Protease-activated receptor 2 activation inhibits N-type Ca2+ currents in rat peripheral sympathetic neurons

Abstract

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca(2+) currents (I(Ca-N)) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca(2+) currents (I(Ca)), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I(Ca). This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca(2+) channel blocker, suggesting the involvement of N-type Ca(2+) channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I(Ca-N) in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca(2+) channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca(2+) channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.

Keywords: N-type Ca2+ channel; celiac ganglion; hypotension; peripheral sympathetic output; protease-activated receptor 2.

Fig. 1.

PAR-2 agonists inhibit I_Ca in rat CG neurons. (A) Left, a representative trace of I_Ca in the presence (●) and absence (○) of 30 nM trypsin. Midle, time course of the effect of 30 nM trypsin on I_Ca. Right, I–V relationship curve of I_Ca measured 10 ms after the onset of the depolarizing pulses in the absence (○) and presence (●) of 30 nM trypsin. (B) Left, a representative trace of I_Ca in the presence (●) and absence (○) of 30 nM BT. Right, time course of BT (30 nM)-induced I_Ca inhibition. (C) Summary of I_Ca inhibition by trypsin or BT. (D) Left, representative traces of I_Ca in the presence (●) and absence (○) of 100 μM SL-NH₂. Middle, time course of 100 μM SL-NH₂ -induced I_Ca inhibition. Right, I–V relationship curve of I_Ca measured 10 ms after the onset of the depolarizing pulses in the absence (○) and presence (●) of 100 μM SL-NH₂. (E) Left, representative traces of I_Ca in the presence (●) and absence (○) of 100 μM LR-NH₂. Right, time course of the 100 μM LR-NH₂ effect on I_Ca. (F) Summary of I_Ca inhibition by SL-NH₂ or LR-NH₂. (G) Concentration response curve for trypsin- or SL-NH₂-induced I_Ca inhibition. n = 7 in all groups.

Fig. 2.

Effect of GDPβS on PAR-2 agonist-induced I_Ca inhibition. (A) Left, a representative trace showing the effect of trypsin (30 nM) on I_Ca. Open (○) and filled circles (●) indicate I_Ca before and after trypsin application, respectively. Right, a representative trace showing the effect of trypsin (30 nM) on I_Ca with GDPβS (2 mM) in the pipette solution. Trypsin was applied to rat CG neurons after 7 min dialysis with GDPβS in the pipette solution. (B) Summary showing the effect of GDPβS on trypsin-induced I_Ca inhibition.(C) Left, a representative trace showing the effect of SL-NH₂ (100 μM) on I_Ca. Open (○) and filled circles (●) indicate I_Ca before and after SL-NH₂ application, respectively. Right, a representative trace showing the effect of SL-NH₂ (100 μM) on I_Ca with GDPβS (2 mM) in the pipette solution. SL-NH₂ was applied to rat CG neurons after 7 min dialysis with GDPβS in the pipette solution. (D) Summary showing the effect of GDPβS on SL-NH₂-induced I_Ca inhibition.

Fig. 3.

Effect of ω-CgTx on I_Ca and PAR-2 agonist-induced I_Ca inhibition. (A) Left, a representative trace showing the effect of consecutive application of ω-CgTx (1 μM) and nifedipine (1 μM) on I_Ca. Right, time course of the effects induced by consecutive application of ω-CgTx (1 μM) and nifedipine (1 μM) on I_Ca. Trace 1, 2, and 3 in the left panel represent the traces recorded at the corresponding time indicated in the right panel, respectively. (B) Contribution of N-type (ω-CgTx-sensitive) and L-type (nifedipine-sensitive) currents to the total I_Ca. (C) Left, a representative trace of the effects induced by consecutive application of ω-CgTx (1 μM) and trypsin (30 nM) on I_Ca. Right, time course of effects induced by consecutive application of ω-CgTx (1 μM) and trypsin (30 nM) on I_Ca.Trace 1, 2, and 3 in the left panel represent the traces recorded at the corresponding time indicated in the right panel, respectively. (D) Summary of I_Ca inhibition by trypsin in the absence and presence of ω-CgTx. (E) Left, a representative trace of the effects induced by consecutive application of ω-CgTx (1 μM) and SL-NH₂ (100 μM) on I_Ca. Right, time course of effects induced by consecutive application of ω-CgTx (1 μM) and SL-NH₂ (100 μM) on I_Ca. Trace 1, 2, and 3 in the left panel represent the traces recorded at the corresponding time indicated in the right panel, respectively. (F) Summary of I_Ca inhibition by SL-NH₂ in the absence and presence of ω-CgTx.

Fig. 4.

Characteristics of PAR-2 agonist-induced I_Ca inhibition in rat CG neurons. (A) Left, a representative trace of I_Ca inhibition by NA (1 μM) application. Right, time course of prepulse facilitation in the absence and presence of 1 μM NA. The I_Ca was evoked every 10 s by a double-pulse voltage protocol consisting of two identical test pulses (0 mV from a holding potential of −80 mV) separated by a large depolarizing conditioning pulse to +80 mV. Prepulse facilitation was calculated as the ratio of the postpulse to prepulse current amplitudes (post/pre) measured isochronally at 10 ms after the start of the test pulse. (B) Left, a representative trace of I_Ca inhibition by trypsin (30 nM) application. Right, time course of prepulse facilitation in the absence and presence of 30 nM trypsin. (A) Left, a representative trace of I_Ca inhibition by SL-NH₂ (100 μM) application. Right, time course of prepulse facilitation in the absence and presence of 100 μM SL-NH₂.

Fig. 5.

Effect of a PAR-2 agonist on repetitive firing of APs in celiac ganglia neurons. (A) Representative traces showing the effect of trypsin (30 nM) on repetitive APs evoked by current injection (100–200 pA, 300 ms) in current-clamp mode. (B) Representative traces showing the effect of SL-NH₂ (100 μM) on repetitive APs evoked by current injection in current-clamp mode. (C) Representative traces showing the effect of consecutive application of ω-CgTx (1 μM) and trypsin (30 nM) on repetitive AP firing evoked by current injection. (D) Representative traces showing the effect of consecutive application of ω-CgTx (1 μM) and trypsin SL-NH₂ (100 μM) on repetitive AP firing evoked by current injection.