©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Angiotensin II Activates the Na/HCO Symport through a Phosphoinositide-independent Mechanism in Cardiac Cells (*)

(Received for publication, March 22, 1995; and in revised form, June 20, 1995)

Trudy A. Kohout Terry B. Rogers (§)

From the Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Angiotensin II (AngII) is a hormone that alters contractility as well as myocyte growth in heart. Since many hormones that regulate cardiac contractility have also been found to modulate intracellular pH (pH) the goal of this study was to determine if AngII altered pH in cultured neonatal rat ventricular myocytes. Changes in pH were monitored in single cells using the fluorescent pH indicator carboxy-seminaphthorhodafluor-1. Application of 100 nM AngII resulted in a rapid, receptor-mediated alkalinization of 0.08 ± 0.02 pH unit. The Na/H exchanger was not involved since the response was HCO(3)-dependent and amiloride-insensitive. Ammonia rebound experiments showed AngII increased the initial rate of recovery from an imposed acid load by 3.15-fold and showed that the hormone led to the selective activation of the Na/HCO(3) symport. In contrast, phorbol ester activation of protein kinase C led to the selective activation of Na/H antiport in these cells. Pharmacological studies showed that the alkalinization was independent of the AngII receptor subtype 1 (AT(1)) phosphoinositide signaling path. In contrast, AngII activation of the symport was blocked by nanomolar AT(2) receptor antagonist PD 123319. Superfusion of the myocytes with exogenous arachidonic acid (5 µM) mimicked the AngII-mediated alkalinization, further suggesting that the AT(2) signaling pathway underlies the response. In summary, while most of the known actions of AngII in heart are mediated through AT(1) receptors, activation of the Na/HCO(3) symport occurs through a distinct alternative path that is likely related to fatty acid production.


INTRODUCTION

Regulation of intracellular pH, pH, is important in all cells, as accumulation of metabolic acid and other perturbations of pH can have profound effects on cellular homeostasis. In heart, acidosis depresses contractility in cardiac myocytes by affecting virtually every step in the excitation-contraction coupling (1) . Hence, in order to preserve the proper functioning of the heart, cardiac myocytes possess pH regulating transporters that maintain pH within very narrow limits(2) . There are three alkalinizing transporters that have been characterized in cardiac myocytes including the Na/H exchanger, the Na/HCO(3) symporter, and the Na-dependent HCO(3)/Cl exchanger (3, 4, 5, 6) . The Na-independent HCO(3)/Cl exchanger acts as an opposing acidifying mechanism in such cells(2, 7) . Although there is some species variation in the expression of exchangers in heart, it is clear that resting pH results from a balance between the intrinsic activities of the exchangers present.

It has been recently recognized that there is a significant functional role for hormonal modulation of these pH-regulating transporters as well. Since cardioactive hormones that regulate contractility, such as alpha(1)-, beta-adrenergic agonists and endothelin, also modulate pH, it appears that the control of pHunderlies part of the inotropic intracellular signaling pathways in heart(8, 9, 10, 11, 12) . Although a frequently seen underlying mechanism involves activation of the Na/H exchanger through PKC(^1)(12, 13) , any pH regulating transporter is a potential target for hormone action. Indeed several recent reports reveal that this is a likely phenomenon in heart, although at present little information is known of the signal transduction mechanisms that bring about these responses(10, 14) .

AngII is a peptide hormone known to regulate contractility as well as to control long term growth in cardiac myocytes. In particular AngII-evoked stimulation of the phosphoinositide/PKC pathway underlies, in part, the Ca mobilization and the hormone-evoked cardiac hypertrophy that have been observed(15, 16, 17) . However, recent studies have revealed that there is a parallel, independent AngII-evoked activation of PLA(2)(18) . The significance of this alternative path remains to be defined.

It is within this context that we sought to determine whether AngII regulates pH in neonatal rat ventricular myocytes. Our results show unexpectedly that AngII induces an intracellular alkalinization by the selective activation of the Na/HCO(3) symport. Further, while the phosphoinositide pathway is not involved, the pH response is mediated by an AT(2) receptor pathway that is likely to involve ARA, possibly through PKC.


