(pho) of 7-35 and at 22 'C.

Transcription

1 J. Physiol. (1983), 345, pp With 6 text-figures Printed in Great Britain THE INTRACELLULAR ph OF FROG SKELETAL MUSCLE: ITS REGULATION IN ISOTONIC SOLUTIONS BY RONALD F. ABERCROMBIE*, ROBERT W. PUTNAM AND ALBERT ROOS From the Department of Physiology and Biophysics and Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. (Received 15 July 1982) SUMMARY 1. The behaviour of intracellular ph (phi) was studied with micro-electrodes in frog semitendinosus muscle which was superfused with Ringer solution and with depolarizing solutions. The electrodes were introduced into the depolarized muscle about 40 min after contracture had subsided. All studies were done at external ph (pho) of 7-35 and at 22 'C. 2. The phi in normal Ringer solution buffered with HEPES was (S.E. of mean) (n = 10); the membrane potential, Vm, was mv. When phi was lowered to about 6-8 by replacing the HEPES by 5 % C02, 24 mm-hco3 (constant pho), it recovered at a very slow rate of ApHi h-1 (n = 6). When all the Na was replaced by N-methyl-D-glucamine (initial phi , initial Vm mv, n = 8), this slow alkalinization was converted into a slow acidification at a rate of ApHi h In muscle depolarized in 15 mm-k (Vm -50 mv), the rate of recovery from CO2 acidification was not increased above that in normal Ringer solution (2-5 mm-k). When, however, the muscle was depolarized in 50 mm-k to about -20 mv, the rate of recovery increased to ApHi h-1 (n = 6) when external Cl was kept constant, or to (n = 9) when [K]. [Cl] product was kept constant. In the absence of Na, phi recovery rate in 50 mm-k was reduced by at least 90 %. 4. Enhanced recovery from C02-induced acidification was also observed in 2-5 mm-k when the fibres were depolarized to about -20 mv in one of two ways: (a) by previous exposure for 60 min to 50 mm-k at constant Cl, or (b) by reduction of external Cl to 5-9 mm in the presence of 0-5 mm-ba. 5. When phi of depolarized fibres (50 mm-k) was lowered to about 6-8 by the weak acid dimethyl-2,4-oxazolidinedione (DMO), it recovered at a rate of 0-12 ApHi h-1 in two experiments. 6. In fibres depolarized in 50 mm-k and constant Cl, either 0-1 mm-sits or 0-5 mm-amiloride slowed phi recovery from CO2 exposure by about 50 %. When the depolarization was achieved at constant [K]. [Cl] product, amiloride slowed ph1 recovery by about 50 %, while SITS had, at most, only a slight effect. * Present address: Department of Physiology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A.

2 176 R. F. ABERCROMBIE, R. W. PUTNAM AND A. ROOS 7. It can be concluded that, in frog muscle, depolarization to about -20 mv accomplished in a number ofways enhances phi recovery from CO2 acidification. Part of this recovery most probably represents the exchange of external Na for internal H. When depolarization is achieved by 50 mm-k at constant Cl, an Na-dependent Cl-HCO3 exchange may also play a role. INTRODUCTION In most cells, hydrogen ions are regulated within fairly narrow limits at a concentration which is below passive electrochemical equilibrium. This regulation of intracellular ph (phi) is accomplished by special systems of coupled ion transport which remove acid from the cell by an energy-requiring process. When an acid load is imposed on the cell, a compensating quantity of H+, OH- or HCO3- is transported across the membrane, and phi returns towards normal. In the past few years, a great deal has been learned about the ionic substrates and the kinetics of the reactions responsible for phi recovery. Thus, an Na-dependent Cl-HCO3 exchange has been implicated in the phi regulation of the snail neurone (Thomas, 1976, 1977), squid axon (Boron & De Weer, 1976a, b; Russell & Boron, 1976), and barnacle muscle fibre (Boron, McCormick & Roos, 1979, 1981). During normal operation of this system, acid equivalents are removed from the cell by an exchange of internal Cl for external HCO3; external Na is needed for this exchange. The system is inhibited by disulphonic stilbenes like SITS that are known