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Intracellular H+mobility was estimated in the rabbit isolated ventricular myocyte by diffusing HCl into the cell from a patch pipette, while imaging pHiconfocally using intracellular ratiometric SNARF fluorescence. The delay for acid diffusion between two downstream regions ≈40 μm apart was reduced from ≈25 s to ≈6 s by replacing Hepes buffer in the extracellular superfusate with a 5 % CO2/HCO3−buffer system (at constant pHoof 7.40). Thus CO2/HCO3−(carbonic) buffer facilitates apparent H+imobility. The delay with carbonic buffer was increased again by adding acetazolamide (ATZ), a membrane permeant carbonic anhydrase (CA) inhibitor. Thus facilitation of apparent H+imobility by CO2/HCO3−relies on the activity of intracellular CA. By using a mathematical model of diffusion, the apparent intracellular H+equivalent diffusion coefficient (DHapp) in CO2/HCO3−‐buffered conditions was estimated to be 21.9 × 10−7cm2s−1, 5.8 times faster than in the absence of carbonic buffer. Facilitation of H+imobility is discussed in terms of an intracellular carbonic buffer shuttle, catalysed by intracellular CA. Turnover of this shuttle is postulated to be faster than that of the intrinsic buffer shuttle. By regulating the carbonic shuttle, CA regulates effective H+imobility which, in turn, regulates the spatiotemporal uniformity of pHi. This is postulated to be a major function of CA in heart.

The intrinsic mobility of intracellular H+ions was investigated by confocally imaging the longitudinal movement of acid inside rabbit ventricular myocytes loaded with the acetoxymethyl ester (AM) form of carboxy‐seminaphthorhodafluor‐1 (carboxy‐SNARF‐1). Acid was diffused into one end of the cell through a patch pipette filled with an isotonic KCl solution of pH 3.0. Intracellular H+mobility was low, acid taking 20‐30 s to move 40 μm down the cell. Inhibiting sarcolemmal Na+‐H+exchange with 1 mmamiloride had no effect on this time delay. Net H+imovement was associated with a longitudinal intracellular pH (pHi) gradient of up to 0.4 pH units. H+imovement could be modelled using the equations for diffusion, assuming an apparent diffusion coefficient for H+ions (DHapp) of 3.78 × 10−7cm2s−1, a value more than 300‐fold lower than the H+diffusion coefficient in a dilute, unbuffered solution. Measurement of the intracellular concentration of SNARF (≈400 μM) and its intracellular diffusion coefficient (0.9 × 10−7cm2s−1) indicated that the fluorophore itself exerted an insignificant effect (between 0.6 and 3.3 %) on the longitudinal movement of H+equivalents inside the cell. The longitudinal movement of intracellular H+is discussed in terms of a diffusive shuttling of H+equivalents on high capacity mobile buffers which comprise about half (≈11 mm) of the total intrinsic buffering capacity within the myocyte (the other half being fixed buffer sites on low mobility, intracellular proteins). Intrinsic H+imobility is consistent with an average diffusion coefficient for the intracellular mobile buffers (Dmob) of ≈9 × 10−7cm2s−1.

1A fall of intracellular pH (pHi) typically depresses cardiac contractility. Among the many mechanisms underlying this depression, an inhibitory effect of acidosis upon the sarcoplasmic reticulum (SR) Ca2+release channel has been predicted, but not so far demonstrated in the intact cardiac myocyte. In the present work, pHiwas manipulated experimentally while confocal imaging was used to record spontaneous ‘Ca2+sparks’ (local SR Ca2+release events) in rat isolated myocytes loaded with the fluorescent Ca2+indicator fluo‐3. In other experiments, whole cell (global) pHior [Ca2+]iwas measured by microfluorimetry (using, respectively, intracellular carboxy SNARF‐1 and indo‐1).2Reducing pHi(i) increased whole cell intracellular [Ca2+] transients induced either electrically or by addition of caffeine, whereas (ii) it decreased spontaneous Ca2+spark frequency. Conversely, raising pHiincreased spontaneous Ca2+spark frequency.3Blocking sarcolemmal Ca2+influx with 10 mmNi2+, or reducing external pH by 1.0 unit, had no effect on the pHi‐dependent changes in spontaneous Ca2+spark frequency.4Decreasing pHiover the range 7.78–7.20, decreased Ca2+spark frequency exponentially as a function of pHi, with frequency declining by ∼33 % for a 0.2 unit fall in pHi. In contrast, over the same pHirange, Ca2+spark amplitude was unaffected. Intracellular acidosis produced a slight slowing of Ca2+spark relaxation.5The results indicate that, in the intact myocyte, a reduced pHidecreases the probability of opening of the SR Ca2+release channel. This phenomenon may contribute to the negative inotropic effects of acidosis.

1. The influence of sarcolemmal Na+‐H+ exchange upon intracellular Na+ activity (aiNa), intracellular pH (pHi), extracellular surface pH (pHs) and tonic tension was investigated in sheep cardiac Purkinje fibres. Intracellular ion activities were measured with liquid sensor ion‐selective micro‐electrodes. A two‐micro‐electrode voltage‐clamp was also used to control membrane potential while simultaneously recording tonic tension. 2. Inhibition of the sarcolemmal Na+‐K+ pump by strophanthidin (10 mumol/l) produced a rise in aiNa, an increase in [Ca2+]i as evidenced by a rise in tonic tension, and a fall in pHi of 0.1‐0.3 units. The intracellular acidosis has been shown previously to be linked to the rise in [Ca2+]i (Vaughan‐Jones, Lederer & Eisner, 1983). 3. Amiloride (1‐2 mmol/l), an inhibitor of Na+‐H+ exchange, produced a small reversible decrease in pHi and aiNa. Both effects became more pronounced in strophanthidin‐exposed fibres. In addition, pHi decreased during application of strophanthidin and this decrease was reversibly inhibited by amiloride. It is concluded that sarcolemmal Na+‐H+ exchange is stimulated following inhibition of the Na+‐K+ pump. 4. In strophanthidin‐exposed fibres, a rise in [Ca2+]i resulted in an intracellular acidosis which could still be observed in the presence of amiloride (1 mmol/l). This suggests that the fall in pHi was not caused by a modulatory effect of [Ca2+]i on sarcolemmal Na+‐H+ exchange. 5. Tetrodotoxin (TTX) produced a small fall in aiNa (ca. 0.5 mmol/l) which was not augmented in the presence of strophanthidin. Furthermore, the effects on aiNa of TTX and amiloride were additive. Thus the influence of amiloride on aiNa does not involve blockade of voltage‐gated Na+ channels. 6. The stoicheiometry of Na+‐H+ exchange, estimated from the rates of change of pHi and aiNa in amiloride, appeared to be electroneutral (1:1). The stoicheiometry was unaffected by changes in pHi. 7. In strophanthidin‐exposed fibres (i.e. aiNa is elevated), the recovery of pHi from an intracellular acidosis (brought about by brief exposure to NH4Cl) was slowed greatly by amiloride (1‐2 mmol/l). The rise in aiNa that occurred during pHi recovery was also reduced by amiloride. It is concluded that Na+‐H+ exchange can be stimulated by a fall in pHi under conditions where aiNa is elevated. However, at a given pHi, its rate of recovery was slower in the presence than in the absence of strophanthidin.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The influence of repetitive activity upon intracellular pH (pHi), intracellular Na+ activity (aNA(i)) and contraction was examined in isolated sheep cardiac Purkinje fibres. Ion‐selective microelectrodes were used to measure intracellular Na+ and H+ ion activity. Twitch tension was elicited by field stimulation or by depolarizing pulses applied using a two‐microelectrode voltage clamp. Experiments were performed in HEPES‐buffered solution equilibrated either with air or 100% O2. 2. An increase in action potential frequency from a basal rate of 0.1 to 1‐4 Hz induced a reversible fall in pHi and a reversible rise in aNa(i). These effects reached a steady state 3‐10 min following an increase in stimulation frequency, and showed a linear dependence on frequency with a mean slope of 0.023 pH units Hz‐1 and 0.57 mmol l‐1 Hz‐1, respectively. The rise in total intracellular acid and aNa(i) associated with a single action potential was estimated as 5.3 mu equiv l‐1 of acid and 3.5 mu equiv l‐1 of Na+. 3. At action potential frequencies greater than 1 Hz, the rate‐dependent rise in aNa(i) was usually accompanied by a positive force staircase. 4. The fall in pHi following a rate increase also occurred when fibres were bathed in Tyrode solution equilibrated with 23 mM‐HCO3‐ plus nominally 5% CO2/95% O2. In these cases, however, the fall in pHi was halved in magnitude. 5. In fibres exposed to strophanthidin (0.5 microM), the rate‐dependent fall in pHi was doubled in magnitude and its time course was more variable than under drug‐free conditions. The rate‐dependent rise in aiNa was also usually larger in strophanthidin. 6. In order to examine the influence of the rate‐dependent acidosis on developed tension, the acidosis was reversed experimentally by adding 2 mmol l‐1 NH4Cl to the bathing solution. This produced a rise in pHi accompanied by a large increase in twitch tension. Such an effect of pHi upon tension was quantitatively similar to that observed in previous work on Purkinje fibres (Vaughan‐Jones, Eisner & Lederer, 1987). 7. It is concluded that the rate dependence of pHi will influence both the magnitude and the time course of an inotropic response to a change in heart rate.