EXPERIMENTAL PROCEDURES

Materials

The pH indicator carboxy-SNARF-1 and nigericin, free acid, were purchased from Molecular Probes (Eugene, OR). The peptides AngII (human) and [Sar^1,Leu^8]AngII were obtained from Bachem California (Torrance, CA). ARA acid and ETYA were purchased from Cayman Chemicals (Ann Arbor, MI). Staurosporine was purchased from LC Laboratories (Woburn, MA) and mouse laminin from Life Technologies, Inc. Amiloride, TPA (12-O-tetradecanoyl phorbol-13-acetate), and DTT were from Sigma. Nifedipine was obtained from Pfizer Corp. (Groton, CT). DuP 753 and PD 123319 were kind gifts from Dupont Merck Corp (Wilmington, DE) and Parke-Davis Pharmaceutical Co. (Ann Arbor, MI), respectively. PC1, a chemically defined, serum-free cell culture medium, was purchased from Hycor Biomedical Inc. (Irvine, CA).

Cell Culture

Cultured neonatal rat ventricular myocytes were prepared from 1-day-old Sprague-Dawley rats and grown in serum-free medium, as described previously(19) . Cells were irradiated with 3500 rads of irradiation after 24 h in culture to prevent the proliferation of non-myocytes as described previously(20) . The media was then changed to a 1:4 dilution of PC1:Dulbecco's modified Eagle's medium supplemented with 5 mM glutamine and 1% gentamicin. Experiments were performed on 2-5-day-old cultures.

Solutions

In experiments myocytes, still attached to coverslips, were superfused with buffer that contained 1.8 mM CaCl(2), 5 mM KCl, 120 mM NaCl, 25 mM HEPES, 1 mM Na(2)HPO(4), 1 mM MgSO(4), and 10 mM glucose, pH 7.4. In bicarbonate-containing HEPES buffer, 25 mM NaHCO(3) was added and NaCl reduced to 100 mM to maintain osmolarity. Sodium-free buffer was composed of 145 mMN-methyl-D-glucamine, 5 mM KCl, 1 mM MgSO(4), 1 mM CaCl(2), and 10 mM glucose. The buffer was then bubbled continuously for 2 h with 5% CO(2) and 95% O(2) to generate N-methyl-D-glucamine HCO(3)(3) . The final pH of the solution was adjusted to 7.4 with HCl. Sodium-containing control experiments were performed in the following buffer: 120 mM NaCl, 25 mM NaHCO(3), 5 mM KCl, 1 mM MgSO(4), 1 mM CaCl(2), and 10 mM glucose, pH 7.4. The latter two solutions were continuously equilibrated with 5% CO(2) and 95% O(2) during experiments to maintain pH 7.4. Chloride-free buffer contained 100 mM sodium gluconate, 5 mM potassium gluconate, 1.8 mM calcium gluconate, 25 mM HEPES, 25 mM NaHCO(3), 1 mM Na(2)HPO(4), 1 mM MgSO(4), and 10 mM glucose, pH 7.4.

Measurement of Intracellular pH

pH was measured with the pH-sensitive fluorescent indicator, carboxy-SNARF-1. Cells, grown on coverslips, were loaded with c-SNARF-1 by incubation with the acetoxymethyl ester form of the indicator (10 µM c-SNARF-1/AM) in 15% pluronic F-127.

The superfusion cell chamber was a small-volume (<500 µl), flow- and temperature-controlled bath developed by Cannell and Lederer(21) . The bath volume was maintained by the use of a small flotation device attached to a piezoresistive element (SensoNor AE-801) and a feedbackflow-control circuit which controlled the outflow pump(21) . Superfusion was maintained at 400 µl/min with a constant temperature of 30 °C. Electrically actuated valves (Lee Valve Co. LFA 1201618H) were used that allowed for rapid changes of bath solutions in less than 10 s.

Fluorescence was measured from single c-SNARF-1-loaded cells using an inverted microscope (Nikon Diaphot) equipped for epifluorescence (Photon Technology International). The excitation wavelength (488 nm) was selected by the monochromator and the emission wavelengths (580 ± 10 and 640 ± 10 nm) were selected by interference filters. A 510-nm dichroic longpass mirror separated the excitation and emission beams in the microscope, and a 610-nm dichroic longpass mirror separated the two emission wavelengths directed to the photomultiplier tubes. Autofluorescence from the cells was found to be negligible.

pH was calculated from the ratio of the emission intensity at 580 nm (I) to that at 640 nm (I). I/I was calibrated in each cell studied by the use of a high K-nigericin method to equilibrate pH with pH solutions of known pH from 6.6 to 7.8 (22) . Calibration solutions contained 150 mM KCl, 5 mM NaCl, 25 mM HEPES, 10 mM glucose, 1 mM EGTA, and 1 µM nigericin. I/I was converted to pH using the experimentally derived calibration curve. After conversion, the calibrated pH signal was filtered by calculating a running average over 40 point intervals. The data acquisition rate was 16 points/s.