inhibitors of anion transport in red blood cells (Cabantchik, Knauf & Rothstein, 1978). A separate system, also capable of removing acid, can operate in the nominal absence of HCO3. It has been described in fertilized sea urchin eggs (Johnson, Epel & Paul, 1976) and in mouse soleus muscle (Aickin & Thomas, 1977), as well as in other cells (Cala, 1980; Kinsella & Aronson, 1980). This system involves an exchange of internal H ions for external Na. It is unaffected by the disulphonic stilbenes, but is inhibited by the diuretic amiloride. In some preparations, the two mechanisms coexist (Aickin & Thomas, 1977; Moody, 1981). While nearly every other cell that has been studied responds to a reduction of its phi by some form of acid removal, frog skeletal muscle seems to be the exception. Bolton & Vaughan-Jones (1977) observed that the phi of frog sartorius fibres, when depressed by 5 % CO2 at constant external ph, ph. = 7-2, did not recover for 25 min. Our observations on the phi behaviour of frog semitendinosus muscle confirm that, under normal conditions, the fibres respond very slowly to a reduction in phi. However, when depolarized by one of several methods, they respond to C02-induced acidification by returning their phi towards more alkaline values. This response requires the presence of external Na. Both amiloride-sensitive and SITS-sensitive recovery components could be demonstrated. Preliminary reports ofsome of this work have been published (Abercrombie & Roos, 1981, 1982). METHODS Preparation and experimental arrangement. The frogs (Rana pipiens, obtained from J. M. Hazen and Co., Alburg, VT or from Lemberger's, Germantown, WI 53022) were kept at room temperature. After the animals were pithed, the dorsal head of the semitendinosus muscle was

3 phi REGULATION IN FROG MUSCLE 177 Normal Ringer (HEP) Normal Ringer (Bic) Normal Ringer, Na-free (HEP) Normal Ringer, Na-free (Bic) 50K, normal Cl (HEP) 50K, normal Cl (HEP) 50K, normal Cl (Bic) 50K, normal [K]. [Cl] (HEP) 50K, normal [K]. [Cl] (Bic) 25K, normal [K]. [Cl] (HEP) 25K, normal [K]. [Cl] (Bic) 25K, normal Cl (HEP) 25K, normal Cl (Bic) 15K, normal [K]. [Cl] (HEP) 15K, normal [K]. [Cl] (Bic) 15K, normal Cl (HEP) 15K, normal Cl (Bic) Low Cl, Ba (HEP) Low Cl, Ba (Bic) 50K, Na-free (HEP) 50K, Na-free (Bic) TABLE 1. Experimental solutions Glucon- Na K NMDG Ba Cl ate (mm) (mm) (mm) (mm) (mm) (mm) DMO (mm) HEP, buffered with 10 mm-hepes to ph Bic, buffered with 24 mm-hco3-5 % CO2 to ph NMDG, N-methyl-D-glucamine; DMO, dimethyl-2,4-oxazolidinedione. All solutions contained 0-1 mm-edta, 2 mm-mg, and 4 mm-ca, and were gassed with either 100 % 02 or with 5 % C02, 95 % 02- isolated. About % of the fibres were removed with fine tweezers leaving a small bundle of about thirty fibres, which was pinned in the chamber at 1-3 times slack length. The phi and membrane potential (Vm) of one of the fibres were monitored while the bundle was superfused with solutions of various compositions. Electrodes and electronics. A conventional micro-electrode filled with 3 M-KCl and a ph-sensitive micro-electrode with recessed tip (Thomas, 1974, 1978) were introduced into the same fibre. The location of the two tips could easily be seen because of the small number of fibres in the preparation. On each experimental day, the ph electrode was calibrated at 22 TC in standards (phthalate at ph 4 and phosphate at ph 7) whose ionic strength was similar to that of frog Ringer solution. The sensitivity of the ph electrode was mv per ph unit. When the ph micro-electrode was transferred from one test solution to another, the change in potential was 80 % complete within one minute. The KCI-filled intracellular electrode had a resistance of MKI. A KCl-filled reference electrode made contact with the external solution. The two KCl-filled electrodes, each connected to a calomel half-cell, and the phi electrode were coupled to separate electrometers (311 K, Analog Devices, Inc., Norwood, Maine) whose input impedance was 1014 Q. By means of operational amplifiers (Burr-Brown Research Corp., Tucson, AZ, 3500 A and Analog Devices 1 18A), the difference between the two intracellular electrodes was obtained, and also that between the two KCI-filled electrodes. These differences represent the potentials due to the intracellular ph and the membrane potential, respectively. The two potentials were recorded on a 10 inch dual-channel chart recorder (1 mv/0 1 inch) (Heath-Schlumberger SR-206). Solutions and chamber. The superfusates are listed in Table 1. They were always buffered to ph with either 5% C02/24 mm-hco3 or 10 mm-n-2-hydroxyethylpiperazine-n'-2-30

