INTRODUCTION

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ELEVATED SYMPATHETIC outflow contributes to the pathogenesis of elevated arterial blood pressure in the spontaneously hypertensive rat (SHR) model of essential hypertension (4, 14, 15, 23, 25, 34, 35). Although the mechanisms remain to be completely elucidated, alterations of neuronal membrane properties may underlie the exaggerated sympathetic outflow. Basal firing rates in central cardiovascular areas of SHR are elevated compared with normotensive rat strains (4, 5, 24), and activation of these areas by stress or direct stimulation elicits exaggerated pressor and sympathetic nerve responses in the SHR (4, 16, 21, 42). Hyperexcitability has been observed in peripheral sympathetic neurons as a loss of spike accommodation (43) that is present in neonatal SHR neurons (22). Additionally, synaptic efficacy is enhanced in the sympathetic ganglion of SHR (27) via increased transmitter release from preganglionic nerve terminals (26), and catecholamine release is elevated at the SHR neuroeffector junction (41). However, the cellular and ionic mechanisms responsible for the elevated excitability of SHR sympathetic neurons remain unclear.

The A-type K⁺ current (I_A), first described by Hagiwara et al. (18) and subsequently characterized by Connor and Stevens (10), is a prime candidate to underlie hyperexcitability in SHR sympathetic neurons. I_A is a transient outward current that activates negative to the threshold for action potential generation and is distinguished by exponential inactivation kinetics after activation. The current is partially inactivated at resting membrane potentials and shows voltage-dependent recovery from inactivation. In terms of cell electrical behavior, these properties allow I_A to provide a rapid but transient hyperpolarizing influence to counter excitatory stimuli by holding the membrane potential away from the threshold (20). In peripheral sympathetic neurons, I_A regulates firing behavior (8), is involved in the integration of synaptic potentials (8), and contributes to action potential repolarization (1, 2). In other preparations, I_A has been suggested to modulate the release of neurotransmitter from nerve terminals (37).

Given the role of I_A in the modulation of neuronal excitability, we examined I_A of sympathetic neurons from SHR and Wistar-Kyoto (WKY) rats to test the hypothesis that alterations in the properties of I_A play a pivotal role in the hyperexcitability of SHR sympathetic neurons and thereby in the development of hypertension in this model. Because I_A has a negative influence on excitability, we expected that the amplitude of I_A would be smaller in SHR neurons. However, I_A amplitudes were not different between adult SHR and WKY neurons when evoked by depolarization from normal physiological potentials. Because the amplitude of I_A depends on multiple properties of the current, we undertook a more detailed characterization of I_A which showed that inactivation gating is altered in adult SHR neurons in a manner consistent with hyperexcitability, but the rats appear to compensate by maintaining a high density of I_A to offset the changes in inactivation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal groups and arterial blood pressures. Male WKY rats and SHR (Harlan Sprague Dawley, Indianapolis, IN) were housed in a temperature- and light-controlled room and maintained on a normal sodium-containing diet with water ad libitum. Sympathetic neurons were isolated from age-matched SHR and WKY rats before hypertension and during established phases of hypertension. The prehypertensive group was 4-6 wk of age, and the hypertensive group was 12-16 wk of age. A separate group of prehypertensive SHR were treated continuously with 25 mg/l enalapril maleate (Sigma Chemical, St. Louis, MO) in drinking water to prevent the development of hypertension. For control purposes, age-matched WKY rats were also treated continuously with enalapril (25 mg/l in drinking water). Neurons were isolated from enalapril-treated animals after 12 wk of age, such that they were age matched with the non-enalapril-treated adults. All treatments and manipulations were approved by the Institutional Animal Care and Use Committee.

