H. G. Glitsch

The Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP production

The role of glycolysis and oxidative phosphorylation in providing the ATP for the cardiac Na+/K+ pump was studied in cardioballs from sheep Purkinje fibres. As an indicator of the pump activity, the pump current Ip was measured at -20mV and 30–33° C by means of whole-cell recording. During intracellular perfusion with a pipette solution containing 5 mM ATP and 15 mM glucose Ip reached a maximum within 8 min and declined to 50% of this value within 27 min after gaining access to the cell interior. Perfusion with an ATP- and glucose-free medium barely enhanced the Ip decline. Inhibition of the oxidative phosphorylation by carbonylcyanide m-chlorophenylhydrazone (CCCP, 2 μM or 20 μM) moderately accelerated the effect of the ATP- and glucose-free pipette solution. Addition of 2 mM iodoacetic acid (an inhibitor of glycolysis) to the latter medium further enhanced the Ip decrease with time. Inhibition of the glycolytic ATP synthesis by 2-deoxy-D-glucose (5 mM) caused a dramatic decline of Ip to half of its maximum within 7.3 min. Pyruvate (5 mM) and inorganic phosphate (2 mM) did not affect the fast Ip decline evoked by the ATP- and glucose-free, 2-deoxyglucose-containing medium, whereas 2 μM CCCP still hastened the fast Ip decrease slightly. This effect of complete metabolic inhibition was reversed by switching to an inhibitor-free pipette solution containing 15 mM ATP. It is concluded that the Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP synthesis.

The dependence of sodium pump current on internal Na concentration and membrane potential in cardioballs from sheep Purkinje fibres

The effect of various intracellular Na concentrations (cNai) and membrane potentials on the Na pump current (IP) was studied in isolated, cultured sheep cardiac Purkinje cells (‘cardioballs’).IP was identified as cardiac steroid sensitive current. The dependence ofIP oncNai was investigated at a membrane potential of −40 mV by means of whote-cell recording from cardioballs internally perfused with media containing various Na concentrations. Internal perfusion with a Na free solution abolishedIP. The amplitude ofIP as a function ofcNai displayed saturation kinetics. Half maximal activation ofIP occured at acNai of about 9 mM. The maximalIP density was estimated to be 1.1μA/cm2. The potential dependence ofIP was studied by conventional whole-cell recording under various ionic conditions. GenerallyIP displayed little voltage dependence at membrane potentials positive to −20 mV.IP declined at more negative potentials. The pump cycle probably includes only one voltage sensitive step. The potential dependence ofIP was more pronounced at lowercNai or lower concentrations of the external pump activator Cs+. The findings are in line with the idea that increasingly steeper ionic gradients against which the cations are pumped strengthen the voltage dependence ofIP in the potential range studied. Other factors probably affecting the pump current-voltage (IP-V) relation are discussed. The results suggest thatIP varies during electrical activity.

Spontaneous tension oscillations in guinea-pig atrial trabeculae

SummarySpontaneous tension oscillations have been recorded from intact guinea-pig auricular trabeculae bathed in Na-poor and/or Ca-rich solutions.The frequency of these oscillations and that of after-contractions (oscillations following an electrically induced contraction) evoked under identical experimental conditions was the same (33°C).The amplitude of the oscillations rose when the [Ca2+]0/[Na+]02-ratio or the intracellular Na-concentration was increased. When the increase of the [Ca2+]0/[Na+]02-ratio was relatively small, tension oscillations only occured after a period of electrical stimulation.The oscillation-frequency increased slightly in media containing 70 instead of 5.4 mM KCl.MnCl2 (3mM) did not affect either the amplitude or the frequency of the oscillations.Caffeine (0.5–2.5 mM) decreased the amplitude and enhanced the frequency of the oscillations. After-contractions were diminished and, at higher concentrations, abolished.It is demonstrated that the membrane potential does not participate in the process causing the tension oscillations. An increased [Ca2+]i is a prerequisite for the occurrence of these oscillations. Characteristics of intracellular Ca-movement probably determine the amplitude and frequency of the spontaneous oscillations of tension.

An identification of the K activated Na pump current in sheep Prkinje fibres

The cell membrane of sheep Purkinje fibres hyperpolarizes transiently on returning to K containing media after several minutes in K free solution. To analyse this ‘K activated response’ voltage clamp experiments and measurements of the internal Na activity (aNai) are performed in fibres bathed in solution containing 0.5–2 mM BaCl2. Compared to the response in Tyrode solution the transient hyperpolarization beyond the resting potential is increased in Ba containing media. The response is blocked by 10−4 M dihydroouabain. During the response, in Ba treated fibres, aNai and a transient outward current decline with the same time constant at all clamp potentials tested. The transient outward current is most probably due to a temporary increase in electrogenic Na pumping. Both the amplitude and the time constant of the pump current show little voltage dependence. The electrogenic fraction of the active Na efflux is estimated to be about 39% and is independent of aNai. Ba ions facilitate the analysis of the pump current in voltage clamped fibres because K depletion is reduced and changes of the I–V relationship by K depletion are minimized. It is concluded that activation of the electrogenic Na pump is mainly responsible for the K activated response of fibres in Ba containing media.

