Effects of Pb2+ on Delayed-Rectifier Potassium Channels in Acutely Isolated Hippocampal Neurons

Michael Madeja1, Ulrich Mubeta hoff1, Norbert Binding2, Ute Witting2, and Erwin-Josef Speckmann1

1 Institut für Physiologie and 2 Institut für Arbeitsmedizin, D-48149 Muenster, Germany

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Madeja, Michael, Ulrich Mubeta hoff, Norbert Binding, Ute Witting, and Erwin-Josef Speckmann. Effects of Pb2+ on delayed-rectifier potassium channels in acutely isolated hippocampal neurons. J. Neurophysiol. 78: 2649-2654, 1997. The effects of Pb2+ on delayed-rectifier potassium currents were studied in acutely isolated hippocampal neurons (CA1 neurons, CA3 neurons, granule cells) from the guinea pig using the patch-clamp technique in the whole cell configuration. Pb2+ in micromolar concentrations decreased the potassium currents in a voltage-dependent manner, which appeared as a shift of the current-voltage relation to positive potentials. The effect was reversible after washing. The concentration-responsiveness measured in CA1 neurons revealed an IC50 value of 30 µmol/l at a potential of -30 mV. The half-maximal shift of the current-voltage relation was reached at 33 µmol/l and the maximal obtainable shift was 13.4 mV. For the different types of hippocampal neurons, the shift of the current-voltage relation was distinct and was 7.9 mV in CA1 neurons, 13.7 mV in CA3 neurons, and 14.2 mV in granule cells with 50 µmol/l Pb2+. The effects described here of Pb2+ on the potassium currents in hippocampal neurons and the differences between the types of hippocampal neurons correspond with the known properties and distributions of cloned potassium channels found in the hippocampus. As a whole, our results demonstrate that Pb2+ in micromolar concentration is a voltage-dependent, reversible blocker of delayed-rectifier potassium currents of hippocampal neurons. This effect has to be taken into consideration as a possible contributing mechanism for the neurological symptoms of enhanced brain activity seen during Pb2+ intoxication.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Exposure to high levels of lead (Pb2+) causes a variety of effects including the impairment of red blood cells, muscles, and intestine and also impairment of the brain. The neurological symptoms are both characteristic of decreased neuronal activity (e.g., coma and paralysis) as well as of enhanced activity (e.g., agitation and seizures). Investigations on the mechanisms of Pb2+ intoxication revealed primarily depressant effects on voltage-operated calcium channels (Audesirk and Audesirk 1991; Büsselberg et al. 1991a,b; Evans et al. 1991), N-methyl-D-aspartate receptors (Alkondon et al. 1990; Uteshev et al. 1993), and adenylate-cyclase (Nathanson and Bloom 1976). The disturbance of calcium channel function by Pb2+ especially has been investigated intensively and is regarded as a main mechanism of Pb2+ neurotoxicity (Audesirk 1993).

The action of Pb2+ on voltage-operated potassium channels, however, has only been studied marginally (Audesirk 1993), although potassium currents are one of the major mechanisms for controlling and setting the neuronal excitation level (Hille 1992). Thus a possible impairment of potassium currents might play a role in the enhancement of neuronal activity seen during Pb2+ intoxication. To fill part of this gap, the effects of Pb2+ on delayed-rectifier potassium channels in mammalian neurons were investigated in the present study. The experiments were done with hippocampal neurons because the hippocampus is of special interest in neurotoxicity due to its possibly marked Pb2+ accumulation (Grandjean 1978, but see also Widzowski and Cory-Slechta 1994). Among the different voltage-operated potassium channels, the delayed-rectifier channels were chosen because they could be investigated without pharmacological blocking of other conductances, a procedure that might interfere with the Pb2+ actions at the potassium channels.

