Myelin basic protein present in the acid extracts of rat hypothalami releases insulin and glucagon from isolated rat pancreatic islets

The insulinotropic and glucagon-releasing activity of acid extracts of rat hypothalami were tested in two bioassay systems using short-time incubation of isolated rat pancreatic islets and a rat insulinoma (RIN) cell line. Release of insulin and glucagon into the incubation medium was measured by radioimmunoassay. The major insulin-releasing and glucagon-releasing activity eluted in a broad zone in Sephacryl S-200 gel filtration in 30% acetic acid corresponding to the molecular weights between approximately 10 and 40 kD. The highest activity was eluted in a zone corresponding to 14 kD and was purified to homogeneity by means of two steps of reversed-phase HPLC. Amino acid analysis and SDS polyacrylamide gel electrophoresis indicated that the biologically active material was the rat small (myelin) basic protein characterized previously by Dunkley & Carnegie (1974). The purified rat hypothalamic material showed insulinotropic and glucagon-releasing activity indistinguishable from that of purified porcine myelin basic protein, and lost its insulinotropic activity when incubated with anti-myelin basic protein immunosorbent. We conclude that the major insulin-releasing and glucagon-releasing activity in acid extracts of the high-molecular-weight fractions of rat hypothalami is myelin basic protein.     

Effect of calmidazolium (R24571) on histamine release from isolated rat mast cells

The effects of selected calmodulin-antagonists, i.e. calmidazolium (R24571), trifluoperazine, cis- and transflupenthixol, chlorpromazine, and imipramine, on rat mast cells and on mast cell histamine release were investigated. The drugs induced histamine release, apparently by cytotoxic effects, with a rank order of potency in accordance with their lipid solubility and with maximal release at calmidazolium (5 mol/l), trifluoperazine (40 mol/l), cis- and trans-flupenthixol (50 mol/l), chlorpromazine (100 mol/l), and imipramine (500 mol/l). Inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was only observed with some of the drugs tested, with maximal inhibition at calmidazolium (2 mol/l), chlorpromazine (25–50 mol/l), and imipramine (100–250 mol/l). The concentration-response curve for histamine release induced by calmidazolium was significantly shifted to the right by antigen (i.e. horse serum) in the medium and the addition of antigen was capable of immediately stopping the release induced by calmidazolium, indicating binding of calmidazolium by antigen. Similar effects on the actions of calmidazolium were observed with phosphatidylserine. The inhibition by calmidazolium of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was significantly counteracted by glucose in the medium. The findings do not confirm an involvement of calmodulin in the histamine release process in rat mast cells. The ability of calmidazolium to bind to proteins and phospholipids in the medium indicates multiple cellular targets for calmidazolium, and the observations with glucose suggest an impaired mitochondrial function to be of major significance. The present investigation, in accordance with other recent studies, indicates that the calmodulin-antagonists are of limited utility as probes for an involvement of calmodulin in cellular responses.     

Cholecystokinin (CCK8) regulates glucagon,insulin, and somatostatin secretion from isolated rat pancreatic islets: interaction with glucose

The effect of CCK₈ on glucagon, insulin and somatostatin release and its interaction with glucose was studied in freshly isolated rat pancreatic islets. While glucose alone inhibited glucagon secretion [half-maximal effect (EC₅₀)=4.6 mM], glucose in the presence of 10 nM CCK₈ increased glucagon release (EC₅₀=6.9 mM). This effect of CCK₈ was dose-dependent at 11.1 mM glucose (EC₅₀=1.0 nM). The dose-response curve for glucose on insulin secretion was shifted to the left by 10 nM CCK₈; the EC₅₀ of glucose was 11.6 and 9.3 mM in the absence and presence of CCK₈, respectively. Glucose alone enhanced somatostatin release; this glucose-induced release was further increased by 10 nM CCK₈. Our data indicate that first, CCK₈ is able to reverse the inhibitory effect of glucose on glucagon secretion, second, CCK₈ sensitizes the beta cell to the insulinotropic effect of glucose, and third, CCK₈ enhances the effect of glucose on somatostatin release.     

