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(Circulation Research. 1997;80:253-260.)
© 1997 American Heart Association, Inc.

Articles

Modulation by pH_o and Intracellular Ca²⁺ of Na⁺-H⁺ Exchange in Diabetic Rat Isolated Ventricular Myocytes

Karine Le Prigent, Dominique Lagadic-Gossmann, Danielle Feuvray

the Laboratoire de Physiologie Cellulaire (K. Le P., D.F.), CNRS ERS 100, Universite Paris XI, Orsay, France, and INSERM-Groupe DRT (D.L-G.), Faculte de Pharmacie, Rennes, France.

Correspondence to Dr D. Lagadic-Gossmann, INSERM-Groupe DRT, 2, avenue Pr. Leon Bernard, Faculte de Pharmacie, 35 043 Rennes cedex, France.

	Abstract

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We have previously shown that diabetes is associated with a decrease in Na⁺-H⁺ exchange activity in rat cardiac papillary muscle. The present work has been carried out in order to elucidate the factors responsible for such an alteration. Thus, we have studied the effects of pH_o and intracellular Ca²⁺ on Na⁺-H⁺ exchange in ventricular myocytes isolated from streptozotocin-induced diabetic rat hearts. pH_i was recorded using carboxy-seminaphthorhodafluor (SNARF-1). The NH₄⁺ (10 mmol/L) prepulse method was used to induce an acid load in order to activate Na⁺-H⁺ exchange in HEPES-buffered Tyrode's solution. Whereas diabetes did not change intracellular buffering power, it significantly decreased acid efflux through Na⁺-H⁺ exchange (acid efflux, 4.32±0.4 [n=32, normal cells] versus 2.5±0.2 [n=43, diabetic cells] meq/L per minute at pH_i 6.9; P<.02). Upon changes of pH_o (at a range of 8.0 to 6.8), acid efflux similarly varied in normal and diabetic cells, thus pointing to an unchanged pH_o sensitivity of Na⁺-H⁺ exchange. Buffering of intracellular Ca²⁺ by pretreatment of the cells with BAPTA-AM (25 µmol/L, Ca²⁺-chelator) resulted in a decrease by 58% of acid efflux in the diabetic group. This decrease was even more marked in normal cells (by 74%). Interestingly, the pH_i dependence of the acid efflux carried by Na⁺-H⁺ exchange then became identical in both groups of cells, thus pointing to a role for intracellular Ca²⁺ in the diabetes-related alterations of the exchange. Inhibition of calmodulin (by 1.5 µmol/L calmidazolium) and of Ca²⁺/calmodulin-dependent protein kinase II (by 2 µmol/L 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine [KN-62]) significantly slowed down pH_i recovery in both normal and diabetic cells. However, the effect of KN-62 was significantly lower in diabetic cells (efflux decreased by 17%) compared with normal cells (decrease by 45%). In conclusion, these data, in light of recent observations showing a decreased [Ca²⁺]_i associated with diabetes in isolated ventricular myocytes, suggest that changes in intracellular Ca²⁺ may play an important role in altering Na⁺-H⁺ exchange activity in diabetic ventricular myocytes. They also point to diabetes-related alterations in the Ca²⁺/calmodulin protein kinase II–dependent phosphorylation of Na⁺-H⁺ exchange.

Key Words: Na⁺-H⁺ exchange • ventricular myocyte • diabetes • intracellular Ca²⁺ • pH_o

	Introduction

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It is now well recognized that the regulation of pH_i is particularly important in the myocardium for two major reasons: (1) a change in pH_i induces a change in contractility,¹ and (2) activation of the Na⁺-H⁺ exchange, one of the major pH_i regulatory mechanisms, after ischemia-induced acidosis, plays a key role in the development of the alterations related to reperfusion. With respect to the latter point, several works have indeed shown that pharmacological inhibition of this exchange resulted in significant protection of the reperfused ischemic myocardium.^{2 3} In this context, the diabetic rat heart appears to be an interesting experimental model, since the significant decrease of Na⁺-H⁺ exchange activity associated with this pathological state^{4 5} has been found to confer some protection to the diabetic heart after an episode of ischemia/reperfusion.⁶ However, the mechanism underlying the slowed activity of the exchanger is still unknown. Therefore, it seems important to elucidate this mechanism in view of the development of new means to inhibit Na⁺-H⁺ exchange and, hence, an improvement in the ability to treat the diseased myocardium. In a previous study, we have shown that the intracellular level of Na⁺ was significantly increased in the diabetic papillary muscle, which led us to suggest that this increase could play a role in the slowing down of the Na⁺-H⁺ exchange activity associated with diabetes.⁷ Besides the sensitivity of the exchanger to Na⁺, it is known that extracellular H⁺ ions and intracellular Ca²⁺ are involved in the modulation of the exchanger. Several works have indeed shown that extracellular H⁺ ions exert an inhibitory action on Na⁺-H⁺ exchange,^{8 9 10} whereas there is growing evidence in favor of a stimulation of the exchange by intracellular Ca²⁺.^{11 12 13 14} Therefore, alterations in the modulation by these ions of Na⁺-H⁺ exchange would appear to explain the reduced activity of the cardiac exchanger observed upon diabetes. In this respect, it is worth noting that diabetes has been found to be associated with a decreased [Ca²⁺]_i in isolated ventricular myocytes.^{15 16}

In the present work, we investigated the modulation by extracellular H⁺ and intracellular Ca²⁺ of pH_i recovery due to Na⁺-H⁺ exchange in myocytes isolated from normal and STZ-induced diabetic rat hearts. To this purpose, pH_i was recorded using the intracellular fluoroprobe carboxy-SNARF-1, and the different solutions were buffered with HEPES in order to turn off the bicarbonate-dependent pH_i-regulating mechanisms, especially the Na⁺-HCO₃^- symport.¹⁷ ß_i, which is needed when calculating acid effluxes,¹⁸ was estimated and compared between both groups. We also tested the effects of calmidazolium, an inhibitor of calmodulin,¹⁹ and the effects of KN-62, an inhibitor of Ca²⁺/CaM kinase II,^{20 21} on the activity of Na⁺-H⁺ exchange.