In this report, it is shown for the first time that Pb2+ can depress delayed-rectifier potassium currents in neurons. The depression is caused by shifts of the current-voltage relation to positive potentials and is distinctive for the differenttypes of hippocampal neurons. Thus effects of Pb2+ on voltage-operated potassium channels have to be taken into consideration as a mechanism possibly contributing to the neurological symptoms of enhanced brain activity seen in Pb2+ poisoning.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Nerve cell isolation

The experiments were carried out on hippocampal neurons of adult guinea pigs (weight: 300-400 g). During ether anesthesia, the brain was removed. The hippocampus was dissected and transverse slices (thickness: 400-500 µm) were cut in parallel to the alvear fibers with a McIlwain tissue chopper. The neurons were isolated acutely according to the technique of Kay and Wong (1986) modified as described by Vreugdenhil and Wadman (1995). In short, the regions of the CA1 neurons, CA3 neurons and granule cells were dissected into sections of ~1 mm2. These tissue pieces were incubated for 75 min at 32°C in oxygenated dissociation solution containing (in mmol/l) 120 NaCl, 5 KCl, 20 piperazine-N,N'-bis[2-ethanesulfonic acid], 1 CaCl2, 1 MgCl2, and 25 D-glucose and 1 mg/ml trypsin (40 U/mg, Merck); pH was 7.0. Thereafter, the tissue was washed with enzyme-free solution and was kept at room temperature. Neurons were isolated by triturating the tissue pieces through a series of pasteur pipettes with decreasing tip diameter. After settling on the bottom of the recording chamber, neurons with bright and smooth appearance and no visible organelles were selected for recording.

Electrophysiological techniques

Voltage-clamp recordings were performed in the whole cell patch-clamp configuration (Hamill et al. 1981). Patch pipettes were pulled from borosilicate glass and had resistances between 2 and 4 MOmega . The pipettes were filled with an intracellular recording solution (in mmol/l) 140 KF, 1 CaCl2, 2 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 0.5 MgATP; pH set at 7.4. The acutely isolated neurons were superfused with an extracellular solution of the following composition (in mmol/l) 130 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 25D-glucose, and 10 HEPES; pH set at 7.4. Pb2+ (1-100 µmol/l; chloride salt), tetraethylammonium (TEA, 100 mmol/l; chloride salt), 4-aminopyridine (4-AP, 5 mmol/l), Ni2+ (0.8 mmol/l; chloride salt), and tetrodotoxin (TTX, 300 nmol/l) were added to the extracellular solution and were applied with a perfusion pipette directly to the recorded neuron. The Pb2+ solution was prepared just before each experiment to avoid precipitation. All substances were applied 30 s before starting the pulse protocols.

Currents were recorded with a List EPC-7 amplifier. Capacitive transients and series resistances were compensated for >80%. After seal formation and membrane rupturing, the nerve cells were allowed to stabilize for 3 min before starting the pulse protocols. Holding potential was -70 mV. Depolarizing voltage pulses were applied to elicit potassium currents. The pulses ranged up to +60 mV in steps of 10 mV and had durations of 600 ms. All experiments were carried out at room temperature (23 ± 1°C). The results were obtained from 36 neurons.

Data acquisition and analysis

Currents were filtered with an 8-pole Bessel filter at a frequency of 1 kHz and were transferred to a computer system (pClamp program, Axon Instruments). Leakage currents were subtracted on-line using the p/4 method (Bezanilla and Armstrong 1977). The amplitudes of the outward currents were measured at the end of the voltage step.

The current-voltage relations were obtained by normalizing the current amplitudes to the value at 0 mV under control conditions. The so-obtained individual or mean values were fitted to the Kuhlmann function y = a(1 - exp(bV))V, where y is the normalized current amplitude, a is the slope of the linear part of the function, b is the factor determining the increase of slope, and V is the voltage difference (measured from the 0 current voltage step at which the potassium currents starts to increase). Further, the function was used to determine the threshold potentials of activation and the Pb2+-induced shift of the current-voltage relations. The threshold potential of activation was determined as the voltage at which the current level had reached 0.05 of the normalized current amplitude. The shift of the current-voltage relations was measured as the voltage difference between the current-voltage relations under control and under Pb2+ at the normalized current of 0.5.

The conductance-voltage relations were obtained by normalizing the conductance data to the value at +60 mV under control conditions. The means of the normalized currents were fitted with the Boltzmann function y = a/[1 + exp((V1/2 - V)/b)], where y is the normalized conductance, a is the maximal conductance, V1/2 is the potential of the half-maximal conductance, V is the voltage, and b is the slope. The shift of the conductance-voltage relations was measured as the voltage difference between the curves under control and under Pb2+ at the normalized conductance of 0.5.

