Control of cytosolic calcium activity during low sodium exposure in cultured chick heart cells

Abstract

We investigated the roles of sodium-calcium exchange, sarcoplasmic reticulum, and mitochondria in Ca(i) homeostasis in cultured chick ventricular cells. Specifically, the influence of low sodium medium on contractile state, calcium fluxes, and cytosolic free [Ca] ([Ca](i)) was examined. [Ca](i) was measured using fura-2. Mean [Ca](i) in control medium was 126 ± 14 nM. Exposure of cells to sodium-free or sodium- and calcium-free medium (choline-substituted) resulted in contracture development, which returned toward the baseline level over 2-3 minutes. The Na(o)-free contracture was associated with a tenfold increase in [Ca](i) (1,280 ± 110 nM) followed by a gradual decrease to a level fourfold above control [Ca](i) (460 ± 58 nM). Na(o)- and Ca(o)-free contracture was associated with a fivefold increase in [Ca](i) (540 ± 52 nM) followed by a rapid decrease to below 80 nM. Sodium-free medium failed to produce an increase in [Ca](i) or contracture in cells preexposed to calcium-free medium, although caffeine, when subsequently added to sodium- and calcium-free medium, was able to elicit a transient increase in [Ca](i) and contracture. Brief, 5-second preperfusion of cells with La3+ (1 mM) or EGTA (1 mM) abolished the Na(o)-free contracture and the increase in [Ca](i). In the presence of 20 mM caffeine, removal of Na(o) resulted in minimal changes in the resting position of the cell although 45Ca uptake and [Ca](i) were increased in response to sodium-free medium; the subsequent decrease in [Ca](i) was greatly slowed. Addition of caffeine during the relaxation phase of the sodium-free contracture produced an additional transient contracture and transient increase in [Ca](i). Ryanodine (1 μM) abolished this effect of caffeine. Caffeine or ryanodine abolished Na(o)- and Ca-free contracture. CCCP (2 μM), a potent oxidative phosphorylation inhibitor, did not significantly affect calcium efflux rate. In the presence of 2 μM CCCP, removal of sodium resulted in an augmented contracture signal and a rise in [Ca](i), followed by a slow decrease. We conclude that removal of extracellular sodium enhances transsarcolemmal entry of calcium via sodium-calcium exchange, but this effect alone does not lead to the development of sodium-free contracture. Calcium displaceable by lanthanum or EGTA appears to contribute to Na(o)-free or Na(o)- and Ca(o)-free contracture. Studies using caffeine and ryanodine suggest that removal of Na(o) leads to release of calcium from the sarcoplasmic reticulum (presumably via calcium-induced calcium release). The released calcium appears to be taken up by both mitochondria and sarcoplasmic reticulum and is in part extruded by the ATP-dependent sarcolemmal calcium pump, causing spontaneous relaxation.