AMERICAN JOURNAL OF PHYSIOLOGY Vol. 230, No. 1, January 1976. Printed
in U.S.A.
Intracellular bicarbonate under different metabolic
of skeletal states
R. N. KHURI, S. K. AGULIAN, AND K. K. BOGHARIAN Department of Physiology, American University of Beirut, Beirut,
AND K. K. BOGHARIAN. different 1976. Intracellular bicarbonate of single muscle fibers in vivo was measured by a direct electrometric method simultaneously with the membrane PD in rats under seven different metabolic states. From the measured intracellular bicarbonate values and the Pcoz, the bicarbonate equilibrium potential and the intracellular pH were calculated. The mean intracellular [HWJ under normal control conditions was 10.3 -t 0.7 mM (SE). The i n t racellular bicarbonate fell significantly in both chronic metabolic acidosis and chronic K+ depletion. In contrast, intracellular bicarbonate was elevated in chronic metabolic alkalosis, K+ loading, and Na+ depletion. Taking intracellular pH as an index of the acid-base status of cells, we find that whereas the calculated cell pH decreased along with the cell bicarbonate in both chronic metabolic acidosis and K+ depletion, cell pH increased along with the bicarbonate only in chronic metabolic alkalosis. Cell pH was unchanged in both chronic K+ loading and Na+ depletion. KRURI,
R. N., S. K. AGULIAN,
muscle
Lebanon
extracellular fluids undergo changes in the same or in opposing directions. The intracellular buffer capacity of skeletal muscle in vivo was calculated from the directly measured intracellular bicarbonate.
Intracellulur bicarbonate of skeletal muscle under metabolic states. Am. J. Physiol. 230(l): 228-232.
METHODS
Electrometric. The construction and characterization of ion-selective double-barreled liquid ion-exchange microelectrodes have been described (15, 16) and their application to bicarbonate microelectrodes recently reported (17). The calibrating standards for intracellular bicarbonate determination contained a constant background of 5 mM chloride to approximate the intracellular Cl- concentration of mammalian skeletal muscle. Since the bicarbonate sensor is sensitive to the level of CO, in solution, the standards were continuously bubbled with 5-6% CO, gas mixture. The standards were maintained at the body temperature of the rat. The blood pH and PcoZ were determined in duplicate by the Radiometer-Copenhagen micro pH and PcoZ electrode systems. AnimaZ. The studies were carried out on SpragueDawley rats maintained on a normal diet, a low-K+ diet (General Biochemicals, no. 170X10), a high-K+ diet with 15% KC1 (General Biochemicals, TD-71051), a lowNa+ diet (General Biochemicals, 411094), and a low-Cldiet (General Biochemicals TD-71110). The states of chronic metabolic acidosis and alkalosis were sustained by substituting for the drinking water of normal diet rats 75 mM ammonium chloride and 75 mM sodium bicarbonate, respectively. All rats were maintained on the dietary and drinking regimens for 6 wk before experiments. The rats were anesthetized by Inactin and placed on an electrically heated table maintained at 38°C. The left carotid artery was cannulated for blood sampling. All the measurements were made on single superficial fibers of the high muscle covered with warm (38°C) mammalian Ringer solution equilibrated with 56% COZ. A reference electrode was made to achieve electrolytic contact with this solution by means of a fine 3 M NaCl salt bridge.
bicarbonate microelectrode; cell bicarbonate; muscle cell bicarbonate; cell pH; H+ electrochemical gradient; HCO,- electrochemical gradient
THE VALIDITY of intracellular pH techniques (glass electrodes, the distribution of weak acids and bases, the CO, method, indicator dyes) is still open to question, we feel that a reliable and direct determination of intracellular [HCOJ in conjunction with the CO, tension can serve to determine the pH of the cell aqueous cytoplasmic phase. Besides its indirect value for the determination of cell pH, intracellular bicarbonate as a buffer base serves as an index of cellular acidbase status. The purpose of this study was to determine directly and simultaneously by means of double-barreled HC03--selective liquid ion-exchange microelectrodes both the intracellular [HCOJ and the membrane PD of single muscle fibers of the rat in vivo under a variety of chronic metabolic states. From the measured intracellular bicarbonate values and the PCQ, the bicarbonate equilibrium potential and the cell pH were calculated. The results obtained demonstrate that both HCO,and H+ ions do not exhibit an electrochemical equilibrium distribution across the muscle fiber membrane, suggesting either active HCO,- influx or active H+ efYlux. The results also delineate when in the different states the acid-base indices of the intracellular and SINCE
RESULTS
Table 1 gives the mean values (*SE) of pH, CO2 tension, and bicarbonate concentration of arterial blood plasma under the six different states as determined by the Radiometer-Copenhagen system at 38OC. The num228
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INTRACELLULAR
BICARBONATE
OF
229
MUSCLE
1. pH, CO, tension, and bicarbonate concentration of arterial blood plasma under different states as determined by Radiometer-Copenhagen microelectrode system
TABLE
PH,U
State
1) Control (4) II) Acidosis
(6)
HI) Alkalosis
(8) IV)
Low K+ (7) V) High K+
(6) VI)
Low Na+ (5)
Values Significance 0.01.
