INTRODUCTION

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ACID-EQUIVALENT EFFLUX from mammalian cardiac cells relies on the activity of membrane ionic carriers, in particular through the activities of both the Na⁺/H⁺ exchanger (4) and a Na⁺-linked influx (3, 8, 14, 19). With respect to the latter mechanism, different modes have been described, requiring or not requiring Cl efflux. Thus a Na⁺-dependent Cl/ exchange has been demonstrated in cultured embryonic chick heart cells (19) as well as in dog vascular smooth muscle cells (8, 9), whereas a Cl-independent Na⁺- symport has been described in sheep cardiac Purkinje fibers (3), guinea pig ventricular myocytes (14), and, more recently, cultured neonatal rat ventricular myocytes (12). Up to now, however, the -dependent alkalinizing carrier has not been characterized in ventricular myocytes of adult rat hearts. The Na⁺ dependency of the carrier has not been demonstrated (11), nor has its possible requirement for Cl been investigated. However, the adult rat heart is used in a number of experimental studies, either as a whole, perfused heart (10, 11, 26) or for isolation of cardiac myocytes (7, 27), and it would be useful to identify the properties of the -dependent alkalinizing mechanism. Furthermore, it may be of interest to determine the relative contribution of the Na⁺/H⁺ exchanger and of the -dependent alkalinizing carrier to intracellular pH (pH_i) regulation in adult rat ventricular myocytes under physiological conditions and especially under pathophysiological conditions.

The present work was designed to investigate the Cl and Na⁺ dependency of alkalinizing influx in ventricular myocytes of adult rat hearts and to determine to what extent this carrier mechanism contributes to acid-equivalent extrusion from adult rat ventricular myocytes after an intracellularly induced acid load. Moreover, we compared the results obtained in ventricular myocytes of normal adult rats with those obtained in myocytes of streptozotocin (STZ)-induced diabetic rats. Indeed, previous studies showed that ventricular myocytes of diabetic rats have a significant decrease in Na⁺/H⁺ exchange activity (15, 18, 22). In addition, because little is as yet known about the modulation of the -dependent process (16, 17, 27), we also examined the effects of intracellular Ca²⁺ (Ca²⁺_i) and of glycolytic inhibition on the carrier activation after the induction of intracellular acidosis. Indeed, such intracellular factors may be of particular importance during ischemia and on reperfusion after an ischemic episode (20, 25, 26). The intracellular fluoroprobe carboxy-seminaphthorhodafluor-1 (SNARF-1) was used to record pH_i, and all experiments were carried out in -buffered solutions.

	METHODS

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All procedures were in accordance with the regulations laid down by the Ministère de l'Agriculture et de la Forêt, France, for the care and use of laboratory animals.

Male Wistar rats weighing between 150 and 180 g were fasted overnight and made diabetic by the injection of STZ (40 mg/kg; Sigma, St. Louis, MO) into the femoral vein. STZ-treated and age-matched control animals were maintained on the same diet until they were used 3-4 wk later. This period of diabetes was chosen because previous studies characterized cardiac alterations during this period (11, 13, 18). The diabetic state was assessed by measurement of nonfasting glucose concentration in blood samples collected at the time of heart excision. Mean glucose levels were 10.2 ± 0.3 and 45.9 ± 2.4 mM for normal (n = 20) and diabetic (n = 20) rats, respectively.

Isolation of rat ventricular myocytes. Briefly, single ventricular cells were obtained from hearts of Wistar rats [250-300 g body wt; anesthetized with thiopental sodium (50 mg/kg body wt ip)] using a combination of enzymatic (collagenase 0.28 mg/ml, Yakult, Japan; protease type XIV 0.05 mg/ml, Sigma) and mechanical dispersion. The composition of the basic solution used for cell isolation and further details of the procedure have been described previously (7). Calcium-tolerant, rod-shaped ventricular myocytes were used on the day of isolation. To deplete intracellular Cl, the cells were suspended in Cl-free Kraftbrühe medium (6) after the dissociation at room temperature until use.