Effect of TMB-8 on histamine release from isolated rat mast cells

TMB-8 was capable of complete inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 (2 microM). The effective concentrations for 50% inhibition (IC50) were 1.2 X 10(-4) M for antigen, 1.6 X 10(-4) M for compound 48/80 both in the absence and in the presence of calcium, and 0.7 X 10(-4) M for the ionophore. The inhibitory action was not affected by increased calcium concentration in the medium from 1 to 2m M. The presence of glucose in the medium counteracted the inhibition by TMB-8 and, for low concentrations of the ionophore (0.25 microM), TMB-8 caused a pronounced enhancement of the histamine release. Our results indicate that TMB-8 primarily exerts its effects by interference with oxidative metabolism rather than by affecting the intracellular calcium availability.     

Myelin basic protein stimulates insulin and glucagon secretion from rat pancreatic islets in vitro and in vivo

The effect of myelin basic protein on insulin and glucagon secretion from rat pancreatic islets was studied in vivo and in vitro. The myelin basic proteins isolated from bovine, human and rat brains all stimulated insulin secretion in a similar fashion. In a static incubation of isolated pancreatic islets, myelin basic protein at doses of 15.6–250 μg in a 0.5-ml reaction volume (1.7 times 10^-8 to 2.7 times 10^-5 M) significantly stimulated hormone release. Maximal stimulation, obtained at the 250-μg dose, was 6.5-fold greater than control for insulin secretion and 6.7-fold greater than control for glucagon secretion. In the case of glucagon no saturation was observed, but saturation was obvious for insulin release at doses of myelin basic protein of 62.5–250μg, larger doses causing permeabilization of the islet membranes as indicated by leakage of acid phosphatase. At a 100-μg dose the time course of insulin secretion induced by myelin basic protein indicated a fast initial release, and after the first 2 h only a little more insulin was released. At the lower doses of myelin basic protein (11 and 33μg) the secretion rate was nearly constant after the first hour. Significant stimulation of glucagon release by myelin basic protein was seen after 60 min, the rate of release being roughly constant at 33-and 100-μg doses thereafter. At the 11-μg dose significant stimulation of hormone release was observed only after a 4-h incubation. Lowering the temperature from 37 to 27 and 20°C reduced both basal and stimulated hormone secretion, the extent of stimulation over the basal level remaining the same at all temperatures only for insulin secretion at a dose of myelin basic protein of 100 μg. A dose of 10 mg myelin basic protein injected intravenously into anaesthetized rats resulted 15 min after injection in a circulating concentration of myelin basic protein of 34.7 μg ml^-1 (1.7 times 10^-6 M) as measured by our radioimmunoassay. It stimulated insulin secretion (P < 0.01), having no significant effect on plasma glucagon levels. Since myelin basic proteins have been shown to display fusogenic properties in cell-free membrane systems, we propose that myelin basic protein exerts its hormone-releasing effect by aggregating and fusing the hormone-containing vesicles to the cell plasma membranes.     

The influence of tetradecanoyl-phorbol-acetate (TPA) on histamine release from isolated rat mast cells

A detailed investigation of the influence of tetradecanoyl-phorbol-acetate (TPA) on isolated rat mast cells was undertaken in order to explore the possible involvement of protein kinase C in histamine release. TPA alone could induce histamine release in a medium without calcium, whereas 1 mM CaCl₂ suppressed the release. TPA in combination with a low concentration of the ionophore A23187 induced a considerable histamine release. Preincubation with TPA followed by incubation with the ionophore induced a similar release at low concentrations of TPA (2.5 nM) whereas the response was reduced at higher concentrations of TPA. The inhibition after preincubation with TPA was almost at a maximum within 2 min and was due to a decreased rate of release. TPA could also increase antigen-induced histamine release. After preincubation the potency of low concentrations of TPA increased, whereas higher concentrations (50 nM) became inhibitory. The effects of preincubation were almost fully expressed after 2 min and were not due to altered kinetics of the release. The interaction of oleoylacetylglycerol (OAG) with the ionophore A23187 and with antigen resembled that of TPA, but OAG was considerably less potent. Preincubation with TPA was inhibitory to the histamine release induced by compound 48/80, particularly in the absence of calcium. The release induced by TPA and the ionophore or antigen was calcium-dependent and energy-requiring, and the effects of TPA persisted after washing the cells before exposure to antigen or the ionophore. Preincubation with the protein kinase C inhibitor isoquinolinesulfonyl-methylpiperazine (H-7) slightly enhanced the histamine release induced by the combination of TPA and the ionophore. The suppression exerted by preincubation with TPA on ionophore- and antigen-induced release was counteracted by H-7. The results indicate that only the inhibitory effects of protein kinase C are affected by H-7. Although not conclusive, the results are compatible with an involvement of protein kinase C in both the enhancing and the inhibitory effects of TPA on mast cell histamine release.Parts of this investigation were presented at the meeting of the European Histamine Research Society in May 1986.     

