Alloxan inhibition of a Ca2+- and calmodulin-dependent protein kinase activity in pancreatic islets (original) (raw)
Related papers
Cell Calcium, 1994
Pancreatic islets contain an aiioxan sensitive, calcium and caimodutin dspendent protein kinase (Ccl&l-PK) which may play an important part in the ceil&r control of insulin secmtkm. We have studied this activity in Mets and the insulin secreting tumor ceil line RiN&f with particular interest in the changes in kinase activity that accompsny stimulation of secMon. initial expertments showed that the C&I-PK activity enriched in microsomai prepsrations from RIN ceils was similar to the islet ceil kinase in terms of apparent endogenous substrates, Ca2+ and caimoduiin dependence, and inactivation by alkxan. For studies of protein substrate specificity, tumor ceil CaM-PK was Mated from other klnase acti* and substantially purified by affinity chromatography with caimodui-. The major protein substrates of CaM-PK (54 kD and 57 kD) co-purified with the kinase acttvity, ming autophosphoryiation of subunits of the enzyme. Exogenous su& &rates phosphoryiated by these preparations included microtubuie-assoctated protein 2, synapsin, and giycogen synthase; this pattern of substrate utiiixation identified the kinase as the Type ii muitifunctionai kinase which has been ex@nsiveiy charactsrixed in brain. A poiycionai antibody to rat brain Cat@PK ii was employed to immunoprecipitate the kinase from RiNm5f ceils incubated with secretagogues to 'measure the effect of stimuktion of secretion on autophosphoryiation of CaM-PK (which mftects kinase activation). ~Giyceraldehyde (22 mM) and depolarizing concentrations of potassium increased autophosphor@+ tion and insulin secretion in a parallel fashion. Potassium stimuiated autwon was dose dependent and saturabie, and was increased to near maximal teveis at times as short as 1 min. These studies dsmonstrate that pancreatic Mets and MN&f ceils contain a Type II CaM-PK which is activated during the secretion process.
The Biochemical journal, 1983
A Ca2+-activated and calmodulin-dependent protein kinase activity which phosphorylates predominantly two endogenous proteins of 57kDa and 54kDa was found in a microsomal fraction from islet cells. Half-maximal activation of the protein kinase occurs at approx. 1.9 microM-Ca2+ and 4 micrograms of calmodulin/ml (250 nM) for phosphorylation of both protein substrates. Similar phosphoprotein bands (57kDa and 54kDa) were identified in intact islets that had been labelled with [32P]Pi. Islets prelabelled with [32P]Pi and incubated with 28 mM-glucose secreted significantly more insulin and had greater incorporation of radioactivity into the 54 kDa protein than did islets incubated under basal conditions in the presence of 5 mM-glucose. Thus the potential importance of the phosphorylation of these proteins in the regulation of insulin secretion is indicated both by activation of the protein kinase activity by physiological concentrations of free Ca2+ and by correlation of the phosphorylatio...
European Journal of Pharmacology, 1998
One component of the mechanism by which imidazoline compounds promote insulin secretion involves closure of ATP-sensitive K+ channels in the beta-cell plasma membrane. Recently, however, it has also been proposed that these compounds may exert important effects on more distal effector systems. In the present work, we have investigated the contribution played by protein kinases A and C to the insulin secretory responses of isolated rat islets of Langerhans treated with efaroxan and RX871024 (1-phenyl-2-(imidazolin-2-yl) benzimidazole). Removal of extracellular Ca2+ or blockade of voltage-sensitive Ca2+ channels prevented stimulation of insulin secretion by efaroxan, confirming a critical role for increased Ca2+ influx in the secretory response. By contrast, inhibition of protein kinases A or C failed to alter efaroxan-induced insulin secretion. RX871024 dose-dependently increased insulin secretion from cultured islets incubated with 20 mM glucose. This effect was unaffected by modulation of protein kinase C, but was significantly attenuated by a selective inhibitor of protein kinase A (Rp-cAMPs). Measurements of cAMP revealed that RX871024 increased the islet cAMP content by more than 3-fold; reaching values similar in magnitude to those elicited by 50 microM 3-isobutyl-1-methyl xanthine. The results reveal that neither protein kinase A nor protein kinase C is obligatory for stimulation of insulin secretion by imidazolines. However, they suggest that a rise in cAMP may contribute to the amplified secretory response observed when cultured islets are incubated with RX871024 in the presence of a stimulatory glucose concentration.
