Evidence for Mechanistic Alterations of Ca2

Altered cytosolic Ca 2 + is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca 2 + regulation. Using human lymphocytes, we studied cytosolic calcium ( Ca i ) and various Ca 2 + transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca 2 + -specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca 2 + signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca 2 + -ATPase (SERCA), was used to study Ca 2 + - store dependent Ca 2 + fluxes. Significant (P < 0.05) elevation of basal Ca i levels was observed in lymphocytes from diabetic subjects. Ca i levels were positively correlated with fasting, plasma glucose and HbAlc. There was also a significant (P < 0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca 2 + influx (as measured both by Fluo-3 fliorescence and C 45 a assays) as a consequence of of thapsigargin-mediated Ca 2 + store depletion. Upon addition of Mn 2 + (a surrogate of Ca 2 + ), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P < 0.05) difference in the Na + / Ca 2 + exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca 2 + sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca 2 + entry. We conclude that cellular Ca 2 + accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na + / Ca 2 + exchange and (3) increased Ca 2 + influx across the plasma membrane.

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