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676 The pH of Spontaneously Beating Cultured Rat Heart Cells Is Regulated by an ATP-Calmodulin-Dependent Na+/H+ Antiport Peter L. Weissberg, Peter J. Little, Edward J. Cragoe Jr., and Alex Bobik Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 We investigated the mechanisms by which spontaneously beating cultured rat ventricular cells regulate intracellular pH (pH,). Specifically, the relative contributions of the Na + /H + antiport, Cr/HCO 3 ~ exchange, ATP, and calmodulin-dependent processes in regulating the pH, of cells loaded with the intracellular fluorescent pH indicator BCECF were investigated. The pH, of ventricular cells bathed in HEPES-buffered medium averaged 7.30±0.02. Subsequent exposure of the cells to CO2-HCO3~-buffered medium resulted in intracellular acidification followed by recovery to pH, levels approximately 0.1 pH units lower than in controls. Recovery was inhibited by the Na + /H + antiport inhibitor 5-(W-ethyl-W-isopropyl)amiloride (EIPA). The recovery from intracellular acidification, induced by a 15-mM ammonium chloride prepulse, was also dependent solely upon activation of the Na + /H + antiport. Recovery was dependent upon extracellular sodium, was completely inhibited by EIPA, and could be modulated by changes in extracellular pH (pHJ. At low pH,, values (6.3) the recovery of pH, was greatly attenuated, while at high pH, (8.0) the recovery process was accelerated. The final pH, to which the cells recovered was also dependent upon pH,,. Preincubation of the cells with 2deoxy-D-glucose to deplete cellular ATP levels reduced pH, by approximately 0.2 pH units and greatly unpaired the cells' ability to recover from 15-mM ammonium chloride-induced acid load. Similarly, preincubation of cells with the calmodulin inhibitors W-7 and trifluoperazine also impaired their ability to recover from the acid load. The C1~-HCO3~ exchange played no role in the cells' ability to recover from intracellular acidosis. However, the presence of HCO3~ significantly increased the resistance of myocardial cells to changes in pH, by approximately doubling their buffer capacity. These results demonstrated that a Na + /H + antiport is the major pH,-regulating system in spontaneously beating rat ventricular cells. The ability of the Na + /H + antiport to regulate myocardial pH, is dependent upon the cells' ability to maintain adequate levels of ATP. The antiport's dependency on ATP, in conjunction with its dependency on calmodulin, suggests that activation of the antiport in ventricular cells involves phosphorylation processes. {Circulation Research 1989;64:676-685) C hanges in intracellular pH (pHj) have important effects on both the contractile and electrical properties of the heart. 12 However, little is known about the mechanisms that regulate myocardial pH;. Myocardial pHj is considerably more alkaline than would be expected from a passive distribution of protons across the cell mem- From the Baker Medical Research Institute and Alfred Hospital Clinical Research Unit, Melbourne, Australia, and Merck Sharp & Dohme Laboratories (E.J.C.), West Point, Pennsylvania. Supported by a Grant-in-Aid from the National Heart Foundation of Australia. P.L.W. received a British Medical Research Council Travelling Fellowship followed by an Alfred Hospital, Edward Wilson Fellowship. Address for correspondence: Dr. A. Bobik, Baker Medical Research Institute, Commercial Road, Prahran, Victoria, Australia. Received October 13, 1987; accepted September 2, 1988. brane. Furthermore, cardiac cells recover rapidly from an imposed acid load. In cultured chick embryo heart cells, recovery from acidosis is dependent on the transmembrane extrusion of protons by the Na+/ H+ antiport.3 Studies on pHj in nonbeating sheep Purkinje cells by use of microelectrodes are also consistent with this observation.4-5 However, the mechanisms involved in pHj regulation in beating mammalian heart cells are unknown. In particular, the relative contributions of the Na + /H + antiport, intracellular HCO3~, and C1"-HCO3~ exchange to pHj regulation have not been investigated, nor have the intracellular processes that influence the activity of these exchange mechanisms. A variety of different conditions may induce acidosis in cardiac cells. During myocardial ischemia, for example, the intracellular acidosis is accom- Weissberg et al Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 panied by a pronounced decrease in energy production relative to demand.6 The same condition does not apply, however, during respiratory