Estimation of Intracellular Buffering Capacity and Acid Extrusion Fluxes (J)

Cellular buffering capacity, beta, was estimated using the ammonia rebound technique(23) . Myocytes were briefly exposed to buffer containing 20 mM NH(4)Cl followed by buffers which contained decreasing amounts of NH(4)Cl. The resulting decreases in pHwere used to estimate the beta of the cell over the pH range 6.7 to 7.8 in the absence and presence of HCO(3)(3, 24) . The calculated betas were plotted as a function of pH and fit with a least-squares linear regression. The derived linear equations describe beta as follows: in 0 HCO(3) beta = -16.86 pH + 140.7; in 25 mM HCO(3) beta = -53.13 pH+ 417.4. These values agree with those seen for guinea pig myocytes incubated under similar conditions(3) .

The proton efflux rate carried by various transporters was calculated from the equation: J = beta(dpH/dt), where beta is the buffering capacity (calculated above) and dpH/dt is the initial rate of pH recovery from an acid load. For the analysis it was necessary to fit experimental data into a continuous curve. The following method was used. The data were divided into four segments. The first three segments were 6-s intervals (100 points); the data points for each of these segments were fit to a line using the least-squares linear regression of the contributing points. The remainder of the recovery was fit with a 2-exponential function. The dpH/dt values were calculated by differentiating the function at 15-s intervals, and its corresponding pH was taken as the mean pH of the interval. The proton fluxes (J) in Table 1were calculated by multipling the dpH/dt value at pH 7.10-7.15 with the appropriate beta. Background loading due to metabolic acid production when all acid extruding transporters are blocked was calculated to be 0.17 ± 0.05 mM/min (Table 1) and was not included in the calculation of fluxes.



Statistics

Data are expressed as the mean ± S.E. (standard error of the mean). In experiments where a cell acted as its own control, the control pH and the experimental pH were compared using a paired Student's t test. Control and experimental J values obtained from sequential experiments performed on the same cell were likewise compared with a paired t test. A p value of <0.05 was considered significant.


RESULTS

Effect of Angiotensin II on pH

The initial goal was to determine if AngII alters pH in cardiac cells. pHwas measured using a microfluorescent technique on actively contracting neonatal rat ventricular myocytes loaded with the dual emission fluorescent indicator carboxy-SNARF-1/AM as described under ``Experimental Procedures.'' Fig. 1A shows that superfusion of 100 nM AngII, in the presence of 25 mM HCO(3), resulted in a rapid and sustained intracellular alkalinization, lasting at least 6 min. The summary of results from many experiments was that AngII caused an increase in pH of 0.08 ± 0.02 pH units (n = 6). Similar results were obtained when the cells were field-stimulated at a constant frequency of 2 Hz (data not shown); therefore, the alkalinization is not the direct result of the increase in beating frequency seen following application of AngII to these cultures(25) . Fig. 1C shows that the AngII effect is strictly dependent on HCO(3). To further characterize the ion dependence of the AngII-activatable acid transporter, myocytes were challenged in the presence and absence of extracellular Na. Fig. 1E shows a control experiment where addition of 100 nM AngII in buffer containing 145 mM extracellular Na resulted in an alkalinization of 0.093 ± 0.003 pH units (n = 3). In a parallel experiment in Fig. 1F, addition of 100 nM AngII in 0 Na-containing buffer had no effect on pH (DeltapH = 0.007 ± 0.003, n = 3). These observations reveal that AngII activates an acid extruding transporter that is HCO(3)- and Na-dependent.


Figure 1: Effects of AngII and phorbol ester on pHi in cultured cardiac cells. Cultured rat myocytes, preloaded with c-SNARF-1 and mounted in a superfusion bath on the stage of a microscope, were superfused with HEPES buffer with (panels A and B) or without (panels C and D) 25 mM HCO(3) added. Cells were also superfused with 25 mM HCO(3) buffer bubbled with 5% CO(2) containing 145 mM Na (panel E) or 0 Na (panel F) as described under ``Experimental Procedures.'' Bars indicate buffer used in experiments in the panels below. Fluorescence of single cells was monitored and pH was determined as described under ``Experimental Procedures.'' In panel A cells were exposed to 100 nM AngII at time indicated (arrow below trace). In panel B similar cells were exposed to 100 nM TPA at the time indicated. Traces in panels C and D show the results of experiments identical to those in A and B, respectively, except that the superfusion buffer did not contain bicarbonate. In panels E and F, cells were stimulated with 100 nM AngII at the indicated time in the presence (panel E) and absence (panel F) of extracellular Na.



