Ha-rus Activates the Na+/H+ Antiporter by a Protein Kinase C-independent Mechanism"

In quiescent Ha-ras-transfected NIH 3T3 cells, ad- dition of serum growth factors, bombesin or 12-0-tetradecanoylphorbol-13-acetate (TPA) leads to a di- methylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent in- hibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independ- ent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras- induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H' antiporter by an as yet unknown protein kinase C-independent mechanism.

In previous publications it has been demonstrated that expression of a transforming Ha-ras oncogene, not, however, of the corresponding protooncogene, leads to a growth factorindependent activation of the Na+/H+ antiporter in NIH 3T3 fibroblasts (1,2). Similar effects have been observed by others following microinjection of ~2 1 ' "~ (3). Stimulation of the Na+/ H' antiporter and the resulting cytosolic alkalinization can be observed under the influence of many growth factors and mitogenic agents and is considered as either essential or at least permissive for the proliferative response (for reviews, see Refs. 4 and 5). In a variety of systems, the stimulation of the Na+/H+ antiporter is mediated via protein kinase C (4,5). This seems to be the case for growth factors which act through a stimulation of a phospholipase C, catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. The primary effect of diacylglycerol is considered to be the activation of protein kinase C (for a review, see Ref. 6). In the system described here, expression of the transforming Ha-ras leads to an increased formation of inositol phosphates (2, 7) and phosphatidic acid (8) suggesting an elevated generation of diacylglycerol which in turn should stimulate protein kinase C. Direct evidence for an activation of protein kinase C in * This work was supported in part by a grant of the Bundesministerium fur Forschung und Technologie of the Federal Republic of Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ll To whom correspondence should be addressed Institute of Medical Chemistry and Biochemistry, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria. ras-transformed cells has been described (9, 10). Thus, available evidence is consistent with the supposition that the stimulation of the Na+/H+ antiporter by the transforming Ha-ras is mediated through protein kinase C. However, the data presented here demonstrate that the Ha-ras-induced activation of the Na+/H+ antiporter is insensitive to staurosporine, a potent inhibitor of protein kinase C (11) and occurs in protein kinase C-depleted cells with the same efficiency as in undepleted controls. Cell Culture-NIH 3T3 fibroblasts were transfected with the transforming human Ha-ras oncogene subjected to transcriptional regulation by glucocorticoids by in vitro recombination with the MMTV-LTR as described by Jaggi et at. (12). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) in the presence of 5% COZ. One day after plating, cells were made quiescent by incubation in DMEM containing 0.5% FCS for 24-48 h.

Materials
Determination of the Cytoplasmic pH-NIH 3T3 cells were grown on coverslips (9 X 18 mm). One day after plating, cells were growth arrested by incubating in low serum (0.5%) medium for at least 48 h.
At this time point average cell count per coverslip was 0.5-1 X lo6 cells. pHi (intracellular pH) was determined by fluorescence spectrophotometry employing BCECF. Fibroblasts attached to rectangular coverslips were washed in a HCOS-free HEPES-buffered saline (HBS) (140 mM NaC1, 5 mM KC1, 1 mM CaCI2, 0.5 mM MgCI2, 5.5 mM glucose, 20 mM HEPES/NaOH, pH 7.4). The cells were loaded with BCECF by incubation in HBS containing 2.5 p~ BCECF acetoxymethylester for 10 min at 37 "C and subsequent washing (two times) with HBS. For fluorescence measurements one coverslip supporting the cells was placed into a 1 X 1 X 3-cm quartz cuvette containing 2 ml of HBS at 37 "C. The coverslip in the cuvette was oriented at a 60" angle relative to the excitation beam, while 0.6 cm2 of the coverglass was illuminated. Fluorescence (excitation, 502 or 440 nm; emission, 530 nm) was detected with a SPEX (Edison, NJ) Fluorolog 2 spectrofluorometer (CM-1) equipped with two excitation monochrometers and a chopper system. The cytoplasmic pHi values were calculated from the ratio of the fluorescence intensities 1602nm/ 1 4 1 0~~. At the end of each experiment the ratio of the fluorescence intensities 1502nm/1440nm was calibrated to pH, using a nigericin calibration procedure as described (28) 140 mM KCl, 10 mM NaCI, 20 mM Hepes/KOH, 10 pg/ml nigericin.
Determination of Protein Kinase C Activity-Protein kinase C was partially purified from NIH 3T3 cells by employing DEAE-cellulose chromatography according to a minor modified method described by Walton et al. (29). Protein was eluted with a gradient from 0.0 to 0.4 M NaCl in elution buffer (20 mM Tris/HCl, 20 mM mercaptoethanol, 0.1 mM EGTA, 0.1 mM EDTA, 2% glycerol, pH 7.4).