Preparation of single neurons. Single superior cervical ganglion (SCG) neurons were isolated by enzymatic dispersion (9). Briefly, after decapitation, both SCG were dissected under iced Hanks' balanced salt solution (HBSS), freed of connective tissue, minced, then transferred into 5 ml of Earl's balanced salt solution modified with 0.5 mg/ml trypsin, 1.0 mg/ml collagenase D (both from Boehringer Mannheim Biochemicals, Indianapolis, IN), 0.1 mg/ml DNase (type I), 20 mM glucose, 10 mM HEPES, pH 7.4 (NaOH), and 0.22 g/l NaHCO₃. SCG fragments were incubated in a shaking water bath at 34°C under an atmosphere of 5% CO₂-95% O₂ for 1 h. Ganglion fragments were then shaken vigorously to disperse single neuronal somata. The enzymatic digestion was stopped with 5 ml of HBSS containing 10% fetal bovine serum (GIBCO BRL, Gaithersburg, MD), 10 mM CaCl₂, and 10 mM HEPES. The resulting cell suspension was centrifuged (50 g for 5 min), and the pellet was resuspended in the modified HBSS described above. Cell preparations were routinely stored at room temperature until use (0.5-8 h), although on occasion cells were stored overnight in a humidified chamber at 4°C; such storage had no discernible effects on the characteristics of I_A (not shown).

Whole cell voltage clamp. Dispersed SCG neurons were voltage clamped in the whole cell configuration of the patch-clamp technique (19) with use of an Axopatch 1-C or 200A amplifier (Axon Instruments, Foster City, CA). Patch electrodes were pulled from N51A borosilicate capillary tubing (Garner Glass, Claremont, CA) with use of a micropipette puller (model P80-PC, Sutter, Novato, CA) and coated with Sylgard (Dow Corning, Midland, MI). Electrodes had resistances of 0.5-3.0 M when filled with internal solution. Membrane currents were filtered at 2.0 kHz with a four-pole low-pass Bessel filter, digitized at 10 kHz with a 12-bit analog-to-digital converter (GW Instruments, Summerville, MA), and stored for analysis on a Macintosh IIci computer. Voltage protocols were generated from a 12-bit digital-to-analog converter (GW Instruments) with use of the S3 data acquisition package (S. R. Ikeda, Guthrie Institute, Sayre, PA). The external solution contained (in mM) 130.0 sodium isethionate, 5.4 KMeSO₄, 4.0 MgCl₂, 1.0 CoCl₂, 10.0 HEPES, 10.0 glucose, and 0.0001 tetrodotoxin. The pH was adjusted to 7.4 with NaOH, and osmolality was adjusted to 300 mosmol/kg with sucrose. The pipette solution contained (in mM) 110 KMeSO₄, 5.0 KCl, 10.0 HEPES, 0.44 EGTA-KOH, 4.0 Na₂ATP, and 0.5 Na₂GTP. The pH was adjusted to 7.4 with KOH, and osmolality was adjusted to 285 mosmol/kg with sucrose. These solutions provided a means to isolate K⁺ currents from other potentially contaminating currents (38). Where possible, I_A was assayed at 30 mV without contamination by other outward currents. For voltage protocols requiring voltage steps positive to 30 mV, I_A was isolated by digital subtraction (see Fig. 2A). All recordings were made at room temperature (24-26°C).

Analysis and statistical evaluation. Current records were analyzed and fitted using IgorPro software (WaveMetrics, Lake Oswego, OR) running on a Macintosh Q630 computer. Current-voltage (I-V) relationships were corrected for linear leakage as determined from hyperpolarizing voltage steps or ramps. Cell membrane capacitances measured by integrating the capacitative transient in response to a +10-mV voltage step were 26.6 ± 8.0 (SD) pF in adult SHR, 28.9 ± 11.2 pF in adult WKY rats, 39.5 ± 17.1 pF in enalapril-treated SHR, 37.6 ± 14.2 pF in enalapril-treated WKY rats, 18.4 ± 5.1 pF in prehypertensive SHR, and 21.3 ± 7.3 pF in prehypertensive WKY rats. Series resistances measured from membrane capacitance and time constants of capacitative transient decay were 4.2 ± 1.4 (SD) M in adult SHR, 3.7 ± 1.2 M in adult WKY rats, 4.0 ± 1.1 M in enalapril-treated SHR, 4.0 ± 1.4 M in enalapril-treated WKY rats, 5.2 ± 1.9 M in prehypertensive SHR, and 5.3 ± 1.9 M in prehypertensive WKY rats. Periods of 200-400 µs at the onset and offset of command pulses were excluded from the current records shown to remove spurious points due to residual capacitative transients. To circumvent contamination of instantaneous I-V relationships by capacitative transients, tail current amplitudes were extrapolated from single-exponential fits of current relaxations 0.6-10 ms after repolarization. Reversal potentials were interpolated by linear regression of instantaneous I-V relationships. I_A parameters measured from multiple neurons isolated from the same rat were averaged for statistical comparisons, thus n values correspond to the number of animals examined. Values are means ± SE, except where noted. Significance was evaluated by ANOVA, with significance between groups determined by Fisher's protected least significant difference post hoc test. P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