Activation of the electrogenic sodium pump in guinea-pig atria by external potassium ions.

1. When cardiac preparations are rewarmed following prolonged hypothermia a transient hyperpolarization occurs in K‐containing media. This hyperpolarization is correlated with the active Na efflux. It might be due to electrogenic Na pumping or to extracellular K depletion brought about by the activity of an electroneutral Na—K exchange pump. In order to distinguish between these mechanisms the effect of various extracellular K concentrations ([K]o) on the membrane potential of guineapig atria was studied before and after hypothermia.

Effects of Na and K Ions on the Active Na Transport in Guinea-Pig Auricles

Summary1.The effect of Na and K ions on active Na transport was studied in guinea-pig auricles by means of flame photometry.2.The Na influx into preparations rewarmed in Tyrodes solution after cooling was estimated to be about 1.05 mmole/l fibre water·min ((l.f.w.·min) or c. 8 pmole/cm2·s. Intracellular Na ions enhanced the active Na efflux over a wide range of concentrations. A decrease in the extracellular Na concentration ([Na]o) had no major effect on the active Na efflux.3.Extracellular K ions initiated an active Na efflux from rewarmed auricles with an elevated [Na]i over a narrow range of K concentrations ([K]o).4.Assuming Michaelis-Menten kinetics the maximal active Na efflux activated by internal Na ions was calculated to be about 4 mmole/l.f.w.·min (30 pmole/cm2·s). Half maximal Na efflux occurred at about 22 mmole/l.f.w. [Na]i. The maximal K-activated active Na efflux was deduced to be about 3.7 mmole/l.f.w.·min (28 pmole/cm2·s) and was half maximal at a [K]o of about 0.2 mM.5.It is tentatively concluded that the maximal active Na efflux from guinea-pig atria is 3–4 times larger than the physiological flux. Under normal conditions active Na efflux in heart is mainly regulated by variations of [Na]i.

Activation of the cAMP–protein kinase A pathway facilitates Na+ translocation by the Na+–K+ pump in guinea-pig ventricular myocytes

1 The effects of the adenylyl cyclase activator forskolin on steady‐state and transient currents generated by the Na+‐K+ pump were studied in guinea‐pig ventricular myocytes by means of whole‐cell voltage clamp at 30 °C. 2 In external solution containing 144 mM Na+ (Na+o) and 10 mM K+ (K+o), steady‐state Na+‐K+ pump current (Ip) activated by 5 mM pipette Na+ (Na+pip) at ‐20 mV was reversibly augmented by forskolin (4 μM) to 133 ± 4 % of the control current (n= 15). The forskolin analogue 1,9‐dideoxyforskolin (10 μM), which does not activate adenylyl cyclases, did not increase Ip (n= 2). Application of the protein kinase A (PKA) inhibitor H‐89 (10 μM) in the continued presence of forskolin reversed the forskolin‐induced elevation of Ip (n= 3). 3 The forskolin effect on Ip persisted in the presence of 50 mM Na+pip which ensured that the internal Na+‐binding sites of the Na+‐K+ pump were nearly saturated. Under these conditions, the drug increased Ip to 142 ± 3 % of the control Ip when the pipette free Ca2+ concentration ([Ca2+]pip) was 0·013 nM (n= 5) and to 138 ± 4 % of the control Ip when free [Ca2+]pip was 15 nM (n= 9). 4 In Na+‐free external solution, Ip activated by 50 mM Na+pip and 1·5 mM K+o was likewise increased by forskolin but to a lesser extent than in Na+‐containing medium (116 ± 3 % of control, n= 10). 5 In order to investigate exclusively partial reactions in the Na+ limb of the pump cycle, transient pump currents under conditions of electroneutral Na+‐Na+ exchange were studied. Transient pump currents elicited by voltage jumps displayed an initial peak and then decayed monoexponentially. Moved charge (Q) and the rate constant of current decay varied with membrane potential (V). The Q‐V relationship followed a Boltzmann distribution characterized by the midpoint voltage (V0·5) and the maximum amount of movable charge (ΔQmax). Forskolin (2‐10 μM) shifted V0·5 to more negative values while ΔQmax was not affected (n= 11). The effects of forskolin on transient pump currents were mimicked by 8‐bromo‐cAMP (500 μM; n= 2) and abolished by a peptide inhibitor of PKA (PKI, 10 μM; n= 5). 6 We conclude that activation of the cAMP‐PKA pathway in guinea‐pig ventricular myocytes increases Na+‐K+ pump current at least in part by modulating partial reactions in the Na+ limb of the pump cycle. Under physiological conditions, the observed stimulation of the cardiac Na+‐K+ pump may serve to shorten the action potential duration and to counteract the increased passive sarcolemmal Na+ and K+ fluxes during sympathetic stimulation of the heart.