RESULTS
Addition of dexamethasone to quiescent NIH 3T3 cells transfected with a MMTV-LTR Ha-ras construct leads to a rapid induction of LTR-ras-transcription. The accumulation of ~21'"" is detectable within 1 h after addition of the glucocorticoid and reaches a maximum after 24 h (12). Some biological and biochemical consequences of p21'"" induction in this system have been described in previous reports (1, 2, 7). As shown in Fig. 1, expression of Ha-ras causes a progressive cytosolic alkalinization which can be blocked by dimethylamiloride (DMA), an inhibitor of the Na+/H+ antiporter. A DMA-sensitive cytosolic alkalinization is also seen after addition of serum growth factors, bombesin or TPA to quiescent 3T3 cells (Fig. 3). The data indicate that Ha-ras  0.04,  0.07,0.09,0.11,0.12,0.14, 0.17,0.24, and 0.4 M. The figure shows the elution profile of protein kinase C activities from controls (0) or
In order to investigate the role of protein kinase C in intracellular alkalinization by Ha-ras, we employed staurosporine which has been shown to represent a powerful inhibitor of protein kinase C (11). The dose-effect relationship for the inhibition of total protein kinase C activity from NIH 3T3 cells by staurosporine is depicted in Fig. 2. Staurosporine (2.5 nM) depresses cytosolic alkalinization by serum growth factors bombesin or TPA (Fig. 3). The DMA-sensitive alkalinization by Ha-ras, however, is not affected by these staurosporine concentrations. As staurosporine had to be present all the time during the induction with dexamethasone ( i e . 20 h), we had to make sure that the inhibitor was still active after this period. Fig. 3 (last column) demonstrates that 20 h after administration of staurosporine, the pHi response to TPA is still completely blocked. The data from Fig. 3 demonstrate that the stimulation of the Na+/H+ antiporter by serum growth factors bombesin and TPA is mediated by a staurosporine-sensitive kinase whereas the Ha-ras-induced activation of the Na+/H+ antiporter occurs by a staurosporine-insensitive mechanism.
Besides protein kinase C, staurosporine may affect other protein kinases (13,14). Protein kinase C-depleted cells were employed in order to obtain more direct information about the role of protein kinase C in the activation of Na+/H+ antiporter by Ha-ras. It is well established that long term exposure to phorbol esters like TPA results in a depletion of the enzyme probably due to proteolytic degradation (15,16). Fig. 4 demonstrates that 24-h exposure to 0.25 pM TPA completely depresses total protein kinase C activity. Restimulation by TPA or addition of bombesin fail to elicit a pH response in protein kinase C-depleted cells (Fig. 5). The Harm-induced alkalinization, however, is not depressed in these cells (Fig. 5). It can be concluded therefore, that the stimulation of the Na+/H+ antiporter by Ha-ras is mediated by a protein kinase C-independent mechanism.

DISCUSSION
The data presented here demonstrate that the stimulation of the Na+/H+ antiporter by Ha-ras is insensitive to concentrations of the protein kinase C inhibitor staurosporine which eliminates the pH response to growth factors or TPA. Furthermore, the DMA-sensitive alkalinization by Ha-ras is not affected by protein kinase C depletion. Thus, the Ha-rasinduced stimulation of the Na+/H+ antiporter occurs by a protein kinase C-independent mechanism.
Protein kinase C-independent activation has been described for several systems including Swiss 3T3 cells stimulated by platelet-derived growth factor or epidermal growth factor + insulin (17,18), for lymphocytes following treatment with interferon, interleukin-la, or interleukin-2 (19)(20)(21) or for smooth muscle cells after exposure to thrombin, angiotensin 11, or serum (22)(23)(24). The detailed mechanism responsible for the activation of the Na+/H+ antiporter in these cases is still obscure. The observation reported here that the Ha-rasinduced activation of the antiporter occurs by such a protein kinase C-independent pathway opens novel aspects for early ras functions.
As outlined in the introduction, evidence obtained with the system employed here (2,7,8), as well as with similar systems studied elsewhere (9, IO), is consistent with a Ha-ras-induced activation of protein kinase C. An activation of protein kinase C has been shown to result in a stimulation of the Na+/H+ antiporter (for reviews, see Refs. 4 and 5). In view of these data, the results presented here, demonstrating that the Harm-induced activation of the antiporter does not involve protein kinase C come as a surprise. It should be emphasized, however, that the alterations in inositol phospholipid metabolism which may be responsible for the activation of protein kinase C by Ha-ras are not ras-specific phenomena but seem to reflect transformation-linked metabolic alterations which are produced by other oncogenes like v-mos as well (7,25). Obviously, Ha-ras activates an additional, still unknown mechanism to stimulate the Na//H+ antiporter. The elucidation of this mechanism may reveal early ras-initiated signals which have not been considered so far and which may be relevant for the stimulation of cellular replication or transformation by this oncogene.