K⁺ currents in general have a negative influence on neuronal excitability. In sympathetic neurons, I_A activates rapidly on depolarization to potentials negative to the action potential threshold, which enables I_A to significantly influence excitability. Decreased amplitudes of I_A could therefore contribute to the hyperexcitability reported in SHR sympathetic neurons (22, 43). To compare I_A in SHR and WKY neurons at physiological membrane potentials, neurons were held at 70 mV, a potential expected during afterhyperpolarizations after action potentials (11), and assayed with a step to 30 mV to mimic a suprathreshold depolarization. Representative currents generated with this protocol are shown in Fig. 1A. Interestingly, I_A recorded from SHR neurons displayed no obvious differences in amplitude or time course compared with those from WKY rats. Figure 1B shows current amplitudes measured from adult SHR (299 ± 37 pA, n = 13) and WKY neurons (304 ± 31 pA, n = 10). Although there was no difference in the current amplitudes when evoked from physiologically relevant membrane potentials, the amplitude of I_A depends on activation and inactivation parameters, and comparisons under such limited conditions could mask alterations in the current. We therefore decided to undertake a complete investigation of the properties of I_A activation and inactivation.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Amplitudes of A-type K+ current (IA) are similar in sympathetic neurons of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). A: membrane currents recorded from WKY and SHR neurons in response to 80-ms steps to 30 mV from 70 mV. IA reached maximal activation within 10 ms and subsequently inactivated over remainder of step. Records lack current relaxations or "tail currents" on repolarization, since inactivation is nearly complete by end of step and there is little to no contribution from slowly activating outward currents such as delayed rectifier K+ current. Records have not been corrected for linear leakage currents. B: peak current amplitudes (means ± SE) summarized for WKY (39 neurons from 10 rats) and SHR (35 neurons from 13 rats). Functional availability of IA was not significantly different between adult SHR and adult WKY neurons.

Current density. We investigated the I-V relationship of I_A after conditioning neurons at 120 mV for 1 s to effect complete recovery of the current from inactivation. Representative currents isolated by digital subtraction are shown in Fig. 2B. At test potentials positive to 40 mV, I_A rapidly activated, reaching maximal amplitude over 7 ms, then decayed over the remainder of the step. Currents from SHR neurons were greater in amplitude than those from WKY neurons. The SHR currents shown were nearly double the amplitude of those from the WKY rats, although on average, SHR currents were 37% greater than the WKY currents. Such a difference could result from differences in cell size. Therefore, I-V relationships are displayed as current density after normalization to cell capacitance, which is proportional to cell surface area (Fig. 2C). The peak I_A density (at +40 mV) in adult SHR neurons was 276 ± 21 pA/pF (n = 13) in adult SHR neurons and significantly (P < 0.05) increased compared with the density in WKY neurons (201 ± 12 pA/pF, n = 10). In contrast to the previous experiment that examined I_A amplitudes at physiological potentials, conditions that maximally activate the current after complete recovery from inactivation demonstrate that I_A density in the SHR is actually elevated.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A: subtraction protocol used to isolate IA from other K+ currents at voltages positive to 30 mV. Holding for 1.0 s (Vpre) at 120 mV results in recovery of IA from inactivation, and subsequent steps to +10 mV elicit rapidly activating IA and a sustained current. Holding at 50 mV inactivates IA, and on depolarization only a slowly activating, sustained current activates. Subtraction of currents evoked from different holding potentials reveals IA in isolation. B: IA elicited by 80-ms voltage steps ranging from 40 to +40 mV (10-mV increments) isolated by digital subtraction. Records contain one-fourth the number of points originally sampled. C: IA density is elevated in adult SHR sympathetic neurons. Current amplitudes measured 7 ms after step were normalized to cell capacitance and plotted against membrane potential for adult SHR and adult WKY neurons. IA was evident at steps positive to 50 mV. D: voltage ranges of activation in adult SHR and WKY neurons. No statistically significant differences were found between SHR and WKY neurons over range of potentials examined. G, conductance; Gmax, maximum conductance. Solid lines, fits of data to a Boltzmann function (see RESULTS). Values in C and D are means ± SE from 39 neurons from 10 WKY rats and 35 neurons from 13 SHR; SE bars were omitted where range was contained within symbols.

