Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell

Abstract

1. The ability of a range of phenothiazines to inhibit activation of brain phosphodiesterase by purified calmodulin was studied. Trifluoperazine, prochlorperazine and 8-hydroxyprochlorperazine produced equipotent dose-dependent inhibition with half-maximum inhibition at 12mum. When tested at 10 or 50mum, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50mum, and produced only a modest inhibition at 100mum. 2. The same phenothiazines were tested for their ability to inhibit activation of brain phosphodiesterase by boiled extracts of rat islets of Langerhans. At a concentration of 20mum, 70-80% inhibition was observed with trifluoperazine, prochlorperazine, 7-hydroxyprochlorperazine or 8-hydroxyprochlorperazine, whereas trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were less effective. 3. The effect of these phenothiazines on insulin release from pancreatic islets was studied in batch-type incubations. Insulin release stimulated by glucose (20mm) was markedly inhibited by 10mum-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20mum. 8-Hydroxyprochlorperazine (20mum) was also a potent inhibitor but 7-hydroxyprochlorperazine (20mum) elicited only a modest inhibition of glucose-stimulated insulin release; no inhibition was observed with trifluoperazine-5-oxide or N-methyl-2-(trifluoromethyl)phenothiazine. 4. Trifluoperazine (20mum) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20mum) decreased insulin release stimulated by glibenclamide in the presence of 3.3mm-glucose. 5. None of the phenothiazines affected basal insulin release in the presence of 2mm-glucose. 6. Trifluoperazine (20mum) did not inhibit islet glucose utilization nor the incorporation of [(3)H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of (32)P from [gamma-(32)P]ATP into endogenous protein substrates. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis resolved several phosphorylated bands, but incorporation was slight. However, calmodulin in the presence of Ca(2+) greatly enhanced incorporation: the predominant phosphorylated band had an estimated mol.wt. of 55000. This enhanced incorporation was abolished by trifluoperazine, but not by cyclic AMP-dependent protein kinase inhibitor protein. 8. These results suggest that islet phosphodiesterase-stimulating activity is similar to, although not necessarily identical with, calmodulin from skeletal muscle; that islet calmodulin may play an important role in Ca(2+)-dependent stimulus-secretion coupling in the beta-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.

Phenothiazines inhibit prolactin secretion in vitro

“…Prochlorperazine, which has also been shown to bind to calmodulin and inhibit its action, was also effective in inhibiting prolactin secretion. The EDso for this drug was ~5/aM, which again is similar to its known binding constant with calrnodulin and the ICso for the inhibition of calmodulin-activated phosphodiesterase [19]. These results suggest that the drugs may be interacting with calmodulin within the cell and that this interaction in some way leads to inhibition of the secretory process.…”

“…It is apparent that the sulphoxides were without inhibitory effect, in the dose range 0.084-84/aM, in the same experiments in which the parent compounds had profound inhibitory effects. Trifluoperazine sulphoxide is known to be considerably less potent as a calmodulin inhibitor [18,19]. The cause of the slight stimutatory effect of the prochlorperazine sulphoxide is not yet known.…”

“…Moreover, the sulphoxide analogue of prochlorperazine, which is also less potent in inhibiting calmodulin-activated phosphodiesterase (unpublished) was also ineffective in inhibiting prolactin secretion. Calmodulin may also be involved in insulin secretion [ 19,23,24], vasopressin-stimulated water flow [25], intestinal ion secretion [26,27] and stimulated thyrotropin secretion [28], in other cellular systems. However, this is the first report of a possible role for calmodulin in basal and stimulated prolactin secretion.…”

Effects of hypophysectomy and growth hormone on cultured islets of Langerhans of the rat

“…The decreased secretory response may be ascribed, at least in part, to a decrease in islet calmodulin content. Since calmodulin does not appear to play a role in glucose-stimulated insulin biosynthesis [22], the decreased rate of insulin biosynthesis in islets from hypophysectomised rats may occur in parallel with rather than consequential to the diminished calmodulin content. As a minimal hypothesis, therefore, we may speculate that hypophysectomy impairs rather specifically the synthesis of proteins involved in secretion, including calmodulin and insulin itself; the diminished secretory response may result from these changes.…”

“…Evidence has been presented [15,22] that calmodulin may play a key role in stimulus-secretion coupling in the B-cell. We therefore measured the concentration of calmodulin in the cultured islets.…”