Sodium-pump potentials and currents in guinea-pig ventricular

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Sodium-pump potentials and currents in guinea-pig ventricular muscles and myocytes Yuji Kasamaki, An Chi Guo, Lesya M. Shuba, Toshitsugu Ogura, and Terence F. McDonald

Abstract: When guinea-pig papillary muscles were depolarized to ca. –30 mV by superfusion with K+-free Tyrode’s solution supplemented with Ba2+, Ni2+, and D600, addition of Cs+ transiently hyperpolarized the membrane in a reproducible manner. The size of the hyperpolarization (pump potential) depended on the duration of the preceding K+-free exposure; peak amplitudes (Epmax) elicited by 10 mM Cs+ after 5-, 10-, and 15-min K+-free exposures were 12.9, 17.7, and 23.2 mV, respectively. Pump potentials were unaffected by external Cl– but suppressed by cardiac glycosides, hyperosmotic conditions, and low-Na+ solution. Using Epmax as an indicator of Na+ pump activation, the half-maximal concentration for activation by Cs+ was 12–16.3 mM. At 6 mM, Cs+ was three times less potent than Rb+ or K+ and five times more potent than Li+. From these findings, and correlative voltage-clamp data from myocytes, we calculate that (i) a pump current of 7.8 nA/cm2 generates an Epmax of 1 mV and (ii) resting pump current in normally polarized muscle (~0.16 µA/cm2) is five times smaller than previously estimated. Key words: sodium pump, cesium, rubidium, sodium pump current. Résumé : Lorsque les muscles papillaires de cobayes ont été dépolarisés à environ –30 mV par la superfusion d’une solution de Tyrode sans K+ et additionnée de Ba2+, Ni2+ et D-600, l’ajout de Cs+ a hyperpolarisé la membrane de manière transitoire et reproductible. Le degré d’hyperpolarisation (potentiel de la pompe) a été fonction de la durée de l’exposition sans K+ précédente; les amplitudes de crête (Ecmax) induites par 10 mM de Cs+ après des expositions sans K+ de 5, 10 et 15 min ont été de 12,9, 17,7 et 23,2 mV, respectivement. Les potentiels de la pompe n’ont pas été influencés par le Cl– externe, mais ils ont été supprimés par les glucosides cardiotoniques, les conditions hyperosmotiques et une solution à faible teneur en Na+. Lorsque l’Ec a été utilisée à titre d’indicateur de l’activation de la pompe à Na+, la concentration mi-maximale nécessaire pour une activation par le Cs+ a été de l’ordre de 12– 16,3 mM. À une concentration de 6 mM, le Cs+ a été trois fois moins puissant que le Rb+ ou le K+ et cinq fois plus puissant que le Li+. D’après ces résultats, et des résultats obtenus sur des myocytes à l’aide de la méthode du potentiel imposé, nous calculons (i) qu’un courant de pompe de 7,8 nA/cm2 produit une Ec de 1 mV et (ii) que le courant de pompe au repos d’un muscle normalement polarisé (~0,16 µA/cm2) est cinq fois plus faible que celui estimé dans nos études antérieures. Mots clés : pompe à sodium, césium, rubidium, courant de la pompe à sodium. [Traduit par la Rédaction]

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Introduction Page and Storm (1965) demonstrated that the Na+–K+ pump in cardiac tissue is electrogenic by showing that a rewarming of cat papillary muscle after hypothermia-induced Na+ loading provoked a hyperpolarization that exceeded the K+ equilibrium potential. Transient hyperpolarizations were later recorded from other cardiac tissues recovering from a variety of interventions (e.g., rapid pacing, inhibition of the pump by removal of external K+) that caused Na+ accumulation (Vassalle 1970; Noma and Irisawa 1975; Gadsby and

Cranefield 1979). Later investigations of Na+–K+ pump electrogenicity in cardiac preparations often included direct measurement of the Na+ pump current and (or) intracellular Na+ activity (for reviews see Gadsby 1984; Glitsch 1982; Eisner and Smith 1992). In the case of studies on multicellular preparations (e.g., Isenberg and Trautwein 1974; Deitmer and Ellis 1978; Gadsby 1980; Eisner and Lederer 1980a, 1980b; Daut and Rüdel 1982; Glitsch 1982; Sejersted et al. 1988), analysis of experimental results required careful consideration of complexities arising from overlapping membrane conductances and accumulation–depletion of ions in

