The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28

The products generated after addition of the ARG-LYS esteropeptidase activity purified from rat brain to synthetic somatostatin-28 were analyzed using radioimmunoassay, HPLC and amino acid analysis. In addition to somatostatin-14, both free arginine and free Lysine were identified together with somatostatin-28. The dipeptide ARG-LYS was not present, which indicates that three peptide bonds were hydrolyzed in order to achieve excision of the doublet. Since it is likely that the octacosapeptide is a precursor for both somatostatin-14 and somatostatin-28, these observations add further support to the hypothesis that the convertase is also involved in the in vivo processing of endogenous somatostatin-28.     

Conversion of somatostatin-28 to somatostatin-14 during maturation of epithelial cells in the porcine jejunum

Fractions of isolated epithelial cells were harvested from a segment of porcine jejunum by ten successive incubations with a chelating buffer. The cell fractions showed a progressive decrease in the activity of the brush-border enzymes, alkaline phosphatase and sucrase, with increasing incubation number but a progressive increase in the ability to incorporate labelled thymidine into DNA. Fractions enriched in cells from the crypt region (fractions 9 and 10) contained higher concentrations per mg protein of somatostatin-like immunoreactivity (1.8-fold), glucagon-like immunoreactivity (5.3-fold) and serotonin (3.0-fold) than fractions enriched in cells from the villus tip (fractions 1 and 2). Analysis of extracts of the fractions by gel filtration/radioimmunoassay showed that somatostatin-28 represented the predominant molecular form of somatostatin-like immunoreactivity in all cell fractions but the relative proportion of somatostatin-14 (and related metabolites) to somatostatin-28 was significantly higher (P less than 0.05) in fractions enriched in villus cells (fraction 1 and 2) than in fractions enriched in crypt cells (fractions 5-10). This result suggests that metabolism of somatostatin-28 to somatostatin-14 takes place during migration of the D cell from the crypt base to the villus tip. Heterogeneity in the somatostatin-14 region of the chromatograms indicates that the peptide may be further metabolized by the action of aminopeptidases.     

High biological activity of the synthetic replicates of somatostatin-28 and somatostatin-25

We have isolated form extracts of ovine hypothalami two molecules characterized as somatostatin-28 and somatostatin-4-28 (referred to as somatostatin-25). They were reproduced by solid hase synthesis. In equimolar ratio and depending upon the experimental conditions, synthetic somatostatin-28 ans somatostatin-25 are 3-14 times more potent than somatostatin-14 to inhibit the basal in vitro secretion of growth hormone or as stimulated by prostaglandin (PGE2). In early studies in vivo, somatostatin-28 and somatostatin-25 are also more potent than somatostatin-14 in inhibiting the secretion of growth hormone acutely stimulated in the rat by injection of morphine; somatostatin-28 is also longer-acting than somatostatin-14. These results suggest that somatostatin-14, as originally isolated, is a biologically active fragment of a larger molecule of greater specific activity; it should be considered as another form of somatostatin with high biological activity present in some tissues and likely secreted y the tissues along with somatostatin-14 and possibly other somatostatin-peptides of diverse sizes.     

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     

Somatostatin receptors on rat cerebrocortical membranes. Structural characterization of somatostatin-14 and somatostatin-28 receptors and comparison with pancreatic type receptors

Somatostatin binding and cross-linking to its receptors on rat cerebrocortical membranes were characterized with [125I-Tyr1]somatostatin-14 and [125I-Leu8, D-Trp22, Tyr25]somatostatin-28. When [125I-Tyr1]somatostatin-14 was cross-linked to its receptors with the photoreactive cross-linker, N-(5-azido-2-nitrobenzoyloxy)succinimide, the hormone was specifically associated with a Mr = 72,000 protein band in the presence or absence of reducing agents. Affinity labeling of the Mr = 72,000 protein band was decreased with increasing concentrations of unlabeled somatostatin-14 and nonhydrolyzable guanine nucleotide analog, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Pretreatment of cerebrocortical membranes with islet-activating protein resulted in a decrease in subsequent labeled somatostatin-14 binding and affinity-labeling of the protein and abolished an inhibitory effect of somatostatin-14 on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. When the affinity-labeled protein was solubilized with Zwittergent 3-12 and adsorbed to wheat germ agglutinin-agarose, it was eluted by N-acetylglucosamine. [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 cross-linking to cerebrocortical and pancreatic membranes with the same photoreactive agent revealed specifically labeled protein bands of a Mr = 74,000 in cerebrocortical membranes and a Mr = 94,000 in pancreatic membranes, respectively. These results suggest that: 1) somatostatin receptor on cerebrocortical membranes is a monomeric glycoprotein with a Mr = 70,000 binding subunit, coupled to guanine nucleotide regulatory protein, and 2) the Mr = 70,000 protein may be a common receptor for somatostatin-28 and somatostatin-14 and is distinct from a common pancreatic type receptor.     