4 178 R. F. ABERCROMBIE, R. W. PUTNAM AND A. ROOS ethanesulphonic acid (HEPES). The Na substitute was N-methyl-D-glucamine (Sigma, St. Louis, MO). The Cl substitute was gluconate (Sigma). Given a stability constant for Ca gluconate of mol11 (Martell & Smith, 1977), the concentration of ionized calcium in the low (5-9 mm)-cl studies was 1-5 mm. The ionized magnesium concentration (stability constant of Mg gluconate = mol1, Martell & Smith, 1977) in these studies was 1-3 mm. The weak acid dimethyl-2,4-oxazolidinedione (DMO) (Eastman, Rochester, NY), used in some experiments to acidify fibres, was recrystallized from benzene. Some solutions contained mm-amiloride (Merck, Sharp & Dohme, West Point, PA) or 0-1 mm-4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS) (I.C.N. Pharmaceuticals, Cleveland, OH). Solutions that contained no CO2 were gassed with 100% 02; those containing C02, with 5% C02-95% 02. The CO2 concentration was accurate to within % absolute. They were drawn into airtight 140 ml syringes from which the muscles were superfused through C02-impermeable Saran tubing (Clarkson Controls & Equipment, Detroit, MI) by a pump (Harvard Apparatus Co., South Natick, MA) at a rate of 0-85 ml/min. The experimental chamber, made of lucite, had a volume of 1.1 ml. Its temperature was maintained at 22 TC by coolant water circulating through its base. Calculation of buffering power. From the increase in phi, ApHi, observed when the 5% CO2 (Pco, = 36-5 mmhg) was replaced by HEPES-buffered medium (Boron, 1977), the intrinsic buffering power, flnt = A[HC03]J/ApH1, was calculated. A CO2 solubility coefficient of mmol 1-1 mmhg-' (Harned & Davis, 1943) and an apparent first pk of carbonic acid of 6-11 (Harned & Bonner, 1945) were used. It was assumed that the Pco, after removal of CO2 was zero. Buffering power was measured at 2-5 mm-k, at 50 mm-k with constant [K]. [Cl] product, and at 50 mm-k with constant external Cl. RESULTS Measurements in normal Ringer solution. The phi of muscle superfused with normal frog Ringer solution (ph 7-35) which was equilibrated with 100 % 02 and buffered with 10 mm-hepes, averaged (S.E. of mean) (n 10); the Vm = was mv. In Fig. 1 A the muscle was exposed to a solution of the same ph, but buffered with 24 mm-hco3/5 % CO2 (95% 02). The phi fell rapidly as CO2 diffused into the cell and, after hydration, yielded protons. Simultaneously, the membrane depolarized. The ph1 then recovered only slightly during the 30 min of continued exposure to CO2. When the original C02-free Ringer solution was re-admitted, the phi and Vm returned to near their original values. In six experiments, phi during CO2 exposure (20-30 min) was ; Vm was mv; the rate of ph1 recovery was ApHi h-1. When the muscle, having been immersed in C02-free medium for about h, 1 was exposed to C02, the membrane potential often fell to as low as -60 mv. This large depolarization was transient: Vm spontaneously recovered during min of continued CO2 exposure. The phi remained depressed during these changes in Vm. Subsequent periods of CO2 acidification, when preceded by brief intervals in C02-free medium, produced only moderate depolarization, as shown in Fig.1 A. Both under control conditions and after acid loading with CO2, the average equilibrium potential for H ions, EH (-1O mv and -32 mv, respectively), was considerably less negative than Vm, i.e. the intracellular H ion concentration was considerably lower than if H ions had been distributed across the cell membrane at electrochemical equilibrium. Measurements in 50 mm-k. A different phi response to CO2 is illustrated in Fig. IB. A similar acid load was imposed as before, but this fibre had been depolarized in 50 mm-k, with constant Cl. Intracellular recordings during the initial period of

5 phi REGULATION IN FROG MUSCLE 179 A B min Vm Vm %C02 5%C02.. r ~~~~~~~~~ phi phi Equilibrium ph K 50 K, constant Cl 6 7 Fig. 1. Response of phj to acidification (5 % C02, pho unchanged at 7 35) in two media. A, normal Ringer solution (2-5 mm-k). Very slight recovery of phi upon acidification. B, 50 mm-k, constant Cl. Significant recovery and post-acidification overshoot. At start of recovery, there is a small inward driving force on HC03- and outward force on H+: EH = EHCO, = -27 mv, while Vm -23 mv. As recovery proceeds, the direction of these forces reverses at the 'equilibrium phi' (6 96). depolarizations were not possible because of contracture. The electrodes were introduced after about 40 min in high K. In contrast to the fibre shown in Fig. 1 A, the depolarized fibre significantly recovered from the C02-induced acidification. The phi, upon removal of the C02, rose to a value more alkaline than the control. This ' overshoot' is the expected consequence of the extrusion of acid from the fibre during the CO2 exposure. In six fibres depolarized to mv at constant Cl, CO2 lowered phi from to The phi then recovered at an average initial rate of ApHi h-1, or about 13 times more rapidly than in resting muscle. The C02 exposure caused a reversible further depolarization of mv (Fig. 1 B). Early in recovery there was an inwardly directed driving force on HCO3 ions and an outward force on H ions which averaged about 12 mv. As recovery continued, the direction of these forces sometimes reversed, as in the case illustrated in Fig. 1 B. At least two mechanisms come to mind that might account for the depolarizationengendered phi recovery. First, the recovery might be linked to the elevation of external K, in analogy with preparations such as the stomach where increased K leads to increased ATPase activity associated with acid secretion (Forte, Machen &

6 180 R. F. ABERCROMBIE, R. W. PUTNAM AND A. ROOS Obrink, 1980; Sachs, Spenney & Lewin, 1978). Secondly, the recovery might be related to the nearly 20-fold increase in internal Cl which is the consequence of maintaining external Cl constant (Boyle & Conway, 1941; Hodgkin & Horowicz, 1959). The elevated internal Cl could initiate phi recovery through the activation of a Cl-HCO3 exchange (Russell & Boron, 1976; Thomas, 1977). These possibilities were considered in the following experiments, illustrated in Fig A B C ] 20Vm Vm ~~20 min -30-5%C02 5%C02 5%C02 p~~jw C I~~ r p 7.3 phi ph, 7'2 7'1.7'0 50 K. constant CI 50 K. constant [K]. [C1] 6'9 12'S K 0-5 Ba, 2-5 K, 5'9 Cl 6'8 Fig. 2. ph1 recovery from C02-acidification (pho unchanged at 7'35) in fibres that had been depolarized to about -20 mv by each of three methods. A, exposure for 1 h to 50 mm-k, constant Cl followed by transfer to normal Ringer solution (2'5 mm-k). Introduction of the micro-electrodes immediately after changing to 2-5 mm-k resulting in some further depolarization. The fibre remained depolarized in 2'5 mm-k. There was a slight upward phi drift in 2-5 mm-k preceding C02 exposure which was less than half the rate of phi rise during C02. Since the buffering power during CO2 exposure was about doubled, the rate of removal of H-equivalents in response to acidification more than quadrupled. B, depolarization in a solution containing 50 mm-k, 5 9 mm-cl (gluconate replacement), i.e. constant [K]. [Cl] product. Prompt phi recovery. C, depolarization by 5.9 mm-cl in presence of 0'5 mm-ba. Prompt phi recovery. Note the post-acidification ph1 overshoot in each of the three cases. Various methods of depolarization. In Fig. 2A, the fibres were soaked for 60 min in a solution containing 50 mm-k and constant Cl. When the bundle was returned to normal Ringer solution (2'5 mm-k), depolarization persisted due to the rectifying properties of the K channels (Hodgkin & Horowicz, 1959). When phi was then lowered by exposing the muscle to 5 % C02, a prompt phi recovery resulted, followed by an overshoot upon CO2 removal. This shows that high external K concentration is not required for recovery. Two additional experiments yielded similar results (mean recovery rate 0'63 + 0'08 ApHi h-1, n = 3). Fig. 2B illustrates an experiment in which K was raised while the [K]. [Cl] product was kept constant (replacement of Cl by gluconate). In nine fibres depolarized in this way, Vm was -21 +I10 mv. Exposure to CO2 lowered phi from 7'33 + 0'03 to 7'00 + 0'02. This mode ofdepolarization, in which internal Cl should remain unchanged, also allowed phi recovery though at a slower initial rate (0'21 +0'03 ApHi h-1) than at constant external Cl. The degree of reversible depolarization was also less than at constant external Cl, namely 0'8 + 0'3 mv. Fig. 2 C illustrates an experiment in which

7 phi REGULATION IN FROG MUSCLE 181 both external K and internal Cl concentrations were left approximately unchanged. The K permeability was reduced with 0 5 mm-ba (Henderson & Volle, 1972; Sjodin & Ortiz, 1975). The membrane potential was now mainly determined by the Cl distribution and, with external Cl lowered to 5-9 mm, the membrane depolarized to about -20 mv. If it is assumed that Cl remained at equilibrium, then the internal A min 0 Vm % C02 5% C phi.7* K, no Na 50 K, constant Cl, no Na Fig. 3. Response of phi to CO2 acidification (pho unchanged at 7-35) in the absence of Na (substitution by N-methyl-D-glucamine). A, 2-5 mm-k. The muscle was superfused with this Na-free solution for 21 h before it was tested with CO2. The rapid initial fall in phi is followed by a plateau-phase acidification (0X081 ApHi h-1). B, 50 mm-k. This muscle was superfused with the depolarizing, Na-free solution for about I h before exposure to CO2. No phi recovery. Cl under these conditions must have been about 2'7 mm. Again, phi recovered promptly from acid loading. Two additional experiments gave similar results (recovery rate ApHi h-'). We conclude that neither elevated external K nor elevated internal Cl are prerequisites for recovery. Measurements in Na-free solutions. In eight muscles, the same studies were done as in normal Ringer solution with the exception that Na was replaced by N- methyl-d-glucamine. Fig. 3A shows a typical experiment. The fibres were superfused with the Na-free HEPES-buffered solution for 2-4 h before their responsiveness to CO2 was tested. The phi was stable during the min of observation in C02-free medium and averaged ; the Vm was mv. These values are the same as in the presence of Na. When now the fibres were acid-loaded with C02, the phi, after the initial rapid fall, continued to decline slowly (plateau acidification) at an average rate of 0' '024 ApHi h-1. The average phi during the plateau acidification was During CO2 exposure, the membrane depolarized to

8 182 R. F. ABERCROMBIE, R. W. PUTNAM AND A. ROOS mv, just as in Na-containing Ringer solution. When the original solution was re-admitted, both Vm and phi returned; the latter leveled off at a value slightly lower than control (see Fig. 3A). We found that Na was required for phi recovery in fibres depolarized with 50 mm-k (Fig. 3B). In the absence of Na, the rate of recovery from CO2 exposure was very slow: ApHi h-1 (n = 5) at constant external Cl and 0-01 ±+ 001 ApHi h-' (n = 3) at constant [K]. [Cl] product. Dependence of phi recovery on Vm. The potential dependence of phi recovery rates in C02-containing solutions of 2-5 mm-, 15 mm- or 50 mm-k is summarized in Fig. 4. The filled circles indicate experiments at constant external Cl; the open circles, those at constant [K]. [Cl] product. The phi recovery rate remained at a low level of about ApHi h-' as long as the potential was more negative than -50 mv. When Rate of recovery in 5% CO2 (6) (ApH, h-1) 0-2 // // (7) 0-1 _ (3) (6)J, (8/ ' Fig. 4. Dependence of phi recovery, on membrane