1. Intracellular pH (pHi) and Na+ activity were recorded (ion‐selective microelectrodes) in guinea‐pig papillary muscle and the sheep cardiac Purkinje fibre while simultaneously recording twitch tension. The effects of intracellular acidosis and alkalosis upon contraction were investigated. 2. A fall of pHi produced by reducing pHo was associated with a fall of twitch tension. Similarly, a rise of pHi produced by raising pHo produced a rise of twitch tension. The time course of the changes in tension correlated with the time course of changes of pHi rather than pHo. These results are consistent with previous work showing that acidosis inhibits contraction and that the inhibition depends upon a fall of pHi. 3. Changes of pHi were produced while maintaining pHo constant at 7.4. Removal of NH4Cl or addition of sodium acetate (pHo 7.4) reduced pHi but this gave either an increase of tension (papillary muscle) or an initial fall followed by a subsequent recovery of tension (Purkinje fibre). The increase or recovery of tension occurred despite the fact that there was an intracellular acid load. Thus, reducing pHi at constant pHo can increase tension whereas reducing pHi at low pHo (6.4, see paragraph 2) inhibits tension. 4. The increase of recovery of tension during intracellular acidosis produced at a constant pHo (7.4) was associated with a rise of intracellular sodium activity (aiNa). Amiloride (1.5 mmol/l), an inhibitor of Na(+)‐H+ exchange, prevented the rise of aiNa during intracellular acidosis and also prevented the recovery of tension. It is concluded that the increase or recovery of tension at low pHi is secondary to a rise of aiNa caused by stimulation of Na(+)‐H+ exchange. A rise of aiNa will elevate Ca2+ via sarcolemmal Na(+)‐Ca2+ exchange and thus will elevate tension. 5. An intracellular acidosis produced by reducing pHo (6.4) does not elevate aiNa in the Purkinje fibre. In papillary muscle, aiNa rises but this occurs slowly and the rise is 50% smaller than that seen when the same intracellular acidosis is induced at normal pHo (7.4). The net depression of tension under these conditions thus correlates with the lack of a large rise of aiNa. 6. Knowing the quantitative dependence of tension upon both aiNa and pHi in the two tissues it is possible to predict the recovery of twitch tension during intracellular acidosis at constant pHo (7.4), using the changes of pHi and aiNa measured under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The dual‐emission pH‐sensitive fluoroprobe carboxy‐SNARF‐1 (carboxy‐seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady‐state pHi in cells bathed in a HEPES‐buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2‐HCO3(‐)‐buffered Tyrode solution (pH 7.4) had a more ‘normal’ pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre‐pulse) was in the range 7‐20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES‐buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM‐NH4Cl pre‐pulse) was inhibited by the Na(+)‐H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N‐methyl‐D‐glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES‐buffered Tyrode solution to one buffered with 5% CO2‐HCO3‐ resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2‐HCO3‐ was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2‐HCO3(‐)‐buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre‐pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4‐diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre‐treatment with 200 microM‐DIDS of a pre‐acidified cell in Na(+)‐free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM‐EIPA. We conclude, that in the presence of CO2‐HCO3‐, a Na(+)‐ and HCO3‐dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The effects of changing PCO2 extracellular pH (pHo) and HCO3‐ on intracellular pH (pHi) were studied in isolated neonatal rat type‐I carotid body cells using the pH‐sensitive fluoroprobe, carboxy‐SNARF‐1. 2. Simulated respiratory acidosis and alkalosis (i.e. changes in PCO2 at constant HCO3‐) led to rapid (half‐time t0.5 = 3 s) monotonic changes in pHi. The relationship between pHi and pHo under these conditions was linear, steep (0.63 pHi/pHo) and remarkably similar to the response predicted from a passive cell model (i.e. a cell lacking pHi regulation). 3. In order to model the above pHi changes (point 2), it was necessary to determine beta i (intrinsic intracellular buffering power). By using small incremental acid loads in the cell (progressive [NH4+]o removal), beta i was determined as a function of pHi to be: beta i = 127.6‐16.04 pHi. 4. Changes in PCO2 at constant pHo (i.e. simultaneously changing HCO3‐) caused rapid transient changes in pHi but did not significantly affect steady‐state pHi over the range 1‐10% CO2. 5. When PCO2 was held constant (5%), changing HCO3‐ and thus pHo (i.e. a simulated metabolic acidosis/alkalosis) led to much slower changes in pHi (t0.5 approximately 1 min). Steady‐state pHi showed an almost identical dependence on pHo (slope 0.68) to that found for simulated respiratory acidosis/alkalosis. Therefore, over the range of pHo, PCO2 and [HCO3‐]o tested, steady‐state pHi appeared to be a unique function of pHo and independent of PCO2 and [HCO3‐]o. 6. The effects on pHi of respiratory acidosis, metabolic acidosis and increases of PCO2 at constant pHo (present work) were compared with previously published work on the ability of similar manoeuvres to increase the carotid sinus nerve (CSN) discharge rate. The two sets of data showed several striking similarities: (i) in both cases, the response to a respiratory acidosis was rapid in onset, maintained and reversible; (ii) in both cases, the speed of response to a metabolic acidosis was significantly slower than in (i) but, again, it was maintained and reversible; (iii) in both cases, increases in PCO2 at constant pHo elicited a rapid response but one which was only transient with no change in the steady‐state value. 7. The close correlation between the effects of changing pHo, PCO2 and [HCO3‐]o on pHi and on CSN discharge suggests that a change in type‐I cell pHi is the first step in the chemoreception of blood pH by the carotid body.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular pH (pHi) was recorded in single, isolated guinea‐pig ventricular myocytes using the pH‐sensitive fluorophore, carboxy‐SNARF‐1 (AM‐loaded). 2. The dual acid extrusion system in this cell (Na(+)‐H+ antiport and Na(+)‐HCO3‐ symport) was activated by inducing an intracellular acid load, produced by addition and subsequent removal of extracellular 10 mM NH4Cl. Under these conditions, it is known that both acid‐equivalent extruders are activated about equally. 3. Application of phenylephrine (100 microM; alpha‐adrenergic agonist) resulted in an inhibition of pHi recovery from an acid load, recorded in HCO3‐buffered medium containing 1.5 mM amiloride (amiloride inhibits Na(+)‐H+ antiport; under these conditions pHi recovery is mediated through only the Na(+)‐HCO3‐ symport carrier). This inhibitory effect of phenylephrine was prevented by the alpha 1‐antagonist, prazosin (0.1 microM) and was unaffected by propranolol (1 microM). 4. Application of phenylephrine in Hepes‐buffered medium (only Na(+)‐H+ antiport is active under these conditions) elicited a stimulation of pHi recovery, again prevented by prazosin (0.1 microM). 5. These results point to an alpha 1 inhibition of Na(+)‐HCO3‐ symport and an alpha 1 stimulation of Na+‐H+ antiport. 6. Both adrenaline (1‐5 microM) and noradrenaline (5 microM) slowed pHi recovery recorded in HCO3(‐)‐buffered solution containing amiloride (1.5 mM). The similarity of this result with that obtained previously using phenylephrine (paragraph 3) suggests that all three agonists inhibit the Na(+)‐HCO3‐ symport through alpha 1 activation. 7. Isoprenaline (1 microM; beta‐adrenergic agonist) slowed pHi recovery in Hepes‐buffered solution but stimulated recovery in a HCO3(‐)‐buffered solution containing amiloride (1.5 mM). These results suggest that beta activation slows Na(+)‐H+ antiport but stimulates Na(+)‐HCO3‐ symport. 8. When both acid‐equivalent extrusion carriers were inhibited in Na(+)‐free, HCO3(‐)‐buffered medium, phenylephrine or isoprenaline had no effect on pHi, ruling out any effect of the adrenergic agonists on background acid‐loading mechanisms. 9. Under physiological conditions (CO2/HCO3(‐)‐buffered solution, no amiloride), when both acid extruders would be activated by an intracellular acid load, application of phenylephrine, adrenaline or noradrenaline were found to slow pHi recovery. In contrast, isoprenaline stimulated pHi recovery under the same conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular Na activity, aiNa, was measured in voltage‐clamped sheep cardiac Purkinke fibres. 2. Increasing [Rb]0 from 0 to 4 mM in K‐free solutions (at a fixed membrane potential) decreased aiNa. Further increases of [Rb]0 (up to 20 mM) had little or no effect. 3. Following exposure to Rb‐free, K‐free solution, the addition of a test concentration of Rb produced an exponential decrease of aiNa. The rate constant of decay of aiNa increased with increasing [Rb]0 over the measured range (0‐20 mM). 4. The accompanying electrogenic Na pump current transient decayed with the same rate constant as aiNa over the range of [Rb]0 examined. During this decay the electrogenic Na pump current was a linear function of aiNa. Increasing [Rb]0 increased the steepness of the dependence of the electrogenic current on aiNa. 5. A constant fraction of the net Na efflux was electrogenic. This fraction was not affected by varying [Rb]0 over the range 0‐20 mM. 6. Using a simple model, it is shown that the dependence of steady‐state aiNa on [Rb]0 is half‐saturated by less than 1 mM‐[Rb]0. The rate constant of decay of aiNa and the slope of the relationship between electrogenic Na pump current and aiNa are, however, better fitted with a lower affinity for Rb (K0.5 = 4 mM‐[Rb]0). 7. Depolarizing the membrane potential with the voltage clamp decreased aiNa; hyperpolarization increased it. These effects persisted in the presence of 10(‐5) M‐strophanthidin. An effect of membrane potential on the net passive Na influx can account for the observations. 8. The effects of membrane potential on the net passive Na influx were examined by measuring the maximum rate of rise of aiNa at different holding potentials after inhibiting the Na‐K pump in a K‐free, Rb‐free solution. Depolarization decreased the Na influx. 9. Using the constant field equation, the net passive Na influx was used to estimate the apparent Na permeability coefficient, PNa. This was between 0.8 x 10(‐8) and 1.5 x 10(‐8) cm sec‐1.