are means t of difference
7.41 kO.1 7.30* 20.01 7.54* 20.02 7.46t kO.01 7.28* 20.01 7.35t 20.01 SE. Numbers from controls:
Pa (‘O., mmHg
39.2 dI.4 39.0 +0.6 48.6* -cl.5 47.5 51.8 55.5* kO.5 56.5* 50.5
[HCO-1,
mM
22.9 20.4 17.8* 20.7 38.7” *1*2 34*7* d.8 24,4 20.3 30.5* 50.5
of rats are in parentheses. * P < o.ooi. -f-P
20.5 kO.01 measured arterial extracellular CO,) and an apparent Values are means * SE, Numbers of observations (fibers:rats) are pK of 6.12 at 38”C, yields an intracellular pH of 7.03 * given in parentheses Significance of difference from controls: 0.03. Thus, under normal control conditions the distribu* P < 0.001. * t P < 0.01. HCOn-7
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230 19.0 t 0.5 mM and 7.20 t 0.01 U, respectively. The increase in intracellular pH is a reflection of the proportionately greater increase in cell [HCOJ than Pco2. There was also a significant (P < 0.01) depolarization of E, to 66.6 t 0.9 mV. Gilbert and Lowenberg (II) reported that frog sartorius muscle fiber E, in vitro depolarizes in response to the elevation of medium pH from 7.4 to 10 pH U. Davies and Keynes (7) have shown that the sodium pump is inhibited in alkaline solutions. With 170 electrometric determinations in potassium deficiency (group ZV), both the intracellular [HCOJ and pH decreased significantly (P < 0.001) to 7.6 * 0.3 mM and 6.78 t 0.03 pH U respectively. In contrast, both the extracellular [HCOJ and pH are elevated (Table 1 for plasma values). This underscores the fact that K+ deficiency is characterized by changes in the acid-base indices of the two major body fluid compartments in opposing directions: intracellular acidosis and extracellular alkalosis. It is interesting to note that the intracellular [HCOJ decreased by one-fourth of the control value, despite the elevation of the CO, tension, The membrane potential rose significantly (P < 0.01) to a mean value of 87.6 +- 0 .6 mV, a hyperpolarization by 9 mV. This is a contrast to the findings of Eckel and Sperlakis (9) who noted depolarization of 20 mV of skeletal muscle E, in K+ deficiency. Our finding, though, is in accord with that of Bolte and Liideritz (1) who reported 12.4 mV hyperpolarization in human skeletal muscle E, in K+ deficiency. Using the chemical CO, method to estimate intracellular [HCOJ in muscle, while some authors (10, 23) reported that K+ depletion is associated with decreased bicarbonate, others (8, 13) concluded there was no change in intracellular [HCOJ. In this study K+ depletion was associated with an intracellular acidosis. While this is in accord with the reported results of some investigators (9, 10, 14, 23), it is in conflict with many others (6, 8, 13, 18) who found intracellular pH of K+-deficient muscle to about the same as that of control. With 151 determinations in chronic K+-loaded rats (group V), the intracellular [HCOJ increased significantly (P < 0.01). However, this increased [HCOJ was associated with a normal intracellular pH of 7.02 t 0.01. The maintenance of intracellular pH within the normal range of values results from the proportionate increase in [HCOJ and Pco2. It is known (19) that the increased Pco2 could itself be the cause of the elevation of the intracellular [HCOJ. The membrane potential was significantly (P < 0.01) depolarized by lo,2 mV. In the Na+depleted rats (‘group VZ), the picture was very similar to the K+-loaded rats in that the intracellular [HCOJ increased while the pH did not change. However, the resting membrane potential in Na+ depletion was the same as in the normal controls. In a series of measurements in rats with chronic Cldepletion, a mean intracellular [HCOJ of 10.2 t 0.6 mM was obtained. This value was not significantly different from the control level. These Cl--depleted rats had the following arterial plasma values: chloride, 93.2 t 1.7 mM; bicarbonate, 30.1 +- 2 . 0 mM; Pcoz, 42.7 t 1.4 mmHg; and pH, 7.46 * 0.01. This picture represents the
KHURI,
AGULIAN,
AND
BOGHARIAN
characteristic extracellular hypochloremic metabolic alkalosis. The calculated intracellular pH of 7.02 is essentially the same as the control value, indicating that Cldepletion is associated with a normal reaction of intracellular fluid. DISCUSSION
