Effect of 17beta-estradiol on the expression of somatostatin genes in rainbow trout (Oncorhynchus mykiss)

In the present study, the effects of 17beta-estradiol (E(2)) treatment on the expression of preprosomatostatin (PPSS) I, PPSS II', and PPSS II" mRNA in the hypothalamus and endocrine pancreas (Brockmann body), as well as the effects of E(2) treatment on plasma somatostatin (SS)-14 and -25 concentrations in sexually immature rainbow trout (Oncorhynchus mykiss), were investigated. E(2) treatment significantly (P < 0.001) depressed both plasma SS-14 and SS-25. In the hypothalamus, E(2) treatment significantly (P < 0.001) decreased the levels of PPSS I and PPSS II" mRNA. However, there was no effect of E(2) treatment on PPSS II' mRNA levels. In the pancreas, E(2) treatment had no significant effect on the levels of either PPSS II' mRNA or PPSS II" mRNA. However, E(2) treatment significantly (P < 0.005) decreased levels of PPSS I mRNA. These data suggest that E(2) acts, in part, to increase plasma growth hormone levels in rainbow trout by decreasing the endogenous inhibitory somatostatinergic tone by inhibiting plasma levels of both SS-14 and SS-25 and hypothalamic levels of mRNA encoding these proteins.     

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)     

Biological activity and receptor binding characteristics to various human tumors of acetylated somatostatin analogs.

Several somatostatin analogs with recently synthesized acetylated N terminus were assayed in vivo for their effects on sodium pentobarbital-stimulated growth hormone (GH) levels in fed male rats and gastrin-releasing peptide (14-27)-stimulated gastrin levels in fasted male rats. The binding characteristics of these analogs to somatostatin receptors were also examined in various human tumors and normal tissues. The analog RC-101-I, injected at a dose of 0.1 micrograms/100 g body wt, significantly suppressed GH release (P less than 0.01) for at least 2 hr. Analog RC-160-II caused the longest inhibition of GH release, greater than that induced by nonacetylated parent analog RC-160, with GH levels showing significant suppression (P less than 0.01) for more than 3 hr. Analogs RC-160-II and RC-101-I and RC-160, injected at a dose of 1.0 micrograms/100 g body wt, significantly (P less than 0.01) suppressed gastrin-releasing peptide (14-27)-stimulated serum gastrin. Analog RC-101-I was active in this test at a dose of 0.1 micrograms/100 g body wt. RC-160-II showed significant binding to somatostatin-14 receptors in all investigated tissues (human colon, human colon cancer, breast cancer, human pancreas and pancreatic cancer, human prostate and prostate cancer, and rat cerebral cortex), but there were marked variations in binding affinities among various normal and cancerous tissues. The highest affinity was found in membranes of colon cancer (Ka = 18.4 nM-1) and breast cancer (Ka = 12.46 nM-1). The binding affinity of RC-160-II to somatostatin receptors in membranes of the breast cancer was similar to that of RC-160. RC-101-I showed higher binding affinity to somatostatin-14 receptors than RC-160 in human breast, pancreatic, and prostate cancer. With the exception of breast cancer tissue, the binding affinity of RC-101-I was significantly lower than that of RC-160-II in membranes of all investigated tissues. It can be concluded that acetylated somatostatin analogs RC-101-I and RC-160-II possess prolonged and enhanced biological activities in suppressing serum GH and gastrin in rats. Significant variations in binding affinities for these analogs in different tissues and various tumors suggest that differences may exist between somatostatin receptors in normal versus malignant tissues. This raises the possibility that some of these analogs could be used more selectively in the treatment of various neoplasms.     