acidosis. These different conditions could profoundly influence the cardiac cells' subsequent ability to recover from intracellular acidosis. Indeed, in some cell lines activation of the Na + /H + antiport appears to be dependent on ATP7 and related phosphorylation processes.8 In the studies reported here, cultured spontaneously beating rat ventricular cells containing the intracellular pH-sensitive fluorophore 2,7biscarboxyethy 1- 5(6)-carboxyfluorscein (BCECF) were used to investigate 1) the relative contributions of the Na + /H + antiport and Cr-HCO 3 " exchange in the maintenance of pH;; 2) the dependence of Na + /H + antiport activity on cellular ATP content; and 3) the relative importance of calmodulinand protein kinase C-dependent processes in the activation of the Na + /H + antiport. Materials and Methods Culture of Cardiac Cells Cardiac cell cultures were prepared from the hearts of 3- to 5-day-old female Wistar Kyoto rats by modification of the method previously used for chick embryo cardiac cultures.9 Briefly, the hearts were removed from decapitated animals and placed into cold Hanks' balanced salt solution. The atria, large vessels, and pericardium were removed, and the ventricles were cut into small (—1-2 ^.1) pieces. The tissue was incubated at 37° C in Medium 199 containing 3 mg/ml collagenase. After 15 minutes, the medium was discarded, and the tissue was dispersed into single cells by successive tryptic digestion in a medium consisting of 0.5% trypsin and 0.01% deoxyribonuclease in calcium-free Dulbecco's medium containing 1 mM MgSO4. The suspended cells were collected into fetal calf serum at 4° C and centrifuged at 150g. The cells were then suspended in Hanks' balanced salt solution containing 2 mM glutamine, 50 mg/l glycine, 12.5 mg/1 hypoxanthine, essential and nonessential amino acids, 100 IU/ml penicillin, and 7.1 mM NaHCO3 and seeded into 90-mm plastic culture dishes. After 75 minutes at 37° C, the medium containing unattached cardiac cells was removed from the dishes, leaving behind fibroblasts, endothelial cells, and smooth muscle cells attached to the plastic dishes. Bromo-deoxyuridine (0.1 mM final concentration) was then added to the medium to inhibit proliferation of any remaining noncardiac cells.10 The medium containing the cardiac cells was then divided into aliquots and placed in 35-mm culture dishes containing two 24x10 mm sterile glass coverslips and incubated at 37° C in 5% CO2 in air. After 12-18 hours, the attached cells were washed with fresh culture medium (containing bromo-deoxyuridine). This technique produced a monolayer of spontaneously beating cells that were used in experiments 48-72 hours after plating. Cardiac IntraceUular pH Regulation 677 Measurement of Intracellular pH The fluorescent pH indicator BCECF was used to monitor changes in cytoplasmic pH in the cultured cardiac cells. BCECF has a slow spontaneous leakage rate, and the intensity of its fluorescence is linearly related to pH from pH 6.5 to 7 . 5 . " l 2 Coverslips to which spontaneously beating cardiac cells were attached were taken from the culture medium and washed three times with 2 ml of physiological salt solution (PSS). The cells were then incubated for 30 minutes at 37° C in PSS containing 1 piM of esterified BCECF (BCECFAM). During this time the (membrane permeable) BCECF-AM entered the cells and was hydrolyzed to free (membrane impermeable) BCECF by intracellular esterases, resulting in entrapment of the fluorescent indicator within the cell. At the end of the incubation, extracellular indicator was removed by washing the coverslips with PSS at 37° C. Examination of these cells under a fluorescence microscope indicated that BCECF fluorescence was evenly distributed throughout the cell cytoplasm. The spontaneous beating of the cell cultures, examined by phase contrast microscopy, was unaffected by BCECF. Coverslips with the BCECF-loaded cells were placed into a vertical holding device that fits into a standard fluorescence cuvette and permits rapid exchange of extracellular medium (~20 ml in 10 seconds) or addition of drugs to the medium without disturbing the cells or their orientation to the excitation beam.13 Fluorescence of the monolayer of cells bathed in the various salt solutions (see "Materials and Methods" and "Results") was measured at 37° C in an LS-5 luminescence spectrometer (Perkin-Elmer, Eden Prairie, Minnesota) with the excitation wavelengths set at 500 or 440 nm (bandpass 10 nm) and the emission wavelengths set at 530 nm (bandpass 5 nm). Under these conditions BCECF fluorescence at 500 nm was maximal and