Journal of Clinical InvestigationVol. 46, No. 6, 1967

Measurement of Intracellular pH of Skeletal Muscle withpH-sensitive Glass Microelectrodes *

NORMANW. CARTER,t FLOYDC. RECTOR,JR., DAVID S. CAMPION,4 ANDDONALDW.SELDIN WITH THETECHNICALASSISTANCEOF ALLEN C. NUNNAND

WINNIE HOWARD(From the Department of Internal Medicine, The University of Texas Southwestern Medical

School at Dallas, Dallas, Texas)

Summary. We used three methods to examine the relationship among in-tracellular pH, transmembrane potential, and extracellular pH. Single-barreled electrodes permitted the determination of resting potential and in-tracellular pH with a minimum of cellular injury. Double-barreled elec-trodes, which incorporated a reference as well as a pH-sensitive electrode ina single tip, facilitated the direct measurement of intracellular pH withoutthe interposition of the transmembrane potential. Triple-barreled electrodespermitted measurement of intracellular pH during the controlled hyper-polarization or depolarization of the cell membrane.

The results of all three methods were in close agreement and disclosed thatthe H+ activity of intracellular and extracellular fluid is in electrochemicalequilibrium at any given transmembrane potential. This implies that thedeterminants of intracellular pH are the transmembrane potential and theblood pH. The actual pH of the normal resting muscle cell is 5.99, as esti-mated from the normal transmembrane potential and blood pH, or as deter-mined by direct measurements of intracellular pH.

Introduction

The measurement of intracellular pH (pH1) ofskeletal muscle has to a large extent involved in-direct techniques that depend on the differentialdistribution of weak acids and bases between in-tracellular and extracellular fluids. Conway andFearon (1), in early studies utilizing the CO2-HCO3- buffer system, found pH1 to be approxi-mately 6.0, with intracellular and extracellular hy-drogen ion activity (H+a) in Donnan equilibrium.Other investigators (2-15), however, using either

* Submitted for publication August 19, 1965; acceptedFebruary 17, 1967.

Supported in part by grants 5 TI AM-5028 and 5 TIHE-5469 from the National Institutes of Health.

t Address requests for reprints to Dr. Norman W.Carter, Dept. of Internal Medicine, The University ofTexas Southwestern Medical School at Dallas, Dallas,Texas 75235.

t:This investigation was supported in part by U. S.Public Health Service international postdoctoral researchfellowship 2 F05-TW-861-02.

the CO2-HCO3- system or the weak acid, 5,5-di-methyl-2,4-oxazolidinedione (DMO), have foundmuch higher values for pHi, ranging from 6.9 to7.1.

Attempts to measure pH, directly with pH-sensitive glass microelectrodes have been ham-pered by the technical difficulties in manufacturingsuitable electrodes. Two separate studies on pH1of skeletal muscle, however, have been published.Caldwell (13, 14), using relatively large micro-electrodes with tip diameters of 50 to 100 ,

found that pHi in crab skeletal muscle was ap-proximately 7.0 and more or less independent ofextracellular pH and transmembrane potentials(Em). Kostyuk and Sorokina (15), utilizingsmaller electrodes (tip diameters of less than1.0 ,u that were insulated either by shellac or byan outer shell of pH-insensitive glass, found pH,in frog skeletal muscle also to be in the range of7.1.

In preliminary studies in this laboratory, pH1

920

INTRACELLULAR PH OF SKELETAL MUSCLE

in rat skeletal muscle, measured with pH-sensitivemicroelectrodes, was found to be different fromthat obtained by Caldwell (14) and Kostyuk andSorokina (15). Therefore, to determine pHi andits relation to Em more precisely, we devisedthree methods. First, double-barreled electrodeswere constructed, consisting of an integral pH-sensitive and reference electrode with a combinedtip diameter less than 1 jA and insulation down tothe terminal 5 to 20 ju of the electrode tip. Thiselectrode had the great advantage of obviatingthe effects of interposed Em on pH, measure-ments, since both electrodes were in the cell cyto-plasm, and of permitting the simultaneous mea-surement of Em. Second, single-barreled pH-sensitive microelectrodes were constructed withtip diameters less than 0.5 /L. The extremely smalltip size of these electrodes minimized disruptionof the cell membrane in the course of puncture.Finally, triple-barreled electrodes were constructed,consisting of pH-sensitive and reference elec-trodes, as well as a third electrode through whichcurrent could be passed. The combined tip diam-eter was approximately 1 ,u. This method pro-vided a means of changing Em experimentallywhile simultaneously measuring pHi and Em. Inall of these methods the electrodes were insulatedwith a special glaze, which greatly decreased in-sulation leaks at the junction of the cell membraneand electrode. In addition, the effects of inade-quate electrode insulation were specifically in-vestigated

The results obtained with all three methodswere in complete agreement and indicated thatH+a of intracellular and extracellular fluid was inelectrochemical equilibrium at all levels of Em.In the normal resting skeletal muscle fiber theEmwas - 89 mv and the pHi approximately 6.0.These results are at great variance with those ob-tained by others utilizing either direct or indirecttechniques. Only with inadequately insulatedelectrodes were we able to duplicate the resultsobtained by Caldwell (14) and Kostyuk and Soro-kina (15).

Methods

Intracellular pH of skeletal muscle was measured inSprague-Dawley rats weighing between 250 and 300 g.Before study, the rats were maintained on a standardlaboratory chow diet and tap water ad libitum. The ratswere anesthetized by an intraperitoneal injection of so-

dium pentobarbital; and the thigh muscles were exposedby removing the skin and subcutaneous tissues of onehind leg. Fascia was carefully dissected from the surfaceof these muscles and care was taken to disturb musclefibers as little as possible. The muscle surface was con-tinuously perfused with castor oil, preheated to main-tain the temperature of the muscle at 370 C and to mini-mize loss of C02. Mineral oil was found to be entirelyunsatisfactory for use with these micro pH electrodes.The siliconized surface of the electrodes avidly holdsmineral oil and precludes any measurement of pH as aresult of very high impedance of the layer of mineral oil.This does not occur with silicone oils or castor oil. Inearly studies, however, it was found that silicone oileventually made micropuncture of muscle fibers diffi-cult because of progressive hardening of the cell mem-brane. For this reason, castor oil was used.

Construction and testing of pH-sensitive microelec-trodes. In manufacturing either single-, double-, ortriple-barreled electrodes the pH-sensitive barrel mustbe adequately insulated so that only the portion of tipthat is inside the muscle cell during puncture is sensi-tive to pH. In addition, the transition from the insu-lated to the uninsulated part of the electrode must beextremely smooth so that a portion of the insulatedarea can also be inserted into the cell without undulydisrupting the cell membrane. This assures that there areno insulation leaks at the junction of the cell membraneand the electrode.

The principle of the insulation technique was to coatCorning no. 0150 pH-sensitive capillary glass with a com-patible glaze, Pemco no. TR-514-A,l which when heatedfluxed to the surface of the glass capillary and completelyblocked pH sensitivity. When this coated capillary washeated and pulled into a microelectrode, the insulatingglaze extended almost to the tip. However, a small por-tion of uninsulated glass was pulled from beneath theglaze as the glass was drawn into a long taper, so thatthe terminal 5 to 20 p of the electrode tip consisted ofuninsulated pH-sensitive glass.

