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 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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Selected References
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- Ashby C. D., Walsh D. A. Characterization of the interaction of a protein inhibitor with adenosine 3',5'-monophosphate-dependent protein kinases. I. Interaction with the catalytic subunit of the protein kinase. J Biol Chem. 1972 Oct 25;247(20):6637–6642. [PubMed] [Google Scholar]
- Ashcroft S. J., Bunce J., Lowry M., Hansen S. E., Hedeskov C. J. The effect of sugars on (pro)insulin biosynthesis. Biochem J. 1978 Aug 15;174(2):517–526. doi: 10.1042/bj1740517. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ashcroft S. J., Crossley J. R. The effects of glucose, N-acetylglucosamine, glyceraldehyde and other sugars on insulin release in vivo. Diabetologia. 1975 Aug;11(4):279–284. doi: 10.1007/BF00422392. [DOI] [PubMed] [Google Scholar]
- Ashcroft S. J. Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia. 1980 Jan;18(1):5–15. doi: 10.1007/BF01228295. [DOI] [PubMed] [Google Scholar]
- Ashcroft S. J., Weerasinghe L. C., Randle P. J. Interrelationship of islet metabolism, adenosine triphosphate content and insulin release. Biochem J. 1973 Feb;132(2):223–231. doi: 10.1042/bj1320223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheung W. Y., Bradham L. S., Lynch T. J., Lin Y. M., Tallant E. A. Protein activator of cyclic 3':5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase. Biochem Biophys Res Commun. 1975 Oct 6;66(3):1055–1062. doi: 10.1016/0006-291x(75)90747-0. [DOI] [PubMed] [Google Scholar]
- Cheung W. Y. Cyclic 3',5'-nucleotide phosphodiesterase. Demonstration of an activator. Biochem Biophys Res Commun. 1970 Feb 6;38(3):533–538. doi: 10.1016/0006-291x(70)90747-3. [DOI] [PubMed] [Google Scholar]
- Cohen P., Burchell A., Foulkes J. G., Cohen P. T., Vanaman T. C., Nairn C. Identification of the Ca2+-dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase. FEBS Lett. 1978 Aug 15;92(2):287–293. doi: 10.1016/0014-5793(78)80772-8. [DOI] [PubMed] [Google Scholar]
- Coll-Garcia E., Gill J. R. Insulin release by isolated pancreatic islets of the mouse incubated in vitro. Diabetologia. 1969 Apr;5(2):61–66. doi: 10.1007/BF01211999. [DOI] [PubMed] [Google Scholar]
- DeLorenzo R. J., Freedman S. D., Yohe W. B., Maurer S. C. Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1838–1842. doi: 10.1073/pnas.76.4.1838. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gopinath R. M., Vincenzi F. F. Phosphodiesterase protein activator mimics red blood cell cytoplasmic activator of (Ca2+-Mg2+)ATPase. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1203–1209. doi: 10.1016/s0006-291x(77)80107-1. [DOI] [PubMed] [Google Scholar]
- Hidaka H., Yamaki T., Totsuka T., Asano M. Selective inhibitors of Ca2+-binding modulator of phosphodiesterase produce vascular relaxation and inhibit actin-myosin interaction. Mol Pharmacol. 1979 Jan;15(1):49–59. [PubMed] [Google Scholar]
- Hille B. Common mode of action of three agents that decrease the transient change in sodium permeability in nerves. Nature. 1966 Jun 18;210(5042):1220–1222. doi: 10.1038/2101220a0. [DOI] [PubMed] [Google Scholar]
- Klee C. B., Krinks M. H. Purification of cyclic 3',5'-nucleotide phosphodiesterase inhibitory protein by affinity chromatography on activator protein coupled to Sepharose. Biochemistry. 1978 Jan 10;17(1):120–126. doi: 10.1021/bi00594a017. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Levin R. M., Weiss B. Binding of trifluoperazine to the calcium-dependent activator of cyclic nucleotide phosphodiesterase. Mol Pharmacol. 1977 Jul;13(4):690–697. [PubMed] [Google Scholar]
- Marcum J. M., Dedman J. R., Brinkley B. R., Means A. R. Control of microtubule assembly-disassembly by calcium-dependent regulator protein. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3771–3775. doi: 10.1073/pnas.75.8.3771. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pipeleers D. G., Marichal M., Malaisse W. J. The stimulus-secretion coupling of glucose-induced insulin release. XV. Participation of cations in the recognition of glucose by the beta-cell. Endocrinology. 1973 Nov;93(5):1012–1018. doi: 10.1210/endo-93-5-1012. [DOI] [PubMed] [Google Scholar]
- Schubart U. K., Erlichman J., Fleischer N. The role of calmodulin in the regulation of protein phosphorylation and insulin release in hamster insulinoma cells. J Biol Chem. 1980 May 10;255(9):4120–4124. [PubMed] [Google Scholar]
- Schulman H., Greengard P. Ca2+-dependent protein phosphorylation system in membranes from various tissues, and its activation by "calcium-dependent regulator". Proc Natl Acad Sci U S A. 1978 Nov;75(11):5432–5436. doi: 10.1073/pnas.75.11.5432. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sieghart W., Theoharides T. C., Alper S. L., Douglas W. W., Greengard P. Calcium-dependent protein phosphorylation during secretion by exocytosis in the mast cell. Nature. 1978 Sep 28;275(5678):329–331. doi: 10.1038/275329a0. [DOI] [PubMed] [Google Scholar]
- Sugden M. C., Ashcroft S. J., Sugden P. H. Protein kinase activities in rat pancreatic islets of Langerhans. Biochem J. 1979 Apr 15;180(1):219–229. doi: 10.1042/bj1800219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sugden M. C., Christie M. R., Ashcroft S. J. Presence and possible role of calcium-dependent regulator (calmodulin) in rat islets of Langerhans. FEBS Lett. 1979 Sep 1;105(1):95–100. doi: 10.1016/0014-5793(79)80894-7. [DOI] [PubMed] [Google Scholar]
- Valverde I., Vandermeers A., Anjaneyulu R., Malaisse W. J. Calmodulin activation of adenylate cyclase in pancreatic islets. Science. 1979 Oct 12;206(4415):225–227. doi: 10.1126/science.225798. [DOI] [PubMed] [Google Scholar]
- Wang J. H., Teo T. S., Ho H. C., Stevens F. C. Bovine heart protein activator of cyclic nucleotide phosphodiesterase. Adv Cyclic Nucleotide Res. 1975;5:179–194. [PubMed] [Google Scholar]
- Weiss B., Levin R. M. Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Adv Cyclic Nucleotide Res. 1978;9:285–303. [PubMed] [Google Scholar]