I_A activation properties. Elevated current density in SHR neurons could result from a change in the voltage dependence of activation of the current that allows a greater fraction of the channels to gate across the voltage range where the current density was measured. We investigated this possibility by transforming I-V relationships to chord conductance after confirming that the instantaneous I-V relationship for I_A was linear (38) (not shown). Chord conductance was calculated as follows: G = I_A/(V_m  E_K), where G is conductance, V_m is the test potential, and E_K is the reversal potential estimated to be 70 ± 1 mV (n = 6 cells) from instantaneous I-V relationships. To obtain the maximum conductance (G_max), the voltage where I_A activation was half-maximal (V_h), and the maximal slope (k) of activation, the resulting activation curves were fit to a modified Boltzmann equation: G = G_max/{1 + exp[(V_m  V_h)/k]}. Mean activation curves for adult SHR and WKY rats are shown in Fig. 2D. The V_h measured in neurons from adult SHR (16.8 ± 1.3 mV, n = 13) was not different from that measured in neurons from WKY neurons (14.0 ± 1.6 mV, n = 10). Complete activation curves could not be obtained for neurons from two SHR, inasmuch as the currents exceeded 20 nA and therefore were beyond the limit of clamp control. The slope (k), a measure of the sensitivity of A-type K⁺ channels to membrane potential, was not different in SHR and WKY neurons. I_A conductance increased e-fold per 14.7 ± 0.7 mV in the adult SHR and 14.6 ± 0.4 mV in neurons from the adult WKY rats. Thus altered activation parameters are not responsible for elevating I_A density in SHR neurons.