Experimental solutions and chemicals. The -buffered Tyrode solution contained (in mM) 117 NaCl, 5.4 KCl, 1 CaCl₂, 1.2 MgCl₂, 11 glucose, and 23 NaHCO₃. All -buffered solutions were equilibrated with 5% CO₂-95% O₂ and had a pH of 7.4 at 37°C. In Na⁺-free, CO₂/-buffered Tyrode solution, NaCl and NaHCO₃ were replaced with 140 mM N-methyl-D-glucamine (NMDG) and the pH was adjusted to 7.4 with HCl under continuous bubbling with 5% CO₂-95% O₂. Cl-free, CO₂/-buffered Tyrode solution contained (in mM) 117 sodium gluconate, 5.4 potassium gluconate, 4 calcium gluconate (hemicalcium salt), 23 NaHCO₃, 1 MgSO₄, and 11 glucose. In Na⁺- and Cl-free, CO₂/-buffered Tyrode solution, sodium gluconate and NaHCO₃ were replaced by 140 mM NMDG and 117 mM gluconic acid. When required, 5 mM 2-deoxy-D-glucose (2DG) was substituted for glucose. When used, the Ca²⁺/calmodulin (CaM) protein kinase II inhibitor KN-62 {1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, Sigma; Refs. 21, 29} was first dissolved in dimethyl sulfoxide (DMSO) before addition to Tyrode solution (final DMSO concn <0.1%). NH₄Cl (Sigma) was added directly to solutions shortly before use. Addition and then removal of NH₄Cl was used to induce an acid load to activate the pH_i-regulatory mechanisms (1). For the experiments carried out in Cl-free solution, 10 mM NH₄Cl was replaced with 10 mM (NH₄)₂SO₄. The nigericin calibration solutions used in this study have been described elsewhere (14).

Loading of cardiac cells with BAPTA-AM. In experiments designed to investigate a possible role of calcium in the modulation of the -dependent process activity, myocytes were incubated for 30 min in the presence of 25 µM of the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; Molecular Probes, Eugene, OR). Cells were then allowed to hydrolyze the ester for at least 1 h before the beginning of the investigation. CaCl₂ was omitted in the external solution. With the use of isolated cardiac myocytes loaded with indo 1, Pucéat and co-workers (23) have previously shown that this BAPTA-AM concentration and the BAPTA-loading protocol efficiently prevent a rise in free Ca²⁺_i.

Measurement of pH_i. The pH_i of single isolated myocytes was monitored using the pH-sensitive fluorescent dye carboxy-SNARF-1 (Molecular Probes; Ref. 2). Cells were loaded with SNARF by incubating them in a 5 µM solution of the acetoxymethyl ester for 10 min at room temperature.

Calculation of sarcolemmal acid efflux. Details of the method for calculating acid efflux (J^e_H) during pH_i recovery in ventricular myocytes have been described previously (14, 24). Briefly, J^e_H was estimated using the following equation: J^e_H = _T × dpH_i/dt, where _T is the total intracellular buffering power and dpH_i/dt is the rate of pH_i recovery at any given pH_i. The efflux through the -dependent process was estimated from pH_i recovery occurring in CO₂/-buffered solution in the presence of amiloride (1 mM; Sigma), an inhibitor of Na⁺/H⁺ exchange (12, 14, 27). Under these conditions, _T is the sum of intrinsic buffering power (_i) plus buffering power due to intracellular CO₂/ (_CO2). _CO2 is given by where []_i is intracellular concentration. []_i is given by where []_o is extracellular concentration and pH_o is extracellular pH. Because we found no difference in _i between normal and STZ-diabetic myocytes, the mean _i value of 31.4 ± 0.75 mM/pH unit (at mean pH_i 7.13 ± 0.019; n = 88 values) was used for both groups of cells (18).

Statistics. All values of pH_i and J^e_H are quoted as means ± SE along with the number of observations, n. Statistical significance was estimated by Student's t-test or analysis of variance followed by Student-Newman-Keuls test to locate differences between groups. Differences were considered significant at the level of P < 0.05.

	RESULTS

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Na⁺ dependency of -dependent alkalinizing transport in ventricular myocytes isolated from adult rat hearts. The Na⁺ dependency of the -dependent pH_i-regulating process has not been previously demonstrated in ventricular myocytes from adult rats (11). The experiment illustrated in Fig. 1 was therefore carried out to test such a dependency in a normal cell. To study the -dependent mechanism alone, 1 mM amiloride was applied throughout the experiment to inhibit Na⁺/H⁺ exchange in -buffered Tyrode solution. The myocyte was subjected to two consecutive intracellular acid loads induced by the NH₄Cl (10 mM)-removal method, first under control conditions and second in the absence of external Na⁺ (replaced by NMDG). In the absence of external sodium, pH_i recovery after intracellular acid load was almost completely inhibited (n = 6 cells), indicating a Na⁺ requirement for the -dependent alkalinizing transporter. In addition, it is worth noting here that when experiments in zero-sodium medium were performed at lower extracellular Ca²⁺ (0.1 or 0.5 mM), the pH_i recovery was still inhibited, thus ruling out any role for Ca²⁺ overload in the observed inhibition. Similar results were obtained in ventricular myocytes isolated from diabetic adult rat hearts (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of external Na+ (Na+o) removal (Na+ replaced by N-methyl-D-glucamine) on intracellular pH (pHi) recovery from intracellular acidification in a normal ventricular myocyte bathed in CO2/-buffered Tyrode solution (pHo 7.4). Amiloride (1 mM) was applied to inhibit Na+/H+ exchange. Cell was acid-loaded twice by NH+4 (10 mM NH4Cl)-prepulse method, 1st pulse under control conditions and 2nd pulse in absence of Na+o. Experiment was carried out in presence of 1 mM Ca2+o.

