Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen-stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells.

Abstract

Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H ÷ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H ÷ exchange system of A431 cells. Stimulation of Na+/H + exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promotors also do not modulate the activation of Na+/ H ÷ exchange. By contrast, the stimulation of Na+/H ÷ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H + antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H + exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca 2+. The inhibition BY PMA of EGF-promoted Na+/H + exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H + exchange by polypeptide growth factors.Stimulation of an amiloride-sensitive Na ÷ uptake is a ubiquitous consequence of the addition of polypeptide growth factors (mitogens) to cells. This increased Na ÷ uptake is primarily due to activation ofNa+/H + exchange (1-7). Recent studies have demonstrated that stimulation of Na+/H + exchange by mitogens leads to an increase in cytoplasmic pH (8)(9)(10)(11). This cytoplasmic alkalinization is likely to have a permissive role in the mitogenic response of cells to growth factors (12).Phorbol diester derivatives that are potent tumor promoters in the mouse skin model system have recently been found to exert a wide variety of biological responses in cells. Phorbol 12-myristate 13-acetate (PMA),~ the most potent compound from this series, is mitogenic for a number of fibroblasts, acting synergistically with growth factors (see reference 13 and references therein), while in other cells PMA induces differentiation (for a recent review, see reference 14). Biochemical investigations suggest that the Ca 2+ and phospholipid-dependent protein kinase (protein kinase C) is the major i Abbreviations used in this paper. EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate. target for PMA action. PMA is a potent protein kinase C activator in vitro, apparently substituting for the natural activator diacylglycerol, which is produced upon hormonal stimulation of phosphatidylinositol turnover (reviewed in reference 15). Binding studies ( 16-18) suggest that protein kinase C is also the cellular phorbol diester "receptor." One of the substrates of activated protein kinase C is the epidermal growth factor (EGF) receptor. Activated protein kinase C catalyzes the phosphor...

Effect of catecholamines on Na/H exchange in vascular smooth muscle cells.

“…Previous studies by Owen (14) and by Whiteley et al (27) have examined the relationship between growth factors and the phorbol ester, TPA, in activating Na/H exchange. These experiments suggested that while TPA alone stimulated Na/H exchange, when TPA was added together with epidermal growth factor it caused inhibition of epidermal growth factor-stimulated Na/H exchange.…”

Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.

“…Fetal calf serum may activate protein kinase C (Moolenaar et al 1981;1983) and so desensitization of receptors may occur which alters the contractile response. TPA may inhibit diacylglycerol formation by epidermal growth factor (Smith, Losonczy, Sahai, Pannerselvam, Fehnel & Salomon, 1983) and TPA may attenuate the stimulation of Na+-H+ exchange by epidermal growth factor (Whiteley, Cassel, Zhuang & Glaser, 1984). Capogrossi et at.…”

Signal transduction by the epidermal growth factor receptor after functional desensitization of the receptor tyrosine protein kinase activity.

“…Associated with this acute desensitization of EGF receptor function is an inhibition of signal transduction by the EGF receptor. EGFstimulated phosphatidylinositol turnover (12)(13)(14)(15), Na+/H' exchange (12,17), and Ca2+ fluxes (12,16) (1)(2)(3)(4)(5)(6)(7)(8)(12)(13)(14)(15)(16)(17). An hypothesis that can account for the apparent desensitization of the EGF receptor caused by PMA is that the phosphorylation of the receptor at Thr654 by protein kinase C causes an inhibition of the EGF receptor tyrosine protein kinase activity (3,4,6,10).…”

“…Phorbol ester also inhibits the increase in cytosolic Ca2+ caused by EGF without affecting basal cytosolic Ca2+ levels (12, 16). Furthermore, the EGF-stimulated Na+/H+ exchange across the plasma membrane is inhibited by phorbol ester (12,17).…”

“…Phorbol ester also inhibits the increase in cytosolic Ca2+ caused by EGF without affecting basal cytosolic Ca2+ levels (12, 16). Furthermore, the EGF-stimulated Na+/H+ exchange across the plasma membrane is inhibited by phorbol ester (12,17).Together, these data indicate that protein kinase C causes an acute desensitization of the function of the EGF receptor.Recently we described a protein kinase activity in cell homogenates that phosphorylates the EGF receptor at threonine-669 (9). An assay for this protein kinase activity was developed using a synthetic peptide substrate corresponding to residues 663-681 of the EGF receptor (peptide T669).…”