Effect of TPA on ion fluxes and DNA synthesis in vascular smooth muscle cells.

Abstract

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...

Stimulation of ribosomal protein S6 kinase activity by pp60v-src or by serum: dissociation from phorbol ester-stimulated activity.

“…Furthermore, they have demonstrated that PMA-stimulated phosphorylation of an Mr 80,000 protein is attenuated upon rechallenge with phorbol ester (12)(13)(14)(15). This is consistent with other results (16)(17)(18)(19)(20)(21)(22)(23) that indicate that prior treatment with phorbol ester attenuates many PMA-induced alterations upon subsequent exposure to the tumor promoter.…”

Angiogenin activates endothelial cell phospholipase C.

“…Well known targets of protein kinase C are an 80-kDa protein of unknown function (26), the epidermal growth factor receptor (27,28), and the Na+/H+ pump-either directly or indirectly (29)(30)(31)(32) (24,34), none of the aforementioned "classic" responses to protein kinase C activation has been observed (35). Nevertheless, interferon-dependent protein kinase activities have been noted with mouse 3T3-F442A and 3T3-C2 fibroblasts.…”

Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels.

“…Na+-H+ counter-transport and sodium-dependent bicarbonate influx A Na+-H+ counter-transport which is of importance for control of pHi has been demonstrated in the smooth muscles of vas deferens (Aickin, 1984) and in cultured smooth muscle cells from aorta (Smith & Brock, 1983;Owen, 1985;Little et al 1986). In the cultured smooth muscle cells the transport was reported to be active under 'resting' conditions inasmuch as EIPA, which inhibits the Na+-H+ countertransport, added to a HEPES-buffered solution caused a gradual acidification (Weissberg et al 1987;Hatori, Fine, Nakamura, Cragoe & Aviv, 1987).…”

“…These results suggest that a Na+-H+ exchange and an INTRODUCTION The control of the intracellular pH (pHi) is on a short-term basis dependent on different intracellular buffers, but is on a longer-term basis dependent on the effective transport of protons over the cell membrane. A Na+-H+ counter-transport system with the potential of controlling pHi by extruding protons in exchange for Na+ moving into the cell along its electrochemical gradient has been demonstrated in many different cells (for review see Roos & Boron, 1981;Mahnensmith & Aronson, 1985) and has also recently been demonstrated in vascular smooth muscles in culture (Smith & Brock, 1983;Owen, 1985; Little, Cragoe & Bobik, 1986;Weissberg, Little, Cragoe & Bobik, 1987). A Na+-HCO3--Cl-counter-transport has also been demonstrated in different cell types mainly from invertebrates (see Boron, 1983) but also in mammalian cells including human carcinoma cells (Rothenberg, Glaser, Schlesinger & Cassel, 1983) and hamster fibroblasts (l'Allemain, Paris & Pouyssegur, 1985).…”