	Materials and Methods

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

Male Wistar rats weighing 150 to 180 g were fasted overnight and made diabetic by an injection of STZ (40 mg/kg, Sigma Chemical Co) into the femoral vein. STZ-treated and age-matched control animals were maintained on the same diet until they were used 3 to 4 weeks later. This period of diabetes was chosen because previous studies have characterized cardiac alterations during this period.^{4 6 16 22} 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 mmol/L for normal rats (n=20) and diabetic rats (n=20), respectively.

Isolation of Rat Ventricular Myocytes
Briefly, single ventricular myocytes were obtained from the hearts of rats (anesthetized with 50 mg·kg^-1 body wt IP pentothal) using a combination of enzymatic (0.28 mg/mL collagenase, Yakult; 0.05 mg/mL protease type XIV, Sigma) and mechanical dispersion. The composition of the basic solution and further details of the procedure have been described previously.²² Ca²⁺-tolerant rod-shaped ventricular myocytes were used on the day of isolation.

Experimental Solutions and Chemicals
HEPES-buffered Tyrode's solution contained (mmol/L) NaCl 140, KCl 5.4, CaCl₂ 1, MgCl₂ 1.2, glucose 11, and HEPES 10, with pH adjusted to 7.4 at 37°C with NaOH. In Na⁺-free Tyrode's solution, NaCl was replaced with 140 mmol/L N-methyl-D-glucamine, and the pH was adjusted to 7.4 at 37°C with HCl. When Tyrode's solution with a pH of 8.0 or 6.8 was needed, HEPPS or PIPES buffer was used in replacement of the HEPES buffer. Ammonium chloride (Sigma) was added directly to solutions shortly before use. Addition and then removal of NH₄Cl was used to induce an acid load in order to activate the pH_i regulatory mechanisms.²³ Because HCO₃^--free solutions were used in the present study, mainly Na⁺-H⁺ exchange was functional during pH_i recovery. Nigericin calibration solutions used in the present study have been described elsewhere.¹⁷ BAPTA-AM was purchased from Molecular Probes. Calmidazolium and the protein kinase inhibitor KN-62 were from Sigma and were added (from stock solutions in dimethyl sulfoxide) to Tyrode's solution immediately before use (final concentration of dimethyl sulfoxide, <0.1%).

Loading of Cardiac Cells With BAPTA-AM
In experiments designed to investigate the role of Ca²⁺ in the Na⁺-H⁺ exchange modulation, myocytes were incubated for 30 minutes in the presence of 25 µmol/L BAPTA-AM. Cells were then allowed to hydrolyze the ester for at least 1 hour before the beginning of the investigation. CaCl₂ was omitted in the external Tyrode's solution. Using indo 1–loaded cardiac cells, Puceat et al²⁴ have previously shown that such a concentration of BAPTA-AM 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).²⁵ Cells were loaded with SNARF by incubating them in a 5 µmol/L solution of the acetoxymethyl ester for 10 minutes at room temperature.

Carboxy-SNARF-1 fluorescence from individual cells was measured with an inverted microscope (Nikon Diaphot) converted to epifluorescence. SNARF-loaded cells were excited with light at 515 nm, and the resulting fluorescence at 590 and 640 nm was measured using two photomultiplier tubes (Nikon). The signals were then digitized at 0.5 kHz (CED 1401 intelligent interface, Cambridge Electronic Design) and stored for later analysis on the hard disk of a computer. The emission ratio 590/640 obtained from intracellular SNARF was calculated and converted to a linear pH scale by use of in situ calibration data obtained at the end of the experiment using the nigericin technique described elsewhere.^{25 26} Finally, the calibrated pH_i signal was averaged over 0.5-second intervals.

Estimation of ß_i at Different pH_i Levels
The method used to estimate ß_i has been described previously.^{17 27} Briefly, a stepwise reduction of external NH₄Cl (from 20 mmol/L) was applied to a selected myocyte. Each reduction in NH₄⁺ resulted in the generation of intracellular H⁺, which was due to dissociation of NH₄⁺ into H⁺ ions and NH₃, with subsequent efflux of NH₃. The resultant changes in pH_i were used to estimate ß_i as follows:

where [NH₄⁺]_i=([H⁺]_ix[NH₄⁺]_o)/[H⁺]_o. The experiments were carried out in the absence of extracellular Na⁺ in order to prevent acid extrusion and Ba²⁺ (1 mmol/L) in order to reduce NH₄⁺ efflux through K⁺ channels.²⁷

Calculation of Sarcolemmal J^e_H
Details of the method for calculating J^e_H during pH_i recovery in ventricular myocytes have been described previously.¹⁷ Briefly, J^e_H was estimated using the following equation: J^e_H=ß_TxdpH_i/dt, where ß_T is the total intracellular buffering power and dpH_i/dt is the rate of pH_i recovery at any given pH_i. In HEPES-buffered medium, ß_T equals ß_i (which was empirically estimated for both normal and diabetic cells in the present study).

Statistics
All data are quoted as mean±SEM along with the number of observations (n). Statistical significance was estimated by Student's t test or ANOVA followed by the Student-Newman-Keuls test to locate differences between groups. Differences were considered significant at a level of P<.05.