dependent on pH|, while the fluorescence at 440 nm (its isosbestic point) was unaffected by pH,.14 The ratio of 500:440 fluorescence values, corrected for cellular autofluorescence at these wavelengths, was used to estimate pHf. Autofluorescence of unloaded cardiac cells represented < 1 % of total fluorescence. Over the pH range of 6.40 to 7.80, the regression line relating the fluorescence ratio (FR) to pH was described by the equation FR=5.82 [pHJ-34.0 (r=0.99) (Figure 1). Known values for pHj of the cardiac cells were obtained as previously described using high-concentration K+ buffers of various pH containing 7 /xM nigericin.12 This fluorescence ratio technique gives consistent estimates of pHj that are not affected by alterations in cell density or leakage of BCECF and obviates the need to calibrate each experiment individually. Measurement of ATP Cellular contents of ATP were measured fluorometrically via the hexokinase reaction.15 Briefly, 678 Circulation Research BCECF Vol 64, No 4, April 1989 Calibration 12 o o o ID- IT) O a c CD 6- ID O C o o <n ID Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 2J 6O 7-0 Intracellular 7-5 pH 1. Relation between apparent intracellular pH of cardiac cells and intracellular BCECF fluorescence intensity ratio. Nigericin and high-potassium buffers of known pH were used to derive the relation. Each value represents mean±SEM of four separate experiments. BCECF, 2,7biscarboxyethyl-5(6)-carboxyfluorescein. FIGURE the cells were washed thoroughly with ice-cold normal saline before extraction of the ATP with ice-cold 0.4 M perchloric acid. After neutralization of the extracts with potassium carbonate, 100 /tl aliquots were assayed for ATP content in 2 ml of buffer containing 100 mM Tris, 5 mM MgCl2, 5 mM glucose, 10 fiM nicotinamide adenine dinucleotide phosphate, and 3.5 units glucose 6-phosphate dehydrogenase. Hexokinase (2.8 units) was added to initiate the reaction, and the changes in fluorescence were monitored in a Perkin-Elmer LS-5 spectrophotofluorometer whose excitation and emission wavelengths were set at 340 and 450 nm, respectively. Standardization of each sample was achieved by monitoring the change in fluorescence after addition of 2 nmol ATP to the reaction cuvette. Cellular protein was measured according to the method of Lowry et al.16 Solutions The PSS used in the study had the following millimolar composition: NaCI 135, KC1 5, CaCl21.8, MgSO4 0.8, glucose 5.5, and N-(2-hydroxyethyl) piperazine-W-2-ethanesulfonic acid 10 (HEPES) adjusted to pH 7.4 at 37° C. When a PSS of different pH values was required, the solution was adjusted with either hydrochloric acid or sodium hydroxide to the appropriate pH. In PSS buffered with CO2HCO3", the sodium chloride concentration was reduced to 115 mM and 20 mM NaHC0 3 was substi- tuted for the HEPES. This solution was equilibrated with 5% CO2 in air to bring its pH to 7.4 at 37° C. When low sodium concentrations were required, the sodium chloride was replaced by equimolar Nmethyl-D-glucamine. In the PSS used for depleting cardiac energy stores, glucose was replaced by 5.5 mM 2-deoxy-D-glucose. The solution used to calibrate fluorescent signals from the cardiac cells was nominally Na+-free PSS containing 140 mM KC1 and 7 fiM nigericin. All experiments were carried out at 37° C. Materials Cell culture reagents were purchased from Commonwealth Serum Laboratories, Parkville, Australia. Tissue culture plates were from Flow Laboratories, Melbourne, Australia. The BCECF-AM was from Molecular Probes, Eugene, Oregon. Nigericin; ouabain; 4-acetamido-4'-isothiocyanostilbene2,2'-disulfonic acid (SITS); l-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride (H-7); trifluoperazine; /V-methyl-D-glucamine; and 2-deoxyD-glucose were from Sigma Chemical, St. Louis, Missouri. N-(6-Aminohexyl)-5-chloro-1 -naphthalenesulfonamide hydrochloride (W-7) was from Seikagaku America, St. Petersburg, Florida. 