J. Phyriol. (1976), 262, pp. 755-771 With 7 text-ftgureM Printed in Great Britain
755
DIRECT MEASUREMENT OF THE INTRACELLULAR pH OF MAMMALIAN CARDIAC MUSCLE
BY D. ELLIS AND R. C. THOMAS From the Department of Physiology, University of Bristol, Bristol BS8 1TD
(Received 21 June 1976) SUMMARY
1. The intracellular pH (pH,) of sheep heart Purkinje fibres and rat, ferret and guinea-pig ventricle has been measured using recessed-tip pHsensitive micro-electrodes. 2. In the absence of CO2 the pHi was approximately 7-2 in all the preparations used. In 5 % CO2 the mean pH, was 7414 in rat and ferret ventricle and 7-02 in sheep Purkinje fibres. 3. The pH, response to an increase or a decrease in the CO, level (at constant external pH) was biphasic with a large transient change followed by a partial recovery to a new sustained pH,. 4. The intracellular buffering capacity was 34-8 + 2-7 m-equiv H+/pH unit per 1. (+ S.E. of mean) in sheep Purkinje fibres, 76-6 + 13-6 in rat ventricle and approximately 69 in ferret ventricle. 5. The pHi of all the preparations tested indicated that H+ ions were not passively distributed across the cell membrane. There was also little or no pH1 change produced by depolarization with high K solutions. 6. Short exposures to hypertonic solutions (100 mm sucrose or 50 mM-KCl) produced a decrease in pHi of approximately 041 pH units. 7. Acetazolamide slowed the pHi response to CO2 changes. 8. Restoration of the pHi after displacement by increasing the C02 was not blocked by ouabain or SITS. 9. The relationship between pHi and cardiac contractility is discussed. INTRODUCTION
Enzyme reactions are well known to be very sensitive to the activity of H+ ions. This may well form the basis for the influence of pH on cardiac contractility, acid solutions decreasing and alkaline solutions increasing contractile strength, respectively (Daly & Clark, 1921). There is some dispute as to whether these effects are due to external or to internal pH
756
D. ELLIS AND R. C. THOMAS changes (see Vaughan Williams & Whyte, 1967 and Cingolani, Mattiazzi, Blesa & Gonzalez, 1970). Our knowledge of how intracellular pH (pHi) affects the contraction of heart muscle is limited by a lack of information on the extent to which the pHi is altered by the methods used to produce these changes. Indeed even the normal value of the pHi is in some doubt. Of the techniques available for measuring the pHi of tissues, only the DMO method (see Waddell & Bates, 1969) has been widely used. It permits pH measurements even in the small cells of heart muscle. However the pH, value obtained probably reflects the over-all pH of all the cellular compartments and the method is unsuitable for following changes in pH1 unless they have a very slow time course. The use of pH-sensitive electrodes was until recently limited to the large cells of crab or frog skeletal muscle (Caldwell, 1954; Kostyuk & Sorokina, 1960) or squid axon (Caldwell, 1958; Bicher & Ohki, 1972). The introduction of the recessed-tip design of glass pH micro-electrode (Thomas, 1974) now makes it possible to use such electrodes even in the small cells of mammalian heart and permits the continuous recording of pHi. The localized measure of the pH1 at the tip of the electrode is likely to produce a closer estimate of the sarcoplasmic pH, that is the pH of the compartment in which the contractile proteins are situated, than would be obtained with the DMO technique. The recessed-tip type of pH electrode has already been used successfully in snail neurones (Thomas, 1974, 1976a, b) and crab leg muscle (Aickin & Thomas, 1975). We have used these electrodes on the smaller cells of rat, guinea-pig and ferret ventricular muscle but most of the work to be described here concerns experiments on rat ventricle and sheep heart Purkinje fibres. A preliminary report of some of the results has already been published (Ellis & Thomas, 1976). Our aims were to measure the normal pH1 of the muscles, to see how this was affected by C02 and various other experimental procedures, to calculate the intracellular buffering capacity of the cells and to compare the results with the better characterized pH, regulatory and buffering mechanisms of the larger, non-mammalian cells previously studied. METHODS
Sheep hearts were obtained from a local slaughterhouse. Immediately after the animal was killed the heart was removed and immersed in a modified Tyrode solution at room temperature which had been equilibrated with a gas mixture of 95 % 02, 5 % C02. During transport to the laboratory and during dissection the solution was continually gassed. Rats and guinea-pigs were killed by a blow to the back of the head and ferrets by an overdose of sodium pentobarbitone. In all cases, free-running thin ventricular
CARDIAC MUSCLE pH
757
bundles (or Purkinje fibres from the sheep hearts) were selected and removed with a little side-wall material attached. This was used to pin the ends of the preparations on to the silicone rubber base of the experimental chamber where it was continually superfused with saline. The preparation was supported at several points along its length by stainless-steel wires running at right angles to it across the base of the chamber. This was found to facilitate penetration by the electrodes without breakage. Stable penetrations seldom lasted longer than 1 hr in ventricular muscle preparations, but in sheep heart Purkinje fibres stable measurements could be obtained for many hours. Solutions. The normal saline equilibrated with nominally 95 % 02, 5 % CO2 contained (mM): Na+ 140, K+ 4, Ca2+ 2, Mg 2+ 1, pyruvate 2, glucose 10, Cl- and HC03reciprocally varied to give a measured pH of 7 40 at 350 C (the concentration of HCO3- was about 20 mm, variations being due to the equilibration gas in different cylinders containing varying amounts of C02; the mean CO2 content was 4- 6 %). The C02-free solutions were saturated with 100% 02, lacked bicarbonate but contained 10 mm of the Na salt of HEPES (2-N-2-hydroxyethyl piperazine-N'-2-ethansulphonic acid). Micro-electrodes. Conventional KCl and pH-sensitive electrodes were prepared as described previously (Thomas, 1974, 1976a). Signal recording. The output from the normal KCl micro-electrode with respect to an agar-Ringer bath reference electrode was displayed on one channel of a potentiometric pen recorder with differential input (Watanabe Multicorder MC 611). The KCl electrode output was also fed, via a unity-gain buffer amplifier, to the low impedance input of a Vibron Electrometer (E.I.L. 62A). The pH-sensitive electrodes were connected to the high impedance (1016 Q) side. The resultant output was also displayed on the pen recorder so that a pH trace could be obtained that was independent of membrane potential changes when the electrodes were intracellular. The cells penetrated by the two electrodes were normally less than 1 mm apart. Low membrane potentials were obtained with a few Purkinje fibres so a minimum acceptance level of 60 mV was employed. However the mean pH1 of the fibres with a low membrane potential was not significantly different from the pHi measured in the normal group. RESULTS
Rat ventricle pHj and the effect of CO2 While most experiments were done on sheep Purkinje fibres, early ones were done on other mammalian preparations. Fig. 1 illustrates the general procedures involved in measuring the pHi of all the cardiac tissues employed. Initially the rat ventricular muscle was superfused with saline equilibrated with 5 % C02, 95 % 02 and the pH-sensitive micro-electrode (bottom trace) and the KCI micro-electrode (top) were extracellular. The pH micro-electrode was then pushed into the tissue where it recorded the membrane potential of a cell minus an offset voltage that was proportional to the pHi. Then the KCl micro-electrode was pushed into the muscle until with some difficulty in this case, a stable membrane potential was recorded from another cell. The KCI micro-electrode output voltage was electronically subtracted from that of the pH electrode. Therefore if the two cells penetrated had approximately the same membrane potential and behaved
758 D. ELLIS AND R. C. THOMAS in the same way to external solution changes, the recording of pHi (as in Fig. 1, bottom trace) would be independent of membrane potential changes. A reasonably accurate measure of pHi was then possible by 0 10 min I
I
20 -
E wU
40
-
60 Em
80
-
69 I
0.
