Activation of Na+/H+ exchange in cultured fibroblasts: synergism and antagonism between phorbol ester, Ca2+ ionophore, and growth factors.

Abstract

The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H' exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H' exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for activating Na+/H+ exchange. However, in contrast to observations in HSWP cells, we were able to activate the Na+/H+ exchanger in mouse 3T3 and human WI-38 cells with PMA alone, suggesting that there is some diversity in the mechanism for activation of Na+/H+ exchange in different types of fibroblasts.Fibroblasts grown in cell culture can be arrested in the Go/Gj phase of the cell cycle by depriving them of growth factors. The re-addition of serum or purified growth factors to the culture medium results in an ordered sequence of biochemical events that lead eventually to an increased cell proliferation. Since activation of Na+/H+ exchange is one of the important early responses ofcultured fibroblasts to mitogenic stimulation (1-5), it is important to understand the mechanism for this activation. Our previous work suggests that in cultured human fibroblasts (HSWP cells) a rise in intracellular Ca2l activity is a necessary step in this process (2, 6, 7).Early studies from our laboratory, utilizing inhibitors of calmodulin (6,8), suggested that the activation of Na+/H+ exchange in HSWP cells was a calmodulin-dependent event.However, subsequent to that study it became apparent that most ofthe calmodulin inhibitors used also can inhibit protein kinase C (9). In addition, with the recent discovery that the site of action of tumor promoters is protein kinase C (9), the early observation that the tumor promoter phorbol 12-myristate 13-acetate (PMA) would stimulate Na influx in 3T3 cells (10) suggested that protein kinase C may be involved in the activation of Na+/H+ exchange. In fact, a recent report (11) suggested that PMA stimulation of Na+/H+ exchange occurs in the absence of a rise in Ca2' activity, which led the authors to suggest that a rise in Ca2' is not a necessary event in fibroblasts for the mitogen activation of Na+/H+ exch...

Cytoplasmic Domain of the Ubiquitous Na+/H+ Exchanger NHE1 Can Confer Ca2+ Responsiveness to the Apical Isoform NHE3

“…The pH i sensitivity of NHE3 is much higher than that of NHE1. This was confirmed by our finding that cells expressing NHE3 possess a much 3 T. Ikeda, unpublished observation. …”

“…Alternatively, it may be located in the homologous NH terminus of the cytoplasmic domain that was important for the maintenance of pH i sensitivity in NHE1 (14). 3 According to our recent study, the cytoplasmic tail (amino acids 659 -815) of NHE1 is not involved in the Ca 2ϩ regulation of NHE1 (7). 3 Identification of this acceptor site(s) would lead to a better understanding of the molecular entity of the H ϩ -modifier site.…”

“…3 According to our recent study, the cytoplasmic tail (amino acids 659 -815) of NHE1 is not involved in the Ca 2ϩ regulation of NHE1 (7). 3 Identification of this acceptor site(s) would lead to a better understanding of the molecular entity of the H ϩ -modifier site. In unstimulated cells expressing NHE1, NHE3, and their chimeras, we observed marked differences in the pH i dependence of 22 …”

“…The electroneutral plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) has been shown to be one of the targets regulated by intracellular Ca 2ϩ (3)(4)(5)(6)(7)(8). NHE1 (9) is a ubiquitous amiloride-sensitive transporter that regulates pH i and cell volume (10,11), and its structure-function relationship has been studied extensively (7,(12)(13)(14)(15)(16)(17).…”

Modulation of cytosolic protein kinase C activity by ferricyanide: priming event seems transmembrane redox signalling

“…This leads to the idea that the Na+/H+ antiporter is a strong candidate for phosphorylation by protein kinase C, although it has yet to be demonstrated. Notwithstanding, there are indications that in certain cells activation of protein kinase C [18] is not sufficient to activate the Na+/H+ antiporter. In the same report it is thought that protein kinase C inhibits some unidentified signal responsible for Na+/H+ activation.…”