The length of the pH-sensitive tip was a function ofthe following three variables: 1) diameter of the capil-lary tubing; 2) thickness of the glaze; and 3) length ofthe taper. The optimal dimension of the Corning no.0150 capillary was found to be 0.8 + 0.05 mmo.d. Theproper thickness of glaze was obtained by closing oneend of the capillary with soft paraffin and dipping in di-luted glaze (200 g of glaze diluted with 450 ml of distilledturpentine). The dipped capillaries were air dried. Thecapillaries were dipped in glaze and air dried a secondtime and then heated at 6000 C for 6 minutes. To pre-vent heat distortion, we sealed small copper wires intothe unglazed end of the capillary so that the capillariescould be suspended in a vertical position during the pe-riod of heating. The ideal length of taper was found tobe 9 to 11 mmfrom the beginning of the taper to theend of the tip.

1 The glaze was supplied in no. 34 oil from Pemco,Division of Glidden Co., Baltimore, Md.

921

CARTER, RECTOR, CAMPION, AND SELDIN

Single-barreled electrodes were pulled by heatingglazed capillaries in a Scientific Instruments pipettepuller. Those electrodes which on subsequent testingwere shown to have closed tips were selected for use.Double-barreled electrodes, one side pH-sensitive, theother serving as a reference electrode, were prepared bycementing a glazed pH capillary and a slightly largercapillary (1.0 ± 0.05 mmo.d.) of Corning no. 0129 leadglass together with epoxy resin. Triple-barreled elec-trodes, consisting of one pH side and two reference sides,were prepared in a similar fashion using two pieces ofthe Corning no. 0129 capillary. The double-barreled ortriple-barreled capillaries were then heated in the pipettepuller until the glass was soft. The double capillarythen was rotated 3600 and the triple capillary was ro-tated 180°; the pipette puller was then released, pullingthe components into either double- or triple-barreledtips. With lead glass capillary slightly larger in diameterthan the pH-sensitive glass, the tip of the reference sidepulled out slightly farther than the pH side. This re-sulted in the pH side being pulled closed while the ref-erence side remained open. All of the electrodes werethen filled with distilled water while heating undervacuum.

The process of pulling the electrodes altered the glassso that the tip resistances were very high (1011 ohms)and the pH sensitivity was poor. However, after theelectrodes were soaked in distilled water at 40 C forapproximately 1 week, the tip resistances fell to approxi-mately 109 ohms and pH sensitivity was regained.

The pH-sensitive side of the electrode was used withdistilled water as the internal reference solution. Al-though distilled water ordinarily has a very low conduc-tivity and hence is a poor reference solution, sufficientelectrolyte was leached from the pH glass within 1 to 2days to raise the conductivity of the water far abovethat of the glass, so that the water functioned as a per-fectly satisfactory reference solution.2 The referencesides of the double- and triple-barreled electrodes werefilled with 2.5 M KCl-0.5 M KNOa by threading smallpolyethylene tubing almost to the tip and displacing thewater. A 34 (B and S)-gauge Ag-AgCl electrode wasinserted into the single reference side of the double elec-trodes and into both reference sides of the triple elec-trodes and sealed with Silastic cement. Before use eachelectrode was siliconized by dipping in a 1: 4 dilutionof General Electric Dri-Film in toluene.

Either the single pH electrode or the pH side of thedouble- or triple-barreled electrodes was placed in aTeflon electrode holder filled with 2.5 M KCl-0.5 MKNOa; an Ag-AgCl electrode in contact with the elec-

2In preliminary tests electrodes filled with distilledwater were shown to have the same stability and pH sen-sitivity as did microelectrodes filled with more conven-tional reference solutions, such as buffered sodium ci-trate or 0.1 N HCl. However, the more conventionalreference solutions rapidly dissolved the electrode tips(within 8 to 12 hours). For this reason distilled waterwas used as the internal reference solution.

trolyte solution in the holder was connected with the inputof a Cary model 31 vibrating reed electrometer. The out-put of the electrometer was connected to a Leeds-North-rup recording potentiometer. When single-barreled elec-trodes were used, a Beckman calomel electrode served asthe reference. With both double- and triple-barreledelectrodes, one of the reference sides of the electrode wasconnected to the low-impedance side of the electrometer.

The micro pH electrodes were calibrated in standardbuffers at room temperature (potassium phthalate pH4.0, sodium-potassium phosphate pH 6.8, potassium phos-phate pH 7.0, sodium-phosphate pH 7.41, potassiumborate pH 10).3 In all electrodes used the relation be-tween electrode voltage and pH was linear over a rangefrom pH 4.0 to 10. Electrodes reading less than 50 mvper pH U were discarded. However, in a few instanceselectrodes with lower slopes were specifically selected forspecial purposes. The initial calibration and testing ofelectrodes were performed in buffers at room temperature.Once the electrodes were selected for use in measuringpH, they were then recalibrated in buffers maintained at370 C.

To make certain that variations in tip potential of thereference side due to differences in the ionic strength ofthe standard buffers and the cytoplasm of the cell wouldnot cause errors in pH measurement, we tested electrodesin a potassium phosphate buffer simulating the internalenvironment of the cell. This buffer had an osmolality of330 mOsmper kg, contained 200 mEq K+ per L, and hada pH of 6.92 at 37° C. All of the electrodes used readthe pH of this buffer within ± 0.02 pH U of 6.92. Inaddition, the effect of a variety of solutions (buffers pH4.0, 6.8, and 7.0; 0.15 M NaCl; 0.15 M KCl; and musclehomogenate) on the tip potential of the reference side ofthe electrode was evaluated by measuring the voltage be-tween the reference side and a Beckman calomel elec-trode. Those electrodes in which the tip potential of thereference side varied by more than 5 mv among thevarious solutions were discarded.

Initially, we were unsuccessful in developing an invitro method of testing the length of the pH-sensitivearea of the microelectrodes. For this reason, we as-certained the adequacy of the insulation of electrodesused in the early experiments presented in this paper byusing the rat renal tubule as previously described (16).When testing the single-barreled electrodes we placedthe reference calomel electrode in a small beaker ofsaline into which the clipped end of the rat's tail was in-serted; the pH sides of the double-and triple-barreledelectrodes were read against their integral referenceelectrodes. Three small cups containing pH buffer stand-ards (pH 4.0, 6.8, and 7.4) in 3% agar were placed inthe peritoneal cavity of the rat; each cup was in elec-trical contact with the peritoneal surface through anopening in the bottom of the cup. We standardized thepH electrodes by reading the voltage in each of thesebuffers.

After calibration of the electrode, a surface tubule of

3 Beckman Instruments, South Pasadena, Calif.

922

INTRACELLULARPH OF SKELETAL MUSCLE

the kidney was punctured by a double-barreled injectionpipette. One side of this pipette was filled with siliconeoil and the other with an isotonic buffer solution. Thetubule was filled with oil and then punctured with the pHelectrode. The oil drop was then split and the buffersolution (pH 6.8 or 7.4) was perfused past the elec-trode tip; the occasional electrode that did not readwithin 0.1 of the known pH of the buffer was discarded.As a final test of the electrode insulation, a second buf-fer (pH 6.0, potassium phosphate), differing in pH fromthe intratubular buffer, was layered over the surface ofthe kidney; if the procedure caused a permanent shift inthe reading greater than 0.1 pH U, the electrode wasdiscarded.

