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Previous reports have suggested that phorbol esters can decrease the affinity of epidermal growth factor (EGF) for its cellular receptors. Investigations of the consequences of the interaction between phorbol esters and EGF, however, have been limited to EGFstimulated Na/H exchange in A431 cells (Whitely, B., D. Cassel, Y.-X. Zuang, and U Glaser, 1984, I. Cell Biol., 99:1162-t 166). In the present study, the effect of the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) on EGF-stimulated ion transport and DNA synthesis was determined in cultured vascular smooth muscle cells (A7r5). It was found that TPA stimulated Na/H exchange when added alone (half-maximal stimulatory concentration, 25 nM). However, when cells were pretreated with TPA and then challenged with EGF, TPA significantly inhibited EGF-stimulated Na/H exchange (78%; half-maximal inhibition [Kd at 2.5 nM). Subsequently the effects of TPA on Na/K/CI co-transport were measured. TPA was observed to inhibit Na/KJCI co-transport (half-maximal inhibitory concentration, 50 nM) and also to inhibit EGF-stimulated Na/KICI co-transport (100%; K~ at 5 nM). Finally, the effects of TPA on DNA synthesis were assessed. TPA had a modest stimulatory effect on DNA synthesis (half-maximal stimulatory concentration, 6 nM), but had a significant inhibitory effect on EGFstimulated DNA synthesis (56%; K~ at 5 nM). These findings suggest that the inhibitory effect of TPA on EGF-receptor functions goes beyond previously reported effects on Na/H exchange in A431 cells and extends to EGF-stimulation of Na/K/CI co-transport and DNA synthesis in vascular smooth muscle cells.The tumor-promoting phorbol esters have recently been demonstrated to exhibit a wide range of biological effects. In this regard, the phorbol esters have been shown to stimulate Na/ H exchange in human epidermoid carcinoma cells A431 (1), in the human leukemic cell line HL-60 (2), in the pre-Blymphocyte cell line 70Z/3 (3), in Swiss 3T3 cells (4), and in HeLa cells, human flbroblasts, and N IE-115 neuroblastoma cells (5). Furthermore, phorbol esters have been found to inhibit Na/K/C1 co-transport in BALB/c-3T3 preadipose cells (6). In addition to these effects upon cellular ion fluxes, many laboratories have observed that phorbol esters are mitogenic for a variety of cell types (7,8).Although the mechanism by which phorbol esters have these effects is not clear, it is known that phorbol esters bind to a specific high affinity membrane receptor (9, 10). This receptor has been postulated to be the calcium-and phospholipid-dependent protein kinase known as protein kinase C (11-12). At present, the physiological substrate for protein kinase C has not been identified; however, recent reports from several laboratories suggest that the epidermal growth factor (EGF) ~ receptor may be a prominent substrate (13-16). These recent observations were not entirely unexpected as previous work by Shoyab et al. indicated that phorbol ester binding to cells resulted in a decreased affinity of EGF for its recept...

Previous studies have indicated that intracellular Ca2+ is involved in fetal bovine serum (FBS)- or growth factor (GF)-stimulated Na+ influx in human foreskin fibroblasts (HSWP). In the present study, 45Ca2+ efflux from serum-deprived HSWP cells was measured in response to 10% FBS or GF [lys-bradykinin, vasopressin, epidermal growth factor, and insulin]. Efflux data were analyzed using a computer program and the best fit indicated the presence of three Ca2+ compartments: a compartment (C1) with a very fast turnover rate, one (C2) with a fast turnover rate, and one (C3) with a slow turnover rate. When serum-deprived cells were treated with 10% FBS, efflux from C2 and C3 increased significantly (p less than 0.05). Similar effects on efflux were observed when serum-deprived cells were treated with individual GFs. Combination of the four GFs produced a higher stimulation than any single factor and a response that was equal to that of FBS. On the other hand, when cells were serum-treated in the presence of the intracellular Ca2+ antagonist, B-(N-N,diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), 45Ca2+ efflux from C2 was substantially reduced. Finally, when cells were treated with the Na+ transport inhibitor amiloride, there was no significant effect on serum-stimulated Ca2+ efflux. These results are consistent with a FBS- or GF-induced mobilization of Ca2+ that can be blocked by intracellular Ca2+ antagonists, and support the hypothesis that the action of these agents on Na+ influx may be via their effects on intracellular Ca2+.