In this study the intracellular bicarbonate in the cytoplasmic aqueous phase (cytosol) of single muscle fibers in vivo was measured by a direct electrometric method. This approach yields direct knowledge of an important index of acid-base composition of cell water. The derived values (2, 4, 5) of intracellular [HCOJ range from 1 to 14.7 mM. These are dependent on several assumptions relating to the existence of different forms of COz as well as on the accuracy of measuring the extracellular space. Thus, the intracellular determination of [HCOJ by indirect chemical methods requires several individual measurements. The total experimental error depends on the experimental errors of each of the different determinations made. With the indirect chemical approach, there is no evidence that the chemical data on intracellular bicarbonate and pH are representative of the cells actually punctured. Our in situ electrometric determination of intracellular [HCOJ is a methodological development which has made acid-base equilibria in cell water accessible to direct analysis. HC03- electrochemical PD AfiHCO,= &jCOa- - &m A large electrochemical PD exists for HCO,- ion across the membrane of single muscle fibers. This PD represents the net driving force favoring passive efflux of HCO,- ions out of the cell and which, in the steady state, may be opposed by an equal and opposite force of active HC03- influx into the cell. The HCO,- electrochemical PD was 56.1 mV under control conditions. Its magnitude underwent a small increase with Na+ depletion but decreased under all the other conditions studied. The greatest decrease was in alkalosis and Kf depletion: about 20% decrease from the normal value. The existence of a net HCO,- electrochemical PD is interpreted to indicate an active HC03- influx (or H+ efflux) across fiber membrane. The observed electrochemical disequilibrium of HCO,- ion across the membrane can be taken only as presumptive evidence for active HCO,- influx and not as conclusive evidence for a pump mechanism. A higher electrochemical potential of HCO,- in intracellular water than in extracellular fluid could also be due to intracellular HCO,- trapping, i.e., difficulty of intracellularly generated HCO,- to equilibrate passively across the cell membrane. This is consistent with the thesis that cell membranes are in general poorly permeable to HCO,- ion. Similarly, we could interpret the results in terms of an H+ electrochemical PD. H+ electrochemical PD AbH+ = 61.5 (pH, - pHi) + Em Under
control
conditions
an electrical
PD of 78.6 mV is
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INTRACELLULAR
BICARBONATE
OF
231
MUSCLE
the force driving H+ into the cell, while the chemical PD of 23.4 mV represents the force driving H+ out of the cell. The algebraic sum of the two forces, i.e., the electrochemical PD of 55.2 mV is the net driving force favoring passive H+ influx into the cell. Under steady-state conditions, this passive influx of H+ must be counterbalanced by an equal and opposite active H+ efflux by a pump mechanism. Thus, the electrochemical disequilibrium distribution of H+ indicates that H+ may be actively extruded from the cell by a pump mechanism located in the cell membrane. This active H+ extrusion mechanism results in a much lower intracellular [H+] than expected on the basis of a Nernst-type passive equilibrium distribution. This study cannot distinguish a passive H+ influxactive H+ efYlux from a passive HC03- efYlux-active HCO,- influx as the underlying pump-leak mechanism in operation. IntraceZZuZar pH. Intracellular pH has been mostly determined by indirect chemical methods (3, 22) involving the distribution of a weak acid (DMO) across cell membranes. The as yet insurmountable problems of adequate insulation of pH glass microelectrodes (20) have prevented reliable intracellular pH measurements by the direct electrometric technique. Accordingly, we have resorted to the approach of calculating the pH of the aqueous cytoplasmic phase from the in situ electrometric [HCO,J in cell water and from the CO, tension of the same intracellular phase. Thus, we use intracellular [HCOJ as an indicator of the intracellular pH of cells. It is certainly safer to assume a homogeneous cytoplasmic water phase with regard to HCO,- ion concentration than with regard to H+ ion concentration. Electrochemical equilibrium of the Nernst type is not a factor in the regulation of intracellular pH. Cell pH seems to be determined mainly by two factors: 1) the intracellular HCOJH,COL buffer system, and 2) an active H+ extruding pump (similar to the Na+ pump). Other factors that control intracellular pH include such buffer systems as the proteinates and the phosphates as well as the rate of production of organic acids. This active H+ pump, by excreting H+ ions, maintains a cytusol pH at or near the neutral level. Although H+ does not exhibit an electrochemical equilibrium distribution across the cell membrane under the various conditions of this study, some pH dependence of the resting membrane potential should be noted. The E, was decreased in alkalosis, the only state in which there was an increase in intracellular pH. Perhaps membrane conductance increases in alkalosis. Under control conditions the muscle cell pH was at the neutral level (7.03 t 0.03). Cells that contain no carbonic anhydrase like muscle have a lower intracellular