	Results

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Effects of Diabetes on J^e_H Carried by Na⁺-H⁺ Exchange in Rat Isolated Ventricular Myocytes
Fig 1 shows representative recordings of pH_i, obtained using carboxy-SNARF-1, in single cardiac ventricular myocytes isolated from a normal and a diabetic rat. The two pH_i traces were superimposed to ease the comparison. Each recording illustrates pH_i recovery from an intracellular acid load, induced by the addition and subsequent removal of 10 mmol/L NH₄Cl²³ in HEPES-buffered Tyrode's solution. Under these conditions, although no difference in steady state pH_i could be found (7.18±0.01 [n=85] versus 7.17±0.01 [n=78] in normal and diabetic myocytes, respectively), pH_i recovery following the acidification was significantly slowed down by diabetes (at pH_i 6.9, dpH_i/dt was 0.115±0.01 pH units/min [n=32 normal cells] versus 0.065±0.004 pH units/min [n=43 diabetic cells]; P<.001).

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of diabetes on pHi recovery following an intracellular acidification in HEPES-buffered Tyrode's solution. pHi was recorded from single isolated myocytes using the fluorescent probe carboxy-SNARF-1. Each single (normal or diabetic) myocyte was acid-loaded by the NH4+ prepulse method (10 mmol/L NH4Cl was added and then removed from the superfusate; pHo 7.4). The two pHi recoveries following the acid load were superimposed to ease the comparison.

Since HEPES was used as an extracellular buffer, Na⁺-H⁺ exchange was the main mechanism regulating pH_i after the acid load.¹⁷ In order to appreciate the effect of diabetes on the J^e_H carried by Na⁺-H⁺ exchange (J^e_H=ß_ixdpH_i/dt), we first estimated ß_i, over the pH_i range of 6.85 to 7.45, using the stepwise removal of external NH₄Cl (from 20 mmol/L; see "Materials and Methods"). The procedure was conducted on the two groups of myocytes (normal [n=12] and diabetic [n=11]). The resulting estimates of ß_i at various values of pH_i are given in Fig 2. It is clear from this figure that diabetes remained ineffective on ß_i in cardiac cells. Moreover, in the pH_i range presently tested, ß_i appeared to be independent of pH_i. Therefore, a mean ß_i value of 31.4±0.75 (mmol/L)·pH unit^-1 (n=88 individual values from 12 normal plus 11 diabetic cells) was used to calculate J^e_H through Na⁺-H⁺ exchange in both groups of cells.

View larger version (17K): [in this window] [in a new window]   Figure 2. Graph showing the effects of diabetes on ßi in rat isolated ventricular myocytes (n=12 normal cells and 11 diabetic cells). Each data point represented an individual ßi value estimated at a given pHi value (corresponding to the midpoint of each stepwise acid load).

Fig 3 illustrates the relationship between pH_i and J^e_H carried by Na⁺-H⁺ exchange calculated in normal (n=32 cells) and diabetic (n=43 cells) myocytes. This figure shows that diabetes results in a leftward shift of the activation curve for Na⁺-H⁺ exchange, which means that its activity is significantly decreased (by 42% at pH_i 6.9, P<.02) in diabetic rat ventricular myocytes.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effects of diabetes on the pHi dependence of JeH (dpHi/dtxßi) carried by Na+-H+ exchange. Each point represents the average efflux (±SEM) calculated at different test pHi levels from 32 normal cells and 43 diabetic cells.

Effects of pH_o on Na⁺-H⁺ Exchange Activity in Diabetic Ventricular Myocytes
In cardiac tissue, as well as in other tissues, it has been shown that extracellular H⁺ ions inhibit Na⁺-H⁺ exchange activity.^{8 9 10} This led us to postulate that in diabetic myocytes, compared with normal myocytes, the observed decrease of Na⁺-H⁺ exchange activity might result from a different pH_o sensitivity, so that the exchange would be more inhibited at physiological pH_o. In order to test this hypothesis, the effects of altering pH_o on Na⁺-H⁺ exchange were examined in both groups of cells. Fig 4 illustrates the effects of changing pH_o from 7.4 to 8.0 (panel A) and from 7.4 to 6.8 (panel B) on pH_i in diabetic myocytes. As previously reported for normal cardiac tissue,^{9 10} increasing pH_o resulted in both an increase of steady state pH_i and a stimulation of the pH_i recovery after the acid load, whereas decreasing pH_o exerted opposite effects. These effects on Na⁺-H⁺ exchange are further outlined in Fig 5, which shows the pH_i dependence of this exchange at the different pH_o levels tested. At pH_o 8.0, compared with pH_o 7.4 (panel A), the pH_i dependence of Na⁺-H⁺ exchange in diabetic cells was shifted to the right (thus pointing to a stimulation of the exchange), whereas at pH_o 6.8 (panel B), it was shifted to the opposite direction (pointing to an inhibition). No significant difference was apparent in the mean change in steady state pH_i recorded upon changing the pH of the superfusing solution between normal and diabetic myocytes (pH_i=-0.28±0.02 [n=6 normal cells] versus -0.28±0.03 [n=8 diabetic cells] pH units, from pH_o 7.4 to 6.8; pH_i=0.18±0.01 [n=9 normal cells] versus 0.15±0.01 [n=7 diabetic cells] pH units, from pH_o 7.4 to 8.0). With respect to the effects of different pH_o levels on Na⁺-H⁺ exchange activity, Fig 6 shows that the increase in J^e_H observed at pH_o 8.0 (relative to the control J^e_H estimated at pH_o 7.4 and at pH_i 6.9) was not significantly different between both normal and diabetic cells, nor was the decrease observed at pH_o 6.8 (relative to control estimated at pH_o 7.4 and at pH_i 6.75). Therefore, the slowing down of Na⁺-H⁺ exchange upon diabetes is unlikely to result from a different pH_o sensitivity of the exchanger.

