Rat SRIF Research Articles

Examination of somatostatin involvement in the inhibitory action of GIP, GLP-1, amylin and adrenomedullin on gastric acid release using a new SRIF antagonist analogue.

1. The effect of a new type 2 selective somatostatin (SRIF) receptor antagonist (DC-41-33) on somatostatin-induced inhibition of pentagastrin-stimulated gastric acid secretion in conscious, chronic gastric fistula equipped rats was studied. 2. Infused intravenously, DC-41-33 dose-dependently inhibits SRIF-induced inhibition of pentagastrin-stimulated gastric acid secretion with an IC50 of 31.6+/-1.2 nmol kg(-1) versus 10 nmol kg(-1) SRIF and blocks the inhibitory effects of SRIF when simultaneously co-infused. Its effectiveness provides additional evidence that SRIF-inhibition of gastric acid release is a SRIF type 2 receptor-mediated process. 3. DC-41-33 is able to completely reverse the inhibitory effect of glucose-dependent insulinotropic polypeptides, GIP and GIP-(1-30)NH2, and glucagon-like polypeptide, GLP-1(7-36)NH2, on pentagastrin-stimulated gastric acid secretion thus confirming that they exert these effects through stimulation of endogenous SRIF release. 4. DC-41-33 only partially blocks potent amylin and adrenomedullin-induced inhibition of gastric acid secretion, therefore suggesting that somatostatin may not function as a primary mediator in the action of these peptides. 5. Our results indicate that DC-41-33, is a potent in vivo inhibitor of exogenous and endogenous SRIF in rats. It represents a new class of SRIF analogues which should eventually provide excellent tools for further evaluating the many physiological roles of SRIF and its five receptor subtypes.

Somatostatin decreases somatostatin messenger ribonucleic acid levels in the rat periventricular nucleus

Growth hormone (GH) secretion from the pituitary is known to be under the dual control of GH-releasing factor (GRF) and somatostatin (SRIF). Hypothalamic SRIF, the major inhibitor of pituitary growth hormone secretion, inhibits its own release by a negative ultrashort-loop feedback mechanism. However, it is not known whether this negative regulation is mediated by inhibition of SRIF mRNA production. GRF may also inhibit its own release, thereby modifying pituitary GH secretion, possibly through an ultrashort-loop feedback mechanism. Thus, SRIF production and GRF release are both regulated by SRIF. Periventricular nucleus (PeN) and mediobasal hypothalamus (MBH) from adult male rats were incubated for 6 h in Waymouth’s medium with either SRIF or the SRIF agonist analog RC 160 (10 −9 to 10 −6 M). Levels of SRIF mRNA were determined by an S1 nuclease protection assay using a 32[P]-labeled rat SRIF riboprobe. SRIF (10 −7 M) and RC 160 (10 −8, 10 −7 M) significantly ( p ≤ 0.01) decreased SRIF mRNA levels in the PeN. The levels of SRIF mRNA in the MBH were not modified by either SRIF or RC 160. SRIF (10 −7 and 10 −6 M) significantly ( p ≤ 0.01 and p ≤ 0.001, respectively) inhibited the release of GRF at 30 min in the MBH. Likewise, the release of GRF was slightly decreased by 10 −7 M RC 160, and significantly inhibited by 10 −6 M ( p ≤ 0.001) at 30 min. At 6 h, the levels of GRF were significantly reduced by 10 −7 M SRIF ( p ≤ 0.05) and by RC 160 (10 −7, 10 −6 M; p ≤ 0.001 and p ≤ 0.05, respectively). In contrast with these results, the SRIF analog was unable to alter SRIF release at 30 min. At 6 h incubation, RC 160 (10 −7 M) significantly ( p ≤ 0.001) reduced SRIF release from MBH fragments. These results demonstrate that SRIF and a SRIF analog decrease SRIF mRNA levels in the PeN and inhibit the release of SRIF from the nerve terminals of the MBH. Thus, SRIF appears to regulate its own gene expression by negative ultrashort-loop feedback. Therefore, when SRIF is secreted from these neurons in response to GRF, it down-regulates the preceding stimulatory input as well as its own secretion.