71 L I 5
7 3
7-
Lp6
L
pH 6-4
I
I
1
%
100% °2
Fig. 1. Pen recording of an experiment to measure pHi in rat ventricular muscle and the response to removal and replacement of external CO2. The upper trace shows the membrane potential (Em) recorded by a conventional micro-electrode. The lower trace shows the voltage difference between the pH and conventional micro-electrodes. The pH electrode was inserted first, then, with some difficulty, the conventional micro-electrode. When both electrodes were intracellular and a stable pH, value obtained the bathing solution was changed from a bicarbonate buffered solution equilibrated with a 5 % C02, 95 % 02 to give a pH of 7-4 to one that was CO2-free but contained 10 mM-HEPES buffer (also at pH 7-4). The pH-sensitivity of the electrode was tested (right) by a reduction of the solution pH to 6-4 (both electrodes extracellular). Before this pH measurement, the muscle was stimiilated by just stiprathreshold pulses once every 10 sec.
CARDIAC MUSCLE pH 759 taking the average of several paired penetrations with the two types of electrode in each experiment. Since C02 crosses cell membranes very easily it is inevitably unimportant factor affecting pHi and we have briefly reported its effects on the pHi of sheep heart Purkinje fibres (Ellis & Thomas, 1976). We have also found that changing C02 has similar effects on the pHi of rat (and ferret) ventricle. Fig. I illustrates the effects of complete removal of external CO2. The pH, showed a large transient decrease followed by a return of the pH1 to a level nearer that in the control solution. The response to re-addition TABLE 1. Summary of pH, and Em values in experiments in the rat, ferret and guinea-pig
Equilibration gas 5% C02,95% 02 Preparation and
temperature Rat ventricle 350 C
,
A,__,____
pH1
Em
pHI
Em
7-14 + 0 03 n= 9
78*7 +1.6
7.20 + 0 03 n= 7 7*17+ 006 n= 7
75.6 +1-7 n= 7 75.4 + 2.8 n= 7
7.14 n =2
84.6
7-20
79.5
n = 9 -
200 C Ferret ventricle 350 C
Equilibration gas 100% 02
n = 2
n =2
n= 2
Guinea-pig ventricle
350C
-
7-16 n =2
75.6
n= 2 Values given are the means ( s.E. of mean) for the number of hearts given by n. The measurements of Em and pH1 for each heart were normally the average from several penetrations.
of C02 was also biphasic with a large decrease in pH, quickly followed by a return to the control level. The mean stabilized pHi values for a number of experiments on rat, guinea-pig and ferret ventricle are given in Table 1. Some other experiments were carried out on rat ventricle at a lower temperature (200 C). The pHi values for this set of experiments were not significantly different from those at 350 C (Table 1). A similar finding using the DMO technique in frog heart muscle has been reported by Reeves & Wilson (1969). A few experiments were carried out on guinea-pig and ferret ventricular preparations. The corresponding pHi values for the same set of experimental conditions are also shown in Table 1. These were very similar to
760 D. ELLIS AND R. C. THOMAS those obtained in the rat. The time course of pHi changes in response to an alteration of the external C02 concentration was very similar in all three preparations. The pHi was consistently higher in the absence than in the presence of C02.
Effect of C02 on pHi in sheep heart Purkinje ftbrea Most of our work on cardiac pHi was carried out on sheep heart Purkinje fibre preparations. The initial penetration of the pH-sensitive electrode into this tissue was more difficult, probably due to the surrounding connective tissue. Once in, however, micro-electrodes often remained intracellular 0 10 min 20
~40
E_ E
60
Em 80
6.8I
7-0 -' L72
100 %°2
Fig. 2. Pen recording of an experiment on a sheep heart Purkinje fibre to show the effect on the Em and pH, of removal and replacement of external C02. Procedure followed was the same as in Fig. 1. The pH electrode showed signs of coming out of the cell on two occasions before a stable penetration was obtained. The effect of C02 removal (with replacement by HEPES buffered saline) and re-addition was then observed. The external pH was maintained at 7-4 throughout.
for several hours and showed very stable pH1 levels. The tendency for the preparation to behave as a good electrical syncytium, which can be observed when the membrane potential is altered, aided the measurement of an accurate value of the pH,. Fig. 2 shows that the responses to removal and re-addition of C02 in
761 CARDIAC MUSCLE pH sheep heart Purkinje fibres were similar to those observed in rat ventricle. The pHi changes were however slower and larger than equivalent changes in rat heart. The mean pHi in sheep heart Purkinje fibres was 7-02 in 5 % C02, 95 % 02 and 7-20 in 100 % 02, i.e. C02 has a larger effect on pH1 than in rat heart where the corresponding values were 7-14 and 7-20. 10 min
m
60 E 70 L E
Em
80 _ 6-8
7-2 6-7 % CO2 1-6 % C02 Fig. 3. Effect of various C02 levels on pH, of a sheep-heart Purkinje fibre. The preparation was initially perfused with the control solution equilibrated with 4-7 % C00, 95.3 % 02. The C02 concentration was then changed, first to 1-6%, returned to the control level and then increased to 6-7 % before finally being returned to the control level again. The external pH was maintained constant at 7-4 throughout by variation of the bicarbonate concentration. The rather large depolarization observed in 6-7 % C02 solution was not a consistent finding.
Smaller alterations in the C02 level of the saline produced similar, but smaller, changes in the pH, as shown in Fig. 3. The external pH was maintained at 7-40 throughout. The mean value of pH1 in 1-6 % C02 was 7-09 + 0-04 (n = 9) and in 6-7 % C02 was 6-94 + 0-03 (n = 4). No change in pH1 was apparent in 100% 02 solutions when the HEPES buffer was substituted by Tris maleate. Intracellular buffering capacity of cardiac tissue When the external C02 concentration was altered the initial change in pHi observed was due to the reaction.
C02 + H O=H
--H+ + HCO3-. Since equal numbers of H+ and HC03- ions are produced it is possible to estimate the internal buffering power from the pHi change assuming that the internal HCO3- level depends only on the pH1 and the C02 level, that the C02 level and the carbonic acid dissociation constant are the same inside and outside the cell and that there is no significant movement of
D. ELLIS AND R. a. THOMAS 762 H+, OH- or HC03- ions across the cell membrane during the period of the initial pH change. From experiments of the type shown in Figs. 2 and 3 the buffering power of sheep heart Purkinje fibres was found to be 34-8 + 2-7 m-equiv H+/pH unit per litre (s.E. of mean, n = 17). 10 min
0
m
10
20 30 E E w
_-
40 _-
50
-
60 1
70 1-
80
-
Em
90 L 6-8 Ir
7-0_
pH_
2_ 7
140 mM -K
Fig. 4. Pen recording to show the effect of a large membrane depolarization on the pHi of a sheep heart Purkinje fibre. The normal K concentration was raised from 4 to 140 mm by replacement of all but 4 mm-Na for the test period indicated by the bar. The fibre was perfused with solutions equilibrated with 5 % C02, 95 % 02 to give an external pH of 7-4 throughout.
In rat ventricle the buffering capacity was estimated using the same method and was found to be 76-6 + 13-6 (S.E. of mean, n = 4). Two experiments in ferret ventricle produced a mean value of 68-8. The speed of the pH, recovery after displacement by C02 changes suggests that the last assumption mentioned above is incorrect so that the values given above are probably higher than the total buffering power of the various cellular constituents. Nevertheless the buffering power is obviously large
AZRDIAC MUSCLE pH
763
and this, taken with the evidently highly active H+ pump, shows that pH, can be regulated with considerable precision and speed. The effect of membrane potential on pHi. The measured pHi values are clearly far from those expected from a passive distribution of H+ ions across the cell membrane, which would give a pHi of about 6-1 for both Purkinje fibres and for rat ventricle. We found no correlation between the normal membrane potential and the pH1, Even with a large depolarization produced by the almost complete replacement of external Na by K (Fig. 4) the maximum pHi change recorded was only 005 units. Any small changes in pH1 observed only appeared immediately after the addition or removal of the high K solution and probably reflected slightly imperfect subtraction, in that depolarization or repolarization possibly had a slightly different time course in the two cells penetrated. Active transport of H+ (or OH- or HCO3-) ions is clearly necessary to account for both the non-equilibrium distribution of H+ ions across the cell membrane and the partial recovery of pHi seen after changes in external C02 (Figs. 1-3).