Recently, we have been successful in developing a satis-factory in vitro method of testing the length of the pH-sensitive area of the microelectrodes. A variety of diffi-culties had to be overcome in developing the in vitro testsystem. For example, it was found that with bufferssolidified with 3%o agar into which the microelectrodescould be inserted a measured distance was entirely un-satisfactory, owing to the fact that when a buffer of adifferent pH was overlaid on the surface of the agarbuffer, the new buffer rapidly seeped down the side of theelectrode and caused a shift in the pH reading thateventually approached the value of the overlaid buffer.We tried several different membranes to obviate theseepage of buffer along the surface of the electrode, in-cluding cellophane, Mylar, silicone rubber, Teflon, andparaffin. All proved to be too tough to puncture withthe fragile microelectrodes.

It was possible, however, to make very thin latex mem-branes that prevented the seepage of buffer and could beeasily punctured by the microelectrodes. These mem-branes were made by placing a very small drop of undi-luted latex injection compound 4 over a 3-mm hole in a 2-X 2-cm square of Parafilm. When dried, these mem-branes were approximately 1.0 As thick. The test systemwas prepared by placing warm pH 6.0 buffer (potas-sium phosphate) containing 3% agar into a glass chamberthat contained an Ag-AgCl reference electrode. Afterthe buffer cooled, one of the latex test membranes wasplaced over the surface of hardened buffer.

The sequence for testing the microelectrodes was asfollows: Step 1. Under microscopic visualization the mi-croelectrode tip was advanced through the latex mem-brane into the agar buffer for a measured distance (5 to15 /u). The depth of penetration was measured with aneyepiece micrometer. The voltage in the pH 6.0 bufferwas read between the pH and reference sides in thedouble-barreled electrodes, and between the pH elec-trode and an Ag-AgCl reference electrode in the agarbuffer when single-barreled electrodes were used. Step 2.pH 7.4 buffer (potassium phosphate) was layered over thesurface of the latex membrane. If the voltage readingwas unaltered, the electrode was considered insulated forthat depth of penetration. However, if the voltagechanged, the electrode was considered inadequately insu-

4 General Biological Supply House, Chicago, Ill.

To Recorder ]-¢ To Qecorder

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FIG. 1. TECHNIQUEFOR SIMULTANEOUSMEASUREMENTOF INTRACELLULAR PH AND TRANSMEMBRANEPOTENTIAL

WITH DOUBLE-BARRELEDELECTRODES.

lated. If the electrode tested uninsulated, it was then pro-gressively advanced into the agar buffer until the correctreading for pH 6.0 buffer was obtained. The measure-ment of the depth of penetration with the eyepie-e mi-crometer gave the length of the uninsulated portion of theelectrode with an accuracy of ± 1.0 ,u. Step 3. The pos-sible effect of the muscle Emon the measurement of in-tracellular pH was simulated by imposing a 90-mv po-tential across the latex membrane by means of referenceelectrodes in the agar buffer and pH 7.4 buffer and a cali-brated potentiometer. With an insulated electrode, noeffect of the imposed membrane potential was seen in thepH electrometer reading.

Simultaneous measurement of pH, and Emwith double-barreled electrodes. Figure 1 depicts the circuit usedwith the double-barreled microelectrodes. By using twoCary model 31 electrometers with separate recorders itwas possible to record intracellular pH and transmem-brane potential simultaneously. A potentiometer wasplaced on the low impedance side of each electrometer sothat the tip potential of the reference side could be ad-justed to zero on the surface of the muscle and the voltagereading of the pH side could be adjusted to appropriatereadings in standard buffers. The transmembrane po-tential was determined by the voltage difference betweenthe reference side of the double electrode inside the muscleand a calomel half cell in electrical contact with the ex-tracellular fluid of the rat through the severed tail. Itwas found that a voltage of ± 500 mv applied between thereference side of the electrode and the calomel half celldid not affect the pH reading of the glass electrode inbuffer solutions. This test showed that there was no elec-trical feedback between the two electrometers and thatthe voltage difference between reference side of the elec-trode and a calomel half cell did not influence the po-tential between the reference and pH sides of the doubleelectrode.

Simultaneous measurement of pH, and Em while ex-perimentally varying Em with triple-barreled electrodes.In order to examine the relationship of intracellular pHand transmembrane potential over a wide range of mem-brane potentials, we used triple-barreled electrodes (onepH barrel and two reference barrels). The second ref-erence barrel was used to apply current across the muscle

923

CARTER, RECTOR, CAMPION, AND SELDIN

membrane in order to vary the transmembrane potential.Each triple-barreled electrode had to be carefully se-

lected and tested. It was necessary to find the maximalcurrent that could pass through the second referencewithout resulting in resistive coupling between the pHside and the first reference electrode. This was easily ac-

complished by placing the electrode in a buffer of knownpH together with two additional calomel reference elec-trodes, one of which was used to read the tip potentialof the first reference electrode and the other to completethe current circuit between the second reference electrodeand the solution. By passing current between the secondreference electrode and its calomel electrode and simul-taneously measuring the tip potential of the first refer-ence and the pH of the buffer solution, it was possible toincrease the current progressively until the tip potentialof the first reference electrode and the pH reading be-gan to vary. This was defined as the maximal toleratedcurrent. During the in vivo experiments, the currentapplied to the second reference electrode was always keptwell below the level that had been shown to influence theother two sides of the electrode. In general, the maxi-mal tolerated current for most triple-barreled electrodeswas in the order of 5 X 10' amp.

Measurement of Em in normal resting skeletal muscle.Since the simultaneously measured Em with both thedouble- and triple-barreled pH electrodes frequentlyyielded varying degrees of membrane depolarization, theprecise Em of normal resting skeletal muscle was mea-

sured with single-barreled Ling-type microelectrodes(17). These were pulled from borosilicate glass havingan outside diameter of 0.9 mmand were filled with 2.5 MKCI-0.5 M KNO3. Electrodes were selected which hadtip resistances between 10 and 50 megohms.

In all experiments, arterial blood pH was determinedwith a Beckman anaerobic electrode at 370 C and a Vibronor Beckman expanded scale pH meter.