The concentration-response curve was determined by fitting the mean currents to the Langmuir equation y = (Km/c)n/[1 +(Km/c)n], where y is the fraction of inhibition of control current, Km is the dissociation constant, c is the concentration of Pb2+, and n is the Hill coefficient. The shifts of the current-voltage relations at different concentrations were fitted to the modified equationy = b(Km/c)m/[1 + (Km/c)m], where y is the shift of the current-voltage relation, b is the maximal possible shift, and m is the Hill coefficient obtained from the fit of the concentration-response curve.

The values are given as means ± SE. The differences of values under control and test conditions were tested for significance using t-test.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Separation of delayed-rectifier potassium currents

Potassium currents were studied without pharmacological blockade of other voltage-operated conductances. This was done to avoid a possible interference of Pb2+ with the several blocking agents at the potassium channel molecule. To obtain the delayed-rectifier potassium currents, 1) amplitudes of outward current were measured at the end of a 600-ms voltage step. During this time, the fast sodium currents and part of the calcium currents were inactivated (cf. Fig. 1A) (Hille 1992). 2) The holding potential was set to -70 mV to further inactivate other voltage-operated channels, especially A-type potassium currents and calcium-activated potassium currents (Numann et al. 1987). 3) Only cells with short neuronal processes were taken because it was found that they had predominantly noninactivating outward currents, whereas the contribution of A-type potassium currents was small. 4) The ATP concentration in the intracellular solution was low, and no protease inhibitors were added, allowing run-down of calcium currents. And 5) pulse protocols were terminated at 0 mV because, up to this potential, the outward currents could be blocked by the potassium channel blockers TEA and 4-AP to <5% of control current (n = 7; Fig. 1A, TEA + 4-AP) (Klee et al. 1995). Thus under the described experimental conditions, the outward currents at the end of the voltage steps can be assumed to be carried mainly through delayed-rectifier potassium channels.


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FIG. 1. Effect of Pb2+ on potassium currents in CA1 neurons. Acutely isolated cells from the guinea pig hippocampus. Original recordings (A), current-voltage relations (B), and conductance-voltage relations (C) of outward currents under control conditions (CTRL; open circle , - - -) and under Pb2+ (50 µmol/l; bullet ------). CTRL 1 and 2 are recordings before application and after wash-out of Pb2+, respectively. TEA + 4-AP shows suppression of outward currents by 100 mmol/l tetraethylammonium (TEA) and 5 mmol/l 4-aminopyridine (4-AP). Irel, potassium current normalized to the control current at 0 mV; MP, membrane potential; Vh, holding potential; Grel, relative conductance normalized to the control value at +60 mV. In C, the calcium channel blocker Ni2+ and the sodium channel blocker tetrodotoxin (TTX) had been added; the Pb2+ concentration was 100 µmol/l. Graphic evaluations show mean values and SE of 5-7 experiments each.

Effects of Pb2+ on potassium currents in CA1 neurons

In CA1 neurons, delayed-rectifier potassium currents were elicited by voltage steps to potentials more positive than -50 mV; the threshold potential (i.e., the potential at which 0.05 of the normalized current at 0 mV was reached) obtained from the fits was -42.8 ± 1.1 (SE) mV (n = 12; Fig. 1). The currents showed a slow inactivation during the 600-ms voltage step, which did not exceed 30% of the peak current amplitude (n = 12). The mean amplitude measured at the end of the voltage pulse was 1.2 ± 0.2 nA (n = 12).

With application of Pb2+, the current-voltage relation was shifted into positive direction; the shift by 50 µmol/l Pb2+ was 7.9 ± 0.8 mV (Fig. 1B; n = 7). The threshold potential of activation was changed to -33.4 ± 1.6 mV (n = 7). The slope of the current-voltage relation was only slightly affected by Pb2+ and was decreased by <17% of control value. In all cases tested, the effects were reversible after removal of Pb2+ (Fig. 1A, CTRL1 and CTRL2; n = 5).