Effects of contraction on pHs All the experiments described were carried out on preparations that were quiescent for most of the time. The pH-sensitive electrodes used, unlike normal microelectrodes, were relatively inflexible and therefore difficult to keep intracellular during the normal strong contractions of ventricular muscle. It was, however, easier to maintain a stable penetration during the relatively weak contractions of Purkinje fibres. We have found that the pH, following short periods of stimulation (4 min at a rate of 120/min) was not greatly affected. The lack of an observable pH, change after a period of stimulation probably justifies the use of quiescent prepara. tions in those experiments. In a few preparations that were spontaneously active throughout part or the whole of an experiment essentially the same pH, and changes in pHi were observed as in quiescent fibres. However transient pHi changes during the course of individual contractions would be too fast to be recorded with this type of electrode.
The effects of external pH on pHi We have found that alteration of the external pH produced only slow and small changes in pHi. An experiment to illustrate this is shown in Fig. 5. The changes in pHi were much smaller and slower than the pH change in the external solution produced by a reduction of the bicarbonate concentration. (The reasons for the small transient changes in pH1 within the first few minutes of a solution change are unknown but were observed in several preparations.) The changes in pHi following a reduction of the external bicarbonate concentration were approximately exponential, the pH1 stabilizing at a
764 D. ELLIS AND R. C. THOMAS new level within 15-40 min. The size of the pHi change was approximately 0-2 pH units for a unit change in external pH. The internal acidification was accompanied by an average depolarization of 3 mV. When the external pH was returned to normal the pHi recovered in a similar manner to the restoration of pHi after the large transient decrease following re-addition of C02 (Figs. 2 and 3). Both processes require some sort of active H+ pumping. The rate of restoration of pHj on return to normal external pH was similar to that for the restoration of pH1 following the re-addition of C02 when the external pH was unaltered. Em 10 min E LU
9goL 6-4
66
6.8-
_pH,
7-0 7.27.4
pH 64 pH 64 Fig. 5. Pen recording of an experiment to illustrate the effect of a change of the extracellular pH (by 1 0 units) on the pH1 of a sheep heart Purkinje fibre. The preparation was perfused with solutions equilibrated with 5 % C02, 95 % 02 throughout. During the periods indicated by the bars the bicarbonate concentration was reduced approximately tenfold to produce an extracellular pH of 6-4. The extent of the pH change in the extracellular solution is shown on the right, at the end of the experiment, where both electrodes were removed from the fibre.
The effect of hypertonicity on pHi In many experimental procedures it is often useful to be able to add compounds to the normal perfusion solution without removing one of the normal constituents, although this makes the test solution hypertonic. To estimate how this type of procedure might affect pH, we have added sucrose (100 mM) or KCI (50 mM) without reducing the NaCl concentration. An experiment of this type is illustrated in Fig. 6. Here the addition of 100 mm sucrose reduced pH, by less than 0.1 pH units. Similarly, extra KCI caused pHi changes of only about 0.1 units. No long-term measurements were carried out to see if this pH change was maintained. Adler, Anderson & Zett (1975) have reported that the addition of 80 mm man-
765 CARDIAC MUSCLE pH nitol to the solution bathing rat diaphragm muscle produced an increase in pH, of approximately 01 pH units. They estimated the pH, from measurements of the distribution of the weak acid DMO and the weak base nicotine after the tissue has been in the vhypertonic test solution for 4 hr. 5 min
70_
El E
..
IJI 100 mM sucrose Fig. 6. Pen recording of an experiment on a sheep heart Purkinje fibre to illustrate the effect of an increase in tonicity of the bathing solution. For the period indicated by the bar the bathing solution was hypertonic due to the addition of 100 mm sucrose to the normal saline. The external pH was maintained at 7-4 throughout with CO2HCO3- buffering.
The effect of acetazolamide Acetazolamide, an inhibitor of carbonic anhydrase, has been shown to slow the effects of C02 addition and removal on the pH1 of snail neurones (Thomas, 1976a). It has, however, been suggested that in muscle carbonic anhydrase might be absent or present in only trace amounts (Van Goor, 1940; Matthews Laszlo, Campbell, Kibby & Freedman, 1968). In sheep heart Purkinje fibres acetazolamide appeared to have some effect, although only a small one, on the pH, responses to C02 addition and removal (Fig. 7). This suggests that carbonic anhydrase is present in cardiac tissue but perhaps only at a low concentration. Effects of ouabain and SITS on the regulation of pHi. Thomas (1976b) has reported that the Na+ and H+ pumps have independent mechanisms in snail neurones. The Na+ pump can be inhibited
D. ELLIS AND R. C. THOMAS by ouabain and the H+ pump by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) but not vice versa. In cardiac Purkinje fibres we have similarly found that the pH, changes occurring when the C02 concentration was altered were unaffected by doses of ouabain (I0-5 M) sufficient to inhibit the Na+ pump completely. However in Purkinje fibres SITS did not affect the pHi responses. Thus in this respect cardiac tissue differs from snail neurones but is similar to mouse skeletal muscle, where SITS has recently been found to be similarly ineffective on the H+ pump (C. C. Aickin and R. C. Thomas, unpublished observations.) 766
Acetazolamide (4 /dM)
15 min
50_
70t' 6-8
i7.01
I~ 100%°2
L 10 100%°2
Fig. 7. Pen recording of an experiment on sheep heart Purkinje fibres showing the effect of 4 IzM acetazolamide on the responses to removal and readdition of CO2 in the gas equilibrated with the perfusion solution. The external pH was maintained at 7-4 throughout. A large artifact is present on both recordings immediately after the addition of acetazolamide, possibly caused by vibration. DISCUSSION
With the use of a method that allows a direct measurement of the pHi of cardiac tissues we have found a non-passive distribution of H+ ions across the cell membranes. This is in accord with previous measurements in crab leg muscle (Caldwell, 1954), frog skeletal muscle (Kostyuk & Sorokina, 1960), squid giant axon (Bicher & Ohki, 1972) and snail neurones (Thomas, 1974). One recent report (Hannan & Wiggins, 1976) has suggested that H+ ions are passively distributed in frog skeletal muscle but this conclusion probably resulted from the type of electrode used (Carter, Rector, Campian & Seldin, 1967) which has previously been shown to be unsuitable for use in tissues where there is a possibility that all of the pH-sensitive glass
767 CARDIAC MUSCLE pH is not intracellular (Paillard, 1972). The observation in the present experiments that depolarization with high K solutions produced no large changes in the pH, supports the finding that H+ ions are not passively distributed. The pH, values obtained in the present experiments on heart muscle are in general equal to, or somewhat larger than, those measured previously with indirect techniques (see Waddell & Bates, 1968; Neely, Whitmer & Rovetto, 1973) and with open tipped electrodes (Lavall6e, 1964). Some differences in the pHi values would be anticipated in view of differences in the techniques used and of the experimental conditions. The experiments by Lavellee (1964) on rat atrial preparations gave a pHi value of 6-91. This rather low figure may have been related to the high rate of stimulation used (200/min), the composition of the perfusion solution, or it may have been influenced by the low sensitivity of his electrodes (approximately