1. Intracellular Na activity (aiNa) was measured in sheep cardiac Purkinje fibres using a recessed‐tip Na+‐sensitive micro‐electrode. The membrane potentials was controlled with a two‐micro‐electrode voltage clamp. Tension was measured simultaneously. 2. Removing external K produced a rise of aiNa and both twitch and tonic tension. On adding 4‐10 mM‐[Rb]0 to reactivate the Na‐K pump aiNa and tension declined. An electrogenic Na pump current transient accompanied the fall of aiNa. 3. The half‐time of decay of the electrogenic Na pump current transient was similar to that of aiNa, (mean tNa0.5/tI0.5 = 0.97 +/‐ 0.03 (S.E.M.; n = 28)). Following the re‐activation of the Na‐K pump, the electrogenic Na pump current transient was linearly related to aiNa. 4. The duration of exposure to K‐free, Rb‐free solutions was varied to change the level of aiNa. On subsequently re‐activating the Na‐K pump with 10 mM‐[Rb]0, the ratio of the charge extruded to the total change of aiNa was constant. It is concluded that the fraction of Na extruded electrogenically is unaffected by changes of aiNa. About 26% of the total Na extrusion appeared as charge transfer. 5. The relationship between tonic tension and aiNa was usually different during Na‐K pump inhibition in a K‐free, Rb‐free solution compared with the relationship during Na‐K pump re‐activation. In general, a given aiNa was associated with a greater level of tonic tension during Na‐K pump inhibition compared with that during pump re‐activation. A similar hysteresis was often seen between twitch tension and aiNa.

Intracellular Na activity (aiNa) was measured with recessed‐tip, Na‐selective micro‐electrodes in voltage‐clamped sheep cardiac Purkinje fibres. Tension was measured simultaneously. aiNa was increased reversibly either by exposing the preparation to K‐free, Rb‐free solution of by adding the cardioactive steroid strophanthidin. An increase of aiNa produced an increase of tonic tension which was larger at depolarized membrane potentials. At sufficiently negative membrane potentials, changes of aiNa (over the range 6‐30 mM) had no effect on tonic tension. Therefore, both an increase of aiNa and a depolarization are required to increase tonic tension. It is concluded that either a low level of aiNa or a large negative membrane potential is sufficient to maintain a low intracellular Ca concentration. Tonic tension was measured as a function of aiNa. At a given membrane potential the relationship can be described empirically by an equation of the form: tonic tension = b(aiNa)y, where y is a constant and b depends on membrane potential. In five experiments y was found to be 3.7 +/‐ 0.7 (mean +/‐ S.E.M.) over a range of potentials from ‐60 to ‐10 mV. Tonic tension was measured as a function of membrane potential. At a given aiNa the relationship can be described approximately as: tonic tension = k exp (aV), where a is a constant and k depends on aiNa. In five experiments a was found to be 0.06 +/‐ 0.01 mV‐1 (mean +/‐ S.E.M.). A depolarization of 10 mV increases tonic tension by the same amount as does an increase of aiNa that is equivalent to a 3.7 mV change of the Na equilibrium potential, ENa. Hence ENa is nearly 3 times more effective than membrane potential in controlling tonic tension. During a prolonged depolarization (several minutes) the initial increase of tonic tension decays gradually. This is associated with a fall of aiNa. The relationship between tonic tension and aiNa is similar to that seen when aiNa is increased by inhibiting the Na pump. It is concluded that the fall of aiNa is responsible for the decay of tonic tension. The changes of tonic tension reported in this paper are consistent with the effects of aiNa and membrane potential on a voltage‐dependent Na‐Ca exchange. The possibility that a voltage‐dependent Ca channel contributes to tonic tension is also discussed.