To investigate if the decrease of potassium currents by Pb2+ is caused by a shift of the current-voltage relation and is not due to a reduction over the whole potential range, current steps to +60 mV were applied under control conditions and under 100 µmol/l Pb2+ (Fig. 1C; n = 5). Because of the positive potentials, in this series of experiments, calcium and sodium conductances were blocked by adding Ni2+ and TTX, which abolished the inward current but did not decrease the outward current. Fitting the mean conductance values under control and under Pb2+ with Boltzmann equations revealed that the maximal conductances were nearly identical (0.93 for control and 0.97 for Pb2+; Fig. 1C). Furthermore, the slope of conductance was similar (reduction by <12% under Pb2+), and the shift of the conductance-voltage relation by 100 µmol/l Pb2+ was 19.7 mV. Thus the data reveal that the effect of Pb2+ is a shift in the voltage dependence of potassium current activation to positive potentials.

The concentration-responsiveness was studied by measuring current-voltage relations under several concentrations of Pb2+ (Fig. 2A). With increasing Pb2+ concentrations, the shifts of the current-voltage relations were augmented (Fig. 2B; n = 5-7). The threshold concentration was found to be at ~1 µmol/l Pb2+ (n = 5). The threshold potential was -41.9 ± 2.7 mV for 1 µmol/l, -36.6 ± 0.7 mV for 10 µmol/l, and -28.9 ± 3.1 mV for 100 µmol/l Pb2+. The mean values of the amounts of current suppression at -30 mV were fitted to a Langmuir equation (Fig. 2C). The fit gave a Hill slope of 1.1 and a Pb2+ concentration needed for a reduction to half of the control value (IC50) of 30 µmol/l. This IC50 value, however, strongly depended on the potential. For example, at a potential of 0 mV, even a concentration of 50 µmol/l Pb2+ did not depress the potassium currents to 50% (Fig. 1B). In contrast, at a potential of -40 mV, the IC50 value was ~10 µmol/l (Fig. 2B). Thus to have a potential-independent parameter for the Pb2+ effect, the shifts of the current-voltage relations at different Pb2+ concentrations were analyzed. The mean shift values were fitted to the modified Langmuir equation. The fitted curve revealed an IC50 value of 33 µmol/l and a maximal obtainable shift of 13.4 mV (Fig. 2D).


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FIG. 2. Dependence of potassium current decrease on the concentration of Pb2+ in CA1 neurons. Acutely isolated cells from the guinea pig hippocampus. A: original recordings of potassium currents under control (CTRL) and under Pb2+ in concentrations of 1, 10, and 100 µmol/l. Calibrations: 500 pA and 100 ms. B: graphic evaluations of current-voltage relations derived from recordings as shown in A. open circle  and - - -, currents under control conditions; bullet  and ------, currents in the presence of Pb2+. Irel, potassium current normalized to the control current at 0 mV; MP, membrane potential; Vh, holding potential. C: dose-response curve for the Pb2+ effects at-30 mV. Mean values were fitted to a Langmuir equation (Hill coefficient: 1.1; dissociation constant: 30 µmol/l). Ifr, fraction of current under control conditions. D: dose-response curve for the shifts of the current-voltage relationships by Pb2+. Mean values were fitted to a modified Langmuir equation with a Hill coefficient of 1.1 (dissociation constant: 33 µmol/l; maximal obtainable shift: 13.4 mV). All symbols represent mean values ± SE of 5-7 cells each.

Pb2+ effects in different types of hippocampal neurons

Current-voltage relations of the other two types of hippocampal pyramidal neurons (CA3 neurons and granule cells) were recorded under control conditions and under 50 µmol/l Pb2+ (Fig. 3). Under control conditions, the threshold potential was -46.9 ± 1.1 mV for the CA3 neurons (Fig. 3A; n = 5) and -52.6 ± 1.1 mV for the granule cells (Fig. 3B; n = 6), thus being more negative than that of the CA1 neurons (Fig. 1B). Also in these neurons, a slow inactivation of the potassium current during the voltage step was found. However, the decrease of the current amplitude was <20% of the peak current in CA3 neurons and <25% in granule cells. The mean amplitude of the potassium current was3.4 ± 0.7 nA for the CA3 neurons (n = 5) and 2.3 ± 0.4 nA for the granule cells (n = 6).