30 mV/pH unit, i.e. only half the theoretical value). On the other hand the differences may reflect a variation of the pHi in different regions of the heart, auricular pHi perhaps being lower than that of ventricle. In the absence of C02 we found that the pHi was similar in all the cardiac preparations we tested, but in the presence of C02 the values were different. This may be due to differences in the buffering capacity of sheep heart Purkinje fibres and of normal ventricular muscle. The calculated buffering powers for the cardiac tissues used were approximately 35 for sheep heart Purkinje fibres and 77 for rat ventricle (and 69 for ferret ventricle), indicating a relatively high buffering capacity. Using the same techniques the buffering power of snail neurones was found to be approximately 30 (Thomas, 1976), for crab leg muscle was 47 (Aickin & Thomas, 1975) and for mouse skeletal muscle 50 (Aickin & Thomas, 1976). There is a large variation in previous estimates of the intracellular buffering capacity of heart muscle. Most of the early work suggested that heart muscle was only poorly buffered in comparison to skeletal muscle (see Brody, 1930) whereas more recently one group have claimed that the reverse is true from in vivo experiments in dog (Clancy & Brown, 1966). They calculated a buffering capacity of about 71 for dog heart. The larger buffering power found in ventricular muscle compared to sheep heart Purkinje fibres possibly reflects the need for better buffering of pH changes in working myocardium compared to the Purkinje fibre. The values obtained in the present experiments are probably slight over-estimates as no allowance has been made for the restoration of the pHi up to the time that the peak C02 effect was observed. A large correction would not be required in sheep heart Purkinje cells because the recovery responses are so slow. However, in the smaller cells of ventricular muscle these responses were more rapid and so a larger correction would be required. Rapid changes of pH have been observed on the outer surface of muscle
768 D. ELLIS AND B. C. THOMAS fibres during activity (Dubuisson, 1950; Dhalla, Yates & Kleinberg, 1973). It was suggested that these might indicate rapid pHi changes associated with contractile activation. The time course of these events would be too fast to be observed with the type of electrode used in the present experiments. However, prolonged depolarization with K-rich solutions, which might be expected to prolong some of the effects of the activation process, produced little or no change in pH,. The apparent lack of effect of temperature on the pHi of rat ventricle is of interest because Reeves & Wilson (1969) also found that the pHi of frog heart muscle was unaffected by temperature. However, they found that both the pH of the surrounding blood and the pHi of skeletal muscle from the same animals were temperature-dependent. An effect of temperature has also recently been found on the pHi of mouse skeletal muscle (C. C. Aickin and R. C. Thomas, unpublished). This could indicate a difference in the major intracellular buffering components of skeletal compared to cardiac muscle (Reeves, 1972). An apparent lack of effect of temperature on pHi in heart muscle might of course only be due to the influence of temperature on processes which tend to change the pH in opposite directions. The relatively small effects of acetazolamide suggest that carbonic anhydrase is present but that its concentration or activity may be fairly low. It has been suggested that a function of the carbonic anhydrase found in snail neurones might be to maximize the buffering capacity of the cells (Thomas, 1976a). Cardiac muscle however appears to be well buffered to C02 changes (Figs. 1-3). The method most commonly used to investigate the effects of pHi on contraction has been to expose the muscle to an altered PCO., keeping the external pH constant by changing the bicarbonate concentration. Only the C02 is assumed to cross the cell membrane, therefore producing a change in pH,. The results of Vaughan Williams & Whyte (1967) in rabbit heart led them to suggest that there was no effect of pHi on contractile force. Their measurements, however, appear to have been made during a steady-state situation approximately 40 min after the change in C02 and bicarbonate. The present results show that there is only a large effect on pH1 during the first 10 min of exposure to an altered C02 level. Therefore if contractile strength is dependent upon pH,, the largest effects should be most noticeable in the first 10 minutes, whereas the effects after 40 min might not appear significant. McElroy, Gerdes & Brown (1958) and Lorkovi6 (1966) have described an inhibition of contraction of guinea-pig and frog heart when exposed to an increase of Pco, with the extracellular pH constant. They observed
769 CARDIAC MUSCLE pH biphasic changes in contractile force, there being an initial decrease followed by a partial recovery. Similarly Cingolani et al. (1970) found a decreased contractility in cat and rat hearts with an increase in Pco,. Their measurements were made within approximately 15 min of an increase in Pco2, so again a large effect on pHi would be expected. Vaughan Williams (1955) showed a biphasic change in the rate of beating of rabbit heart when the Pco, and bicarbonate were altered to maintain a constant external pH. The rate of beating declined to a minimum within approximately 5 min and recovered to near the control level within a further 10 min. A biphasic change in rate in the opposite direction occurred on return to the normal Pco,. These effects might well have been due to the pHi changes we have observed. It is generally agreed that external pH has a large effect on cardiac contraction but these experiments suggest that internal pH changes can also influence contractility, although the effects might only be small and perhaps transient due to high internal buffering capacity and active proton pumping. Such internal pH changes might affect the cell membrane permeability to Ca and/or the uptake and release of Ca by the sarcoplasmic reticulum (Carvalho & Leo, 1967). Our results show that the use of the recessed tip design of micro-electrode permits a direct measure of the pH, even in the small cells of mammalian ventricular muscle. This is the only technique available so far for giving a continuous measure of the pH,. The indirect methods can be used for studying slow changes in the pHi but, as has been described above, the pH changes in response to C02 are rapid and biphasic. We are grateful to the Medical Research Council for financial support and to Dr M. J. Purves and Miss C. C. Aickin for reading the manuscript. REFERENCES ADLEpR, S., ANDERSON, B. & ZETr, B. (1975). Effect of osmolarity on intracellular pH of rat diaphragm muscle. Am. J. Phy8iol. 228, 725-729. AIcmNw, C. C. & THoNs, R. C. (1975). Micro-electrode measurement of the internal pH of crab muscle fibres. J. Phy8iol. 252, 803-815. AIcRmN, C. C. & Thoii&s, R. C. (1976). Intracellular pH of mouse soleus muscle. J. Physiol. 260, 25-26P. BIcEmR, H. I. & OEmii, S. (1972). Intracellular pH electrode experiments on the giant squid axon. Biochim. biophy8. Acta 255, 900-904. BRODY, H. (1930). The carbon dioxide dissociation curve of frog heart muscle. Am. J. Phy8iol. 93, 190-196. CAT.wELL, P. C. (1954). An investigation of the intracellular pH of crab muscle fibres by means of micro-glass and micro-tungsten electrodes. J. Physiol. 126, 169180. CALDwELL, P. C. (1958). Studies on the internal pH of large muscle and nerve fibres. J. Physiol. 142, 22-62.
