). Inhibition of the intracellular transport either by monensin or by preincubation at reduced temperature resulted in an increase of prosomatostatin-derived peptides in the Golgi-enriched fractions. Finally, immunogold labeling using antibodies raised against S-28(1-12) and S-14 epitopes revealed the presence of these forms almost exclusively in the Golgi-enriched fraction mainly at the surface of saccules and vesicles. Together these data demonstrate that in rat neural cells, prosomatostatin proteolytic processing at both monobasic and dibasic sites is initiated at the level of the Golgi apparatus.     

Somatostatin inhibits insulin-like growth factor-I receptor expression in the gill of a teleost fish (Oncorhynchus mykiss)

Rainbow trout gill tissue was used to examine the role of somatostatin (SS) on insulin-like growth factor-I (IGF-I) receptor expression. In vivo implantation of fish with somatostatin-14 (SS-14) reduced expression of IGF-I receptor mRNAs as well as [(125)I]-IGF-I binding. In vitro incubation of gill filaments with SS-14 or various SS isoforms, including SS-28 and [Tyr(7), Gly(10)]-SS-14-containing peptides, directly inhibited IGF-I receptor mRNA expression. SS-14 also inhibited [(125)I]-IGF-I binding in vitro. These data indicate that SSs inhibit the mRNA and functional expression of IGF-I receptors in gill, and suggest that SSs regulate growth in an extrapituitary manner by reducing sensitivity to IGF-I.     

Differences between somatostatin-28 and somatostatin-14 with respect to their biological effects in healthy humans and acromegalics

In order to compare the effects of somatostatin-28 (SS-28) with those of somatostatin-14 (SS-14) in humans, we administered both compounds randomly in 5 healthy persons and 3 patients with active acromegaly. Blood glucose, growth hormone, insulin, glucagon, TSH, FSH, LH and prolactin were estimated after arginine, TRH and LHRH stimulation in the normals and without stimulation in the acromegalics. Both substances were administered in doses of 25, 50, 200 and 250 micrograms. Our results indicate that SS-28 is at least 5 times more potent in man than SS-14 as far as inhibition of growth hormone, insulin, glucagon and prolactin secretion is concerned. On the other hand SS-28 is at least 2 times more potent than SS-14 in the inhibition of TSH, FSH and LH. If this difference in potency is calculated on the basis of equimolarity, the action of SS-28 becomes even much greater. According to these findings, SS-28 appears to be either the main hormone and SS-14 a fragment of it with a lesser degree of biologic activity, or the prohormone with special properties.     

Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines

The central nucleus of the amygdala (Ce) mediates cardiovascular and autonomic changes associated with defense or fear responses. At least 16 different neuropeptides have been identified within nerve terminals within the Ce. The role that these peptides play in the Ce regulation of cardiovascular and autonomic function has been assessed. Neuropeptides were microinjected into the region of the Ce and mean arterial pressure (MAP), heart rate (HR) and plasma catecholamine concentrations were measured. Five of the 16 peptides caused changes of MAP and HR. Thyrotropin releasing factor (TRF) and calcitonin gene-related peptide (CGRP) induced increases of MAP and HR. Angiotensin-II (A-II) and somatostatin-28 (SS-28) injection produced increases of MAP and decreases of HR. Bombesin (Bom) injections into the Ce induced an increase of MAP but did not alter HR. Corticotropin releasing factor (CRF), TRF and CGRP were the only peptides found to increase plasma catecholamine concentrations. These results support the conclusion that the Ce contains several peptides that could be involved in the regulation of cardiovascular and autonomic nervous system function. A role of the amygdala in mediating the observed effects of CRF, TRF, CGRP, A-II, SS-28, and Bom is suggested by these studies.     

