Effects of acidosis on mechanical function and Ca2+ exchange in rabbit myocardium.

Effects of acidosis on mechanical function and Ca2’ exchange in rabbit myocardium PHILIP

A. POOLE-WILSON

AND

GLENN

A. LANGER

Departments of Physiology and Medicine and Los Angeles County Cardiovascular Research Laboratory, University of California at Los Angeles, Center for the Health Sciences, Los Angeles, California 90024; and Cardiothoracic Institute and National Heart Hospital, London WlN 2DX, England POOLE-WILSON, PHILIP A., AND GLENN A. LANGER. Effects of acidosis on mechanical function and Ca2+ exchange in rabbit myocardium. Am. J. Physiol. 236(4): H525-H533, 1979 or Am. J. Physiol.: Heart Circ. Physiol. 5(4): H525-H533, 1979.-The effects of acidosis on myocardial function and calcium exchange have been studied in the isolated but arterially perfused interventricular septum of the rabbit. Temperature was 28’C and stimulation rate 48 beats/min. Acidosis was induced either by increase of the per&sate PCO~ (pH reduced from 7.35 to 6.68) or by decrease of the bicarbonate-chloride ratio (pH 7.35 to 6.72). The effect on calcium efflux was assessed by introduction of acidosis at different times during the washout of 45Ca2+ from the muscle. The uptake of 47Ca2+ was recorded directly with a NaI crystal and counter. An increase of perfusate Pco2 caused a rapid fall in developed tension. The efflux of slowly exchanging 45Ca2+ and the uptake of 47Ca2+ were inhibited. There was no rapid displacement of calcium from the muscle. Decrease of the bicarbonate-chloride ratio caused a slower fall of developed tension and neither the efflux nor uptake of calcium were altered. These results suggest that developed tension and calcium exchange in the myocardium are more responsive to acidosis within the cell or cell membrane than to extracellular acidosis.

and was absent at stimulation rates above 30 beats/min. This study was undertaken to define more precisely the effects of pH on calcium exchange in the isolated nonischemic myocardium. Acidosis induced by an increase of perfusate PCO~ (respiratory acidosis) was compared with that caused by a decrease of the bicarbonatechloride ratio (metabolic acidosis). To detect small changes in the uptake of calcium, we have devised a new and more sensitive technique utilizing 47Ca2’, a highenergy gamma emitter. METHODS

Perfused septal preparation. The experimental procedure for the arterial perfusion of the interventricular septum of the rabbit has been described previously (11, 23). Adult male New Zealand White rabbits (2-3 kg) were heparinized and anesthetized with sodium pentobarbital. The thorax was rapidly opened. The heart was excised and placed in warm oxygenated perfusate. The septal artery was cannulated within 2-4 min. The tissue was mounted between two pairs of forceps and the apex was connected to a transducer (Statham UC4P, Oxnard, carbon dioxide; pH; contractility CA). Tension and the rate of change of tension were recorded (Devices M4, Welwyn Garden City, England). The tissue was perfused either by the hydrostatic pressure obtained from a reservoir of perfusate raised lOOSEVERALEXPERIMENTALOBSERVATIONS onheartmuscle 150 cm above the preparation or by a perfusion pump indicate that a reduction of perfusate pH may inhibit calcium exchange across the cell membrane. Acidosis (Harvard Apparatus, model 975). Flow was measured by promotes recovery of mechanical function in the myotiming effluent drops of a constant size and varied becardium after a period of ischemia (28) or hypoxia (2, 7, tween 1.0 and 1.8 (ml/min)/g tissue wet wt; flow was 22), inhibits calcium uptake on reoxygenation after a maintained constant in each septum. The perfusate was period of hypoxia (22), diminishes the uptake of 45Ca2+ warmed by a heating coil immediately adjacent to the induced in the rat by isoprenaline (6), and reduces the cannula. The septa were maintained at a temperature severity of the calcium paradox (l), that is, the conse- between 27°C and 28OC. Septa weighed 0.8-1.5 g and were stimulated at a rate of 36-48 beats/min. Experiquences of perfusing the myocardium with a solution containing no calcium. Although the mechanisms of ments were begun after a period of at least 1 h had been these phenomena may be in part related to a reduction allowed for equilibration of the muscle. of calcium influx at a low pH, there is only one report of Solutions and chemicals. The perfusate contained (in the effect of acidosis alone on myocardial calcium ex- mM): Na+, 142; K+, 5.0; Ca2’, 1.5; Mg2+, 1.0; Cl-, 124; change. Morgenstern et al. (15) measured the uptake of HzPO4-, 0.4; HCOa-, 28; and D-glucose, 5.6. The solution 45Ca2+by guinea pig atria over a period of 10 min and in the reservoir was equilibrated and continuously bubdemonstrated that the uptake was probably greater un- bled with CO2-02 gas mixtures containing 5-30s COZ. der alktiine conditions (low Pco2; pH 8.0 compared to The 45Ca2+was obtained from New England Nuclear, pH 7.0). Their technique was, however, relatively insen- Boston, MA; 47Ca2+and 51Cr- labeled ethylenediaminetetraacetic acid (EDTA) were obtained from the Radisitive; the reduced uptake was difficult to demonstrate 0363-6135/79/0000-0000$01.25