View larger version (13K): [in this window] [in a new window]   Figure 4. Effects of different pHo levels on pHi recovery from an acid load in diabetic isolated ventricular myocytes. A, Effect of an alkaline medium. B, Effect of an acidic medium. Each cell was acid-loaded twice by the NH4+ prepulse method as follows: first pulse, under control conditions (ie, at pHo 7.4); second pulse, at the test pHo. No difference was observed between two consecutive pHi recoveries under control conditions. Different durations of NH4+ pulse were used in order to get overlapping of the pHi recoveries recorded at the different pHo levels.

View larger version (14K): [in this window] [in a new window]   Figure 5. Representative pHi dependence of Na+-H+ exchange at extracellular pH (pHe) 8.0 (A) and at pHe 6.8 (B) in diabetic ventricular myocytes. pHi recoveries recorded in Fig 4 were used to estimate JeH at the different pHe levels.

View larger version (16K): [in this window] [in a new window]   Figure 6. Comparison of the effects of extracellular pH (pHe) on JeH carried by Na+-H+ exchange between normal and diabetic ventricular myocytes. The effect of the two test pHe levels (6.8 and 8.0) on JeH carried by Na+-H+ exchange is given relative to the control JeH (obtained from the same cell at pHe 7.4) estimated at pHi 6.9. Cell numbers are as follows: n=9 normal and 7 diabetic cells at pHe 8.0; n=6 normal and 8 diabetic cells at pHe 6.8.

Dependence of Na⁺-H⁺ Exchange on Intracellular Ca²⁺
It has been suggested that Na⁺-H⁺ exchange in spontaneously beating cultured rat heart cells is predominantly calmodulin dependent,⁹ which points to modulation of the exchanger by intracellular Ca²⁺. Besides, the basal intracellular Ca²⁺ level has been shown to be significantly decreased in stimulated as well as in quiescent ventricular myocytes isolated from STZ-diabetic rats.^{15 16} In this context, we hypothesized that the activity of cardiac Na⁺-H⁺ exchange might be slowed down in diabetes through an altered modulation by intracellular Ca²⁺. If the decrease in intracellular Ca²⁺ did account for the reduced activity of Na⁺-H⁺ exchange in diabetic cells, then in the presence of an intracellular Ca²⁺ buffer, the exchange should have displayed a similar activity in both normal and diabetic myocytes. Cells were thus loaded with BAPTA-AM (25 µmol/L) and superfused with a Ca²⁺-free solution in order to buffer intracellular Ca²⁺ ions. Under these conditions, BAPTA-loaded cells had a more acid steady state pH_i compared with control (nontreated; see above) cells in both groups (pH_i 7.10±0.01, n=11 normal treated cells; pH_i 7.13±0.02, n=8 diabetic treated cells; P<.05). This acidification might result from a decrease in basal Na⁺-H⁺ exchange activity. Indeed, under steady state conditions, Na⁺-H⁺ exchange in ventricular myocytes has been shown to exhibit an activity, though low, in order to counterbalance background acid-loading mechanisms.²⁸ In this context, a slowing down of the exchange would then induce a decrease of steady state pH_i. On the other hand, Fig 7A shows that in a normal cell treated with the Ca²⁺ chelator, pH_i recovery after an acid load was markedly slowed down compared with that recorded in a control normal cell. Similar qualitative effects were observed in the diabetic group. However, in Fig 7B, it appears that the leftward shift, induced by the BAPTA treatment, of the pH_i dependence for Na⁺-H⁺ exchange was more pronounced in normal than in diabetic myocytes; at pH_i 6.9, J^e_H was thus decreased by 74% in normal cells versus 58% in diabetic cells. Moreover, it is worth noting that when intracellular Ca²⁺ was buffered to a low level, J^e_H carried by the exchange then became identical in both groups. It is important to stress that the BAPTA-loading protocol used in the present study did not significantly change ß_i in either group of cells (at pH_i 6.9, ß_i=33.8 and 31.3 (mmol/L)·pH unit^-1 in normal [n=5] and diabetic [n=5] BAPTA-loaded cells, respectively), thus pointing to direct effects on Na⁺-H⁺ exchange. Therefore, these results suggest that the alterations in the intracellullar Ca²⁺ level previously reported in diabetic cells^{15 16} might, at least in part, account for the slowing down of Na⁺-H⁺ exchange.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effects of buffering intracellular Ca2+ on Na+-H+ exchange activity in normal and diabetic ventricular myocytes. A, Representative pHi recoveries from an acid load obtained from a control (nontreated) cell and from a BAPTA-AM–loaded cell. These two cells were isolated from normal rat hearts. B, Effect of buffering intracellular Ca2+ on the pHi dependence of JeH carried by Na+-H+ exchange in both normal (N) and diabetic (D) cells. Cell numbers are as follows: control, n=32 normal and 43 diabetic cells; BAPTA-loaded: n=11 normal and 8 diabetic cells.