Results

Testing of electrodes. When the double-bar-reled electrodes were tested in various standardbuffers, the relation between pH and the voltage

reading was linear over a range from pH 4.0 to10.0. The calibration curve for each electrode was

highly reproducible during the course of an ex-

periment and from day to day.The results of the in vitro test for adequacy of

insulation are shown in Table I for typical insu-lated and inadequately insulated electrodes. Thevoltage readings in standard buffers pH 6.0 and7.4 were obtained first. The electrode was thenadvanced through the latex membrane into theagar buffer, pH 6.0, to a depth of 10 p. The volt-age reading in the agar buffer was always ap-

proximately the same as that obtained in the stand-ard pH 6.0 buffer. While the tip was still in theagar buffer, the latex membrane was overlaid withpH 7.4 buffer. This had no effect on the voltagereading of the insulated electrode, which continuedto read pH 6.0. With the inadequately insulatedelectrode the voltage shifted to 86 mv, indicatingan apparent pH of 7.24. The next step in thetest was to impose a potential difference of 90 mvacross the latex membrane with the agar bufferbeing negative relative to the. superficial buffer.The imposition of the transmembrane potentialhad no effect on the voltage reading of the insu-lated electrode, but shifted the voltage of the in-adequately insulated electrode from + 86 to + 101mv. This caused a shift in the apparent pH from7.24 to 6.97. Inadequately insulated electrodes,therefore, are subject to two sources of error: 1)they are influenced by the pH of the fluid on bothsides of the membrane, and 2) they are partiallyaffected by the transmembrane potential. Onlyelectrodes with uninsulated tips less than 20 u

long were used for the measurement of pHi.Weexamined the ability of these well-insulated

TABLE I

In vitro test of double-barreled microelectrodes

Insulated electrode Uninsulated electrode

Apparent ApparentDepth of pH of agar Depth of pH of agar

penetration Electrometer reading buffer penetration Electrometer reading buffer

p my pU mvBuffer pH 6.0 +1571 +155B58 mv/pH U 755 mv/pH U

Buffer pH 7.4 + 76J + 78JAgar buffer pH 6.0 10 +155 6.04 42 +157 5.96Overlaid buffer pH 7.4 10 +155 6.04 42 + 86 7.24Transmembrane potential 10 +155 6.04 42 +101 6.97

of 90 mv imposed

924

INTRACELLULAR PH OF-SKELETAL MUSCLE

double-barreled microelectrodes to measure pHof biologic fluids by comparing the pH of bloodand muscle homogenates obtained with these elec-trodes with that obtained with Beckman macro-electrodes. As shown in Table II, the measure-ment of blood pH with the microelectrode agreedvery closely with that obtained with the Beckmanelectrode. The greatest discrepancy between thetwo readings was 0.16 pH U; the average differ-ence was 0.02. The results obtained in musclehomogenates are also listed in Table II. Again,the results obtained with the microelectrode closelyagreed with those obtained with the Beckmanelectrode. These results indicate that the micro-electrodes are nearly as accurate as the Beckmanelectrode in measuring the pH of biologic fluidsand that these electrodes are not subject to anyunique artifacts in the presence of proteins orother macromolecules.

Measurements with the double-barreled elec-trodes. Simultaneous measurements of blood pH,pHi, and Emof skeletal muscle were obtained in

TABLE II

Comparison of pH measurements of blood and muscle homog-enate made with doubl-barreled electrodes and with

Beckman glass electrodes

Double-barreled

Experi- micro- Beckmanment electrodes* electrode A pH

Blood1 7.37 7.37 0.002 7.33 7.38 -0.053 7.39 7.35 +0.044 7.50 7.45 +0.055 7.46 7.48 -0.026 7.76 7.60 +0.167 7.38 7.32 +0.068 7.39 7.47 -0.089 7.39 7.39 0.00

10 7.34 7.34 0.001 1 7.48 7.44 +0.0412 7.45 7.42 +0.0313 7.36 7.42 -0.0614 7.42 7.42 0.0015 7.44 7.44 0.0016 7.40 7.40 0.00

Mean +0.02 :1= 0.04

Muscle homogenates1 7.02 7.10 -0.082 7.15 7.14 +0.013 7.10 7.20 -0.104 6.97 7.02 -0.05

* Each value represents a single measurement obtainedwith a different double-barreled electrode.

5.5p-

cc 6.0-J

3 6.50

crc- 7.0z

a0

8 *

I iS/ : ~~~~~*I

75j0 10 20 30 40 50 60 70 80 90

TRANSMEMBRANEPOTENTIAL (-mV)

FIG. 2. RELATION BETWEENINTRACELLULAR PH (pH,)AND TRANSMEMBRANEPOTENTIAL (EM) AS MEASUREDWITH DOUBLE-BARRELEDELECTRODES. The solid line is cal-culated from the Nernst equation, Em= 61.5 (pH, -pHbl.od), assuming electrochemical equilibrium of H' inintracellular and extracellular fluid and a blood pH of7.40. The solid dots represent values obtained with elec-trodes tested in the renal tubule. The open circles rep-resent values obtained with electrodes tested in thein vitro system.

57 normal rats. The blood pH was 7.41 ± 0.06(SD). The pHi varied widely from 5.77 to 7.14,with an average of 6.48. The Em ranged from- 30 to - 92 mv.

In Figure 2, the relation between Emand pH1is plotted. The solid line represents the relationbetween pH1 and Em, estimated from the Nernstequation, assuming Ha in intracellular and extra-cellular fluid to be in thermodynamic equilibrium.The solid dots are results obtained in earlier ex-periments with electrodes tested in the renal tu-bule, whereas the open circles represent values ob-tained with electrodes whose insulation was checkedin the in vitro system. It is evident that the pointsscatter about the solid line, suggesting that H+a isin fact in electrochemical equilibrium.

The wide scatter in Emfrom - 30 to - 92 mvindicates that micropuncture with these micro-electrodes tends to cause an electrical leak, thusdepolarizing the membrane. If Ha were in equi-librium across the cell membrane, any artifactualchange in Emmight in itself alter pHi. The truepH1, therefore, can only be ascertained in thosemeasurements where Emwas normal. To deter-mine the normal Em of resting skeletal muscle,we next made measurements using Ling-type mi-croelectrodes. The average Em in 259 measure-ments in 12 rats was - 88.9 + 3.9 mv, a valuevery similar to that obtained by others (18, 19).

In view of the value of - 89 mv for the normal

925

CARTER, RECTOR, CAMPION, AND SELDIN

TABLE III

Intracellular pH and transmembrane potential of normal rat skeletal muscle measured with double-barreled electrodes

Membrane Intracellular pHBlood pH Electrode potential

Rat no. (pHb) no. (Em) Measured Calculated* Differencet

1 7.39

2 7.42

3 7.41

4 7.41

5 7.38

6 7.44

123

4

5

6

789

10

11

12

13

7 7.42 14

15

16

17

Mean 4 SD

mv869090909092

868885929089

898585

8887

899087

929090879090

909085909090909089878585

88.7 A 2.2

6.295.925.946.085.966.28

5.875.805.925.775.866.02

5.975.905.88

5.826.00

5.925.976.05

5.955.946.086.205.946.28

6.085.866.035.966.286.285.965.925.946.105.855.80

5.99 it 0.14

5.995.935.935.935.935.90

6.025.996.045.925.965.97

5.966.036.03

5.986.00

5.935.925.97

5.945.995.986.035.985.98

5.965.966.045.965.965.965.965.965.976.016.046.04

5.97 i: 0.04

+0.30-0.01+0.01+0.15+0.03+0.38-0.15-0.19-0.12-0.15-0.10+0.05

+0.01-0.13-0.15

-0.160.0

-0.01+0.05+0.08

+0.01-0.04+0.10+0.17-0.04+0.30

+0.12-0.10-0.01

0.00+0.32+0.32

0.00-0.04-0.03+0.09-0.19-0.24

+0.02 4- 0.15

* Calculated equilibrium intracellular pH = pHb -(Em/61.5).t Difference = measured pH - calculated pH.

