calyculin-a and Leukemia--Basophilic--Acute

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) 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.

Topics: Animals; Boron Compounds; Calcium; Calcium Signaling; Cations, Divalent; Cells, Cultured; Enzyme Inhibitors; Ion Transport; Leukemia, Basophilic, Acute; Manganese; Marine Toxins; Membrane Potentials; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rats; Thapsigargin

Effects of okadaic acid (OA) and calyculin-A (CL-A), selective inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), on the release of serotonin from the rat basophilic leukemia cell line (RBL-2H3) were investigated. Both OA and CL-A induced the long-lasting release of serotonin in an extracellular Ca(2+)-independent manner. CL-A did not increase intracellular Ca2+ concentration in the fura-2-loaded cells. CL-A was 100-fold more potent than OA in inducing the release, suggesting that PP1 is a dominant protein phosphatase in regulating RBL-2H3 cells. The CL-A-induced release of serotonin was completely inhibited by the nonselective protein kinase inhibitors, staurosporine and K-252a. CL-A induced phosphorylation of several cellular proteins in RBL-2H3 cells, which could be inhibited by staurosporine. These findings suggest that the release of serotonin is subject to tonic, Ca(2+)-independent, inhibition by PP1 in RBL-2H3 cells.

Topics: Alkaloids; Animals; Calcium; Ethers, Cyclic; Leukemia, Basophilic, Acute; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Rats; Serotonin; Staurosporine; Tumor Cells, Cultured