Gastrointestinal somatostatin: distribution, secretion and physiological significance

Somatostatin-like immunoreactivity (SLI) has been found throughout the gastrointestinal tract in all species examined. In the stomach it is mainly present in endocrine-type D-cells whereas in the intestine there is also an extensive distribution in enteric neurones. In all regions of the gastrointestinal tract multiple forms of somatostatin exist. A precursor (prosomatostatin) has been partially sequenced, three forms with 20 (SS-20), 25 (SS-25) and 28 (SS-28) amino acids completely sequenced, and somatostatin-14 (SS-14) demonstrated by radioimmunoassay. Both SS-14 and SS-28 exert a wide range of actions on the gastrointestinal tract and there is strong supportive evidence for a role in the regulation of gastric acid and gastrin secretion, gastrointestinal motility and intestinal transport. Both in vivo and in vitro studies on the secretion of gastric SLI into the vasculature have shown that nutrients initiate the process but that subsequent events are regulated by a complex interplay between hormonal and neuronal pathways. GIP is one of the most potent hormonal secretagogues. In the stomach, acetylcholine, opioid peptides and substance P are probably involved in parasympathetic inhibitory pathways and gastrin releasing peptide in stimulatory pathways. The sympathetic nerves are also stimulatory. Regulation of secretion of intestinal SLI has not been so extensively studied. Although SLI is also found in the gastrointestinal lumen the significance is unclear. Despite these advances the exact route of delivery of somatostatin to its target organs is uncertain and paracrine, endocrine and neural pathways may all be involved.     

Hypersomatostatinemia in chronic renal failure

Plasma concentrations of somatostatin-like immunoreactivity (SLI) were determined in uremic patients on maintenance hemodialysis. Plasma SLI levels were significantly (p less than 0.001) elevated in 26 diabetic uremic patients (67.1 +/- 6.8 pg/ml, mean +/- SE) and in 24 non-diabetic uremic patients (43.5 +/- 7.2 pg/ml), when compared with 60 healthy subjects (5.0 +/- 0.7 pg/ml). Paired pooled plasma from uremic patients before and after hemodialysis was subjected to a reverse-phase octadecasilyl-silica (C-18) cartridge and then the extract was gel filtered on a Sephadex G-25 column (1.6 X 90 cm). Both elution profiles showed two peaks of SLI which coeluted with synthetic somatostatin (SS)-28 and SS-14 markers, respectively. The SS-28-like immunoreactivity (LI) peak, which was estimated by using SS-14 as a reference standard, was 3-fold larger than that for SS-14 LI. On the basis of immunoequivalency of the two components in the present assay, SS-28 LI constitutes approximately 75% of circulating somatostatin. In conclusion, plasma SLI is substantially high in uremic patients of both diabetic and non-diabetic etiology and the SS-28 is a predominant form of circulating SLI in these patients, probably, in part, for a lower clearance of this molecule.     

Overexpression in Escherichia coli, purification, and characterization of Sphingomonas sp. A1 alginate lyases

A bacterium Sphingomonas sp. A1 produces three kinds of alginate lyases [A1-I (66 kDa), A1-II (25 kDa), and A1-III (40 kDa)] from a single precursor, through posttranslational processing. Overexpression systems for these alginate lyases were constructed in Escherichia coli cells by controlling of the lyase genes under T7 promoter and terminator. Expression levels of A1-I, A1-II, and A1-III in E. coli cells were 3.50, 3.04, and 2.13 kU/liter of culture, respectively, and were over 10-fold higher than those in Sphingomonas sp. A1 cells. Purified A1-I, A1-II, and A1-III from E. coli cells were monomeric enzymes with molecular masses of 63, 25, and 40 kDa, respectively. The depolymerization pattern of alginate with A1-I and A1-II indicated that both enzymes cleaved the glycosidic bond of the polymer endolytically and by beta-elimination reaction. A1-II preferred polyguluronate rather than polymannuronate and released tri- and tetrasaccharides, which have unsaturated uronyl residues at the nonreducing terminal, from alginate as the major final products. A1-I acted equally on both homopolymers and produced di- and trisaccharides as the final products.     