Copyright

0 1979 the American

Physiological

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H526 ochemical

P. A. POOLE-WILSON

Centre,

Amersham, England. of pH. The pH of the perfusate was measured on a sample drawn anaerobically into a glass syringe from a stopcock adjacent to the cannula but prior to the heating coil, so that the sample was obtained at room temperature. Room temperature was recorded and varied between 23OC and 25OC. The pH was measured on a BMSI and PHM 72 (Radiometer, Copenhagen). Buffer standards (Instrumentation Associates) were used to calibrate the electrode. PCO~ was measured on a Severinghaus-type electrode (Radiometer). The electrode was calibrated with two gas mixtures whose CO:! content was measured on a Scholander apparatus. Measurements of pH and PCO~ were initially made at both 25OC and 38OC. Values at 25OC could be accurately calculated from those at 38OC by use of known values for the solubility of CO2 at different temperatures (24) and a blood gas calculator (Radiometer BCGl) set for the extracellular fluid; thus all PCO~ measurements were made at 38°C only. The pH was adjusted for the exact temperature of the tissue. The perfusate was prepared with a known bicarbonate concentration. All three variables in the Henderson-Hasselbalch equation were, therefore, known and could be used to assess the accuracy of measurements. The calculated bicarbonate concentration of the control perfusate varied between 25.5 and 27.5 mM when the expected value was 28 mM. Experiments with 45Ca2’. Muscles were labeled with 45Ca2+ for periods of 5-55 min. Short labeling periods were used to preferentially label rapidly exchanging calcium in those experiments in which acidosis was introduced early in the washout. At the start of the washout, the perfusate was switched to unlabeled solution. Four effluent drops were caught in planchets at recorded times during the washout. The time taken for the drops to form on each occasion was also noted. Activity of 45Ca2’ in the planchets was counted with a Geiger-Miiller probe and counter (Nuclear-Chicago, Ultrascaler II). The activity was expressed as (counts/min)/min of effluent flow, to take account of any small random changes of flow that might occur during the washout. Total tissue calcium was measured by atomic absorption spectrometry. Experiments with 47Ca2’. The isotope 47Ca2’ is a highenergy (1.3 MeV), gamma-emitting isotope with a half time of 4.5 days. It has the advantage that radioactivity from the whole septum can be continuously recorded and the thickness of the muscle (- 4 mm) is not a limiting factor. Previous measurements (11) of calcium uptake in this preparation have been made with a G-M probe and 45Ca2+. This technique only detected radioactivity in a thin layer of superficial cells and there were problems because of low sensitivity and accumulation of calcium on the outer surface of the septum. With 47Ca2+, these difficulties are overcome but several precautions are necessary. Accumulation of the isotope on metallic parts of the apparatus was eliminated by using plastic forceps to hold the muscle, a glass thermistor needle, and platinum stimulation electrodes. The radioactivity was recorded by a sodium iodide crystal (5 x 8 cm) and counter (J and P Engineering, Reading, England). The crystal was placed 2 mm in front of the septum, and the whole apparatus was surrounded by 10 cm of lead. The effluent

