Effect of pH upon Ca(2+)-ATPase activity of rat pancreatic islets: its possible contribution to the inhibitory effect of different insulin secretagogues.

This work was undertaken in an attempt to elucidate the possible mechanism by which insulin secretagogues produce a fast and transient drop in the Ca(2+)-ATPase activity of the pancreatic islet membrane. For this purpose, the enzyme activity was measured in either homogenates or partially purified membranes of islets previously incubated under different experimental conditions. Ca(2+)-ATPase activity measured in homogenates of islets preincubated with 8 mM glucose decreased significantly compared to control islets incubated with 2.8 mM glucose. The inhibition was also observed when the enzyme activity was measured in homogenates of islets preincubated with 2.8 mM glucose plus 20 mM propionic acid as well as with glucose 2.8 mM in a buffer equilibrated with a gas mixture of O2 and either 12% or 30% CO2. Ca(2+)-ATPase activity decreased significantly in partially purified islet membranes preincubated for 3 min with glucose (2 and 8 mM), 15 mM KCl and 2 mM tolbutamide. These substances did not affect the Ca(2+)-ATPase activity when added directly to the enzyme assay medium. The enzyme activity also decreased when measured in membranes preincubated at pH 6.5. The addition of 1 mM ATP to the preincubation medium protected the Ca(2+)-ATPase activity from the inhibition induced by glucose, KCl and tolbutamide as well as from the one produced by acidic pH in the medium. On account of these results, we suggest that insulin secretagogues, as well as either acidification of B-cell cytosol or islet membrane incubation medium, produce changes at the islet membrane level which promote a decrease in the Ca(2+)-ATPase activity. A shift of the E1-E2 equilibrium of the phosphoenzyme towards E1 may account for such decreased activity. Changes in Ca(2+)-ATPase activity could either favour the decrease or the increase in the cytosolic concentration of Ca2+ in B-cells. Therefore, negative and positive modulation of its activity might allow Ca(2+)-ATPase to play a role in the switch-on and -off mechanism for intracellular Ca2+ signal regulation of B-cell secretion of insulin.

Correlation between Ca(2+)-ATPase activity of rat islet cells and insulin secretion.

Using medium with a low ionic strength, a low concentration of Ca2+ and Mg2+ and devoid of K+, we have measured Ca(2+)-ATPase activity in the homogenates of rat islets preincubated for 3 min with several hormones in the presence of 3.3 mmol glucose/l. Insulin secretion was also measured in islets incubated for 5 min under identical experimental conditions. Islets preincubated with glucose (3.3 mmol/l) and glucagon (1.4 mumol/l) plus theophylline (10 mmol/l), ACTH (0.11 nmol/l), bovine GH (0.46 mumol/l), prolactin (0.2 mumol/l) or tri-iodothyronine (1.0 nmol/l) have significantly lower Ca(2+)-ATPase activity than those preincubated with only 3.3 mmol glucose/l. All these hormones increased the release of insulin significantly. Dexamethasone (0.1 mumol/l) and somatostatin (1.2 mumol/l) enhanced the Ca(2+)-ATPase activity while adrenaline (10 mumol/l) did not produce any significant effect on the activity of the enzyme. These hormones decreased the release of insulin significantly. These results demonstrated that islet Ca(2+)-ATPase activity was modulated by the hormones tested. Their inhibitory or enhancing effect seemed to be related to their effect on insulin secretion; i.e. those which stimulated the secretion of insulin inhibited the activity of the enzyme and vice versa. Hence, their effect on insulin secretion may be due, in part, to their effect on enzyme activity and consequently on the concentration of cytosolic Ca2+. These results reinforce the assumption that Ca(2+)-ATPase activity participates in the physiological regulation of insulin secretion, being one of the cellular targets for several agents which affect this process.

Effect of different stimulators and a blocker of insulin release on islet Na+, K+-ATPase activity.

The effect of several insulin secretagogues and a blocker upon islet Na+, K+-ATPase activity was studied using rat islet homogenates. None of the agents tested modified the enzyme activity when added directly to the enzyme assay. Activity of Na+, K+-ATPase measured in islets preincubated during 3 min with glucose 3.3, 8 or 16.6 mM, as well as with 15 mM KIC or 1.2 microM somatostatin, did not significantly change. The presence of glucagon (1.4 microM) plus theophylline (10 mM) in the preincubation medium significantly enhanced activity while tolbutamide (1.48 mM) or gliclazide (76 microM) significantly decreased such activity. These results suggest that Na+, K+-ATPase activity would not be a main common step involved in the mechanism by which glucose, KIC, glucagon + theophylline and somatostatin exert their effect on insulin secretion. Conversely, the enzyme might contribute to the stimulatory effect of gliclazide and tolbutamide on insulin release. Such effect would be secondary to the release of some cellular mediator rather than a direct action of these compounds on the enzyme. Such effect would later favor a rise in the cytosolic concentration of calcium which might trigger the release of insulin.

Characteristics of a Ca2+-ATPase activity measured in islet homogenates.

Ca2+-ATPase activity was measured in rat islet homogenates, in a medium of low ionic strength containing a low concentration of Ca2+ and Mg2+ and devoid of K+. The enzyme activity was highly sensitive to inhibition by compound 48/80 (a calmodulin inhibitor), stimulated by 120 nM calmodulin and slightly affected by 10 mM NaN3. The addition of Mg2+ to the assay medium promotes the disappearance of apparent Ca2+-ATPase activity. Ouabain (0.1 mM) did not modify this ATPase activity. The enzyme showed two kinetic components for Ca2+ as well as for ATP: one with high apparent affinity and low maximum velocity and the other with low apparent affinity and high maximum velocity. Incubation of islet homogenates in this assay medium with [gamma-32P]ATP in the presence of proteolytic inhibitors, results in the appearance of a single labelled band of 130 kDa, identified by gel electrophoresis. The incorporation of 32P into this band was similar in the presence of either 2.8 or 50 microM Ca2+ and susceptible to hydroxylamine attack. The results indicate that, under the conditions described above, the Ca2+-ATPase activity evidenced in the islet homogenates had characteristics resembling those of the enzyme which catalyzes the outward Ca2+ transport. On the other hand, the method could provide a useful tool to test the effect of different agents which affect insulin secretion upon the islet plasma membrane Ca2+-ATPase activity.

Effect of different insulin secretagogues and blocking agents on islet cell Ca2+-ATPase activity.

Plasma membrane Ca2+-ATPase activity was measured in rat islet homogenates. The enzyme was inhibited, in a dose-dependent manner, when the islets were preincubated for 5 min with different concentrations of glucose (2 to 16 mM). This inhibition disappeared almost entirely after 15 min incubation, regardless of the glucose concentration in the medium. Simultaneous measurement of insulin in the medium revealed a stimulatory effect of glucose upon insulin secretion. The Ca2+-ATPase activity was also inhibited when the islets were preincubated for 3 min with other stimulators of insulin secretion such as gliclazide (76 microM), tolbutamide (1.5 mM), glucagon (1.4 microM) + theophylline (10 mM) and ketoisocaproic acid (15 mM). Conversely, the activity of the enzyme was significantly enhanced when the islets were preincubated briefly with the insulin secretion blocker, somatostatin (1.4 microM). Neither glucose nor any of the other substances tested when added directly to the enzyme assay medium modified significantly the Ca2+-ATPase activity measured in the islet homogenates. These results would suggest that the activity of the islet plasma membrane is modulated by one or more of the intracellular metabolites produced when the islets are challenged by the insulin stimulator or blocking agents.