Somatostatin antisense oligodeoxynucleotide-mediated stimulation of lymphocyte proliferation in culture

We have previously shown that somatostatin (SRIF) is synthesized in B and T lymphocytes of rat spleen and thymus and released into the medium of cultured lymphocytes. To determine the role of SRIF in the control of lymphocytes proliferation, the expression of SRIF in normal lymphocytes was inhibited using a 3'-terminal phosphorothioate-modified antisense oligonucleotide complementary to a sequence that includes the translation start site of the rat SRIF messenger RNA. Spleens were obtained from adult male rats, and their lymphocytes were cultured for 24 or 72 h to measure SRIF content and cell proliferation, respectively. For the proliferation studies, [3H]thymidine was incorporated during the final 18 h. The lymphocytes were incubated with 15-30 micrograms/ml SRIF antisense and control antisense. SRIF antisense (25 micrograms/ml) increased lymphocyte proliferation 15-fold (P < or = 0.001), reaching a plateau (25- to 30-fold increase) between 25-30 micrograms/ml SRIF antisense. SRIF was extracted from lymphocytes and measured by RIA. Levels of SRIF content were almost undetectable with 10 micrograms/ml antisense and were significantly lower (P < or = 0.01) with 25 micrograms/ml antisense. When RC 160 (10(-5) M), a SRIF agonist analog, was used in the incubation, the stimulation of cell proliferation exerted by the SRIF antisense was completely abolished. Control antisense had no effect on proliferation or SRIF content. These findings indicate that 1) lymphocytes in culture are able to incorporate SRIF antisense; and 2) SRIF antisense inhibits the expression of lymphocytic SRIF, which leads to lymphocyte proliferation. In conclusion, cell proliferation is dramatically increased by eliminating the expression of SRIF from the lymphocytes, which indicates that in vitro SRIF is acting in a paracrine and/or autocrine fashion to inhibit lymphocyte proliferation.

Somatostatin antisense oligodeoxynucleotide-mediated stimulation of lymphocyte proliferation in culture.

We have previously shown that somatostatin (SRIF) is synthesized in B and T lymphocytes of rat spleen and thymus and released into the medium of cultured lymphocytes. To determine the role of SRIF in the control of lymphocytes proliferation, the expression of SRIF in normal lymphocytes was inhibited using a 3'-terminal phosphorothioate-modified antisense oligonucleotide complementary to a sequence that includes the translation start site of the rat SRIF messenger RNA. Spleens were obtained from adult male rats, and their lymphocytes were cultured for 24 or 72 h to measure SRIF content and cell proliferation, respectively. For the proliferation studies, [3H]thymidine was incorporated during the final 18 h. The lymphocytes were incubated with 15-30 micrograms/ml SRIF antisense and control antisense. SRIF antisense (25 micrograms/ml) increased lymphocyte proliferation 15-fold (P < or = 0.001), reaching a plateau (25- to 30-fold increase) between 25-30 micrograms/ml SRIF antisense. SRIF was extracted from lymphocytes and measured by RIA. Levels of SRIF content were almost undetectable with 10 micrograms/ml antisense and were significantly lower (P < or = 0.01) with 25 micrograms/ml antisense. When RC 160 (10(-5) M), a SRIF agonist analog, was used in the incubation, the stimulation of cell proliferation exerted by the SRIF antisense was completely abolished. Control antisense had no effect on proliferation or SRIF content. These findings indicate that 1) lymphocytes in culture are able to incorporate SRIF antisense; and 2) SRIF antisense inhibits the expression of lymphocytic SRIF, which leads to lymphocyte proliferation. In conclusion, cell proliferation is dramatically increased by eliminating the expression of SRIF from the lymphocytes, which indicates that in vitro SRIF is acting in a paracrine and/or autocrine fashion to inhibit lymphocyte proliferation.

Effect of intravenous or intracerebroventricular injections of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on GH release in conscious, freely moving male rats.

