EVIDENCE FOR A PHORBOL ESTER-INSENSITIVE PHOSPHORYLATION STEP IN CAPACITATIVE CALCIUM-ENTRY IN RAT THYMIC LYMPHOCYTES

Experiments were undertaken to investigate the regulation of capacitat ive Ca2+ entry by phorbol ester-sensitive protein kinase C and serine( threonine protein phosphatase activity, The thapsigargin-activated Ca2 + entry pathway was probed in control cells and cells treated with pho sphatase type 1/2A inhibitors, okadaic acid and calyculin A, or with t he phorbol ester, phorbol 12-myristate 13-acetate. The permeability st ate of this pathway was monitored in the presence or absence of these agents using fluorometric measurements of intracellular Ca2+ concentra tion, unidirectional Mn2+ entry, and membrane potential and unidirecti onal measurements of Ca2+ uptake using Ca-45(2+). The results of these studies demonstrate that modification of the phosphorylation state of target protein(s) on serine/threonine amino acid residues by inhibiti on of phosphatase type 1/2A inhibits the capacitative Ca2+ entry pathw ay in rat thymic lymphocytes, Importantly, the capacitative Ca2+ entry pathway in rat thymic lymphocytes is not modulated by activation of p horbol ester-sensitive protein kinase C.