Received September 28, 1998. Y. Kasamaki,1 A.C. Guo, L.M. Shuba, T. Ogura, and T.F. McDonald.2 Department of Physiology and Biophysics, Dalhousie University, Halifax, NS B3H 4H7, Canada. 1

Present address: Nihon University School of Medicine, 2nd Department of Internal Medicine, 30-1, Oyaguchi-Kamimachi, Itabashiku, Tokyo, Japan.

2

Author for correspondence (e-mail: [email protected]).

Can. J. Physiol. Pharmacol. 77: 339–349 (1999)

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tortuous extracellular clefts (Attwell et al. 1979; Eisner and Smith 1992). These complications were greatly eased when isolated cardiomyocytes were used to study Na+ pump currents (Gadsby et al. 1985; Cohen et al. 1987; Gadsby and Nakao 1989; Nakao and Gadsby 1989; Bielen et al. 1991a; Shattock and Matsuura 1993). However, voltage-clamp studies of pump currents in cardiomyocytes are not free of technical difficulties (Gadsby and Nakao 1989; Bielen et al. 1991b; Carmeliet 1992; Stimers et al. 1993), and extrapolation of the results to the situation in multicellular tissue may not be completely straightforward. The objective of the present study was to investigate whether certain aspects of Na+ pump activity in guinea-pig papillary muscles can be assessed by measurement of the changes in resting potential caused by reactivation of the pump following periods of K+-free inhibition. Ba2+ (5 mM) was used to set the resting potential near –30 mV, Ni2+ and D600 were included to inhibit nonpump membrane currents, and monovalent cations (generally Cs+) were added to the K+-free bathing solution to reactivate the pump. The results demonstrate that reactivations of the pump provoke transient hyperpolarizations that are reproducible on multiple trials conducted over several hours. The “pump potentials” were inhibited by cardiac glycosides and hyperosmotic solution, and their amplitudes were dependent on the duration of the preceding pump inhibition, the Na+ concentration of the superfusate, and the concentration and species of activating cations. Methodological concerns are addressed with experiments on guinea-pig ventricular myocytes, and the pump potential data are related to earlier observations on pump current and intracellular Na+ concentration ([Na+]i).

Methods Papillary muscles General Male guinea-pigs (250–350 g) were killed by cervical dislocation, and the hearts were quickly removed and immersed in oxygenated (100% O2) Tyrode’s solution at room temperature. Papillary muscles (diameter ≤ 0.8 mm) were excised from the right ventricles, mounted horizontally in a 0.25-mL perfusion chamber, and superfused with oxygenated Tyrode’s solution at 6–9 mL/min. Membrane potentials were recorded with 3 M KCl-filled microelectrodes (resistance 8–12 MΩ) connected to a high-input impedance amplifier (model 750, WP Instruments, New Haven, Conn.) via an Ag–AgCl pellet. The reference electrode located downstream of the muscle was a flowing 3 M KCl, Ag–AgCl unit with frittered glass junction. Electrical signals were recorded on a chart recorder (model RS3400, Gould, Cleveland, Ohio), and recordings were scanned into a PC as required for the preparation of figures. The data reported here were obtained from experiments at 36°C, in which impalements were continuous throughout the observation periods.

Solutions Normal Tyrode’s solution contained (in mM) NaCl, 140; KCl, 4.6; CaCl2, 2.4; MgCl2, 1.2; glucose, 10; and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (Hepes), 5 (pH 7.4 with NaOH). Cl–-free Tyrode’s solution contained (in mM) Na+-methanesulfonate, 140; KOH, 4.6; CaOH2, 2.4; MgOH2,1.2; glucose, 10; and Hepes, 5 (pH 7.4 with methanesulfonic acid). K+-free (Na+ loading) solution was a modified Tyrode’s solution (KCl omitted, NaCl reduced to 130 mM) that was supplemented with 5 mM

