Stimulation of the T3-T Cell Receptor-associated Ca2’ Influx Enhances the Activity of the Na+/H+ Exchanger in a Leukemic Human T Cell Line*

Three monoclonal antibodies reactive with different structural domains of the T3-T cell receptor complex of the human T cell leukemia line, HPB-ALL, were previously shown to activate a membrane potential-sensitive, La3+-inh1bitable Ca2’ influx (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) CeZZ 40, 583-590). OKT3 (anti-T3), WT-31 (anti-re- ceptor constant region), and T40/25 (anti-receptor variable region) also enhance the activity of the Na+/ H+ exchanger in these cells. The associated rise in pHi was dependent on the presence of external Ca2+ and Na+, was inhibited by dimethylamiloride and La3+, and was maintained for at least 20 min. Phorbol esters, which are co-mitogenic in T cells and activate protein kinase C, also stimulated the exchanger, but by a mech- anism not requiring an elevation in cytoplasmic Ca”; the rise in pHi rapidly peaked and returned to baseline levels within 20 min. Pretreatment with phorbols pre-vented an increase in pHi by OKT3 although a transient additive effect was observed when the two were added simultaneously. Receptor function was maintained in the presence of phorbol esters as OKT3 still stimulated a Ca” influx. These data demonstrate the existence of two interdependent pathways to activate Na+/H+

Stimulation of undifferentiated or quiescent cells with growth factors and mitogens-is accompanied by rapid alterations in the flux rates of several critical cations, often leading to dramatic changes in the intracellular ionic milieu. Many cell systems have been described in which binding of a specific mitogen to its receptor initiates a series of membrane transport events which eventually lead to increases in cytosolic pH, [Na+], and free [Ca2+] (Rosoff and Cantley, 1983;Rosoff et al., 1984;Cantley et al., 1984;Rosoff and Cantley, 1985a;Rozengurt and Heppel, 1975;Rozengurt, 1981;Rothenburg et al., 1983;Mix et al., 1984;Moolenaar et al., 1981;Moolenaar et al., 1984a). In LPS1-treated 70Z/3 pre-B lymphocytes and lectin and antibody-stimulated T cells, these changes appear to be rate-limiting for either differentiation or proliferation to proceed (Rosoff and Cantley, 1983;Rosoff et al., 1984;Mastro and Smith, 1983;DeCoursey et al., 1984;Truneh et al., 1985;Weiss et aL, 1984, a and b).
The increase in [Ca2+]i may be accomplished by at least two independent pathways: the opening of a membrane calcium channel which allows extracellular Ca2+ to flow down its concentration gradient as occurs in IgE-stimulated, antigenprimed mast cells (Beaven et al., 1984) or release of Ca2+ from a microsomal storage pool via an inositol 1,4,5-trisphosphatemediated mechanism Joseph et al., 1984, a and b; . The latter pathway is coupled to the phosphatidylinositol turnover cycle which, in addition to inositol trisphosphate, generates diacylglycerol. This latter compound is essential for the activation of protein kinase C, the calcium-activated, phospholipid-dependent protein kinase (Berridge, 1984;Nishizuka, 1984). It is also possible that a mutual interaction between the two exists.
The rise in cell Na+ and pH is due to the increased activity of the Na+/H+ antiporter (Rosoff et al., 1984;Boron, 1984;PouyssBgur, 1983,1984). We and others have shown that tumor-promoting phorbol esters, which directly activate protein kinase C by substituting for endogenous diaclyglycerol, can stimulate Na+/H+ exchange by a calcium-independent pathway and can, in some cells, induce proliferation or differentiation (Rosoff et al., 1984;Moolenaar et al., 1984b;Besterman and Cuatrecacas, 1984;Burns and Rozengurt, 1983). The antiport may also be activated by a Ca2+-dependent pathway as demonstrated by Villereal and his colleagues (Owen and Villereal, 1982;Villereal, 1981). Quiescent T lymphocytes may be induced to proliferate by treatment with lectins, calcium ionophores, or anti-T3 antibodies, in the presence of monocytes or phorbol esters. These agents cause an increase in cell Ca2+ (Tsien et al., 1982;Weiss et al., 1984a;Metcalfe et al., 1980;Akerman and Anderson, 1984). Treatment with mitogenic lectins such as con A has been reported to cause an increase in pHi as well Gerson et al., 1982;Hesketh et al., 1985). Neither monocytes nor phorbol esters cause an increase in [Ca2+']i (Tsien et al., 1982). These agents apparently provide a second parallel signal for the induction of cell growth.
