Release Of Immunoreactive Somatostatin Research Articles

Release of immunoreactive somatostatin, vasoactive intestinal polypeptide (VIP), and galanin during propulsive complexes in isolated pig ileum

We studied the release of immunoreactive somatostatin, VIP, and galanin during net aboral propulsive complexes (NAP) in isolated, perfused, 80-cm segments of porcine ileum. Net aboral propulsive complexes were induced by controlled infusion of liquid (perfusion medium, 3.5 ml/min) into the proximal opening of the ileum segment. In response to liquid infusion, the ileum segments generated propulsive complexes rapidly propagating along the entire segment in the aboral direction, resulting in emptying of the luminal contents. The NAPs occurred with an average interval of 7 minutes. The concentrations of galanin, somatostatin, and VIP in the venous effluent, which in control experiments without luminal infusion did not change, increased significantly (by 63.6 ± 23.7%, 43.8 ± 31.8%, and 38.8 ± 14.6%, respectively) during NAPs and emptying. Atropine (10 −6 mol/l) and hexamethonium (10 −5 mol/l) abolished both NAP generation and peptide responses. It is concluded that the enteric neruopeptides, somatostatin, VIP, and galanin, all of which have pronounced intestinal motor effects, may participate in the generation of net aboral propulsive complexes in the ileum of the pig, possibly mainly in descending relaxation.

GABAergic Influences on Somatostatin Secretion from Hypothalamic Neurons Cultured in Defined Medium

The effects of GABAergic influences intrinsic to the hypothalamus on the secretion of somatostatin were studied using cultured fetal rat hypothalamic neurons. The existence of GABAergic neurons within the cultures was confirmed by immunocytochemistry. These neurons appeared to be actively secreting GABA as antagonism of GABAA receptors with bicuculline and picrotoxinin caused a dose-dependent increase in the release of immunoreactive somatostatin (SRIF), which was Ca(2+)-dependent. Although exogenous GABA inhibited SRIF secretion at concentrations of 10(-6) M and greater, muscimol, a GABAA agonist, inhibited SRIF release at 10(-8) M, whereas baclofen, a GABAB agonist, required concentrations two orders of magnitude greater to produce an effect. Phaclofen, a GABAB antagonist, was inactive (10(-8)-10(-4) M). A GABA uptake inhibitor, SKF 89976A, produced a dose-dependent inhibition of SRIF release. These results, therefore, support a role for intrahypothalamic GABA neurons in the regulation of SRIF secretion in the neonatal rat, predominantly via a type A receptor, and provide further evidence for a neuroendocrine role for GABA in controlling growth hormone secretion.

Serotonin, but neither noradrenaline nor GABA, inhibits capsaicin-evoked release of immunoreactive somatostatin from slices of rat spinal cord

It has been suggested that somatostatin is involved in nociceptive transmission in the dorsal horn and that it is contained in small primary afferent neurons. In the present experiments, to elucidate neural systems inhibiting the release of somatostatin from the primary afferent terminals, we examined the effects of serotonin, noradrenaline and γ-aminobutyric acid on the capsaicin-evoked, dorsal-rhizotomy-sensitive and tetrodotoxin-insensitive release of immunoreactive somatostatin, 98.7% of which was somatostatin itself, from the dorsal-half slices of lumbar and cervical enlargements of rat spinal cord. Serotonin (30–100 μM) suppressed the evoked release in a concentration-dependent manner, and the suppression was antagonized by methysergide (100 μM). The evoked release of immunoreactive somatostatin was not inhibited by noradrenaline (100 μM) or γ-aminobutyric acid (100 μM). The present results suggest that the serotonergic systems exert an inhibitory effect on the release of somatostatin from the central terminals of primary sensory neurons.