5-(N-EthylN-isopropyl)amiloride (EIPA) was synthesized by Dr. E.J. Cragoe. All other chemicals were of analytical or tissue culture grade and were purchased from local chemical suppliers. Statistics Results are expressed as the mean±SEM. Statistical significance was evaluated by twotailed Student's t test. Results Determinants of Basal Intracellular pH The basal pHj of spontaneously beating rat cardiac cells at 37° C in HEPES-buffered PSS (pH 7.4) averaged 7.30±0.02 (n=ll). This basal pH; was to some extent maintained by the Na + /H + antiport since the addition of 200 fiM EIPA, a potent inhibitor of Na + /H + exchange, caused a gradual time-related fall in pH, of between 0.01 and 0.02 pH( units/min over the ensuing 5 minutes. However, the contribution of Na + /H + exchange to the maintenance of normal pHf could be demonstrated more easily when the cells were changed to a bicarbonate-buffered PSS (Figure 2). When the extracellular medium was changed from HEPESbuffered PSS (pH 7.4) to the CO2-HC(V-buffered PSS (pH 7.4), pHj fell rapidly as dissolved carbon dioxide entered the cells. Intracellular pH then recovered to a new equilibrium about 0.1 pH units below the original pHj. The pH; under these conditions averaged 7.19±0.04 (n=6). The maintenance of pHj under these conditions was greatly dependent on Na + /H + exchange since EIPA (200 /iM) prevented any recovery from the initial (carbon dioxideinduced) fall in pHj (Figure 2). Weissberg et al Cardiac Intracellular pH Regulation 679 PSS CO2/HCO3 PSS 7-8 n \ c 3 7-4 ^ X a t CO 2 /HCO 3 PSS+ EIPA 70 6-6-1 CO 'c PSS + EIPA 2OOv*4 Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 X a 7-6 -1 1min 2. Role of Na+/H+ antiport in regulation of intracellular pH in CO2-HCO3'-buffered PSS medium. Cells equilibrated in HEPES-buffered PSS (pH 7.4) were perfused with COrHCO{-buffered PSS (pH 7.4). Top panel depicts changes in intracellular pH; bottom panel shows effect of inhibition of Na+IH+ exchange with 200 fiM EIPA on intracellular pH. PSS, physiological salt solution; EIPA, 5(S-ethyl-N-isopropyl)amiloride. I a 7-2 - FIGURE + Na /H* Antiport and Recovery From Intracellular Acidosis To examine the importance of Na + /H + exchange in regulation of pHj, we compared the rates of recovery of pH; after acidification by a 15-mM NH^Cl prepulse in the absence and presence of bicarbonate. Upon exposure of the cells to 15 mM NH,C1 in HEPES-buffered PSS, pHj rose rapidly from 7.30±0.03 to 7.74±0.02 (n=5). Despite the continual presence of ammonium chloride, pHs gradually returned towards the basal level, attaining a pH| of 7.38±0.04 5 minutes after the initial exposure to ammonium chloride. Upon removal of the ammonium chloride by perfusion with HEPES-buffered PSS (pH 7.4), pHj fell rapidly to 6.67±0.04 (n=5). In HEPES-buffered PSS (pH 7.4) containing 135 mM Na + , the recovery from acidosis was rapid; the half-time for recovery averaged 0.625±0.025 minutes (Figure 3). The recovery was prevented by the Na + /H + exchange inhibitor EIPA. Recovery from the ammonium chloride-induced acidosis was also dependent on extracellular sodium concentration ([Na],,). Reduction of the [Na]0 by substitution of an equimolar amount of Nmethyl-D-glucamine for sodium in the HEPES- - 6-8 6-4-1 FIGURE 3. Representative tracings demonstrating effects of addition and removal of 15 mM NH4C1 on intracellular pH of cells equilibrated in HEPES-buffered PSS (pH 7.4). Top panel depicts transient fall in intracellular pH upon perfusion of ammonium chloride-exposed cells with HEPES-buffered PSS (pH 7.4). Bottom panel shows effect of 200 yM EIPA on recovery phase of intracellular pH. PSS, physiological salt solution; EIPA, 5(N-ethyl-Nisopropyl)amiloride. buffered PSS (pH 7.4) reduced the initial rate of recovery (Figure 4). In a nominally Na+-free HEPESbuffered PSS (pH 7.4), there was no recovery from the acidosis. Eadie-Hofstee analysis of the dependence of pHj recovery rate on [Na]0 under these conditions gave a Km for extracellular sodium of 9.8±1.6 mM and a maximum recovery rate of 0.84±0.09 pH units/min (Figure 4). The dependence on sodium and the sensitivity to EIPA demonstrate that in the absence of bicarbonate, Na + /H + exchange is wholly capable of mediating complete recovery from acidosis. Since a membrane HCO3"-C1" transport mechanism has been shown to exist in mammalian Purkinje fibers,1718 we also examined how it might influence the cells' ability to recover from acidosis. 680 Circulation Research Vol 64, No 4, April 1989 PSS 1-0 • 7-8 -i c I CO 0-8 'E I a. Na 0 7-4 3 I m >» "cD u co CD O O CD 70 "o 0-4- 0-2 CD CO (£ 1n*i Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 40 80 120 160 Extracellular sodium (mM) FIGURE 4. Effects of extracellular Na+ concentration ([Na+]o) on recovery of cells from acidosis. Left panel is a composite of several tracings in which recovery ofintracellular pHfrom a 15-mM NH4Cl-induced acidosis was measured in HEPES-buffered PSS (pH 7.4) containing various concentrations of sodium (Nao). Right panel shows relation between initial rates of recovery measured from above experiments and [Na+]o. PSS, physiological salt solution. Upon exposure of the cells to 15 mM NH4CI in CO2-HC<V-buffered PSS (pH 7.4), pH; rose from 7.14±0.04 to 7.48±0.04 (n=6, p<0.0l). This