Inactivation properties. The amplitude of I_A when elicited from a holding potential of 70 mV was not different between SHR and WKY neurons, despite elevation of the maximal current density in the SHR (Fig. 1B). A viable explanation for this apparent contradiction is that a greater fraction of I_A was inactivated by holding at 70 mV, which masked the elevated current in the SHR. This was confirmed by measuring the amplitude of I_A elicited at a constant test potential from a range of holding potentials with use of a double-pulse protocol. Specifically, neurons were conditioned by 1-s pulses to voltages ranging from 120 to 40 mV to allow inactivation to reach a steady state, and the fraction of current remaining was then measured by a subsequent test pulse to 30 mV. Representative currents generated with this double-pulse protocol are shown in Fig. 3A and demonstrate that current amplitude decreased as the conditioning prepulse was progressively depolarized. Thus the fraction of channels inactivated, and therefore unable to pass current, increased with depolarization. The SHR currents were approximately double the amplitude of the WKY currents but are scaled to the same size to compare the inactivation kinetics. Single-exponential fits of current decay over the potential range 40 to +10 mV confirmed that inactivation time courses were not different between SHR and WKY neurons (not shown). Moreover, the time course of recovery from inactivation was also not different between SHR and WKY neurons (not shown). Figure 3B shows current amplitudes measured 7 ms after the test pulse, plotted against the conditioning potential, and normalized to maximum to emphasize the fraction of current elicited from each conditioning potential. To obtain the voltage at which 50% of the current inactivated (half-maximal inactivation potential, V_h), the maximal current amplitude (I_max), and the maximal slope of inactivation (k), the data were fit to a modified Boltzmann function: I_A = I_max/{1 + exp[(V_h  V_m)/k]}. The V_h for adult SHR neurons was 81.1 ± 1.7 mV (n = 15) and significantly (P < 0.05) shifted to more negative voltages compared with the V_h for adult WKY neurons (V_h = 75.1 ± 1.4 mV, n = 10). The slope factor, k, a measure of the sensitivity of I_A inactivation to membrane potential, was not different between SHR and WKY neurons. I_A amplitudes decreased e-fold per 6.9 ± 0.4 mV change in membrane potential in adult SHR neurons compared with 6.5 ± 0.5 mV in adult WKY neurons. To demonstrate that the shift of inactivation could be offset by increased current density, we normalized inactivation curves to cell capacitance (Fig. 3C). This procedure emphasizes the actual amount of the current available to influence cell excitability at various holding potentials. Inactivation curves normalized to cell capacitance for SHR and WKY neurons virtually superimpose over a range of potentials from 70 to 40 mV.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   IA undergoes steady-state inactivation at more negative voltages in adult SHR than in WKY neurons. A: currents from SHR and WKY neurons at 30 mV after 1-s prepulses to 120 (trace 1), 80 (trace 2), and 60 mV (trace 3). Vertical calibration bars are different for SHR and WKY currents. Records contain one-fourth the number of points originally sampled and are not leak subtracted. B: normalized current amplitudes (IA/Imax) from different holding potentials for SHR and WKY neurons. Inactivation curve for IA of SHR neurons is shifted approximately 6 mV to more hyperpolarized potential compared with adult WKY neurons. Solid lines, fits of data to a Boltzmann function (see RESULTS). Numbers (1-3) represent prepulse potential for current records in A. C: inactivation curves normalized to cell capacitance demonstrate that amount of current available in SHR neurons is not different across range of holding potentials from 75 to 40 mV, despite shift of inactivation to more hyperpolarized potentials. Values in C and D are means ± SE from 42 neurons from 10 WKY rats and 52 neurons from 15 SHR; SE bars were omitted where range was contained within symbols.

Primary alterations of I_A properties. The shift of I_A inactivation to more negative voltages in SHR neurons is consistent with hyperexcitability and could be a primary alteration associated with the development of hypertension in the model. To test this possibility, we examined inactivation of I_A in neurons isolated before the onset of hypertension and after chronic enalapril treatment to prevent hypertension in adult SHR. Figure 4 shows V_h for adult, prehypertensive, and enalapril-treated adult rats. The voltage range of inactivation in prehypertensive SHR neurons was similar to that in neurons from prehypertensive WKY and adult WKY rats, where V_h was 74.5 ± 3.3 mV (n = 6), 75.8 ± 3.2 mV (n = 4), and 75.1 ± 1.4 mV (n = 10), respectively. Because the inactivation of I_A was not shifted to negative voltages in SHR neurons before the onset of hypertension, the shift is likely associated with the onset of hypertension but could be secondary to elevated blood pressure. This possibility was examined with neurons isolated from enalapril-treated rats. The shift of approximately 6 mV in V_h was still evident in SHR after chronic enalapril treatment, V_h = 80.8 ± 1.2 mV (n = 7), suggesting that the shift of inactivation in adult SHR neurons is not a secondary alteration to hypertension. Enalapril did not have a direct effect on A-type channels, since the inactivation V_h did not change in the enalapril-treated WKY rat. After chronic enalapril treatment, inactivation V_h was 74.3 ± 1.4 mV (n = 7) in adult WKY neurons.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Shift of IA inactivation to more negative voltages is a primary alteration in SHR sympathetic neurons. Half-inactivation voltages (Vh) are summarized for neurons of both strains isolated from adult, young prehypertensive, and enalapril-treated animals. Values are means ± SE of number of animals in parentheses. * Statistical significance (P < 0.05) between means as determined by ANOVA. Total number of cells examined ranged from 16 (young SHR) to 52 (adult SHR).

