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 12μm. When tested at 10 or 50μm, 7-hydroxyprochlorperazine was a similarly potent inhibitor. However, trifluoperazine-5-oxide and N-methyl-2-(trifluoromethyl)phenothiazine were ineffective at concentrations up to 50μm, and produced only a modest inhibition at 100μm. 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 20μm, 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 10μm-trifluoperazine or -prochlorperazine and further inhibited at a concentration of 20μm. 8-Hydroxyprochlorperazine (20μm) was also a potent inhibitor but 7-hydroxyprochlorperazine (20μm) 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 (20μm) markedly inhibited insulin release stimulated by leucine or 4-methyl-2-oxopentanoate in the absence of glucose, and both trifluoperazine and prochlorperazine (20μm) 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 (20μm) did not inhibit islet glucose utilization nor the incorporation of [3H]leucine into (pro)insulin or total islet protein. 7. Islet extracts catalysed the incorporation of 32P from [γ-32P]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 Ca2+ 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 Ca2+-dependent stimulus–secretion coupling in the β-cell; and that calmodulin may exert part at least of its effect on secretion via phosphorylation of endogenous islet proteins.
Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell
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Juan J. Gagliardino, Donna E. Harrison, Michael R. Christie, Elma E. Gagliardino, Stephen J. H. Ashcroft; Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell. Biochem J 15 December 1980; 192 (3): 919–927. doi: https://doi.org/10.1042/bj1920919
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