1. Intracellular Na activity, aiNa, was continuously measured in crab (Carcinus maenas) muscle fibres using a recessed‐tip Na+ ‐sensitive glass micro‐electrode. Experiments could last up to several hours. AiNa remained stable during prolonged experiments. The mean resting aiNa was 8‐4 +/‐ 0‐02 mM (S.E. of mean for eighty‐nine fibres) and the mean resting membrane potential was 65‐3 mV +/‐ 0‐3 (S.E. of mean for eighty‐nine fibres). 2. Reducing [Na]o to 1/10 normal (maintaining ionic strength with equivalent amounts of either Li or Tris) caused a large and rapid fall of aiNa. There appeared to be two components of the effect, a fast and slow. The initial fast rate of decrease was about 3‐5 m‐mole/min decreasing to half this value in about 1 min. The rate of decrease of aiNa was not linearly related to aiNa. The size of the fast change of aiNa was related to the magnitude of the Na gradient across the membrane. 3. High concentrations (2 x 10‐4m) of ouabain caused a very slow rise of aiNa by 1 or 2 mn/hr. This was equivalent to a net Na influx of between 1 and 10 p‐mole/cmi. sec, depending on whether or not a correction was applied to account for the increased surface area of the fibre caused by the invaginating cleft system. 4. The response to low Nso was virtually insensitive to the removal of Ko or to prolonged reatment with high concentrations of ouabain (2 x 10‐4 m; 100 min) and so could not readily be attributed to active Na/K pumping. 5. The response of aiNa to low Nao was reversibly inhibited by high concencentrations of Mn (50 mm) and by low concentrations of La (3‐1 mm). La itself stimulated a rapid fall of aiNa in normal Nao.

1. Ion‐sensitive electrodes (made with a chloride‐sensitive ion‐exchange resin) were used to measure the internal chloride activity (aiCl) of frog sartorius fibres at 25° C.

Tension was measured in voltage clamped sheep cardiac Purkinje fibres while simultaneously measuring the intracellular Na activity (aiNa) with a recessed‐tip, Na‐selective micro‐electrode. Inhibiting the Na‐K pump either by exposing the preparation to a K‐free solution or by adding the cardioactive steroid strophanthidin increased both aiNa and twitch tension and resulted in the development of tonic tension, after‐contractions and a transient inward current (ITI). The increase of twitch tension was present at lower aiNa than that required to produce the other phenomena. The relationship between the magnitude of the twitch tension and aiNa was always non‐linear. Twitch tension increased steeply with aiNa at first but the relationship flattened off at higher aiNa and tension eventually decreased. Over the steep range, the relationship between tension and aiNa could be represented as: twitch tension = b (aiNa)y where y had a mean value of 3.2. Changing membrane potential or [Ca2+]o changed b but had little effect on y. Mn (2 mmol/l) greatly decreased twitch tension but, at least initially, had little effect on tonic tension. The steep relationship between twitch tension and aiNa was seen, irrespective of whether the Na‐K pump was inhibited either by exposure to K‐free solution or to strophanthidin and whether the relationship was measured either when aiNa was increasing or after it had reached a steady state. The steep dependence of twitch tension on aiNa observed in the present work means that manoeuvres which produce even small changes of aiNa will have significant effects on contraction.

Intracellular Cl activity (aiCl), and intracellular pH (pHi) were measured in isolated sheep cardiac Purkinje fibres using a liquid ion exchanger Cl‐selective micro‐electrode and a glass recessed‐tip, pH‐selective micro‐electrode. Removal of external Cl (glucuronate substituted) produced a fall in aiCl from about 20 to about 4 mmol/l: the residual level is probably caused by intracellular interference on the Cl‐sensitive electrode. Re‐exposure of the fibre to increased levels of external Cl produced, in the steady state, increased levels of aiCl. The dependence of steady‐state aiCl upon external Cl activity, aoCl, was roughly hyperbolic with 50% recovery occurring at an aoCl of about 9.5 mmol/l. At all levels of external Cl tested, Cl was accumulated to a level much higher than that predicted for passive electrochemical equilibrium. Exposure of a Cl‐depleted fibre to various levels of external Cl produced an exponential rise with time in aiCl. The initial rate‐of‐rise in aiCl was estimated to be a saturating function of aoCl, with a half‐maximal effect occurring at an aoCl of about 33 mmol/l. The rate‐of‐rise was about 10‐fold greater than that predicted from constant‐field theory using published values for PCl, the Cl permeability coefficient. Steady‐state aiCl was essentially insensitive to changes in external HCO3 concentration, [HCO3]o, if these changes were made at a constant external pH, pHo, i.e. when a reduction in [HCO3]o was accompanied by a simultaneous reduction in the partial pressure of CO2, PCO2. In contrast, if PCO2 was maintained constant, then a change in [HCO3]o (thus producing a change in pHo) resulted in an inverse change in aiCl. This change in aiCl was also accompanied by a change in pHi: when aiCl increased, pHi decreased and vice versa. The anion‐exchange inhibitor, DIDS (4,4‐diisothiocyanato‐stilbene disulphonic acid) abolished the effect on aiCl of changes in [HCO3]o and pHo (at constant PCO2). Furthermore DIDS reduced the influence of pHo upon pHi. Both the fall of aiCl in Cl‐free solution and the subsequent reuptake of Cl following re‐exposure to Cl‐containing solution were slowed by a reduction in [HCO3]o (constant pHo, reduced PCO2). Both reuptake and wash‐out of Cl were saturating functions of [HCO3]o with half‐maximal effect occurring at an [HCO3]o of 1‐1.3 mmol/l. The reuptake of Cl was little affected by removal of external Na (bis,2‐hydroxy ethyl, dimethyl ammonium substituted). The reuptake of Cl was unaffected by amiloride (1 mmol/l) but slowed by piretanide (1 mmol/l).(ABSTRACT TRUNCATED AT 400 WORDS)