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FIG. 3. Effects of Pb2+ on potassium currents in CA3 neurons (A) and granule cells (B). Acutely isolated cells from the guinea pig hippocampus. open circle  and - - -, currents under control conditions (CTRL); bullet  and ------, currents in the presence of Pb2+ (50 µmol/l). Irel, potassium current normalized to the control current at 0 mV. Symbols represent mean values ± SE of 5-6 cells each. MP: membrane potential. Vh, holding potential. Insets: original recordings; duration of voltage steps: 600 ms; amplitude of control current at 0 mV for the CA3 neuron and for the granule cell: 4.6 nA and 1.7 nA, respectively.

Correspondingly, in all types of neurons, the basic effect of Pb2+ was a shift of the current-voltage relations into positive direction. With 50 µmol/l Pb2+, the threshold potential of activation was changed to -29.3 ± 1.3 mV in the CA3 neurons (n = 5) and to -36.6 ± 1.6 mV in the granule cells (n = 6). The mean of the slopes of the current-voltage relation was increased to 1.07 ± 0.16 of control value inthe CA3 neurons and to 1.21 ± 0.11 in the granule cells(n = 5-6); however, the changes were statistically not significant.

In contrast to these aforementioned findings and corresponding with the changes in threshold potentials, the amount of shift was different for the various hippocampal neuron types. The means of shifts with 50 µmol/l Pb2+ for the CA3 neurons and granule cells were 13.7 ± 2.6 mV(n = 5; Fig. 3A) and 14.2 ± 2.0 mV (n = 6; Fig. 3B), respectively. Thus the shifts of these neurons were larger than those found for the CA1 neurons (cf. Fig. 1B). The differences in mean shift of the CA3 neurons and granule cells to those of the CA1 neurons were statistically significant with P < 0.02.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The results of the present study have shown that delayed-rectifier potassium currents of hippocampal nerve cells are reduced by Pb2+. Up until now, however, no or only negligible effects of Pb2+ on these potassium currents in neurons have been described in literature. Thus in dorsal root ganglion cells, Evans et al. (1991) reported reductions of the delayed-rectifier potassium currents by only 4% with 1 µmol/l Pb2+ at a potential of 0 mV. In Aplysia neurons, the potassium currents were not affected with Pb2+ in concentrations up to 200 µmol/l at a Vc of +20 mV (Büsselberg et al. 1991a). Furthermore, potassium currents in human neuroblastoma cells were found not to be significantly altered at a potential of +50 mV by 10 µmol/l Pb2+ (Reuveny and Narahashi 1991). Finally, Talukder and Harrison (1995) found reductions of delayed-rectifier currents in cultured rathippocampal neurons only at concentrations of >100 µmol/lPb2+, although significant shifts of the current-voltage relation of transient outward currents were described in the lower micromolar concentration range.

On the basis of past results, it was concluded that Pb2+ has no significant effect on neuronal potassium channels, whereas the present study has shown that Pb2+ in micromolar concentrations exerts marked effects on hippocampal potassium channels. Three lines of observation may explain this discrepancy. First, most of the above-mentioned investigations dealt primarily with calcium channels. Thus only screening tests were performed on potassium currents investigating the effects of Pb2+ at only one potential, which was quite positive in all cases. Because the basic effect of Pb2+ is a shift of the current-voltage relation, the small effects are probably due to the decreasing Pb2+ efficacy at positive going potentials. Therefore, more pronounced effects might have existed at more negative potentials in the preparation used in past research. Second, the appearance of the different potassium channel types in the above-mentioned cells has not been studied until now. Therefore, it is possible that some of the investigated cell types contained other and possibly less Pb2+-sensitive potassium channels compared with the channels studied here of the hippocampus. This idea is in line with investigations on calcium channels that showed that the calcium currents of cortical neurons were more sensitive to Pb2+ than those of neuroblastoma cells or snail neurons (Audesirk 1993). Third, several agents, e.g., TEA and Cd2+ were used to block other voltage-operated conductances in some of the papers mentioned here. These substances might interfere with the binding site of Pb2+ at the potassium channel. Thus the number of Pb2+ vulnerable channels would be decreased and, with that, the efficacy of Pb2+ could be reduced.