Em, only those measurements of pH1 in which thesimultaneous Emwas between - 85 and - 93 mvwere used to establish the normal pH1. Thirty-eight measurements that were found to fit thesecriteria are listed in Table III. pH1 ranged from5.8 to 6.3, with an average of 5.99 ± 0.14 (SD).The calculated equilibrium pH, for the averageEmof - 88.9 mv and blood pH of 7.41 is 5.97.The close agreement between the observed pH1and the calculated equilibrium value strongly

suggests that H+ is in electrochemical equilibriumacross the muscle cell membrane.

Since these results are at such marked variancewith those of Caldwell (14) and Kostyuk andSorokina (15), the question of inadequate elec-trode insulation was specifically investigated. Onthe basis of the in vitro test, electrodes with pH-sensitive tips ranging from 5 to 80 ,u in lengthwere selected. The values of pH1 obtained withthese various electrodes were plotted against the

926

INTRACELLULAR PH OF SKELETAL MUSCLE

length of the pH-sensitive tip as shown in Figure3. Only those values of pH, in which the simul-taneous Emwas - 85 mv or greater are plotted.Those electrodes with pH-sensitive tips of 20 juor less gave pHi of approximately 5.9. Thoseelectrodes with pH-sensitive tips greater than 50 p,gave pHi of approximately 7.0. Those electrodeswith sensitive tip lengths between 20 and 50 u

gave intermediate pHi between 6.0 and 7.0. Themeasured value of pHi, therefore, is clearly afunction of the adequacy of electrode insulation.Those electrodes shown to have adequate insula-tion by in vitro testing yielded a pHi value of ap-proximately 5.9, whereas those shown to be inade-quately insulated yielded a value for pH1 moreclosely related to that reported by Caldwell (14)and Kostyuk and Sorokina (15).

Measurements with single-barreled electrodes.The low membrane potential obtained in many ofthe punctures with the double-barreled electrodesis undoubtedly the consequence of an electricalleak due to the comparatively large size of theelectrode tip (0.5 to 1.0 /L). To minimize thissource of error, we measured pHI separately withsingle-barreled electrodes whose tip size was mi-nute (0.1 is).

The experiments with the single-barreled pH-sensitive electrodes were specifically designed todetermine if H+a is in electrochemical equilibriumacross the muscle cell membrane. The potentialdifference between the -single-barreled pH elec-trode and the extracellular reference electrode wasmeasured first while both were in extracellularfluid and then after the microelectrode had beenadvanced into the muscle cell. If the microelec-trode had a theoretic slope of 61.5 mv per pH Uand if Ha was in electrochemical equilibrium,then there should have been no change in the po-tential reading between the two when the micro-electrode was advanced from the extracellular intothe intracellular fluid. However, the microelec-trodes used in these experiments had somewhatless than theoretic slope. This means that even ifH a was in electrochemical equilibrium, a smallpotential change would have occurred when themicroelectrode was advanced into the cell; thischange can be predicted from the electrode slopeand the normal resting Em of - 89 mv (TableIV, column 4). On the other hand, if H+a wasnot in electrochemical equilibrium and pH1 was

7.5

701L0._.

L 6.5

I 6.0

z 5.5

0

0 *

.

-0 0e * S

0

~0 10 20 30 40 50 60 70 80MICRONS

LENGTHOF pH SENSITIVE TIP(Determined in vitro)

FIG. 3. RELATIONSHIP OF MEASURED INTRACELLULAR

PH TO THE MEASUREDLENGTH OF THE PH-SENSITIVE TIPOF DOUBLE-BARRELEDELECTRODES. All measurements ofpH, were accompanied by simultaneously measured trans-membrane potentials of -85 mv or higher.

7.0 as reported by others, the observed voltagechange would have been much higher. This valuecan be calculated from an assumed pHi of 7.0, thenormal resting Emof - 89 mv, and the electrodeslope (Table IV, column 5).

The observed potential changes with eight dif-ferent electrodes are shown in Table IV, column3. In every instance the observed potential changeclosely approximated the predicted value for elec-trochemical equilibrium (Table IV, column 4)and was markedly different from the potentialchange predicted from a pH1 of 7.0 (Table IV,column 5).

One of the possible difficulties in single-barreledelectrode experiments, where potential change issmall, is determining whether or not the electrodetip is inside the cell. Weused two techniques toobviate this difficulty. First, the muscle surfacewas carefully observed microscopically during thepuncture procedure. Almost invariably the cellmembrane could be seen to dimple and then returnto its normal position as the electrode entered thecell. This type of microscopically observed punc-ture with the Ling-type microelectrode invariablygave a high sustained Em. The second means ofsurmounting this difficulty was to select pH-sen-sitive electrodes with a low slope and thus amplifythe predicted voltage change. As shown in TableIV, electrode number 4 had a slope of 45 mv andthe predicted voltage change was 22.3 mv. Whenthe muscle was punctured, sudden changes in thepotential occurred as the membrane was pene-trated, and the observed potential changes were

927

Ir-

CARTER, RECTOR, CAMPION, AND SELDIN

TABLE IV

Single-barreled electrode measurements

Electrode Calculated A Et Calculated AEtnumber Number of Measured A E* assuming Ha+ assuming non-

and slope measurements (mean 4 SD) equilibrium equilibrium of H.+

Insulated electrodes1 3 16.3 7.1 66.4

56.5 mv Range 5-252 10 14.2 16.1 68.9

50.3 mv i 3.23 10 3.7 4.9 65.8

58.0 mv + 4.74 11 17.7 23.7 71.0

45.0 mv i 4.15 6 7.7 13.6 68.2

52.0 mv :1: 3.96 3 8.8 8.5 66.8

55.5 mv Range 7.5-107 7 18.9 18.0 69.4

49.0 mv i 5.18 8 1.5 0.5 64.6

61.0 mv =1: 1.2

Poorly insulated electrodes9 8 68.6 25.2 71.4

44 mv i 2.510 9 82.7 16.5 69.0

50 mv 4.9

* A E is the difference between the potential of the single-barreled electrode in extracellular fluid (ECF) and thatmeasured within the muscle fiber.

t A E = 89 mv - (electrode slope X 1.45) where 89 mv is the assumed Emand 1.45 is equivalent pH units at 370 C(89/61.5 = 1.45). Ha+ = intra- and extracellular hydrogen ion activity.

t A E = 89 mv - (electrode slope X 0.4) where 89 mv is the assumed Emand 0.4 is equal to the difference betweenpH of ECFand an assumed intracellular pH (pHi) of 7.0 (see text).

again equal to the value predicted for electro-chemical equilibrium.