1. The mechanism of the rate‐dependent decrease in intracellular pH (pHi) and its recovery were studied in isolated sheep cardiac Purkinje fibres. Intracellular Na+ activity (aiNa) and pHi were measured using ion‐selective microelectrodes. Twitches were elicited by field stimulation or by depolarizing pulses applied using a two‐microelectrode voltage clamp. 2. A 3 Hz train of short (50 ms) depolarizing voltage‐clamp pulses induced a reversible fall in pHi which was accompanied by a reversible increase in aiNa. A train of longer (200 ms) pulses also produced a fall in pHi which was now paralleled by a decrease in aiNa. These observations indicate that the rate‐dependent acidosis is not dependent upon a rise in aiNa. 3. Neither the fall in pHi nor the increase in aiNa seen upon an increase in action potential frequency was inhibited by amiloride (1 mmol l‐1) which indicates that Na+‐H+ exchange is not involved in the generation of the acidosis. Furthermore, the rate‐dependent acidosis was not abolished in Na+‐free solution (Li+ or N‐methyl glucamine substituted) indicating that other Na+‐requiring processes (such as Na+‐Ca2+ exchange) are not a necessary requirement. Rate‐dependent pHi changes were also unaffected by the stilbene compound DIDS indicating no participation by Cl‐‐HCO‐3 exchange. 4. The rate‐dependent acidosis was inhibited by the organic calcium antagonist D600 (20 mumol l‐1) which also inhibited twitch tension. This suggests that the acidosis is related to the activation by Ca2+ of developed tension. D600 also inhibited the rate‐dependent rise in aiNa (field stimulation). 5. The rate‐dependent acidosis was not inhibited by cyanide (2 mmol l‐1) but it was blocked by iodoacetate (0.5 mmol l‐1) and by 2‐deoxyglucose (DOG) (10 mmol l‐1, applied in glucose‐free solution). These results suggest that the acidosis is generated metabolically via stimulation of glycolysis, following an increase in contraction. Contributions from aerobic metabolism are likely to be small. 6. Twitch tension was inhibited by ryanodine (10 mumol l‐1) but the drug had little inhibitory effect on the rate‐dependent acidosis. A tonic component of tension was observed, however, in the presence of ryanodine. The lack of effect of ryanodine upon the rate‐induced acidosis is discussed. 7. The half‐time of pHi recovery from the frequency‐dependent acidosis was consistently shorter than that from an intracellular acid load induced by adding and then removing external NH4Cl (10 mmol l‐1).(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular pH was recorded (double‐barrelled pH‐selective microelectrodes) in single ventricular myocytes and whole papillary muscles isolated from guinea‐pig heart. Both preparations were acid‐loaded by various manoeuvres (addition and removal of external NH4Cl or CO2) in order that a comparison could be made of the size and speed of intracellular pH changes and hence of the apparent intracellular buffering power (beta). 2. For the same acid‐loading procedure, the size of intracellular pH (pHi) changes was about threefold larger in the isolated myocyte than in whole papillary muscle. The rate of initial acid loading as well as the subsequent rate of pHi recovery (caused by acid extrusion from the cell) were also threefold faster in the myocyte. Estimates of apparent intrinsic (non‐CO2) buffering power, based upon the size of pHi changes during acid loading, were 15‐20 mmol l‐1 for the myocyte and about 70 mmol l‐1 for whole muscle. This latter value is similar to previous estimates of beta in heart. 3. When acid extrusion was reduced by applying a high dose of amiloride (1 mmol l‐1), then the size of the pHi change during acid loading increased greatly in papillary muscle but changed much less in the myocyte; beta now appeared to be about 30 mmol l‐1 in whole muscle but remained essentially unchanged in the myocyte. 4. We conclude that previous values for beta in cardiac muscle have been greatly overestimated because of the presence of sarcolemmal acid extrusion. Paradoxically, this error in estimating beta is far less evident in the isolated myocyte. We suggest that this is because a much more rapid acid loading is achievable in the myocyte so that acid loading will be blunted less by acid extrusion than in whole muscle. We present a simple mathematical model that demonstrates this phenomenon. We conclude that beta in ventricular muscle is likely to resemble that measured in the isolated myocyte, i.e. 15‐20 mmol l‐1. 5. Slow acid loading in whole ventricular muscle will also affect the kinetics of pHi changes. The model indicates that the rate of pHi recovery from an acid load in papillary muscle does not reflect the pHi dependence of acid extrusion. Instead, it is heavily influenced by the slow rate of acid loading. This emphasises that great care should be taken when interpreting the kinetics of pHi changes in multicellular ventricular preparations.