Can. J. Physiol. Pharmacol. Vol. 77, 1999 BaCl2, 5 mM NiCl2, and 10 µM D600. Cl–-free K+-free (Na+ loading) solution was a modified Cl–-free Tyrode’s solution (0 mM KOH, 130 mM Na+-methanesulfonate) that was supplemented with 5 mM BaOH2, 5 mM NiOH2, and 10 µM D600. Pump-reactivating solution was (i) K+-free (Na+ loading) solution that contained 2–40 mM CsCl, RbCl, LiCl, or KCl or (ii) Cl–-free K+-free solution that contained 10 mM Cs added as CsOH. In some of the experiments, the Na+ concentration of the Na+-loading and pump-reactivating solutions was reduced to 75 or 20% normal (isosmotic replacement by sucrose).

Predepolarization equilibration All muscles were stimulated at 1 Hz (1-ms pulses, 1.2 times threshold, bipolar electrode) for 60 min in normal Tyrode’s solution, treated with 5 mM Ni2+ and 10 µM D600 for 10 min, and rested in the latter solution for 5 min prior to application of K+-free solution. In the case of experiments with Cl–-free solutions, an extra 60-min period with Cl–-free Tyrode’s (1-Hz stimulation) followed the initial 60-min equilibration.

Ventricular myocytes Cell isolation Single guinea-pig ventricular myocytes were obtained by enzymatic dispersion. Excised hearts were sequentially perfused with warmed (37°C) oxygenated normal Tyrode’s solution, Ca2+-free Tyrode’s solution (CaCl2 omitted), Ca2+-free Tyrode’s solution containing collagenase (0.05–0.1 mg/mL; Yakult, Tokyo, Japan), and storage solution. The ventricles were cut into chunks, and cells were dispersed by mechanical agitation and kept in storage solution at room temperature. All experiments were carried out within 9 h of cell isolation.

Electrophysiology An aliquot of storage solution containing myocytes was transferred to the experimental chamber positioned on top of an inverted microscope stage (Nikon Diaphot, Tokyo, Japan). The chamber was perfused with normal Tyrode’s solution heated to 35– 36°C. Pipettes pulled from thick-walled borosilicate glass capillaries (Jencons, Bedfordshire, U.K.) had a resistance of 2–3 MΩ when filled with pipette solutions. The voltage-clamp amplifier was an EPC-7 (List Medical Electronic, Darmstadt, Germany), and a flowing 3 M KCl, Ag–AgCl reference electrode was used to minimize changes in liquid junction potential. Membrane currents were recorded on a video cassette recorder through an A/D PCM-2-B adapter (Medical Systems Corp., Greenvale, N.Y.) prior to computer analysis using pCLAMP 6 software (Axon Instruments Inc., Foster City, Calif.). Currents were filtered at 3 kHz and digitized at 8 kHz. Resting potentials were recorded with 3 M KCl-filled microelectrodes (50–70 MΩ resistance).

Solutions Myocytes were superfused with the normal or modified Tyrode’s solutions listed above. The pipette solution used in voltage-clamp experiments contained (in mM) NaCl, 10 (15 or 25); KCl, 30 (25 or 15); KOH, 106; aspartic acid, 106; MgATP, 5; ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5; and Hepes, 5 (pH 7.2 with KOH). In some experiments, NaCl was raised to 15 or 25 mM, and KCl was reduced to 25 or 15 mM. The storage solution (see Isenberg and Klöckner 1982) contained (in mM) KCl, 30; KOH, 80; glutamic acid, 50; KH2PO4, 30; MgSO4, 3; taurine, 20; glucose, 10; EGTA, 0.5; and Hepes, 10 (pH 7.4 with KOH).