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.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Somatostatin Binding Sites in Human and Monkey Brain: Localization and Characterization

A radioiodinated analogue of somatostatin 28, 125I [Leu8,D-Trp22,Tyr25] SS-28, was used to localize and characterize somatostatin binding sites in both human and monkey brain. High-affinity binding sites (approximately 1 nM) were found in cerebral cortex. The highest binding was in cerebral cortex with intermediate binding found in hippocampus, striatum, and amygdala and low binding in hypothalamus and brainstem. There was a rough correlation between somatostatin receptor binding and concentrations of somatostatin-like immunoreactivity (SLI) in human brain. Somatostatin receptors were stable for up to 24 h in an animal model simulating human autopsy conditions and there was no correlation between postmortem interval and receptor binding in human brain. Pharmacologic characterization in human cortex showed that there was a correlation between the inhibition of receptor binding by somatostatin analogues and their known abilities to inhibit growth hormone secretion. These findings demonstrate that a highly specific membrane-associated receptor for somatostatin is present in both monkey and human brain. Examination of somatostatin receptor binding in Alzheimer's disease and Huntington's disease may improve understanding of the role of somatostatin in both these illnesses.     

High affinity binding sites for a somatostatin-28 analog in rat brain

Using an iodinated analog of a large (28 residues) and biologically active form of somatostatin, ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵]SS-28, it was possible to demonstrate saturable and high affinity binding sites (dissociation constant = 0.46 ± 0.04 nM) in rat cortical membranes. Somatostatin, somatostatin-28, as well as two potent analogs, [D-Trp⁸] somatostatin and [D-Trp²²] somatostatin-28, could completely displace the radiogland in the nanomolar range whereas the inactive analog Des-Trp⁸-somatostatin and the unrelated peptide GnRH showed no affinity for these binding sites; octa- and nona-peptide analogs of somatostatin were inactive. High binding was found in hippocampus, amygdala, tuberculum olfactorium, caudate-putamen and cortex; moderate binding in midbrain and hypothalamus, and no binding in the cerebellum. These results suggest that specific somatostatin receptors can be measured within the brain with ¹²⁵I[Leu⁸,D-Trp²²,Tyr²⁵] SS-28 as radioligand.     

Comparison of somatostatin-28 and somatostatin-14 clearance by the perfused rat liver

The hepatic clearances of somatostatin (SS)-28 and SS-14 by the perfused rat liver were compared, using a recirculating, plasma-free, erythrocyte-containing perfusion system. The disappearance rate constant, half time, clearance, and hepatic extraction ratio when 1.2 nM SS-28 was added to the perfusate were 0.0221 +/- 0.0051 min-1, 36.6 +/- 7.6 min, 0.34 +/- 0.08 mL/min, and 17.2 +/- 3.9%, respectively. The corresponding values obtained when SS-14 was added to the perfusate were 0.0405 +/- 0.0022 min-1, 17.3 +/- 1.0 min, 0.71 +/- 0.05 mL/min, and 35.4 +/- 2.6%, respectively. The differences between the SS-28 and SS-14 indices were all statistically significant. In addition, the perfusates with SS-28 added were eluted on Sephadex G-25 fine columns and somatostatinlike immunoreactivity (SLI) was determined. No SS-14 was found in perfusate containing SS-28 at both 5 and 30 min after the beginning of the perfusion. To investigate whether or not the liver plays an important role in the clearance of SS-28 or the conversion of SS-14 in vivo, the plasma disappearance of 2 micrograms SS-28 was compared in the whole rat and the functionally hepatectomized model. The half time of plasma SS-28 was 1.43 +/- 0.12 min in the whole rat, significantly shorter than the 2.20 +/- 0.14 min in the hepatectomized model. Gel filtration of plasma extract samples at 0.5 min after the SS-28 injection showed two major peaks of SLI: a first peak corresponding to SS-28 and a second peak coeluted in the position of SS-14 in both the whole rat and the hepatectomized model. At 4 min after the SS-28 injection, the first peak disappeared and only a small second peak was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differential expression of two somatostatin genes during zebrafish embryonic development

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.     