1. Intrinsic, intracellular H+ buffering power (beta) was estimated in the isolated sheep cardiac Purkinje fibre at various values of intracellular pH (pHi) in the range 6.2‐7.5 and for various values of extracellular pH (pHo) in the range 6.5‐8.5. Buffering power was calculated from the fall of pHi (recorded with an intracellular pH‐selective microelectrode) induced by addition and removal of extracellular, permeant weak acids and bases (NH4Cl, trimethylamine chloride, sodium propionate). Experiments were performed under conditions nominally free of CO2‐HCO3. 2. beta was estimated firstly following acid loads induced by NH4Cl removal (10‐20 mM) under conditions where Na(+)‐H+ exchange was operational (i.e. in Na(+)‐containing Tyrode solution). At constant pHi, the value of beta appeared to double (from a control level of 39.7 mM) as pHo was increased from 7.5 to 8.5. Notably, raising pHo in this range greatly accelerated pHi recovery from an intracellular acid load, indicating stimulation of acid extrusion. It is likely that this stimulation results in an overestimation of beta because it blunts the intracellular acid load. The apparent elevation of beta at high pHo may therefore be an artifact. 3. Estimates of beta were compared (NH4Cl removal) before and after inhibiting Na(+)‐H+ exchange in Na(+)‐free solution or with amiloride (1 mM). The acid load was larger and in many (but not all) cases the apparent value of beta decreased after inhibition of acid extrusion. This indicates that, if Na(+)‐H+ exchange is operational, it can result in an overestimate of beta. In amiloride, beta was 26.6 +/‐ 1.4 mM (n = 8) at a mean pHi of 6.84 +/‐ 0.03. 4. Small stepwise reductions of external NH4Cl (from 40 to 0 mM), in the presence of Na(+)‐free solution plus 5 mM‐BaCl2 at constant pHo, resulted in small stepwise reductions of pHi (approximately 0.1 units). When these were used to calculate beta, we observed that beta increased roughly linearly as pHi became more acid. For a pHi of 7.2, beta approximately 20 mM. 5. An almost identical relationship between beta and pHi was found when using the method of sodium propionate addition (10‐50 mM): amiloride (1 mM) was present and pHi was manipulated to various test levels by changing pHo. This confirms that beta varies inversely with pHi and also that it is independent of pHo. We conclude that the apparent variation of beta with pHo observed earlier was indeed an artifact.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The inhibition of acid extrusion via Na(+)‐H+ exchange caused by reducing pHo (extracellular pH) was examined in the sheep cardiac Purkinje fibre. Intracellular pH (pHi) and intracellular Na+ activity (alpha 1 Na) were recorded using ion‐selective microelectrodes. Acid extrusion via Na(+)‐H+ exchange was estimated from the pHi recovery rate (multiplied by intracellular buffering power, beta) in response to an internal acid load induced by 20 mM‐NH4Cl removal (nominally CO2‐HCO3‐free media). 2. At a given pHi, acid extrusion decreased sigmoidally with decreases of pHo in the range 8.5 to 6.5 (50% inhibition of efflux occurred at a pHo between 7.0 and 7.5). This inhibition was associated with a parallel decrease in Na+ influx as evidenced from a decrease in the rise of alpha i Na measured during acid extrusion, suggesting inhibition of Na(+)‐H+ exchange. 3. The background acid‐loading rate (estimated by adding 1 mM‐amiloride to inhibit Na(+)‐H+ exchange and recording the initial rate of fall of pHi) was found to be unaffected in the steady state by changes of pHo. We therefore conclude that the slowing of pHi recovery at low pHo is due to direct inhibition of Na(+)‐H+ exchange rather than to an increase of background acid loading. 4. Reducing pHo (constant pHi) inhibited acid efflux by producing a parallel shift of the efflux versus pHi relationship to lower values of pHi, consistent with a decrease in the apparent internal H+ ion affinity (pKi) of the system. 5. Raising pHi (constant pHo) also inhibited acid efflux, but this was associated with a rise in the pHo required for 50% maximal inhibition of acid efflux (pKo), consistent with an increase in apparent affinity for external H ions. Thus reduction of pHo reduces pKi (point 4) while reduction of pHi reduces pKo (point 5). 6. Inhibition by elevated Ho+ was not linearly related to the decrease in chemical driving force for Na(+)‐H+ exchange, nor was it related to a reversal of the transmembrane H+ gradient. We found that efflux still occurred when pHo less than pHi. 7. Efflux was not a unique function of the transmembrane H+ ratio (i.e. pHo‐pHi). At appropriate values of pHi and pHo, acid efflux could be kept constant despite a four‐fold change in the transmembrane H+ ratio. 8. Inhibition by low pHo was a saturating function of Ho+ ions with a Hill coefficient of 1.2.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Ischaemia was simulated in the isolated sheep cardiac Purkinje fibre and guinea‐pig papillary muscle by immersing the preparations in paraffin oil. Ion‐selective microelectrodes recorded potassium (Ks+) and pH (pHs) in the thin film of Tyrode solution trapped at the fibre surface while other microelectrodes recorded intracellular pH (pHi), membrane potential and action potentials (AP) (evoked by field stimulation), or membrane current (two‐microelectrode voltage clamp in shortened Purkinje fibres). Twitch tension was also monitored. The paraffin oil model reproduced the salient characteristics of myocardial ischaemia, i.e. a decrease of twitch tension; a decrease of pHi and pHs; a rise in Ks+ (by 2‐3 mM); a depolarization of diastolic membrane potential; considerable shortening of the AP (up to 30% within 4 min). 2. The sulphonylurea compounds, glibenclamide (200 microM) and tolbutamide (1 mM), known inhibitors of the KATP channel, completely blocked the ischaemic rise of Ks+ and prevented AP shortening. Ischaemic tension decline was notably less pronounced in the presence of sulphonylureas. 3. The ischaemic increase of slope conductance (Purkinje fibre) was prevented by 1 mM‐tolbutamide and 200 microM‐glibenclamide. 4. Sulphonylureas did not affect resting membrane potential, the AP or the current‐voltage relationship under non‐ischaemic conditions (this also indicates that ischaemic Ks+ accumulation is not fuelled by the background K+ current [iK1] which was shown, as expected, to be Ba2+ sensitive). 5. In a normally perfused preparation, reducing intracellular ATP by inhibiting glycolysis with 2‐deoxyglucose (DOG) produced a similar AP shortening plus a membrane hyperpolarization, both of which were inhibited by tolbutamide or glibenclamide. The AP shortening was not related uniquely to the fall of pHi observed under these conditions since experimentally reducing pHi (by reducing pHo in the absence of DOG) lengthened rather than shortened the AP. 6. The possibility that the ischaemic rise in Ks+ might be the cause of AP shortening was excluded by the observation that, in a normally perfused Purkinje fibre, experimentally reducing pHi (by an amount similar to that seen during ischaemia) completely neutralized the AP‐shortening effect of an elevated Ko+ (from 4.5 to 6.5 mM). Furthermore, the sulphonylurea‐sensitive AP shortening seen during DOG treatment could not have been associated with a Ks+ rise since, in these particular experiments, the fibres were well perfused and diastolic membrane potential hyperpolarized.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular pH (pHi) was recorded in isolated sheep cardiac Purkinje fibres using liquid sensor ion‐selective microelectrodes in conjunction with conventional (3 M‐KCl) microelectrodes (to record membrane potential). 2. In HEPES‐buffered solution (pH0 7.4), pHi recovery from an intracellular acid load (20 mM‐NH4Cl removal) was blocked by 1 mM‐amiloride, consistent with the inhibition of Na(+)‐H+ exchange. Replacement of the HEPES buffer with CO2‐HCO3‐ caused a transient acidosis followed by an amiloride‐resistant recovery of pHi to more alkaline levels (n = 43). This implies the presence of a HCO3(‐)‐dependent pHi regulatory mechanism. 3. Comparison of the membrane potential with the equilibrium potential for HCO3‐ ions (EHCO3) estimated during amiloride‐resistant pHi recovery, showed that for polarized fibres (membrane potential Em approximately ‐80 mV), there was a net outward electrochemical driving force for HCO3‐ ions. Hence the amiloride‐resistant pHi recovery cannot be explained in terms of passive HCO3‐ influx through membrane channels. 4. Removal of external Na+ (Na0+ replaced by N‐methyl‐D‐glucamine) inhibited HCO3(‐)‐dependent pHi recovery, whereas removal of external Cl‐ (leading to depletion of internal Cl‐; Cl0‐ replaced by glucuronate) or short‐term removal of extracellular K+ had no inhibitory effect. We suggest that a Na(+)‐HCO3‐ co‐influx causes the recovery. Replacement of external Na+ with Li+ greatly reduced HCO3(‐)‐dependent pHi recovery indicating that Li0+ cannot readily substitute for Na0+ on the co‐transport. 5. The stilbene drug DIDS (4,4‐diisothiocyano‐stilbene‐disulphonic acid, 500 microM) slowed HCO3(‐)‐dependent pHi recovery. 6. Depolarization of the membrane potential in high K0+ (44.5 mM) solution or with 5 mM‐BaCl2 had no effect upon the rate of HCO3(‐)‐sensitive pHi recovery. This observation, when coupled with the fact that activation of HCO3(‐)‐dependent pHi recovery was associated with no consistent change of membrane potential, suggests that the Na(+)‐HCO3‐ co‐influx is electroneutral and voltage insensitive. 7. HCO3(‐)‐dependent pHi recovery was unaffected by the Na(+)‐K(+)‐2Cl‐ co‐transport inhibitor, bumetanide (150 microM). 8. The contribution of Na(+)‐H+ exchange and Na(+)‐HCO3‐ co‐transport to net acid efflux was assessed. At a pHi of 6.6, we estimate that the co‐transport should account for 20% of total acid equivalent efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Intracellular pH (pHi) was recorded in isolated guinea‐pig ventricular myocytes using the pH‐sensitive fluoroprobe, carboxy‐SNARF‐1 (carboxy‐seminaphthorhodafluor). 2. Addition and removal of 10 mM NH4Cl was used to induce an intracellular acid load in a myocyte perfused with HCO3(‐)‐buffered solution containing amiloride. Under these conditions, subsequent pHi recovery is known to rely upon Na(+)‐HCO3‐ co‐transport into the cell. The application of 0.5‐5 microM adrenaline resulted in an inhibition of this pHi recovery. 3. In HEPES‐buffered solution, where acid extrusion is mediated primarily by Na(+)‐H+ antiport, pHi recovery from an acid load was stimulated by the application of adrenaline. 4. In HCO3‐/CO2‐buffered solution (no amiloride), when both acid‐aquivalent extruders are activated by an intracellular acidification, adrenaline was found to slow pHi recovery. 5. When both carriers were inhibited in Na(+)‐free, HCO3(‐)‐buffered medium, adrenaline had no effect on pHi, ruling out any effect of the catecholamine on background acid loading. 6. The voltage clamp technique was used to test if the inhibitory effect of adrenaline on amiloride‐resistant, HCO3(‐)‐dependent pHi recovery was due to an efflux of HCO3‐ ions through catecholamine‐activated anion channels. During pHi recovery, membrane depolarization, sufficient to reverse the electrochemical driving force acting on HCO3‐, had no effect upon pHi recovery rate. 7. The above results show that adrenaline has direct but opposite effects on Na(+)‐HCO3‐ co‐transport and Na(+)‐H+ antiport. In the presence of this agonist, the pHi dependence of Na(+)‐HCO3‐ symport was shifted to the left along the pHi axis by 0.13 +/‐ 0.03 units (n = 4) whereas that for Na(+)‐H+ antiport was shifted in the opposite direction by only 0.07 +/‐ 0.01 units (n = 3). Following an acid load, the net effect of adrenaline under physiological conditions was, therefore, a slowing of pHi recovery. 8. The application of extracellular ATP (ATPo, 10‐50 microM) mimicked the effects of adrenaline on both Na(+)‐H+ exchange and Na(+)‐HCO3‐ symport. 9. Adenosine (50 microM) and ADP (50 microM) did not induce any inhibition of Na(+)‐HCO3‐ symport, suggesting that the inhibition induced by ATP was not mediated through P1 or P2‐purinergic receptors. 10. We conclude that Na(+)‐H+ antiport and Na(+)‐HCO3‐ symport are both coupled to adrenaline and ATPo receptors. Activation of these receptors switches acid‐equivalent extrusion from a situation dependent on both HCO3‐ and H+ ions to one nearly exclusively dependent upon H+.