increase in pHj (0.29±0.01 pH units) was significantly less than the corresponding rise in HEPES-buffered PSS (0.44±0.01 pH units, p<0.01 for difference). After 5 minutes pHj had completely returned to basal levels (pH 7.17±0.04) (Figure 5). Upon perfusion of the cells with COrHCCV-buffered PSS, pH; rapidly fell to 6.71 ±0.04, a value similar to that observed with the HEPES-buffered PSS. However, in the CO2-HC(V-buffered PSS, the magnitude of the fall in pHj (0.46±0.03) was significantly less than that in HEPES-buffered PSS (0.70±0.01, p<0.01 for difference). Recovery from intracellular acidosis was unaffected by the presence of HC03~ in the medium and could be completely inhibited by EIPA (Figure 5). Furthermore, the anion exchange inhibitor SITS (300 fiM) had no inhibitory effect on the recovery (not shown). These results indicated that Na + /H + exchange is the major pHj regulating mechanism responsible for the cells' ability to recover from acidosis. Although C1"-HCO3" exchange plays no part in the recovery from acidosis, the CO2-HCO3~ does protect the cell from large fluctuations in pH, by increasing the cell's buffering capacity. The intrinsic buffering capacity (pi)19 of the cells in the HEPES-buffered PSS (pH 7.4), derived from the magnitude of the alkalinization (approximately 0.40 pH units) that occurred upon substitution of HEPES-buffered PSS (pH 7.4) for COrHCCVbuffered PSS (pH 7.4), averaged 20.7 mM/pH. The total buffering capacity in the HCO3~-buffered PSS, calculated from the estimated intracellular HC0 3 concentration according to Roos and Boron,19 averaged 41 mM/pH. Thus, the total buffering capacity of the cell was approximately doubled by the presence of bicarbonate. Extracellular pH and Na+/H+ Antiport Activity Since reductions in extracellular pH (pH,,) are known to greatly increase the concentration of acidic metabolites within the heart,2021 the effects of alterations in pH,, on Na + /H + exchange were also examined. In these experiments, Na + /H + antiport activity was assessed by measurement of the rate of recovery from an NH^Cl-induced acidosis in HEPESbuffered PSS (pH 6.3-8.0). Under these conditions the fall in pH,, induced by superfusion of the cells with the different HEPES-PSS buffers, was similar, averaging 6.66±0.05 (Figure 6). Na + /H + antiport activity was greatly attenuated when pHo was low and was enhanced when pHo was elevated. The initial rates of recovery (pH units per minute) from this level of acidosis (NaHE) were related to pH,, by the equation NaHE=0.755[pH o ]-4.63 (n=13, r=0.93). The final pHs values achieved under these conditions were also dependent on pH,, (Figure 6). Dependency of Na+/H+ Exchange on Metabolic Energy Previous studies in a number of cell lines have provided conflicting evidence as to the dependency of Na + /H + antiport activity on metabolic energy.7,22.23 Therefore, we assessed the energy dependency of the antiport in cardiac cells by studying their ability to recover from an ammonium Weissberg et al P8S • 7-4, I a = 70- P8S+E1PA 200(lM 7-6 • Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 I a 7-2. 2 6*- FIGURE 5. Tracings depicting effects of addition and removal of 15 mM NH4Cl on intracellular pH of cells equilibrated in CO2-HCOf-buffered PSS (pH 7.4). Top panel demonstrates transient nature of fall in intracellular pH upon perfusion of ammonium chloride-exposed cells with COrHCOf-buffered PSS (pH 7.4). Bottom panel shows effect of 200 fiM EIPA on recovery from acidosis. PSS, physiological salt solution; EIPA, 5(N-ethyl-N-isopropyl)amiloride. chloride-induced acidosis after 30 minutes of incubation in HEPES-buffered PSS (pH 7.4) containing 5.5 mM 2-deoxy-r>glucose instead of glucose. This procedure reduced cellular ATP content by 90%, from 18.4±0.3 nmol/mg protein (n=3) to 1.7±0.1 nmol/mg protein (n=3). Under these conditions the cells' ability to recover from the 15-mM NH»C1induced acidosis was markedly attenuated (Figure 7). Furthermore, basal pHi in these cells was reduced by approximately 0.2 pH units, presumably by the inability of the antiport to extrude protons. To examine whether this effect was due to a reduction in the transmembrane sodium gradient via inhibition of the Na-K pump due to ATP depletion, we examined the effect of 3 mM ouabain on the recovery process. Incubation of the cells for 60 minutes in ouabain did not affect their subsequent ability to recover from an acid load (Figure 7). Calmodulin Dependence of the Na+/H+ Antiport The inhibition of Na + /H + antiport activity by 2-deoxy-D-glucose-induced ATP depletion raised the possibility