pH than cells containing cytoplasmic carbonic anhydrase, e.g., kidney cell (21). With K+ deficiency a mild extracellular alkalosis (0.05 pH U increase) was accompanied by a severe intracellular acidosis (0.25 pH U decrease)+ In contrast, in metabolic acidosis and alkalosis intracellular and extracellular fluids underwent pH changes in the same direction. In metabolic acidosis intracellular and extracellular pH decreased by 0.19 and 0.11 U, respectively. In metabolic alkalosis the intracellular and extracellular pH increased by 0.17 and 0.13 U, respectively. A comparison of the magnitude of changes of intracellular with extracellular pH for the same change in CO2 tension can be used to compare the intracellular buffer capacity with that of blood. In both acidosis and alkalosis, blood exhibited a smaller pH change, suggesting that blood has a larger buffer capacity than intracellular fluid. IntraceZZuZar buffer capacity. A buffer is a substance which minimizes pH change in a solution. The buffer capacity of a solution is an indication of its effectiveness in minimizing pH change. The HCO,--CO, buffer system has a volatile btier. The buffer capacity of a tissue is better measured in vivo than in vitro. The buffer capacity in vivo includes the contribution of the H+ pump, There are two measures of intracellular buffer capacity. First is the buffer capacity index which measures the ability to buffer C02. This CO2 buffer capacity is given by the ratio, p = -Alog PCOJApHi. For OUT recently reported (17) experiments on acute respiratory acidosis, we obtain a value of 5.6 over the PcoZ range of 40-146 mmHg. This value is in close agreement with a CO, buffer capacity of 5.1 reported by Clancy and Brown (4) in dog skeletal muscle and 5J reported by Heisler and Piiper (12) in rat diaphragm muscle. The intracellular CO, buffering capacity was the same as the blood buffering capacity for CO,. This is also borne out by the observation that with 20% CO, the fall of intracellular pH of 0.36 U is quite comparable to the blood pH fall of 0.34 u. The second index of intracellular buffer capacity is given by the ratio: A[HCO,-]JApHi. The units of this ratio are termed Slykes if the base is in millimoles per liter. This would yield for rat skeletal muscle in vivo an intracellular buffer capacity of 19.7 Slykes with respiratory acidosis (17), and 15.8 Slykes with metabolic acidosis. These buffer values obtained from direct electrometric determination of intracellular base concentration are in reasonable agreement with a buffer value of 20 meq/pH U obtained from indirect chemical methods on rat diaphragm (12) and dog skeletal muscle (4). Received
for publication
23 January
1975.
REFERENCES 1. BOLTE, H. D., AND B. L~~DERITZ. Membrane potentials in experimental potassium deficiency. PfZuegers Arch. 301: 43-49,1968. 2. BURNELL, J. M. In vivo response of muscle to changes in CO, tension or extracellular bicarbonate. Am. J. Physiol. 215: 13761383, 1968. 3. BVTLER, T. C., D. T. POOLE, AND W, J, WADDELL. Acid-labile carbon dioxide in muscle: its nature and relationship to intracel-
lular pH. Proc. Sot. 4. CLANCY, R. L., AND skeletal and cardiac 1966. 5. CONWAY, E. J., AND malian muscle and London 103: 274-289,
Exptl. Biol. Med. 125: 972-974, 1967. E. B. BROWN. In vivo CO, buffer curves of muscle. Am. J. Physiol. 211: 1309-1312, P. J. FEARON. The acid-labile the pH of the muscle fibre. 1944.
CO, in mamJ. Physiol.,
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232 6. DARROW, D, C., R. E. COOKE, AND F. E. COVILLE. Kidney electrolytes in rats with alkalosis associated with potassium deficiency. Am. J. Physiol. 172: 55-59, 1953. 7. DAVIES, R. E., AND R. D. KEYNES. A coupled sodium-potassium pump. In: Membrane Transport and Metabolism, edited by A. Kleinzeller and A, Kotyk. New York, 1960, p. 336-340. 8. ECKEL, R. E., A. W. BOTSCHNER, AND D. H. WORD, The pH of the K-deficient muscle. Am. J. Physiol. 196: 811-818, 1959. 9. ECKEL, R. E., AND N, SPERLAKIS. Membrane potentials in Kdeficient muscle. Am. J. Physiol. 205: 307-312, 1963. 10. GARDNER, L. I., E. A. MACLACHLAN, AND H. BERMAN. Effect of potassium deficiency on carbon dioxide, cation, and phosphate content of muscle. J. Gen. Physiol. 36: 153-159, 1952. 11. GILBERT, D. L., AND W. E. LOWENBERG. Effect of pH on the resting membrane potential of frog sartorius muscle. J. CeZZuZar Comp. Physiol. 63: 359-364, 1964. 12. HEISLER, N., AND J. PIIPER. Determination of intracellular buffering properties in rat diaphragm muscle. Am. J. Physiol. 222: 747-753, 1972. 13. HUDSON, J. B., AND A. S. RELMAN. Effects of potassium and rubidium on muscle cell bicarbonate. Am. J. Physiol. 203: 209214, 1962. 14. IRVINE, R. 0. H., S* J. SAUNDERS, M. D. MILNE, AND M. A. CRAWFORD. Gradients of potassium and hydrogen ion in potassium-deficient voluntary muscle. C&z. Sci. 20: 1-18, 1960. 15. KHWRI, R. N., S. K. AGULIAN, AND A. KALLOGHLIAN. Intracellu-
KHURI,
16.