Measurements

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G. A. LANGER

from the septum was caught in a drop collector and returned by a suction system to the storage area for perfusate. Perfusate was not recycled. The storage area for perfusate was kept 1.5 m from the septum and surrounded by a further 10 cm of lead. With these precautions, no accumulation of 47Ca2+ could be detected and the background counts were constant for 5 h at approximately 4-10s of the total counts. The same precautions and techniques were used for experiments with 51Crlabeled EDTA. Preliminary experiments were undertaken to determine whether geometric factors were important. Elongation of the septum by as much as 4 mm at the apex and corners did not alter the amount of radioactivity recorded. The 47Ca2’ in the perfusate and whole septum was counted in a well gamma counter (ICN, Middlesex, England). Calculations. Results are expressed as means t SE. Differences between groups were assessed by Student’s t test. The quantitation of the net gain or loss of calcium that would result from the changes of 45Ca2+ efflux during experimental interventions was made by a previously described method (11). The radioactivity in the effluent drops, expressed as (counts/min)/min of perfusate flow, was plotted against time on a linear scale. A projected curve was drawn between the points prior to and after the experimental intervention. In some experiments (e.g., Fig. 1) a projection was not necessary because a control curve was available for comparison. The area between the projected curve and the curve delineated by the experimental points was measured by cutting out the area and comparing the weight of the piece of paper with that of a known area. The number of counts represented by the area was calculated. To estimate the net change of calcium from these counts, the specific activity of the 45Ca2+ at the beginning of the experimental intervention must be estimated. This specific activity is determined from the specific activity of the perfusate, completeness of labeling, and the extent of the washout of the isotope at the time of the experimental intervention. The latter two factors must relate to the monoexponential kinetic compartment in which the flux changes are occurring; these factors are readily calculated from the rate constant, the duration of labeling, and the duration of the washout at the start of the experimental intervention. The amount may be expressed as a percentage of the total calcium in that kinetic compartment. We obtained values for the rate constants and calcium content of each kinetic compartment from the previous study of Shine et al. (25), in which the same techniques were used on the same preparation. The absolute amount of calcium calculated for each experiment is not precise because the value would be expected to vary according to how closely an individual septum approximated the mean values derived by Shine et al. The values are, however, close approximations and are used only to indicate whether the component of calcium is within a more or less rapidly exchangeable fraction. The method is sufficiently sensitive to detect a rapid increase in calcium efflux of less than 30 pmol/kg tissue wet wt if the experimental intervention is introduced 8 min after the start of the washout (Fig. 1).


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washout of 45Ca2+are shown in Fig. 1. The septum was labeled with 45Ca2+for 45 min. After 7 min of washout, Mechanical response to acidosis. The effects of a 30% CO2 was introduced for a period of 10 min. The efflux of 45Ca2-twas rapidly reduced, but increased on respiratory and metabolic acidosis on the mechanical performance of the septum have been previously de- reversion to the control perfusate. After a delay of 50 min the same muscle was again labeled for 45 min and a scribed by us in detail (23). Similar results were obtained second washout was performed under control conditions. in this study. Increase of the CO2 in the gas mixture from 5% to 30% reduced the perfusate pH from 7.35 t 0.01 to A direct comparison of the two curves (Fig. 1) shows that was less than control during the entire 6.68 t 0.01 (n = 10). Developed tension after 1 min had the efflux of 45Ca2+ fallen to 29.6 * 2.6% of control and after 10 min, when period of respiratory acidosis. The control and experimental curves were identical prior to the acidosis. The developed tension was relatively steady, to 28.0 t 2.4% experiment was repeated in eight septa and there was a of control (Fig. 4). Reduction of the bicarbonate-chloride ratio at a constant PCO~ (gas mixture contained 5% COZ, similar reduction of efflux in all of them. The intervention Pco~, 43 t 2 mmHg) reduced the perfusate pH to 6.72 was introduced 6-12 min after the washout was begun. t 0.03 (n = 5). Developed tension after 1 min was greater The amount by which the efflux had been reduced by a than 95% of control and after 10 min was 78 t 4% of respiratory acidosis in comparison to control conditions could be quantified by integration of the area between control. Alterations in the differential of tension with respect to ti .me were similar to alterations in developed the experimental and control curves (METHODS, Fig. 1). The mean decrease of calcium efflux over the experimentension., and acidosis had no effect on resting tension. tal period was 142 t 18 pmol/kg tissue wet wt (n = 8). In six experiments, a respiratory acidosis was introduced while the muscle was unstimulated and remained The mean duration of exposure to an elevated Pco2 was quiescent for a period of 60-90 s. The size of the first beat 9 min. In this calculation it is assumed that the diminuon restimulation was similar to that which occurred when tion of efflux was due to changes in phase 2 alone (METHthe continuously stimulated muscle was exposed to 30% ODS), and the effect of a small change in phase 3 during a respiratory acidosis (see below) has not been included CO2 for the identical period of time, The experiments indicate that the negative inotropic effect of 30% CO2 is in this calculation. In a similar manner, 30% CO2 was introduced 38 min not beat dependent. Efflux of 45Ca2’. The effects of 30% CO2 on calcium after the washout was begun. The duration of labeling efflux were determined by introduction of 30% CO2 at with 45Ca2+was 55 min. A small reduction of efflux occurred over a period of 10 min (Fig. 2). In five experithree different times during the washout of 45Ca2+from the septum. In a group of five experiments 30% CO2 was ments, the fall of 45Ca2+efflux represented 19 t 4 ,umol/ introduced 3 min after the washout of 45Ca2+was begun. kg tissue wet wt if we assume that all the calcium arises There was no detectable change in the efflux of 45Ca2+. at this time from the washout of phase 3 (METHODS). The effects of a metabolic acidosis were studied in four To preferentially label rapidly exchanging calcium in the septum, the duration of labeling was 5-8 min. To achieve experiments. Acidosis (pH, 6.72 t 0.02, Pco~, 43 t 2 the greatest accuracy, all effluent drops were collected mmHg, n = 4) was introduced 7 min after the washout and the counting time was chosen to give a minimum of was begun and was continued for 10 min. There was no discernible effect on the efflux of 45Ca2+in any experi5,000 counts in each planchet. The effects of respiratory acidosis later during the ment. RESULTS