Tissue-Specific Alterations in Somatostatin mRNA Accumulation in Streptozocin-Induced Diabetes

In streptozocin-induced diabetes in rats, there is a marked increase in the content and release of immunoreactive somatostatin (SLI) from the pancreas and upper gut. To elucidate whether these SLI changes are associated with alterations in somatostatin gene transcription, we measured somatostatin mRNA (SmRNA) accumulation in these and other SLI-producing tissues. Pancreas, stomach, jejunum, hypothalamus, and cerebral cortex were removed from control rats, 6-wk-diabetic rats, and diabetic rats treated with insulin for 6 wk. Total RNA was isolated by centrifugation through CsCl and fractionated on agarose gels. A sensitive radiodensitometric hybridization assay was used to determine SmRNA levels in absolute amounts by in vitro synthesized sense-strand RNA as a quantitative standard and antisense cRNA as a specific probe. SLI was determined by radioimmunoassay. SmRNA exhibited size heterogeneity between the different control and diabetic tissues. A 2- to 3-fold increase in total SmRNA was found in pancreas and stomach of the diabetic rats that suppressed toward normal with insulin treatment. These two tissues also exhibited significant 1.6- to 2.6-fold increases in SLI, respectively. The remaining tissues showed no diabetes-related changes in SLI or SmRNA. We conclude that in insulinopenic diabetes, tissue SLI and SmRNA accumulation undergo parallel changes; are increased in pancreas and upper gut, reflecting augmented somatostatin synthesis; are reciprocally related to insulin acting directly or indirectly on somatostatin-producing cells; and are unchanged in the lower gut and brain, suggesting tissue-specific regulation of somatostatin gene transcription in diabetes.

Somatostatin: evidence for a role in thermal nociception

In barbiturate-anaesthetized spinalized cats, antibody microprobes were used to investigate the release of immunoreactive somatostatin (irSS) in the lumbar dorsal horn in response to cutaneous stimuli. In the absence of applied stimulation, a significant basal release of irSS was present in the region of the substantia gelatinosa. Such release was not increased by innocuous or noxious cutaneous mechanical stimuli nor by innocuous thermal stimuli, but was increased by noxious thermal stimulation. The magnitude of this noxious heat-evoked release was estimated by comparing in vivo microprobes with those used to detect known concentrations of somatostatin in vitro. Pairs of microprobes were used to detect simultaneous release of both irSS and immunoreactive substance P in the substantia gelatinosa. The results support the putative role of somatostatin in the spinal transmission of thermal nociceptive information.

Release of immunoreactive somatostatin in the spinal dorsal horn of the cat

Antibody microprobes were used to investigate the possible release of immunoreactive somatostatin (irSS) within the lumbar spinal cord of anaesthetized cats. A basal release of irSS was detected in the region of the substantia gelatinosa of the dorsal horn. By comparison with in vitro standards the concentration of SS detected in this region was estimated at 10 −7M. This release of irSS was not significantly altered by electrical stimulation of large myelinated primary afferent fibres but was increased when unmyelinated afferents were additionally stimulated. Release of irSS was also detected at the spinal cord surface. The results support a role for somatostatin in nociceptive transmission in the spinal cord.

12- O-tetradecanoyl phorbol-13-acetate (TPA) stimulates somatostatin release from isolated perfused rat stomach

Effect of TPA (12- O-tetradecanoyl phorbol-13-acetate), a potent tumor promoter, on immunoreactive somatostatin release was investigated using the isolated perfused rat stomach. TPA at the concentration as low as 20nM significantly stimulated the somatostatin release from isolated perfused rat stomach. The integrated net output of somatostatin induced by TPA was dose-dependent in a range of 5–50nM TPA. Since TPA is known to activate C-kinase specifically at a low dose (<20nM), these findings suggest that C-kinase system may be involved in the regulation of somatostatin release in rat stomach.