17.
18.
18. 20. 21,
22.
23.
AGULJAN,
AND
BOGHARIAN
lar potassium in cells of the distal tubule. Pfluegers Arch. 335: 297-308, 1972, KHURI, R., J. J. HAJJAR, S. AGULIAN, K. BOGHARIAN, A. KALE LOGHLIAN, AND H. BIZRI. Intracellular potassium in cells of the proximal tubule of Necturus macuZosus. Pfluegers Arch. 338: 7380, 1972. KHWRI, R. N,, K, K. BOGHARIAN, AND S. K. AGULIAN. Intracellular bicarbonate in single skeletal muscle fibers. Pfluegers Arch. 349: 285-294, 1974. MILLER, R. B., I. TYSON, AND A. S, RELMAN. pH of isolated resting skeletal muscle and its relation to potassium content. Am. J, Physiol. 204: 1048-1054, 1963. NICHOLS, G. Serial changes in tissue CO, content during acute respiratory acidosis. J. Chin. Invest. 37: 1111-1122, 1958. PAILLARD, M. Direct intracellular pH measurement in rat and crab muscle. J. Physiol., London 223: 297-319, 1972. STRUYVENBERG, A., R. B. MORRISON, AND A. S. RELMAN. Acidbase behavior of separated canine renal tuble cells. Am. J. Physiol, 214: 1155-1162, 1968. WADDELL, W. J,, AND T. C. BUTLER. Calculation of intracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO). Application to skeletal muscle of the dog. J. Clin. Invest. 38: 720-729, 1959. WILSON, A, F., AND D. H. SIMONS, Relationship between potassium, chloride, intracellular and extracellular pH in dogs. CZin. sci. 39: 731-745, 1970.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 1, 2019.
in U.S.A.
Intracellular bicarbonate under different metabolic
of skeletal states
R. N. KHURI, S. K. AGULIAN, AND K. K. BOGHARIAN Department of Physiology, American University of Beirut, Beirut,
AND K. K. BOGHARIAN. different 1976. Intracellular bicarbonate of single muscle fibers in vivo was measured by a direct electrometric method simultaneously with the membrane PD in rats under seven different metabolic states. From the measured intracellular bicarbonate values and the Pcoz, the bicarbonate equilibrium potential and the intracellular pH were calculated. The mean intracellular [HWJ under normal control conditions was 10.3 -t 0.7 mM (SE). The i n t racellular bicarbonate fell significantly in both chronic metabolic acidosis and chronic K+ depletion. In contrast, intracellular bicarbonate was elevated in chronic metabolic alkalosis, K+ loading, and Na+ depletion. Taking intracellular pH as an index of the acid-base status of cells, we find that whereas the calculated cell pH decreased along with the cell bicarbonate in both chronic metabolic acidosis and K+ depletion, cell pH increased along with the bicarbonate only in chronic metabolic alkalosis. Cell pH was unchanged in both chronic K+ loading and Na+ depletion. KRURI,
R. N., S. K. AGULIAN,
muscle
Lebanon
extracellular fluids undergo changes in the same or in opposing directions. The intracellular buffer capacity of skeletal muscle in vivo was calculated from the directly measured intracellular bicarbonate.
Intracellulur bicarbonate of skeletal muscle under metabolic states. Am. J. Physiol. 230(l): 228-232.
METHODS
Electrometric. The construction and characterization of ion-selective double-barreled liquid ion-exchange microelectrodes have been described (15, 16) and their application to bicarbonate microelectrodes recently reported (17). The calibrating standards for intracellular bicarbonate determination contained a constant background of 5 mM chloride to approximate the intracellular Cl- concentration of mammalian skeletal muscle. Since the bicarbonate sensor is sensitive to the level of CO, in solution, the standards were continuously bubbled with 5-6% CO, gas mixture. The standards were maintained at the body temperature of the rat. The blood pH and PcoZ were determined in duplicate by the Radiometer-Copenhagen micro pH and PcoZ electrode systems. AnimaZ. The studies were carried out on SpragueDawley rats maintained on a normal diet, a low-K+ diet (General Biochemicals, no. 170X10), a high-K+ diet with 15% KC1 (General Biochemicals, TD-71051), a lowNa+ diet (General Biochemicals, 411094), and a low-Cldiet (General Biochemicals TD-71110). The states of chronic metabolic acidosis and alkalosis were sustained by substituting for the drinking water of normal diet rats 75 mM ammonium chloride and 75 mM sodium bicarbonate, respectively. All rats were maintained on the dietary and drinking regimens for 6 wk before experiments. The rats were anesthetized by Inactin and placed on an electrically heated table maintained at 38°C. The left carotid artery was cannulated for blood sampling. All the measurements were made on single superficial fibers of the high muscle covered with warm (38°C) mammalian Ringer solution equilibrated with 56% COZ. A reference electrode was made to achieve electrolytic contact with this solution by means of a fine 3 M NaCl salt bridge.