potential. Experiments were done in solutions containing 2-5 mm-k (Vm mv), 15 mm-k (Vm mv), and 50 mm-k (Vm -20 mv), either with constant [K]. [Cl] product (O --- 0), or with constant Cl (I *.The vertical lines indicate + I S.E. of mean. Vm the membrane was depolarized to -20 mv (50 mm-k), recovery rate increased about 10-fold. Recovery was greater when external Cl was kept constant than when [K]. [Cl] product was kept constant. Drug 8ens3itivity. The effect of various drugs on the rate of phi recovery in fibres depolarized in 50 mm-k and challenged with 5 %/ C02 is summarized in Fig. 5. Either external Cl or [K]. [Cl] product was kept constant. The recovery rate was substantially greater at constant Cl. The diuretic amiloride slowed recovery by about 50 %/. At constant external Cl, the disulphonic stilbene, SITS, a known blocker of anion movements (Cabantchik et al. 1978), also slowed recovery by about 50 /%. SITS and amiloride in combination reduced phi recovery rate by 65 /, i.e. less than the sum

9 li Rate of recovery in 5% C02 (ApH W') (6) phi REGULATION IN FROG MUSCLE K, constant CI 50 K, constant [K]. [CII 03 - (9) amiloride SITS 02d r o losits T r ( SITS + 2mm sits amiloride amiloride SITS (4) (4) amiloride Na-free (5) Af1Na-free (3) Fig. 5. Sensitivity of ph1 recovery in fibres depolarized by 50 mm-k to SITS, amiloride, and removal of Na. The vertical lines indicate +1I5.E. of mean. When SITS and amiloride were used together, SITS was first applied for about 20 min; the medium was then changed to one containing amiloride as the only inhibitor. N-methyl-D-glucamine was the Na replacement. All solutions contained 50 mm-k. In the five groups on the left, external Cl was kept constant. The recovery rates were, from left to right: 0-33 ±+007, , , and 0 03 ± 0-02 ApHi h-1. In the five groups on the right, in which the [K]. [Cl] product was kept constant, the recovery rates were , 0-18±-001, , and ApHi h-'. The numbers of experiments are shown in parentheses. ofthe separate effects of the two drugs. When the [K]. [Cl] product was kept constant, SITS had, at most, only a slight effect on recovery, both by itself and in the presence of amiloride. Fig. 5 shows that Na removal was more effective than SITS and amiloride, either alone or in combination. In two studies, fibres depolarized in HEPES-buffered 50 mm-k (constant Cl) were acid-loaded in the nominal absence of HCO3, by substituting 30 mm of the Na salt of the weak acid DMO (pk' 6 1) for an equal amount of NaCl. The phi initially - fell to 6-8, approximately the same value as with 5 % CO2. The rate of phi recovery in both experiments was 0-12 ApHi h-1 (Fig. 6), less than when phi was reduced with C02, and about the same as the recovery rate after a CO2 challenge in the presence of SITS. This would be expected since HCO3 ions were nominally absent in the DMO experiments, and because the intracellular buffering power during CO2 and DMO application was nearly the same. As in the case of C02, DMO produced an additional and reversible depolarization of 3 or 4 mv (Fig. 6). Buffering power. At normal K (2-5 mm) and in solutions containing 50 mm-k at constant [K]. [Cl] product, the intrinsic buffering power (mmol per 1 fibre water per

10 184 R. F. ABERCROMBIE, R. W. PUTNAM AND A. ROOS DMO V min Im 50 K, constant Cl Fig. 6. phi recovery from acidification (ph. unchanged at 7 35) by the weak acid DMO in fibres depolarized in HEPES-buffered 50 mm-k-containing solution (constant Cl). In the DMO solution, 30 mm-nacl was replaced by the same concentration of Na DMO. unit change in phi) was the same: mm, n = 5, and mm, n = 18, respectively. Bolton & Vaughan-Jones (1977), using the same method, found a buffer value of 35 mm in fibres exposed to normal K at ph The starting phi of their muscles, 7 05, was significantly lower than ours (7 18), and fell upon exposure to 5% CO2 by only 0-17 unit, as compared to 0-38 in our work. At 50 mm-k and constant Cl, we found that the buffering power was reduced to mm, n = 19. This 27 % reduction is due to