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D. ELLIS AND R. C. THOMAS
CARTER, R. L., RECTOR, F. C., CAMPIAN, D. S. & SELDIN, D. W. (1967). Measurement of intracellular pH of skeletal muscle with pH-sensitive glass micro-electrodes J. clin. Inve8t. 46, 920-933. CARVALwo, A. P. & LEO, B. (1967). Effects of ATP on the interaction of Ca++, Mg++ and K+ with fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle. J. gen. Phy8iol. 50, 1327-1352. CLANcY, R. L. & BROWN, E. B. (1966). In vivo CO2 buffer curves of skeletal and cardiac muscle. Am. J. Phyaiol. 211, 1309-1312. CINGoLANI, H. E., MAT&iAzzi, A. R., BLESA, E. S. & GONZALEZ, N. C. (1970). Contractility in isolated mammalian heart muscle after acid-base changes. Circulation Re8. 15, 393-405. DALY, I. DE BURGH & CLARx, A. J. (1921). The action of ions upon the frog's heart. J. Physiol. 54, 367-383. DHALLA, N. S., YATES, J. C. & KTLINBERG, I. (1973). Oscillations of intramuscular pH in perfused rat heart. Can. Jnl Physiol. & Pharmacol. 51, 234-238. DuIssON, M. (1950). Some chemical and physical aspects of muscle contraction and relaxation. Proc. R. Soc. B. 137, 63-70. ELLs, D. & THOMAS, R. C. (1976). Microelectrode measurement of the intracellular pH of mammalian heart cells. Nature, Lond. 262, 224-225. HANNAN, S. F. & WIGGINS, P. M. (1976). Intracellular pH of frog sartorius muscle. Biochim. biophy8. Acta 255, 900-904. KoSTYUK, P. G. & SOROKINA, Z. A. (1960). On the mechanism of the ion distribution between cell protoplasm and the medium. In Membrane Transport and Metabolism, ed. KLEINZELLER, A. & KOTYK, A., pp. 193-203. London: Academic Press. LAVATLRE, M. (1964). Intracellular pH of rat atrial muscle fibres measured by glass micropipette electrodes. Circulation Re8. 15, 185-193. LoRKovi6, H. (1966). Influence of changes in pH on the mechanical activity of cardiac muscle. Circulation Res. 19, 711-720. MArrlws, C. M. E., LASZLO, G., CAMPBELL, E. J. M., KIBBY, P. M. & FREEDMAN, S. (1968). Exchange of CO2 in arterial blood with body CO2 pools. Re8p. Phyeiol. 6, 29-44. MCELROY, W., JR, GERDES, A. & BROWN, E. B., JR (1958). Effect of CO2 bicarbonate and pH on the performance of isolated guinea pig hearts. Am. J. Phy8iol. 195, 412-416. NEELY, J. R., WHITMER, J. R. & Rov-ETTo, M. J. (1973). Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. Circulation Re8. 37, 733-741. PAILLARD, M. (1972). Direct intracellular pH measurement in rat and crab muscle. J. Phyeiol. 223, 297-319. REEVES, R. B. (1972). An imidazole alphastat hypothesis for vertebrate acid base regulation: tissue carbon dioxide content and body temperature in bullfrogs. Reep. Physiol. 14, 219-236. REEVES, R. B. & WILsoN, T. L. (1969). Intracellular pH in bullfrog striated and cardiac muscle as a function of body temperature. Fedn Proc. 28, 782. THOMAS, R. C. (1974). Intracellular pH of snail neurones measured with a new pHsensitive glass micro-electrode. J. Physiol. 238, 159-180. THOMAS, R. C. (1976a). The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J. Physiol. 255, 715-735. THOMAS, R. C. (1976b). The ionic mechanism of the H+ pump in a snail neurone. Nature, Lond. 262, 54-55. VAN GOOR, H. (1940). Die Verbreitung und Bedeutung der Carbonanhydrase. Enzymologia 8, 113-128. 1
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VAUGHAN WILLIAMS, E. M. (1955). The individual effects of C02, bicarbonate and pH on the electrical and mechanical activity of isolated rabbit auricles. J. Physiol. 129, 90-110. VAUGHAN WILLIAMS, E. M. & WHYTE, J. M. (1967). Chemosensitivity of cardiac muscle. J. Phy8iol. 189, 119-137. WADDELL, W. J. & BATES, R. G. (1969). Intracellular pH. Phy8iol. Rev. 49, 285-329.
755
DIRECT MEASUREMENT OF THE INTRACELLULAR pH OF MAMMALIAN CARDIAC MUSCLE
BY D. ELLIS AND R. C. THOMAS From the Department of Physiology, University of Bristol, Bristol BS8 1TD
(Received 21 June 1976) SUMMARY
1. The intracellular pH (pH,) of sheep heart Purkinje fibres and rat, ferret and guinea-pig ventricle has been measured using recessed-tip pHsensitive micro-electrodes. 2. In the absence of CO2 the pHi was approximately 7-2 in all the preparations used. In 5 % CO2 the mean pH, was 7414 in rat and ferret ventricle and 7-02 in sheep Purkinje fibres. 3. The pH, response to an increase or a decrease in the CO, level (at constant external pH) was biphasic with a large transient change followed by a partial recovery to a new sustained pH,. 4. The intracellular buffering capacity was 34-8 + 2-7 m-equiv H+/pH unit per 1. (+ S.E. of mean) in sheep Purkinje fibres, 76-6 + 13-6 in rat ventricle and approximately 69 in ferret ventricle. 5. The pHi of all the preparations tested indicated that H+ ions were not passively distributed across the cell membrane. There was also little or no pH1 change produced by depolarization with high K solutions. 6. Short exposures to hypertonic solutions (100 mm sucrose or 50 mM-KCl) produced a decrease in pHi of approximately 041 pH units. 7. Acetazolamide slowed the pHi response to CO2 changes. 8. Restoration of the pHi after displacement by increasing the C02 was not blocked by ouabain or SITS. 9. The relationship between pHi and cardiac contractility is discussed. INTRODUCTION
Enzyme reactions are well known to be very sensitive to the activity of H+ ions. This may well form the basis for the influence of pH on cardiac contractility, acid solutions decreasing and alkaline solutions increasing contractile strength, respectively (Daly & Clark, 1921). There is some dispute as to whether these effects are due to external or to internal pH
756
D. ELLIS AND R. C. THOMAS changes (see Vaughan Williams & Whyte, 1967 and Cingolani, Mattiazzi, Blesa & Gonzalez, 1970). Our knowledge of how intracellular pH (pHi) affects the contraction of heart muscle is limited by a lack of information on the extent to which the pHi is altered by the methods used to produce these changes. Indeed even the normal value of the pHi is in some doubt. Of the techniques available for measuring the pHi of tissues, only the DMO method (see Waddell & Bates, 1969) has been widely used. It permits pH measurements even in the small cells of heart muscle. However the pH, value obtained probably reflects the over-all pH of all the cellular compartments and the method is unsuitable for following changes in pH1 unless they have a very slow time course. The use of pH-sensitive electrodes was until recently limited to the large cells of crab or frog skeletal muscle (Caldwell, 1954; Kostyuk & Sorokina, 1960) or squid axon (Caldwell, 1958; Bicher & Ohki, 1972). The introduction of the recessed-tip design of glass pH micro-electrode (Thomas, 1974) now makes it possible to use such electrodes even in the small cells of mammalian heart and permits the continuous recording of pHi. The localized measure of the pH1 at the tip of the electrode is likely to produce a closer estimate of the sarcoplasmic pH, that is the pH of the compartment in which the contractile proteins are situated, than would be obtained with the DMO technique. The recessed-tip type of pH electrode has already been used successfully in snail neurones (Thomas, 1974, 1976a, b) and crab leg muscle (Aickin & Thomas, 1975). We have used these electrodes on the smaller cells of rat, guinea-pig and ferret ventricular muscle but most of the work to be described here concerns experiments on rat ventricle and sheep heart Purkinje fibres. A preliminary report of some of the results has already been published (Ellis & Thomas, 1976). Our aims were to measure the normal pH1 of the muscles, to see how this was affected by C02 and various other experimental procedures, to calculate the intracellular buffering capacity of the cells and to compare the results with the better characterized pH, regulatory and buffering mechanisms of the larger, non-mammalian cells previously studied. METHODS
Sheep hearts were obtained from a local slaughterhouse. Immediately after the animal was killed the heart was removed and immersed in a modified Tyrode solution at room temperature which had been equilibrated with a gas mixture of 95 % 02, 5 % C02. During transport to the laboratory and during dissection the solution was continually gassed. Rats and guinea-pigs were killed by a blow to the back of the head and ferrets by an overdose of sodium pentobarbitone. In all cases, free-running thin ventricular
CARDIAC MUSCLE pH
757
bundles (or Purkinje fibres from the sheep hearts) were selected and removed with a little side-wall material attached. This was used to pin the ends of the preparations on to the silicone rubber base of the experimental chamber where it was continually superfused with saline. The preparation was supported at several points along its length by stainless-steel wires running at right angles to it across the base of the chamber. This was found to facilitate penetration by the electrodes without breakage. Stable penetrations seldom lasted longer than 1 hr in ventricular muscle preparations, but in sheep heart Purkinje fibres stable measurements could be obtained for many hours. Solutions. The normal saline equilibrated with nominally 95 % 02, 5 % CO2 contained (mM): Na+ 140, K+ 4, Ca2+ 2, Mg 2+ 1, pyruvate 2, glucose 10, Cl- and HC03reciprocally varied to give a measured pH of 7 40 at 350 C (the concentration of HCO3- was about 20 mm, variations being due to the equilibration gas in different cylinders containing varying amounts of C02; the mean CO2 content was 4- 6 %). The C02-free solutions were saturated with 100% 02, lacked bicarbonate but contained 10 mm of the Na salt of HEPES (2-N-2-hydroxyethyl piperazine-N'-2-ethansulphonic acid). Micro-electrodes. Conventional KCl and pH-sensitive electrodes were prepared as described previously (Thomas, 1974, 1976a). Signal recording. The output from the normal KCl micro-electrode with respect to an agar-Ringer bath reference electrode was displayed on one channel of a potentiometric pen recorder with differential input (Watanabe Multicorder MC 611). The KCl electrode output was also fed, via a unity-gain buffer amplifier, to the low impedance input of a Vibron Electrometer (E.I.L. 62A). The pH-sensitive electrodes were connected to the high impedance (1016 Q) side. The resultant output was also displayed on the pen recorder so that a pH trace could be obtained that was independent of membrane potential changes when the electrodes were intracellular. The cells penetrated by the two electrodes were normally less than 1 mm apart. Low membrane potentials were obtained with a few Purkinje fibres so a minimum acceptance level of 60 mV was employed. However the mean pH1 of the fibres with a low membrane potential was not significantly different from the pHi measured in the normal group. RESULTS
Rat ventricle pHj and the effect of CO2 While most experiments were done on sheep Purkinje fibres, early ones were done on other mammalian preparations. Fig. 1 illustrates the general procedures involved in measuring the pHi of all the cardiac tissues employed. Initially the rat ventricular muscle was superfused with saline equilibrated with 5 % C02, 95 % 02 and the pH-sensitive micro-electrode (bottom trace) and the KCI micro-electrode (top) were extracellular. The pH micro-electrode was then pushed into the tissue where it recorded the membrane potential of a cell minus an offset voltage that was proportional to the pHi. Then the KCl micro-electrode was pushed into the muscle until with some difficulty in this case, a stable membrane potential was recorded from another cell. The KCI micro-electrode output voltage was electronically subtracted from that of the pH electrode. Therefore if the two cells penetrated had approximately the same membrane potential and behaved
758 D. ELLIS AND R. C. THOMAS in the same way to external solution changes, the recording of pHi (as in Fig. 1, bottom trace) would be independent of membrane potential changes. A reasonably accurate measure of pHi was then possible by 0 10 min I
I
20 -
E wU
40
-
60 Em
80
-
69 I
0.
