ph1 recovery following acidification. 90 % or complete removal of external micro-electrodes, fell on application of (NH4)2SO4 and increased

Transcription

1 J. Phy8iol. (1977), 273, pp With 12 text-figure Printed in Great Britain AN INVESTIGATION OF THE IONIC MECHANISM OF INTRACELLULAR ph REGULATION IN MOUSE SOLEUS MUSCLE FIBRES BY C. CLAIRE AICKIN AND R. C. THOMAS From the Department of Physiology, University of Bristol, Bristol BS8 1TD (Received 17 June 1977) SUMMARY 1. Intracellular ph (ph1) of surface fibres of the mouse soleus muscle was measured in vitro by recessed-tip ph-sensitive micro-electrodes. ph1 was displaced in an acid direction by removal of external (NH4)2SO4 after a short exposure, and the mechanism of recovery from this acidification was investigated. 2. Removal of external K caused a very slow acidification (probably due to the decreasing Na gradient) but had no effect on the rate of ph, recovery following acidification. This indicates that K+-H+ exchange is not involved in the phi regulating system. 3. Short applications of 104 M ouabain had no obvious effect on ph, and did not alter the rate of ph1 recovery following acidification. This suggests that there is no direct connexion between the regulation of ph1 and the Na pump. 4. Reduction of external Ca from 10 to 1 mm caused a transient fall in ph1, but the rate of ph1 recovery following acidification was unaffected. This suggests that Ca2+-H+ exchange is not involved in the ph1 regulating system. 5. An 11% reduction in external Na caused a significant slowing of ph1 recovery following acidification. 90 % or complete removal of external Na almost stopped ph1 recovery. This suggests that Na+-H+ exchange is involved in ph1 regulation. 6. Amiloride (104 M) reversibly reduced the rate of ph1 recovery to much the same extent as removal of external Na. Its effect was not additive to that of removal of external Na. 7. Internal Na ion concentration ([Na+]i), measured using Na+sensitive micro-electrodes, fell on application of (NH4)2SO4 and increased on its removal. The increase transiently raised [Na+]i above the level

2 C. AICKIN AND R. C. THOMAS recorded before (NH4)2S04 application. This overshoot of [Na+]i was almost completely inhibited by amiloride. This is consistent with the involvement of Na+-H+ exchange in the ph, regulating system. 8. Removal of external C02 or application of SITS (10-4M) caused some slowing of the rate of ph1 recovery following acidification by removal of (NH4)2S04. The effect of SITS was additive to that of Na-free Ringer or amiloride. These results suggest that Cl-HCO3- exchange is also involved in the ph, regulating system and that it is a separate mechanism. Under the conditions used, Cl-HCO3- exchange formed about 20 % of the ph1 regulating system. 9. Decreasing the temperature from 37 to 28 'C not only caused an increase in phi, but also considerably slowed the rate of ph1 recovery following acidification. We have calculated a Q10 for Na+-H+ exchange of 1-4 and for Cl-HCO3- exchange, We conclude that the ph1 regulating system is comprised of two separate ionic exchange mechanisms. The major mechanism is Na+-H+ exchange, which is probably driven by the transmembrane Na gradient. The other mechanism is Cl-HCO3- exchange, which probably requires metabolic energy. INTRODUCTION Notwithstanding the wide agreement that H+ ions are far from equilibrium across the muscle cell membrane, it has only been possible to speculate about the way this disequilibrium might be maintained. This is probably due to the limitations of the indirect methods of measurement of intracellular ph (ph,) generally used, where only a single determination is possible with each preparation, these being made after long equilibration periods. Recent continuous recordings of ph1 (Thomas, 1974, 1976a; Aickin & Thomas, 1975, 1977a; Boron & De Weer, 1976a, b; Ellis & Thomas, 1976; Russell & Boron, 1976) using ph-sensitive micro-electrodes, have shown that changes in ph1 can occur very rapidly, too rapidly to be followed by the indirect methods. The ph1 of mammalian skeletal muscle has generally been found to be around 7 0 (see Waddell & Bates, 1969). In an earlier study of ph1 of mouse soleus muscle (Aickin & Thomas, 1977a) we found a mean value of 7 07 at external ph 7 40 (C02/HC03- buffered Ringer) at 37 'C with a membrane potential (Em) of 71x8 mv. Thus ph1 is almost one ph unit more alkaline than would be expected from a purely passive distribution of H+ ions. Two possible mechanisms which could hold ph1 so far from equilibrium are the transport of H+ (or OH- or HCO3-) ions across the fibre membrane and the internal consumption of H+ ions. When acid is added to the fibre interior the immediate change in ph1 will be determined

3 IONIC MECHANISM OF ph, REGULATION 297 by the internal buffering power, but any recovery of ph1 must be due to one of the mechanisms mentioned above. Several ionic mechanisms have been put forward for the transport of H+ ions and some of these have been collected together in Fig. 1. Perhaps the most widely suggested mechanism in mammalian skeletal muscle is K+-H+ exchange (as in Fig. 1 a). This was deduced both from the considerable decrease in muscle K content on acidosis (Goto, 1918) and from the fall in phi seen on K+ depletion first described by Gardner, MacLachlan & Berman (1952) and later the subject of a considerable volume of work using indirect methods for measurement of ph1 (for references see Waddell & Bates, 1969; Cohen & Iles, 1975). One of the conclusions of this work Outside K+ K+ Ca2+ Na+ HCO3- H+ Na+/H+ H+ H+ Cl- H Inside Fig. 1. Diagram of some of the mechanisms proposed for the phi regulating system. The possible energy sources are not shown; schemes a, b, and e require metabolic energy, but c and d could be driven by the Ca or Na gradient. was that active H+ extrusion was impaired by K+ depletion (Irvine, Saunders, Milne & Crawford, 1960). An alternative interpretation of these results was that H+ ions share the outward limb of the Na pump (Fig. 1 b) as proposed by Woodbury (1965), since removal of external K inhibits the Na pump. Support for this hypothesis was seen in the fall in ph, on application of ouabain (Williams, Withrow & Woodbury, 1971) and in the reduction in the Na efflux on decreasing ph1 (Keynes, 1965). Ca2+-H+ exchange (Fig. 1 c) is known to occur in isolated mitochondria (Bartley & Amoore, 1958; Chappell, Greville & Bicknell, 1962) and could also occur across the cell membrane. Na+-H+ exchange (Fig. 1 d), thought to occur in many secretary and excretory tissues (Teorell, 1939; Pitts & Alexander, 1945; Parsons, 1956; Whitlock & Wheeler, 1969; Frazier & Vanatta, 1973), has recently been shown to be responsible for most of the intracellular alkalinization during activation of sea urchin eggs after fertilization (Johnson, Epel & Paul, 1975). The last mechanism shown in Fig. 1, Cl-HCO3- exchange (Fig. 1 e), is favoured for acid secretion in the stomach (e.g. Durbin & Heinz, 1958; Kasbekar & Durbin, 1965) and has been studied in great detail in erythrocytes where it is known as the 'chloride

4 298 C. C. AICKIN AND R. C. THOMAS shift'. It has recently been suggested that it is involved in the regulation of ph1 in snail neurones (Thomas, 1976b) and shown to be the mechanism in the squid giant axon (Russell & Boron, 1976). In an earlier paper (Aickin & Thomas, 1977a) we showed that there was a large and rapid acidification on removal of external NH4+ and NH3 after short applications. The extent of this acidification was dependent on the ph1 immediately before the removal of external NH4+ and NH3, and on the internal buffering power. The acidification was followed by a fairly rapid recovery in ph1 which can most easily be explained by effective transport of H+ ions across the fibre membrane. This acid loading technique has recently been used by Boron & De Weer (1976b) to investigate the ph, regulating system in the squid giant axon, and we have now used it to test on soleus muscle the five possibilities shown in Fig. 1. A preliminary report of some of this work has been communicated to the Physiological Society (Aickin & Thomas, 1977b). METHODS These were generally the same as described previously (Aickin & Thomas, 1977 a). The experiments were performed on surface fibres of the mouse soleus muscle. As before, the external Ca concentration was higher than that in normal mammalian Ringer to increase the stability of the membrane potential. When Em stabilized below 60 mv at the beginning of an experiment, the fibre was judged to be too damaged and the electrodes were withdrawn. But if Em later fell below 60 mv, the experiment was continued. Solutions. Throughout most of the experiments the muscle was continually superfused with Ringer equilibrated with nominally 5% C02, 95 % 02 and buffered to ph 7 40 with NaHCO, at 37 'C. The mean percentage C02 was 5-18 and Ringer in equilibrium with this had the following composition (mm): NaCl, 119; KCl, 4; CaCl2, 10; MgCl2, 1; NaHCO3, 23; NaH2PO4/Na2HPO4 at ph 7-4; 0.1, glucose, 10. Ringer containing 10 mm-(nh4)2s04 had 15 mm less NaCl than the normal Ringer to maintain the osmotic strength constant. (NH4)2SO4 was added in appropriate amounts from a 1 m stock solution immediately before use. In a few experiments C02-free Ringer was used, equilibrated with 100 % 02 and buffered with 10 mm- HEPES (2-N-hydroxyethyl piperazine N'-2-ethanesulphonic acid) adjusted to ph 7*4 by adding NaOH. Some experiments were conducted in part at 28 0C by turning off the secondary heating close to the bath. This caused a slight fall in the ph of the Ringer, to Micro-electrodes and electrical arrangements. These were the same as described previously (Aickin & Thomas, 1977a), with two exceptions: firstly that some of the ph-sensitive micro-electrodes were made using a gas cylinder to provide much greater pressure for the glass-glass seal than was possible using a hand-operated syringe (Thomas, 1974), and secondly, in some experiments Na+-sensitive microelectrodes (Thomas, 1970) were used. Calcuaions. The rate constants for the recovery of ph1l following an acid load were calculated by least-squares fit of the semilog plot of ph, against time.

5 IONIC MECHANISM OF ph, REGULATION 299 RESULTS Application of an acid load The procedure used is illustrated in Fig. 2. Both a ph-sensitive and a conventional micro-electrode were inserted into the same fibre of the mouse soleus muscle as previously described (Aickin & Thomas, 1977a). After the membrane potential (Em) had stabilized, the preparation was exposed to Ringer containing 10 mm-(nh4)2so4. This causes a rapid alkalinization followed by a relatively slow acidification during which the fibre presumably accumulates NH4+ as discussed earlier (Aickin & Thomas, 1977a). On removal of external (NH4)2SO4 most of the accumulated NH4+ leaves the fibre as NH3, thus loading the fibre with acid by the following reaction NH4+ = NH3 + H+ Following the acid load there is a fairly rapid recovery in internal ph (ph,) complete in min. This recovery can most easily be explained by the transport of H+, OH- or HCO3- ions across the fibre membrane. For convenience this will be called the 'ph, regulating system' since this makes no assumptions about the ionic species transported. The effect of removal of external K The first mechanism (Fig. 1 a), clearly requires external K+ ions. Fig. 3 shows that on removal of external K (substituted by Na) there was a very slow internal acidification, ph, falling by 0*06 ph units in 1 hr. The mean fall in ph, after 1 hr exposure to K-free Ringer was 0x (S.D. of an observation, n = 5). This acidification was fairly rapidly reversed when external K was replaced. Removal of external K also caused an unexpected depolarization, in contrast to the hyperpolarization observed by Akaike (1975) in rat soleus. Perhaps this difference due to the elevated Ca level in our Ringer. The rate of ph, recovery following an acid load, however, did not appear to be affected by the absence of external K. The rate constant for the recovery in K-free Ringer was min-1 in the experiment shown in Fig. 3, compared with min' in normal Ringer. This clearly rules out the exchange of external K+ for internal H+ as the mechanism of the ph1 recovery. It is of interest that external NH4+ appeared to substitute for K at the external site of the Na pump since the large hyperpolarization seen on application of (NH4)2SO4 in K-free Ringer was completely abolished by the presence of 104 M ouabain. The increase in the rate of acidification during the exposure to (NH4)2SO4 in K-free Ringer was also inhibited by ouabain. This is good evidence for the hypothesis of NH4+ entry causing

6 1oo ~~~~~~~10 min_ 300 C. C. AICKIN AND R. C. THOMAS > 40 E E E m0meqv phi j 20 m-equiv NH+4 Fig. 2. Pen recordings of the beginning and end of an experiment showing the effect on the membrane potential (Em) and ph, of application and removal of (NH4)S04. The top trace shows the potential recorded by the voltage electrode and the bottom trace that recorded by the phsensitive micro-electrode minus that recorded by the voltage electrode. Initially both electrodes were extracellular. The ph-sensitive microelectrode was inserted into the fibre first and when it had stabilized attempts were made to penetrate the same fibre with the voltage electrode. The first attempt was a failure, seen in the mirrored decay of Em in the ph-trace (see Aickin & Thomas, 1977a). The second attempt succeeded. When Em had stabilized the fibre was exposed to Ringer containing 10 m -(NH4)2S04 for 10 min. Throughout the experiment the preparation was superfused with Ringer equilibrated with 5% CO, 95% O2, buffered to ph 7-4 with NaHCO3. At the end of the experiment both electrodes were withdrawn from the fibre, the potentials recorded by both electrodes returning to those recorded before penetration. The ph-sensitive microelectrode was then calibrated by exposure to Ringer of ph 6-4.

7 IONIC MECHANISM OF ph, REGULATION 301 the 'acid shuttle' during exposure to external NH4+ and NH3 (Boron & De Weer, 1976a). The low Em recorded in the last half of the experiment shown in Fig. 3 was probably largely due to the decreased ionic gradients across the fibre membrane, caused by the inhibition of the Na pump. Although this low Em, and the declining Em seen in some other experiments, was a little worrying, it clearly demonstrates the great stability of the ph1, and its independence of Em. If the preparation was left in normal Ringer Em remained stable for many hours. 0 I I 10 F 30 min > ~~~~Em E30 40?-4 E 50-60L F H r20 m-equiv NH+ K-free Ringer 20 m-equiv NH+ Fig. 3. Pen recordings of an experiment showing the effect of removal of external K (substituted by Na) on ph, and Em and on the rate of recovery of ph, following acidification on removal of (NH4)2SO4. The control exposure to, and recovery from, 10 mm-(nh4)2so4 is shown on the right. The first break in the record was for a period of 10 min and the second for 40 min. The effect of ouabain The lack of effect of removal of external K on ph, recovery following acidification not only suggests that K+-H+ exchange was not involved in the ph, regulating system, but also that the mechanism in which H+ ions share the outward limb of the Na pump (Fig. 1 b) was unlikely to be involved since removal of external K inhibits the Na pump. This was confirmed by experiments in which ouabain (104 M) had no effect on the ph, recovery following an acid load. In experiments where ouabain was applied without previous intracellular acidification no obvious effect was seen on ph,. But only short application of ouabain could be made, since after min the membrane potential had decayed considerably and

8 302 C. C. AICKIN AND R. C. THOMAS the fibres seemed to be in poor condition (as judged by their milky and blown up appearance). This effect of ouabain is hard to explain, since fibres survived for hours in K-free Ringer min 30 _ > 40 Em 60 i 80 A dt FpHi [- 7-4,, s 20 m-equiv NH+ 20 m-equiv NH4 10% Ca (Na) Fig. 4. Pen recording of part of an experiment showing the effect of reduction of external Ca to 10% of its level in normal Ringer (substituted by Na) on ph, and Em and on the rate of recovery of ph1 following an acid load (part of this experiment, lasting about 90 min, is not shown: during the part not shown there was another exposure to (NH4)2SO4 and a brief exposure to 10% Ca (Na)). Effect of reduction of external Ca If Ca2+-H+ exchange was involved in the ph, regulation system (Fig. 1 c), then ph1 recovery should be inhibited by removal or reduction of external Ca. Fig. 4 shows an experiment where external Ca was reduced to 1 mm (10 % of its level in the normal Ringer) extra NaCl being added to maintain tonicity. There was an immediate fall in ph1 of 0-14 units (mean (S.D. of an observation, n = 4)) but ph1 then slowly recovered, and in this experiment stabilized at a higher value. On return to normal external Ca there was a small acidification followed by a fairly rapid alkalinization, often to a higher ph1 than was recorded before the exposure to low Ca as seen in this experiment. Reduction in external Ca also caused an immediate and considerable fall in Em (to a mean value mv in 10 % Ca) and there was a continued slow fall throughout the exposure. Complete recovery of Em only occurred after brief exposure to low external Ca Ḋuring exposure to (NH4)2SO4 in low Ca, there was a very much faster internal acidification as compared with the control, shown on the left (Fig. 4). This accelerated acidification suggests a faster entry of NH4+ (see above), and in this case may reflect a general increase in the permeability of the membrane. However, the recovery in ph, following the acid