fibre swelling. The degree of swelling can be calculated by applying the equation [K]1. [Cl]i = [K]0. [Cl]0 to the new steady state reached after K is raised to 50 mm at normal ( mm) Cl. In this equation, [K]i = (140 V+X)/(V+AV) and [Cl]i = (3-0 V+X)/(V+AV); the intracellular K and Cl concentrations in normal Ringer solution are taken as 140 and 3 0 mm, respectively. V is the fibre's original water volume and A V and X are the volume of water and the number of millimoles of K and Cl that enter upon depolarization. Since the entering KCI solution is isotonic, X/A V = 125 mrm. The equation yields a value for AVIV of Fibre water thus increases by 50%, with resulting reduction in the concentration of impermeant cellular constituents by 1-1/1 5 or 33 %. This figure is in reasonable agreement with the observed reduction in buffering power. Rate of removal of H-equivalents and fibre volume. The rate of ph1 recovery during CO2 exposure can be converted to the rate of removal of H-equivalents by means of * ph

11 phi REGULATION IN FROG MUSCLE the intracellular buffering power. To compare this removal at 50 mm-k and constant [K]. [Cl] product with that at constant external Cl, it is practical to consider H extrusion from the entire fibre, rather than per unit surface area, since swelling in constant Cl increases surface area. The swelling, while not affecting the intrinsic buffer content, 26 V, which is the product of the fibre's original water volume and buffering power, will increase the fibre's bicarbonate buffer content from 2-3 [HCO3]i V to 2-3 [HCO3]i (V+ A V), assuming phi remains unchanged. Because the phi during CO2 exposure at constant Cl was somewhat lower than at constant [K]. [Cl] product, the average [HCO3]i during CO2 exposure was - 9 mm in the former case and - 13 mm in the latter. Thus, the total buffer content under the two conditions happened to be nearly the same: [26 + (2-3 x 9 x 1-5)] V = 57 V at constant Cl, and [26 + (2-3 x 13)] V = 56 V at constant [K]. [Cl] product. It follows that the rates of H extrusion per fibre bear nearly the same relationship to each other as the rates of phi recovery. 185 DISCUSSION The intracellular ph of resting frog muscle is higher than if the H ions were at equilibrium. The existence of an H ion gradient, maintained in the face of acidifying influences such as metabolic acid production, H influx and HCO3 efflux (assuming these ions to be permeant) strongly suggests the existence of a mechanism that removes H-equivalents. This mechanism functions slowly as judged from the sluggish phi recovery from CO2-induced acidification. Because depolarized muscle requires Na for phi recovery (see below), it is reasonable to assume that the slow recovery in resting muscle is also Na-dependent. The absence of a perceptible downward drift of phi after at least 2 h in nominally CO2-free, Na-free Ringer solution (see Fig. 3A) indicates, therefore, that metabolic acid production and H influx in resting muscle are very small. When phi is reduced during CO2 exposure, these two factors should be even less significant, because the inward driving force on H is less and glycolysis is depressed (see Roos & Boron, 1981). The plateau acidification observed during CO2 exposure in Na-free Ringer solution (Fig. 3A) is, therefore, most likely the result of an outward leak of HCO3. Assuming constant field, and a mean fibre diameter of 90,sm, we derived from the average plateau slope (0-069 ApHi h-1) a HCO3 permeability of 7-4 x 10-8 cm s-1. Woodbury (1971) estimated a permeability of about 4 x The actual rate of 'active' phi recovery of resting muscle in Na-containing Ringer solution during exposure to 5 % CO2 must be taken as the sum of the acidification rate in the absence of Na, ApHi h-1, and the observed alkalinization rate in its presence, 0-025, and thus amounts to about If the absence of Na had not entirely suppressed the removal of H-equivalents, both HCO3 permeability and rate of ' active' phi recovery from CO2 would be underestimates. The above reasoning assumes that the prior superfusion for 2-4 h with Na-free medium had reduced internal Na activity to a negligible level, so that acidification due to exchange of internal Na for external H can be excluded. Depolarized fibres were found to recover briskly from acidification (CO2 exposure) by two distinct mechanisms, both of which require Na: one that is amiloride-sensitive and is observed either when external Cl or when the [K]. [Cl] product is kept constant, and a second one that is SITS-sensitive, and is much more striking at constant