Effect of synthetic C-terminal fragments of hGH on insulin release by isolated islets.

Synthetic fragments representing the C-terminal end of the growth hormone molecule have been tested for their direct in vitro effects on insulin release by isolated rat islets of Langerhans. hGH 177-191 caused a dose-related potentiation of glucose-induced insulin release, whereas the peptide by itself caused no stimulation of insulin release from the islets. The rate curves constructed for insulin secretion as a function of extracellular glucose concentration showed that the Km for glucose is not altered in the presence of the peptide, but that the Vmax of secretion is increased. Significant potentiation of insulin release by the peptide was seen only at high extracellular concentrations of glucose. Measurement of cAMP levels in islets showed that the peptide caused no significant alteration of cAMP levels while still potentiating insulin release. It was therefore concluded that the mechanism of potentiation of insulin release by the peptide may be independent of the changes in cAMP levels in islets. hGH 172-191, too, caused potentiation of glucose-stimulated insulin release from islets, whereas hGH 179-191 was not active in this report.     

Myelin basic protein stimulates insulin and glucagon secretion from rat pancreatic islets in vitro and in vivo.

The effect of myelin basic protein on insulin and glucagon secretion from rat pancreatic islets was studied in vivo and in vitro. The myelin basic proteins isolated from bovine, human and rat brains all stimulated insulin secretion in a similar fashion. In a static incubation of isolated pancreatic islets, myelin basic protein at doses of 15.6-250 micrograms in a 0.5-ml reaction volume (1.7 X 10(-6) to 2.7 X 10(-5) M) significantly stimulated hormone release. Maximal stimulation, obtained at the 250-micrograms dose, was 6.5-fold greater than control for insulin secretion and 6.7-fold greater than control for glucagon secretion. In the case of glucagon no saturation was observed, but saturation was obvious for insulin release at doses of myelin basic protein of 62.5-250 micrograms, larger doses causing permeabilization of the islet membranes as indicated by leakage of acid phosphatase. At a 100-micrograms dose the time course of insulin secretion induced by myelin basic protein indicated a fast initial release, and after the first 2 h only a little more insulin was released. At the lower doses of myelin basic protein (11 and 33 micrograms) the secretion rate was nearly constant after the first hour. Significant stimulation of glucagon release by myelin basic protein was seen after 60 min, the rate of release being roughly constant at 33- and 100-micrograms doses thereafter. At the 11-micrograms dose significant stimulation of hormone release was observed only after a 4-h incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hydroxycitric acid on serotonin release from isolated rat brain cortex.

There is evidence that hydroxycitric acid (HCA), an extract of dried fruit rind of South Asian trees of the genus Garcinia cambogia, can reduce food intake in experimental animals. In the present study, we investigated the effect of HCA on basal and potassium-depolarization evoked increase in radiolabeled serotonin ([3H]-5-HT) release from rat brain cortex slices in vitro. HCA (10 microM-1 mM) altered the baseline of spontaneous tritium efflux but had no significant effect on potassium-evoked release of [3H]-5-HT. When applied on its own, HCA (10 microM-1 mM) elicited a concentration-dependent increase in efflux of [3H]-5-HT reaching a maximum at 300 microM. We conclude that HCA can increase the release of radiolabeled 5-HT from the isolated rat brain cortex.     

Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum.

The dynamic release of somatostatin-like immunoreactivity (SLI) from duodenum into the lumen was studied in the isolated, vascularly perfused rat duodenum. The luminal release of duodenal SLI was stimulated by a cholinergic agonist, carbachol, and the carbachol-induced release of SLI was completely blocked by atropine, but not by hexamethonium. These data suggest that the luminal release of SLI from rat duodenum is under the control of a cholinergic muscarinic stimulation. The ratio of somatostatin-14 to somatostatin-28 in picograms was about 1 during basal release but increased to approximately 2 during carbachol stimulation.     