Isolation of products and intermediates of pancreatic prosomatostatin processing: use of fast atom bombardment mass spectrometry as an aid in analysis of prohormone processing

Major products and an intermediate in the proteolytic processing pathway of preprosomatostatin I from anglerfish (Lophius americanus) were purified and characterized. Proteolytic mapping by fast atom bombardment mass spectrometry was used to rapidly locate regions of the peptides whose masses deviated from those deduced from the cDNA sequence. Amino acid analysis and partial Edman sequencing were also used to confirm the structures. The protein structural data indicate a Glu for Gly substitution at position 83 of preprosomatostatin I (aPPSS-I, numbering from the initiator Met) relative to the cDNA sequence. Two of the peptides isolated, aPPSS-I (26-52) (7.5 nmol X g-1) and aPPSS-I (26-92) (49.5 nmol X g-1), define signal cleavage as occurring between Cys-25 and Ser-26. A partial sequence was obtained from fragment ions in the mass spectrum of a peptide corresponding to aPPSS-I (94-105) (58 nmol X g-1). The 14-residue somatostatin [SS-14 corresponding to aPPSS-I (108-121)] has previously been isolated [Noe, B. D., Spiess, J., Rivier, J. E., & Vale, W. (1979) Endocrinology (Baltimore) 105, 1410-1415]. Taken together, these peptides suggest a pathway for prosomatostatin I processing in which the residues corresponding to SS-14 and the immediately preceding 14 residues are cleaved from the prohormone via endoproteolysis (order of cleavage not determined). The fragment aPPSS-I (94-105) was isolated in lower yield than SS-14 and may represent a secondary site of cleavage. Subsequent cleavage at arginine-53 results in the minor peptide aPPSS-I (26-52).(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential suppression of insulin-stimulated proliferation of cultured hepatocytes by somatostatin: Evidence for receptor-mediated growth regulation

The role of somatostatin (SS-14) in the regulation of rat liver regeneration was examined by using thymidine incorporation into hepatocyte DNA labeled with tritiated thymidine, a nuclear-labeling index, and the binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes isolated at various times after partial hepatectomy. The data demonstrated no suppressive effect of SS-14 on insulin and glucagon-stimulated thymidine incorporation into hepatocyte DNA as early as 2 h after partial hepatectomy. These data were substantiated by a nuclear labeling index studies. At 2 h, ¹²⁵I-tyr¹¹-SS-14 binding to its specific sites on isolated hepatocytes was undetectable. There was a time-dependent increase in binding of ¹²⁵I-tyr¹¹-SS-14 to hepatocytes obtained at various times after partial hepatectomy. There was a significant decrease in the number of binding sites after partial hepatectomy as determined by Scatchard analysis. The data were supported by autoradiography analysis of affinity labeled ¹²⁵I-tyr¹¹-SS-14-binding protein complex followed by SDS-PAGE. SS-14 also inhibited intracellular cAMP in hepatocytes obtained at 18 h after hepatectomy. The data are consistent with the hypothesis that SS-14 participates via its own receptor in the regulation of the liver regeneration. © 1995 Wiley-Liss, Inc.     

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.     

Neuropeptide Y- and somatostatin-like immunoreactivities in ganglioneuroma, ganglioneuroblastoma and neuroblastoma