Adaptive changes of I_A properties. The elevated current density in SHR neurons would be expected to blunt excitability and, therefore, could be an adaptive response. To test this hypothesis, we used the protocol described above (see Current density) to measure the current density of neurons isolated from prehypertensive SHR and enalapril-treated SHR. Current densities of adult and prehypertensive neurons are grouped by rat strain in Fig. 5 to distinguish between age- and hypertension-related changes in the current. In WKY rats, current density was 290 ± 45 pA/pF (n = 4) at 4-6 wk of age and decreased by ~44% by 12 wk of age. In contrast, I_A density did not decrease with age in SHR neurons. Prehypertensive SHR neurons displayed a current density of 324 ± 46 pA/pF (n = 6), which was not different from the density of adult hypertensive SHR neurons. After chronic enalapril treatment, I_A density decreased by ~50% to 219 ± 19 pA/pF (n = 5) in adult SHR neurons, a level not different from that of adult WKY neurons. The current density in neurons from enalapril-treated adult WKY rats was 236 ± 21 pA/pF (n = 7), which was not different from the density in untreated WKY adult neurons, suggesting that enalapril did not have a direct effect on the I_A density of sympathetic neurons. These data suggest that the increased I_A density observed in the adult SHR may be a secondary adaptation to elevated blood pressure.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Elevated IA density in adult SHR sympathetic neurons is secondary to hypertension. Current densities of neurons isolated from adult, prehypertensive, and enalapril-treated SHR and WKY rats are compared. Values are means ± SE of number of animals in parentheses. Values were measured from digitally subtracted currents elicited by steps to +40 mV from a holding potential of 120 mV. * Statistical significance at P < 0.05 as determined by ANOVA. Total number of cells tested ranged from 18 (young SHR) to 39 (adult SHR).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The objective of this study was to determine whether properties of I_A expressed in SHR sympathetic neurons are altered in a manner consistent with the neuronal hyperexcitability observed in this model of essential hypertension. Accordingly, we describe two alterations in I_A of SHR neurons compared with currents of age-matched, normotensive WKY neurons. The more negative range of I_A inactivation observed in SHR neurons than in WKY neurons appeared to be compensated by an elevated current density.

The properties of I_A in SCG neurons isolated from adult normotensive WKY rats observed in the present study compare well with previous reports of I_A in SCG neurons from adult and 5-wk-old Sprague-Dawley rats (2, 17, 28, 29, 38), with the different concentrations of external divalent cations employed in the previous studies taken into account (30). One notable difference between the present study and previous reports involves changes in I_A properties with age. In contrast to other reports that I_A density increases with age (32, 33), we observed a decrease in I_A density as rats aged from 4 to 16 wk. Although the reason for this discrepancy remains unclear, it likely reflects the considerable difference in ages examined in each study. The present study compared postweanling with adult rats, whereas previous studies examined I_A over the embryonic and immediately postnatal periods. McFarlane and Cooper (31) reported that the expression of I_A in postnatal neurons in culture requires the presence of unidentified trophic factor(s). Thus one possible explanation for the decline in I_A density with age is the loss of the trophic factors that support the expression of I_A.