that an ATP-dependent process such Cardiac IntraceUular pH Regulation 681 as phosphorylation was required for Na + /H + antiport activation. Therefore, we investigated the role of calmodulin and other kinase-dependent processes in activation of the antiport. A 30-minute preincubation of the cells with the calmodulin-selective antagonist W-7 (200 /iM)24-25 completely impaired their ability to recover from a 15-mM NH4Cl-induced acid load in HEPES-buffered PSS (pH 7.4) (Figure 8). Lower concentrations caused partial impairment of recovery. The IC50 for inhibition of recovery by W-7 was 50 fiM. To test whether this was a direct (extracellular) effect of W-7 on the Na + /H + antiport, similar to that observed with EIPA, we also assessed its acute effects on the ability of cells to recover from the acid load. When ammonium chloride was removed from cells not previously exposed to W-7 with PSS containing W-7 (200 /xM), the initial rate of recovery from acidosis was unimpaired. However, after approximately 2 minutes the rate of recovery fell dramatically, and pH, gradually commenced to fall (not shown). After 30 minutes of exposure to 200 ^.M W-7, the fall in pHj averaged 0.50±0.05 pH units. Preincubation of the cells for 30 minutes with the structurally unrelated calmodulin antagonist trifluoperazine (up to 60 /xM)26 also had similar effects on the ability of cardiac cells to recover from an acid load (Figure 8). Fifty percent inhibition by trifluoperazine occurred at 12 /iM; at 60 fiM, the highest concentration used, trifluoperazine inhibited pH, rate of recovery by 82±3%. These results were consistent with the exertion of the inhibitory effect of calmodulin antagonists on the antiport by indirect mechanisms, presumably inhibition of phosphorylation. In contrast to the effects of W-7, the related sulfonamide derivative H-7, known to inhibit protein kinase C, cyclic AMP, and cyclic GMP-dependent protein kinase systems at a concentration of 200 /iM,27-28 had only a marginal effect on the ability of the cells to recover from the same acid load (Figure 8). Discussion We have demonstrated that an ATP-calmodulindependent Na + /H + antiport is the major p H r regulating mechanism in synchronously beating mammalian myocardial cells. Recovery from intracellular acidosis is mediated solely by this antiport. Our finding that preincubation with 2-deoxyr>glucose or the calmodulin antagonists greatly attenuated activation of the antiport during acidosis indicated that activation of the antiport is dependent on cellular metabolic energy and may involve ATP-dependent phosphorylation processes. The ineffectiveness of the sulfonamide derivative H-7, a potent inhibitor of several protein kinase systems including those dependent on cyclic AMP, cyclic GMP, and diacylglycerols,27-28 suggested that regulation of the antiport in cardiac cells is predominantly calmodulin dependent. It has been generally assumed that Na + /H + exchange in cells is an ATP-independent process 682 Circulation Research Vol 64, No 4, April 1989 PSS NH4CI 7-8i pHo 8-OT 8.0 X a I a 7-4 -I o o CD ~ c to 7-0 6.3 6-0-1 6-0 1 mm 6-5 70 7-5 8-0 8-5 Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 Extracellular pH + + FIGURE 6. Effects of extracellular pH on Na /H antiport-mediated recovery from acidosis. Left panel is a composite of tracings showing recovery of intracellular pH in acidotic (15-mM NH^Cl-induced) cells under conditions of various extracellular pH. Right panel shows relation between extracellular pH and final intracellular pH achieved by cells in above experiments. PSS, physiological salt solution; pH0, extracellular pH. driven by the inward electrochemical gradient for sodium and that allosteric regulation of the antiport by intracellular protons is the major mechanism by which it functions to maintain pH; homeostasis.29 Our results in beating myocardial cells were consistent with this hypothesis. The activity of the antiport was greatly increased by intracellular acidification. Furthermore, the extent of the increase in activity was dependent on [Na]0, particularly at concentrations below 15 mM. However, the inability of the antiport to maintain pHj when the cells were incubated with 2-deoxy-r>glucose, a procedure that depletes cellular ATP, strongly indicated that this allosteric regulation by intracellular protons is subject to additional regulating mechanisms such as phosphorylation. Although our evidence for involvement of a phosphorylation process in the regulation of the antiport is indirect and other mechanisms may contribute to the observed effects, reports on