J Mol

Cell

Cardiol

24, 1307-1320

Mechanism Metabolic

(1992)

of the Increase Inhibition: Direct Voltage-dependent Rafael

Division of Cardiolou,

in Intracellular Sodium During Evidence Against Mediation by Sodium Channels

Mejia-Alvarez

Department

and

Eduardo

Marban

of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

(Received 13 April

1992, accepted in revised form 3 June 1992)

R. MEJ~A-ALVAREZ AND E. MARBAN. Mechanism of the Increase in Intracellular Sodium During Metabolic Inhibition: Direct Evidence Against Mediation by Voltage-dependent Sodium Channels. Journal of Molecular and Cellular Cardiology (1992) 24, 1307-1320. During ischemia or metabolic inhibition, intracellular Nat conccntration ([Na+],) increases considerably. Elevation of [Na+], figures critically in the mechanism of cellular injury by promoting Car’ influx via the Na+-Cap+ exchanger, but the exact mechanism of this intracellular Na’ accumulation remains unknown. To test directly the hypothesis that voltage-dependent Nat channels are involved, we measured Nat currents (c) in isolated guinea-pig ventricular myocytes using the patch-clamp technique. The cell-attached configuration was used in order to avoid disturbing the intracellular milieu. Metabolic inhibition was induced by exposing the cells to either iodoacetate (IAA, I mM) to inhibit glycolysis or 2,4-dinitrophenol (DNP, 0.2 mM) to uncouple oxidative phosphorylation. The amplitude ofIN,, was measured in multichannel patches before and during exposure to IAA or DNP, by depolarizing the cell to different membrane potentials from a holding potential of - 135 mV. Analysis of current-voltage relations before and during metabolic inhibition revealed a modest but significant reduction of peak INa at test potentials positive to - 40 mV with DNP; no change was observed with IAA. The voltage dependence of steady-state parameters of inactivation was not altered by either intervention; specifically, no steady-state (“window”) current was induced. Although we cannot exclude the possibility that other factors not explored here might lead to different conclusions during genuine &hernia, metabolic inhibition alone does not up-regulate the function of Na’ channels. Thus, we conclude that other mechanisms underlie the accumulation of intracellular Na’ observed during metabolic inhibition. KEY WORDS: Sodium

current;

Ventricular

myocytes;

Introduction Metabolic inhibition produces marked alterations of intracellular cation homeostasis, which are just as marked or even greater in ischemia. Intracellular sodium and calcium increase significantly during metabolic inhibition or during ischemia; the accumulation of intracellular calcium figures prominently in the pathogenesis of the associated cellular injury [I]. The increase in cell calcium comes about at least partially due to Ca2+ influx via Na+-Ca2+ exchange [2, 31, driven by the concomitant increase of intracellular Naf concentration ( [Na+li) [m. Although the

Patch-clamp;

Glycolysis;

1 I 1307 + 14 008.00/O

phosphorylation.

exact mechanism of the [Na+li accumulation is still unknown, two general possibilities need to be considered: decreased Na+ efflux or increased Na+ influx. During ischemia, ATP production and the free energy of ATP hydrolysis are diminished, so that decreased Na’ efflux might result simply from Na’/K+ ATPase inhibition. This mechanism seems unlikely to be primary in hypoxia or metabolic inhibition since the time course of ATP depletion lags behind that of [Na+], accumulation [A; furthermore, there is no simple correlation between ATP and [Na+]; in either metabolic inhibition or ischemia [5, 6, 81. i2s

Please address all correspondence to: E. Marban, Division of Cardiology, University, 720 N. Rutland Avenue, Baltimore, MD 21205, USA. ‘This work was performed with the support of NIH grant no. HL 44065. 0022-2828/92/

Oxidative

844 Ross Building,

Ql992

The Johns

Academic

Hopkins

Press Limited

1308

R. Mejia-Alvarez

regards an increased Na+ influx mechanism, a fall in intracellular pH ( pHi) might drive Na+ into the cells via Na’-H’ exchange [2, 7, 9, 201. Experiments with organic Na’-H+ exchange blockers support the notion that this exchanger attempts to neutralize intracellular acidosis by extruding protons from the cell at the expense of the normal Na+ gradient, both physiologically [II, 121 and during ischemia [IO] or hypoxia [9]. Despite the attractive features of Na+-H+ exchange as a possible pathway for Na + influx, the evidence for its involvement rests largely upon the effects of high concentrations of amiloride, which is a non-specific blocker [1.%15]. An alternative mechanism that has been put forth to explain an increase of Na+ influx is modification of voltage-dependent Na’ channel activity. The opening ofjust one Na+ channel can admit more than 10 million Na+ ions per s into the cytoplasm, and the density of these channels is known to be extremely high in heart cells [lq. Indeed, several pieces of evidence suggest the involvement of NaC channels: lidocaine, a Na+ channel blocker, inhibits the increase in [Na+]; during ischemia [18]; likewise, R-56865, a putative blocker of voltage-dependent Na+ channels, prevents the rise in [Na’li and the cellular damage produced by hypoxia in ventricular myocytes [19]; tetrodotoxin has been found to protect against neural injury provoked by hypoxia and reoxygenation [20]; finally, lysophosphatidylcholine, a toxin that reportedly accumulates in ischemia, increases dramatically the open probability (PO) of cardiac Na+ channels [21]. Nevertheless, the gradual depolarization that occurs during ischemia would tend to inactivate Na’ channels unless their gating properties are altered to favor steady-state current. A provocative precedent for such modification can be found in the work of Bhatnagar e! al. [22], who have demonstrated that the steady-state kinetics of Naf channels are modified during free radical-induced oxidative stress in frog myocardial cells. Under these circumstances, the noninactivating Na+ “window” current increases 12-fold in amplitude, and its peak is shifted 10 mV towards more negative potentials. Such modifications in Z,* would be expected to allow persistent Na+ entry into the cell at the resting potential during oxida-