FIG. 1. Washout of 45Ca2+ from septum. After a washout under experimental conditions (cZosed circZes) muscle was relabeled and a second experiment was performed under control conditions (cZosed triangles). Introduction of 30% CO2 8 min after start of experimental washout caused a reversible reduction of efflux. Prior to the intervention the two washout curves were superimposable.

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H528

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Uptake of 47Ca2’. The uptake of 47Ca2+ by the septum under control conditions is shown in Fig. 3. After an initial rapid uptake, presumed to be largely into the vascular and extracellular spaces, a slow steady uptake occurred for the next 4.5 h. During the last 2 h of this period there was a small fall in developed and resting tension. At the end of the experiment the muscle was removed from the apparatus and the radioactivity was compared with that of a known volume of perfusate. The calcium content calculated from the tissue counts of 47Ca2’ was 3.3 mmol/kg wet wt. The calcium within the septum was, therefore, 80% labeled, indicating that there exists some calcium in the septum that exchanges only slowly with the extracellular fluid. A similar conclusion has been previously reported from studies of 45Ca2+ efflux (25) The effect of 30% CO2 on the uptake of 47Ca2’ is depicted in Figs. 4 and 5. The acidosis was introduced 10 6

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after 40 and 160 min of the uptake, respectively. A reversible inhibition of the uptake of calcium and the absence of any immediate net displacement are readily apparent. The same result was demonstrated in 12 septa, and Fig. 6 depicts the mean results from 6 experiments in which acidosis was introduced at 30 min. To make a comparison with seven control experiments, the counts at 20 min have been normalized to 100%. Exposure to 30% CO2 depressed the myocardial uptake of 47Ca2’. This reduction was evident irrespective of whether the septum was beating or not. Introduction of 30% CO2 (Fig. 7) during a period of almost complete quiescence inhibited the uptake in a manner similar to that in the beating septum; the same result was obtained in three septa. In Fig. 6 the counts increased between 30 and 58 min of the uptake from 111 t 2% to 134 t 2% under control conditions and to 122 t 3% in the presence of 30% CO2 (P < 0.01 for any point after 35 min). There was therefore

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FIG. 2. Washout of *%a2’ from septum. Increase of perfusate CO2 38 min after start of washout caused a small reduction of efflux.

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FIG. 3. Uptake of 47Ca2+ by septum under control conditions. After an initial rapid increase of counts, isotope is taken up more slowly and does not reach equilibrium after 4.5 h. A small decline of resting and developed tension occurred after 3 h in this experiment.

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FIG. 4. Uptake of 47Ca2+ by septum. Exposure of septum to 30% CO2 40 min after start of experiment caused a decrease in uptake of isotope. Rate of uptake increased on return to control conditions. Small fall in counts immediately after exposure to 30% CO2 was not seen in most muscles.