Effects of I_A alterations on sympathetic excitability. In general terms, K⁺ currents provide a hyperpolarizing influence on the membrane and, therefore, inhibit cell excitability (20). Several characteristics of I_A make this current particularly well suited to regulate neuronal firing behavior. First, I_A rapidly activates at voltages negative to the threshold for action potential generation. In peripheral sympathetic neurons, this allows I_A to participate in the integration of synaptic potentials and determine whether the membrane reaches threshold (8). Second, I_A is an inactivating current; therefore, during continuous depolarization the channels underlying the current pass into an inactivated, nonconducting state. Inactivation may be observed in the decay of the whole cell currents (Figs. 1A and 2B). In sympathetic neurons that fire repetitively, the current time course can influence the time the membrane remains below threshold, thereby modulating the firing frequency (7, 20). At membrane potentials encountered by sympathetic neurons, the majority of A-type K⁺ channels are inactivated (Fig. 3B, WKY) and a small fraction is actually "available" to influence cell excitability. The "availability" of the channels can be increased if the neuron is hyperpolarized, which would effect recovery of some of the channels from inactivation. In WKY sympathetic neurons an afterhyperpolarization to approximately 70 mV would be expected to result in recovery of as much as 45% of the current (Fig. 3B). On the other hand, because the inactivation of I_A is shifted to more negative voltages in SHR, recovery from inactivation could be achieved in only 35% of I_A. Taken in isolation, the hyperpolarized shift of I_A inactivation in SHR is consistent with enhanced excitability. However, the elevated I_A density observed in adult SHR neurons would be expected to enhance the inhibitory influence of I_A on excitability and, therefore, counter the effects of shifted inactivation.

Role of I_A in SHR neuronal hyperexcitability and elevated sympathetic outflow. The elevated sympathetic outflow observed in SHR may result from multiple mechanisms. One mechanism that appears to underlie the onset of hypertension is the increased sympathetic outflow detectable in prehypertensive SHR (13, 40). The underlying mechanism for this effect may be related to the hyperexcitability observed in sympathetic neurons cultured from neonatal SHR (22). The shift of I_A inactivation reported here is unlikely to be involved in the increased sympathetic outflow of prehypertensive SHR or the heightened excitability of neonatal SHR, since the inactivation shift was not present in the young prehypertensive rats examined in this study. However, the increase of sympathetic outflow with age is greater in SHR than in WKY rats (13), and at ~7 wk of age, sympathetic outflow increases dramatically in the SHR (6, 13, 39). This "burst" of sympathetic activity is coincident with a rapid increase in blood pressure (39) and the negative shift of I_A inactivation reported here for the SHR (6, 13, 40). Thus the possibility exists that the same alteration occurring in central cardiovascular areas could contribute to the onset of hypertension in the SHR.

Primary and secondary changes in I_A. At least two possibilities could explain the changes in I_A of SHR sympathetic neurons. First, the alterations may reflect that different Kv gene products underlie I_A in the two strains or a change in the proportion of the different gene products. Of the Kv gene superfamily, the biophysical properties of the Kv4.1, Kv4.2, and Kv3.4 gene products most closely resemble the native I_A of rat sympathetic ganglia (12). Because the Kv4 and Kv3.4 gene products have different activation ranges, it seems unlikely that the alterations of I_A between SHR and WKY neurons represent an alteration of the proportion of Kv3.4 and Kv4 gene expression, since the activation voltage range of I_A was not different between SHR and WKY rats. -Subunits can associate with rapidly inactivating Kv channels and modulate their inactivation properties (36). Considering that current density was elevated in the SHR, possibly because of increased channel expression, it is attractive to speculate that mismatched stoichiometry of - and -subunits contributes to the altered inactivation of I_A in SHR neurons. However, when the current density was lowered in SHR neurons after enalapril treatment, the inactivation properties of I_A did not change, which decreases the possibility that -subunits are involved in the alteration of I_A in SHR neurons. Alternatively, the negative shift of inactivation displayed by SHR neurons may stem from mutation(s) or posttranslational modification(s) that alters the primary structure of the channel protein. Because these changes were not prevented by normalizing the blood pressure by enalapril treatment, they appear to be primary changes. The elevation of current density, on the other hand, appears to be secondary to elevated blood pressure, elevated nerve activity in SHR, or some other aspect of the SHR model of essential hypertension, since it was prevented by enalapril treatment. Changes in current density could occur as a result of changes in the probability of channel opening, single-channel conductance, or channel number via increased gene expression, mRNA stability, or decreased channel degradation. A molecular approach to quantify RNA levels similar to that used by Dixon and McKinnon (12) or quantitative Western blots using selective antibodies directed against Kv gene products may help elucidate these questions.

Perspectives