The effect of inadequate electrode insulation wasalso tested in these experiments. The resultsfrom two inadequately insulated electrodes areshown at the bottom of Table IV. In contrast tothe results obtained with the insulated electrodes,there was a marked change in voltage when theinadequately insulated electrodes penetrated themuscle membrane. It is not immediately apparentwhy a poorly insulated electrode, which shouldbe reading predominantly pH of the extracellularfluid, should record such a large voltage changewhen the cell is punctured. Although the reasonis not completely clear, it was repeatedly demon-strated in the in vitro test system that inadequatelyinsulated electrodes read most of the transmem-brane potential (70 to 80 mv out of 90), eventhough the electrode was predominantly detectingthe pH of the overlaid buffer. Thus, the poorly

insulated electrodes behaved the same in the invitro test system as during muscle puncture. Ifthe voltage change obtained when puncturingmuscle with these electrodes is corrected for anEm of - 89 mv, the calculated pHi is approxi-mately 7.0. Thus, the inadequately insulatedsingle electrodes gave results very similar to theinadequately insulated double-barreled electrodes.

Measurements with the triple-barreled elec-trodes. The experiments with double-barreledelectrodes summarized in Figure 2 suggested thatelectrochemical equilibrium of H a existed overa very wide range of Em. However, in these stud-ies Emfell because of an electrical leak incident tothe micropuncture. To determine whether, infact, thermodynamic equilibrium of Ha obtainedover a wide range of Emindependent of membraneinjury, we conducted experiments with triple-barreled electrodes through which a current couldbe passed to alter Em. After puncturing a single

928

INTRACELLULARPH OF SKELETAL MUSCLE

TABLE V

Effect of changing transmembrane potential on intracellular pH as determinedwith a triple-barreled electrode (study 3)

Initial Initial New New Theoretical TheoreticalpHi Em Current Em pHi cell pHi* electrode pHt

mu amp mu6.15 -73 0

10-8 -19 7.02 7.03 7.416.15 -73 0

6 X 10-9 -43 6.71 6.65 6.859 X 10-9 -23 7.13 7.00 7.36

6.15 -75 010-8 -27 7.06 6.94 7.2510-7 +15 7.69 7.62 8.24

6.22 -71 0-2 X 10-8 -149 4.74 4.94 4.41-4 X 10-8 -233 3.25 3.57 2.45

0 -47 6.64 6.61 6.78(Recovery)

* Calculated on the basis of equilibrium distribution of H+ across cell membrane.t Calculated on the basis that the change in Emcauses an electrical artifact in the pH electrode; the electrode slope

was used in these calculations (see text).

muscle fiber and obtaining a stable Em greaterthan - 70 mv, current was applied through thethird barrel of the electrode so as to either raiseor lower Em. In these experiments, care wastaken to select triple-barreled electrodes whosevoltage change per unit pH change was less thanthe theoretical value of 61.5 mv. This greatlyfacilitated the differentiation of electrical artifactsfrom true changes in pH,. For example, if Hisrapidly achieved electrochemical equilibrium, a61.5-mv change in Em would cause a 1.0-Uchange in pH,. If the pH electrode had a per-fect slope, an electrical artifact of 61.5 mv wouldalso read as a 1.0-U change in pH,. However, ifthe slope of the electrode were significantly lessthan the theoretical value, for example 50 mv perpH U, then an electrical artifact of 61.5 mv wouldbe read as a 1.2-U change in pH,.

The data from a representative triple-barreledelectrode experiment are presented in Table V.The initial Emwas - 73 mv and pH1 was 6.15.The cell membrane was both depolarized and hy-perpolarized by the passage of curtent through theelectrode. Em was varied between + 15 and- 233 mv. When the membrane potential waschanged to a new value, the pH, shifted to a newsteady state almost instantaneously. The exactrate at which pH1 changed could not be ascertainedbecause the limiting response time of the high re-sistance electrodes and the recording system was

of the order of 10 to 15 seconds. The pHi rangedfrom 7.69 in the maximally depolarized state to3.25 in the maximally hyperpolarized state. Inevery instance, the measured pH1 was close to theequilibrium pHi calculated from the observed Emand was different from the value that would havebeen anticipated from an electrical artifact af-fecting the micro pH electrode. This can beseen more clearly in Figure 4, where the actualpHi falls closer to the line predicted from the Em

3.0

:t 4.0

IL

7i5.0

oV 6.0

4 7.0

+40 0 40 60 120 160 200 240 260Tr'ansmembv'ane Potenrdiat (-PnV)

FIG. 4. EFFECT OF CHANGINGTRANSMEMBRANEPOTEN-TIAL ON INTRACELLULAR PH AS DETERMINED WITHTRIPLE-BARRELED ELECTRODES. The solid line is calcu-lated on the basis of electrochemical equilibrium acrossthe cell membrane, which would give a slope of 1 pH Uper 61.5-mv change. The dotted line is calculated on thebasis of the electrode slope (see text). The electrodeused in this study had a slope of 43 mv per pH U.

929

I.1

.1.0

11.,

Ifee 0

.0.1

El ec I r-o act,& --./ .0

Scope rane

Scope

.1

... t 2 . I

CARTER, RECTOR, CAMPION, AND SELDIN

3.0O

-34.0_

50

6.0

z 70_ /

8.0+40 0 40 80 120 160 200 240 280Tranrsrnembr'ane Polenrtat (-mV)

FIG. 5. RELATIONSHIP BETWEEN INTRACELLULAR PHAND TRANSMEMBRANEPOTENTIAL WHENEM IS EXPERI-

MENTALLY VARIED WITH TRIPLE-BARRELED ELECTRODES.Data from five experiments are plotted, each with a dif-ferent symbol.

than to the line predicted from the electrical char-acteristics of the micro pH electrode. This rela-tion suggests that the observed changes in pH1are true changes in pH rather than electrode arti-facts. The data from five similar experiments are

plotted in Figure 5. The solid line representstheoretical pH, at each Em, assuming that HWa inintracellular and extracellular fluid is in electro-chemical equilibrium and assuming an extracellularpH of 7.4. The experimental points are closelyclustered along the theoretical line. From thesedata we concluded that HW is in rapid electro-chemical equilibrium across the muscle cell mem-

brane and that, consequently, the pH, is deter-mined by both the Emand the pH of extracellularfluid.

Discussion

The results of our studies support Conway's hy-pothesis that in resting skeletal muscle intracellu-lar Ha is in electrochemical equilibrium with ex-

tracellular HW (20). Moreover, variations in Emproduce predictable changes in pH,. It follows,therefore, that with the knowledge of blood pHand Em, pHi of muscle can be calculated. In a

separate series of experiments utilizing Ling-type electrodes the resting Em of normal ratmuscle was found to be - 89 mv. With this valueof Emand a blood pH of 7.4, the calculated pH1would be 5.97. Direct measurement of pH, ob-tained with double-barreled electrodes in whichthe membrane was not significantly depolarized

(as indicated by Embetween - 85 and - 93 mv,Table III) disclosed an average pHi of 5.99 ±0.14, which is in excellent agreement with thevalue of 5.97 predicted for thermodynamic equi-librium.

The value of 5.99 for the pH, of normal ratskeletal muscle obtained in the present studies isfar below the value of approximately 7.0 reportedin other studies that utilized either glass electrodesor the partition of weak acids or bases (21). Ina preliminary report from our laboratory, pH1was given as 6.79 (22). The validity of thoseearlier measurements, however, is questionable forthe following three reasons: first, the adequacy ofelectrode insulation could not be sufficiently estab-lished; second, the tip diameters were greaterthan 1.0 /%; and third, the simultaneous Emwasnot recorded.