Effects of Inhibiting Calmodulin and Ca²⁺/CaM Kinase II on Na⁺-H⁺ Exchange Activity
The fact that the diabetes-related intracellular Ca²⁺ level alterations might underlie the change in Na⁺-H⁺ exchange activity raised the possibility that alterations in the dependencies of Na⁺-H⁺ exchange on calmodulin and/or on Ca²⁺/CaM kinase II might occur in diabetic myocytes, since both pathways are known to be dependent on the intracellular Ca²⁺ level. In order to test this hypothesis, we used calmidazolium (1.5 µmol/L)¹⁹ and KN-62 (2 µmol/L)^{20 21} to respectively inhibit calmodulin and CaM kinase II and compared their effects between normal and diabetic groups. Fig 8 shows the typical experiments carried out in order to investigate the effects of these inhibitors on pH_i recovery due to Na⁺-H⁺ exchange in normal ventricular myocytes. When calmidazolium (panel A) or KN-62 (panel B) was applied to the cell, this resulted in both an acidification of steady state pH_i and a clear slowing of pH_i recovery following an acid load, thus pointing to a slowing down of Na⁺-H⁺ exchange. This slowing is further stressed in Fig 9, where the pH_i dependence of Na⁺-H⁺ exchange is significantly shifted to the left by both inhibitors. Similar qualitative effects were observed in diabetic myocytes. However, whereas the degree of inhibition by calmidazolium was not different between both normal and diabetic myocytes (Fig 10), it appeared that KN-62 inhibited J^e_H through the exchange more markedly in normal myocytes (by 45%, relative to control estimated at pH_i 6.85) than in diabetic myocytes (by 17%, relative to control estimated at pH_i 6.85; a significant difference has also been found at pH_i levels other than 6.85). It is important to point out here that these effects of calmidazolium and KN-62 on steady state pH_i and pH_i recovery could have involved effects on the background acid loading mechanisms (metabolic H⁺ production and/or Cl^--HCO₃^- exchange) rather than effects on Na⁺-H⁺ exchange. In order to rule out such a hypothesis, we tested the effects of both inhibitors on pH_i when Na⁺-H⁺ exchange was blocked by the application of 1 mmol/L amiloride. As expected, this maneuver elicited a slow decrease of steady state pH_i. However, under these conditions, addition of either calmidazolium or KN-62 to the superfusion solution did not change the rate of pH_i decrease despite the inhibition of Na⁺-H⁺ exchange (data not shown). Therefore, this indicated that (1)-calmidazolium and KN-62 per se did not acidify the cells via effects on background acid loading mechanisms, and (2) the effects of these inhibitors on steady state pH_i and pH_i recovery following an acid load were due to inhibition of Na⁺-H⁺ exchange. In conclusion, taken altogether, these results are in favor of alterations in the modulation by CaM kinase II of Na⁺-H⁺ exchange in ventricular myocytes isolated from diabetic rat hearts.

View larger version (16K): [in this window] [in a new window]   Figure 8. Effects of the calmodulin inhibitor, calmidazolium (1.5 µmol/L, A), and of the inhibitor of the Ca2+/CaM protein kinase II, KN-62 (2 µmol/L, B), on pHi recovery due to Na+-H+ exchange in normal ventricular myocytes. Each myocyte was acid-loaded twice: first pulse, under control conditions; second pulse, in the presence of inhibitor. No difference was observed between two consecutive pHi recoveries under control conditions.

View larger version (14K): [in this window] [in a new window]   Figure 9. Effects of calmidazolium (1.5 µmol/L, A) and KN-62 (2 µmol/L, B) on pHi dependence of Na+-H+ exchange in normal cells. Cell numbers are as follows: for panel A, n=6 cells; for panel B, n=6 cells. *P<.01.

View larger version (16K): [in this window] [in a new window]   Figure 10. Comparison of the effects of calmidazolium and KN-62 on Na+-H+ exchange between normal and diabetic myocytes. The percentage of effect was calculated relative to the control efflux estimated at pHi 6.85 for each cell. Cell numbers are as follows: calmidazolium, n=6 normal and 6 diabetic cells; KN-62, n=6 normal and 8 diabetic cells. **P<.03.

	Discussion

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In a previous study, we showed that pH_i recovery following an intracellular acid load was markedly slowed down in papillary muscles isolated from diabetic rat hearts.⁴ Since, in HCO₃^--buffered medium, the differences in the time course of pH_i recovery between normal and diabetic muscles were abolished by amiloride, we then suggested that the activity of the amiloride-sensitive Na⁺-H⁺ exchange was altered. A similar finding was also reported in diabetic whole heart⁶ and cardiac sarcolemmal vesicles.⁵ The present work corroborates these results by showing that in isolated ventricular myocytes, diabetes induces a significant slowing down of pH_i recovery following an acid load in HEPES-buffered medium. Since no change in ß_i has been found to be associated with diabetes (Fig 2), calculation of acid fluxes firmly demonstrates that the activity of the cardiac Na⁺-H⁺ exchange is markedly decreased by this pathological state, with a reduction by 42% of the J^e_H estimated at pH_i 6.9 when compared with normal (Fig 3).

The activity of the ubiquitous or "housekeeping" form of Na⁺-H⁺ exchanger (the NHE-1 type, which is the major isoform in the myocardium)²⁹ has been shown to rely on the inward Na⁺ gradient³⁰ and on intracellular and extracellular H⁺ ions.^{8 9 10 30} Several other factors control the activity of the exchanger, such as membrane composition and fluidity,³¹ growth factors,³² neurotransmitters,³³ and second messengers.^{34 35}

In this context, several cellular alterations associated with diabetes might account for the depressed activity of Na⁺-H⁺ exchange. For instance, the altered membrane composition,³⁶ through a change in the microenvironment of the exchanger, may induce a shift of its affinity for extracellular and intracellular H⁺ and Na⁺. Extracellular H⁺ ions have been shown to exert an inhibitory action on the cardiac exchanger.¹⁰ A diabetes-induced change of the pH_o dependence would thus appear to be a good candidate to underlie Na⁺-H⁺ exchange depression. However, the present results are not in favor of such a hypothesis. Indeed, we have found that an increase or a decrease in the pH of the superfusing solution elicited similar relative (to control) changes in steady state pH_i and in J^e_H carried by the exchanger in both normal and diabetic myocytes (Fig 6). It is worth noting that we have previously detected an increase in aⁱ_Na in diabetic papillary muscles.⁷ This increase may well contribute to the depressed activity of the exchanger through a decrease of the transmembrane Na⁺ gradient. On the other hand, the increased aⁱ_Na itself could also play an important and direct inhibitory role, possibly by competing with H⁺ binding to the internal transport site of the exchanger.³⁷ However, such a role remains to be demonstrated in cardiac tissue.