It has recently been reported from this laboratory and others that monoclonal antibodies directed against the T3-T cell receptor complex of human T lymphocytes induce a membrane potential-sensitive calcium influx that is La3+inhibitable (Oettgen et aE., 19%, Weiss et al., 1984, a and c;. In at least one case, treatment with these agents is also accompanied by an increase in phospha-tidylinositol turnover and release of CaZ+ from intracellular stores . These authors have suggested that resulting increased liberation of diacylglycerol activates protein kinase C which in turn may affect the Ca2+ influx. We have utilized monoclonal antibodies that bind to distinct epitopes residing on the T3-T cell receptor complex: OKT3 reacts with the T3 complex present on all mature human T cells and WT-31 and T40/25 which react with the constant and variable domains, respectively, of the 90-kDa heterodimeric T cell receptor for antigen on HPB-ALL cells (Meuer et al., 1984;Oettgen et al., 1984;Tax et al., 1983;Kappler et al., 1983). In this report, we show that these monoclonal antibodies activate Na+/H+ exchange that is dependent on the presence of extracellular Ca" and is inhibited by dimethylamiloride and La3'. Phorbol esters stimulate the antiporter by a calcium-independent pathway in these cells and can block the effects of anti-T3 antibodies on Na+/H'+ exchange. These data provide further insight into the triggering process of T lymphocytes and suggest a mechanism for internal regulation of these pathways.

Cells
HPB-ALL cells were used for all experiments. They are a human T cell leukemia line, the characteristics of which have been described elsewhere (Minowada, 1983). They were maintained in continuous suspension culture in RPMI-1640 medium supplemented to 5% with fetal calf serum (v/v), 10 mM Na+-pyruvate, 10 mM Hepes, 100 units/ ml of penicillin G, and 100 pg/ml streptomycin sulfate at 37 "C in humidified air, 5% COZ. Reagents-Dimethycarboxyfluorescein diacetate and its free acid were from Molecular Probes Inc., Junction City, Oregon. Phorbol esters were from L. C. Services Corp., Woburn, MA. DMA was kindly provided by Dr. E. J. Cragoe, Jr. of Merck Research Labs, West Point, PA. It was kept in a stock solution at 23 mM in dimethyl sulfoxide at -20 "C. Quin2/AM (the acetomethoxy ester of Quin2) was from Sigma and stored as a 30 mM stock solution in dimethyl sulfoxide, frozen at -20 "C. All other chemicals used were of reagent grade.
Determination of pHi Intracellular pH measurements were made using the pH-sensitive fluorescent dye, DMCF, using a modification of our previously reported method (Rosoff et al., 1984). This compound has a pK, of approximately 7.05 which makes it ideal for the study of pHi. HPB-ALL cells were collected, washed, and resuspended to 5 X lo6 cells/ ml in Buffer A (145 mM NaCl, 4 mM KCl, 1 mM NaH2P04, 0.8 mM MgS04, 1.8 mM CaC12, 25 mM Hepes, 10 mM glucose, pH 7.4). DMCFdiacetate was added to 50 p M from a 0.5 mM stock made up in Buffer A (DMCF-diacetate powder was first dissolved in 50 pl of absolute ethanol, then brought up to the appropriate stock concentration in Buffer A) and incubated for 45 min at 37 "C. At the end of the loading period, the cells were washed 3 times in fresh Buffer A (37 "C), resuspended in 1 ml of the same buffer, and kept on ice as a stock suspension. Cells kept in this manner for up to 4 h did not exhibit any loss of viability as determined by the exclusion of trypan blue.