It is important, therefore, to consider the pos-sible sources of error in our present method. Thefirst is a loss of selectivity of the micro pH elec-trode. Although the Corning 0150 glass has beenshown to be sensitive only to He when used withinthe physiologic pH range (23), it is possible thatsome selectivity is lost in the process of manu-facturing the microelectrodes. This seems un-likely, however, since it was shown that theseelectrodes accurately measured pH of buffer solu-tions despite wide variations in ionic composition.A second possibility is that the high protein con-tent of intracellular fluid in some way influencesthe behavior of the micro pH electrodes. Thisalso appears unlikely, since these electrodes gavethe same pH readings of blood and muscle ho-mogenates (Table II) as did Beckman macro pHelectrodes. A third possibility is that the elec-trodes were inadequately insulated. This can beexcluded by the fact that those electrodes whichwere shown to be adequately insulated by in vitrotesting gave a pH, of approximately 6.0, whereasonly those electrodes shown by the in vitro test tobe inadequately insulated gave higher values,which were similar to those reported by others. Afourth possibility is that there were electrical arti-facts due either to changes in the tip potential ofthe reference electrode or to an effect of Em onthe over-all behavior of the double-barreled elec-trode. We minimized errors in tip potentialby selecting electrodes with small tip potentialsthat remained constant in different test solutions

930

INTRACELLULAR PH OF SKELETAL MUSCLE

of varying ionic compositions. The fact that thedouble-barreled microelectrodes gave the samepH reading of blood and of muscle homogenatesas did Beckman macro pH electrodes is evidencethat the tip potential was not in any important wayaltered by biologic solutions. It is unlikely thatthe normal Em of skeletal muscle influences pHreadings obtained with the microelectrodes, sincea transmembrane potential of - 90 mv was dem-onstrated to have no effect on the behavior of well-insulated electrodes in the in vitro test system(Table I).

A final possibility is some type of artifact re-sulting from cellular injury. Micropuncture ofcells may cause two types of injury. First, thedisruption of the integrity of the cell membranemay give rise to an electrical leak, which lowersEm. If an electrical leak is the only disturbancecreated by the micropuncture, this would not nec-essarily alter pH1 unless the latter were, in partat least, determined by potential. If, in fact, itcould be shown that H+ were in electrochemicalequilibrium across the cell membrane, than a fallin Emas a result of an electrical leak would raisepH, but should not alter the relationship amongpH,, extracellular pH, and Em. The results withboth single- (Table IV) and double-barreledelectrodes (Figure 2, Table III) suggest thatH+a is in electrochemical equilibrium at all valuesof Em. This point was much more securely es-tablished by experiments with triple-barreledelectrodes in which the cell membrane was notonly depolarized but also hyperpolarized. Thiswas accomplished not as a result of fortuitouselectrical leaks incident to micropuncture, butrather by the controlled passage of current throughthe electrode (Figures 4 and 5, Table V). An in-duced variation in Em from + 30 to - 230 mvwas invariably followed by a rapid restoration ofelectrochemical equilibrium of He.

Micropuncture might cause a second form ofinjury resulting in intracellular acid-base changesthat could alter the relation between pHi andblood pH. Acute cellular injury might alkalinizethe cell by accelerating hydrolysis of creatine phos-phate (24) and by permitting seepage of extracel-lular fluid into the cell. On the other hand, in-jury might result in localized acidification aroundthe electrode tip, perhaps by increased productionof lactic acid. It is highly unlikely, however, that

in the presence of significant acid-base changesdue to cellular injury, pHi would respond in sucha rapid and sensitive way to alterations in Emaswas observed with the triple-barreled electrodes.Moreover, studies with single-barreled electrodesin which the minute tip diameter minimized cel-lular injury also indicated electrochemical equi-librium (Table IV).

In summary, none of these possible sources oferror account for the lower pH, obtained in ourstudies or for the fact that pH, is in electrochemi-cal equilibrium with pH of extracellular fluid.

The question may then be asked why other in-vestigators using either glass microelectrodes orbuffer partition techniques found a pH, of ap-proximately 7.0, which was not in electrochemicalequilibrium with extracellular pH (pHe). Inview of the fact that we obtained a pHi of ap-proximately 7.0 with electrodes proven to be in-adequately insulated by our in vitro test, it istempting to speculate that the electrodes used byboth Caldwell (14) and Kostyuk and Sorokina(15) were also uninsulated. However, their pub-lished results showing that the electrodes were notaffected by alterations in pHe (other than thosechanges produced by variations in Pco2) militatesomewhat against this conclusion.

In attempting to evaluate the difference in pH1obtained by others using buffer partition tech-niques and by us using glass microelectrodes, itis helpful to consider the nature of pHi. As dis-cussed by Caldwell (24), it is extremely unlikelythat there is any single homogeneous value forpH1 in a complex, highly organized structure suchas a skeletal muscle cell. First, the interior ofsubcellular particles (e.g., nuclei, mitochondria,microsomes) might have a pH entirely differentfrom that of the cell cytoplasm due to metabolicprocesses, Donnan effects, or possible other fac-tors. Second, the cell cytoplasm itself, which con-sists of a high concentration of polyelectrolytes,most likely has no uniform pH. The pH in themicro-environment surrounding charged macro-molecules, which has been termed surface pH(pH8), may differ markedly from the pH of thebulk solution. According to Danielli (25), pH8is a function of the isoelectric point of the mole-cule, the ionic strength of the solution, and thepH of the bulk phase. If the isoelectric point ofthe polyelectrolytes is higher than the bulk phase

931

CARTER, RECTOR, CAMPION, AND SELDIN

pH, pH. will be alkaline relative to the bulk solu-tion, whereas pH. will be relatively acid if theisoelectric point is lower than the bulk phase pH.Therefore, in a solution containing many differentpolyelectrolytes with differing isoelectric pointsthere will be a single value for the pH of theaqueous bulk phase but many different values forpH8.

The measured value for pHi will depend on themethod employed. For example, in protein solu-tions the pH obtained with the glass electrode maydiffer markedly, sometimes as much as 1 to 2 U,from that obtained with various acid-base indi-cators (25). Since the volume of water surround-ing polyelectrolytes is of molecular dimension,both macro and microglass electrodes measurebulk phase pH, whereas the acid-base indi-cators may measure some integrated pH, whichis perhaps disproportionately influenced by pH,.Acid-base indicators that are positively chargedin the ionized form will be attracted by Donnanforces to negatively charged polyelectrolytes andwill give a pH value more acid than the bulk phasepH; indicators that are negatively charged in theionized form (e.g., the CO2-HCO3- and DMOsystems) will be preferentially attracted to posi-tively charged polyelectrolytes and will give a pHmore alkaline than the bulk phase pH. Althoughthe bulk of muscle protein, consisting of actin andmyosin, has isoelectric points below 6.0 and wouldthus be negatively charged in a solution with abulk phase pH of 6.0, electrophoretic studies showthat a significant portion of the protein is positivelycharged (26). Whether the concentration ofthese basic proteins is sufficiently high to accountfor the difference in the bulk phase pH of 6.0 mea-sured with our glass electrodes and the pH 7.0 ob-tained with CO2-HCO3- and DMOmethods isnot known. However, the fact that some of thesepositively charged proteins that migrate towardsthe cathode are extremely basic, having isoelectricpoints as high as 11.0 (e.g., histones), makes itlikely that the intracellular pH measured withnegatively charged acid-base indicators would besignificantly more alkaline than either the bulkphase pH or the average pH8.5

5In a complex system of polyelectrolytes acid-baseindicators such as COG-HCO8- and DMOdo not mea-sure either the bulk phase pH or the average cell pH.

It is reasonable to assume, therefore, that in thecell cytoplasm each different protein has a differ-ent pH, depending upon its isoelectric point, andthat each pH5 is in Donnan equilibrium with thesingle bulk phase pH. The glass microelectrodesmeasure only the bulk phase pH, whereas acid-base indicators, such as the C02-HC03- systemor DMO, measure neither the bulk phase pH northe average pH,. Although the glass electrodemeasures only the bulk phase pH rather than theaverage or integrated cell pH, the question as towhether or not H+ ions are in electrochemicalequilibrium across the cell membrane can be an-swered only by knowing the H+,, in the bulk phaseand not on the basis of any average H+W through-out the cell cytoplasm. The results of our studiesindicate that the bulk phase pH, measured withthe microelectrodes, is indeed in electrochemicalequilibrium with extracellular pH, and that whenEmis altered there is a rapid change in measuredpH, with reestablishment of electrochemicalequilibrium.

The speed with which electrochemical equi-librium was restored after a transient disequi-librium produced by changing Em with triple-barreled electrodes was surprising and unexpected.In those studies the change in pHi after a changein Emwas almost instantaneous. Any delay ob-served between the change in Emand pHi couldby fully accounted for by the response time of themicro pH electrode and the recording equipment.This rapid change in pHi indicates that largeamounts of either H+ or OH- or HCO- ions canbe transferred across the muscle cell membrane ina very short time.

References

1. Conway, E. J., and P. J. Fearon. The acid-labileC02 in mammalian muscle and the pH of themuscle fiber. J. Physiol. (Lond.) 1944, 103, 274.

2. Fenn, W. 0. The carbon dioxide dissociation curveof nerve and muscle. Amer. J. Physiol. 1928, 85,207.

3. Stella, G. The combination of carbon dioxide withmuscle: its heat of neutralization and its dissocia-tion curve. J. Physiol. (Lond.) 1929, 68, 49.

However, as pointed out by Caldwell (24) and by Adler,Roy, and Relman (12), they do measure the averageHCO8- and OH- concentrations in the cell.

932

INTRACELLULAR PH OF SKELETAL MUSCLE

4. Wallace, W. M., and A. B. Hastings. The distri-bution of the bicarbonate ion in mammalian muscle.J. biol. Chem. 1942, 144, 637.

5. Gardner, L. I., E. A. MacLachlan, and H. Berman.Effect of potassium deficiency on carbon dioxide,cation, and phosphate content of muscle with anote on the carbon dioxide content of humanmuscle. J. gen. Physiol. 1952, 36, 153.

6. Eckel, R. E., A. W. Botschner, and D. H. Wood.The pH of K-deficient muscle. Amer. J. Physiol.1959, 196, 811.

7. Miller, R. B., I. Tyson, and A. S. Relman. pH ofisolated resting skeletal muscle and its relation topotassium content. Amer. J. Physiol. 1963, 204,1048.

8. Waddell, W. J., and T. C. Butler. Calculation of in-tracellular pH from the distribution of 5,5-di-methyl-2,4-oxazolidinedione (DMO). Applicationto skeletal muscle of the dog. J. clin. Invest. 1959,38, 720.

9. Irvine, R. 0. H., S. J. Saunders, M. D. Milne, andM. A. Crawford. Gradients of potassium and hy-drogen ion in potassium-deficient voluntary muscle.Clin. Sci. 1961, 20, 1.

10. Robin, E. D., R. J. Wilson, and P. A. Bromberg.Intracellular acid-base relations and intracellularbuffers. Ann. N. Y. Acad. Sci. 1961, 92, 539.

11. Bittar, E. E., M. F. Watt, V. R. Pateras, and A. E.Parrish. The pH of muscle in Laennec's cirrhosisand uremia. Clin. Sci. 1962, 23, 265.

12. Adler, S., A. Roy, and A. S. Relman. Intracellularacid-base regulation. I. The response of musclecells to changes in CO2 tension or extracellularbicarbonate concentration. J. clin. Invest. 1905,44, 8.

13. Caldwell, P. C. An investigation of the intracellularpH of crab muscle fibres by means of micro-glassand micro-tungsten electrodes. J. Physiol. (Lond.)1954, 126, 169.

14. Caldwell, P. C. Studies on the internal pH of largemuscle and nerve fibers. J. Physiol. (Lond.) 1958,142, 22.

15. Kostyuk, P. G., and Z. A. Sorokina. On the mecha-nism of hydrogen ion distribution between cellprotoplasm and the medium. Membrane Trans-port and Metabolism, Proceedings of a Symposium,Prague, 1960. A. Kleinzeller and A. Kotyk, Eds.New York, Academic Press, 1960, p. 193.

16. Rector, F. C., N. W. Carter, and D. W. Seldin.The mechanism of bicarbonate reabsorption in theproximal and distal tubules of the kidney. J. clin.Invest. 1965, 44, 278.

17. Ling, G., and R. W. Gerard. The normal membranepotential of frog sartorius fibers. J. cell comp.Physiol. 1949, 34, 383.

18. Adrian, R. H. The effect of internal and externalpotassium concentration on the membrane poten-tial of frog muscle. J. Physiol. (Lond.) 1956, 133,631.

19. Pillat, B., 0. Kraupp, G. Giebisch, and H. Stormann.Die Abhangigkeit des elektrischen Ruhepotentialsdes isoliert durchstrbmten Sdugetiermuskels vonder extracellularen Kaliumkonzentration. PfluigersArch. ges. Physiol. 1958, 266, 459.

20. Conway, E. J. Nature and significance of concen-tration relations of potassium and sodium ions inskeletal muscle. Physiol. Rev. 1957, 37, 84.

21. Bittar, E. E. Cell pH. Washington, Butterworths,1964.

22. Carter, N. W. Direct measurement of intracellularpH with glass microelectrodes (abstract). Clin.Res. 1961, 9, 177.

23. Dole, M. The Glass Electrode: Methods, Applica-tions, and Theory. New York, John Wiley, 1941,p. 129.

24. Caldwell, P. C. Intracellular pH. Int. Rev. Cytol.1956, 5, 229.

25. Danielli, J. F. On the pH at the surface of ovalbu-min molecules, and the protein error with indi-cators. Biochem. J. 1941, 35, 470.

26. Jacob, J. J. C. The electrophoretic analysis of pro-tein extracts from striated rabbit muscle. Bio-chem. J. 1947, 41, 83.

933