Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.

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1 Previously we have described a monovalent cation (MC) current that could be unmasked by the removal of extracellular divalent cations in vascular smooth muscle cells (SMC) and cardiac myocytes, but speci®c and potent inhibitors of MC current have not been found, and the mechanism of its intracellular regulation remains obscure. 2 Here we show that small MC current is present in intact cells and could be dramatically upregulated during cell dialysis. MC current in dialyzed cells strongly resembled monovalent cation current attributed to Ca 2+ release-activated Ca 2+ -selective (CRAC) channels, but its activation did not require depletion of Ca 2+ stores, and was observed when the cells were dialyzed with, or without BAPTA. 3 Intracellular free Mg 2+ inhibits MC current with K d =250 mM. 4 Extracellular (but not intracellular) spermine eectively blocked MC current with K d =3 ± 10 mM, while store-operated cations (SOC) channels and capacitative Ca 2+ in¯ux were not aected. 5 Spermine eectively inhibited MC current-induced SMC depolarization, and prevented Ca 2+ paradox-induced vascular contracture. 6 Both, MC and SOC currents were inhibited by 2-aminoethoxydiphenyl borate (2-APB). 7 It is concluded that MC current could be regulated by intracellular Mg 2+, and low concentrations of extracellular spermine could be used to discriminate it from SOC current, and to assess its role in cellular function.

Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1-5 microM) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 microM of adenosine 3'-phosphate 5'-phosphosulfate, a selective P(2)Y(1) receptor antagonist. ATP alone did not cause release of intracellular Ca(2+), but produced a significant Ca(2+) response in the presence of norepinephrine. In contrast, the P(2)X(1) receptor agonists P(1),P(6)-diadenosine-5' hexophosphate and alpha,beta-methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 microM. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress.