Hypertension induced by angiotensin II and a high salt diet involves reduced SK current and increased excitability of RVLM projecting PVN neurons

Abstract

Although evidence indicates that activation of presympathetic paraventricular nucleus (PVN) neurons contributes to the pathogenesis of salt-sensitive hypertension, the underlying cellular mechanisms are not fully understood. Recent evidence indicates that small conductance Ca(2+)-activated K(+) (SK) channels play a significant role in regulating the excitability of a key group of sympathetic regulatory PVN neurons, those with axonal projections to the rostral ventrolateral medulla (RVLM; i.e., PVN-RVLM neurons). In the present study, rats consuming a high salt (2% NaCl) diet were made hypertensive by systemic infusion of angiotensin II (AngII), and whole cell patch-clamp recordings were made in brain slice from retrogradely labeled PVN-RVLM neurons. To determine if the amplitude of SK current was altered in neurons from hypertensive rats, voltage-clamp recordings were performed to isolate SK current. Results indicate that SK current amplitude (P < 0.05) and density (P < 0.01) were significantly smaller in the hypertensive group. To investigate the impact of this on intrinsic excitability, current-clamp recordings were performed in separate groups of PVN-RVLM neurons. Results indicate that the frequency of spikes evoked by current injection was significantly higher in the hypertensive group (P < 0.05-0.01). Whereas bath application of the SK channel blocker apamin significantly increased discharge of neurons from normotensive rats (P < 0.05-0.01), no effect was observed in the hypertensive group. In response to ramp current injections, subthreshold depolarizing input resistance was greater in the hypertensive group compared with the normotensive group (P < 0.05). Blockade of SK channels increased depolarizing input resistance in normotensive controls (P < 0.05) but had no effect in the hypertensive group. On termination of current pulses, a medium afterhyperpolarization potential (mAHP) was observed in most neurons of the normotensive group. In the hypertensive group, the mAHP was either small or absent. In the latter case, an afterdepolarization potential (ADP) was observed that was unaffected by apamin. Apamin treatment in the normotensive group blocked the mAHP and revealed an ADP resembling that seen in the hypertensive group. We conclude that diminished SK current likely underlies the absence of mAHPs in PVN-RVLM neurons from hypertensive rats. Both the ADP and greater depolarizing input resistance likely contribute to increased excitability of PVN-RVLM neurons from rats with AngII-Salt hypertension.

Fig. 1.

Comparison of SK current among PVN-RVLM neurons from NT and HT rats. A: representative traces from a neuron of a NT rat showing outward tail current following step (100 ms) depolarization of V_m (−60 to +10 mV) in the absence (black trace) and presence (gray trace) of apamin (100 nM). B: outward tail current in a neuron from a HT rat in the absence (black trace) and presence (gray trace) of apamin (100 nM). Note: Insets in A and B show the net SK current obtained by subtraction (control - apamin). C: summary data show the amplitude (left) and density (right) of SK current for cells in each treatment group. All recordings were performed with TTX (0.5 μM) and TEA (1.0 mM) in the bath and with 8-(4-chlorophenylthio) 3′,5′-cyclic adenosine monophosphate (8CPT-cAMP, 50 μM) in the intracellular solution. *P < 0.05, **P < 0.01 vs. NT (unpaired Mann-Whitney test).

Fig. 2.

Effect of SK channel blockade on excitability of PVN-RVLM neurons from NT and HT rats. A: voltage traces showing representative responses of PVN-RVLM neurons from NT rats to a 200 pA depolarizing current injection in the absence (top, control) and presence (bottom) of the SK channel blocker apamin (100 nM). Note that traces were recorded from 2 different neurons. B: representative responses of 2 different PVN-RVLM neurons from HT rats to depolarizing current injections in the absence (top, control) and presence (bottom) of apamin. C: graphs showing the relationship between the amplitude of injected current and the frequency of evoked discharge for cells from NT (left) and HT (right) rats in the absence (control) and presence of apamin. Note that the maximum discharge frequency achieved was lower in the NT (left) than HT (right) group under control conditions and that apamin increased discharge in the NT group (left) but not the HT group (right). D: the slope of the linear portion of current-discharge response (0–125 pA) curves is shown for neurons from NT and HT rats in the absence (control) and presence of apamin. Note that under control conditions the slope was significantly greater for neurons of the HT group than the NT group. Apamin significantly increased the slope of the response of the NT group but not the HT group (left). Interspike intervals (ISIs) plotted for trains of current-evoked action potentials revealed that average ISI and spike-frequency adaptation (ISI prolongation) were greater (right) among neurons from NT than HT rats. *P < 0.01, **P < 0.001 apamin vs. control groups from NT rats (2-way ANOVA). †P < 0.05, ††P < 0.01 HT vs. NT groups in the absence of apamin (2-way ANOVA). #P < 0.05 vs. NT groups in the absence of apamin (1-way Kruskall-Wallis ANOVA).

Fig. 3.

Effect of SK channel blockade on V_t and depolarizing R_input of PVN-RVLM neurons from NT and HT rats. A: representative voltage traces from two different PVN-RVLM neurons from NT rats showing the response to ramp current injection in the absence (top, control) and presence (bottom) of the SK channel blocker apamin (100 nM). B: representative voltage traces from 2 different PVN-RVLM neurons from HT rats showing the response to ramp current injection in the absence (top, control) and presence (bottom) of apamin. In A and B, membrane potential depolarized gradually and action potential discharge began at a discrete voltage threshold (V_t). C: summary data showing that subthreshold depolarizing input resistance (R_input, top) and spike frequency (middle) were greater under control conditions for neurons in the HT than NT group. Apamin increased both R_input and spike frequency only in the NT group. V_t (bottom) was similar across groups under control conditions and was unchanged by apamin. *P < 0.05 vs. NT groups under control condition; NS, P > 0.05 (1-way Kruskall-Wallis ANOVA).

Fig. 4.

Effect of SK channel blockade on the mAHP and the ADP of PVN-RVLM neurons from NT and HT rats. A: representative voltage traces showing the effect of SK channel blockade with apamin (100 nM) on the mAHP recorded in neurons from NT rats. In the absence of apamin (top), a train of action potentials was induced by a depolarizing current pulse (150 pA, 500 ms). Following termination of action potential firing, a mAHP was observed. In the presence of apamin (bottom), the mAHP disappeared and ADP was revealed. B: representative voltage traces showing the effect of apamin (100 nM) on the mAHP and ADP of cells from HT rats. In the absence (top) and presence (bottom) of apamin, an ADP was observed. C: summary data showing the maximum ADP amplitude (left) and decay time constant (right) of cells from NT and HT rats. NS, P > 0.05 (1-way Kruskall-Wallis ANOVA).