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
✍ Scribed by Bill Hendey; Mark D. Mamrack
- Publisher
- John Wiley and Sons
- Year
- 1991
- Tongue
- English
- Weight
- 938 KB
- Volume
- 146
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
PMA and thrombin were examined for their ability to activate Naf/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 NaC/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 pM). 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 pM) 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 p, M H-7. The thrombin-induced activation of Na+/H+ exchange was inhibited by 200 pM TMB8 or pretreatment with BAPTA. PMA caused translocation 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 I3HI-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 corre-WS-1 cells. a cell lates with a mitogenic response.
strain derived from human embrvonic skin, are growth arrested for 48 hours in serumfree media. The addition of thrombin to these quiescent fibroblasts is sufficient to elicit a mitogenic response (Hendey and Mamrack, 1988). Thrombin causes the rapid production of inositol phosphates a transient increase in intracellular free calcium (Ca2'+J, and a rise in pH (pH,) (Hendey and Mamrack, 1988;Hendey et al., 1989). The rapid rise in Ca2+i is primarily the result of release from internal stores, presumably due to the action of inositol-1,4,5-tris phosphate (IP3) (Hendey et al., 1989). The rise in calcium correlates with the activation of the Na+/H+ exchange and inhibition of. the thrombin-induced Ca2+i transient inhibits the rise in pH,.
How the Ca2+i transient induced by thrombin activates the Na+/H+ exchanger is unknown. Calcium could interact with the exchanger directly, or through a calmodulin-dependent pathway (Owen and Villereal, 1982). Since thrombin stimulates phosphoinositide breakdown in WS-1 cells (Hendey and Mamrack, 19881, another possible mode of activation of the Na+/H+ exchange would involve the Ca2+/phospholipid-
dependent family of protein kinases (PKC). Activation of PKC in a variety of cell types correlates with increased Na+/H+ exchange (Moolenaar et al., 1984; Moolenaar, 1986). The phosphorylation of the Na+/H+ exchanger either by a Ca2+icalmodulin-dependent protein kinase or PKC has been proposed as a mechanism for increasing the affinity of the exchanger for intra-