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.
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Research Article|
December 15 1980
Evidence for the participation of calmodulin in stimulus–secretion coupling in the pancreatic β-cell
Juan J. Gagliardino;
Juan J. Gagliardino
1Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford, U.K.
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Donna E. Harrison;
Donna E. Harrison
1Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford, U.K.
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Michael R. Christie;
Michael R. Christie
1Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford, U.K.
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Elma E. Gagliardino;
Elma E. Gagliardino
1Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford, U.K.
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Stephen J. H. Ashcroft
Stephen J. H. Ashcroft
1Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Oxford, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1980 London: The Biochemical Society
1980
Biochem J (1980) 192 (3): 919–927.
Citation
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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