The HCO(3) dependence suggests that the Na/H exchanger is not involved. This hypothesis was explored in parallel experiments with the phorbol ester, TPA, a PKC activator that stimulates particular isoforms of the Na/H exchanger in many cells including cardiac myocytes(6, 13, 26) . As shown in Fig. 1B, application of 100 nM TPA resulted in a rapid increase in pH (DeltapH = 0.09 ± 0.01, n = 6) that is nearly identical to that seen with AngII. However, in contrast to the response for AngII, the TPA-evoked alkalinization was the same in the absence or presence of HCO(3) (Fig. 1D, DeltapH = 0.06 ± 0.01, n = 8). Thus, the underlying mechanisms of alkalinization evoked by AngII and TPA are distinct.

Experiments with a Na/H exchange inhibitor, amiloride, provided further evidence for the lack of involvement of this antiporter. A double-pulse protocol was developed so that the effects of the inhibitor could be followed on a single cell. In these sets of experiments, cells were challenged with 10 nM AngII instead of 100 nM. Although the pH response was submaximal under these conditions, the protocol permitted reversal of the AngII effect. Fig. 2A shows the responses of a cell to two consecutive applications of 10 nM AngII. After the initial challenge with 10 nM AngII (DeltapH = 0.034 ± 0.002, n = 5), the cell was washed by constant superfusion with hormone-free buffer for 15 min to reverse the initial pH increase. Following this, a second challenge of 10 nM AngII resulted in a nearly identical pH response (DeltapH = 0.040 ± 0.003, n = 5). Thus, there is little ``run down'' of the cell or desensitization of the myocytes to AngII under the double-pulse protocol. In parallel experiments amiloride (1 mM) was added after the first AngII stimulation. As shown in Fig. 2B, the AngII response in amiloride-treated cells was similar to the control value (DeltapH = 0.033 ± 0.003, n = 3 compared to the control 0.04 pH units). Control experiments were performed with TPA (Fig. 2C) which revealed that 1 mM amiloride completely blocked the TPA-evoked alkalinization (DeltapH = 0.003 ± 0.007, n = 3) as expected if the Na/H exchanger was inhibited. The amiloride-insensitive nature of the AngII effect is further evidence that activation of the Na/H exchanger is not an underlying mechanism.


Figure 2: Effect of amiloride on AngII- and TPA-evoked alkalinizations. Myocytes were superfused with HEPES/bicarbonate buffer as described under ``Experimental Procedures'' and Fig. 1. Panel A shows the effects of AngII (10 nM) on pH in a double-pulse protocol. AngII was first applied over several minutes as indicated by the time bar. At the end of this exposure, the cell was superfused for 15 min with buffer to reverse AngII alkalinization (data not recorded; note break in trace). Reapplication of 10 nM AngII resulted in a nearly identical pH response by the same cell. Shown is a representative trace of five experiments where the first stimulation with 10 nM AngII yields an average DeltapH = 0.034 ± 0.002, and the second challenge yields a DeltapH = 0.040 ± 0.003 pH unit (n = 5). Panel B shows an identical experiment except that 1 mM amiloride was added prior to the second AngII application as shown by the time bar. Panel C shows pH traces from experiments on two different cells; on the left, 100 nM TPA was added to the myocyte resulting in an alkalinization of 0.09 ± 0.01 pH unit (n = 6). On the right, 100 nM TPA was added during superfusion with 1 mM amiloride resulting in a complete inhibition of the alkalinization. The double-pulse protocol could not be used with TPA because the response to this phorbol was not readily reversible.