Cl-independent Na⁺- cotransport in adult rat ventricular myocytes. As yet, the Cl dependency of the Na⁺- and -dependent transport has not been studied in ventricular myocytes of adult rat heart. Thus, if Na⁺- and -dependent transport in the adult rat heart is mediated by a Na⁺-dependent Cl/ exchanger, depletion of intracellular Cl should inhibit it. To test this, cells were incubated in Cl-free solution, external Cl being replaced by gluconate (see METHODS). Figure 2A shows a control NH⁺₄ pulse in the presence of Cl. After external Cl was removed, an intracellular alkalinization developed (pH_i = 0.165 ± 0.006 pH unit, n = 8 cells), tending towards a plateau after ~10 min. This acute alkalinization, which was unaltered by removal of external sodium (n = 3 cells; data not shown), has been previously observed in guinea pig ventricular myocytes and attributed to activation of influx in exchange for Cl efflux via a Na⁺-independent Cl/ exchanger (14). Figure 2A also shows that the rate of pH_i recovery after an acid load induced in Cl-free solution was not noticeably different from that seen in the control situation. In a total of four cells, acid efflux during the pH_i recovery phase calculated at pH_i 7.0 was 2.21 ± 0.07 meq · l¹ · min¹ in presence of external Cl and was not significantly different (2.48 ± 0.19 meq · l¹ · min¹) after 40 min in Cl-free solution.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effects of removal (Cl replaced by gluconate) on pHi recovery due to Na+- and -dependent transport in CO2/-buffered solution + 1 mM amiloride. In A, normal cell was acid-loaded twice by NH+4-prepulse method, 1st pulse under control conditions and 2nd pulse in absence of . In B, normal cell was stored in Cl-free Kraftbrühe medium ~3 h before acid loading. In all experiments, NH+4 pulse was induced by adding (and then removing) 10 mM (NH4)2SO4.

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Comparison of pH_i regulation in ventricular myocytes isolated from normal and diabetic rat hearts. Figure 3A shows representative recordings of pH_i and pH_i changes induced by the addition and subsequent removal of 10 mM NH₄Cl (1) in CO₂/-buffered Tyrode solution. The two pH_i traces obtained in ventricular myocytes isolated from normal and STZ-induced diabetic rat hearts were superimposed to facilitate comparison. No difference exists in steady-state pH_i values between the two groups [7.06 ± 0.019 (n = 45) and 7.06 ± 0.017 (n = 36) in normal and diabetic myocytes, respectively]. These pH_i values are in good agreement with those recorded in diabetic papillary muscles (15). However, pH_i recovery from the intracellular acidification was significantly slowed down in diabetic cardiac cells [dpH_i/dt = 0.092 ± 0.005 (n = 12) and 0.062 ± 0.006 pH units/min (n = 12) in normal and diabetic myocytes, respectively, at pH_i 6.9; P < 0.05].

View larger version (16K): [in this window] [in a new window]   Fig. 3.   A: effects of diabetes on pHi recovery from intracellular acidification in CO2/-buffered Tyrode solution. pHi recordings were obtained from either a normal or a diabetic isolated myocyte. The 2 traces were superimposed to ease comparison. Each single myocyte was acid loaded by NH+4 (10 mM NH4Cl)-prepulse method. B: effects of diabetes on pHi recovery due to -dependent mechanism alone (in presence of 1 mM amiloride). Superimposed pHi traces were obtained from a normal cell and from a diabetic cell.