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a 52% reduction in the increase of counts in the period in which 30% CO2 was present. The amount of total tissue calcium labeled after 60 min of the uptake under control conditions was 1.87 t 12 mmol/kg wet wt. The extracellular space of the septum was 41% (18) and the perfusate calcium concentration was 1.5 mmol/l. The amount of intracellular calcium labeled between 30 and 58 min of the uptake was 0.32 mmol/kg wet wt under control conditions and 0.15 mmol/kg wet wt in the presence of 30% COZ.’ The decrease in influx of calcium caused by 30% CO2 over this period cannot therefore have been less than 170 ,umol/kg wet wt. This estimate is a gross approximation and inevitably an underestimate because no account is taken of the reduction of efflux already shown to occur, and because many calcium stores will have been saturated before introduction of 30% COS. Nevertheless, the total reduction in efflux caused by 30% CO2 over a lo-min period was 161 ,umol/kg wet wt and the fall of influx was greater than 170 pmol/kg wet wt over a 28 min period. A metabolic acidosis in which perfusate pH was reduced by an amount similar to that caused by 30% CO2 had no effect on 47Ca2+uptake. The same result was obtained in five experiments. Because efflux was unal-

tered by a metabolic acidosis, influx must also have been unchanged. Figure 8 shows the effect of a more severe metabolic acidosis. In this experiment the decline of tension was considerable and after 15 min there appeared to be a small reduction of uptake that increased on reversion to the control perfusate. In three experiments an attempt was made to maintain the perfusate pH constant while altering the ratio of CO2 and bicarbonate. The result of such an experiment is shown in Fig. 9. Tension fell rapidly and then slowly increased toward normal. An overshoot was apparent on return to control conditions. In association with the fall of tension there was a reduction in the uptake of 47Ca2+, despite perfusate pH being constant throughout the experiment. The fall of the 47Ca2’uptake caused by 30% CO2 might have resulted in part from a change in the extracellular space of the septum, particularly because the muscle was contracting less vigorously (Fig. 4). Changes of the extracellular space, therefore, were measured with the same techniques as for 47Ca2+but with the isotope 51Cr-EDTA. The use of 51Cr-EDTA as an extracellular marker has been previously validated (21). The uptake of the isotope under control conditions is shown in Fig. 10.


H530

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n=7

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FIG. 6. Uptake of 47Ca2’ by septum under control conditions in 7 experiments and with introduction of 30% CO2 in 6 experiments. Uptake of 47Ca2” is reduced by 30% CO2 (P < 0.01 for points after 35 min). Counts at 20 min have been normalized to 100%.

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FIG. 7. Uptake of 47Ca2+ by septum. Inhibition of uptake of 47Ca2’ by 30% CO2 occurred even in a quiescent preparation. Stimulator was turned off after 26 min. Muscle beat spontaneously at 4 beats/min before 30% CO2 was introduced.

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Equilibration of the septum is not complete for 20 min but thereafter there is only a slow increase in counts over a period of 2.5 h. The 30% CO2 causes a small and rapid increase of the extracellular space that reverts to normal when control conditions are restored. In five experiments the increase with 30% CO2 was 7.8 t 1.3% of the extracellular space. The extracellular space of this preparation estimated from the distribution volume of 51Cr-EDTA after 8 min is 41.2 t 2.2 ml of water/100 g wet wt (18). These changes could, therefore, cause asmall increase of the tissue counts of 47Ca2+in the presence of 30% CO2.

The magnitude of the increase would be less than 3% of the total tissue counts if 30% CO2 were introduced after 30 min of 47Ca2+uptake. In several experiments a small increase of this size was observed. DISCUSSION

These experiments demonstrate that a respiratory acidosis rapidly diminishes developed tension in the myocardium and inhibits both influx and efflux of calcium. The decline in developed tension is not beat dependent


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CA2+

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FIG. 9. An increase of perfusate PCO~ at constant pH was introduced during uptake of 47Ca2+. Developed tension fell, but slowly returned toward control value. Uptake of 47Ca2+ was reduced.

