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WS‐1 human fibroblasts contain distinct calcium and protein kinase C‐mediated pathways for activation of Na + /H + exchange: Contrasting effects of thrombin and PMA
Author(s) -
Hendey Bill,
Mamrack Mark D.
Publication year - 1991
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041460214
Subject(s) - staurosporine , thrombin , protein kinase c , bapta , chemistry , cytoplasm , calcium , amiloride , sodium–hydrogen antiporter , extracellular , microbiology and biotechnology , intracellular , intracellular ph , biochemistry , biophysics , kinase , sodium , biology , platelet , organic chemistry , immunology
PMA and thrombin were examined for their ability to activate Na + /H + exchange in growth‐arrested WS‐1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na + /H + exchanger. However, PMA and thrombin activated Na + /H + exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA‐induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H‐7 (100 μM) No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 μM) or the intracellular calcium‐chelator BAPTA. In contrast, the thrombin‐induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 μM H‐7. The thrombin‐induced activation of Na + /H + exchange was inhibited by 200 μM TMB8 or pretreatment with BAPTA. PMA caused translo‐cation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth‐arrested WS‐1 human fibroblasts. Unlike thrombin, PMA did not stimulate [ 3 H]‐thymidine incorporation in cells serum‐deprived for 48 hours. In addition, PMA inhibited thrombin‐induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na + /H + exchange by distinct pathways, but only the thrombin‐induced pathway correlates with a mitogenic response.