Evidence for the presence of dihydro (reduced) somatostatin-14 in the guinea pig brain

Analysis of somatostatin-like immunoreactivity (SLI) in guinea pig brain by HPLC and radioimmunoassay revealed an unexpected peak of SLI eluting at a retention time slightly later than standard somatostatin-14. The following evidence argues that this peak represents dihydro (H2) somatostatin-14. (1) The peak had the same retention time as standard [H2]somatostatin. (2) The possibility of a reduction artefact due to tissue processing was excluded by adding exogenous somatostatin-14 or 125I-labeled N-Tyr-somatostatin-14 to tissue and observing that no corresponding reduced peptides were generated. (3) Mild oxidation of brain extracts with H2O2 decreased, whereas mild reduction with dithiothreitol increased, the proposed peak of [H2]somatostatin. (4) Reaction of tissue extracts with iodoacetamide decreased the size of the proposed [H2]somatostatin peak but resulted in generation of a new peak co-eluting with standard carboxymethylated somatostatin-14. The proportion of the [H2]somatostatin peak in five brain regions, the hypothalamus, amygdala, cerebral cortex, brainstem and cerebellum, ranged from 6 to 20% of total SLI. The probability of somatostatin-14 existing endogenously in reduced or oxidized forms may have implications for its biological function in the guinea pig.     

Processing of somatostatin-28 to somatostatin-14 by rat hypothalamic synaptosomal membranes

The conversion of synthetic somatostatin-28 (S-28) to somatostatin- 14 (S-14, SRIF) by subcellular fractions of rat hypothalamus has been investigated. The conversion products were identified by two techniques: (1) two separate RIAs using antibodies directed toward the central (RIA R149) or the N- terminal (RIA S39) region of the S-14 molecule, (2) gel chromatography of the reaction mixture followed by analysis of the column fractions by RIA R149. Maximal S-28 to S-14 converting activity was observed with the particulate fraction of the lysed synaptosomal pellet sedimenting at the density interface 8–16% Ficoll in 0.32 M sucrose in a discontinuous sucrose/ Ficoll gradient. Concomitant with conversion, degradation of total somatostatin-like immunoreactivity (SLI) was also observed with this fraction ($\text{t}\text{1}\text{2}\text{～ 24}\text{min}$). Relatively little converting activity was found in the remaining subcellular fractions. These data suggest that hypothalamic synaptosomes contain membrane bound enzymes which are able to catalyze the conversion of S-28 to S-14. Tissue specific differences in this converting activity may account for the reported variability in the S-28:S-14 ratios in different tissues.     

Radioautographic localization of somatostatin-14 and somatostatin-28 binding sites in the rat brain

Somatostatin-14 (S14) and its precursor, somatostatin-28 (S28), are widely distributed throughout the rat brain, suggesting that they could act as neurotransmitter or neuromodulator in the central nervous system. The present study was undertaken to study the localization of S14 and S28 receptors in the rat brain determined by "in vitro" radioautography. The study performed on slide mounted frozen brain section with iodinated S14 and S28 analogs revealed an identical distribution of binding sites for the two forms of somatostatin. A good correlation could be observed between receptor distribution and immunohistologically localized neuropeptides except for striatum and hypothalamus. However, receptors were not detectable in the hypothalamus and were found in low concentration in the caudate-putamen nucleus, two regions containing high amounts of S28 and S14, suggesting a high occupancy of receptors in these areas by endogenous peptides or an inverse correlation between receptor and peptide concentrations.     

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.     