pHi was determined in a Kontron Uvikon 810 dual beam spectrophotometer utilizing the specific pH-dependent absorbance properties of this fluor. The parent compound of DMCF, carboxyfluorescein, has a peak absorbance at Ae5 , , , , , and an isosbestic point at A465 nm (Thomas et al., 1982). To determine the analogous spectral properties of DMCF, the hydrolyzed acid was suspended to 0.5 pM in 1 ml of a solution containing 130 mM KCl, 1 mM MgCl2, and one of the following buffers: 25 mM Mes ( t o pH 6.5), 25 mM Hepes (to pH 6.9, 7.1, 7.3, and 7.5), and 25 mM Tris base (to pH 7.9 and 8.3) and absorbance scans were run. The results are shown in Fig. 1. There is an absorbance peak at Ass and an isosbestic point at h470 Therefore, these values were used for all further measurements and calculations. The ratio of absorbance at &05nm/470,,,,, (after subtracting away the absorbance at these wavelengths of an equal concentration of cells that had not been loaded with the dye) was used to determine pHi by comparison to a standard calibration curve generated by permeabilizing DMCF-loaded HPB-ALL cells with 5 pg/ml nigericin in variable pH/KCl buffers as described (Rosoff et al., 1984;Thomas et al., 1982). The calibration curve used for this report is shown in the inset of Fig. 1. For each experiment 1 X lo6 DMCF-loaded HPB-ALL cells were suspended in 1 ml of the appropriate buffer in 1-ml disposable polystyrene microcuvettes (American Scientific) in a 37 "C constant temperature cuvette chamber and allowed to equilibrate for 5 min. Data were collected using the multiple wavelength program feature of the Uvikon 810 with a 2-9 integration time for Aa0 mr hso~,, &om, and XeO, (2 nm bandwidth). Absorbance spectra from 550 nm to 400 nm for the appropriate buffer and any added reagents were baseline-subtracted for each experiment. For experiments done in the absence of extracellular Ca2+ or Na+, Buffers B and C were used, respectively (Buffer B: 145 mM NaCl, 4 mM KCl, 1.0 mM NaH2P04, 0.8 mM MgSO,, 10 mM glucose, 25 mM Hepes, 0.5 mM EGTA, pH 7.4; Buffer C: 145 mM choline Cl' , 3 mM KCl, 1 mM KH2P04, 0.8 mM MgS04, 1.8 mM CaC12, 10 mM glucose, 25 mM Hepes acid, pH 7.4).

Determination of [Ca"Ji
HPB-ALL cells were collected, washed, and loaded with the permeant acetomethoxy ester of Quin2 and fluorescence intensity was measured as described (Oettgen et al., 1985).

WAVELENGTH (nm)
FIG. 1. Absorbance spectra for dimethylcarboxylfluorescein: dependence on pH. DMCF (free acid) was diluted to 0.5 pM in buffers of varying pH as described under "Materials and Methods" and absorbance scans were performed. Inset: calibration curve of &a nm/407 ,,,,, uersus pH in permeabilized HPB-ALL cells. monoclonal antibodies directed against different regions of the T3-T cell receptor complex ih HPB-ALL cells stimulate a membrane potential-sensitive Ca" influx (Oettgen et al., 1985) that is inhibited by both EGTA and La3+. Both OKT3, which binds to an invariant heterotrimer present on all mature human T cells (Meuer et al., 1984), and WT-31, which recognizes a constant region domain of the 90-kDa heterodimer antigen receptor, are mitogenic for resting T cells (Tax et al., 1983). Increases in Na+/H+ exchange have been shown to be intimately associated with cell activation; in at least one case, stimulation of this antiport appears to be essential for the induction of differentiation (Rosoff and Cantley, 1983;Rosoff et al., 1984). In addition, the presence of extracellular Na' has been shown to be necessary for mitogen activation of T cells (Deutsch et al., 1981(Deutsch et al., , 1984. We therefore wished to see if specific stimulation of the T3-T cell receptor complex with these monoclonal reagents could stimulate the Na+/H+ antiport in these cells. OKT3, T40/25, and WT-31 all caused a rapid and sustained increase in pHi within 30 s after addition to a suspension of DMCF-loaded HPB-ALL cells (Fig. 2). The pH change was detected as an increase in the absorbance ratio at nm/470 nm of DMCF (see "Materials and Methods"). The resting pHi in these cells is approximately 7.15 +. 