Calmodulin-activated protein kinase activity in rat pancreatic islet cell membranes
Archives of Biochemistry and Biophysics, 1982
A calmodulin-activated protein kinase activity was identified in pancreatic islet cell membranes, which phosphorylated an endogenous protein of molecular weight 57,000 as determined by sodium dodecyl sulfate electrophoresis. Calmodulin activation of the protein kinase activity was dose dependent and saturable, with half-maximal activation occurring at 360 nM calmodulin. Trifluoperazine inhibited calmodulin activation of the protein kinase but had negligible effects on basal activity. The 50% inhibition occurred at 40 @M trifluoperazine. Subcellular fractionation of islet cells demonstrated that the calmodulin-activated activity was enriched in a light-particle fraction. This fraction was enriched in endopiasmic reticulum, but there was no correlation between protein kinase activity and endoplasmic reticulum marker enzyme activity among various subcellular fractions. The function of the kinase activity and the identity of the substrate are unknown, but the kinase activity may be involved in pancreatic stimulus-secretion coupling.
Molecular and Cellular Biochemistry, 1994
The effects of the diabetogenic agent, alloxan, on membrane potential, input resistance and electrical activity of normal mouse pancreatic [t-cells were studied. Tetraethylammonium (TEA), quinine and Glyburide were used to block K § and to elucidate the mechanisms underlying alloxan's effects on [~-cell membrane potential. Exposure of the islet to alloxan (75-100 ~tM) in the presence of glucose (I t raM), produced a rapid (15 see), transient inhibition of electrical activity, often accompanied by hyperpolarization of the membrane, and this was followed by recovery of the burst pattern. This early effect of alloxan was followed after approximately 15 rain by a complete inhibition of electrical activity and hyperpolarization. The inhibition accompanied by hyperpolarization was associated with a decrease in input resistance, indicating increased K+-conductance. Both the transient and delayed effects of alloxan were blocked by glucose (33 mM), quinine and glyburide but not by other conditions which induce continuous electrical activity such as elevated external [K § (10 mM), ouabain, K + removal, or TEA (20 mM). The transient inhibition induced by alloxan may be due to a direct competition with glucose transport/metabolism since it did not occur when alpha-keto isocaproic acid (KIC) was used to induce electrical activity. The delayed inhibition may reflect indirect effects of accumulation of this agent or its metabolites within the cell. Since both effects of alloxan are blocked by glyburide they appear to involve activation of the ATP-sensitive K+-channel (K-ATP).
Effect of different insulin secretagogues and blocking agents in islet cell Ca2+-ATPase activity
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988
Plasma raemla.ane Caz+.ATPase activity was meastwed in rat islet homogenates. The enzyme was inhibited, in a dose-del~ndent manner, when the islets were imdncuhated for 5 mln wilh differex~t concentrations of glucose (2 to 16 raM). This inklhifion disappeared almost enl~rely alter 15 m[n inenbatinn, regardless of the glucose concentration in the medium. Simuitaneotm m,easuRment of insulin in the medium revealed a slimulatory effect of glucose upon insulin secrettom The CaZ+-A'rPase activity was also inhibited whe~ the islets were pt-eincubaeed for 3 mill with other sfnallainrs of insulin secretitm such as glldazide (76 aM), tolbutamide (1-.~ mM), glucagon (1A FM)+ theophylline (10 raM) and ketoisocapmic acid (15 raM). Conversely, the aefivlty of the enz,jme was significantly enhanced when the islets were preinanbated with file Insulin seerelinn blocker, somatost~n (1.4 ~tM). Neither giueose nor any el the other substan~ tested when added diree'dy to the eazyme assay medinm m~llfled significantly the Cs~+-A'I'P"~,e aetivily measm~ in the islet homogenates. The~, remits would mggest that the activity of the islet plasma membrane is modulated by one or move ef the intracelinlar metabolites pmdmed when the islets are challenged by the insnlin stlmulatm' or I~ddng agents.
Journal of Diabetes Mellitus, 2015
Background: Catalase deficiency (acatalasemia) is sensitive to alloxan, and the administration to acatalasemic mice develops hyperglycemia under mild conditions. However, the mechanism is still poorly understood. Methods: Alloxan was used to induce the oxidative stress and intraperitoneally administered to acatalasemic and normal mice. The blood samples of these mice after 1, 3, 5 and 7 days were examined. The pancreatic islets 7 days after alloxan administration were isolated, and the insulin released under 3 mM and 20 mM glucose was examined. Results: After alloxan administration, increase of oxidative markers in blood and pancreatic apoptosis in acatalasemic mice were observed immediately. Insulin in blood was lowered after 3 days, and the insulin in acatalasemic mice was lower than that in normal mice. Hyperglycemia in the acatalasemic mice was observed after 3 days. The pancreatic islets after 7 days were isolated. A reduction of the insulin released from the islets under glucose stimulation was observed. The stimulation indexes of the normal and acatalasemic mice were 1.4 ± 0.6 and 0.7 ± 0.3, respectively. Conclusions: Alloxan induced a deterioration of glucose-dependent insulin secretion ability from the islets, and the deterioration mostly contributed to hyperglycemia, rather than apoptosis.