bicarbonate microelectrode; cell bicarbonate; muscle cell bicarbonate; cell pH; H+ electrochemical gradient; HCO,- electrochemical gradient
THE VALIDITY of intracellular pH techniques (glass electrodes, the distribution of weak acids and bases, the CO, method, indicator dyes) is still open to question, we feel that a reliable and direct determination of intracellular [HCOJ in conjunction with the CO, tension can serve to determine the pH of the cell aqueous cytoplasmic phase. Besides its indirect value for the determination of cell pH, intracellular bicarbonate as a buffer base serves as an index of cellular acidbase status. The purpose of this study was to determine directly and simultaneously by means of double-barreled HC03--selective liquid ion-exchange microelectrodes both the intracellular [HCOJ and the membrane PD of single muscle fibers of the rat in vivo under a variety of chronic metabolic states. From the measured intracellular bicarbonate values and the PCQ, the bicarbonate equilibrium potential and the cell pH were calculated. The results obtained demonstrate that both HCO,and H+ ions do not exhibit an electrochemical equilibrium distribution across the muscle fiber membrane, suggesting either active HCO,- influx or active H+ efYlux. The results also delineate when in the different states the acid-base indices of the intracellular and SINCE
RESULTS
Table 1 gives the mean values (*SE) of pH, CO2 tension, and bicarbonate concentration of arterial blood plasma under the six different states as determined by the Radiometer-Copenhagen system at 38OC. The num228
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 1, 2019.
INTRACELLULAR
BICARBONATE
OF
229
MUSCLE
1. pH, CO, tension, and bicarbonate concentration of arterial blood plasma under different states as determined by Radiometer-Copenhagen microelectrode system
TABLE
PH,U
State
1) Control (4) II) Acidosis
(6)
HI) Alkalosis
(8) IV)
Low K+ (7) V) High K+
(6) VI)
Low Na+ (5)
Values Significance 0.01.
are means t of difference
7.41 kO.1 7.30* 20.01 7.54* 20.02 7.46t kO.01 7.28* 20.01 7.35t 20.01 SE. Numbers from controls:
Pa (‘O., mmHg
39.2 dI.4 39.0 +0.6 48.6* -cl.5 47.5 51.8 55.5* kO.5 56.5* 50.5
[HCO-1,
mM
22.9 20.4 17.8* 20.7 38.7” *1*2 34*7* d.8 24,4 20.3 30.5* 50.5
of rats are in parentheses. * P < o.ooi. -f-P
20.5 kO.01 measured arterial extracellular CO,) and an apparent Values are means * SE, Numbers of observations (fibers:rats) are pK of 6.12 at 38”C, yields an intracellular pH of 7.03 * given in parentheses Significance of difference from controls: 0.03. Thus, under normal control conditions the distribu* P < 0.001. * t P < 0.01. HCOn-7
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230 19.0 t 0.5 mM and 7.20 t 0.01 U, respectively. The increase in intracellular pH is a reflection of the proportionately greater increase in cell [HCOJ than Pco2. There was also a significant (P < 0.01) depolarization of E, to 66.6 t 0.9 mV. Gilbert and Lowenberg (II) reported that frog sartorius muscle fiber E, in vitro depolarizes in response to the elevation of medium pH from 7.4 to 10 pH U. Davies and Keynes (7) have shown that the sodium pump is inhibited in alkaline solutions. With 170 electrometric determinations in potassium deficiency (group ZV), both the intracellular [HCOJ and pH decreased significantly (P < 0.001) to 7.6 * 0.3 mM and 6.78 t 0.03 pH U respectively. In contrast, both the extracellular [HCOJ and pH are elevated (Table 1 for plasma values). This underscores the fact that K+ deficiency is characterized by changes in the acid-base indices of the two major body fluid compartments in opposing directions: intracellular acidosis and extracellular alkalosis. It is interesting to note that the intracellular [HCOJ decreased by one-fourth of the control value, despite the elevation of the CO, tension, The membrane potential rose significantly (P < 0.01) to a mean value of 87.6 +- 0 .6 mV, a hyperpolarization by 9 mV. This is a contrast to the findings of Eckel and Sperlakis (9) who noted depolarization of 20 mV of skeletal muscle E, in K+ deficiency. Our finding, though, is in accord with that of Bolte and Liideritz (1) who reported 12.4 mV hyperpolarization in human skeletal muscle E, in K+ deficiency. Using the chemical CO, method to estimate intracellular [HCOJ in muscle, while some authors (10, 23) reported that K+ depletion is associated with decreased bicarbonate, others (8, 13) concluded there was no change in intracellular [HCOJ. In this study K+ depletion was associated with an intracellular acidosis. While this is in accord with the reported results of some investigators (9, 10, 14, 23), it is in conflict with many others (6, 8, 13, 18) who found intracellular pH of K+-deficient muscle to about the same as that of control. With 151 