The present study was designed to examine the effects of intracerebroventricular injection of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP) on GH secretion in freely moving conscious male rats and to determine whether the central action of GHRP is mediated by increased GHRH and/or somatostatin (SRIF) release. A significant and dose-related suppression of natural fluctuations in plasma GH occurred immediately after intracerebroventricular injection of GHRP at the doses of 1, 3 and 10 mu g/rat and was sustained for more than 1 h. An intracerebroventricular injection of GHRP also suppressed plasma GH rises after an intravenous injection of [D-Ala2, Nle27] human GHRH (1-28)agmatine (hGHRH analog), but the GH suppression by intracerebroventricular GHRP was significantly blunted in rats treated with antirat SRIF gamma-globulin (SRIF-ab). In addition, the suppression by intracerebroventricular GHRP of hGHRH-analog-induced GH release was observed even in rats treated with anti-rat GHRH goat gamma-globulin (GHRH-ab) which does not cross-react with hGHRH analog, and again its suppression was significantly attenuated by simultaneous treatment with SRIF-ab and GHRH-ab. Furthermore, intracerebroventricularly injected GHRP was effective enough to inhibit the central-nervous-system (endogenous GHRH)-driven natural GH-secretory surges in SRIF-ab-treated rats. When 10 mu g/kg GHRP was injected intravenously every 1 h during a 7-hour observation period in control rats without antibody treatment, plasma GH levels were increased with the peak 10 min after each injection during the surge period of the GH-secretory rhythm, but the plasma GH response to GHRP was frequently absent during its trough period. In addition, in SRIF-ab-treated rats, basal GH levels as well as plasma GH peaks after GHRP injections were markedly elevated and the obvious plasma GH rises after GHRP could be observed even during the trough period. In GHRH-ab-treated rats, basal GH levels were lower, and the plasma GH response to GHRP was significantly attenuated as compared with those in control rats. Plasma GH peaks after GHRP injections in rats simultaneously treated with SRIF-ab and GHRH-ab were higher than those in rats given GHRH-ab alone, but its difference in GH response was obviously smaller than that observed between SRIF-ab-treated and control rats, suggesting a crucial role of endogenous GHRH in GHRP-induced GH release. To further clarify the interaction of GHRH and GHRP in GH release, hGHRH analog was injected intravenously alone or simultaneously with GHRP in rats treated with SRIF-ab and GHRH-ab. The peak GH values after simultaneous injection of hGHRH analog and GHRP were significantly higher than the sum of GH peaks after each injection of hGHRH analog and GHRP. In conclusion, an intracerebroventricular injection of GHRP suppresses GH secretion by a central mechanism which is likely to include increased SRIF release, reduced GHRH release or both. We have confirmed that intravenous injection of GHRP can stimulate GH release by itself in an immunologically nullified condition of circulating endogenous GHRH and SRIF in rats, and that endogenous SRIF modestly suppresses the GH-stimulating effect of intravenously injected GHRP while both endogenous and exogenous GHRH markedly enhance GH release by intravenously injected GHRP in a synergistic manner.

Growth hormone-releasing factor increases somatostatin release and mRNA levels in the rat periventricular nucleus via nitric oxide by activation of guanylate cyclase.

Previous work has shown that growth hormone-releasing factor (GRF) stimulates cGMP production and somatostatin [somatotropin (growth hormone)-release-inhibiting factor, SRIF] release without altering cAMP accumulation by fragments of median eminence incubated in vitro. Therefore, this study was undertaken to evaluate the effect of GRF and cGMP on SRIF mRNA and SRIF release in the periventricular nuclei of male rats in vitro. SRIF mRNA levels were determined in explants of periventricular nuclei incubated for 6 hr in Waymouth's medium in the presence of various substances. Steady-state levels of SRIF mRNA were measured by an S1 nuclease protection assay using a 32P-labeled rat SRIF RNA probe. SRIF release and cGMP formation were measured at 30 min and 6 hr by RIA. SRIF mRNA levels and SRIF release were significantly (P < 0.025) increased (approximately 2-fold) by 1 microM dibutyryl cGMP, whereas sodium butyrate had no effect. This augmentation was not influenced by cycloheximide, an inhibitor of protein synthesis. Sodium nitroprusside (10 microM), an activator of the guanylate cyclase pathway via its release of nitric oxide, augmented (P < 0.001) SRIF mRNA levels and significantly increased (P < 0.05) SRIF release. GRF (1 nM) increased SRIF mRNA (P < 0.001) and stimulated the release of SRIF at 30 min (P < 0.05) and 6 hr (P < 0.01). This stimulation was abolished by 10 microM NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of nitric oxide synthase, but not by NG-monomethyl-D-arginine (D-NMMA, the inactive isomer). GRF also increased cGMP formation. This effect was completely blocked by incubation with L-NMMA but not D-NMMA. These results indicate that GRF releases nitric oxide. The nitric oxide diffuses to the adjacent SRIF neurons, where it activates guanylate cyclase, leading to increased formation of cGMP. This cGMP increases SRIF mRNA and SRIF release in the periventricular nuclei of male rats.

Insulin-like growth factor I modulates hypothalamic somatostatin through a growth hormone releasing factor increased somatostatin release and messenger ribonucleic acid levels