1. Intracellular pH (pHi) was recorded ratiometrically in isolated guinea‐pig ventricular myocytes using the pH‐sensitive fluoroprobe, carboxy‐SNARF‐1 (carboxy‐seminaphthorhodafluor). 2. Following an intracellular acid load (10 mM NH4 Cl removal), pHi recovery in HEPES‐buffered Tyrode solution was inhibited by 1.5 mM amiloride (Na(+)‐H+ antiport blocker). In the presence of amiloride, switching from HEPES buffer to HCO3‐/CO2 (pHo of both solutions = 7.4) stimulated a pHi recovery towards more alkaline levels. 3. Amiloride‐resistant, HCO(3‐)‐dependent pHi recovery was inhibited by removal of external Na+ (replaced by N‐methyl‐D‐glucamine), whereas removal of external Cl‐ (replaced by glucuronate, leading to depletion of internal Cl‐), removal of external K+, or decreasing external Ca2+ by approximately tenfold had no inhibitory effect. These results suggest that the amiloride‐resistant recovery is due to a Na(+)‐HCO3‐ cotransport into the cell. 4. The stilbene derivative DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid, 500 microM) slowed Na(+)‐HCO(3‐)‐dependent pHi recovery. 5. Intracellular pH increased in Cl(‐)‐free solution and this increase still occurred in Na(+)‐free solution indicating that it is not caused via Na(+)‐HCO3‐ symport and is more likely to be due to Cl‐ efflux in exchange for HCO3‐ influx on a sarcolemmal Cl(‐)‐HCO3‐ exchanger. The lack of any significant pHi recovery from intracellular acidosis in Na(+)‐free solution suggests that this exchanger does not contribute to acid‐equivalent extrusion. 6. Possible voltage sensitivity and electrogenicity of the co‐transport were examined by using the whole‐cell patch clamp technique in combination with SNARF‐1 recordings of pHi. Stepping the holding potential from ‐110 to ‐40 mV did not affect amiloride‐resistant pHi recovery from acidosis. Moreover, following an intracellular acid load, the activation of Na(+)‐HCO3‐ co‐influx (by switching from HEPES to HCO3‐/CO2 buffer) produced no detectable outward current (outward current would be expected if the coupling of HCO3‐ with Na+ were > 1.0). 7. Intracellular intrinsic buffering power (beta i) was assessed as a function of pHi (beta i computed from the decrease of pHi following reduction of extracellular NH4 Cl in amiloride‐containing solution). beta i in the ventricular myocyte increases roughly linearly with a decrease in pHi according the following equation: beta i = ‐28(pHi) +222.6. 8. Comparison of acid‐equivalent efflux via Na(+)‐HCO3‐ symport and Na(+)‐H+ antiport showed that, following an intracellular acidosis, the symport accounts for about 40% of total acid efflux, the other 60% being carried by the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Liquid ion‐exchanger Cl‐ ‐sensitive micro‐electrodes were used to make continuous measurements of the intracellular Cl activity, aCli, of quiscent sheep cardiac Purkinje fibres in vitro. 2. aCli was higher than that expected from a passive distribution, (which would have been about 5 mM). It was 3‐‐4 times hiable; EC1 was about 35 mV positive to Em. It was over twice as high in the nominal absence of bicarbonate/CO2 (when the buffer‐system was HEPES/O2) but was not always so stable, and ECl was about 20 mV positive to Em. 3. Experiments designed to assess the maximum possible error likely to occur in the measurement of aCli showed that this could not be large and that the estimates of ECl were accurate to within 8 mV. 4. The ability of Cl to move down both concentration and potential gradients was established by demonstrating a loss of aCli in Cl‐free solutions and a gain when Em was depolarized positive to ECl in high‐K solutions. In both cases, the changes were complete within about 100‐‐160 min. 5. The decline of aCli in Cl‐free solutions (glucuronate‐substituted) was not significantly affected by changes of [Ca]o from 0 to 12 mM or by the depolarizations of Em of up to 60 mV that sometimes occurred in low or zero [Ca]o. 6. Only 2‐‐3 mM‐aClo was sufficient to impede substantially the ready loss of aCli in HEPES‐buffered solutions. 7. In high‐K solutions (45 mM), Cl appeared to be passively distributed since, at equilibrium, Em and ECl differed by less than 2 mV. 8. In HEPES‐buffered Tyrode, ECl of quiescent papillary muscle of the guinea‐pig was, on average, 39 mV positive to Em. 9. It is concluded that liquid ion‐exchanger Cl‐ ‐sensitive micro‐electrodes are suitable for studying the Cl regulation of sheep Prukinje fibres, and probably of other cardiac tissues. The measurements of resting aCli are quite accurate when using either HEPES or bicarbonate‐buffered Tyrode. The results are discussed in relation to estimates of the apparent membrane Cl permeability under various conditions and the possible existence of an inwardly directed ‘Cl pump'.

1. The intracellular Cl activity, alpha iCl was measured inside quiescent sheep cardiac Purkinje fibres, bathed in normal Tyrode at pH 7.40, buffered with approximately 22 mM‐bicarbonate/approximately 5% CO2 + 95% O2. The measurements were made using liquid ion‐exchanger Cl‐sensitive micro‐electrodes. 2. After internal Cl levels had been depleted by prolonged exposure to Cl‐free media (glururonate‐substituted) when external Cl was restored, there was a rapid re‐accumulation of Cl inside the fibres to levels that were much higher than those expected for a passive Cl distribution. Such a process can be conveniently defined as an active inward Cl pump. 3. The inward‐pumping was noticeably temperature‐sensitive (Q10 approximately 2.6), its rate was reduced about eighteenfold in the nominal absence of external bicarbonate/CO2 and it was substantially inhibited by the drug SITS (4‐acetamido‐4'‐isothiocyanato‐stilbene‐2,2'‐disulphonic acid). 4. The fall of alpha iCl in Cl‐free solution was slow and was also equally temperature‐sensitive and substantially inhibited by SITS, but was only slightly impaired in the nominal absence of external bicarbonate/CO2. 5. pHi was measured using recessed‐tip pH‐sensitive micro‐electrodes, and in some experiments both pHi and alpha iCl were monitored simultaneously. When alpha iCl slowly declined in Cl‐free solution then pHi slowly became alkaline. Upon restoring external Cl, then there was, as usual, a rapid recovery of a high alpha iCl and this was accompanied by a rapid re‐acidification of pHi. Both the recovery of alpha iCl and pHi were exponential with virtually the same time constant. 6. Both the slow alkalinization of pHi in Cl‐free solution and the rapid re‐acidification upon restoring external Cl were substantially inhibited by the drug SITS. 7. When [k]O was raised to 45 mM or more (by removing equivalent amounts of [Na]O), there was a large depolarization of Em and a slow rise of alpha iCl, which was not accompanied by a large change of pHi. The rise of alpha iCl appeared to be unaffected by SITS. 8. It is suggested that a Cl/CHO‐3 exchange mechanism can operate reversibly across the membrane of quiescent Purkinje fibres, and that it can account, at least in part, for the high levels of alpha iCl measured in the resting state. It is also concluded that Cl can cross the membrane in other ways, especially in high‐K solution possibly by moving passively through conductance channels that are open under these conditions.