9 IONIC MECHANISM OF phi REGULATION 303 load was unaffected (rate constant for the recovery in 10 % Ca was min-' compared with min-1 for the control). In some experiments Mg was used as the Ca substitute in an attempt to stabilize the fibre membrane. Although the changes in ph1 were qualitatively the same, there was a much greater fall in Em and in most cases the preparation contracted, knocking the electrodes out. Attempts to remove external Ca completely caused considerable contraction of the whole preparation. Effect of reduction or removal of external Na If Na+-H+ exchange (Fig. 1 d) is involved in the ph1 regulating system then a decrease in external Na would be expected to reduce the rate of ph1 recovery. When external Na was reduced or removed there was an immediate and maintained small acidification which was reversed as soon 40 -I - > 50 E- E min 60F E N J ~- phi I7 0- l 7.2 L m-equiv NH+ 20 m-equiv NH+,, 20 m-equiv NH+ Low Na (K) Fig. 5. Pen recordings of part of an experiment to show the effect of an 11% reduction in external Na (substituted by K). The three breaks in the records are each 25 min periods during which both Em and ph, remained virtually constant. as the normal Na was replaced. But the recovery of phi following an acid load was significantly slowed by even small reductions in the external Na, as shown in Fig. 5. In this experiment external Na was decreased by only 16 mm (11 % reduction approximately), substituted by K, but the rate constant for the ph, recovery was reduced by 57 % (rate constants for ph1 recovery in normal Ringer were and 0*164 min- and in decreased Na, min-). The lower Em in decreased Na is unlikely to be responsible for the decreased -rate of ph1 recovery since at a lower Em the tendency for H+ ions to enter the fibre would be less. The increased K itself also seems unlikely to be responsible. We have confirmed this by using 16 mm Li to replace the Na, instead of K, when we saw the same decrease in the rate of ph, recovery.

10 304 C. C. AICKIN AND R. C. THOMAS Complete or 90 % removal of external Na (substituted by Li) almost stopped recovery of ph, following an acid load (see Fig. 1 of Aickin & Thomas, 1977b). This inhibition was fully reversible immediately the normal Na was replaced. However, complete removal of external Na (substituted by Li) usually caused enough contraction to knock the electrodes out of the fibre, presumably due to Na+-Ca2+ exchange, and nearly always caused a large reduction in Em which was only partially reversible. In later experiments we were partly able to overcome these problems by Em %.170 E "E min 6-6_ 68t ph X I7.AL m-equiv NH+ 20 m-equiv NH+ 4~~~ m-amiloride Fig. 6. Pen recordings of part of an experiment showing the effect of a short application of amiloride (104 M) on the recovery of p11. increasing external K before removing external Na. When other Na substitutes (BDAC - bis(2-hydroxyethyl)dimethylammonium chloride - or choline following 40 min pre-treatment with 10-4 M-atropine) were used however, contraction of the whole preparation occurred. Effect of amiloride on ph1 recovery The 'K-sparing' diuretic, amiloride has recently been shown by Johnson et al. (1976) to completely block Na+-H+ exchange during activation of sea urchin eggs after fertilization, as seen in the inhibition of both acid efflux and Na accumulation. Less direct evidence for amiloride inhibiting

11 IONIC MECHANISM OF phi REGULATION 305 Na+-H+ exchange has been given in preparations ranging from human kidney to toad skin (Bull & Laragh, 1968; Emilo & Menano, 1975). Fig. 6 shows that the application of amiloride on removal of (NH4)2S04 caused a considerable slowing of the rate of phi recovery in mouse soleus muscle following the acid loading. Some inhibition was seen at 104 M but the maximal effect was obtained at 10-4 M. This inhibition was reversed as soon as the drug was removed from the superfusate. The effect of application of amiloride was very similar to that of removal of external 30 r min ~50 E 60 - Em '1'70j 801 _ 6.6F 68F: M -amiloride 20 m-equiv NH+ 20 m-equiv NH+ Na-free Ringer 23 K Fig. 7. Pen recordings of part of an experiment to show that the effect of amiloride was not additive with removal of external Na (substituted by Li). Before the removal of external Na, external K was increased to 23 mu in an attempt to minimize contraction. The artifact shortly after removal of amiloride was due to movement around the tip of the voltage electrode, and was stopped by slightly withdrawing the electrode. The break in the record was for a period of 15 min during which both Em and ph, remained unchanged. Na, suggesting that amiloride and removal of external Na might be acting on the same process. Experiments such as that shown in Fig. 7 support this, since the effect of application of amiloride was not additive to that of application of Na-free Ringer. There is some evidence that ethacrynic acid has a similar action to amiloride in some preparations (Gee, 1976). We have seen some slowing of the rate of ph, recovery following an acid load on application of

12 306 C. C. AICKIN AND B. C. THOMAS ethacrynic acid (10-4 M) but the effect was not as great as with application of amiloride (10-4 M) and was irreversible or only very slowly reversible. Effect of application of an acid load on [Na+]i The effects of removal of external Na and application of amiloride suggest that Na+-H+ exchange (Fig. 1 d) was involved in the phi regulating system. To confirm this, we have used the Na+-sensitive micro-electrode described by Thomas (1970). The same protocol was adopted for placing both electrodes in the same fibre as was used with the ph-sensitive microelectrode, as shown in Fig. 8. On penetration with the voltage electrode, an 05 - r Cs20 min u 20- ~30 E 40 E 50 Em LQ '5 Z rit20e Rigri tenprietlcambrhe]1sestvem Fig. 8. Pen recordings of the beginning of an experiment to show the effect of the application and removal of (NH4)2S04 on [Na+]j. The top trace again shows the potential recorded by the voltage electrode and the lower trace now shows that recorded by the Na+-sensitive micro-electrode minus that recorded by the voltage electrode. Although the Nat-sensitive microelectrode records the Na+ activity, the potential derived from the electrode was converted into mm~since the electrode was calibrated in the solutions of known concentrations. The break in the record was for a period of about 40 min during which the muscle was dissected and allowed to equilibrate with the Ringer in the experimental chamber. The Nat-sensitive microelectrodes were tested before the preparation was placed in the experimental chamber to avoid unnecessary damage to the preparation.