pH_i dependence of J^e_H carried by Na⁺- cotransport in ventricular myocytes from normal and diabetic rats. Figure 4 plots J^e_H as a function of pH_i for normal and diabetic cells (see METHODS for determination). Total efflux carried when both Na⁺- cotransport and Na⁺/H⁺ exchanger were operational was estimated under control conditions in CO₂/-buffered solution. J^e_H carried by the Na⁺- cotransport alone was estimated in CO₂/-buffered solution in the presence of amiloride (1 mM). Comparison of the relationship between pH_i and total J^e_H between normal (n = 12 cells) and diabetic (n = 12 cells) myocytes clearly demonstrates that total acid-equivalent efflux is significantly decreased in diabetic rat ventricular myocytes over the pH_i 6.75-6.90 range. Thus at pH_i 6.75 average total J^e_H is decreased by ~31% in diabetic rat ventricular myocytes. On the other hand, the relationship between J^e_H and pH_i obtained for the two groups of cells in the presence of amiloride when acid-equivalent efflux was carried by the Na⁺- cotransport shows no difference between normal (n = 12) and diabetic (n = 12) cells. Therefore, these results indicate that Na⁺- cotransport activity is unaffected by diabetes over the pH_i range studied. As a consequence, the observed decrease in total J^e_H from the diabetic myocytes essentially results from the significant decrease in Na⁺/H⁺ exchange activity previously shown (15, 18, 22).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effects of diabetes on pHi dependence of acid efflux [JeH = (rate of pHi recovery) × (total intracellular buffering power)] measured without (filled symbols; total JeH) or with (open symbols; JeH carried by Na+- cotransport) amiloride in -buffered medium. Each point represents average efflux (± SE) calculated at a test pHi from 12 normal cells (circles) or 12 diabetic cells (squares). * P < 0.05 vs. normal.

Effects of Ca²⁺_i buffering and inhibition of Ca²⁺/CaM protein kinase II on Na⁺- cotransport activity. Recent reports have shown that the other Na⁺-dependent alkalinizing carrier, the Na⁺/H⁺ exchanger, is modulated by Ca²⁺_i, so that increasing Ca²⁺_i induces a stimulation of the exchange (31). In particular, the control of Na⁺/H⁺ exchange activity may depend on a phosphorylation by the Ca²⁺/CaM-dependent protein kinase II (5). If calcium did underlie part of the modulation of the activity of the Na⁺- cotransport, then the presence of an Ca²⁺_i buffer would lead to a change in its activity in both normal and diabetic ventricular myocytes. Cells were therefore loaded with BAPTA-AM (25 µM) and superfused with a Ca²⁺-free solution to buffer Ca²⁺_i to a low level (23). Figure 5 shows that BAPTA treatment induced no change in the pH_i dependence of the acid efflux carried by the cotransport (estimated in HCO₃-CO₂ buffer + amiloride). This is in favor of the hypothesis that Ca²⁺_i has no influence on Na⁺- cotransport activity.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effects of buffering Ca2+ (in Ca2+-free solution) on pHi dependence of JeH carried by Na+- cotransport in normal and diabetic ventricular myocytes. Control: n = 12 normal () and 12 diabetic () cells; BAPTA-loaded: n = 11 normal () and 8 diabetic () cells.

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View larger version (11K): [in this window] [in a new window]   Fig. 6.   A: effects of the inhibitor of the Ca2+/calmodulin-dependent protein kinase II, KN-62 (2 µM), on pHi recovery due to Na+- cotransport in a normal ventricular cell. Myocyte was acid loaded twice, 1st pulse under control conditions, 2nd pulse in presence of inhibitor. B: in same ventricular cell, pHi dependence of JeH carried by Na+- cotransport in absence () or presence () of KN-62.

Effect of 2DG, an inhibitor of glycolysis, on Na⁺- cotransport activity. Glycolytic metabolism, through glycolytic ATP production, has been shown to be important for the regulation of various membrane proteins, including the Na⁺/H⁺ exchanger (33, 34). No data are so far available concerning the possible modulatory influence of glycolysis on Na⁺- cotransport. To investigate this possibility, we used the glycolytic inhibitor 2DG (5 mM) instead of glucose in CO₂/-buffered Tyrode solution containing 1 mM amiloride. This procedure has been shown to greatly reduce cellular ATP content (33, 34). As illustrated in Fig. 7A, the first observation was that the presence of 2DG resulted in a rapid decrease in steady-state pH_i, which stabilized after ~5 min (pH_i = 0.09 ± 0.017 pH_i unit; n = 13 cells). This decrease likely resulted from reduced basal activity of the cotransporter, which has been shown to participate in steady-state pH regulation (14). Furthermore, pH_i recovery from an intracellular acid load was slowed significantly in the presence of 2DG [dpH_i/dt = 0.030 ± 0.007 pH unit/min in the presence of 2DG (n = 8 cells) and 0.047 ± 0.004 pH unit/min under control conditions (n = 8 cells), at pH_i 6.9; P < 0.05].