71 L I 5
7 3
7-
Lp6
L
pH 6-4
I
I
1
%
100% °2
Fig. 1. Pen recording of an experiment to measure pHi in rat ventricular muscle and the response to removal and replacement of external CO2. The upper trace shows the membrane potential (Em) recorded by a conventional micro-electrode. The lower trace shows the voltage difference between the pH and conventional micro-electrodes. The pH electrode was inserted first, then, with some difficulty, the conventional micro-electrode. When both electrodes were intracellular and a stable pH, value obtained the bathing solution was changed from a bicarbonate buffered solution equilibrated with a 5 % C02, 95 % 02 to give a pH of 7-4 to one that was CO2-free but contained 10 mM-HEPES buffer (also at pH 7-4). The pH-sensitivity of the electrode was tested (right) by a reduction of the solution pH to 6-4 (both electrodes extracellular). Before this pH measurement, the muscle was stimiilated by just stiprathreshold pulses once every 10 sec.
CARDIAC MUSCLE pH 759 taking the average of several paired penetrations with the two types of electrode in each experiment. Since C02 crosses cell membranes very easily it is inevitably unimportant factor affecting pHi and we have briefly reported its effects on the pHi of sheep heart Purkinje fibres (Ellis & Thomas, 1976). We have also found that changing C02 has similar effects on the pHi of rat (and ferret) ventricle. Fig. I illustrates the effects of complete removal of external CO2. The pH, showed a large transient decrease followed by a return of the pH1 to a level nearer that in the control solution. The response to re-addition TABLE 1. Summary of pH, and Em values in experiments in the rat, ferret and guinea-pig
Equilibration gas 5% C02,95% 02 Preparation and
temperature Rat ventricle 350 C
,
A,__,____
pH1
Em
pHI
Em
7-14 + 0 03 n= 9
78*7 +1.6
7.20 + 0 03 n= 7 7*17+ 006 n= 7
75.6 +1-7 n= 7 75.4 + 2.8 n= 7
7.14 n =2
84.6
7-20
79.5
n = 9 -
200 C Ferret ventricle 350 C
Equilibration gas 100% 02
n = 2
n =2
n= 2
Guinea-pig ventricle
350C
-
7-16 n =2
75.6
n= 2 Values given are the means ( s.E. of mean) for the number of hearts given by n. The measurements of Em and pH1 for each heart were normally the average from several penetrations.
of C02 was also biphasic with a large decrease in pH, quickly followed by a return to the control level. The mean stabilized pHi values for a number of experiments on rat, guinea-pig and ferret ventricle are given in Table 1. Some other experiments were carried out on rat ventricle at a lower temperature (200 C). The pHi values for this set of experiments were not significantly different from those at 350 C (Table 1). A similar finding using the DMO technique in frog heart muscle has been reported by Reeves & Wilson (1969). A few experiments were carried out on guinea-pig and ferret ventricular preparations. The corresponding pHi values for the same set of experimental conditions are also shown in Table 1. These were very similar to
760 D. ELLIS AND R. C. THOMAS those obtained in the rat. The time course of pHi changes in response to an alteration of the external C02 concentration was very similar in all three preparations. The pHi was consistently higher in the absence than in the presence of C02.
Effect of C02 on pHi in sheep heart Purkinje ftbrea Most of our work on cardiac pHi was carried out on sheep heart Purkinje fibre preparations. The initial penetration of the pH-sensitive electrode into this tissue was more difficult, probably due to the surrounding connective tissue. Once in, however, micro-electrodes often remained intracellular 0 10 min 20
~40
E_ E
60
Em 80
6.8I
7-0 -' L72
100 %°2
Fig. 2. Pen recording of an experiment on a sheep heart Purkinje fibre to show the effect on the Em and pH, of removal and replacement of external C02. Procedure followed was the same as in Fig. 1. The pH electrode showed signs of coming out of the cell on two occasions before a stable penetration was obtained. The effect of C02 removal (with replacement by HEPES buffered saline) and re-addition was then observed. The external pH was maintained at 7-4 throughout.
for several hours and showed very stable pH1 levels. The tendency for the preparation to behave as a good electrical syncytium, which can be observed when the membrane potential is altered, aided the measurement of an accurate value of the pH,. Fig. 2 shows that the responses to removal and re-addition of C02 in
761 CARDIAC MUSCLE pH sheep heart Purkinje fibres were similar to those observed in rat ventricle. The pHi changes were however slower and larger than equivalent changes in rat heart. The mean pHi in sheep heart Purkinje fibres was 7-02 in 5 % C02, 95 % 02 and 7-20 in 100 % 02, i.e. C02 has a larger effect on pH1 than in rat heart where the corresponding values were 7-14 and 7-20. 10 min
m
60 E 70 L E
Em
80 _ 6-8
7-2 6-7 % CO2 1-6 % C02 Fig. 3. Effect of various C02 levels on pH, of a sheep-heart Purkinje fibre. The preparation was initially perfused with the control solution equilibrated with 4-7 % C00, 95.3 % 02. The C02 concentration was then changed, first to 1-6%, returned to the control level and then increased to 6-7 % before finally being returned to the control level again. The external pH was maintained constant at 7-4 throughout by variation of the bicarbonate concentration. The rather large depolarization observed in 6-7 % C02 solution was not a consistent finding.
Smaller alterations in the C02 level of the saline produced similar, but smaller, changes in the pH, as shown in Fig. 3. The external pH was maintained at 7-40 throughout. The mean value of pH1 in 1-6 % C02 was 7-09 + 0-04 (n = 9) and in 6-7 % C02 was 6-94 + 0-03 (n = 4). No change in pH1 was apparent in 100% 02 solutions when the HEPES buffer was substituted by Tris maleate. Intracellular buffering capacity of cardiac tissue When the external C02 concentration was altered the initial change in pHi observed was due to the reaction.
