Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogen c stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [³H]‐thymidine. We have determined previously that pretreatment with the tumor‐promoting phorbol ester 12–0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) inhibits mitogen‐stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen‐stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen‐stimulated inositol phosphate release occurs at 1–2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate‐stimulated inositol phosphate release and Na influx. In addition, Orthovanadate was determined to increase intra‐cellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura‐2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus‐mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA‐mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [³H]‐thymidine. These results are contrasted with those obtained in WI‐38 cells. A cell‐type in which (1) orthovanadate does not stimulate inositol phosphate release, (2) TPA has minimal or no inhibitory effect on early growth factor induced cellular responses (i.e., inositol phosphate release, intracellular Ca mobilization, activation of Na/H exchange), (3) TPA stimulates Na/H exchanger activity, without activating inositol phosphate release, and (4) TPA promotes (unlike in HSWP cells) the incorporation of [³H]‐thymidine.