Tissue-specific posttranslational processing of pre-prosomatostatin encoded by a metallothionein-somatostatin fusion gene in transgenic mice

The somatostatins are neuropeptides of 14 and 28 amino acids that inhibit the release of growth hormone and other hypophyseal and gastrointestinal peptides. These neuropeptides are cleaved posttranslationally from a common precursor, pre-prosomatostatin. We report here the production and processing of pre-prosomatostatin by transgenic mice carrying a metallothionein-somatostatin fusion gene. The most active site of somatostatin production, as determined by hormone concentrations in the tissues, is the anterior pituitary, a tissue that does not normally synthesize somatostatin-like peptides. Anterior pituitary processed pre-prosomatostatin almost exclusively to the two biologically active peptides, somatostatin-14 and somatostatin-28, whereas the liver and kidney synthesized much smaller quantities of predominantly a 6000 dalton somatostatin-like peptide. The growth of the transgenic mice was normal despite high plasma levels of the somatostatin-like peptides. These studies indicate that proteases which cleave prosomatostatin to somatostatin-28 and somatostatin-14 are not specific to tissues that normally express somatostatin.     

Solid phase synthesis of somatostatin-28 II. A new biologically active octacosapeptide from anglerfish pancreatic islets

Somatostatin-28 II, an octacosapeptide recently isolated from anglerfish pancreatic islets, was synthetized by the solid phase method along with its somatostatin-14 II and somatostatin-28 II-(1-12) corresponding domains. Homogeneity of the synthetic peptides was demonstrated by analytical RP-HPLC, thin layer chromatography and electrophoresis. The peptides were further characterized by amino acids analysis, fast atomic bombarding mass spectrometry and/or 252Cf plasma desorption mass spectrometry. Synthetic somatostatin-28 II and somatostatin-14 II displace equally well the potent agonist (Tyr0,D-Trp8)-somatostatin-14 from its specific binding sites on anterior pituitary cells membranes. Both peptides activate adenylate cyclase from dispersed rat anterior pituitary cells.     

Somatostatin Precursor in the Rat Striatum: Changes After Local Injection of Kainic Acid

The molecular forms of somatostatin contained in the rat striatum were separated by size-exclusion HPLC. Three major peaks of somatostatin-like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin-14 (SS-14) and somatostatin-28 (SS-28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 microgram) into the left striatum resulted in a persistent decrease (65-85%) of all three forms of somatostatin. In the contralateral--not injected--striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS-14 and SS-28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin-containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS-14 and SS-28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin-containing perikarya in the striatum.     

Multiple preprosomatostatin sorting signals mediate secretion via discrete cAMP- and tetradecanoylphorbolacetate-responsive pathways.

We have previously detected a sorting signal in the amino-terminal 78 residues of rat preprosomatostatin (rPPSS) that targets the precursor into a regulated secretory pathway or pathways allowing proteolytic maturation (Sevarino, K. A., Stork, P., Ventimiglia, R., Mandel, G., and Goodman, R. H. (1989) Cell 57, 11-19). To further localize this signal, we constructed three rPPSS expression vectors that code for substitutions or mutations spanning that portion of rPPSS implicated in sorting, and the precursors were expressed in RIN 5F cells. Fractionation of the intracellular products revealed that accurate processing to somatostatin-14 (SS-14) was not affected by any of the mutations. Examination of the secreted products showed no reduction in processing efficiency, indicating that none of the mutations blocked sorting from constitutive into regulated secretion. Finally, we examined the response to two separate secretogogues, cAMP and 12-O-tetradecanoylphorbol-13-acetate (TPA). Clones expressing two of the three mutant precursors displayed the same stimulation of SS-14 secretion by exogenously administered cAMP and TPA as cells expressing wild-type rPPSS, indicating that targeting specifically to the secretory pathway, or pathways, responsive to cAMP and TPA was not disrupted. However, cells expressing the mutant precursor containing a substitution of the amino-terminal 34 residues of rPPSS by the amino terminus of the vesicular stomatitis virus G protein displayed greatly reduced stimulation of SS-14 secretion by TPA, with a less than compensatory increase in response to cAMP, when compared to cells expressing wild-type rPPSS. In conjunction with our previous studies with anglerfish preprosomatostatins, we conclude that 1) the sorting signal(s) in rPPSS necessary for cAMP-responsive secretion are redundant and probably reside within both mature peptide regions and extrapeptide regions; 2) two or more distinct regulated secretory pathways utilized by secreted peptides can be demonstrated in transfected endocrine cells and targeting to these pathways can be separately mediated by at least two different types of sorting signals within the neuropeptide precursor itself; and 3) pro-region conformation plays little role in prosomatostatin-processing site recognition.