13 IONIC MECHANISM OF phi REGULATION 307 internal sodium ion concentration ([Na+]i) often rose slightly but usually recovered. The mean [Na+]i recorded in normal Ringer (142 Na) was mm (S.D. of an observation, n = 9) with a range mm. This was equivalent to a mean Na+ activity of 8-6 mm (assuming an activity coefficient for our Ringer solution of 0.75) and similar to that recently reported for Purkinje fibres (Ellis, 1977). The mean Em recorded in these experiments was mv mi _% > 40- E - 50 Em E [ E 50 - E ' 20 L [Na+Ji z i 1 20 m-equi NH+4 20 m-equiv H+ 4s 4 s ~~~10-4 m-amiloride Fig. 9. Pen recordings of the beginning of an experiment to show the effect on [Na+], of application of amiloride following acid loading. Application of 10 mm-(nh4)2so4 caused a maintained decrease in [Na+]i presumably due both to the lower Na level in the (NH4)2SO4 Ringer and to stimulation of the Na pump by external NH4+. But on removal of external (NH4)2SO4 there was not only a recovery, but also an overshoot in [Na+]i. If this overshoot in [Na+]j was solely due to Na+-H+ exchange being involved in the recovery of ph,, then it should be blocked by application of amiloride. Fig. 9 shows an experiment where 10-4 M amiloride was applied on removal of (NH4)2SO4 (cf. Fig. 6). The overshoot was reduced in size, but not completely abolished. The rate constant for the rise in [Na+]i was also decreased, by an average of 42 % in three experiments. It is possible that this effect is simply due to amiloride reducing passive

14 308 C. C. AICKIN AND R. C. THOMAS Na influx. If so, it should cause an increase in Em, and none was observed. Furthermore, when amiloride was removed, [Na+]i rose slightly and then returned to the value recorded before the application of (NH4)2SO4. Effect of removal of external C02 on phi recovery The last mechanism (Fig. 1 e), Cl-HCO3- exchange, requires external HCO3- ions. Thus if this anion exchange is involved in the ph1 regulating system, some slowing of the recovery of ph1 following an acid load 60 7 Em E 70- EE80Ḻ -= min ~ LIIL 20 m-equiv NH' 20 m-equiv 4J NH M-amiloride 10-4M-amiloride C02-free Ringer Fig. 10. Pen recording of part of an experiment showing the effect of removal of external CO2 on the rate of recovery of ph, following acidification. In both cases the initial part of the recovery was recorded in the presence of 10-4 M-amiloride. should be seen on removal of external C02. Fig. 10 shows an experiment where the ph, recovery, briefly inhibited by amiloride, in normal Ringer was compared with that in C02-free Ringer. The rate of recovery of ph1, both with and without inhibition of amiloride, was decreased in the absence of external C02. From seven experiments we have found a mean decrease in the rate constant of the ph1 recovery of % (S.D. of an observation). However, because the buffering power is reduced by the removal of external C02 (Aickin & Thomas, 1977a), this reduction in the rate constant of the ph1 recovery represents a greater reduction in the rate of effective H+ ion transport by the ph1 regulating system. In red blood cells Cl-HC03- exchange can be irreversibly blocked by

15 IONIC MECHANISM OF ph1 REGULATION 309 SITS (4-acetamide-4'-isothiocyanostilbine-2-2'-disulphonic acid) (Knauf & Rothstein, 1971). More recently SITS has been shown to block ph1 recovery in the snail neurone (Thomas, 1976b) and acid extrusion in the squid giant axon (Russell & Boron, 1976). We have found that application of SITS (10-4 M) to soleus muscle fibres produces some slowing of the ph1 recovery from acidification, with an average decrease in the rate constant of the ph1 recovery of about 30 %. 60 >70FEM 30 min 6-8 F phi CL7: = M-SITS 20 m-equiv NH+ 20 m-equiv NH' 4 20 m-equiv NH l 10-4 M-amiloride 10-4 M-amiloride Fig. 11. Pen recordings of part of an experiment showing the effect of amiloride and the combined effect of amiloride and SITS on the recovery of ph, following acidification by removal of (NH4)2S04. The break in the records was for a period of 40 min during which there was another exposure to (NH4)S04. Effect of application of both amiloride, or Na-free Ringer and SITS The foregoing results clearly suggest that both Na+-H+ exchange (Fig. 1 d) and Cl--HCO3- exchange (Fig. 1 e) are involved in the recovery of ph1 following acidification. If these two mechanisms were essentially independent, then the effects of application of amiloride or Na-free Ringer and the effect of application of SITS would be additive. Fig. 11 shows an experiment where the effect of amiloride (10-4 M) was compared with the effect of amiloride and SITS (104 M) together. The usual slow recovery of ph1 was seen in the presence of amiloride, but on application of amiloride and SITS together there was no recovery, and a continued slow acidification occurred. The same effect was seen when SITS was used in combination with Na-free Ringer. Thiocyanate, shown to inhibit acid secretion in the stomach (Davenport, 1940) by its suggested action on a 'Cl-dependent proton pump' (Durbin & Heinz, 1958), had no effect on the rate of ph1 recovery by itself and was not additive with amiloride. These results suggest that the ph1 regulating system in mouse soleus muscle is comprised of two completely separate exchange systems. From

