Some effects of carbon dioxide on intracellular potassium in frog muscle

141

Respiration Physiology (1982) 47. 141 150 Elsevier Biomedical Press

S O M E EFFECTS OF C A R B O N D I O X I D E O N I N T R A C E L L U L A R P O T A S S I U M IN F R O G M U S C L E

F. H U G U E N I N * The Physiological Laboratory, Downing Street, Cambridge, CB2 3EG and the Physiological Department of the University, Biihlplatz 5, 3012 Bern, Switzerland

Abstract. The short-term effect of carbon dioxide on resting potential (Era), intracellular potassium activity (ak) and intracellular pH (PHi) has been investigated in frog skeletal muscle. The external pH was kept constant in the range 6.8-7.6 by addition of HCO~ in presence o f CO 2. Measurements were done in single or surface fibres o f the semi-tendinosus muscle. CO 2 reduced quickly E m and a k at high but not at low Pco 2. This effect persisted in presence of ouabain (10-5 M). These actions of CO2 are largely accountable for by osmotic swelling, but additional factors are involved.

Carbon dioxide Intracellular pH Intracellular potassium activity

Resting potential Skeletal muscle

Carbon dioxide influences E m and a~ in frog skeletal muscle (Meves and V61kner, 1958; Huguenin et al., 1980). One possible mechanism contributing to this effect would be an osmotic swelling: CO 2 buffering would produce H C O 3 ions and thereby increase the osmolar content of the cell; this would in turn cause water entry, a dilution of internal potassium, a decrease in a iK and finally a decrease in Em. The aim of this work was to obtain evidences supporting this hypothesis. To this purpose the effect of CO 2 on E m and a in was measured at P co2' s ranging from 22 to 588 m m Hg. In order to estimate the quantity of H C O 7 produced by CO 2, the pH~ was measured and the intracellular H C O £ concentration calculated. The possible effect of intracellular CO2 buffering on a~ and Em was then computed and compared with present and previous observations. The agreement between predictions and observations suggests that a substantial part of the effect of CO 2 o n E m and a ni might be the osmotic consequence of intracellular CO 2 buffering. However, this mechanism cannot explain all features of the present observations Accepted Jbr publication 4 November 1981 * Present address: Physiologisches Institut, Biihlplatz 5, 3012 Bern, Switzerland. 0034-5687/82/0000-0000/$02.75 © Elsevier Biomedical Press

142

F. H U G U E N I N

and additional factors are considered. Among these factors appear the well known CO:-induced K + shifts between intra- and extracellular fluids (Fenn and Cobb, 1934; Lad6 and Brown, 1963). Part of this work has been performed in Cambridge and part in Berne. Some of these results have been reported in preliminary form (Huguenin and Malachowski, 1979).

Methods MATERIAL

Results were obtained on skeletal muscles of the frog (R. temporaria). The experiments were done on surface fibres of the semitendinosus muscle, except for two experiments on single fibres of the semi-tendinosus and the ilio-fibularis muscles. All measurements were made at room temperature (20 + 3 °C).

MEASUREMENT

O F E m A N D aiK

The technique has been described in detail previously (Huguenin et al., 1980). Briefly a muscle was lightly stretched in an experimental chamber, 0.5 ml in volume, and superfused with solutions at a flow rate of 2 ml. min -~. A voltage-sensitive and a liquid ion-exchanger K + -sensitive (Corning code 477317) glass micro-electrodes were inserted 120 + 24/~m apart into a surface fibre. This allowed the continuous i monitoring of E m and a}~. Some experiments were performed in presence of ouabain. Control measurements showed that 10 -5 M ouabain did not affect the response of the K +-sensitive microelectrodes (K+-electrodes) between 1.5 and 500 mM K + concentration. Single muscle fibres were prepared and their E m measured as described by Hodgkin and Horowicz (1959). During impalement of single fibres, the solution flow as stopped.

MEASUREMENT

O F pH i

pH~ was measured in the same way as a~, but with pH-sensitive glass microelectrodes (Thomas, 1978a) instead of K+-electrodes.

SOLUTIONS

Their composition is given in table 1. They have been computed so as to have an ionic strength of 0.13 M and an ionized calcium concentration of about 0.9-1.4 raM.

10

10

2.5

10

2.5

2.5

2.5

2.5

2

3

4

5

6

7

8

9

87

119

79

1 I0

111

117

76

76

109

Na +

7.5

1.8

8.0

3.6

1.8

2.8

7.4

7.4

3.2

Ca 2+

98

-

98

11

87

11

11

87

C1

25

-

-

25

25

109

-

-

-

HCO 3 -

1.43

1.43

1.43 1.57

1.57

1.57

1.57

Hepes-

Hepes 1.43

0.82

1.80

0.82

0.82

1.80

HPO42

0.87

1.20

0.87

0.87

1.20

H2PO 4 -

S o l u t i o n 8 c o n t a i n s 0.1 m M N a p h o s p h a t e b u f f e r at p H 7.6.

2.5

K +

1

Ref.

TABLE 1

-

39

48

10

1

4

44

44

13

SO42 -

87

111

23

350

4

449

99

27

Sucrose

Composition of solutions (mM)

4

4

4

4

4

4

4

4

4

D-Glucose

7.6

7.6

7.6

7.6

6.8

7.0

6.8

6.8

7.0

pH

97

-

-

22

22

588

-

-

-

Pco 2 (mm Hg)

233

231

235

235

644

232

646

-

230

Osmolality (mosmol/kgH20)

+

r-

¢3 t'~ r" t"

7~

7.

Z

~7

144

F. HUGUENIN

CO2-containing solutions were equilibrated with CO2/O 2 mixtures prepared by British Oxygen Company or Carba (Berne, Switzerland) and having nominal P c o 2 values of 22, 97 and 588 mm Hg. The osmolality was measured from the freezing point depression with an osmometer Fiske (QF model 330 D), and it was ascertained by calculation that the small differences of osmolality among solutions (table 1) could not account for the effects of CO2 on aK reported here.

STATISTICS

Mean results are given as X + SEM. The significance of differences has been evaluated by means of Students' 't'-test for paired or unpaired data. Differences have been considered statistically significant when P < 0.05, n indicates the number of measurements.

Results THE EFFECT OF AN APPROXIMATELY PHYSIOLOGICAL Pco 2

In this section the effect of increasing P c o 2 from 0 to 22 mm Hg has been investigated. The latter value lies not far from that measured in the arterial blood of the frog at 20 °C, 12.7 mm Hg (Howell et al., 1970). The external pH was kept constant at 7.6 by addition of HCO3- in presence of CO 2. This pH value too is 'close' to that of frog arterial blood, 7.87. The effect of CO 2 was followed during 10 min. In 4 surface fibres, E m was -85.3 + 3.6 mV in absence of CO 2, and -86.3 + 3.5 mV after 10 min in presence of CO2 (solutions 6 and 8: table 1). The difference between these values is not significant. In order to assess the possibility that measurements on surface fibres underestimate the effect of CO 2, the experiment was repeated on single fibres. In 4 fibres having a diameter of 115+_10 /am, E m went from -87.2+_2.2 in CO2-free saline to -86.2 +- 3.2 mV in CO2-containing solution. The difference is not significant either. Although CO2 had no statistically significant effect in C1--free solution (n = 7; P > 0.1), it induced a highly variable depolarization in most cases. Figure 1 shows two characteristic r e s u l t s ~ one particular fibre. The influence of CO 2 on ak was then continuously recorded during 10 min in 4 surface fibres in C1--containing solutions (6 and 8 ; table 1). No significant change was present. The K + equilibrium potential, E K, was -104.0 + 2.1 mV in absence of CO2, and - 104.4 +- 2.0 mV in presence of CO 2. These figures correspond to ak values of 116.9 and 118.8 mM. The effect of CO 2 on pH~ was then continuously monitore~l d ~ i n g 10-50 min in the presence of C1-. Figure 2 shows an original recording.

PCO 2 2 2 m m H g

I

I

pH 7 . 6

- 3 0 --

-40

oo o

--

/

-50

S\

/

\ \

/

-60

> E

j

O

/

\

/

E -70 ILl

°/

-8,0 -

-90

-

-100

-

\

0

\

\

/•

--O

o

ID

•

I

I

I

I

-lO

o

+10

+ 20

TiME

(min)

Fig. 1. The effect of a nearly physiological P c o 2 on E m in a single muscle fibre in absence of chloride. Both solutions (7 and 9; table 1) had same pH and contained 3. I 0 - 7 M tetrodotoxin in order to prevent action potentials a n d twitches. E m was measured by consecutive impalements about 80 # m apart. Fibre diameter: 125 #m. Two consecutive experiments were conducted: O, first run; O, second run.

0

- -

-20 - -

E LC -60 - -

30rain

I -80

--

I

Em

-100

23

-6.8 '

pHi f " "

m

72

-

t...,

L...

L

J

--

I

I PC02 22rnmHg

76

CAl,.

• PH 0 7 6

Fig. 2. Recording showing the effect of a nearly physiological P c o 2 on pH i and E m in a surface muscle fibre. The external p H (pHo) remained constant at 7.6. Initially both voltage' and p H micro-electrodes were intracellular. After I0 min in absence of CO2, the CO2-containing solution was switched on for 30 min, and off again. The p H micro-electrode was disimpaled at arrow 1, which hyperpolarized the fibre. (arrow 2). At arrow 3, the volta.ge micro-electrode was withdrawn. The p H micro-electrode was then calibrated (CAL) with 2 solutions of p H 7.54 and 6.16. In this figure as in fig. 3, the lower trace follows the upper trace. This was to allow the pens on the recorder to move freely past each other. Solutions 6 and 8 (table 1).

146

F. HUGUENIN

Within 10 min, C O 2 lowered p H i from an initial steady value (7.26 + 0.05, n = 11) to a new steady value (7.04 + 0.05). The pH~ drop was fully reversible 20 min after CO 2 removal. This p H i decrease corresponds to an increase in intracellular H C O 7 concentration from 0 to 7.1 mM. The latter figures were estimated with the Henderson-Hasselbalch equation, on the assumption of identical external and internal Pco2's, apparent dissociation constant of carbonic acid (6.23) and solubility coefficient of CO2 (0.05 raM/ram Hg). These p H i measurements are in agreement with previous observations of Bolton and Vaughan-Jones (1977) on the sartorius.

THE EFFECT

OF Pco 2 =

97 mm Hg

In these experiments, the external p H was kept constant at 7.0. The effect of CO 2 on p H i was continuously monitored during 30 min. As previously, two steady values could be recorded: 6.96 + 0.06 in absence, and 6.54 + 0.05 (n = 8) in presence of CO 2 (solutions 1 and 4: table 1). Twenty minutes were necessary for a steady p H i to be reached in presence of CO 2. The effect of CO 2 was but partly reversible since p H i measured 25 min after CO 2 removal (6.80 + 0.06) was lower than before CO 2 application. The p H i values measured in absence and in presence of CO 2 correspond to calculated internal H C O 3 concentrations of 0 and 9.9 mM. In previously published experiments at same P¢o2 and p H (Huguenin et al., 1980), it was reported that CO 2 reduces E m and E K in surface fibres by 1.3 + 0.5 mV and 1.4 + 0.6 mV, respectively, within 5 min.

THE EFFECT OF Pco 2 = 588 mm Hg In this section the external p H was kept constant at 6.8. This low value was required because it is impossible to make up solutions of normal ionic strength with both a higher p H and a Pco2 of 588 m m Hg. The movement artifacts caused by such high Pco2'S in normal Ringer make microelectrode measurements nearly impossible. This difficulty was circumvented by using K*-rich hypertonic solutions and by limiting the CO 2 exposure to 4 rain. Muscles were equilibrated for one hour in a CO:-free, isotonic solution, and for half an hour in a similar hypertonic solution. E m and a iK were then continuously monitored. Solution numbers were 2, 3 and 5 (table 1). On CO 2 exposure, E m w e n t within 4 min from - 6 6 . 9 + 2.0 to - 5 9 . 2 + 2.9 mV (n = 11). On CO2 removal, it came back to - 6 4 . 6 + 2.9 mV. CO 2 also displaced E K within 4 min from - 8 3 . 9 + 1.5 to - 8 0 . 7 + 2.5 mV. Four minutes after CO 2 removal, E K was -82.7 + 2.3 mV. These figures correspond to a k changes from 212.1 to 186.8, and then to 202.2 m M . Part of the effect of C O 2 o n Era and a iK might result from an inhibition of the Na + p u m p (Keynes, 1965). If such were the case, the effect should be lessened by prior application o f ouabain which blocks the N a + pump. Therefore the responses