the properties of this antiport in other cell lines support a phosphorylation hypothesis. Recently, phorbol esters, known activators of protein kinase C, have been shown to stimulate Na + / H+ exchange in fibroblasts,30 lymphocytes,31 and myoblasts.32 In the latter instance, the effect of the phorbol esters on the antiport was to shift its pHj dependence to a more alkaline pHj. The inability of the myocardial cells to recover from an acid load after preincubation with 2-deoxy-r>glucose or the calmodulin antagonists could well have been due to a shift in- the pHf dependence of the antiport to a more acidic pHj. Such an effect has recently been reported for a cultured human cell line.7 This ability of processes, presumably phosphorylation, to alter the activity of the antiport in cardiac cells would represent an important mechanism by which cardiac cells regulate and maintain intracellular pH during periods of increased metabolic demand. In this context it is interesting to note that isoproterenol has been recently reported to indirectly stimulate the Na + /K + pump of isolated cardiac myocytes of rabbits by increasing the electroneutral influx of sodium.33 Our finding that Cr-HC0 3 ~ exchange in beating heart cells does not contribute to the recovery from acidosis was consistent with recent observations in cardiac Purkinje strands 1718 and cultured chick embryo cardiac cells.3 Nevertheless, intracellular HC03~ does contribute significantly to the overall buffering capacity, thereby increasing the cells' ability to withstand shifts of pH; to more alkaline and acidic values. The intracellular buffering capacity of 42 mM/pH unit, observed under these conditions, is similar to that reported for sheep heart Purkinje fibers34 and other muscle.19 The observation that pH, was consistently approximately 0.1 pH units lower in HCCV-COj-buffered PSS than in nominally HCO3~-free PSS was consistent with previous observations on myocardial pH.34 This has been attributed to an HCO3"-CO2 shuttle movement in which the continuous efflux of HC0 3 " down its electrochemical gradient imposes a constant acid load on the cell by continuously dissociating carbonic acid.33 However, it is still not clear why the Na + /H + antiport did not return pHj to control levels. Na + /H + antiport activity in cardiac cells was not impaired by the presence of CO2HC0 3 " in the incubation medium since initial rates of recovery were identical to those observed in HEPES-buflfered medium. Identical initial rates of Weissberg et al Cardiac Intracellular pH Regulation • dd load 683 acid load I H-7 7. Comparison of effects of preincubation of cells with 3 mM ouabain and 5.5 mM 2-deoxy-D-glucose (2-DG) on the cells' subsequent ability to activate Na+l H+ antiport after a 15-mM NH4Cl-induced acid load. Arrow indicates time at which ammonium chloride was removed from medium, thereby inducing acidosis. Intracellular pH recovery was measured in HEPES-buffered media containing ouabain or 2-deoxy-D-glucose. FIGURE Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 pH, recovery in HEPES-buffered and HCCV-COjbuffered media have also been reported in cardiac Purkinje fibers despite a higher intracellular buffer capacity when the fibers are incubated in HCO3~COr-buffered medium.18 Vanheel et al18 attributed this higher-than-predicted initial recovery rate from acidosis to a transient lowering of intracellular bicarbonate concentration during the initial phase of the ammonium chloride-induced acidosis. A similar mechanism may also have been responsible for the apparent equalization of pHj recovery rates in the cultured heart cells. Clearly, this aspect of pHj control, together with the reasons as to why the steady-state pH, is lower in cardiac cells incubated in the HCO3~-CO2-buffered medium, requires further investigation. However, the physiological consequence of this latter effect is that pH, will be lower in a respiratory acidosis than in an equivalent metabolic acidosis. Reductions in pH, are known to induce graded reductions in the transmembrane cardiac calcium current36 and also reduce the affinity of myofibrils for calcium.37 The effects of these reductions due to increasing levels of intracellular acidosis would explain why the cardiac inotropic state is depressed more by respiratory acidosis than by an equivalent metabolic acidosis. An elevation in myocardial Na + /H + antiport activity in respiratory failure could also account for the increased toxicity of digoxin in these subjects.38 Under these conditions, sodium influx via the Na + /H + antiport is increased in response to