Alterations in homeostasis of intracellular Ca²⁺ have been recently demonstrated in diabetic cardiomyopathy, leading to a decrease in [Ca²⁺]_i in quiescent or stimulated myocytes.^{15 16} Although not yet established in the heart, recent reports have shown that the isoform NHE-1 of the Na⁺-H⁺ exchanger is modulated by intracellular Ca²⁺, so that increasing [Ca²⁺]_i induces a stimulation of the exchange.¹⁴ In this context, we have supposed that the reduced [Ca²⁺]_i level related to diabetes might be responsible for the depressed Na⁺-H⁺ exchange activity. Although the present results do not provide direct evidence in support of this hypothesis, experiments carried out with BAPTA-loaded cells would tend to favor it. Indeed, when intracellular Ca²⁺ was buffered, J^e_H carried by Na⁺-H⁺ exchange was not only significantly reduced in both normal and diabetic myocytes, but it then became similar in both groups (Fig 7B). Thus, two interesting and important points can be drawn from these data: (1) the basal activity of Na⁺-H⁺ exchange appears to be controlled by the basal level of intracellular Ca²⁺ in cardiac tissue, and (2) the decrease in activity of Na⁺-H⁺ exchange would stem from the diabetes-related alterations in [Ca²⁺]_i level. With respect to the latter point, in order to fully validate this hypothesis, experiments determining that increasing [Ca²⁺]_i stimulates the cardiac exchanger should have been quoted. Unfortunately, under our experimental conditions, such experiments carried out with the Ca²⁺ ionophore, ionomycin, appeared inconclusive, in that ionomycin was found to elicit effects on pH_i recovery independent of external Ca²⁺ (data not shown).

Recent studies indicate that the control of NHE-1 activity by Ca²⁺ may go through at least two different pathways: (1) through direct binding of Ca²⁺/calmodulin to the exchanger and/or (2) through phosphorylation by Ca²⁺/CaM kinase II. With respect to the first pathway, it has been reported that NHE-1 in fibroblasts is a Ca²⁺/calmodulin-binding protein.³⁸ After an increase in [Ca²⁺]_i (induced by ionomycin addition), the direct binding of Ca²⁺/calmodulin to NHE-1 then stimulates the exchange.¹⁴ Moreover, it is worth noting that inhibition of calmodulin by either W-7 or trifluoperazine has been shown to impair pH_i recovery from an acid load in spontaneously beating cultured rat heart cells.⁹ On the other hand, Fliegel et al³⁹ have evidence indicating that activation of Ca²⁺/CaM kinase II results in a direct phosphorylation of the cardiac Na⁺-H⁺ exchanger, which might then lead to its activation. In this context, we have tested the effects of calmidazolium, an inhibitor of calmodulin,¹⁹ and of KN-62, an inhibitor of CaM kinase II,^{20 21} on the activity of the cardiac exchanger in normal and diabetic myocytes in order to find out which of these pathways could be affected by diabetes. Whereas it was known that the purified protein of the cardiac Na⁺-H⁺ exchange could be directly phosphorylated by CaM kinase II,³⁹ no evidence was so far available concerning the regulation, by this kinase, of the exchanger in a cellular system. To our knowledge, this study is the first to demonstrate that inhibition of CaM kinase II under acid-load conditions results in a significant (by 45%) reduction of Na⁺-H⁺ exchange activity in cardiac ventricular myocytes (Figs 9B and 10). This raises the interesting possibility of a basal control of the cardiac NHE-1 exchange activity by direct CaM kinase–dependent phosphorylation. With respect to the HCO₃^--dependent acid extruder that works in parallel with Na⁺-H⁺ exchange in physiological buffer solution,¹⁷ preliminary results indicate that this mechanism is not under control of the CaM kinase II pathway.⁴⁰ This suggests that under physiological conditions, any agonist that activates this pathway might influence pH_i regulation after an acid load through an effect on mainly Na⁺-H⁺ exchange. It could be argued that the action of KN-62 on Na⁺-H⁺ exchange would involve unspecific effects on [Ca²⁺]_i rather than effects on CaM kinase II. Although [Ca²⁺]_i was not recorded in the present study, one could have expected an increase in [Ca²⁺]_i due to the intracellular acidification elicited by the application of KN-62. Under such conditions and according to our hypothesis (ie, a regulation of the exchanger by Ca²⁺), a stimulation of Na⁺-H⁺ exchange would have occurred. That was clearly not the case. Comparison of the effects of KN-62 between normal and diabetic myocytes shows that the regulatory pathway involving CaM kinase II is most likely affected by diabetes (since KN-62 reduced Na⁺-H⁺ exchange activity by only 17% in diabetic myocytes compared with 45% in normal cells), thus supporting the hypothesis that the decrease in steady state [Ca²⁺]_i may alter the Ca²⁺/CaM kinase II activity. However, whether this pathway is affected because of the decrease in basal [Ca²⁺]_i related to diabetes^{15 16} and/or because of alterations in CaM kinase II activity remains to be fully elucidated. Moreover, it would be interesting to quantify Na⁺-H⁺ exchanger phosphorylation in both groups in order to readily confirm the involvement of this process. In this respect, it is worth noting that a recent report has suggested that the elevated Na⁺-H⁺ exchanger activity in lymphoblasts from human hypertensive patients may be related to increased exchanger phosphorylation.⁴¹ In the present work, the results obtained with calmidazolium appear rather intriguing. As expected, we found that inhibition of calmodulin resulted in a negative effect on Na⁺-H⁺ exchange in ventricular myocytes. However, this effect was found to be identical in normal and diabetic cells, whereas some differences could have been expected because CaM kinase II is known to depend on calmodulin. The absence of different effects of calmidazolium between both groups of cells could come from the use of a too-low dose (1.5 µmol/L). In this regard, it should be emphasized here that Weissberg et al⁹ have found a nearly complete inhibition of cardiac Na⁺-H⁺ exchange with 200 µmol/L W-7 and 60 µmol/L trifluoperazine, two other calmodulin inhibitors, versus an inhibition by 40% with 1.5 µmol/L calmidazolium in the present work (we have also tested a higher dose of calmidazolium [5 µmol/L] in normal cells, but a toxic effect was then observed). Clearly, further work will be required to clarify this point.