0.05, in good agreement with data reported elsewhere using a different technique to measure basal pHi in nontransformed T cells . The pHi increased by about 0.15 pH unit after treatment with the antibodies. WT-82, a monoclonal reagent reactive with the T8-T lymphocyte surface antigen that is expressed on these cells, had no effect on pHi in HPB-ALL cells ( Fig. 2A). The increase in pHi was sustained for at least 20 min (see below) and was initiated on the same time scale as the antibody-stimulated Ca2+ influx (Oettgen et al., 1985). In addition, the absolute increase in pHi measured here is similar to that noted in LPS-treated 70Z/3 pre-B lymphocytes (Rosoff, et al., 1984), epidermal growth factor-stimulated A431 cells (Rothenburg et al., 1983), and serum-stimulated fibroblasts (Burns and Rozengurt, 1983). The fact that the anti-T3-T cell receptor complex antibodies induce an increase in intracellular pH does not, by itself, mean that this is due t,o enhanced Na+/H+ exchange. We therefore examined the ability of the antibodies to increase pHi in the presence of DMA, a potent and specific inhibitor of the Na+/H+ antiport (Zhuang et al., 1984). DMA alone caused a rapid decrease in pHi to a new steady state value of 6.60 after about a 4-min incubation at 37 "C ( Fig. 2 A ) , suggesting that the antiporter is in a partially active state in the unstimulated cells. Although there was some variability in the rate of decrease in pHi with the addition of DMA between experiments, the eventual steady state pH of 6.60 was reproducibly achieved and addition of antibodies against the T3-T cell receptor complex caused no significant deviation from this low value. These results are in agreement with our observations on the effect of amiloride on pHi in 70Z/3 cells (Rosoff et al., 1984). The concentration of DMA used (50 PM) was well below the Kr for inhibition of protein kinase C as recently reported by Besterman et al. (1985). The basal pHi was not affected by exposure to 1% dimethyl sulfoxide, the solvent in which DMA was dissolved (data not shown). These data all suggest that the antibodies are stimulating the Na+/H+ antiport to produce a rise in pHi.
Further evidence that these reagents are causing an increase in Na+/H+ exchange is provided by the dependence of the increase in pHi on external Na+. DMCF-loaded HPB-ALL cells were placed in a Na+-free, choline C1+ buffer (Buffer C), allowed to equilibrate to the 37 "C temperature of the cuvette chamber for 5 min, treated with OKT3, WT-31, or T40/25, and the pHi was followed. The results are shown in Fig. 3. Elimination of extracellular Na+ by itself without antibody treatment caused a decrease in pHi to approximately 6.6, very similar to the decrease observed in the presence of DMA (Figs. 2 and 3). As in the case of DMA addition, the scatter in the data was greater in the Na+-free medium; however, the antibodies against the T3-T cell receptor complex did not appear to cause a significant increase in cytoplasmic pH under these conditions. The regulation of cytoplasmic pH in unstimulated cells is dependent on extracellular Na+. The antibodyinduced alkalinization is also dependent on extracellular Na+, indicating the role of the plasma membrane Na+/H+ exchange system in this regulation.