Drugs D600 and ouabain (Sigma, St. Louis, Mo.) were prepared as 0.01 M aqueous stock solutions. Strophanthidin (Sigma) was made up as a 0.1 M stock solution in dimethyl sulfoxide (DMSO), and © 1999 NRC Canada



Kasamaki et al. Fig. 1. Dependence of the amplitude of Cs+-evoked hyperpolarization on the duration of the preceding K+-free exposure. The solutions used in these and all other experiments contained 5 mM Ba2+, 5 mM Ni2+, and 10 µM D600. (A) Hyperpolarizations evoked by 5-min applications of 10 mM Cs+ after 15 min of Na+-pump inhibition (and consequent loading of cells with Na+) induced by superfusion with K+-free solution. The hyperpolarizations quickly reached a peak when the Na+ pump was reactivated on addition of Cs+, and gradually decayed as continued pumping lowered intracellular Na+ and consequently the Na+ pump current generating the hyperpolarization. (B) Responses to 5-min Cs+ applications after 15- and 5-min loadings. (C) Basal hyperpolarization detected by withdrawal of 10 mM Cs+ from a nonloaded muscle.

used at a final concentration of 100 µM (0.1% DMSO). Concentrations of DMSO up to 0.4% are reported to have no significant effect on Na+ pump current in guinea-pig ventricular myocytes (Gadsby and Nakao 1989).

Statistics Results are expressed as means ± SE; Student’s t-test was used for comparisons of mean values, with significance set at p < 0.05.

Results Figure 1A illustrates the effects of the procedures used to depress membrane conductance, impose Na+ loads, and reactivate the Na+–K+ pump in quiescent guinea-pig papillary muscles. Superfusion with K+-free loading solution depolarized the muscle membrane, and switching to 10 mM Cs+ solution caused a 25-mV hyperpolarization that decayed by about 50% during the 5-min treatment. The conditions during both K+-free exposure and Cs+ treatment (i.e., solutions

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supplemented with 5 mM Ba2+, 5 mM Ni2+, and 10 µM D600) were designed to optimize the recording of changes in membrane potential caused by changes in electrogenic Na+ pumping. The Ba2+ ions imposed a strong blockade of inward-rectifying K+ channels that is unlikely to have been affected by addition of monovalent cations. Although studies on K+ activation of Na+ pump current in myocytes suggest that 1–2 mM Ba2+ is sufficient for this purpose (e.g., Gadsby and Nakao 1989; Stimers et al. 1990; Shattock and Matsuura 1993; Kinard et al. 1994), the 5 mM concentration was used to ensure minimal leak of K+ and to secure the lowest possible resting potential under K+-free conditions. Along with 10 µM D600, the inclusion of 5 mM Ni2+ minimized noninactivated or hyperpolarization-reactivated Na+ and Ca2+ channel currents (cf. Fozzard and Hanck 1992; McDonald et al. 1994). Ni2+ also minimized any membrane current flow through Na+–Ca2+ exchangers and Ca2+-activated nonselective cation channels (Ehara et al. 1989; Kimura et al. 1987). Neither the amplitude nor voltage-dependence of Na+ pump current is affected by 5 mM Ba+ (Schweigert et al. 1988) or 5 mM Ni2+ (Bielen et al. 1991a). Effects of Na -loading time, external Na , and external Cl on Cs -induced hyperpolarization Earlier studies on cardiac Purkinje fibres have indicated that (i) [Na+]i increases with the duration of Na+ pump inhibition (Deitmer and Ellis 1978; Eisner et al. 1981a) and (ii) Na+ pump current is proportional to [Na+]i (Eisner et al. 1981b; Glitsch et al. 1982). More recent studies on mammalian ventricular myocytes have established that pump current has a sigmoidal dependence on dialysate Na+ concentration ([Na+]pip), with half-maximal activation at [Na+]pip of 11– 21 mM (Nakao and Gadsby 1989; Shattock and Matsuura 1993; Whalley et al. 1993). These findings indicate that if the amplitude of papillary muscle hyperpolarization is proportional to Na+ pump current, it should be affected in a predictable manner by the duration of the preceding Na+loading period. Figure 1A illustrates that 5-min applications of 10 mM Cs+ after each of the three 15-min loadings elicited similar-sized (ca. 23 mV) hyperpolarizations. However, when shorter 5-min loadings were interspersed with 15-min loadings in another muscle, the hyperpolarizations after the 5min loadings were much smaller than after the longer loadings (Fig. 1B). In the absence of any loading, the “basal” hyperpolarization (Fig. 1C) was 6.3 ± 0.7 mV (n = 4). The effects of loading duration on Cs+-induced hyperpolarizations were evaluated in 62 muscles that were depolarized–loaded for 5, 10, or 15 min. The following parameters were measured (see Fig. 2A): RP (resting potential in the absence of Cs+), Epmax (RP minus the potential at peak hyperpolarization), Ep·t (the area circumscribed by the hyperpolarization (Ep) during 5-min application of Cs+), and ∆Ep/∆t (the mean rate of decay of Ep measured during the 2.5 min after the time of Epmax). RP declined from –89.3 ± 0.3 mV (n = 62) to –36.8 ± 0.7 mV in 18 muscles exposed to K+-free loading solution for 5 min, to –32.0 ± 0.7 mV in 18 muscles exposed for 10 min, and to –32.8 ± 0.8 mV in 26 muscles exposed for 15 min (Fig. 2B). In these muscles, Cs+-induced Epmax was 12.9 ± 0.8, 17.7 ± 1.0, and 23.2 ± 0.9 mV, respectively (Fig. 2C). Measurements on these © 1999 NRC Canada