The effects of neurotensin, vasoactive intestinal polypeptide and other neuropeptides on the secretion of somatostatin from cerebral cortical cells

We have examined the effects of several neuropeptides on the release of immunoreactive somatostatin from cerebral cortical cells in vitro. Neurotensin and vasoactive intestinal polypeptide induced large increases in somatostatin release, whereas cholecystokinin, gonadotropin releasing hormone and Met-enkephalin induced only modest increases. Thyrotropin releasing hormone and insulin had no effect. These results demonstrate a complex interaction amongst neuropeptides in the cerebral cortex, which must be considered in future studies of the roles of peptides in cortical function.

Effects of cysteamine and antibody to somatostatin on islet cell function in vitro. Evidence that intracellular somatostatin deficiency augments insulin and glucagon secretion.

In this study we have characterized the effects of cysteamine (CHS) on the cellular content and release of immunoreactive somatostatin (S-14 LI), insulin (IRI), and glucagon (IRG) from monolayer cultures of neonatal rat islets. Incubation of cultures with 0.1-10 mM CHS for 1 h led to an apparent, dose-dependent reduction of cellular S-14 LI that was 50% of control at 0.3 mM, 87% at 1 mM, and 95% at 10 mM. IRI content was unaffected by CHS up to 1 mM, but at 10 mM 90% loss of IRI occurred. All concentrations were without effect on IRG content. The loss of S-14 LI and IRI was completely reversible with time, but with different recovery rates for the two hormones (48 h for S-14 LI, and 72 h for IRI). Released S-14 LI rose progressively with increasing doses of CHS from 21 +/- 2.5 pg/ml per hour to 41 +/- 1.4 pg/ml per hour at CHS concentrations of 5 mM and 10 mM. IRI and IRG secretion were both also significantly enhanced (by 55% and 88%, respectively), despite the elevated medium S-14 LI. Since CHS reduced cellular S-14 LI but augmented medium S-14 LI, the relative effects of CHS (1 mM) and immunoneutralization with antibody to S-14 LI on IRI and IRG secretion were tested. Anti S-14 LI alone stimulated basal IRG (67%) but not IRI. Cultures rendered S-14 LI deficient with both CHS and anti-S-14 LI exhibited threefold and 2.3-fold potentiation of IRG and IRI secretions, respectively, greater than that expected from the separate effects of the two agents. Increasing medium glucose from 2.8 mM to 16.7 mM stimulated IRI release by 86% and suppressed IRG by 53%. CHS (1 mM) and anti-S-14 LI further augmented stimulated IRI release, by 30%; although 16.7 mM glucose suppression of IRG was still maintained under these conditions, the quantitative IRG response was significantly greater. These results suggest that CHS induces an apparent loss of islet S-14 LI, and at high doses, of IRI as well, but has no effect on A cells. Complete islet S-14 LI deficiency augments IRI and IRG secretion over a wide range of glucose concentrations, suggesting a physiological role of D cells on B cell and A cell regulation. D cell modulation of B cells requires cellular but not extracellular S-14 LI, being mediated probably though direct intracellular communication, whereas the A cells seem to be regulated by both direct contact as well as through locally secreted S-14 LI.

The in situ release of immunoreactive somatostatin from the rabbit spinal dorsal horn evoked by noxious heat stimuli

The in situ release of immunoreactive somatostatin from the rabbit spinal dorsal horn evoked by noxious heat stimuli

The in situ release of immunoreactive somatostatin from the rabbit spinal dorsal horn evoked by noxious heat stimuli

The in situ release of immunoreactive somatostatin from the rabbit spinal dorsal horn evoked by noxious heat stimuli

Dopamine stimulates somatostatin release from perifused hypothalamic cells.