Characterization of pH-regulating Transporters in Rat Cardiocytes

A reasonable hypothesis was that AngII alkalinized cardiocytes through an activation of an acid extruding transporter. Several alkalinizing transporters are differentially distributed in cardiac cells from a variety of species(3, 4, 5, 27) . Experiments were designed to not only identify the acid extruding transporters expressed in cultured cardiac cells, but to also assess their relative contributions to the total cellular proton efflux. By imposing an intracellular acid load with an ammonia rebound protocol (see ``Experimental Procedures''), it was possible to adjust the conditions so that the contribution of each exchanger to the total flux of H equivalents, J, could be quantitated. The results are shown in Table 1. In the presence of 25 mM HCO(3), where the Na/H exchanger and the HCO(3) transporters are functional, the maximum J at the peak of the acid load (pH = 7.10-7.15) was 3.00 ± 0.34 mM/min, consistent with the values seen in other cardiac cells(8, 11) . Under conditions where the Na/H exchanger is inactive but HCO(3)dependent transporters are functional (25 mM HCO(3) and 1 mM amiloride), J was decreased to about 50% of the total flux value. Conversely, in the absence of HCO(3) when only the Na/H exchanger should be active, J was also about 50% of the total (Table 1). Thus, the contribution to the total flux of H equivalents was equal between the Na/H exchanger and the HCO(3)-dependent transporters. Experiments in Fig. 1, E and F, showed that the HCO(3)-dependent transporter stimulated by AngII was also Na-dependent, thereby implicating the presence of the Na/HCO(3) and/or the Na-dependent HCO(3)/Cl transporters. To determine the contribution of the Na-dependent HCO(3)/Cl exchanger, cells were incubated for 3 h in Cl-free buffer to deplete intracellular Cl(28) . The total flux measured under these conditions was nearly identical to that seen in the presence of Cl (Table 1). Thus if the Nadependent HCO(3)/Cl exchanger is present in neonatal rat heart, its contribution to the total acid efflux is not significant. Since the Na/HCO(3) symport and the Na/H exchanger have been shown to be the principal alkalinizing transporters in mammalian heart cells(3, 29) , the most reasonable conclusion from the results reported here is that these two transporters are the major ones expressed in cultured neonatal myocytes as well. Further, the results in Table 1reveal that they contribute equally to the acid extrusion.

Activation of Na/HCO Symport by Angiotensin II

Based on the results of Table 1and Fig. 1and 2, an appealing hypothesis is that AngII increases pH through an activation of the Na/HCO(3) symport. A series of ammonia rebound experiments were performed to test this hypothesis more directly. The tracing in the control ammonia rebound experiment in Fig. 3A shows the normal rate of recovery from an intracellular acid load. In a second ammonia rebound experiment on the same cell 100 nM AngII was added at the peak of the acid load (Fig. 3A, arrow) resulting in a marked increase in the rate of acid extrusion. In this particular experiment the initial recovery rate was stimulated 2.76-fold over control by AngII. Over many experiments there was a 3.15 ± 0.43-fold (n = 13, p < 0.0005) stimulation of rate of recovery by AngII. In contrast, in the absence of HCO(3), when only the Na/H exchanger is active, AngII did not stimulate the rate of acid load recovery (Fig. 3C). AngII-stimulated pH recovery rate under these conditions was 0.86 ± 0.08-fold (n = 10, p = NS), a value not statistically different from control. It was clear that the Na/H exchanger was active under these conditions since parallel experiments revealed that 100 nM TPA increased the rate of acid recovery 2.20 ± 0.36-fold (n = 10) in the absence of HCO(3) (data not shown). Taken together, these data provide evidence that AngII selectively stimulates the Na/HCO(3) symport.


Figure 3: AngII stimulates rate of pH recovery from an acid load. Panel A shows the pHversus time trace for a cell exposed to a 2-min pulse of 10 mM NH(4)Cl (indicated by bar above trace) in HEPES buffer containing 25 mM HCO(3). After a 10-min wash, the cell was exposed to an identical second pulse of NH(4)Cl except that at the point of maximum acid load (marked with an arrow) 100 nM AngII was added to the cells. The AngII-stimulated rebound is labeled above the trace. The overshoot of the recovery phase is accentuated by the dotted line marking basal pH. In panel B, the Jversus pHrelation was plotted for the control () and 100 nM AngII-stimulated (bullet) traces shown in panel A. Fluxes and pH are calculated as described under ``Experimental Procedures.'' Panel C shows results of parallel experiments as in panel A that were performed in the absence of HCO(3). Panel D displays the Jversus pH relation from the results in panel C.



The mechanism of the AngII effect was studied in more detail by analyzing the net proton efflux, J, as a function of pH. As shown in Fig. 3B, AngII had several effects on the pH-flux relation. There was a marked stimulation in the symport activity at pH in the range of 7.10 to 7.15. Second, there was a shift in the curve to the right in the region of resting pH, indicative of the activation of the symporter by AngII at resting pH. This is also illustrated by the overshoot seen in the recovery phase of the NH(4)Cl pulse response following AngII application (Fig. 3A) and is consistent with the AngII-induced alkalinization observed in resting cells (Fig. 1A).