C02 + H O=H
--H+ + HCO3-. Since equal numbers of H+ and HC03- ions are produced it is possible to estimate the internal buffering power from the pHi change assuming that the internal HCO3- level depends only on the pH1 and the C02 level, that the C02 level and the carbonic acid dissociation constant are the same inside and outside the cell and that there is no significant movement of
D. ELLIS AND R. a. THOMAS 762 H+, OH- or HC03- ions across the cell membrane during the period of the initial pH change. From experiments of the type shown in Figs. 2 and 3 the buffering power of sheep heart Purkinje fibres was found to be 34-8 + 2-7 m-equiv H+/pH unit per litre (s.E. of mean, n = 17). 10 min
0
m
10
20 30 E E w
_-
40 _-
50
-
60 1
70 1-
80
-
Em
90 L 6-8 Ir
7-0_
pH_
2_ 7
140 mM -K
Fig. 4. Pen recording to show the effect of a large membrane depolarization on the pHi of a sheep heart Purkinje fibre. The normal K concentration was raised from 4 to 140 mm by replacement of all but 4 mm-Na for the test period indicated by the bar. The fibre was perfused with solutions equilibrated with 5 % C02, 95 % 02 to give an external pH of 7-4 throughout.
In rat ventricle the buffering capacity was estimated using the same method and was found to be 76-6 + 13-6 (S.E. of mean, n = 4). Two experiments in ferret ventricle produced a mean value of 68-8. The speed of the pH, recovery after displacement by C02 changes suggests that the last assumption mentioned above is incorrect so that the values given above are probably higher than the total buffering power of the various cellular constituents. Nevertheless the buffering power is obviously large
AZRDIAC MUSCLE pH
763
and this, taken with the evidently highly active H+ pump, shows that pH, can be regulated with considerable precision and speed. The effect of membrane potential on pHi. The measured pHi values are clearly far from those expected from a passive distribution of H+ ions across the cell membrane, which would give a pHi of about 6-1 for both Purkinje fibres and for rat ventricle. We found no correlation between the normal membrane potential and the pH1, Even with a large depolarization produced by the almost complete replacement of external Na by K (Fig. 4) the maximum pHi change recorded was only 005 units. Any small changes in pH1 observed only appeared immediately after the addition or removal of the high K solution and probably reflected slightly imperfect subtraction, in that depolarization or repolarization possibly had a slightly different time course in the two cells penetrated. Active transport of H+ (or OH- or HCO3-) ions is clearly necessary to account for both the non-equilibrium distribution of H+ ions across the cell membrane and the partial recovery of pHi seen after changes in external C02 (Figs. 1-3).
Effects of contraction on pHs All the experiments described were carried out on preparations that were quiescent for most of the time. The pH-sensitive electrodes used, unlike normal microelectrodes, were relatively inflexible and therefore difficult to keep intracellular during the normal strong contractions of ventricular muscle. It was, however, easier to maintain a stable penetration during the relatively weak contractions of Purkinje fibres. We have found that the pH, following short periods of stimulation (4 min at a rate of 120/min) was not greatly affected. The lack of an observable pH, change after a period of stimulation probably justifies the use of quiescent prepara. tions in those experiments. In a few preparations that were spontaneously active throughout part or the whole of an experiment essentially the same pH, and changes in pHi were observed as in quiescent fibres. However transient pHi changes during the course of individual contractions would be too fast to be recorded with this type of electrode.
The effects of external pH on pHi We have found that alteration of the external pH produced only slow and small changes in pHi. An experiment to illustrate this is shown in Fig. 5. The changes in pHi were much smaller and slower than the pH change in the external solution produced by a reduction of the bicarbonate concentration. (The reasons for the small transient changes in pH1 within the first few minutes of a solution change are unknown but were observed in several preparations.) The changes in pHi following a reduction of the external bicarbonate concentration were approximately exponential, the pH1 stabilizing at a
764 D. ELLIS AND R. C. THOMAS new level within 15-40 min. The size of the pHi change was approximately 0-2 pH units for a unit change in external pH. The internal acidification was accompanied by an average depolarization of 3 mV. When the external pH was returned to normal the pHi recovered in a similar manner to the restoration of pHi after the large transient decrease following re-addition of C02 (Figs. 2 and 3). Both processes require some sort of active H+ pumping. The rate of restoration of pHj on return to normal external pH was similar to that for the restoration of pH1 following the re-addition of C02 when the external pH was unaltered. Em 10 min E LU
9goL 6-4
66
6.8-
_pH,
7-0 7.27.4
pH 64 pH 64 Fig. 5. Pen recording of an experiment to illustrate the effect of a change of the extracellular pH (by 1 0 units) on the pH1 of a sheep heart Purkinje fibre. The preparation was perfused with solutions equilibrated with 5 % C02, 95 % 02 throughout. During the periods indicated by the bars the bicarbonate concentration was reduced approximately tenfold to produce an extracellular pH of 6-4. The extent of the pH change in the extracellular solution is shown on the right, at the end of the experiment, where both electrodes were removed from the fibre.
The effect of hypertonicity on pHi In many experimental procedures it is often useful to be able to add compounds to the normal perfusion solution without removing one of the normal constituents, although this makes the test solution hypertonic. To estimate how this type of procedure might affect pH, we have added sucrose (100 mM) or KCI (50 mM) without reducing the NaCl concentration. An experiment of this type is illustrated in Fig. 6. Here the addition of 100 mm sucrose reduced pH, by less than 0.1 pH units. Similarly, extra KCI caused pHi changes of only about 0.1 units. No long-term measurements were carried out to see if this pH change was maintained. Adler, Anderson & Zett (1975) have reported that the addition of 80 mm man-
765 CARDIAC MUSCLE pH nitol to the solution bathing rat diaphragm muscle produced an increase in pH, of approximately 01 pH units. They estimated the pH, from measurements of the distribution of the weak acid DMO and the weak base nicotine after the tissue has been in the vhypertonic test solution for 4 hr. 5 min
70_
El E
..
IJI 100 mM sucrose Fig. 6. Pen recording of an experiment on a sheep heart Purkinje fibre to illustrate the effect of an increase in tonicity of the bathing solution. For the period indicated by the bar the bathing solution was hypertonic due to the addition of 100 mm sucrose to the normal saline. The external pH was maintained at 7-4 throughout with CO2HCO3- buffering.