This paper has focused on delayed-rectifier potassium channels. A pharmacological separation from other voltage-operated conductances was avoided because an interference of the blocking agents (especially of divalent cations for blocking calcium channels) with Pb2+ at the ion channel molecule cannot be excluded. However, with the protocol used here, there is evidence that other voltage-operated conductances do not contribute to the outward currents. Thus the almost complete blockade of the outward current by TEA and 4-AP indicates that it is due to a potassium current. The lack of a long-lasting inward current under these conditions indicates that at least most calcium currents have run-down. Furthermore, a significant contribution of a calcium-dependent potassium current to the whole potassium current is unlikely because the outward currents are not reduced after adding the calcium channel blocker Ni2+ and because, in principle, the same effects of Pb2+ were found with and without blocked calcium channels. Therefore, it can be assumed that the studied outward currents are due to the activation of potassium channels.

The effect of Pb2+ described in this paper is primarily a shift of the current-voltage relation of the outward currents to more positive potentials. Divalent cations have been found to shift current-voltage relations in the same direction, presumably through a change of the ion channel surface potentials (Hille 1992; Kostjuk et al. 1982). For the effect of Pb2+ on the potassium channels, however, a change of surface potentials appears unlikely because comparable effects on surface potentials have been reported only with divalent cation concentrations in the millimolar range (Hille et al. 1975), whereas the effects of Pb2+ on cloned potassium channels were found in the micromolar range (Madeja et al. 1995). Furthermore, application of the divalent cations magnesium and calcium to cloned neuronal potassium channels expressed in oocytes of Xenopus laevis could not mimic the Pb2+ effect, which, for the cloned channels, was also a shift of the current-voltage relation to more positive potentials (Madeja et al. 1995).

The different shifts of the current-voltage relation in the three types of hippocampal neurons is probably due to different distributions and properties of the potassium channel types in these neurons. Experiments on several cloned hippocampal potassium channels expressed in oocytes of X. laevis have shown distinct sensitivities for the different channel types (Madeja et al. 1995). The shift of the current-voltage relation by 50 µmol/l Pb2+ was 28 mV for the Kv1.1 channel (Madeja et al. 1995), 18 mV for the Kv1.2 channel, and 2 mV for the Kv2.1 channel. Thus the shift in the hippocampal neurons is in this range and corresponds to a mixed expression of the delayed-rectifier potassium channels. Furthermore, the small shift in the CA1 neurons is obviously due to the missing expression of Kv1.1 channels in this cell type (Kues and Wunder 1992). Thus the CA1 neurons do not have the most sensitive potassium channel tested so far and predominantly contain channels more insensitive to Pb2+.

In summary, Pb2+ in micromolar concentrations has been found to be a voltage-dependent, reversible blocker of delayed-rectifier potassium channels of hippocampal neurons. Concerning the clinical semiology of Pb2+ poisoning, this effect has to be taken into consideration as a possible contributing mechanism for the neurological symptoms of enhanced brain activity seen during severe Pb2+ intoxication.

    ABBREVIATIONS

Pb2+ lead
PIPES piperazine-N, N'-bis[2-ethanesulfonic acid]
HEPES N-[2-Hydroxyethyl]piperazine-N[prime]-[2-ethanesulfonic acid];
TEA tetraethylammonium
4-AP 4-aminopyridine
Ni2+ nickel
TTX tetrodotoxin

    ACKNOWLEDGEMENTS

  We are very grateful to M. Vreugdenhil and W. J. Wadman for the introduction into the technique of acute isolation and investigation of hippocampal neurons. We thank G. Goder for help in the fitting procedures.

    FOOTNOTES

  Address for reprint requests: M. Madeja, Institut für Physiologie, Robert-Koch-Str. 27 A, D-49149 Muenster, Germany.

  Received 19 September 1996; accepted in final form 3 July 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society




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