Carbon monoxide inhibits capacitative calcium entry in human platelets

Abstract

The cytosolic calcium concentration in human platelets is elevated by several agonists via receptor-operated mechanisms involving both Ca 2+ release from intracellular stores and Ca 2+ entry. In order to get a mechanistic insight in the effect of carbon monoxide (CO)-containing solutions, this work examines the changes in [Ca 2+] i induced by 100 μM adenosine 5′diphosphate (ADP), 0.1 IU/ ml thrombin, 0.5 μM thapsigargin or 0.5 μM ionomycin in human platelets. In a saline solution bubbled with CO, the increase of [Ca 2+] i produced by thrombin was 72±4% of the response evoked in the control solution (CO-free) and the response elicited by ADP was 64±8% of the control. When a mixture of 5% CO/95% N 2 was used, the responses were 70±7% of control for thrombin and 79±6% of control for ADP. The mobilization of stored calcium produced by thrombin in a calcium-free solution and the increase of [Ca 2+] i produced by subsequent introduction of 1 mM extracellular calcium were both reduced in the presence of CO (82±6% and 78±5% of control, respectively). Similar reductions in the presence of CO were found when platelets were stimulated by ADP (62±8% and 60±8% for mobilization in calcium-free media and calcium entry, respectively). Although the change in [Ca 2+] i induced by ionomycin in the presence of extracellular calcium was almost the same in the absence or presence of CO (97±5% of control), the entry induced by depletion of reservoirs with the ionophore undergoes a significant reduction in a solution bubbled with CO (84±5% of control). In agreement with the concept that CO has a direct inhibitory effect on capacitative calcium entry, a reduction to 47±6% of control was obtained when sarco/endoplasmic reticulum ATPase was blocked by thapsigargin. Diverse mechanisms could be responsible for the effect of CO on calcium entry. On the one hand, a decrease in the calcium release from intracellular stores or an increase in the rate of its back-sequestration could occur, being the reduction of capacitative calcium entry an indirect consequence of a diminished emptying of reservoirs. On the other hand, CO could have a direct inhibitory effect on the pathway that produces the calcium entry. The decrease in the Ca 2+ signal in the presence of CO evoked by receptor-independent emptying of reservoirs indicates that a direct effect of CO on capacitative calcium entry participates in the antiaggregatory properties of CO. The proposal that CO inhibits directly store-operated calcium influx widens the potential mechanisms by which heme oxygenase regulates cell functions.