Mechanism of Angiotensin II Activation of the Na/HCO Symport

Preliminary experiments with the antagonist 100 nM [Sar^1, Leu^8]AngII, a well characterized AngII receptor antagonist, demonstrated that the AngII response was receptor-mediated (data not shown). The specificity of the response was characterized by exploiting AngII receptor subtype-specific antagonists(30) . When PD 123319 (100 nM), a potent AT(2) receptor antagonist, was used in double-pulse experiments it did not alter resting pH, but it did completely inhibit the AngII (10 nM) response (Fig. 4B and Table 2). In parallel experiments with the nonpeptide AT(1) antagonist, DuP 753, it was found that this agent also inhibited the AngII response (Fig. 4C and Table 2). Although these results might implicate both receptor subtypes in the response, they should be interpreted with caution since DuP 753 can block the AT(2)-like signaling path in intact cultured myocytes in the 100 nM range(18) .


Figure 4: Effects of receptor-specific blockade on the AngII-evoked pH response. Panel A depicts the pH responses in the double-pulse protocol with AngII in the presence of HEPES/HCO(3) buffer as described in Fig. 2. Bars on top of panel indicate interval in which the cell is superfused with AngII; bottom traces are aligned so that application of AngII coincide with these bars. Panel B depicts results from a similar experiment in which the second AngII stimulation is preceded by the application of 100 nM PD 123319 (an AT(2) receptor subtype antagonist), as indicated by the bar below trace. Panel C shows an experiment in which 10 nM DuP 753 (an AT(1) receptor type antagonist) is present during the second AngII challenge. For the experimental results displayed in panel D, myocytes were incubated for 1 h at 37 °C in HEPES/HCO(3) buffer with 1 mM DTT. Cells were then loaded with c-SNARF-1 and placed in a superfusion chamber where they were challenged with 10 nM AngII in the continued presence of 1 mM DTT. Values for pH on the y axis were calculated as described under ``Experimental Procedures.'' Summary data are displayed in Table 2.





Additional experiments were pursued to more clearly define the receptor specificity of the AngII effect. DTT is known to selectively inactivate AT(1) receptors in isolated membranes(30) . In addition, recent studies by Lokuta and Rogers (18) have shown that it will block the AT(1)/phosphoinositide response in intact cardiac cells while sparing the AT(2) signaling path. As seen in Fig. 4D, DTT-treated cells still displayed an AngII-mediated alkalinization. Over many experiments there was no difference in the AngII response in the presence or absence of DTT (Table 2). It is important to note that control experiments demonstrated that DTT did not alter spontaneous beating behavior, resting pH, or the proton fluxes observed in NH(4)Cl pulse experiments compared to nontreated cells (data not shown). As an additional control it was found that DTT completely blocked the contractile responses of the myocytes to 100 nM Ang II (Fig. 5), further functional evidence that the AT(1) pathway was inhibited under these incubation conditions. Taken together, these data indicate that the AT(1)/phospholipase C signaling path is not involved and further underscore the importance of the AT(2)-like signaling mechanism in the alkalinization response.


Figure 5: Effects of DTT on spontaneous beating behavior of cultured myocytes. Cells were mounted in a superfusion chamber as described in Fig. 1and under ``Experimental Procedures.'' Contractile beating behavior of a single cell was monitored using a video dimensional analysis system as described previously(43) . Panels A and B show the contractile behavior of a cell before and 1.5 min after application of 100 nM AngII, respectively. Panels C and D depict the results from parallel experiments in which the cultures were preincubated for 1 h with DTT prior to the superfusion experiment. Shown is the beating behavior before (panel C) and 1.5 min after (panel D) application of AngII. DTT levels were maintained throughout this protocol.



Since stimulation of AT(2) receptors increases ARA production (18) , it is possible that AngII increases pH through elevations in this fatty acid. To examine this issue, the effects of exogenous ARA on pH were studied. As shown in Fig. 6A, application of 5 µM ARA led to a rapid increase in pHwith a magnitude nearly identical to that seen with 10 nM AngII. The threshold dose of ARA was in the range of 2 µM. The results from a large number of experiments revealed an increase of DeltapH = 0.044 ± 0.01 (n = 6) with 5 µM ARA. Similar to the AngII effect, Fig. 6B shows that the ARA-induced alkalinization was strictly HCO(3)-dependent (DeltapH = -0.010 ± 0.003, n = 3). The specificity of the ARA response was further characterized in Fig. 6C by the use of the non-metabolizable analogue ETYA (5 µM) (31, 32) which evoked a nearly identical pH response (DeltapH = 0.030 ± 0.003, n = 3). These data suggest that ARA mobilization may underlie, in part, the effects of AngII on pH in the cardiac myocytes.