Presence of somatostatin-28 like immunoreactivity in the human pancreas

Specific antisera against somatostatin-28 were prepared by absorption of somatostatin-28 antisera with sepharose 4B-somatostatin-14. Indirect immunofluorescence techniques using somatostatin-14 antisera and specific antisera against somatostatin-28 were carried out to elucidate the time of occurrence of somatostatin-28 in the fetal pancreatic islets and to ascertain whether somatostatin-28 was present in the adult pancreatic islets or not, and further to examine whether cells reacting with specific antisera against somatostatin-28 are identical to those reacting with somatostatin-14 antisera or not. Somatostatin-28 like immunoreactivity occurred in the fetal pancreatic islets at 11th week's gestation and was found in all fetal pancreatic islets examined in the present study. It was also found in the adult pancreatic islets. Furthermore, cells reacting with specific antisera against somatostatin-28 in the fetal and adult pancreatic islets were identical to those reacting with somatostatin-14 antisera. Thus, the present study elucidated the presence of somatostatin-28 like immunoreactivity in the human pancreas. However, it could not be decided whether cells reacting with somatostatin-28 antisera contain either only somatostatin-28 or both somatostatin-28 and somatostatin-14; in other words, whether somatostatin-14 is produced from somatostatin-28 or not, since somatostatin-14 antisera had a cross-reactivity to both somatostatin-14 and somatostatin-28.     

Subcellular distribution of somatostatin-14, somatostatin-28 and somatostatin-28 (1-12) in rat brain cortex and comparisons of their respective binding sites in brain and pituitary

Subcellular distribution and binding characteristics of the three endogenous peptides somatostatin-14 (SRIF-14), somatostatin-28 (SRIF-28) and somatostatin-28(1-12) (SRIF-28(1-12] derived from preprosomatostatin were investigated in the rat brain cortex. The three peptides are predominantly recovered from a crude mitochondrial pellet (P2), containing the pinched off nerve endings. Specific high affinity binding sites for 125I-N-Tyr-SRIF-14 and 125I-N-Tyr-SRIF-28 are present on pituitary and brain membranes. Under the same conditions, 125I-N-Tyr-SRIF-28(1-12) binding is undetectable. Moreover, SRIF-28(1-12) does not displace 125I-N-Tyr-SRIF-14 or 125I-N-Tyr-SRIF-28 binding. SRIF-28 is more potent than SRIF-14 to displace 125I-N-Tyr-SRIF-28 binding to brain and pituitary membranes, while both peptides are equipotent to displace 125I-N-Tyr-SRIF-14 binding. Finally, the regional distribution of 125I-N-Tyr-SRIF-14 and 125I-N-Tyr-SRIF-28 binding sites in the brain is identical. In conclusion, the present results are consistent with a neurotransmitter and neurohormonal role for SRIF-14 and SRIF-28. The function of SRIF-28(1-12) in brain remains to be elucidated. Additionally, a differential role for SRIF-14 and SRIF-28 both in adenohypophysis and brain cannot be ascertained at the present time.     

Glucose-induced changes in somatostatin-14 and somatostatin-28 released from rat hypothalamic fragments 'in vitro'

Previous data suggest that somatostatin is present and released from the hypothalamus in several molecular forms, basally and after K+ or electrical stimulation. In order to evaluate the proportions of somatostatin-14 (S14) and somatostatin-28 (S28) released during a stimulus which may be more closely related to the control of growth hormone secretion 'in vivo', we studied the molecular forms of somatostatin released from hypothalamic fragments ' in vitro', during incubations with different glucose concentrations (1.35 and 22mM), which we have previously shown to be inversely related to somatostatin release. Sephadex G-50 chromatography demonstrated that both forms are released in the same proportions (S14: 70%; S28: 30%) during incubation with different glucose concentrations; there is a parallel increase in both forms when low glucose is used. Although on a molar basis less S28 is released than S14, the higher potency, longer duration of action and higher affinity for pituitary receptors of S28 suggests that it may be of major physiological importance.     

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)     

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.     

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.     

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)     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

The intestinal mucosa preferentially releases somatostatin-28 in pigs

We studied the molecular forms of somatostatin-like immunoreactivity (SLI), newly released from isolated perfused preparations of the porcine antrum, stomach, pancreas and upper small intestine: Perfusion effluents were concentrated by Sep-Pak C-18 adsorption, eluted with ethanol, dessicated, and subjected to gel filtration with subsequent radioimmunoassays for somatostatin-14 and N-terminal somatostatin-28 immunoreactivity. All the SLI newly released from the stomach and antrum eluted at the position of somatostatin-14, and such was also the case for more than 95% of the SLI newly released from the pancreas, while 68 -/+ 7% and 75 -/+ 8% of the SLI newly released from the isolated perfused jejunum and ileum, respectively, corresponded to somatostatin-28. By reverse phase HPLC the identity of these peptides with synhetic somatostatin-14 and -28 was established.     

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.