Emiocytosis of B granules from saurian pancreatic islets perifused in vitro

Splenic islets of Anolis carolinensis incubated in an in vitro perifusion system with media containing a non-stimulatory concentration of glucose had the same ultrastructural appearance as islets removed from an intact animal. When horseradish peroxidase was included in this medium, no profiles suggestive of emiocytotic release of B granules were observed despite basal insulin secretion. With cytochalasin B and a high concentration of glucose, the B cells exhibited margination of their secretory granules and moderate degranulation, but rarely were profiles suggestive of emiocytosis seen. In the presence of cytochalasin B, glucose and the other insulin secretagogues used, the B cells were more degranulated than with only cytochalasin B and glucose present, but profiles indicative of emiocytosis were infrequently encountered. Emiocytosis of the B granules was demostrated convincingly with the inclusion of peroxidase and the above agents. Virtually all the B cells had at least one secretory granule fused with the limiting membrane and surrounded by the peroxidase, and some B cells exhibited multiple release of granules. From these observations it is concluded that emiocytosis plays a major role in the secretion of insulin from B cells of Anolis. The B cells from control perifusions, as well as from intact animals, did not contain a microfilamentous cell web. Granular or filamentous masses were not demonstrated in B cells from splenic islets perifused with cytochalasin B. The mechanism of emiocytotic release of insulin from the B cells of Anolis is discussed with reference to these observations.     

Glucagon-induced somatostatin release from perifused rat hypothalamus: calcium dependency and effect of cysteamine treatment

Somatostatin (SRIF) release from rat hypothalamus was investigated in vitro with a perifusion system. Glucagon (1 microM) and high potassium concentrations (56 mM) stimulated SRIF release in a calcium-dependent manner. Pretreatment of the rat with cysteamine (30 mg/100 g body weight, 7 h earlier) significantly reduced SRIF release from the hypothalamus in glucagon- and high potassium-stimulated states as well as in the basal state. SRIF release from rat hypothalamus was also stimulated by both dibutyryl cyclic AMP (1 mM) and theophylline (3 mM). These results suggest that glucagon, acting in a calcium-dependent manner and possibly through the adenylate cyclase-cyclic AMP system, stimulates SRIF release from rat hypothalamus and that cysteamine treatment reduces releasable SRIF in the hypothalamus.     

The effect of secretin on the glucose-induced insulin release from isolated pancreatic islets in mice

A simple and rapid method for isolation of pancreatic islets from mice using Percoll as a separation medium is described. Increasing concentrations of secretin from 10(-11) to 10(-6) M were without effect on the insulin release from the islets at 5 X 5 mM glucose, whereas significant increases were found at 12 mM glucose. Secretin did not elicit additional increases of the insulin release at 5 X 5 and 12 mM glucose with 5 mM theophylline. The mechanism by which secretin acts on the pancreatic islets is discussed. Based upon the above observations it is suggested that secretin apparently represents a modulator of the insulin release from the pancreatic islets.     

A-cell-hyperplasia of islets of Langerhans on rabbits after immunisation with glucagon (author's transl)

Rabbits are immunized with glucagon for the production of anti-glucagonsera. Pancreatic islets of 9 animals are investigated histochemically. In the islets of immunized rabbits is a hyperplasia of A-cells detected. Singular islets consist only of A-cells.     

The effect of calcium-blockers nicardipine, darodipine, PN-200-110 and nifedipine on insulin release from isolated rat pancreatic islets

The effect of the calcium-blockers nicardipine, darodipine, PN-200-110 on insulin release from pancreatic islets was studied using nifedipine as a reference compound. All drugs at a concentration of 10(-6)M significantly inhibited insulin release in response to both low (5.5 mM) and high (22 mM) glucose. The present observations support previous reports that calcium blockers of the dihydropyridine series are effective inhibitors of glucose-induced insulin release.     

Influence of DPPE on histamine release from isolated rat mast cells

A second messenger function for histamine has been proposed based on the effects of the anti-estrogen drug DPPE (N,N-diethyl-2-(4-(phenylmethyl)phenoxy) ethanamine.HCl). The ability of DPPE to inhibit concanavalin A-induced histamine release led to the present investigation of its influence on the mast cell response to a wider selection of secretagogues. DPPE was an efficient inhibitor of antigen-induced release, while responses to compound 48/80 were virtually unaffected. Responses to the ionophore A23187 could be enhanced as well as inhibited, whereas the influence of DPPE on the combination of the ionophore and the phorbol ester TPA was variable and small. These results seem to exclude an involvement of a DPPE-sensitive histamine-mediated signal system of common importance in mast cell histamine release.