Figure 6: Effect of arachidonic acid on pH. Myocytes were superfused with 5 µM ARA in HEPES buffer in the presence (panel A) or absence (panel B) of 25 mM HCO(3). Bars above traces indicate interval during which myocyte is exposed to ARA. Panel C shows that application of 5 µM ETYA in the presence of 25 mM HCO(3) resulted in an alkalinization of nearly identical magnitude as elicited by ARA. Summary data are displayed in Table 3. DeltapH values are reported as the means ± S.E. for three to six experiments.





It has been shown that the cardiac AT(2)/ARA signal transduction path depends on extracellular Ca in a characteristic manner; i.e. the response is blocked by extracellular EGTA but is insensitive to the Ca channel inhibitor nifedipine(18) . Thus if the AngII/pH response is related to ARA production, then a similar dependence on extracellular Ca should prevail. Paired pulse AngII stimulation experiments were performed in which 2 mM EGTA was added to the superfusion buffer (free extracellular [Ca]o = 100 nM) after the first AngII stimulation. Under these conditions the second AngII response was completely inhibited (Table 2). In contrast, addition of 1 µM nifedipine, which inhibits L-type Ca channels, had no effect on the AngII-mediated alkalinization (Table 2). Thus the profile of Ca dependence for the AngII/pHresponse is consistent with a role of ARA in the pathway.

Although the alkalinizing characteristics of TPA are distinct from those of ARA and AngII, PKC may still be involved in the response of these latter signaling molecules. To test this hypothesis, cells were preincubated with a PKC inhibitor, staurosporine (10 nM, 45 min at 37 °C), prior to loading with c-SNARF-1/AM. Spontaneous contractions of the cultured cardiac cells were maintained during this incubation. Control experiments indicated that this treatment inhibited PKC, as the effects of TPA were reduced to 10% of the normal response (Table 3). Under similar conditions, staurosporine inhibited the AngII- and ARA-mediated alkalinizations by 91 and 98% of control, respectively (Table 3). These results suggest that PKC, or perhaps another kinase, mediates the effects of TPA and AngII. Furthermore, they also suggest that the action of ARA is not direct but is also mediated by a kinase.


DISCUSSION

It is clear that intracellular pH is a crucial factor in cardiac myocyte function, as it is in many cells(1, 33, 34) . Thus it is not surprising to observe that there is a wide array of acid transporters which are responsible for the control and maintenance of pH(2) . Recent studies in heart show that hormone-evoked changes in acid transporter activities are also important mechanisms in the regulation of contractility(8, 9, 11) . It is within this context that studies were initiated to determine if myocyte pH is regulated by AngII, a cardioactive hormone that alters contractility as well as cardiac cell growth(15, 17, 25, 35) . An initial finding was that AngII rapidly alkalinizes myocytes through a receptor-mediated process. More focused experiments revealed the unexpected finding that, although the Na/H exchanger is a widely seen target for alpha(1)-adrenergic, endothelin signaling, and activated PKC in heart(8, 9, 11, 13) , AngII-evoked alkalinization is mediated by a selective activation of the Na/HCO(3) symport. Finally, it is likely that a previously described AT(2)-like signal transduction pathway involving fatty acid production is responsible for the hormone-evoked alkalinization.

Several observations demonstrate that the AngII-evoked alkalinization is caused by a direct activation of an acid transporter rather than indirectly through changes in contractility(25, 36, 37) . When the beating frequency was fixed with field stimulation or when contractility was completely blocked with nifedipine, AngII-evoked alkalinization was still observed. In addition, when cells were pretreated with DTT, under conditions which block the AngII-mediated increase in frequency, an identical alkalinization was still observed. Finally, flux studies revealed that an activation of an acid transporter was a likely mechanism underlying the alkalinization. Taken together, these data support the view that the action of AngII on heart cells includes a second messenger-mediated activation of one or more acid transporters.

It has been shown that in mammalian heart pH regulation is accomplished principally through two alkalinizing transporters, the Na/H exchanger and the Na/HCO(3) symport (3, 29) . Several results in the present study support the conclusion that AngII selectively activates the Na/HCO(3) symport. First the AngII alkalinization is strictly Na- and HCO(3)-dependent, as well as amiloride-insensitive. Although the lack of specificity of amiloride adds an element of caution, control experiments revealed that the Na/H exchanger was in fact inhibited under the conditions used. In addition, through the use of ammonia rebound protocols it was possible to measure the acid flux generated by each transporter and to demonstrate the selective activation of a HCO(3)-dependent proton flux by AngII. While it is possible that another previously unidentifed cardiac Na- and HCO(3)-dependent transporter is involved, the evidence reported here taken together with the work of other groups support the conclusion that the Na/HCO(3) symport underlies the response.