147

CO z A N D I N T R A C E L L U L A R K + PCO 2

410

-

~85

-

I

588mmHg

3

I

pH 6 8 E 83.2 -~"

3 7

5

-

f

-

I

1£5.g -

7'6 L-0

-

-20

-

-40

-

-60

-

-80

-

6o

4 -40 I

7

- 2 0

-4-"

-

E I~

t.d

I 2

4min

I

I

CAL

j

4

Fig. 3. The effect of a very high Pco 2 on ak and E m in a surface muscle fibre in the presence of 10-5 M ouabain. The external pH was kept constant at 6.8. Initially both electrodes were extracellular. The voltage electrode was inserted first, then the K +-electrode (arrow 1). Arrow 2 shows the effect of inserting the K+-electrode on Em. Seven minutes later, the CO2-containing solution was switched on for 4 rain, and off again. Later the K +-electrode was withdrawn (arrow 3), which affected Ern (arrow 4). Finally, the voltage electrode was disimpaled and the calibration (CAL) took place. Solutions 3 and 5 (table 1).

to CO 2 were reinvestigated in presence of 10 -5 M ouabain. Figure 3 shows a typical recording. CO 2 decreased E m from --62.0 + 2.9 to --52.9 + 2.4 mV within 4 min (lower curve). On CO: removal, E m came back to -58.1 + 2.6 mV within 4 min. As for E K (upper curve), it went from - 7 9 . 9 + 2.5 to - 7 4 . 8 + 2.4 mV within 4 min of C02 exposure and came back to - 7 6 . 6 + 2.8 mV within 4 min of CO2 removal. These E K figures correspond to changes in a K from 181.0 to 147.8, and then to 158.8 mM. C o m p a r i n g the data in absence and in presence of ouabain shows that the drug tended to make E K less negative, but did not influence significantly the E K changes caused by CO 2. The same conclusion holds for E,~ measurements.

Discussion

Carbon dioxide influences Era, a~ and pH~ in frog skeletal muscle. While no change in Em or ak can be detected at nearly physiological Pco2 in presence of C1 , a clear decrease appears at very high Pco 2. The effect of CO 2 persists in presence of ouabain.