The increase in pHi stimulated by OKT3, WT-31, and T40/ 25 appears to result from the stimulated Ca2+ influx. The effects of antibody treatment on pHi in the absence of extracellular Ca2+ are shown in Fig. 3. Preventing the influx of Ca2+ that is associated with antibody treatment by removal of its external source completely eliminated the stimulation of Na+/H+ exchange. Removal of extracellular Ca2+ had no effect on pHi by itself over the 15-min time course of the experiment. We have previously shown that La3+ inhibits the antibody-stimulated Ca2+ influx (Oettgen et al., 1985). This cation also blocked the ability of the antibodies to induce an increase in pHi (data not shown). These results suggest that the rise in pHi is a process initiated or dependent upon the Ca2+ influx stimulated by receptor activation. Phorbol esters are potent co-mitogens for lectin and anti-T3 antibody stimulated mitogenesis in T lymphocytes (Meuer et al., 1984;Tsien et al., 1982). They replace the obligatory requirement for monocytes/macrophages to be present in the culture system. We have previously shown that phorbol esters enhance amiloride-sensitive Na+/H+ exchange in 702/3 pre-B lymphocytes and that this enhancement appears to be essential for the successful induction of differentiation (Rosoff et al., 1984). Phorbol esters stimulate Na+/H+ exchange in other types of cells as well (Burns and Rozengurt, 1983;Moolenaar et al., 1984b;Besterman and Cuatrecacas, 1984). The increase in exchange activity is presumed to be mediated via activation of protein kinase C Nishizuka, 1984). We have shown that TPA has no effect on [CaZ+li in HPB-ALL cells (Oettgen et al., 1985). Phorbol esters have also been reported to have little effect on [Ca2+Ii in quiescent, nontransformed T cells as well (Tsien et al., 1982;Hesketh et al., 1985). We therefore wished to determine the effects of TPA on pHi in HPB-ALL cells.

Phorbol Esters Stimulate Na+/H+ Exchange by a Mechanism Which Does Not Require Elevation of
DMCF-loaded HPB-ALL cells were exposed to either 50 nM TPA or PDD (a biologically inactive phorbol ester) and cytoplasmic pH was monitored. The results are shown in Fig.  4. TPA stimulated an increase in pHi that was independent of the presence of extracellular Ca2+, but was dependent on the presence of external Na+. In addition, as with the rise in pHi effected by OKT3, WT-31, and T40/25 antibodies, the effects of TPA on pHi were completely blocked by the addition of 50 PM DMA. Curiously, unlike the response to the antibodies where pH< remained elevated for at least 20 min (see below), after 8-10 min of exposure to TPA, the pHi returned to the prestimulated level. The biologically inactive phorbol, PDD, and 0.1% dimethyl sulfoxide (v/v), the solvent in which both TPA and PDD were dissolved, had no effect on pHi. These data suggest that there are at least two separate mechanisms for stimulation of the Na+/H+ antiport in these cells: one via a Ca2+-dependent pathway through stimulation of the T3-T cell receptor complex-associated Ca2+ channel and the second via a Ca2+-independent pathway mediated by protein kinase C.

Pretreatment with Phorbol Esters Prevents OKT3 Stimulation of Na+/H+
Exchange-It has recently been reported that even though phorbol esters cause a small increase in cytoplasmic pH in A431 human epidermoid carcinoma cells, pretreatment with phorbol esters blocks the epidermal growth factor stimulation of Na+/H+ exchange (Whitely et al., 1984). We therefore wished to see if pretreatment of HPB-ALL cells with TPA would abolish the effect of anti-T3-T cell receptor antibodies on Na+/H+ exchange.
When added simultaneously, TPA and OKT3 had apparent additive effects on the pHi of DMCF-loaded HPB-ALL cells (Fig. 5). However, as with TPA treatment alone, the pH; rapidly increased and then returned to baseline levels within 10 min. When these cells were exposed to 50 nM TPA for 15 min at 37 "C (by which time the pH; had returned to baseline) and then treated with OKT3, there was complete inhibition 6 . 4~" " ' 1 ' 1 " " " ' of the ability of the antibody to stimulate Na+/H+ exchange (Fig. 5). The converse condition produced opposite results: preincubation with OKT3 did not block the effects of subsequently added TPA on pHi. It can be seen from Fig. 5 that a small increase in pHi occurs when TPA is added subsequent to OKT3 pretreatment but, as observed with simultaneous addition of the two agents, the pH, returns to baseline after 8-10 rnin. Thus, the effect of phorbol esters on pHi is transient whereas that of the antibodies is more prolonged. With OKT3 addition alone, the pHi remained elevated above that of control cells for at least 20 min. The results in Fig. 5 indicate that while TPA enhances the effect of OKT3 on pH; at short times after addition, it eliminates the response at longer times, thus converting a step increase in pH; into a transient pulse.