On the temperature dependence of the Na pump in sheep Purkinje fibres

The temperature dependence of cardiac active Na transport is studied in voltage clamped sheep Purkinje fibres by means of simultaneous measurements of the membrane current (I) and the intracellular Na activity (aNai). During activation of the Na pump a transient outward current (ΔI) andaNai decline exponentially with an identical time constant (τ). The transient outward current and the decline inaNai are blocked by 10−4 M dihydroouabain (DHO). Lowering the temperature from 42°C to 17°C prolongs τ. The electrogenic fraction (e.f.) of the active Na efflux remains unaffected. The Q10 value of the active Na transport derived from the changes of τ varies within the temperature range studied. The Q10 amounts to ∼1.2 between 42°C and 35°C, to ∼2.4 between 35°C and 22°C and to ∼2.1 between 35°C and 17°C. Correspondingly the activation energy of the active Na transport is not constant between 42°C and 17°C. It is calculated to be 3.4 kcal/mol between 42°C and 35°C, 15.9 kcal/mol between 35°C and 22°C and 12.4 kcal/mol between 35°C and 17°C. Variations in temperature change the maximal rate constant of the active Na transport, whereas the sensitivity of the Na pump towards the extracellular K concentration (Ko) is little affected. The unidirectional active Na efflux of a fibre as a function of the intracellular Na concentration (Nai) at 35°C and 22°C is derived from the experemental data. The relationship is linear over the narrow Nai range studied but seems to be more complex when a wider Nai range is considered.

Change of Na+ pump current reversal potential in sheep cardiac Purkinje cells with varying free energy of ATP hydrolysis.

1. The Na(+)‐K+ pump current, Ip, of cardioballs from isolated sheep cardiac Purkinje cells was measured at 30‐34 degrees C by means of whole‐cell recording. 2. Under physiological conditions Ip is an outward current. Experimental conditions which cause a less negative free energy of intracellular ATP hydrolysis (delta GATP) and steeper sarcolemmal gradients for the pumped Na+ and Cs+ ions evoked an Ip in the inward direction over a wide range of membrane potentials. The reversal of the Ip direction was reversible. 3. The inwardly directed Ip increased with increasingly negative membrane potentials and amounted to ‐0.13 +/‐ 0.03 microA cm‐2 (mean +/‐ S.E.M.; n = 6) at ‐95 mV. 4. The reversal potential (Erev) of Ip was studied as a function of delta GATP at constant sarcolemmal gradients of the pumped cations. 5. In order to vary delta GATP the cell interior was dialysed with patch pipette solutions containing 10 mM ATP and different concentrations of ADP and inorganic phosphate. The media were composed to produce delta GATP levels of about ‐58, ‐49 and ‐39 kJ mol‐1. 6. A less negative delta GATP shifted Erev to more positive membrane potentials. From measurements of Ip as a function of membrane potential Erev was estimated to be ‐195, ‐115 and ‐60 mV at delta GATP levels of approximately ‐58, ‐49 and ‐39 kJ mol‐1, respectively. The calculated Erev amounted to ‐224 mV at delta GATP approximately ‐58 kJ mol‐1, ‐126 mV at delta GATP approximately 49 kJ mol‐1 and ‐24 mV at delta GATP approximately ‐39 kJ mol‐1. 7. Possible reasons for the discrepancy between estimated and calculated Erev values are discussed. 8. Shifting delta GATP to less negative values not only altered Erev but also diminished Ip at each membrane potential tested. The maximal Ip (Ip,max), which can be activated by external Cs+ (Cs+o), decreased under these conditions, whereas [Cs+]o causing half‐maximal Ip activation remained unchanged. Similarly, the voltage dependence of Ip activation by Cs+o was unaffected. 9. It is concluded that Erev of Ip varies with delta GATP at constant sarcolemmal gradients of the pumped cations. This agrees with thermodynamic considerations.

Properties of an inward rectifying K channel in the membrane of guinea-pig atrial cardioballs.

Single channel outward current fluctuations are recorded in excised (outside-out) membrane patches of isolated atrial cells in culture (cardioballs) from hearts of adult guinea-pigs. The ionic channel displays a high selectivity to K ions. Accordingly the reversal potential of the single channel current is close to the K equilibrium potential. The open channel conductance is unaffected by the membrane potential but depends on the K concentration of the outside solution (19.7pS at 2 mM K0 to 30.7pS at 20 mM K0). The open state probability (P0) of the channel shows a marked voltage dependence. P0 amounts to c.0.9 at −40mV and decreases to c.0.1 at +40mV.Under the assumption of no channel interaction a macroscopic steady state current voltage relationship is reconstructed from the single channel data. The relationship displays inward-going rectification. The rectification is due to the voltage dependence of P0. TheI–V curve displays a negative slope at membrane potentials positive to −15 mV.In bathing solutions containing Ba ions (0.2 mM) P0 is reduced by rapid closures which interrupt the open state events. The unit channel conductance is unaffected by Ba ions. The channel block exerted by Ba ions is augmented with increasing membrane hyperpolarization.The results suggest that the channel studied may represent a background K conductance.