Parallel experiments with phorbol ester, TPA, further defined the selectivity of AngII action. This agent can activate distinct Na/H exchanger isoforms via PKC in several cell types, including rat heart myocytes(13) . The phorbol ester effects reported here were consistent with the view that PKC-mediated Na/H exchange activation had occurred. Thus, although the increases in resting pH evoked by AngII and TPA are of nearly identical magnitude and time course, the underlying molecular mechanisms are clearly distinct.

Since there is little information on intracellular signaling pathways that regulate the Na/HCO(3) symport, a focus of this study was to identify such underlying mechanisms. A series of experiments in the present study revealed that the AngII stimulation of the Na/HCO(3) symport is also independent of the AT(1)-phospholipase C path. The AT(2) receptor ligand, PD 123319 (100 nM), completely inhibited the alkalinization at doses that have no effect on AngII evoked phosphoinositide turnover (18) . DTT pretreatment, which has been shown to selectively inhibit the AT(1)-phospholipase C pathway, had no effect on the AngII-evoked alkalinization. There was one conflicting pharmacological result; the AT(1) receptor antagonist DuP 753 also inhibited alkalinization. However, these data should be interpreted with caution since detailed studies in a previous report reveal that low doses of this antagonist can partially inhibit the AT(2)-like pathway in intact heart cell cultures(18) . Thus the accumulated evidence support the view that the signaling pathway that leads to increases in pH is independent of the AT(1)/phospholipase C system.

The observations that ARA, and a nonmetabolizable analogue, ETYA, evoke a HCO(3)-dependent alkalinization are consistent with previous reports that describe a role for ARA as an important signaling molecule in heart. For example, studies have shown that this fatty acid can alter contractility, ion channel activity, and Ca homeostasis in heart cells (31, 38, 39, 40) . It is not yet known what role DeltapH may play in these various responses. Interestingly, some of these actions appear to be mediated by PKC(39, 40) . Consistent with this view, experiments with staurosporine reported here suggest that activation of PKC appears to be necessary for ARA-, as well as for AngII-induced alkalinization. Although control experiments revealed that PKC was inhibited under the incubation conditions used, these data should be interpreted with caution since staurosporine can also inhibit other kinases(41) . However, taken together these data extend the results of others that implicate kinases in the action of ARA in heart.

Alkalinization is a signaling motif that has been seen in cardiac cells with alpha(1)-adrenergic agonists, TPA, endothelin, and AngII (8, 9, 11, 12, 13, 42) . Since activation of the Na/H exchanger is frequently a mechanism underlying these responses, it was unexpected to find in the present study that AngII had no effect on this antiporter. The physiological significance of signaling pathways that favor one transporter compared to another is not defined at present, although the selective action of AngII reported here underscores the importance of such a diversity. In part the answer may lie in the notion that activation of transporters subserve a metabolic role within the cell beyond that of pH regulation alone.

In summary, these studies provide new insight into the regulation of pH. AngII stimulates intracellular alkalinization through an unusual pathway that is independent of phosphoinositide signaling and the Na/H exchanger and is likely to involve products of fatty acids such as ARA. Since it has been established that several of the PKC isoforms found in heart are activated by fatty acids, focused experiments with PKC isoforms are warranted to further define the action of ARA and AngII in this context.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL-28138 and Pol HL-27867 and NIH Training Grant GM-08181 (to T. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Maryland School of Medicine, Biomedical Research Facility, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-3169; Fax: 410-706-6676.

(^1)
The abbreviations used are: PKC, protein kinase C; AngII, angiotensin II; ARA, arachidonic acid; c-SNARF-1, carboxy-seminaphthorhodafluor-1; ETYA, 5,8,11,14-eicosatetraynoic acid; TPA, 12-O-tetradecanoyl phorbol-13-acetate; DTT, dithio-threitol; PLA(2), phospholipase A(2); DuP 753, 2-n-butyl-4-chloro-5-hy-droxymethyl-1-[(2`-(1H-terazol5-yl)biphenyl-4-yl) methyl]imidazole, potassium salt; PD 123319, 1,{[4-(dimethylamino)-3-methylphenyl] methyl}-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo (4,5-c)pyri-dine-6-carboxylic acid, ditrifluoroacetate.


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