and

E. Marban

tive stress. In principle, a similar mechanism could explain the increase in Na+ influx during or after metabolic inhibition, particularly since oxidative stress has been implicated as a component of reoxygenation-induced injury

v31. To investigate directly the idea that voltage-dependent Na+ channels are modified such that they might contribute to the increase in [Nat],, we studied the effects of metabolic inhibition on Z,, using the patchOur results indicate that clamp technique. during metabolic inhibition induced by exposing the cells to 2,4-dinitrophenol (DNP) or iodoacetic acid (IAA), ZNa is slightly reduced or unaffected. By exclusion, this observation provides an indirect argument in favor of Na+-H+ exchange (or another coupled transport mechanism) to explain the pathophysiologically important increase in [Na’],.

Methods Cell isolation Male guinea pigs weighing approximately 250 g were anesthetized by intraperitoneal injection of Na-pentobarbital (97 mg), after which hearts were excised rapidly. Single ventricular myocytes were isolated by a variant of the enzymatic dissociation technique described by Mitra and Morad [24]. Briefly, the heart was initially perfused with normal Tyrode solution (in mM, NaCl 140, KC1 5, MgCl, 1, CaCI, 1, glucose 10, HEPES 10, pH= 7.4 adjusted with NaOH) for 3-4 min. After the heart fully recovered its contractility, Ca ‘+-free Tyrode solution was perfused for about 6 min. After 7 min of enzymatic digestion (collagenase type I, 262.5 pug/ml; protease type XIV, 57.5,uglml; both from Sigma, St. Louis, MO, USA) the heart was rinsed with low Ca’+ (0.2 mM) Tyrode solution. Finally, the ventricular portion of the heart was placed in high K+ solution (in mM, KC1 20, K-glutamate 120, EGTA 0.1, glucose 10, HEPES 10, pH= 7.4 adjusted with KOH) and cut into small fragments. The cells were dispersed by gentle agitation and stored at room temperature. Until storage, the entire isolation process was carried out with O,saturated solutions at 37°C.

Effects

Electrical

of Metabolic

Inhibition

recordings

To study INa, the isolated ventricular myocardial cells were allowed to settle to the bottom of a small experimental chamber and continuously superfused with high K’ solution at a rate of l-4 ml/min. The cell-attached configuration [,%I was chosen for two reasons: first, to avoid dialysis of the intracellular milieu, which might bias the cellular response to metabolic inhibition; second, to enable the simultaneous evaluation of gating and unitary permeation properties (see Results). All experiments were performed within 4-6 h of cell isolation at room temperature (21°C). Patch-clamp pipettes were made of borosilicate glass by a Flaming-Brown programmable pipette puller (model P-87, Sutter Instruments Company, San Francisco, CA, USA), coated with Sylgard (Dow Corning Corporation, Midland, MI, USA) and firepolished. In order to increase the number of Na’ channels per patch to enable assessment of the “macroscopic” behavior of INa, fairly large pipettes (0.5-l MQ resistance) were favored. Ag/AgCl electrodes were used to connect electrically the pipette and the bath solution. An Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA) with an IH-1 integrating headstage was used. Currents were digitized at 10 kHz with a 12 bit A/D converter (model TL- 1 DMA Labmaster, Axon Instruments, Foster City, CA, USA) and lowpass filtered at 2 kHz (4-pole Bessel). Pipette capacitance was compensated by injection of approximately 5 pF of capacitive current. The cells were stimulated at 0.3 Hz by 70 ms depolarizing pulses to different membrane potentials from a holding potential (HP) of - 135 mV (unless otherwise indicated). The data were collected and stored for further analysis in an IBM-compatible personal computer using custom software. Electrical recordings were first collected 5-10 min after seal formation. This delay was sufficient for stabilization of the well-recognized shift of steady-state activation and inactivation to more negative potentials [16].