The effect of acetazolamide Acetazolamide, an inhibitor of carbonic anhydrase, has been shown to slow the effects of C02 addition and removal on the pH1 of snail neurones (Thomas, 1976a). It has, however, been suggested that in muscle carbonic anhydrase might be absent or present in only trace amounts (Van Goor, 1940; Matthews Laszlo, Campbell, Kibby & Freedman, 1968). In sheep heart Purkinje fibres acetazolamide appeared to have some effect, although only a small one, on the pH, responses to C02 addition and removal (Fig. 7). This suggests that carbonic anhydrase is present in cardiac tissue but perhaps only at a low concentration. Effects of ouabain and SITS on the regulation of pHi. Thomas (1976b) has reported that the Na+ and H+ pumps have independent mechanisms in snail neurones. The Na+ pump can be inhibited
D. ELLIS AND R. C. THOMAS by ouabain and the H+ pump by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) but not vice versa. In cardiac Purkinje fibres we have similarly found that the pH, changes occurring when the C02 concentration was altered were unaffected by doses of ouabain (I0-5 M) sufficient to inhibit the Na+ pump completely. However in Purkinje fibres SITS did not affect the pHi responses. Thus in this respect cardiac tissue differs from snail neurones but is similar to mouse skeletal muscle, where SITS has recently been found to be similarly ineffective on the H+ pump (C. C. Aickin and R. C. Thomas, unpublished observations.) 766
Acetazolamide (4 /dM)
15 min
50_
70t' 6-8
i7.01
I~ 100%°2
L 10 100%°2
Fig. 7. Pen recording of an experiment on sheep heart Purkinje fibres showing the effect of 4 IzM acetazolamide on the responses to removal and readdition of CO2 in the gas equilibrated with the perfusion solution. The external pH was maintained at 7-4 throughout. A large artifact is present on both recordings immediately after the addition of acetazolamide, possibly caused by vibration. DISCUSSION
With the use of a method that allows a direct measurement of the pHi of cardiac tissues we have found a non-passive distribution of H+ ions across the cell membranes. This is in accord with previous measurements in crab leg muscle (Caldwell, 1954), frog skeletal muscle (Kostyuk & Sorokina, 1960), squid giant axon (Bicher & Ohki, 1972) and snail neurones (Thomas, 1974). One recent report (Hannan & Wiggins, 1976) has suggested that H+ ions are passively distributed in frog skeletal muscle but this conclusion probably resulted from the type of electrode used (Carter, Rector, Campian & Seldin, 1967) which has previously been shown to be unsuitable for use in tissues where there is a possibility that all of the pH-sensitive glass
767 CARDIAC MUSCLE pH is not intracellular (Paillard, 1972). The observation in the present experiments that depolarization with high K solutions produced no large changes in the pH, supports the finding that H+ ions are not passively distributed. The pH, values obtained in the present experiments on heart muscle are in general equal to, or somewhat larger than, those measured previously with indirect techniques (see Waddell & Bates, 1968; Neely, Whitmer & Rovetto, 1973) and with open tipped electrodes (Lavall6e, 1964). Some differences in the pHi values would be anticipated in view of differences in the techniques used and of the experimental conditions. The experiments by Lavellee (1964) on rat atrial preparations gave a pHi value of 6-91. This rather low figure may have been related to the high rate of stimulation used (200/min), the composition of the perfusion solution, or it may have been influenced by the low sensitivity of his electrodes (approximately
30 mV/pH unit, i.e. only half the theoretical value). On the other hand the differences may reflect a variation of the pHi in different regions of the heart, auricular pHi perhaps being lower than that of ventricle. In the absence of C02 we found that the pHi was similar in all the cardiac preparations we tested, but in the presence of C02 the values were different. This may be due to differences in the buffering capacity of sheep heart Purkinje fibres and of normal ventricular muscle. The calculated buffering powers for the cardiac tissues used were approximately 35 for sheep heart Purkinje fibres and 77 for rat ventricle (and 69 for ferret ventricle), indicating a relatively high buffering capacity. Using the same techniques the buffering power of snail neurones was found to be approximately 30 (Thomas, 1976), for crab leg muscle was 47 (Aickin & Thomas, 1975) and for mouse skeletal muscle 50 (Aickin & Thomas, 1976). There is a large variation in previous estimates of the intracellular buffering capacity of heart muscle. Most of the early work suggested that heart muscle was only poorly buffered in comparison to skeletal muscle (see Brody, 1930) whereas more recently one group have claimed that the reverse is true from in vivo experiments in dog (Clancy & Brown, 1966). They calculated a buffering capacity of about 71 for dog heart. The larger buffering power found in ventricular muscle compared to sheep heart Purkinje fibres possibly reflects the need for better buffering of pH changes in working myocardium compared to the Purkinje fibre. The values obtained in the present experiments are probably slight over-estimates as no allowance has been made for the restoration of the pHi up to the time that the peak C02 effect was observed. A large correction would not be required in sheep heart Purkinje cells because the recovery responses are so slow. However, in the smaller cells of ventricular muscle these responses were more rapid and so a larger correction would be required. Rapid changes of pH have been observed on the outer surface of muscle
768 D. ELLIS AND B. C. THOMAS fibres during activity (Dubuisson, 1950; Dhalla, Yates & Kleinberg, 1973). It was suggested that these might indicate rapid pHi changes associated with contractile activation. The time course of these events would be too fast to be observed with the type of electrode used in the present experiments. However, prolonged depolarization with K-rich solutions, which might be expected to prolong some of the effects of the activation process, produced little or no change in pH,. The apparent lack of effect of temperature on the pHi of rat ventricle is of interest because Reeves & Wilson (1969) also found that the pHi of frog heart muscle was unaffected by temperature. However, they found that both the pH of the surrounding blood and the pHi of skeletal muscle from the same animals were temperature-dependent. An effect of temperature has also recently been found on the pHi of mouse skeletal muscle (C. C. Aickin and R. C. Thomas, unpublished). This could indicate a difference in the major intracellular buffering components of skeletal compared to cardiac muscle (Reeves, 1972). An apparent lack of effect of temperature on pHi in heart muscle might of course only be due to the influence of temperature on processes which tend to change the pH in opposite directions. The relatively small effects of acetazolamide suggest that carbonic anhydrase is present but that its concentration or activity may be fairly low. It has been suggested that a function of the carbonic anhydrase found in snail neurones might be to maximize the buffering capacity of the cells (Thomas, 1976a). Cardiac muscle however appears to be well buffered to C02 changes (Figs. 1-3). The method most commonly used to investigate the effects of pHi on contraction has been to expose the muscle to an altered PCO., keeping the external pH constant by changing the bicarbonate concentration. Only the C02 is assumed to cross the cell membrane, therefore producing a change in pH,. The results of Vaughan Williams & Whyte (1967) in rabbit heart led them to suggest that there was no effect of pHi on contractile force. Their measurements, however, appear to have been made during a steady-state situation approximately 40 min after the change in C02 and bicarbonate. The present results show that there is only a large effect on pH1 during the first 10 min of exposure to an altered C02 level. Therefore if contractile strength is dependent upon pH,, the largest effects should be most noticeable in the first 10 minutes, whereas the effects after 40 min might not appear significant. McElroy, Gerdes & Brown (1958) and Lorkovi6 (1966) have described an inhibition of contraction of guinea-pig and frog heart when exposed to an increase of Pco, with the extracellular pH constant. They observed
769 CARDIAC MUSCLE pH biphasic changes in contractile force, there being an initial decrease followed by a partial recovery. Similarly Cingolani et al. (1970) found a decreased contractility in cat and rat hearts with an increase in Pco,. Their measurements were made within approximately 15 min of an increase in Pco2, so again a large effect on pHi would be expected. Vaughan Williams (1955) showed a biphasic change in the rate of beating of rabbit heart when the Pco, and bicarbonate were altered to maintain a constant external pH. The rate of beating declined to a minimum within approximately 5 min and recovered to near the control level within a further 10 min. A biphasic change in rate in the opposite direction occurred on return to the normal Pco,. These effects might well have been due to the pHi changes we have observed. It is generally agreed that external pH has a large effect on cardiac contraction but these experiments suggest that internal pH changes can also influence contractility, although the effects might only be small and perhaps transient due to high internal buffering capacity and active proton pumping. Such internal pH changes might affect the cell membrane permeability to Ca and/or the uptake and release of Ca by the sarcoplasmic reticulum (Carvalho & Leo, 1967). Our results show that the use of the recessed tip design of micro-electrode permits a direct measure of the pH, even in the small cells of mammalian ventricular muscle. This is the only technique available so far for giving a continuous measure of the pH,. The indirect methods can be used for studying slow changes in the pHi but, as has been described above, the pH changes in response to C02 are rapid and biphasic. We are grateful to the Medical Research Council for financial support and to Dr M. J. Purves and Miss C. C. Aickin for reading the manuscript. REFERENCES ADLEpR, S., ANDERSON, B. & ZETr, B. (1975). Effect of osmolarity on intracellular pH of rat diaphragm muscle. Am. J. Phy8iol. 228, 725-729. AIcmNw, C. C. & THoNs, R. C. (1975). Micro-electrode measurement of the internal pH of crab muscle fibres. J. Phy8iol. 252, 803-815. AIcRmN, C. C. & Thoii&s, R. C. (1976). Intracellular pH of mouse soleus muscle. J. Physiol. 260, 25-26P. BIcEmR, H. I. & OEmii, S. (1972). Intracellular pH electrode experiments on the giant squid axon. Biochim. biophy8. Acta 255, 900-904. BRODY, H. (1930). The carbon dioxide dissociation curve of frog heart muscle. Am. J. Phy8iol. 93, 190-196. CAT.wELL, P. C. (1954). An investigation of the intracellular pH of crab muscle fibres by means of micro-glass and micro-tungsten electrodes. J. Physiol. 126, 169180. CALDwELL, P. C. (1958). Studies on the internal pH of large muscle and nerve fibres. J. Physiol. 142, 22-62.
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