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and there is no immediate displacement of calcium from the tissue. In comparison, a metabolic acidosis of similar severity causes only a slow decline in developed tension, and no effect on calcium exchange can be detected. In 1879 Klug (8) demonstrated that CO2 reduced myocardial mechanical function, and in 1926 Smith (26) observed that for a given change of extracellular pH a more rapid decline of function was caused by a respiratory rather than a metabolic acidosis. Similar results were obtained in this study and have been previously reported (4, 16, 23). Because intracellular pH is altered by changes of acid-base balance in a similar manner to

developed tension (5, 20) it has been argued that intracellular pH is the controlling factor (4,5,23,26) affecting the mechanical function of the heart. In a previous study (23) we showed that both influx and efflux of potassium are also more affected by changes of intra- rather than extracellular pH, and the present study demonstrates that calcium exchange is affected in a similar manner. A respiratory acidosis but not a metabolic acidosis decreases both calcium influx and efflux, and calcium uptake can be reduced at a constant extracellular pH if the perfusate PCOZand bicarbonate are increased (Fig. 9). In this experiment it is probable that because CO2


H532

P. A. POOLE-WILSON

AND

G. A. LANGER

$ 8,000 \cn s s u 4,000

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FIG. 10. Top: uptake of 51Cr-EDTA by septum under control conditions. Bottom: introduction of 30% CO2 increased counts of “‘Cr-EDTA. Increase occurred over a period of 5 min and was reversed on returning to control conditions.

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diffuses more rapidly into the cell than bicarbonate ions (or hydrogen ions out of the cell) ($20) intracellular pH initially falls and both developed tension and calcium uptake are consequently reduced (Fig. 9). A metabolic acidosis (Fig. 10, pH 6.4) can, if it is more severe than the respiratory acidosis, (pH 6.7) depress both developed tension and calcium uptake. Under these circumstances it is presumed that a metabolic acidosis does, over a period of 30 min, appreciably lower intracellular pH. These observations are pertinent to two problems, the mechanism by which alteration of intracellular pH exerts an effect on the mechanical function of otherwise normal myocardium and the mechanism by which an acidosis alters the response of the myocardium to pathological conditions. Many effects of a change of intracellular pH on subcellular organelles in cardiac muscle have been reported (for references see Refs. 19 and 27). The physiological significance of these observations is problematic because in most of these studies the bicarbonate-CO2 buffer system is not used, the ionic concentrations often differ substantially from those likely to be present in the cytosol, and it is not possible to determine exactly the calcium activity at which many of the experiments are performed. The uptake of calcium by the myocardium reported in this paper is complex. The initial rapid uptake is probably into the vascular and extracellular spaces (Fig. 3). However, the uptake of the extracellular marker 51Cr-EDTA (Fig. 10) indicates that equilibration of molecules in the perfusate with the extracellular space may be slower than previously thought (25). Although most of the uptake is complete within 5 min, there is a small further increase for 20 min. It is possible to account for this observation by entry of the marker into the intracellular fluid, but the increase does not continue after 20 min and could be better explained as entry into less accessible parts of the extracellular space, such as T tubules. The uptake of calcium into the whole extracellular space may, therefore,

not be totally complete for up to 20 min. Thereafter, the increase must be due to exchange with intracellular calcium. Part of this exchange is slow (Fig. 3), a result compatible with studies of calcium efflux (25). Because the removal or replenishment of calcium in the extracellular space causes a rapid change in mechanical function in the myocardium, calcium exchange directly concerned with tension development probably resides in a rapidly exchanging compartment (25) and this would be saturated early in the uptake of calcium. Because in these experiments calcium influx was only studied after 30 min of exposure to the isotope and the effect was similar whether the muscle was stimulated or quiescent, it is most unlikely that calcium directly involved in the contractile process was being studied. Nevertheless, both influx and efflux of calcium from intracellular compartments, which account for most of the intracellular myocardial calcium, were decreased. If these calcium channels are partially blocked by an acidosis, it is reasonable to suppose that those channels relating to calcium exchange immediately involved in excitation-contraction coupling are similarly affected and may in part account for the negative inotropic effect of an intracellular acidosis. This argument is supported by the finding that slow calcium current is also reduced by acidosis (3, 9). Studies on isolated preparations of sarcolemma have indicated that the binding of calcium is pH sensitive (14, 29) though in the physiological range the effect may be small (14). In the present experiments we could not demonstrate over the physiological pH range any displacement of calcium from the tissue or alteration in the size of a rapidly exchanging calcium store such as has been described for lanthanum (12) and manganese (10, 13). Unlike verapamil (13), respiratory acidosis caused a decline in developed tension that was not beat dependent. These observations would best be explained if acidosis exerted its major effect by inhibition of calcium