The fact that OKT3 can no longer increase pHi after preincubation of HPB-ALL cells with TPA could be due to modification of the T3-T cell receptor complex with a corresponding functional inhibition. We therefore measured the ability of OKT3 to stimulate a Ca" influx in QuinP-loaded HPB-ALL cells as previously described (Oettgen et ul., 1985).
The results are shown in Fig. 6. It is clear that prolonged exposure to TPA had no effect on the ability of OKT3 to increase [Ca"], a function associated with stimulation of the T3-T cell receptor complex and a prerequisite for antibody stimulation of Na+/H" exchange. The [Ca"+]i rose from a baseline value of 150 nM to about 300 nM, as previously observed (Oettgen et d., 1985). Both WT-31 and T40/25 antibodies were also able to stimulate a Caz+ influx after TPA preincubation (data not shown). These results suggest that the inhibitory action of phorbol esters is directed against a step distal to the T3-T aell receptor complex-associated Ca2+ influx and argues against a feedback regulatory role of protein kinase C on the T3-T cell receptor complex.

DISCUSSION
In this paper we show that monoclonal antibodies directed against the variable and constant domains of the T cell receptor (T40/25 and WT-31, respectively) and the associated T3 antigen (OKT3) of the human T cell leukemia line HPB-ALL rapidly stimulate Na+/H+ exchange, leading to an increase in pHi. The antibody-induced rise in pHi is dependent on the presence of extracellular Na+ and Ca2+ and is blocked by dimethylamiloride, an inhibitor of Na+/H+ exchange (Figs. 2 and 3), and La3+, an inhibitor of Ca" channels. We have previously demonstrated that these same antibodies activate a membrane potential-sensitive Ca" influx in HPB-ALL cells that produces a rise in cytosolic free [Ca"] (Oettgen et ul., 1985). These data therefore suggest that stimulation of the T3-T cell receptor complex-associated Ca2+ influx is a necessary prerequisite for activation of the antiport in T lymphocytes.
We also show that TPA, a potent tumor-promoting phorbol ester, stimulates DMA-inhibitable Na+/H+ exchange in these cells (Fig. 4). This agent, which does not activate the T3-T cell receptor Ca" channel (Oettgen et at., 1985), enhances antiporter activity by a mechanism which does not require elevation of cytoplasmic Ca". TPA and OKT3 appear to have

T3-T Cell Receptor Activation
of Na+/H+ Exchange additive, but transitory, effects on pHi when added to HPB-ALL cells simultaneously (Fig. 5 ) . However, when the cells are preincubated with TPA prior to the addition of the antibody, OKT3 is incapable of stimulating an increase in pHi. The converse situation gives different results: preincubation with OKT3 does not prevent further activation of Na+/H+ exchange by TPA. The fact that phorbol esters can enhance Na+/H+ exchange suggests a role for receptor-stimulated phosphatidylinositol turnover which has been shown to increase in response to anti-T3 and T cell receptor antibodies (Irnboden and Stobo, 1985). These data also suggest the existence of a feedback regulatory pathway mediated by protein kinase C.
It is not surprising that the antibody-stimulated Ca2+ influx should lead to enhanced Na+/H+ exchange by a Ca2+-dependent mechanism. Several investigators have shown that treatment of splenic and thymic lymphocytes Gerson and Kiefer, 1982;Hesketh et al., 1985) with the T cell mitogen, con A, produces an increase in pHi. As con A treatment causes a rise in [CaZ+li that is dependent on the presence of extracellular Ca2+ (Tsien et al., 1982;Hesketh et aL, 1985), it is reasonable to suppose that a pathway similar to the one described here is operative in these nontransformed cells. However, it has yet to be proven that con A is exerting its effects by stimulating the antigen receptor. It has been suggested that the increase in Na+/H+ exchange resulting from serum or growth factor stimulation of quiescent fibroblasts is due to activation of a Ca2+-calmodulin-dependent pathway resulting from a primary increase in [Caz*]i (Villereal, 1981;Owen and Villereal, 1982;Vincentini et al., 1984). These workers have shown that the calcium ionophore A23187 activates an miloride-sensitive Na+ influx into treated cells that is blocked by the calmodulin inhibitor, trifluoperazine, but not by the phospholipase C inhibitor, mepacrine. The interpretation of these data is complicated by the relatively nonspecific effects of these drugs on a variety of Ca2+ and lipid-dependent enzymes (Nishizuka, 1984). Siffert et al. (1984) also show that A23187 stimulates amiloridesensitive Na+/H+ exchange in platelets by measuring H+ efflux from treated cells. Using the spectrophotometric methodology to measure intracellular pH as described here, we have been unable to use Ca" ionophores to study their effects on pHi since they work by exchanging H+ for Ca". However, these results suggest that there may be at least two complementary pathways for increasing the activity of the Na+/H+ antiport in quiescent cells.