determinations in chronic K+-loaded rats (group V), the intracellular [HCOJ increased significantly (P < 0.01). However, this increased [HCOJ was associated with a normal intracellular pH of 7.02 t 0.01. The maintenance of intracellular pH within the normal range of values results from the proportionate increase in [HCOJ and Pco2. It is known (19) that the increased Pco2 could itself be the cause of the elevation of the intracellular [HCOJ. The membrane potential was significantly (P < 0.01) depolarized by lo,2 mV. In the Na+depleted rats (‘group VZ), the picture was very similar to the K+-loaded rats in that the intracellular [HCOJ increased while the pH did not change. However, the resting membrane potential in Na+ depletion was the same as in the normal controls. In a series of measurements in rats with chronic Cldepletion, a mean intracellular [HCOJ of 10.2 t 0.6 mM was obtained. This value was not significantly different from the control level. These Cl--depleted rats had the following arterial plasma values: chloride, 93.2 t 1.7 mM; bicarbonate, 30.1 +- 2 . 0 mM; Pcoz, 42.7 t 1.4 mmHg; and pH, 7.46 * 0.01. This picture represents the
KHURI,
AGULIAN,
AND
BOGHARIAN
characteristic extracellular hypochloremic metabolic alkalosis. The calculated intracellular pH of 7.02 is essentially the same as the control value, indicating that Cldepletion is associated with a normal reaction of intracellular fluid. DISCUSSION
In this study the intracellular bicarbonate in the cytoplasmic aqueous phase (cytosol) of single muscle fibers in vivo was measured by a direct electrometric method. This approach yields direct knowledge of an important index of acid-base composition of cell water. The derived values (2, 4, 5) of intracellular [HCOJ range from 1 to 14.7 mM. These are dependent on several assumptions relating to the existence of different forms of COz as well as on the accuracy of measuring the extracellular space. Thus, the intracellular determination of [HCOJ by indirect chemical methods requires several individual measurements. The total experimental error depends on the experimental errors of each of the different determinations made. With the indirect chemical approach, there is no evidence that the chemical data on intracellular bicarbonate and pH are representative of the cells actually punctured. Our in situ electrometric determination of intracellular [HCOJ is a methodological development which has made acid-base equilibria in cell water accessible to direct analysis. HC03- electrochemical PD AfiHCO,= &jCOa- - &m A large electrochemical PD exists for HCO,- ion across the membrane of single muscle fibers. This PD represents the net driving force favoring passive efflux of HCO,- ions out of the cell and which, in the steady state, may be opposed by an equal and opposite force of active HC03- influx into the cell. The HCO,- electrochemical PD was 56.1 mV under control conditions. Its magnitude underwent a small increase with Na+ depletion but decreased under all the other conditions studied. The greatest decrease was in alkalosis and Kf depletion: about 20% decrease from the normal value. The existence of a net HCO,- electrochemical PD is interpreted to indicate an active HC03- influx (or H+ efflux) across fiber membrane. The observed electrochemical disequilibrium of HCO,- ion across the membrane can be taken only as presumptive evidence for active HCO,- influx and not as conclusive evidence for a pump mechanism. A higher electrochemical potential of HCO,- in intracellular water than in extracellular fluid could also be due to intracellular HCO,- trapping, i.e., difficulty of intracellularly generated HCO,- to equilibrate passively across the cell membrane. This is consistent with the thesis that cell membranes are in general poorly permeable to HCO,- ion. Similarly, we could interpret the results in terms of an H+ electrochemical PD. H+ electrochemical PD AbH+ = 61.5 (pH, - pHi) + Em Under
control
conditions
an electrical
PD of 78.6 mV is
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INTRACELLULAR
BICARBONATE
OF
231
MUSCLE
