Specificity of inhibition of calcium- and calmodulin-dependent protein kinase by alloxan

Norling, Laura L.; Colca, Jerry R.; Brooks, Charles L.; Kloepper, Robert F.; McDaniel, Michael L.; Landt, Michael

doi:10.1016/0304-4165(84)90068-0

Cited by 11 publications

(8 citation statements)

References 21 publications

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“…Phosphodiesterase activity (nmol of cyclic AMP hydrolysed) in the presence of: quent homogenization and assay; and islets prepared from rats injected with alloxan also showed decreased Ca2+-calmodulin-dependent protein kinase activity (Norling et al, 1984). Ca2+-calmodulin-dependent protein kinase in brain extracts was also inhibited by alloxan, but the amount of activity extractable from brain was not diminished by administration of the drug in vivo (Norling et al, 1984), consistent with previous views on the mechanism of tissue specificity.…”

Section: Discussionsupporting

confidence: 86%

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans

Harrison¹,

Poje²,

Ročić³

et al. 1986

Biochemical Journal

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Dehydrouramil hydrate hydrochloride (DHU), a stable analogue of alloxan, inhibited the phosphorylation of an endogenous protein of Mr 53,000 catalysed by a Ca2+-calmodulin-dependent protein kinase in extracts of islets of Langerhans. The concentration of DHU required for 50% inhibition was 0.09 mM. DHU did not inhibit islet cyclic AMP-dependent protein kinase and caused only slight inhibition of Ca2+-phospholipid-dependent protein kinase. Inhibition of Ca2+-calmodulin-dependent protein kinase was neither prevented nor reversed by dithiothreitol. DHU did not affect the ability of calmodulin to activate cyclic AMP phosphodiesterase. In intact islets, pre-exposure to DHU impaired the insulin-secretory response to glucose and blocked the potentiatory effect on insulin secretion of forskolin, an activator of adenylate cyclase, and of tetradecanoylphorbol acetate (TPA), an activator of Ca2+-phospholipid-dependent protein kinase. The increase in islet cyclic AMP elicited by forskolin was not affected by DHU. The data are consistent with the hypothesis that protein phosphorylation catalysed by a Ca2+-calmodulin-dependent protein kinase may play a central role in the regulation of insulin secretion.

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Section: Discussionsupporting

confidence: 86%

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans

Harrison¹,

Poje²,

Ročić³

et al. 1986

Biochemical Journal

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“…The possibility that calmodulin per se acts on an unknown link of the exocytosis process cannot be excluded. In any event, the development of the digitonin-permeabilized islet system will allow detailed testing of these hypotheses under various conditions with inhibitors of the protein kinase activity such as calmodulin antagonists, diphenylhydantoin and alloxan (Landt et al, 1982;Norling et al, 1984;Colca et al, 1983b). The present studies demonstrate the usefulness of the digitonin-permeabilized cells in the study of protein phosphorylation and insulin release in intact cellular membranes.…”

Section: Discussionmentioning

confidence: 64%

Protein phosphorylation in permeabilized pancreatic islet cells

Colca¹,

Wolf²,

Comens³

et al. 1985

Biochemical Journal

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A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.

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“…We also tested polyglutamate (GLU~=70) and polyaspartate (ASP,=~0), both from Sigma, and we found them to be potent inhibitors of AED-induced exocytosis (see Table I). In addition we explored any possible effects of the protein kinase inhibitors diazepam (13) from Serva (Heidelberg, Federal Republic of Germany) and alloxan (directed against Ca 2+-CaM-dependent protein kinase [46]), as well as of the metalloprotease inhibitor phenanthroline (44) from Janssen Pharmaceutica (Beerse, Belgium). They were applied in concentrations up to 10 times above those indicated in the literature, combined with subsequent AED triggering.…”

Section: Inhibitor Experiments With Cellsmentioning

confidence: 99%

Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

Momayezi¹,

Lumpert²,

Kersken³

et al. 1987

The Journal of Cell Biology

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Specificity of inhibition of calcium- and calmodulin-dependent protein kinase by alloxan

Cited by 11 publications

References 21 publications

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans

Effects of dehydrouramil on protein phosphorylation and insulin secretion in rat islets of Langerhans

Protein phosphorylation in permeabilized pancreatic islet cells

Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

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