148

v. HUGUENIN

These observations support the hypothesis that CO2 causes an osmotic swelling of muscle. In presence of CO 2, the non-bicarbonate buffers should be titrated inside the cell according to the reaction: CO 2 + H20 + B) BH + H C O f (l) where B and BH are respectively buffer bases and their conjugate acids. This titration should, by producing H C O 3 ions from a dissolved gas, increase the quantity of osmotically active particles; this should cause a water entry into the cell, decrease a in and E m. A t Pcoz'S of 22 and 97 m m Hg, the results indicate a production of 7-10 m M H C O 3 by reaction (1) within 10-20 min. Since muscle water behaves as if it were contained in a simple osmometer (Dydyfiska and Wilkie, 1963), it can be calculated from the van 't Hoff's osmotic equation that a iK should decrease by 3-4 mM. This is consistent within experimental error with the absence of significant change at Pco 2 22 m m Hg and with the previous observation (Huguenin et al., 1980) that a Pco 2 of 97 m m Hg reduces a iK by 5 m M within 5 rain. Other factors remaining constant, a decrease of a K by 4 m M should reduce E m by 0.6 mV according to the constant field theory. The E m measurements in presence of C1 - indicate no significant effect at Pco2 22 m m Hg or a depolarization by 1.3 mV within 5 min at Pco2 97 m m Hg (Huguenin et al., 1980). Predictions and observations are therefore of similar magnitude. The incomplete reversibility of the a K decrease on CO 2 removal, at Pco 2 588 m m Hg, may be due to an additional phenomenon, a loss o f K + . The mechanism of this loss is not entirely clear, although a link between the ratios [H+~]/[H+o] and [K~+]/[Ko +] has long been recognized (Fenn and Cobb, 1934; Brown and Goott, 1963). According to mathematical models developed by Burton (1980), this loss would be a logical consequence of p H i regulation and interdependence of transm e m b r a n e ionic gradients; however, another factor, an inhibition by CO 2 of the Na + p u m p and therefore of active K + transport has also to be considered (Keynes, 1965). The effect of CO 2 o n E m in C1--free medium, when present, (fig. 1) is unlikely caused by an osmotic decrease of a~. It is more probably due either to a block of inward-rectifier K ÷ channels by the lowered pH i (Blatz, 1980), or to a small flow of H C O 7 down its electrochemical gradient, i.e. an ionic current depolarizing the membrane. The experiments indicate no measurable pH~ recovery in frog skeletal muscle during CO 2 administration (fig. 2). This contrasts with observations on snail neurones (Thomas, 1978b), mouse soleus muscle (Aickin and Thomas, 1977), barnacle muscle (Boron et al., 1979) or m a m m a l i a n cardiac muscle (Ellis and Thomas, 1976). Two mechanisms might explain this discrepancy: an extremely slow ionic pH~ regulation, or production of acids through metabolism or intracellular organelles in presence of CO 2. The present pH~ measurements allow to compute the non-bicarbonate buffer

c o 2 AND INTRACELLULAR K +

149

value of sarcoplasm in the semi-tendinosus muscle, i.e. the ratio _ A(HCO~)/ApH i (see for example K a r m a n n and Held, 1972). Its value ranges from 24 to 32 m M / p H . A value of 35 m M / p H has been previously measured in the frog sartorius (Bolton and Vaughan-Jones, 1977). It is interesting to mention that these measured buffer values are relatively close to that, 40.3 m M / p H , calculated from the chemical composition of muscle (Reeves and Malan, 1976). At Pco2 97 m m Hg, the effect of CO2 on pH~ was but partly reversible. This might be due to a loss of r i C O 7 by muscle in presence of CO 2. The same mechanism has been invoked by Heisler and Piiper (1972) and by Boron et al. (1979) in order to explain the intracellular acidification observed at high Pco2 in rat diaphragm or in barnacle muscle. If frog muscle behaves like barnacle muscle, this H C O £ loss might occur through a H C O z - C 1 - effiux through exchange mechanism, or as a passive H C O f efflux through C1- channels. The inhibitory effect of SITS (4-acetamide-4'-isothiocyanostilbine-2.2'-disulphonic acid) on both H C O T - C 1 - exchange (Boron et al., 1979) and Cl- permeability (Dfrrscheidt-K~ifer, 1980) does not allow to distinguish between these possibilities. In conclusion, these observations show that marked Pco2 increases reduced E m and a~. Several mechanisms seem involved. The present data in vitro are consistent with the idea that, in vivo, cells play a major role in buffering extracellular fluid and that, in respiratory acidosis, the associated shifts of H + or H C O f between cells and extracellular fluid are partly balanced by movements of K + that preserve electroneutrality (Pitts, 1968). Should the extracellular fluid have constant osmolality, pH, [K +] and [C1-], one would expect an osmotic swelling of muscle and a decrease of extracellular volume during acute hypercapnia in vivo. This, however, is not the case (Held and Steiner, 1971).

Acknowledgements M y thanks are due to Mr. P. Reynolds for technical assistance, Prof, S. Weidmann and G, Malachowski for helpful discussion and to Prof. D. R. Held for reading the manuscript. Prof. R . D . Keynes granted me hospitality. This work has been partly supported by the 'Schweizerische Stiftung ffir Medizinisch-Biologische Stipendien'.