342 Fig. 2. Analysis of the transient hyperpolarizations evoked by 5min applications of 10 mM Cs+ following initial K+-free, Na+loading periods of 5, 10, or 15 min. (A) Record of transient hyperpolarization showing the parameters that were measured: RP, resting membrane potential in the absence of Cs+; Epmax, RP minus the potential at peak hyperpolarization; Ep·t, the area circumscribed by the hyperpolarization (Ep); ∆t, the 2.5-min interval following Epmax; and ∆Ep, the change in potential during ∆t. (B) RP measured just before addition of Cs+ after first-loading durations of 0 (control), 5, 10, and 15 min. The number of muscles is given within the bars. (C) Effects of the duration of the first loading period on Epmax evoked by Cs+. *p < 0.05, **p < 0.01. (D) Plots of relative Ep·t (left) and ∆Ep/∆t (right) versus Epmax. Each symbol represents a measurement from a single muscle.

responses to Cs+ indicate that there was a direct relationship between Ep·t, ∆Ep/∆t, and Epmax (Fig. 2D). The dependence of Epmax on loading time suggests that the magnitude of Epmax is dependent on [Na+]i. We sought further evidence of this by measuring hyperpolarizations in muscles superfused with low-Na+ solution to reduce Na+ accumulation. In nine muscles that were depolarized and loaded for 10 min in 75% Na+ solution, Epmax (13.6 ± 0.9 mV) was significantly smaller (p < 0.01) than the 17.7 ± 1.0 mV (n = 18) recorded under standard conditions. In one additional muscle, four standard cycles of 7.5 min loading, 5 min Cs+ were separated by test (20% Na+) cycles of similar duration. Epmax was 14.4 ± 1.1 mV with 100% Na+, and 4.2 ± 0.7 mV with 20% Na+. The equilibrium potential for Cl– in cardiac tissue is near –50 mV (Seyama 1979), and therefore falls within the volt-

Can. J. Physiol. Pharmacol. Vol. 77, 1999 Fig. 3. Reproducibility of the Epmax evoked by 10 mM Cs+. (A) Epmax on three successive trials with Cs+ following K+-free Na+-loading periods that were either 5, 10, or 15 min long. Trial 1 followed the initial depolarizing loading period. (B) Relative amplitudes of Epmax on the last five of six trials with Cs+ after 15-min loading periods. The first of these six followed the initial depolarizing loading period. The data are from complete experiments on eight muscles.

