Inhibition by calyculin A and okadaic acid of the Ca2+ release-activated Ca2+ entry pathway in rat basophilic leukemia cells: Evidence for regulation by Type 1/2A serine/threonine phosphatase activity

摘要

Using a combination of fluorescence measurements of intracellular Ca2+ ion concentration ([Ca2+](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca2+ entry stimulated by activation of the Ca2+ release-activated Ca2+ (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca2+](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca2+ rise associated with activation of CRAC uptake and inhibited Mn2+ entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca2+ stores is accompanied by a large depolarisation which is absolutely dependent upon Ca2+ entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid. (c) 2005 Elsevier B.V. All rights reserved.