12 186 R. F. ABERCROMBIE, R. W. PUTNAM AND A. ROOS external Cl. In the absence of Na, phi recovery under either condition is reduced by at least 90 %. It is probable that the ionic basis of the amiloride-sensitive recovery is an Na-H exchange, and that of the SITS-sensitive recovery, an Na-dependent Cl-HCO3 exchange. Since the internal Cl remains unchanged at constant [K]. [Cl] product, while it increases at constant external Cl, the absence of a striking effect of SITS under the former condition may well be due to internal Cl not being high enough to allow a Cl-HCO3 exchange (Russell & Boron, 1976; Thomas, 1977). The phi recovery from CO2 acidification in depolarized muscle fibres can be ascribed in part to the elimination of the outward HCO3 leak due to the membrane being depolarized beyond EHCO,: 59 x ( ) c -30 mv. This would account for 20 or 33 % of the observed recovery, depending upon whether external Cl or [K]. [Cl] product was kept constant. The inward driving force on HCO3, usually present during the early part of recovery from CO2 acidification in 50 mm-k (see Fig. 1 B) and averaging about 12 mv, is too small for HC03 influx to make a significant contribution to phi. The remaining % of the recovery rate might be controlled by membrane potential in a number of ways. For instance, either Na-H or Cl-HCO3 exchange, or both, might be electrogenic. If more than one H were removed for each Na entering and/or more than one HCO3 were entering for each Cl leaving, then depolarization would favour the exchange reactions and thus might increase their rate. Another possibility is that the carrier proteins responsible for the exchanges possess an electric dipole moment that deforms them as the electric field across the membrane changes. Such deformation could affect the rates of the ionic exchange mediated by the carriers, even if the exchanges were electrically neutral. Finally, there might be an indirect link between the exchangers and intracellular parameters such as cyclic AMP, internal Ca, protein phosphorylation, etc. which, themselves, could be affected by membrane potential. In that case, hormonal or other factors might also affect the exchange and thus the rate of phi recovery. Only further experiments will allow a choice between these possibilities. We thank Merck, Sharp & Dohme for the gift of amiloride, and Janice J. Wuelling and Susan J. Eads for their meticulous typing of the various versions of the manuscript. We are grateful to Paul De Weer for his careful reading of a late version of the manuscript, which led to several improvements, and especially to the referee for her outstanding review of this and the following paper. This work was supported by the National Institutes of Health Grant and Research Career Award HR to A. R., and a Muscular Dystrophy Post-doctoral Fellowship to R.W. P. REFERENCES ABERCROMBIE, R. F. & Roos, A. (1981). Effect of depolarization on behavior of intracellular ph (ph1) in frog muscle. Biophys. J. 33, 62a. ABERCROMBIE, R. F. & Roos, A. (1982). Recovery of intracellular ph (ph1) in frog skeletal muscle: effects of membrane voltage and of inhibitors. Biophys. J. 37, 335a. AICKIN, C. C. & THOMAS, R. C. (1977). An investigation of the ionic mechanism of intracellular ph regulation in mouse soleus muscle fibres. J. Physiol. 273, BOLTON, T. B. & VAUGHAN-JONES, R. D. (1977). Continuous direct measurement of intracellular chloride and ph in frog skeletal muscle. J. Physiol. 270,

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