age range traversed by Ep. To determine whether any basal Cl– conductance in muscle cells has a significant effect on pump potentials, pump activation was examined under conditions expected to depress Cl– conductance. Muscles pretreated for 60 min with Cl–-free Tyrode’s solution (to deplete intracellular Cl–) were exposed to Cl–-free K+-free (100% Na+) solution for 15 min, and then tested with 10 mM Cs+. Unlike Purkinje fibres (which depolarize to ca. –30 mV when superfused with low-Cl– solution (Gadsby and Cranefield 1979)), papillary muscles did not depolarize in Cl– -free Tyrode’s, and the steady resting potential reached during K+-free exposure was not different than under control Cl– conditions. Addition of 10 mM Cs+ after 15-min loadings evoked an Epmax (24.3 ± 1.0 mV, n = 7 muscles) that was not significantly different from control Epmax (23.2 ± 0.9 mV, n = 26 muscles). The absence of effect of Cl– removal on pump-induced hyperpolarization in papillary muscles contrasts with the strong augmentation of hyperpolarization recorded from rabbit sinoatrial node (Noma and Irisawa 1975; Seyama 1979). The reproducibility of 5-min Cs+ responses was assessed on successive loading, 10 mM Cs+ trials, in which loading duration was fixed at 5 min (n = 5 muscles), 10 min (n = 6 muscles), or 15 min (n = 15 muscles). When Epmax on the first trial in each muscle was designated as 100%, Epmax (second trial) after 5-, 10-, and 15-min loadings was 97.2 ± 2.8, 98.1 ± 7.3, and 87.5 ± 2.1%, and Epmax (third trial) was 89.1 ± 6.1, 85.8 ± 2.2, and 84.6 ± 3.7%, respectively (Fig. 3A). In eight muscles subjected to six consecutive 15 min loading, 5 min Cs+ cycles, Epmax on trials 3 to 6 © 1999 NRC Canada



Kasamaki et al. Fig. 4. Inhibition of Cs+-induced hyperpolarization by strophanthidin and hyperosmotic solution. (A) Inhibition after a 2-min pretreatment with 100 µM strophanthidin (Str). Washout of strophanthidin led to gradual recovery of the hyperpolarizing response as tested with 10 mM Cs+ and then with 10 mM Rb+. (B) Inhibition caused by elevating the osmolality of the Cs+ solution with 60 mM sucrose (Su). Left, example record; right, percent inhibition calculated by comparing the mean response on two hyperosmotic tests with the interposed control response (see example record); n = 4 muscles.

ranged from 91.5 ± 6.9 to 96.4 ± 8.1% of Epmax on the second trial (Fig. 3B). Inhibition by cardiac glycosides and hyperosmotic sucrose solution To confirm that the hyperpolarizations elicited by 10 mM Cs+ were due to reactivations of the Na+ pump, we investigated whether they were inhibited by interventions that inhibit Na+ pump activity. Pump potentials in control muscles loaded for 10 min were compared with those in test muscles to which 20 µM ouabain was applied for 2 min prior to Cs+ solution. Epmax was 18.4 ± 1.6 mV (n = 17 trials on 12 muscles) under control conditions, and only 2.4 ± 0.7 mV (n = 12 muscles) under ouabain conditions. As was the case with ouabain, a 2-min pretreatment with 100 µM strophanthidin had no effect on the depolarized RP, but suppressed Cs+-induced hyperpolarizations (n = 4 muscles). The record in Fig. 4A indicates that washout of this glycoside for 7 min led to a partial recovery of sensitivity to 10 mM Cs+, and washout for an additional 10 min led to a large hyperpolarization upon addition of 10 mM Rb+. The latter was 50% larger than the control hyperpolarization produced by 10 mM Cs+, a difference that may have reflected the longer period of pump inhibition prior to the reactivation by Rb+, and (or) stronger activation of the pump by Rb+ than Cs+ (cf. Eisner and Lederer 1980a) (also see below).

343 Fig. 5. Concentration–response relationship for Cs+. Epmax was measured on a test application of Cs+, and expressed relative to the Epmax obtained on the preceding (second) control response to 10 mM Cs+ (see schematic). All superfusates contained 75% Na+, and the osmolality of the 20–40 mM Cs+ test solutions was the same as that of the 10 mM Cs+ solution (lower Cs+ solutions slightly hyposmotic). The curve fitting the data (circles) is a hyperbolic function with maximum = 2.22 and K0.5 = 12 mM; the number of muscles is given in parentheses. 䉲, mean Epmax value corrected for relative changes in pump current caused by the dependence of pump current on voltage (see text, and Table 1 (column 10)). The curve fitting the data has a maximum of 2.63 and K0.5 16.3 mM.