Insulin-like growth factor I (IGF-I) has been shown to participate in feedback inhibition of growth hormone (GH) secretion at the level of both the pituitary and hypothalamus. Therefore, we tested the possible involvement of IGF-I on somatostatin (SRIF) and GH-releasing factor (GRF) release in median eminence (ME) fragments and periventricular nucleus (PeN) of male rats. The levels of SRIF messenger ribonucleic acid (mRNA) were also determined in PeN incubated in vitro with IGF-I. The ME's were incubated in Krebs-Ringer bicarbonate glucose buffer in the presence of various concentrations of IGF-I (10 −7 to 10 −11 M) for 30 min. SRIF and GRF released into the medium were quantitated by RIA. The release of SRIF and GRF from the ME's was stimulated significantly ( P < 0.025 and P < 0.05, respectively) by 10 −9 M IGF-I. To determine whether the effect of IGF-I on SRIF release is mediated by GRF release in the ME, a specific GRF antibody (ab) (1 : 500) was used concomitantly with IGF-I (10 −9 M). The release of SRIF induced by IGF-I was blocked by the GRF ab ( P < 0.001), but not by normal rabbit serum used at the same dilution. To determine the effect of IGF-I on the regulation of SRIF mRNA levels, SRIF mRNA was determined in PeN explants incubated in the presence of IGF-I (10 −8 to 10 −10 M) for 2 to 6 h. Levels of SRIF mRNA were determined by a S 1 nuclease protection assay using a 32P-labelled rat SRIF riboprobe. IGF-I (10 −8 M) caused a significant increase in SRIF mRNA levels ( P < 0.05) at 6 h of incubation. This effect of IGF-I on SRIF mRNA was also blocked by GRF ab. IGF-I (10 −8 M) stimulated significantly the release of SRIF from the PeN at 30 min and 6 h. GRF release was increased significantly by 10 −10 M and 10 −8 M IGF at 30 min and 6 h, respectively. These results suggest that the effect of IGF-I on SRIF release is due to its combined effects on GRF-mediated SRIF release and increased SRIF mRNA levels.

Cloning and expression of a rat somatostatin receptor enriched in brain.

The tetradecapeptide somatostatin (SRIF) is a hormone release-inhibiting substance that mediates diverse effects in brain and peripheral organs via specific receptors. A cDNA encoding a rat SRIF receptor was identified by use of degenerate oligonucleotide primers and polymerase chain reaction amplification of cDNA prepared from transcripts expressed in rat brain. The complete cDNA encodes a protein of 391 amino acids with seven potential transmembrane domains. Expression of the cDNA product in transfected COS-7 cell lines provides the same high affinity of binding to [125I-Tyr11]SRIF-14 as that of rat cerebral cortex tissues. However, the binding of [125I-Tyr11]SRIF-14 to cloned rat SRIF receptor is not displaced by MK678, a SRIF analog that partially displaces [125I-Tyr11]SRIF-14 binding sites in membranes of rat cerebral cortex. Northern analysis and in situ hybridization indicate that mRNA (4.0 kilobases) for cloned rat SRIF receptor is preferentially expressed in rat brain regions such as cerebral cortex and hippocampus with no detectable expression in most peripheral organs. This pattern contrasts with the exclusive peripheral expression of a recently cloned human SRIF receptor. The cDNA probe of rat receptor detects mRNA from mouse brain but not from human cerebral cortex and cerebellum.

Hypothalamic growth hormone-releasing factor (GRF) participates in the negative feedback regulation of growth hormone secretion

Effects of growth hormone (GH) excess on immunoreactive hypothalamic GH-releasing factor (GRF) and somatostatin (SRIF) were studied in rats. Hypothalamic GRF content significantly reduced after 7-day daily treatment with 160 μg of rat GH or after inoculation of GH-secreting rat pituitary tumors, MtT-F 4 for 9 or 13 days and GH 3 for 3 months. Basal and 59mM K +-evoked release of GRF from incubated hypothalami diminished, more than the content, by 43–51% in MtT-F 4 tumor- or by 67–83% in GH 3 tumor-bearing rats. In contrast, there was a small but significant increase in content or release of SRIF in rats harboring the GH 3 or MtT-F 4 tumor, respectively. These results indicate the existence of a negative feedback loop via hypothalamic GRF as well as SRIF in control of GH secretion.

Developmental regulation of somatostatin gene expression in the brain is region specific.

Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.

Effects of substance P and neurotensin on somatostatin levels in rat portal plasma.

A sensitive and specific RIA system has been developed for measuring somatostatin (SRIF) in plasma. On chromatographic analysis of the plasma of portal blood of rats, two peaks of SRIF-like immunoreactivity were found, one (large form) near the position of albumin and the other (small form) in the same position as synthetic SRIF. The nature of the large form is unknown, but the small form was immunologically and physicochemically indistinguishable from synthetic cyclized SRIF. After iv injection of substance P (10 micrograms/100 g BW) into rats, there was a significant increase in the portal plasma level of SRIF from the basal values of 277 +/- 75 to 1130 +/- 111 pg/ml at 15 min. On the other hand, the pancreatic SRIF concentration decreased from 0.27 +/- 0.01 to 0.16 +/- 0.04 ng/mg wet wt at 15 min and was maintained at a low level up to 30 min after the injection. Injection of substance P did not affect the SRIF concentration in the stomach. Similar results were obtained after iv administration of neurotensin (3 micrograms/100 g BW). These findings suggest that increases in the portal plasma levels of SRIF in rats treated with substance P or neurotensin may reflect, at least in part, the release of SRIF from the pancreas. The physiological significance of SRIF secretion remains to be clarified, but this study suggests that SRIF can be released from the digestive organs, including the pancreas, into the portal vein and may act on the tissues in the peripheral area.