Neuropeptide Y (NPY)- and somatostatin (SS)-like immunoreactivities (LI) were investigated in tumor tissues of one ganglioneuroma (GN), 3 ganglioneuroblastomas (GNB) and one neuroblastoma (NB) by radioimmunoassay. NPY-LI was detected from all 5 tumor tissues (16.4-1247 pmol/g wet tissue). Sephadex G-50 column chromatography and reverse phase high performance liquid chromatography (HPLC) revealed that most of the NPY-LI in tumor extracts was eluted in an identical position to synthetic human NPY except one GNB (case 2). In this case, most of the NPY-LI was eluted in a higher molecular weight region than synthetic human NPY in Sephadex G-50 column chromatography and in a more hydrophobic position in HPLC. SS-LI was detected from 4 tumor extracts except one GNB (case 2) (21.3-787 pmol/g wet tissue). Sephadex G-25 column chromatography and reverse phase HPLC revealed that SS-LI in tumor extracts was eluted just after the void volume and then in the same positions as SS-28 and SS-14. These results suggest that NPY, SS-14 and SS-28 exist in tumor tissues of GN, GNB and NB, and most of the NPY-LI in one GNB was a higher molecular and more hydrophobic form of NPY-LI.     

Cysteamine-induced reduction in gastrointestinal somatostatin: evidence for a region-specific loss in immunoreactivity

Administration of cysteamine (beta-mercaptoethylamine; 2-aminoethanethiol) to rats has been shown to decrease the levels of somatostatin-like immunoreactivity (SLI) in the gastrointestinal tract and pancreas but its mode of action is unclear. In the current study the effect of cysteamine on gastrointestinal and pancreatic SLI has been studied using two antisera with different regional specificities. In addition, the in vitro effect of cysteamine on SS-14 and SS-28 has been studied by high-performance liquid chromatography (HPLC). Characterization of the two antisera (AS 26.3.2 and AS 1001) with a range of analogs of SS-14 revealed that both were directed against the midportion of the molecule but that AS 1001 was also sensitive to changes at the N- and C-termini. Tissue extracts from cysteamine-treated rats measured with AS 26.3.2 showed no significant change for the stomach, jejunum or pancreas but duodenal levels were reduced. With AS 1001 SLI levels were reduced in all tissues. Gel permeation chromatography of stomach extracts measured with AS 1001 showed a reduction in both SS-14 and SS-28. With AS 26.3.2 an increase in SLI eluting prior to the SS-14 peak occurred explaining why no significant reduction in total SLI was detected. With duodenal extracts the elution profiles with AS 1001 reflected the large reduction in total SLI whereas with AS 26.3.2 a smaller reduction occurred. Both SS-14 and SS-28 were reduced. HPLC analysis of SS-14 and SS-28 following incubation with cysteamine in vitro showed a time-dependent decrease in both somatostatin species with absorbance at 280 nm was measured. New peptide peaks which developed were not all detectable by radioimmunoassay with either antibody. The results suggest that cysteamine causes a change in the structure of somatostatin which probably first involves a reduction of the disulphide bridge and then the N- and C-terminal regions of the molecule thus making it unmeasurable by antisera sensitive to changes in these regions.     

Mechanisms for direct inhibition of canine gastric parietal cells by somatostatin

To examine the potential mechanisms by which somatostatin inhibits gastric acid secretion we studied its effects on isolated canine gastric parietal cells. Using 125I-[Leu8-D-Trp22-Tyr25]somatostatin-28 as ligand, we identified somatostatin-binding sites in parietal cell-enriched fractions of fundic mucosa. Two binding sites with respective dissociation constants of 3.2 X 10(-9) and 2.1 X 10(-7) M were identified. Somatostatin-14 and -28 were equally potent both in displacing bound ligand and in inhibiting parietal cell activity as measured by [14C]aminopyrine uptake. Pertussis toxin reversed the ability of somatostatin to inhibit the uptake of [14C]aminopyrine and production of cAMP by parietal cells stimulated with histamine and forskolin but not with dibutyryl cAMP or pentagastrin. Furthermore, somatostatin had no effect on parietal cell membrane inositol phospholipid turnover or changes in protein kinase C (Ca2+/phospholipid-dependent enzyme) activity induced by carbachol or pentagastrin. These data indicate that somatostatin directly inhibits parietal cell activity via mechanisms both dependent on and independent of the pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.