We studied the release of immunoreactive somatostatin (IR-SRIF) from hypothalamic cells that were obtained from rats and dispersed with the aid of collagenase. Twenty-four hours after dispersion, cells were placed in a column supported by a matrix of preswollen Biogel P2 and perifused. Fractions were collected on ice and subsequently assayed for SRIF. SRIF release was stimulated markedly by potassium depolarization (KCl, 56 mM), by the Na+-K+-ATPase inhibitor ouabain (10(-4) M), and by dopamine at concentrations as low as 10(-11) M. The stimulatory effects of membrane depolarization were calcium dependent and were not observed in the absence of exogenous calcium in the perifusion medium or in the presence of EDTA (0.05 M). Metoclopramide, the dopamine antagonist, abolished the stimulatory effect of dopamine. In conclusion, release of IR-SRIF by dispersed rat hypothalamic cells can be studied in a simple perifusion apparatus. Release is stimulated by membrane depolarization in a calcium-dependent manner and by dopamine at physiological concentrations.

Characterisation of somatostatin and TRH release by rat hypothalamic and cerebral cortical neurons maintained on a capillary membrane perfusion system.

We have investigated the nature of the immunoreactive somatostatin (SS) and thyrotropin-releasing hormone (TRH) produced by long-term capillary perfusion cultures of rat fetal cortical and hypothalamic cells. Dispersed cortical and hypothalamic cells from 17-day-old fetal rats were injected on to the outer surfaces of separate capillary membrane perfusion systems. Recirculating nutrient medium (Minimum Essential Medium with added glucose, antibiotics and 10% fetal calf serum) was then perfused via the capillary lumen at a rate of 1.5 ml/min and was changed three times weekly. Medium reservoirs, gaseous exchange coils and capillary columns were maintained in a 95% air/5% CO2 environment with 100% humidity. After 6 and 12 days in continuous perfusion, both cortical and hypothalamic cells demonstrated immuno-reactive SS release following 60 mMK+ depolarization (5- to 7-fold increase from basal secretion levels of 15-20 pg/3 X 10(7) cells/10 min). This response was clearly calcium dependent since it was abolished during washes with Ca2+-free Krebs-Ringer bicarbonate solution. Affinity purified material from pooled neuronal perfusates showed three distinct peaks of somatotropin release-inhibiting factor (SRIF) immunoreactivity following polyacrylamide gel chromatography on Biogel-P10. The dominant form coeluted with synthetic tetradecapeptide-somatostatin (SS-14) and a smaller amount (c 30%) coeluted with synthetic SS-28. (The SS-14 antibody used showed equimolar cross reactivity with SS-28). A larger form of immunoreactive material was also detected with an apparent molecular weight of about 11,500 daltons. Hypothalamic and cortical perfusates produced similar electrophoretic patterns of immunoreactive SS (ISS).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucagon, Neurotensin, and vasoactive intestinal polypeptide on somatostatin release from perifused rat hypothalamus.

The effects of neuropeptides on the release of immunoreactive somatostatin (SRIF) from the rat hypothalamus were examined in vitro using a perifusion system. Twelve hypothalamic halves of male rats were placed on a Sephadex G-25 column and continuously eluted with Krebs-Ringer bicarbonate buffer, poH 7.4, at 37 C. A high potassium concentration (56 mM) stimulated SRIF release in a calcium-dependent manner. The infusion of glucagon (10(-7), 5 x 10(-7), and 10(-6) M) resulted in a dose-related increase in the release of SRIF. Neurotensin (10(-6) M) also stimulated SRIF release, whereas vasoactive intestinal polypeptide (10(-7) and 10(-6) M) inhibited SRIF release. SRIF release was not affected by cholecystokinin-octapeptide (10(-6) M), cholecystokinin-tetrapeptide (10(-6) M), or tRH (10(-6) M). These findings suggest that SRIF release from the rat hypothalamus is influenced by glucagon, neurotensin, and vasoactive intestinal polypeptide.