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exchange through calcium channels in the cell membrane. The pH-sensitive site may be on the inner aspect of the bilipid layer or within the glycocalyx of the-cell membrane. The site however must not be accessible to hydrogen ions in the extracellular fluid, so that tension development and calcium exchange in the myocardium are more responsive to changes of intra- than extracellular pH. Acidosis does not alter d.eveloped tension in skele btal muscle as i t does in cardia .C muscle (1 7) because skeletal muscle is insensitive to extracellular calcium. Inhibition of myocardial calcium exchange may account for the advantageous effects of acidosis under pathological experimental conditions. Recent studies have indicated that during hypoxia acidosis (2, 7, 22) delays the onset of rise of resting tension and increases the recovery of developed tension on reoxygenation. The exposure of the myocardium to an increased PCO~ prior to a period of ischemia increases recovery of normal mechanical function on reperfusion (28). The calcium paradox is delayed and inhibited in the presence of an acidosis (1). The accumulation of calcium& the myocar-

dium of rats exposed to isoprenaline is almostI abolished by increase of the Pcoz (6). These ph.en.omena have been interpreted (1, 6, 22) as indicative of an inhibition by acidosis of calcium influx into the cell and particularly into the mitochondria. There has, however, been no previous demonstration of this inhibition in ventricular myocardium under control conditions. From Figs. 4, 5, and 6 it is evident that because only small changes occur, a particularly sensitive technique is necessary to demonstrate the phenomena. Under pathological conditions large gains in tissue calcium occur and if these are inhibited by an acidosis they can be detected by lessprecise techniques. Part of this work was conducted during the tenure of a BritishAmerican Research Fellowship by P. A. Poole-Wilson. The investigation was supported by Public Health Service Grant HL-11351-08 and a grant from the British Heart Foundation. Address requests for reprints to P. A. Poole-Wilson, Cardiothoracic Institute, 2 Beaumont St., London WIN 2DX, England. Received

19 June

1978; accepted

in final

form

22 November

1978.

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Ca2+ exchange: effects on mechanical and electrical function of guinea-pig myocardium.

Endothelin reverses the effects of acidosis on the intracellular Ca2+ transient and contractility in ferret myocardium.

Effect of pH on ionic exchange and function in rat and rabbit myocardium.

Effect of ouabain in therapeutic concentrations on K+ exchange and contraction of human and rabbit myocardium.

Calcium exchange in rabbit myocardium during and after hypoxia: role of sodium-calcium exchange.

The effects of cocaine on intracellular Ca2+ handling and myofilament Ca2+ responsiveness of ferret ventricular myocardium.

Effects of hypoxia, acidosis, and simulated ischemia on repriming of caffeine contracture in rat myocardium.

Effects of lemakalim on changes in Ca2+ concentration and mechanical activity induced by noradrenaline in the rabbit mesenteric artery.

Na-H exchange in myocardium: effects of hypoxia and acidification on Na and Ca.

Effects of allocryptopine on outward potassium current and slow delayed rectifier potassium current in rabbit myocardium.

Creatine kinase release, potassium-42 content, and mechanical performance in anoxic rabbit myocardium.

Mechanical properties of skinned rabbit psoas and soleus muscle fibres during lengthening: effects of phosphate and Ca2+.

Effects of ibutilide fumarate, a novel antiarrhythmic agent, and its enantiomers on isolated rabbit myocardium.

Prolonged propionyl-L-carnitine pre-treatment of rabbit: biochemical, hemodynamic and electrophysiological effects on myocardium.

Sarcolemmal Na(+)-Ca2+ exchange activity and exchanger immunoreactivity in developing rabbit hearts.

Exchange transfusion in newborn infants: effects on granulocyte function.

Effects of endothelin on cytosolic Ca2+ level and mechanical activity in rat uterine smooth muscle.

The effects of Na+ replacement on intracellular pH and [Ca2+] in rabbit salivary gland acinar cells.

Effects of Ca2+ and ionophore A23187 on protein synthesis in intact rabbit reticulocytes.

Effects of diltiazem and verapamil on the mechanical performance of the rabbit myocardium perfused with an oxygenated and hypoxic medium [proceedings].

Influence of acidosis on rumen function.

Effects of K+ channel blockers on the action potential of hypoxic rabbit myocardium.

Heparin-mediated reductions of the toxic effects of protamine sulfate on rabbit myocardium.

Effects of α-methylnorepinephrine on cardiac function and myocardium at early stage of resuscitation in rabbits.