16 310 C. 0. AICKIN AND R. C. THOMAS six experiments, like the one shown in Fig. 11, we have calculated that Cl-HCO3- exchange forms a mean of 21x % (S.D. of an observation) of the ph1 regulating system under these conditions. Effect of temperature on ph1 recovery Fig. 12 illustrates part of an experiment to show the effect of temperature on the rate of recovery of ph,. Comparison of the ph1 recovery at the two temperatures clearly shows a considerable decrease in the rate of recovery with decreased temperature. In this case the decrease was by 65 %. The acidification seen on increasing temperature (0.18 ph unit) was similar to the difference in the mean ph1 values at these two temperatures reported previously (Aickin & Thomas, 1977a) min 40 I E 50 I Em E Temperature 28-C 37 "C ~~. 68k ~~~phi m-equiv NH+ 20 m-equiv NH+ Fig. 12. Pen recordings of part of an experiment showing the effect of increasing the temperature of the experimental chamber from 28 0C to 37 0C, on ph, and the rate of recovery of phi following acidification on removal of (NH4)2SO0. The break in the records was for a period of about 55 min during which there was another exposure to (NH,,)SO,S, and the recovery in ph, was blocked by application of amiloride. At the arrow, the ph-sensitive micro-electrode started to come out of the fibre, and it was then pushed back in again, causing the rapid decay in Em. From three experiments we have calculated an average Q10 for the temperature range 'C of 2-6 for the uninhibited ph1 recovery, and of 6-9 for the recovery in the presence of amiloride. If Na+-H+ exchange is completely inhibited by amiloride, then the Q10 of 6-9 is that of Cl-HCO3- exchange. Since we have calculated that Cl--HC03- exchange forms about 20 % of the ph1 recovery at 37 'C, then by proportion, the Q10 for Na+-H+ exchange is 1-4.

17 IONIC MECHANISM OF ph1 REGULATION 311 These values can only be taken as rough estimates, particularly that for Na+-H+ exchange since we did not compare the effect of inhibition of Cl-HCO3- exchange or complete inhibition of the ph1 regulating system at the two temperatures. However, it is clear that the Q10 for Na+-H+ exchange must be less than 2-6 (that for the uninhibited ph1 recovery) since the Q10 for amiloride inhibited ph, recovery was so much larger. DISCUSSION These results emphasize how well intracellular ph is controlled in mammalian skeletal muscle, seen in the rapid recovery of ph, following quite a large acid displacement (e.g. Fig. 2). The main conclusions to be drawn from these results concern the ionic mechanisms involved in this recovery of ph1, for convenience called the ph, regulating system. The schemes of Fig. 1 will be considered in turn. (a) K+-H+ exchange The lack of effect of removal of external K on the rate of recovery of phi following an acid load rules out K+-H+ exchange as the mechanism of the ph1 regulating system. It also suggests that the very slow acidification seen on application of K-free Ringer is not due to a primary effect on the ph, regulating system, but to a secondary effect. This is most probably the increasing internal Na (due to inhibition of the Na pump) and the resultant decreasing transmembrane Na gradient (see later). This secondary effect is the most probable explanation for the fall in ph1 on K depletion seen in earlier reports (for references see Waddell & Bates, 1969; Cohen & Iles, 1975), although in some of the in vivo experiments, an elevated Pco2 in compensation for an extracellular alkalosis (Miller, Tyson & Relman, 1963) may also have been partly responsible. (b) H+ ions sharing the outward limb of the Na pump The lack of effect of both removal of external K and application of high concentrations of ouabain on the ph1 recovery following an acid load, indicates that the Na pump is not involved in the phi regulating system. Roos (1975) has also reported that ouabain has no effect on the ph, of rat diaphragm, and more recently Thomas (1976b) has shown that ouabain has no effect on the recovery of ph1 following injection of HCl into snail neurones. Previous results (e.g. Williams et al. 1971) where ouabain was reported to cause a fall in phi, may well have been due to secondary effects, since ph1 was not determined until at least 1 hr after application of ouabain.

18 312 C. C. AICKIN AND R. C. THOMAS (c) Ca2+-H+ exchange Although we were unable to record from fibres exposed to Ca-free Ringer, our results with reduced external Ca suggest that Ca2+-H+ exchange is unlikely to be involved in the phi regulating system, at least at the fibre surface membrane. Even though reduction in external Ca probably increased membrane permeability (suggested from the decrease in Em and accelerated acidification during exposure to (NH4)2SO4), the ph, recovery following an acid load was unaffected. The transient effects on ph1 of altering external Ca may be due to the close intracellular interrelationship between Ca2+, Na+ and H+ ions. As shown in these experiments, ph, is dependent on the transmembrane Na gradient, itself affected in heart muscle by changes in external Ca (see Ellis, 1977). In snail neurones ph1 is also related to internal Ca through Ca2+-H+ exchange, probably at the mitochondrial membrane (Meech & Thomas, 1977). (d) Na+-H+ exchange Our results suggest that Na+-H+ exchange is the main mechanism of the ph1 regulating system, since as little as an 11 % reduction in external Na gave a significant decrease in the rate of phi recovery following an acid load (see Fig. 7) and on complete or 90 % removal of external Na there was only very slow recovery of phi. Amiloride also greatly reduced the rate of ph1 recovery following acidification. This inhibition seemed to be mediated through the same system, since the effects of removal of external Na and application of amiloride were not additive. This effect of amiloride does not appear to be the same as its action in frog skin, where the inhibited Na influx is current generating (see Bentley, 1968), since the ph1 regulating system appears to be electroneutral. Experiments using Na+-sensitive micro-electrodes add further evidence to the involvement of Na+-H+ exchange in the ph1 regulating system. Application of (NH4)2SO4 caused a decrease in [Na+]1 probably due both to the decrease in external Na and stimulation of the Na pump. But on removal of external (NH4)2SO4 the overshoot in [Na+]t, which could be mostly inhibited by amiloride, strongly suggested an extra influx of Na occurring via Na+-H+ exchange. The finding that amiloride caused a considerable decrease in the rate constant for the increase in [Na+]i seen on application of an acid load is also in agreement with this. Na+-H+ exchange has also been suggested to be responsible for intracellular alkalinization in sea urchin eggs (Johnson et al. 1976) where it can be inhibited both by removal of external Na and by the presence of amiloride, and in snail neurones (Thomas, 1977) where it is not inhibited by amiloride.

