Influence of thiol groups, calcium, and glucose metabolism on cholinergic-induced insulin release and on methylscopolamine binding to muscarinic receptors in pancreatic islets of the rat

Abstract

Short-term regulation of [3H]methylscopolamine binding to muscarinic receptors and acetylcholineinduced stimulation of insulin release was investigated in pancreatic islets of the rat. Binding of methylscopolamine was reversible; 47% of label was displaced 10 min and 70% 30 min after addition of unlabelled substance. 0.1 mM chloromercuribensoic acid, when present during binding incubations, inhibited binding by 54%, whereas acetylcholine-induced insulin release was unaffected by the presence of the thiol reactant. Pre-incubation for 60 min in a calcium-deprived medium or in the presence of 50 \g=m\mtrifluoroperazine likewise inhibited binding. Pre-incubation with 1.0 mm 3-isobutyl-l-methylxanthine or 16.7 m glucose failed to influence subsequent binding although acetylcholine-induced insulin release was 4-fold enhanced by priming with glucose. We conclude that 1) binding to muscarinic receptors is influenced by thiol interaction, 2) short-term alterations in calcium fluxes influence binding, whereas short-term changes in cyclic AMP (cAMP) or glucose metabolism do not, 3) a priming effect of glucose on insulin secretion is not mediated by changes in receptor binding. The parasympathetic nervous system exerts an important influence on insulin secretion. Cephalic phase insulin secretion may thus be mediated to a major part by vagai stimulation ; its absence leads to delayed insulin secretion out of phase with mealinduced hyperglycaemia (Trimble et al. 1981). The receptors mediating the cholinergic effect are muscarinic in nature since acetylcholine-induced insu¬ lin secretion is readily blocked by atropine (Froh¬ man et al. 1967).To gain further insight into mechanisms behind acetylcholine-induced insulin release, we previous¬ ly studied binding of [3H]methylscopolamine to rat pancreatic islets. A high-affinity, rapid and specific binding of the muscarinic antagonist could be demonstrated (Grill & Östenson 1983). Half-maxi¬ mal inhibition of acethylcholine-induced release by methylscopolamine occurred at concentrations which were not lower than those causing halfmaximal displacement of label, suggesting that the binding parameter measured reflected an initial step in acetylcholine-induced insulin secretion.The aim of the present study was to gain further insight into islet regulation of muscarinic receptor binding and its role for insulin release. To this end we have investigated the effects on binding of factors of importance for insulin release such as the functional state of membrane thiol groups, per¬ turbations in cyclic AMP and calcium levels as well as changes in glucose metabolism. Additionally the dissociation of [3H]methylscopolamine was studied in order to further validate our procedure for measuring binding. Material and Methods Material and animah[3H]methylscopolamine chloride (S.A. 85 Ci/mmol) and [14C]sucrose (S.A. 673 mCi/mmol) was obtained from New England Nuclear (Dreieichenhain, FRG). Unlabel¬ led methylscopolamine nitrate was from Pharmacia, Uppsala, Sweden. Acetylcholine chloride, p-chloromerc...

Glucose-, calcium- and concentration-dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets

“…At a non-stimulatory concentration of glucose (3 mM), a small and progressive increase in insulin release was caused by 100 gM-, but not by 1 sM-ACh. This observation agrees both with our previous reports that 1 ,UM-ACh is ineffective [8,1 1] and with reports by others that high concentrations of muscarinic agonists slightly stimulate release by islets incubated at sub-stimulatory concentrations of glucose [21,22]. The kinetics, magnitude and Ca2"-dependency of the release of insulin evoked by 100 1uM-ACh in the presence of 3 mM-glucose are strikingly similar to those of insulin release induced by 20 mM-arginine or by a moderately elevated (14 mM) concentration of K+ [11,36].…”

“…Glucose has no short-term effects on the number and affinity ofmuscarinic receptors, but prolonged incubation of islets in a Ca2l-free medium has been shown to decrease methylscopolamine binding to these receptors [21]. However, Ca2l omission did not cause a generalized decrease in ACh efficiency.…”

“…There is no dispute that muscarinic agonists do not induce insulin release in a glucose-free medium [12,19,20]. However, several groups have observed a stimulation in the presence of only 1.7-3 mM-glucose [5,15,[20][21][22], whereas others consistently failed to observe such an effect at substimulatory concentrations of glucose [7,8,11,16,23,24]. Finally, whereas there is general agreement that muscarinic agonists are very efficient in the presence of stimulatory concentrations of glucose, their effects in the presence of a maximally effective concentration of the sugar have not been characterized.…”