In summary, the present study shows that since intracellular intrinsic buffering power, ß_i, remains unaffected by diabetes, this pathological state is clearly associated with a significant decrease of Na⁺-H⁺ exchange activity in rat isolated ventricular myocytes. This decrease may result from the diabetes-related alterations in [Ca²⁺]_i level, since buffering of intracellular Ca²⁺ was shown to abolish differences between both groups of cells. An altered CaM kinase II–dependent phosphorylation of the exchanger might underlie this effect, although this does not rule out any other alterations, such as a pretranslational modification of NHE-1 associated with diabetes.

	Selected Abbreviations and Acronyms

 

ßi = intracellular intrinsic H+–buffering power aiNa = intracellular Na+ activity CaM kinase II = calmodulin-dependent protein kinase II JeH = acid efflux KN-62 = 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine SNARF = seminaphthorhodafluor STZ = streptozotocin

	Acknowledgments

 
This study was supported by the "Programme DSPT.5-Biologie, Medecine et Sante, M.E.S.R., France." We wish to thank Francoise James for her technical assistance.

	Footnotes

 
Reprint requests to K. Le Prigent, Laboratoire de Physiologie Cellulaire, Universite Paris XI, Bat. 443, 91405 Orsay cedex, France.

Received August 12, 1996; accepted November 7, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Orchard CH, Kentish JC. Effects of changes of pH on the contractile function of cardiac muscle. Am J Physiol. 1990;258:C967-C981.[Abstract/Free Full Text]

2. Fliegel L, Frohlich O. The Na⁺/H⁺ exchanger: an update on structure, regulation and cardiac physiology. Biochem J. 1993;296:273-2853.

3. Karmazyn M, Moffat MP. Role of Na⁺/H⁺ exchange in cardiac physiology and pathophysiology: mediation of myocardial reperfusion injury by the pH paradox. Cardiovasc Res. 1993;27:915-924.[Free Full Text]

4. Lagadic-Gossmann D, Chesnais JM, Feuvray D. Intracellular pH regulation in papillary muscle cells from streptozotocin diabetic rats: an ion-sensitive microelectrode study. Pflugers Arch. 1988;412:613-617.[Medline] [Order article via Infotrieve]

5. Pierce GN, Ramjiawan B, Dhalla NS, Ferrari R. Na⁺-H⁺ exchange in cardiac sarcolemmal vesicles isolated from diabetic rats. Am J Physiol. 1990;258:H255-H261.[Abstract/Free Full Text]

6. Khandoudi N, Bernard M, Cozzone P, Feuvray D. Intracellular pH and role of Na⁺/H⁺ exchange during ischaemia and reperfusion of normal and diabetic rat hearts. Cardiovasc Res. 1990;24:873-878.[Medline] [Order article via Infotrieve]

7. Lagadic-Gossmann D, Feuvray D. Intracellular sodium activity in papillary muscle from diabetic rat hearts. Exp Physiol. 1991;76:147-149.[Abstract]

8. Aronson PS, Suhm MA, Nee J. Interaction of external H⁺ with the Na⁺/H⁺ exchanger in renal microvillus membrane vesicles. J Biol Chem. 1983;258:6767-6771.[Abstract/Free Full Text]

9. Weissberg PL, Little PJ, Cragoe EJ Jr, Bobik A. The pH of spontaneously beating cultured rat heart cells is regulated by an ATP-calmodulin–dependent Na⁺/H⁺ antiport. Circ Res. 1989;64:676-685.[Abstract/Free Full Text]

10. Vaughan-Jones RD, Wu M-L. Extracellular H⁺ inactivation of Na-H exchange in the sheep cardiac Purkinje fibre. J Physiol (Lond). 1990;428:441-466.[Abstract/Free Full Text]

11. Vicentini LM, Villereal ML. Activation of Na⁺/H⁺ exchange in cultured fibroblasts: synergism and antagonism between phorbol ester, Ca²⁺ ionophore, and growth factors. Proc Natl Acad Sci U S A. 1985;82:8053-8056.[Abstract/Free Full Text]

12. Grinstein S, Cohen S. Cytoplasmic [Ca²⁺] and intracellular pH in lymphocytes. J Gen Physiol. 1987;89:185-213.[Abstract/Free Full Text]

13. Hendey B, Mamrack MD, Putnam RW. Thrombin induces a calcium transient that mediates an activation of the Na⁺/H⁺ exchanger in human fibroblasts. J Biol Chem. 1989;264:19540-19547.[Abstract/Free Full Text]

14. Wakabayashi S, Bertrand B, Ikeda T, Pouyssegur J, Shigekawa M. Mutation of calmodulin-binding site renders the Na⁺/H⁺ exchanger (NHE1) highly H⁺-sensitive and Ca²⁺ regulation-defective. J Biol Chem. 1994;269:13710-13715.[Abstract/Free Full Text]

15. Noda N, Hayashi H, Miyata H, Suzuki S, Kobayashi A, Yamazaki N. Cytosolic Ca²⁺ concentration and pH of diabetic rat myocytes during metabolic inhibition. J Mol Cell Cardiol. 1992;24:435-446.[Medline] [Order article via Infotrieve]

16. Lagadic-Gossmann D, Buckler KJ, Le Prigent K, Feuvray D. Altered Ca²⁺ handling in ventricular myocytes isolated from diabetic rats. Am J Physiol. 1996;270:H1529-H1537.[Abstract/Free Full Text]

17. Lagadic-Gossmann D, Buckler KJ, Vaughan-Jones RD. Role of bicarbonate in pH recovery from intracellular acidosis in the guinea-pig ventricular myocyte. J Physiol (Lond). 1992;458:361-384.[Abstract/Free Full Text]

18. Roos A, Boron WF. Intracellular pH. Physiol Rev. 1981;61:296-434.[Free Full Text]

19. Veigl ML, Klevit RE, Sedwick WD. The uses and limitations of calmodulin antagonists. Pharmacol Ther. 1989;44:191-239.

20. Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, Hidaka H. KN-62,1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin-dependent protein kinase II. J Biol Chem. 1990;265:4315-4320.[Abstract/Free Full Text]

21. Okazaki K, Ishikawa T, Inui M, Tada M, Goshima K, Okamoto T, Hidaka H. KN-62, a specific Ca⁺⁺/calmodulin-dependent protein kinase inhibitor, reversibly depresses the rate of beating of cultured fetal mouse cardiac myocytes. J Pharmacol Exp Ther. 1994;270:1319-1324.[Abstract/Free Full Text]

22. Jourdon P, Feuvray D. Calcium and potassium currents in ventricular myocytes isolated from diabetic rats. J Physiol (Lond). 1993;470:411-429.[Abstract/Free Full Text]

23. Boron WF, De Weer P. Intracellular pH transients in squid giant axons caused by CO₂, NH₃ and metabolic inhibitors. J Gen Physiol. 1976;67:91-112.[Abstract/Free Full Text]

24. Puceat M, Clement O, Scamps F, Vassort G. Extracellular ATP-induced acidification leads to cytosolic calcium transient rise in single rat cardiac myocytes. Biochem J. 1991;274:55-62.

25. Buckler KJ, Vaughan-Jones RD. Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH-measurement in small isolated cells. Pflugers Arch. 1990;417:234-239.[Medline] [Order article via Infotrieve]

26. Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehrlich ascites tumour cells utilising spectroscopic probes generated in situ. Biochemistry. 1979;18:2210-2218.[Medline] [Order article via Infotrieve]

27. Vaughan-Jones RD, Wu M-L. Intracellular intrinsic buffering power varies with pH_i in the sheep cardiac Purkinje fibre. J Physiol (Lond). 1990;425:429-448.[Abstract/Free Full Text]

28. Lagadic-Gossmann D, Vaughan-Jones RD. Background acid loading in guinea-pig isolated ventricular myocyte. J Mol Cell Cardiol. 1993;25:S29. Abstract.

29. Fliegel L, Dyck JRB. Molecular biology of the cardiac sodium/hydrogen exchanger. Cardiovasc Res. 1995;29:155-159.[Medline] [Order article via Infotrieve]

30. Mahnensmith RL, Aronson PS. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ Res. 1985;57:773-788.

31. Dudeja PK, Foster ES, Brasitus TA. Modulation of rat distal colonic brush-border membrane Na⁺-H⁺ exchange by dexamethasone: role of lipid fluidity. Biochim Biophys Acta. 1987;905:485-493.[Medline] [Order article via Infotrieve]

32. Paris S, Pouyssegur J. Growth factors activate the Na⁺/H⁺ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H⁺. J Biol Chem. 1984;259:10989-10994.[Abstract/Free Full Text]

33. Puceat M, Vassort G. Neurohumoral modulation of intracellular pH in the heart. Cardiovasc Res. 1995;29:178-183.[Medline] [Order article via Infotrieve]

34. Lagadic-Gossmann D, Vaughan-Jones RD, Richmond P. Effects of elevating cAMP on acid extrusion in the guinea-pig isolated ventricular myocyte. J Physiol (Lond). 1993;459:418P. Abstract.

35. Wu M-L, Vaughan-Jones RD. Effect of metabolic inhibitors and second messengers upon Na⁺-H⁺ exchange in the sheep cardiac Purkinje fibre. J Physiol (Lond). 1994;478.2:301-313.

36. Pierce GN, Kutryk MJB, Dhalla NS. Alterations in Ca²⁺ binding by and composition of cardiac sarcolemmal membrane in chronic diabetes. Proc Natl Acad Sci U S A. 1983;80:5412-5416.[Abstract/Free Full Text]

37. Grinstein S, Goetz JD, Rothstein A. ²³Na⁺ fluxes in thymic lymphocytes, II: amiloride sensitive Na⁺/H⁺ exchange pathway: reversibility of transport and asymmetry of the modifier site. J Gen Physiol. 1984;84:585-600.[Abstract/Free Full Text]

38. Bertrand B, Wakabayashi S, Ikeda T, Pouyssegur J, Shigekawa M. The Na⁺/H⁺ exchanger isoform (NHE1) is a novel member of the calmodulin-binding proteins: identification and characterization of calmodulin-binding sites. J Biol Chem. 1994;269:13703-13709.[Abstract/Free Full Text]

39. Fliegel L, Walsh MP, Singh D, Wong C, Barr A. Phosphorylation of the C-terminal domain of the Na⁺/H⁺ exchanger by Ca²⁺/calmodulin-dependent protein kinase II. Biochem J. 1992;282:139-145.

40. Le Prigent K, Lagadic-Gossmann D, Feuvray D. Na⁺-HCO₃^- symport activity in diabetic ventricular myocytes. J Mol Cell Cardiol. 1996;28:A67. Abstract.

41. Ng LL, Sweeney FP, Siczkowski M, Davies JE, Quinn PA, Krolewski B, Krolewski AS. Na⁺-H⁺ antiporter phenotype, abundance, and phosphorylation of immortalized lymphoblasts from humans with hypertension. Hypertension. 1995;25:971-977.[Abstract/Free Full Text]

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