intracellular acidosis, and in the absence of an adequate Na + /K + pump to extrude the sodium, intracellular sodium concentration increases. This increase in sodium would in turn stimulate calcium entry via Na+-Ca2+ exchange. Such an interaction between the Na + /H + antiport, the Na+-K+ pump, and Na+-Ca2+ exchange has FIGURE 8. Comparison of effects of preincubation of cells with calmodulin inhibitors W-7 (200 fj.M) and trifluoperazine (TFP) (60 fjM) and protein kinase C inhibitor H-7 (200 nM) on their subsequent ability to activate Na+/H+ antiport after a 15-mM NH^l-induced acid load in HEPES-buffered PSS (pH.7.4). Arrow indicates time at which cells were perfused with ammonium chloridefree HEPES-buffered PSS (pH 7.4) to induce acid load. Intracellular pH recovery was measured in HEPESbuffered media containing H-7, TFP, or W-7. PSS, physiological salt solution. recently been proposed to play an important role in the positive inotropic effects of cardiac glycosides.3940 In this context, it is interesting to note that both the electrocardiographic and inotropic effects of digoxin can be antagonized by amiloride.4142 These effects were formerly attributed to the diuretic's potassium-sparing properties. However, partial inhibition of Na + /H + exchange could also contribute to the antagonistic effect of amiloride. Intracellular acidosis due to the retention of acidic metabolites is also an early event in the progress of ischemia.20 This is accompanied by a fall in extracellular pH. Our results indicated that under these conditions proton extrusion by the Na + /H + antiport will be impaired and pH, will fall, thereby depressing myocardial contractile function. After transient ischemia in which reperfusion occurs before the depletion of high-energy phosphates such as ATP and cytidine 5'-triphosphate (CTP), the Na + /H + antiport would rapidly extrude accumulated protons and restore pHf to normal. During more severe ischemia in which ATP levels are reduced, restoration of Na + /H + antiport activity under these conditions will depend upon the synthesis of adequate cellular ATP. In conclusion, our results demonstrated that an ATP-calmodulin-dependent Na + /H + antiport in beating mammalian heart cells is the sole pHregulating system. The antiport is stimulated by intracellular acidosis and inhibited by extracellular acidosis. The ability of the antiport to respond to an intracellular acidosis is dependent on adequate high-energy phosphate stores and calmodulin- 684 Circulation Research Vol 64, No 4, April 1989 Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 dependent processes. This raises the interesting possibility that only phosphorylated Na + /H + antiport proteins are capable of translocating intracellular protons in exchange for extracellular sodium. It is tempting to speculate that mechanisms that regulate the extent of phosphorylation and dephosphorylation of Na + /H + antiport proteins maintain intracellular pH within normal limits. Such a mechanism would in essence be capable of responding to an increased acid load by recruiting additional antiport units activated by phosphorylation. Furthermore, the involvement of calmodulin in the antiport's activation process suggests that calcium may also be an important determinant of antiport activity. Clearly, further work will be required to substantiate these hypotheses and understand precisely how the Na + /H + antiport is involved in pH, regulation of the normal and impaired myocardial cell. Acknowledgment The technical assistance of Miss Annette Grooms is gratefully acknowledged. References 1. 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Jounela A, Pyorala K: Effect of amiloride on digitalisinduced electrocardiographic changes. Ann Clin Res 1975; 7:66-70 42. Waldorff S, Hansen PB, Kjaergard H, Buch, J, Egeblad H, Steiness E: Amiloride-induced changes in digoxin dynamics and kinetics: Abolition of digoxin-induced inotropism with amiloride. Clin Pharmacol Ther 1981 ;30:172-176 + + KEY WORDS • intracellular pH • Na /H antiport • ATP heart cells • BCECF • calmodulin Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 The pH of spontaneously beating cultured rat heart cells is regulated by an ATP-calmodulin-dependent Na+/H+ antiport. P L Weissberg, P J Little, E J Cragoe, Jr and A Bobik Downloaded from http://circres.ahajournals.org/ by guest on August 1, 2017 Circ Res. 1989;64:676-685 doi: 10.1161/01.RES.64.4.676 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1989 American Heart Association, Inc. All rights reserved. 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