We have shown that phorbol esters can activate Na+/H+ exchange in 702/3 pre-B lymphocytes. This activation appears to be a requirement for the induction of differentiation (Rosoff and Cantley, 1983;Rosoff et al., 1984). Phorbol esters have been shown to have similar effects on several other cell types as well (Besterman et al., 1984;Moolenaar et al., 1984b). In TPA-treated 702/3 cells, this activation does not require a prior elevation of cytoplasmic Ca2+ and actually decreases the [Ca2+Ii (Rosoff and Cantley, 1985b). A decrease in 1CaZ+lj has also been observed in TPA-treated splenic T cells (Tsien et al., 1982) and neutrophils (Lagast et al., 1984;Korchak et aL, 1984). These data suggest that the Na+/H+ antiport can be directly activated by protein kinase C via a pathway that does not require elevation of cytoplasmic Ca2+. By substituting for endogenously produced diaclyglycerol, TPA lowers the K,,, for Caz+ of protein kinase C to the [Ca2++Ii found in many unstimulated cells (Nishizuka, 1984). Our results with HPB-ALL cells also support this idea. TPA treatment leads to a rapid and transient increase in pHi that is dependent on Ndi, inhibitable by DMA, and does not involve an increase in cytoplasmic [Ca2+].
Resting T cells may be induced to proliferate by anti-T3 antibodies, the calcium ionophore A23187, or mitogenic lectins such as con A, but only when a second stimulus such as TPA or monocytes are simultaneously present (Chang et al., 1982;Mastro and Smith, 1983). Since the first signals all produce a rise in [Ca2+], as well as pH:, this argues that these changes alone are not sufficient to induce mitogenesis in these cells. The importance of the enhanced Na+/H+ exchange activity in mitogen activation of resting T cells is suggested by studies which have demonstrated the requirement for extracellular Na+ for con A and phytohemagglutinin-induced DNA synthesis (Deutsch et al., 1981;Deutsch et al., 1984) and the close association of mitogen treatment and increases in ouabain-insensitive Na+ uptake (Segal et ai., 1979). Protein kinase C apparently performs an integral role in co-mitogenesis, distinct from its synergistic action on Na+/H+ exchange. It is of course possible that the ability of TPA to attenuate the Na+/H' exchange activation is an essential step in the proliferative response ( i e . a pulse of Na+/H+ exchange activation rather than continuous activation may be necessary for a proper cellular response to the stimulatory signal). However, it seems likely that protein kinase C causes addi-tionaI cellular responses not involving Na+/H+ exchange.
The ability of TPA to attenuate the activation of Na+/H+ exchange in HPB-ALL cells suggests a possible feedback mechanism mediated by C kinase. TPA-activated protein kinase C has been reported to decrease the tyrosine kinase activity and cellular response of both the epidermal growth factor receptor (McCaffrey et al., 1984;Iwashita and Fox, 1984;Cochet et ai., 1984;Whitely et al., 1984) and the insulin receptor (Jacobs et al., 1983;Takeyama et al., 1984). We have also found that TPA inhibits phosphatidylinositol turnover in 70213 pre-B lymphocytes (Rosoff and Cantley, 1985b). The data presented in Fig. 5 show that the ability of the anti-T3-T cell receptor complex antibodies to stimulate Ca2+ influx is not affected by pretreatment with TPA. Thus, the step at which TPA attenuates antibody-activated Na'/H+ exchange is subsequent to the receptor-activated Ca2+ influx. Further work is required to determine the complex regulation of Na+/ H+ exchange and its role in T cell activation.