the force driving H+ into the cell, while the chemical PD of 23.4 mV represents the force driving H+ out of the cell. The algebraic sum of the two forces, i.e., the electrochemical PD of 55.2 mV is the net driving force favoring passive H+ influx into the cell. Under steady-state conditions, this passive influx of H+ must be counterbalanced by an equal and opposite active H+ efflux by a pump mechanism. Thus, the electrochemical disequilibrium distribution of H+ indicates that H+ may be actively extruded from the cell by a pump mechanism located in the cell membrane. This active H+ extrusion mechanism results in a much lower intracellular [H+] than expected on the basis of a Nernst-type passive equilibrium distribution. This study cannot distinguish a passive H+ influxactive H+ efYlux from a passive HC03- efYlux-active HCO,- influx as the underlying pump-leak mechanism in operation. IntraceZZuZar pH. Intracellular pH has been mostly determined by indirect chemical methods (3, 22) involving the distribution of a weak acid (DMO) across cell membranes. The as yet insurmountable problems of adequate insulation of pH glass microelectrodes (20) have prevented reliable intracellular pH measurements by the direct electrometric technique. Accordingly, we have resorted to the approach of calculating the pH of the aqueous cytoplasmic phase from the in situ electrometric [HCO,J in cell water and from the CO, tension of the same intracellular phase. Thus, we use intracellular [HCOJ as an indicator of the intracellular pH of cells. It is certainly safer to assume a homogeneous cytoplasmic water phase with regard to HCO,- ion concentration than with regard to H+ ion concentration. Electrochemical equilibrium of the Nernst type is not a factor in the regulation of intracellular pH. Cell pH seems to be determined mainly by two factors: 1) the intracellular HCOJH,COL buffer system, and 2) an active H+ extruding pump (similar to the Na+ pump). Other factors that control intracellular pH include such buffer systems as the proteinates and the phosphates as well as the rate of production of organic acids. This active H+ pump, by excreting H+ ions, maintains a cytusol pH at or near the neutral level. Although H+ does not exhibit an electrochemical equilibrium distribution across the cell membrane under the various conditions of this study, some pH dependence of the resting membrane potential should be noted. The E, was decreased in alkalosis, the only state in which there was an increase in intracellular pH. Perhaps membrane conductance increases in alkalosis. Under control conditions the muscle cell pH was at the neutral level (7.03 t 0.03). Cells that contain no carbonic anhydrase like muscle have a lower intracellular
pH than cells containing cytoplasmic carbonic anhydrase, e.g., kidney cell (21). With K+ deficiency a mild extracellular alkalosis (0.05 pH U increase) was accompanied by a severe intracellular acidosis (0.25 pH U decrease)+ In contrast, in metabolic acidosis and alkalosis intracellular and extracellular fluids underwent pH changes in the same direction. In metabolic acidosis intracellular and extracellular pH decreased by 0.19 and 0.11 U, respectively. In metabolic alkalosis the intracellular and extracellular pH increased by 0.17 and 0.13 U, respectively. A comparison of the magnitude of changes of intracellular with extracellular pH for the same change in CO2 tension can be used to compare the intracellular buffer capacity with that of blood. In both acidosis and alkalosis, blood exhibited a smaller pH change, suggesting that blood has a larger buffer capacity than intracellular fluid. IntraceZZuZar buffer capacity. A buffer is a substance which minimizes pH change in a solution. The buffer capacity of a solution is an indication of its effectiveness in minimizing pH change. The HCO,--CO, buffer system has a volatile btier. The buffer capacity of a tissue is better measured in vivo than in vitro. The buffer capacity in vivo includes the contribution of the H+ pump, There are two measures of intracellular buffer capacity. First is the buffer capacity index which measures the ability to buffer C02. This CO2 buffer capacity is given by the ratio, p = -Alog PCOJApHi. For OUT recently reported (17) experiments on acute respiratory acidosis, we obtain a value of 5.6 over the PcoZ range of 40-146 mmHg. This value is in close agreement with a CO, buffer capacity of 5.1 reported by Clancy and Brown (4) in dog skeletal muscle and 5J reported by Heisler and Piiper (12) in rat diaphragm muscle. The intracellular CO, buffering capacity was the same as the blood buffering capacity for CO,. This is also borne out by the observation that with 20% CO, the fall of intracellular pH of 0.36 U is quite comparable to the blood pH fall of 0.34 u. The second index of intracellular buffer capacity is given by the ratio: A[HCO,-]JApHi. The units of this ratio are termed Slykes if the base is in millimoles per liter. This would yield for rat skeletal muscle in vivo an intracellular buffer capacity of 19.7 Slykes with respiratory acidosis (17), and 15.8 Slykes with metabolic acidosis. These buffer values obtained from direct electrometric determination of intracellular base concentration are in reasonable agreement with a buffer value of 20 meq/pH U obtained from indirect chemical methods on rat diaphragm (12) and dog skeletal muscle (4). Received
for publication
23 January
1975.
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