Whalley et al. (1993) have reported that hyperosmotic solution inhibits Na+ pump current in mammalian ventricular myocytes. We ascertained that switches from standard to hyperosmotic solution had little effect on resting membrane potential under K+-free conditions (not shown) and then compared the effects of standard and hyperosmotic (+60 mM sucrose) 10 mM Cs+ solutions. Figure 4B indicates that hyperosmotic solution depressed Epmax by 25 ± 5% (n = 4 muscles). Dependence of pump potentials on cation concentration and species The relationship between Epmax and external Cs+ concentration was examined in muscles that were subjected to two control cycles of 10 min loading, 5 min 10 mM Cs+, and then a single test cycle in which the Cs+ concentration ranged from 2 to 40 mM (Fig. 5, top). The osmolality of the 20–40 mM Cs+ test solutions was adjusted to the osmolality of the 10 mM Cs+ test solution by lowering the sucrose concentration of 75% Na+ solutions. The normalized data in Fig. 5 (open circles) are well-fitted with a rectangular hyperbola that places the concentration of Cs+ for half-maximal activation (K0.5) at 12 mM. When corrections were made to account for the voltage dependency of Na+ pump current (see below), the K0.5 increased to 16.3 mM (Fig. 5, triangles). In comparison with Cs+, Rb+ and K+ are stronger activators of Na+ pump current in cardiac Purkinje strands, and Li+ is a considerably weaker one (Eisner and Lederer 1980a). The records in Fig. 6A were obtained from muscles treated with 10 mM Cs+, 6 mM Rb+, or 40 mM Li+ after 15-min © 1999 NRC Canada



344 Fig. 6. Relative potency of monovalent cations in activating pump potentials in papillary muscles. (A) Membrane potentials elicited by 10 mM Cs+, 6 mM Rb+, and 40 mM Li+ after exposure of muscles to K+-free solution for 15 min. (B) Relative Epmax elicited by test applications of 6 mM Cs+, Li+, Rb+, and K+ after 15 min K+-free loading (100% Na+). Epmax elicited by monovalent cation (MC) was normalized by reference to Epmax on the second control trial with 10 mM Cs+ that preceded the test. Each muscle was tested once with Cs+, Li+, Rb+, or K+, and the number of muscles is given below the bars. (C) Pump potential elicited by 40 mM Rb+ after a 7.5-min loading with K+-free, 75% Na+ solution.

loadings. The amplitudes and rates of decay of the pump potentials suggest a potency order of 6 mM Rb+ > 10 mM Cs+ > 40 mM Li+ for reactivation of the pump. A quantitative determination of the relative potencies of these cations was obtained by testing Na+-loaded (15 min) muscles with a 6 mM concentration of Cs+, Rb+, K+, or Li+. These tests were conducted after two control cycles of 15 min loading, 5 min Cs+ (10 mM) reactivation to secure a reference response (10 mM Cs+ after the second loading) (Fig. 6B). The results indicate that the order of potency for 6 mM monovalent cation was K+ ~ Rb+ > Cs+ >> Li+ (Fig. 6B). We used a saturating 40 mM concentration of Rb+ (Eisner and Lederer 1980a; Sejersted et al. 1988) to obtain maximal activation of external pump sites. In five muscles superfused with 100% Na solution and loaded for 15 min, the Rb+ solution evoked pump potentials with Epmax of 46 to 56 mV (not shown). After a more moderate loading regimen (7.5 min, 75% Na+), the pump potentials were about 30% smaller. The record in Fig. 6C illustrates that they decayed in a multiphasic manner to Cs+ >> Li+, is also in agreement with measurements of Na+ pump current in sheep Purkinje fibres (Eisner and Lederer 1980a, 1980b). The concentration of Cs+ required for half-maximal activation of pump potential was 12 mM (raw data) or 16.3 mM (correction for voltage as in Table 1, D3B). This result is in excellent agreement with the K0.5 of 14.2 mM Cs+ reported for reactivation of sheep Purkinje fibre pump current by Eisner and Lederer (1980a), and with the similar K0.5 they measured for the decline of intracellular Na+ activity (Eisner et al. 1981a). On the other hand, these K0.5 values are about four times larger than the 3.2 mM value reported by Glitsch et al. (1989b) for the activation of pump current by Cs+ in sheep Purkinje fibre cardioballs. Although this disparity has been rationalized on the basis that depletion of cations during pumping in the tissue may have caused the high K0.5 (see Eisner and Lederer 1980a, 1980b; Glitsch et al. 1989b), there are several reasons for believing that this may not be the central point. First, whereas the tight packing of cells in sheep Purkinje fibres can lead to significant problems with ion accumulation and depletion (Attwell et al. 1979; Eisner