View larger version (12K): [in this window] [in a new window]   Fig. 7.   A: effects of application of 2-deoxyglucose (2DG; 5 mM in glucose-free solution) on pHi recovery in a normal ventricular myocyte. Myocyte was acid loaded twice, 1st pulse under control conditions and 2nd pulse in presence of the metabolic inhibitor. B: representative pHi dependence of Na+- cotransport under control conditions () and in presence of 2DG (). pHi recoveries illustrated in A and recorded in same cell were used to estimate JeH.

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	DISCUSSION

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The present work provides evidence for the existence of a Cl-independent Na⁺- cotransport contributing to pH_i recovery from an intracellular acid load in normal and diabetic adult rat ventricular myocytes. Na⁺- cotransport accounts for 33% of the total acid-equivalent efflux from normal adult myocytes after intracellular acidification at pH_i 6.75 in CO₂/-buffered solution. In addition, the activity of this carrier, which is not affected either by decreasing Ca²⁺_i or by inhibiting CaM-kinase II, is downregulated by inhibition of glycolysis.

Characterization of Na⁺- cotransporter in adult rat ventricular myocyte. A Na⁺- symport carrier has been demonstrated in sheep cardiac Purkinje fiber (3), guinea pig ventricular myocyte (14), and cultured neonatal rat ventricular myocytes (12). Here, we demonstrate the Na⁺ dependency of the alkalinizing -dependent process operating in adult rat ventricular myocytes. In addition, the Na⁺- cotransporter displays no absolute requirement for Cl as demonstrated by others in cardiac cells of different species (8, 9, 19). The cotransporter is obviously expressed in various cardiac cell types of several mammalian species (3, 12, 14). However, the cotransporter may be, in some aspects, different in the adult rat ventricular myocyte (present work) from that in the guinea pig myocyte, because opposite responses to some agonists' stimulation (namely epinephrine and extracellular ATP) have been observed (17, 27). Our results allow us to exclude the possibility, in explaining these opposite responses, of a different mode of -dependent alkalinizing carrier between these two species.

Functional importance of Na⁺- cotransport in adult rat ventricular myocytes. In normal myocytes, the contribution to the total acid efflux of the Na⁺- cotransporter and of the Na⁺/H⁺ exchanger after intracellular acidification (the latter being calculated from the difference between total acid efflux and sensitive acid efflux; see Fig. 4) was estimated to be 31 and 69%, respectively, at pH_i 6.9 and 33 and 67%, respectively, at pH_i 6.75. These results are in accordance with those obtained in the guinea pig ventricular myocyte, in which the efficiency of the Na⁺- cotransport was estimated to be ~40% at pH_i 6.9 (14). Terzic et al. (27) suggested that -dependent pH_i recovery from an acid load could potentially substitute for the Na⁺/H⁺ exchanger when the latter is inhibited, as in some pathophysiological states. We examined this possibility in ventricular myocytes from diabetic rats, in which we have demonstrated a significant decrease in Na⁺/H⁺ exchange activity (15, 18). It can be inferred from Fig. 4 that acid efflux carried by Na⁺- cotransport remains nearly identical in both normal and diabetic cells. However, in diabetic myocytes, the contribution of the Na⁺- cotransport is increased up to 38% at pH_i 6.9 in comparison to normal cells. Moreover, the contribution of this mechanism even becomes predominant over the Na⁺/H⁺ exchanger contribution at a lower pH_i (e.g., pH_i 6.75), reaching ~58% of the total acid efflux. Unfortunately, the methods used here in isolated myocytes did not allow us to assess the contribution of the two carriers at lower pH_i values, such as those that may be reached during ischemia (down to ~6.0) and in the initial stage of reperfusion after an ischemic episode.

Modulation of Na⁺- cotransport activity. In contradistinction to the recently reported role for Ca²⁺_i in the control of Na⁺/H⁺ exchange activity (18), the present study shows no change in the activation of Na⁺- cotransport under conditions of Ca²⁺_i buffering induced by pretreatment of the cells with a Ca²⁺ chelator in Ca²⁺-free medium. Furthermore, the presence of a specific inhibitor against Ca²⁺/CaM kinase II, KN-62 (29), was without effect on the cotransport activity. These negative results seem to exclude the possibility that a Ca²⁺-dependent pathway, in particular through Ca²⁺/CaM kinase II-dependent phosphorylation, may be involved in the control of cotransport basal activity.