Effects of neurotransmitters and cyclic AMP on somatostatin release from cultured cerebral cortical cells

The influence of cortical neurotransmitters and cyclic AMP on the release of immunoreactive somatostatin (IRS) from cultured cortical cells was examined. Cells were obtained by mechanoenzymatic dispersal of telencephalons of 17-day-old rat embryos and were maintained as monolayers in minimum essential medium with 10% heat-inactivated horse serum. After the cultures had stabilized morphologically and in cellular IRS content they were subjected to rapid sequential changes of a buffered salt solution with or without test substances added. The amount of somatostatin released was measured by a specific radioimmunoassay. Acetylcholine and the GABA antagonist, picrotoxin, both stimulated IRS release. The cholinergic stimulation was predominantly muscarinic. GABA and histamine, to a lesser extent, were inhibitory and norepinephrine and serotonin produced no net change in IRS release. Both cAMP and theophylline (DMX) stimulated IRS release. These results confirm the potential of intrinsic cortical somatostatinergic neurons to respond to endogenous neurotransmitters and further establishes somatostatin as a cortical neuromodulator.

Somatostatin release from rat hypothalamus in vitro: effects of melatonin and serotonin.

The release of immunoreactive somatostatin (SRIF) from explants of rat medial basal hypothalamus, which were maintained in culture for 24 h, was quantitated by a sensitive RIA. Validity of the specific SRIF assay has been previously established by chromatographic criteria using gel and high pressure systems and by the demonstration of immunological parallelism. After 24 h of culture in medium containing heat-inactivated fetal calf serum, hypothalamic fragments were incubated in serum-free medium, and the release of immunoreactive SRIF was quantitated. Melatonin at concentrations of 10(--8) and 10(--7) M stimulated SRIF release, and no significant increases were observed at concentrations of 10(--9) M or less or at concentrations of 10(--6) M or greater. Serotonin oxalate at concentrations of 10(--8)--10(--5) M significantly inhibited SRIF release. The serotonin antagonist cyproheptadine at a concentration of 10(--5)M had no effect on basal SRIF release but abolished the inhibitory effect of 10(--7) M serotonin. Finally, when hypothalami were incubated with melatonin and serotonin, each at 10(--7) M, SRIF release was unchanged compared to control values. The results suggest that the brain indoleamines, melatonin and serotonin, may modulate GH secretion by effects on SRIF release at a hypothalamic level.

Stimulation by bombesin of immunoreactive somatostatin release into rat hypophysial portal blood.

Bombesin was injected into the cerebral ventricle of male rats anesthetized with urethane to study its effect on plasma levels of immunoreactive somatostatin (IRS) in hypophysial portal and jugular blood. An intraventricular injection of bombesin (0.2 and 2 micrograms/rat) caused a significant and dose-related increase in plasma IRS in hypophysial portal blood but not in jugular blood. Although bombesin placed into the cerebral ventricle is known to stimulate glucagon and epinephrine release, an iv injection of glucagon (100 micrograms/100 g BW) or epinephrine (2.5 micrograms/100 g BW) did not cause any significant changes in plasma IRS levels in hypophysial portal and jugular blood, suggesting that these substances do not mediate bombesin stimulation of portal IRS release. Pretreatment with naloxone (75 micrograms/100 g BW, iv) failed to affect the portal IRS release induced by bombesin (2 micrograms/rat), indicating that the opiate receptor is not likely to be involved in this reaction. To ascertain whether IRS released by bombesin into hypophysial portal blood is biologically active, the effect of bombesin on the plasma GH level was then examined. Bombesin (2 micrograms/rat) injected intraventricularly completely suppressed the rise of plasma GH after the intraventricular injection of beta-endorphin (1 microgram/rat) or the iv injection of prostaglandin E1 (5 micrograms/100 g BW). Bombesin thus appears to stimulate the secretion of IRS, and probably biologically active somatostatin as well, from the hypothalamus into hypophysial portal blood, thereby inhibiting GH release from the anterior pituitary.