Data analysis To eliminate tial functions

capacitative were fitted

transients exponento the baseline and

on Na’

Channels

1309

subtracted from the single-channel records. Best-fit curves were calculated by a nonlinear, least squares method (Levenberg-Marquardt algorithm). Averaged data are expressed as mean f S.E.M. Statistical significance was assessed using paired samples I test and Wilcoxon test [Zq. Solutions and chemicals The bath solution used during the electrical recordings contained (in mM): KC1 20, Kglutamate 120, MgCl, 1, dextrose IO, EGTA 1, HEPES 10, pH= 7.4, adjusted with KOH. The isotonic K’ served to zero the cell membrane potential and thus enabled explicit quantification of the trans-patch potential. To record Na+ channels the pipette solution consisted of (in mM): NaCl200, BaCl, 1, CaCl, 2, HEPES 5, pH= 7.4, adjusted with NaOH; the high [Na’] increased the signal to noise ratio, while Ba2+ suppressed K+ current (notably I KATP [301).For K+ channel recordings the pipette contained (in mM): KC1 140, CaCl, 1, HEPES 10, pH= 7.4, adjusted with KOH. MgATP, IA.4 and DNP were purchased from Sigma.

Results

Metabolic

inhibition

induced hv Il.NP

or IA.4

In order to induce metabolic inhibition, we blocked either glycolysis or oxidative phosphorylation. Glycolysis was inhibited using IAA, which impairs the production of 1,3bisphosphoglycerate by inactivating the enzyme glyceraldehyde 3-phosphate dehydrogenase [27]. Oxidative phosphorylation was uncoupled by DNP [28] which dissipates the proton gradient across the inner membrane of the mitochondria [29]. To confirm the effectiveness of these two drugs as metabolic inhibitors under our experimental conditions, we monitored the activity of Z,..,, 1301. This channel provides a convenient bioassay for the subsarcolemmal ATP concentration, since its PO increases dramatically when [ATP] falls to micromolar levels [.%I. The result of such experiments is shown in Figure 1. In the control, patches were generally either devoid of activity or contained only the inward rectifier Zk,. With a variable latency after addition

1310

R. Mejia-Alvarez

(a)

and

E. Marban

Control

DNP

(b)

200

Membrane -100

-75

IAA I rw

PM

potential

(mV)

-50

-10 I K,ATP

Current

(PA)

of either 0.2 mM DNP [Fig. 1 (a), left] or 1 mM IAA [Fig. l(a), right] to the bath solution, a new type of single-channel activity became evident. This effect was observed 7-23 min (13.6 f 3.1 min; n = 5) after addition of either metabolic inhibitor. The new type of channel activity was identified as IK,ATP based on the observations that: (a) activity was completely blocked by 1 mM ATP (upon excision into the inside-out configuration, data not shown), and (b) the single-channel conductance equalled 95 pS [ 140 mM KC1 in the pipette solution, Fig. 1 (b)].

FIGURE 1. ATP sensitive K’ channel activity as an assessment of the efficacy of metabolic inhibition. Singlechannel activity was recorded in the cell-attached configuration, using 120 ms depolarizing pulses to -80 mV from - 100 mV of HP. (a) No single-channel activity was recorded in control conditions. After 20 min of exposure to 200 PM DNP in the extracellular solution (bottom, left) a new type of single-channel activity became evident. Single-channel opening events are displayed as downward deflections. The same phenomenon was observed when 1 rnM IAA was added to the extracellular solution (right). (b) The single-channel conductance calculated from the unitary current-voltage relation was 95 pS (140 rnM KC1 in the pipette solution). Filled circles reprrsent pooled data from five experiments. No single-channel openings were resolved at positive potentials.

The consistent activation of Z,,,,, within the first 23 min of exposure to either IAA or DNP provides strong evidence that Na+ channels would be exposed to similarly severe metabolic rundown in the subsarcolemmal region adjacent to the ionic channels within the time frame of our experiments. Efects of metabolic inhibition

on I,

amplitude

Figure 2(a) shows records of Na+ channel activity obtained in the cell-attached configuration from a ventricular myocyte.

Effects

of Metabolic

Inhibition

on Na’

Channels DNP

(a )

-

I

WM

_

-4OmV

-20

200

mV

5 PA L Elms

(b)

-120

Membrane

potentlal

-80

-40 +--..

m-0

Control

o-.-o

DNP 200 PM

(mV)

0

Current

40

+

(PA)

FIGCRL 2. Efiect of mrtaholic inhibition induced by DNP on Ix,,. (a) Na’ channel current rtwrdinqs nbtainvd with the cell-attached configuration. G~rrcnt~ ww viicited by 70 ms drpolarizin,q pulses to dilfcrcnt mcmbranc potentials (indicated hetwwn columns) from a HP 01 - 135 mV. The left hand column shows singlr-channl-I activity in control conditions. ‘Thr riqht hand , olnmrr shows the effect of 200~~ DNP. ihi 1-L’ cur\c