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and Smith 1992), the 100-fold larger extracellular space in guinea-pig ventricular muscles greatly eases this situation (Daut 1982). Secondly, Daut (1982) concluded that there is no significant depletion of low millimolar K+ during pump activation in guinea-pig ventricular tissue, and Gadsby (1980) reached the same conclusion from observations on rabbit Purkinje fibres (where cells are less tightly packed than in sheep fibres). Finally, our results (and those of Eisner and Lederer (1980a)), were obtained with external Cs+, which as a relatively weak activator, seems unlikely to undergo a significant fractional depletion. An alternative possibility is that the disparity is due to alterations caused by the experimental conditions used to examine the activation of pump currents in the cardioballs. Resting pump current and pump potential The resting pump current in normally polarized muscles is likely to be much larger than the 0.05 µA/cm2 calculated for depolarized muscles bathed in 10 mM Cs+ (Table 1, row 7). An upper estimate is provided by the 0.33 µA/cm2 calculated for the 40 mM Rb+-induced hyperpolarization to ca. –85 mV of muscles estimated to have an [Na+]i of 22 mM (Table 1, row 1). A more likely 0.16 µA/cm2 is calculated by setting resting [Na+]i at 10 mM and deriving pump determinants for RP ~85 mV in a muscle superfused with 5.4 mM K+ solution (Table 1, row 8). Since R m under these conditions is 7.2– 9 kΩ·cm 2 (Weidmann 1970; Isenberg and Trautwein 1974), inhibition of a resting current of 0.16 µA/cm2 would depolarize the membrane by 1.3 mV. To obtain experimental information on this point, we superfused four guinea-pig ventricular myocytes with 4.6 mM K+ solution and found that 5 min treatment with 100 µM ouabain depolarized the cells by 2.3 ± 0.3 mV from control –89.7 ± 0.6 mV. Considering that Rm was perhaps 20% higher under 4.6 mM than 5.4 mM K+ conditions, and that there was probably a small drop in the K+ equilibrium potential as a result of a loss of intracellular K+ during the 5-min oubain treatment, the result is in excellent agreement with the calculated effect of inhibiting a pump current of 0.16 µA/cm2 (and with an earlier measurement of 1.5 mV depolarization caused by 50 µM strophanthidin (5.4 mM K+) in these myocytes (Levi 1991)). Both the preferred (0.16 µA/cm2) and upper (0.33 µA/cm2) estimates of resting pump current are considerably lower than the previous estimate of 0.8 µA/cm2 for guinea-pig ventricular muscle at ca. –95 mV (3 mM K+) (Daut and Rüdel 1982), and therefore resolve a large discrepancy with the 0.15 µA/cm2 value reported for sheep Purkinje fibres at –60 mV (Eisner et al. 1981a) (also see Cohen et al. 1987). Calculated resting pump current increased from 0.05 µA/cm2 in depolarized muscle to 0.16 µA/cm2 in normally polarized muscle, despite a significant voltage-dependent decline in pump current (Table 1). One possible explanation for the increase is that fractional activation of the pump by 5.4 mM K+ is smaller than the 1.0 value used in Table 1 (row 8) to calculate resting pump current in normally polarized muscle; another is that fractional activation by 10 mM Cs+ is larger than the 0.42 value used to calculate resting pump current in depolarized muscles (an unlikely fractional activation of 1.0 would increase the current from 0.05 to 0.13 µA/cm2). However, the primary factor is likely to be the increase in driving force on Na+ at –90 versus –30 mV, with resultant increase © 1999 NRC Canada



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in Na+ influx, and therefore pump turnover. In this regard, Eisner et al. (1981b) found that the rate of increase in intracellular Na+ activity rose by ~130% when the membrane potential of sheep Purkinje fibres superfused with K+-free solution was increased from –10 to –60 mV.

Acknowledgements The authors thank Gina Dickie for excellent technical assistance. The study was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Nova Scotia.

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