Noradrenaline stimulates somatostatin release from incubated slices of the amygdala and the hypothalamic preoptic area

Neurotransmitter effects were studied on in vitro release of immunoreactive somatostatin (SRIF) from slices prepared from several regions of the rat brain: mediobasal hypothalamus (MBH), preoptic anterior hypothalamic area (POA) and amygdaloid complex (AMY). Potassium (K +, 56 mM) stimulated SRIF release in all structures tested in a calcium dependent manner. Morphine, dopamine, GABA and serotonin did not modify SRIF release in any structure; noradrenaline (NA) was not effective on MBH slices, but elicited a dose-dependent stimulation of SRIF release from POA and AMY (ED 50 = 6.4nMand3.6 ±nM respectively). Conveerse orders of potency of adrenergic agonist were observed in both structures (POA, adrenalin > noradrenaline > isoproterenol; AMY, isopreterenol > adrenaline > noradrenaline). Phentolamine blocked NA-induced SRIF release in the POA while propanolol was ineffective. On the contrary, propanolol, but not phentolamine, antagonized NA stimulation in the amygdala. The data suggest that NA acting through specific receptors modulate SRIF release from POA and AMY. In POA, NA effect seems mediated through α adrenergic receptors while in AMY, β receptors are involved. The possibility that these interactions of NA with SRIF release are correlated with effects of NA on growth hormone secretion or on epileptic events is discussed.

Truncal vagotomy abolishes the somatostatin response to insulin-induced hypoglycemia in man.

The mechanism whereby insulin-induced hypoglycemia stimulates release of immunoreactive somatostatin (SRIF-LI) into the peripheral circulation in man is unknown. We have measured the plasma SRIF-LI response to insulin-induced hypoglycemia in 16 healthy subjects and five subjects with prior truncal vagotomy. Mean nadir of plasma glucose was similar in the two groups (37 +/- 5 and 34 +/- 2 mg/dl in the control group). Hypoglycemia induced a brisk rise in plasma pancreatic polypeptide (hPP) concentrations in healthy subjects (maximum concentration 1563 +/- 245 pg/ml) whereas in none of the postvagotomy subjects was there a significant change in hPP concentrations, indicative of completeness of the truncal vagotomy. In healthy subjects SRIF-LI concentrations rose for a basal of 168 +/- 19 pg/ml to a maximum of 254 +/- 30 at 39 +/- 4 minutes (p less than 0.005) with an incremental area of 2.8 +/- 1 ng . min/ml above basel. In vagotomized subjects, the mean basal SRIF-LI concentration of 166 +/- 22 pg/ml was not significantly different from that in healthy subjects. After insulin injection, SRIF-LI concentration fell with a net decrement of -3.2 +/- 1 ng . min /ml below the basal. It is concluded that the SRIF-LI response to insulin-induced hypoglycemia is dependent upon vagal integrity. Section of the vagus unmasks a suppressive effect of insulin action or its metabolic or hormonal consequences on the concentration of SRIF-LI in plasma.

Effect of capsaicin pretreatment on capsaicin-evoked release of immunoreactive somatostatin and substance P from primary sensory neurons

Release of immunoreactive somatostatin (I-SRIF) and immunoreactive substance P (I-SP) was studied from slices prepared from upper dorsal horn (UDH) and lower dorsal plus ventral horn (LDH-VH) of rat spinal cord. Superfusion with capsaicin (10 microM) led to release of I-SRIF and I-SP from UDH slices but not from LDH-VH slices. The capsaicin-evoked release of I-SP was 6 fold higher than that of I-SRIF. A pulse of 60 mM K+ applied after the capsaicin pulse caused release of I-SRIF and I-SP from UDH as well as LDH-VH slices. Pretreatment of rats with capsaicin (125 mg/kg, s.c.) led to a nearly 40% depletion of I-SP in slices from UDH only. Capsaicin-evoked release from these slices was reduced by 81% for I-SRIF and by 79% for I-SP. Release evoked by K+ remained unchanged. These results indicate that capsaicin causes release of both I-SRIF and-I-SP and that this release is most likely restricted to primary sensory neurons. The marked reduction of the release of I-SP after systemic capsaicin pretreatment may well represent one of the, or even the reason for the insensitivity of capsaicin pretreated rats towards chemogenic pain.