References Aickin, C.C. and R.C. Thomas (1977). Micro-electrode measurement of the intraceIlular pH and buffering power of mouse soleus muscle fibres. J. Physiol. (London) 267: 791-810. Bolton, T. B. and R. D. Vaughan-Jones (1977). Continuous direct measurement of intracellular chloride and pH in frog skeletal muscle. J. Physiol. (London) 270: 801-833. Blatz, A. L. (1980). Chemical modifiers and low internal pH block inward-rectifier K channels. Fed. Proc. 39: 2073. Boron, W.F., W.C. McCormick and A. Roos (1979). pH regulation in barnacle muscle fibers: dependence of intracellular and extracellular pH. Am. J. Physiol. 237: C 185-C 193.

150

F. HUGUENIN

Brown, E.B., Jr., and B. Goott (1963). Intracellular hydrogen ion changes and potassium movement. Am. J. Physiol. 204: 765-770. Burton, R. F. (1980). The role of intracellular buffers in acid-base disturbances: mathematical modelling. Respir. Physiol. 39:45 61. D6rrscheidt-Kfifer, M. (1980). The role of amino groups in contraction initiation and in other membrane parameters of frog skeletal muscle..1. Physiol. (London) 305: 81P. Dydynska, M. and D.R. Wilkie (1963). The osmotic properties of striated muscle fibres in hypertonic solutions. J. Physiol. (London) 169:312 329. Ellis, D. and R.C. Thomas (1977). Direct measurement of the intracellular pH of mammalian cardiac muscle. J. Physiol. (London) 262: 755-771. Fenn, W.O. and D.M. Cobb (1934). The potassium equilibrium in muscle. J. Gen. Physiol. 17: 629656. Heisler, N. and J. Piiper (1972). Determination of intracellular buffering properties in rat diaphragm muscle. Am. J. Physiol. 222: 747-753. Held, D. R. and C. A. Steiner (1971). Effect of acute respiratory acidosis on arterial plasma osmolality. Respir. Physiol. 12: 25-35. Hodgkin, A. L. and P. Horowicz (1959). The influence of potassium and chloride ions on the membrane potential of single muscle fibres. J. Physiol. (London) 148 : 127-160. Howell, B.J., F.W. Baumgardner, K. Bondi and H. Rahn (1970). Acid-base balance in cold-blooded vertebrates as a function of body temperature. Am. J. Physiol. 218: 600-606. Huguenin, F. and G. Malachowski (1979). Carbon dioxide increases the weight of frog skeletal muscle. J. Physiol. (London) 300:17P. Huguenin, F., W. Reber and T. Zeuthen (1980). Carbon dioxide, membrane potential and intracellular potassium activity in frog skeletal muscle. J. Physiol. (London) 303: 139-152. Karmann, U. and D.R. Held (1972). Equations treating the pH and (HCO3-) of buffered media as functions of Pco 2. Respir. Physiol. 15: 343-349. Keynes, R.D. (1965). Some further observations on the sodium efflux in frog muscle. J. Physiol. (London) 178: 305-325. Lad6, R. I. and E. B. Brown, Jr. (1963). Movement of potassium between muscle and blood in response to respiratory acidosis. Am. J. Physiol. 204: 761-764. Meves, H. and K.G. V61kner (1958). Die Wirkung von CO 2 auf das Ruhemembranpotential und die elektrischen Konstanten der quergestreiften Muskelfaser. Pfliigers Arch. Ges. Physiol. 265 : 457-476. Pitts, R.F. (1968), Physiology of Kidney and Body Fluids. 2nd edn. Chicago, Year Book Medical Publishers Incorporated, pp. 170-178. Reeves, R.B. and A. Malan (1976). Model studies of intracellular acid-base temperature responses in ectotherms. Respir. Physiol. 28:49 63. Thomas, R. C. (1978a). Ion-sensitive Intracellular Microelectrodes. London, New York, San Francisco, Academic Press, pp. 32-44 and 75 77. Thomas, R. C. (1978b). Comparison of the mechanisms controlling intracellular pH and sodium in snail neurones. Respir. Physiol. 33: 63-73.