19 IONIC MECHANISM OF ph1 REGULATION 313 (e) Cl--HCO3- exchange Although complete removal of external Na and application of amiloride greatly slowed the ph1 recovery following acidification, some recovery was still seen. A fully inhibited ph, regulating system however, should lead to a slow intracellular acidification dependent on the passive influx of H+ ions. This discrepancy can be explained in two ways. First, that Li partially substituted for Na (as mentioned in Results we were unable to test this by using another substitute) and that amiloride did not give a complete block. Secondly, that some other mechanism was involved. The effect of both removal of external C02 and application of SITS in slowing the ph1 recovery following an acid load supports the latter explanation and suggests that Cl-HC03- exchange is indeed involved. Cl-HC03- exchange has also been suggested or shown to be involved in the ph1 regulating system of both snail neurones (Thomas, 1976b) and squid giant axons (Boron & De Weer, 1976b; Russell & Boron, 1976). The mechanism for the ph1 regulating system in mouse solews muscle Na+-H+ and Cl-HC03- exchange have both recently been directly shown to be involved in intracellular alkalinization in invertebrate preparations (Russell & Boron, 1976; Johnson et al. 1976; Thomas, 1977). The results discussed above strongly suggest that both mechanisms are also involved in the recovery of phi following acidification in mouse soleus muscle. The additive effects of application of SITS and Na-free Ringer or of SITS and amiloride provide evidence for them being entirely separate mechanisms with Cl-HC03- exchange, under the conditions of our observations, accounting for about 20 % of the ph1 regulating system. Experiments where the rate of ph1 recovery was compared at two temperatures also provide evidence for Na+-H+ and Cl-HC03- exchange being separate mechanisms, since the calculated Q10 for the ph, recovery in normal Ringer was considerably lower than that for ph1 recovery in the presence of amiloride. This independence of Na+-H+ and Cl-HC03- exchange is in contrast to the tightly linked system found in snail neurones (Thomas, 1977). The calculated Q10 for Na+-H+ exchange (1-4) suggests that this is a passive process and the great reduction in the rate of ph1 recovery following an acid load on only 11 % reduction of external Na agrees with this. In addition the transmembrane Na gradient is quite large enough to provide the energy for this exchange (electrochemical gradient for Na is about 160 mv compared with 20 mv for H+ ions). Johnson et al. (1976) and Thomas (1977) have also suggested that Na+-H+ exchange is driven by the Na gradient. The calculated Q10 for Cl-HC03- exchange (6.9) on the

20 314 C. C. AICKIN AND R. C. THOMAS other hand, suggests that this is an active process, requiring metabolic energy. Russell & Boron (1976) have shown that Cl-HCO3- exchange requires ATP in the squid giant axon and Cl-HCO3- ATPases have been found in various tissues (e.g. Sachs, Mitch & Hirschowitz, 1965; Simon, Kinne & Knauf, 1972). We wish to thank Professor A. Roos, Dr D. Ellis and Dr J. W. Deitmer for criticism of an early draft of this paper, the M.R.C. for monies, and Merck, Sharp and Dohme for the gift of amiloride and ethacrynic acid. C. C. Aickin is an M.R.C. Scholar. REFERENCES AICKIN, C. C. & THOMAS, R. C. (1975). Micro-electrode measurement of the internal ph of crab muscle fibres. J. Phy8iol. 252, AIcKIN, C. C. & THOMAS, R. C. (1977a). Micro-electrode measurement of the intracellular ph and buffering power of mouse soleus muscle fibres. J. Phy8iol. 267, AicxN, C. C. & THOMAS, R. C. (1977b). The effect of external Na and amiloride on ph1 recovery in mouse soleus muscle. J. Physi-ol. 269, 80-81P. AAIKE, N. (1975). Contribution of an electrogenic sodium pump to membrane potential in mammalian skeletal muscle fibres. J. Phyaiol. 245, BARTLEV', W. & AMOORE, J. E. (1958). The effects of manganese on the solute content of rat-liver mitochondria. Biochem. J. 69, BENTLEY, P. J. (1968). Amiloride: A potent inhibitor of sodium transport across the toad bladder. J. Phyaiol. 195, BORON, W. F. & DE WEER, P. (1976a). Intracellular ph transients in squid giant axons caused by CO8, NH8 and metabolic inhibitors. J. gen. Physiol. 67, BORON, W. F. & DE WEER, P. (1976b). Active proton transport stimulated by C02/HCO3-, blocked by cyanide. Nature, Lond. 259, BuLL, M. B. & LARAGH, J. H. (1968). Amiloride. A potassium-sparing natriuretic agent. Circulation 37, CHAPPEL, J. B., GREVILLE, G. D. & BICKNELL, K. E. (1962). Stimulation of respiration of isolated mitochondria by manganese ions. Biochem. J. 84, 61P. COHEN, R. D. & ILES, R. A. (1975). Intracellular ph: Measurement, control and metabolic interrelationships. Critical Reviews in Clinical Laboratory Sciences 6, DAVENPORT, H. W. (1940). The inhibition of carbonic anhydrase and of gastric acid secretion by thiocyanate. Am. J. Physiol. 129, DTYRBIN, R. P. & HEINZ, E. (1958). Electromotive chloride transport and gastric acid secretion in the frog. J. gen. Phyaiol. 41, ELLIS, D. (1977). The effects of external cations and ouabain on the intracellular sodium activity in sheep heart Purkinje fibres. J. Physiol. 273, ELLIS, D. & THOMAS, R. C. (1976). Direct measurement of the intracellular ph of mammalian cardiac muscle. J. Physiol. 262, EMILO, M. G. & MENANO, H. P. (1975). The excretion of hydrogen ion by the isolated amphibian skin: Effects of antidiuretic hormone and amiloride. Biochim. biophys. Acta 382, FRAzIER, L. W. & VANATTFA, J. C. (1973). Characteristics of H+ and NR4+ excretion by the urinary bladder of the toad. Biochim. biophy8. Acta 311,