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3‐36 h) before use. [Ca2+]i was measured using the Ca(2+)‐sensitive fluoroprobe indo‐1. Indo‐1 was loaded into cells using the esterified form indo‐1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated‐patch (amphotericin B) whole‐cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L‐type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non‐selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (‐40 to ‐60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Acid extrusion through Na(+)‐H+ exchange was studied in the sheep cardiac Purkinje fibre (bathed in Hepes‐buffered solution, nominally free of CO2‐HCO3‐) by examining (i) intracellular pH (pHi) recovery from an intracellular acid load (induced by 20 mM NH4Cl prepulse) and (ii) the rate of rise of intracellular Na+ activity (aiNa) following the ammonium prepulse (used as an estimate of apparent Na+ influx on Na(+)‐H+ exchange). The pHi and aiNa were recorded using ion‐selective microelectrodes. 2. The pHi recovery and rise of aiNa were both greatly slowed in the presence of 2‐deoxyglucose (DOG; glucose‐free solution), an inhibitor of glycolysis, indicating inhibition of Na(+)‐H+ exchange. 3. Cyanide moderately slowed pHi recovery rate but did not significantly affect the rise of aiNa. Estimates of beta 1 (intracellular buffering power) indicated an increase of approximately 50% in the presence of cyanide; such an increase accounts for most of the observed slowing of pHi recovery. It is concluded that oxidative inhibition with cyanide does not inhibit Na(+)‐H+ exchange. 4. Intracellular ATP, measured from luciferin‐luciferase luminescence, was reduced by a similar amount (approximately 70%) by either DOG or cyanide. This suggests that, if intracellular ATP (ATPi) reduction is the cause of exchanger inhibition by metabolic inhibitors, then ATPi generated glycolytically is more important for activation of the exchange. 5. 3‐Isobutyl‐1‐methylxanthine (IBMX; a non‐specific phosphodiesterase inhibitor which can elevate intracellular [cAMP]) slowed acid extrusion and reduced apparent Na+ influx by a similar amount, whereas addition of sodium nitroprusside (to elevate intracellular [cGMP]) had no effect, suggesting that raising intracellular [cAMP] downregulates Na(+)‐H+ exchange, whereas raising intracellular [cGMP] does not. 6. Application of trifluorperazine (TFP; a non‐specific calcium‐calmodulin inhibitor) completely reversed the inhibitory effects of IBMX upon pHi recovery and aiNa. Under control conditions (no IBMX), TFP had no effect on pHi recovery or upon resting pHi. 7. The phorbol ester 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) had no significant effect on pHi recovery or apparent Na+ efflux. 8. We conclude that inhibition of glycolysis or elevation of cAMP produces downregulation of Na(+)‐H+ exchange in the cardiac Purkinje fibre. Possible reasons for the lack of inhibitory effect of oxidative inhibitors are discussed.

1. An acid‐induced rise in the intracellular calcium concentration ([Ca2+]i) of type I cells is thought to play a vital role in pH/PCO2 chemoreception by the carotid body. In this present study we have investigated the cause of this rise in [Ca2+]i in enzymatically isolated, neonatal rat type I cells. 2. The rise in [Ca2+]i induced by a hypercapnic acidosis was inhibited in Ca(2+)‐free media, and by 2 mM Ni2+. Acidosis also increased Mn2+ permeability. The rise in [Ca2+]i is dependent, therefore, upon a Ca2+ influx from the external medium. 3. The acid‐induced rise in [Ca2+]i was attenuated by both nicardipine and methoxyverapamil (D600), suggesting a role for L‐type Ca2+ channels. 4. Acidosis depolarized type I cells and often (approximately 50% of cells) induced action potentials. These effects coincided with a rise in [Ca2+]i. When membrane depolarization was prevented by a voltage clamp, acidosis failed to evoke a rise in [Ca2+]i. The acid‐induced rise in [Ca2+]i is a consequence, therefore, of membrane depolarization. 5. Acidosis decreased the resting membrane conductance of type I cells. The reversal potential of the acid‐sensitive current was about ‐75 mV. 6. A depolarization (30 mM [K+]o)‐induced rise in [Ca2+]i was blocked by either the removal of extracellular Ca2+ or the presence of 2 mM Ni2+, and was also substantially inhibited by nicardipine. Under voltage‐clamp conditions, [Ca2+]i displayed a bell‐shaped dependence on membrane potential. Depolarization raises [Ca2+]i, therefore, through voltage‐operated Ca2+ channels. 7. Caffeine (10 mM) induced only a small rise in [Ca2+]i (< 10% of that induced by 30 mM extracellular K+). Ca(2+)‐induced Ca2+ release is unlikely, therefore, to contribute greatly to the rise in [Ca2+]i induced by depolarization. 8. Although the replacement of extracellular Na+ with N‐methyl‐D‐glucamine (NMG), but not Li+, inhibited the acid‐induced rise in [Ca2+]i, this was due to membrane hyperpolarization and not to the inhibition of Na(+)‐Ca2+ exchange or Na(+)‐dependent action potentials. 9. The removal of extracellular Na+ (NMG substituted) did not have a significant effect upon the resting [Ca2+]i, and only slowed [Ca2+]i recovery slightly following repolarization from 0 to ‐60 mV. Therefore, if present, Na(+)‐Ca2+ exchange plays only a minor role in [Ca2+]i homeostasis. 10. In summary, in the neonatal rat type I cell, hypercapnic acidosis raises [Ca2+]i through membrane depolarization and voltage‐gated Ca2+ entry.

1. The fall of intracellular pH (pH1) following the reduction of extracellular pH (pH0) was investigated in guinea‐pig isolated ventricular myocytes using intracellular fluorescence measurements of carboxy‐SNARF‐1 (to monitor pH1). Cell superfusates were buffered either with a 5% CO2‐HCO3‐ system or were nominally CO2‐HCO3‐free. 2. Reduction of pH0 from 7.4 to 6.4 reversibly reduced pH1 by about 0.4 pH units, independent of the buffer system used. 3. In HCO3(‐)‐free conditions, acid loading in low pH0 was not dependent on Na(+)‐H+ exchange or on the presence of Na+. It was unaffected by high‐K+ solution, by voltage‐clamp depolarization, by various divalent cations (Zn2+, Cd2+, Ni2+ and Ba2+) and by the organic Ca2+ channel blocker diltiazem, thus ruling out proton influx through H(+)‐or Ca(2+)‐conductance channels or influx via a K(+)‐H+ exchanger. The fall also persisted in the presence of glycolytic inhibitors, or the lactate transport inhibitor, alpha‐cyano‐4‐hydroxy cinnamate. 4. In HCO3(‐)‐free conditions, acid loading in low pH0 was reversibly inhibited (by up to 85%) by Cl(‐)0 removal and was slowed by the stilbene drug DBDS (dibenzamidostilbene disulphonic acid). In contrast, the Cl(‐)‐HCO3‐exchange inhibitor DIDS (4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid) had no inhibitory effect. Acid loading is therefore mediated by a novel Cl(‐)‐dependent, acid influx pathway. 5. After switching to CO2‐HCO3(‐)‐buffered conditions, acid loading was doubled. It was still not inhibited by Na(+)‐free or high‐K+ solutions but was once again inhibited (by 78%) in Cl(‐)‐free solution. The HCO3(‐)‐stimulated fraction of acid loading was inhibited by DIDS. 6. We propose a model of acid loading in the cardiomyocyte which consists of two parallel carriers. One is Cl(‐)‐HCO3‐exchange, while we suggest the other to be a novel Cl(‐)‐OH‐exchanger (although we do not rule out the alternative configuration of H(+)‐Cl‐co‐influx). The proposed dual acid‐loading mechanism accounts for most of the sensitivity of pH1 to a fall of pH0.