21 IONIC MECHANISM OF ph, REGULATION 315 GARDNER, L. I., MAcLACHLAN, E. A. & BERMAN, H. (1952). Effect of potassium deficiency on carbon dioxide, cation and phosphate content of muscle. J. gen. Physiol. 36, GEE, J. D. (1976). Fluid secretion by the malpighian tubules of the tsetse fly Glo88ina morsitan8: the effects of ouabain, ethacrynic acid and amiloride. J. exp. Biol. 65, GOTO, K. (1918). Mineral metabolism in experimental acidosis. J. biol. Chem. 36, IRVINE, R. 0. H., SAUNDERS, S. J., MILNE, M. D. & CRAWFORD, M. A. (1960). Gradients of potassium and hydrogen-ion in potassium-deficient voluntary muscle. Clin. Sci. 20, JOHNSON, J. D., EPEL, D. & PAUL, M. (1976). Intracellular ph and activation of sea urchin eggs after fertilization. Nature, London. 262, KASBEKAR, D. K. & DURBIN, R. P. (1965). An adenosine trophosphatase from frog gastric mucosa. Biochim. biophys. Acta 105, KEYNES, R. D. (1965). Some further observations on the sodium efflux in frog muscle. J. Physiol. 178, KNAuF, P. A. & ROTHSTEIN, A. (1971). Chemical modification of membranes. I. Effect of sulphydryl and amino reactive reagents on anion and cation permeability of the human red blood cell. J. gen. Physiol. 58, MEECH, R. W. & THOmAS, R. C. (1977). The effect of calcium injection on the intracellular sodium and ph of snail neurones. J. Physiol. 265, MILLER, R. B., TYSON, I. & RELMAN, A. S. (1963). ph of isolated resting skeletal muscle and its relation to potassium content. Am. J. Phyaiol. 204, PARSONS, D. S. (1956). The absorption of bicarbonate-saline solutions by the small intestine and colon of the white rat. Q. J1 exp. Physiol. 41, PrrTS, R. F. & ALEXANDER, R. S. (1945). The nature of the renal tubular mechanism for the acidification of the urine. Am. J. Physiol. 144, Roos, A. (1975). Intracellular ph and distribution of weak acids across cell membranes. A study of D- and L-lactate and of DMO in rat diaphragm. J. Phygiol. 249, RUSSELL, J. M. & BORON, W. F. (1976). Role of chloride transport in regulation of intracellular ph. Nature, Lond. 264, SACHS, G., MIrCH, W. E. & HIRSCHowrrz, B. I. (1965). Frog gastric mucosal ATPase. Proc. Soc. exp. Biol. Med. 119, SIMON, B., KnINE, R. & KNAUF, H. (1972). The presence of a HCO3- ATPase in glandula submandibularis of rabbit. Pflugers. Arch. ges. Physiol. 337, TEORELL, T. (1939). On the permeability of the stomach mucosa for acids and some other substances. J. gen. Physiol. 23, THOMAS, R. C. (1970). New design for sodium-sensitive micro-electrode. J. Physiol. 210, 82-83P. THOMAS, R. C. (1974). Intracellular ph of snail neurones measured with a new phsensitive glass micro-electrode. J. Physiol. 238, THOMAS, R. C. (1976a). The effect of carbon dioxide on the intracellular buffering power of snail neurones. J. Physiol. 255, THOMAS, R. C. (1976b). Ionic mechanism of the H+ pump in a snail neurone. Nature, Lond. 262, THOMAS, R. C. (1977). The role of bicarbonate, chloride and sodium ions in the regulation of intracellular ph in snail neurones. J. Physiol. 273, WADDELL, W. J. & BATES, R. G. (1969). Intracellular ph. Physiol. Rev. 49, WHInLOCK, R. T. & WHEELER, H. 0. (1969). Hydrogen ion transport by isolated rabbit gall bladder. Am. J. Physiol. 217,

22 316 C. C. AICKIN AND R. C. THOMAS WImiAms, J. A., Wmmow, C. D. & WOODBuTRY, D. M. (1971). Effects of ouabain and diphenyihydantoin on transmembrane potentials, intracellular electrolytes, and cell ph of rat muscle and liver. J. Physiol. 212, WOODBURY, J. W. (1965). Regulation of ph. In Physiology and Biophysicw, ed. RUCH, T. C. & PATTON, H. D., pp Philadelphia and London: Saunders.