Suppression of Peripheral Sympathetic Activity Underlies Protease-Activated Receptor 2-Mediated Hypotension

Protease-activated receptor (PAR)-2 is 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 some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, omega-conotoxin GVIA (CgTx), a selective N-type Ca2+ channel (I(Ca-N)) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by omega-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of I(Ca-N) which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

Influence of omega-Conotoxin GVIA, Nifedipine and Cilnidipine on Catecholamine Release in the Rat Adrenal Medulla

The present study was designed to establish comparatively the inhibitory effects of cilnidipine (CNP), nifedipine (NIF), and omega-conotoxin GVIA (CTX) on the release of CA evoked by cholinergic stimulation and membrane depolarization from the isolated perfused model of the rat adrenal medulla. CNP (3 micrometer), NIF (3 micrometer), and CTX (3 micrometer) perfused into an adrenal vein for 60 min produced greatly inhibition in CA secretory responses evoked by ACh (5.32 x 10(-3) M), DMPP (10(-4) M for 2 min), McN-A-343 (10(-4) M for 2 min), high K+ (5.6 x 10(-2) M), Bay-K-8644 (10(-5) M), and cyclopiazonic acid (10(-5) M), respectively. For the CA release evoked by ACh and Bay-K-8644, the following rank order of potency was obtained: CNP > NIF > CTX. The rank order for the CA release evoked by McN-A-343 and cyclopiazonic acid was CNP > NIF > CTX. Also, the rank orders for high K+ and for DMPP were NIF > CTX > CNP and NIF > CNP > CTX, respectively. Taken together, these results demonstrate that all voltage-dependent Ca2+ channels (VDCCs) blockers of cilnidipine, nifedipine, and omega-conotoxin GVIA inhibit greatly the CA release evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization without affecting the basal release from the isolated perfused rat adrenal gland. It seems likely that the inhibitory effects of cilnidipine, nifedipine, and omega-conotoxin GVIA are mediated by the blockade of both L- and N-type, L-type only, and N-type only VDCCs located on the rat adrenomedullary chromaffin cells, respectively, which are relevant to Ca2+ mobilization. It is also suggested that N-type VDCCs play an important role in the rat adrenomedullary CA secretion, in addition to L-type VDCCs.

Calcium Ions are Involved in Modulation of Melittin-induced Nociception in Rat: I. Effect of Voltage-gated Calcium Channel Antagonist

Melittin-induced nociceptive responses are mediated by selective activation of capsaicin-sensitive primary afferent fibers and are modulated by excitatory amino acid receptor, cyclooxygenase, protein kinase C and serotonin receptor. The present study was undertaken to investigate the peripheral and spinal actions of voltage-gated calcium channel antagonists on melittin-induced nociceptive responses. Changes in mechanical threshold and number of flinchings were measured after intraplantar (i.pl.) injection of melittin (30microg/paw) into mid-plantar area of hindpaw. L-type calcium channel antagonists, verapamil [intrathecal (i.t.), 6 or 12microg; i.pl.,100 & 200microg; i.p., 10 or 30 mg], N-type calcium channel blocker, omega-conotoxin GVIA (i.t., 0.1 or 0.5microg; i.pl., 5microg) and P-type calcium channel antagonist, omega-agatoxin IVA (i.t., 0.5microg; i.pl., 5microg) were administered 20 min before or 60 min after i.pl. injection of melittin. Intraplantar pre-treatment and i.t. pre- or post-treatment of verapamil and omega-conotoxin GVIA dose-dependently attenuated the reduction of mechanical threshold, and melittin-induced flinchings were inhibited by i.pl. or i.t. pre-treatment of both antagonists. P-type calcium channel blocker, omega-agatoxin IVA, had significant inhibitory action on flinching behaviors, but had a limited effect on melittin-induced decrease in mechanical threshold. These experimental findings suggest that verapamil and omega-conotoxin GVIA can inhibit the development and maintenance of melittin-induced nociceptive responses.

Influence of Cilnidipine on Catecholamine Release in the Perfused Rat Adrenal Medulla

The present study was attempted to investigate the effect of cilnidipine (FRC-8635), which is a newly synthesized novel dihydropyridine (DHP) type of organic Ca2 channel blockers, on secretion of catecholamines (CA) evoked by acetylcholine (ACh), high K, DMPP and McN-A-343 from the isolated perfused rat adrenal gland. Cilnidipine (1~10microM) perfused into an adrenal vein for 60 min produced relatively dose- and time-dependent inhibition in CA secretory responses evoked by ACh (5.32 10 3 M), DMPP (10 4 M for 2 min) and McN-A-343 (10 4 M for 2 min). However, lower dose of cilnidipine did not affect CA secretion by high K (5.6 10 2 M), higher dose of it reduced greatly CA secretion of high K. Cilnidipine itself did fail to affect basal catecholamine output. In the presence of cilnidipine (10microM), the CA secretory responses evoked by Bay-K-8644 (10microM), an activator of L-type Ca2 channels and cyclopiazonic acid (10microM), an inhibitor of cytoplasmic Ca2 -ATPase were also inhibited. Moreover, omega-conotoxin GVIA (1microM), a selective blocker of the N-type Ca2 channels, given into the adrenal gland for 60 min, also inhibited time-dependently CA secretory responses evoked by Ach, high K, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid. Taken together, these results demostrate that cilnidipine inhibits CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors from the isolated perfused rat adrenal gland without affecting the basal release. However, at lower dose, cilnidipine did not affect CA release by membrane depolarization while at larger dose inhibited that. It seems likely that this inhibitory effect of cilnidipine is exerted by blocking both L- and N-type voltage-dependent Ca2 channels (VDCCs) on the rat adrenomedullary chromaffin cells, which is relevant to inhibition of both the Ca2 influx into the adrenal chromaffin cells and intracellular Ca2 release from the cytoplasmic store. It is thought that N-type VDCCs may play an important role in regulation of CA release from the rat adrenal medulla.

Nimodipine as a potential pharmacological tool for characterizing R-type calcium currents

Nimopidine, one of dihydropyridine derivatives, has been widely used to pharmacologically identify L-type Ca currents. In this study, it was tested if nimodipine is a selective blocker for L-type Ca currents in sensory neurons and heterologous system. In mouse dorsal root ganglion neurons (DRG), low concentrations of nimodipine (10 muM) further reduced the "residual" currents in DRG neurons from alpha1E knock-out mice, after blocking L-, N- and P/Q-type Ca currents with 10 muM nimodipine, 1 muM omega-conotoxin GVIA and 200 nM omega-agatoxin IVA, indicating inhibitory effects of nimodipine on R-type Ca currents. Nimodipine (>10 muM) also produced the inhibition of both low-voltage-activated calcium channel currents in DRG neurons and alpha1B and alpha1E subunit based Ca channel currents in heterologous system. These results suggest that higher nimodipine (>10 muM) is not necessarily selective for L-type Ca currents. While care should be taken in using nimodipine for pharmacologically defining L-type Ca currents from native macroscopic Ca currents, nimodipine (>10 muM) could be a useful pharmacological tool for characterizing R-type Ca currents when combined with toxins blocking other types of Ca channels.

Calcium channel blockers suppress the responses of rat dorsal horn cell to nociceptive input

Calcium ions are implicated in a variety of physiological functions, including enzyme activity, membrane excitability, neurotransmitter release, and synaptic transmission, etc. Calcium antagonists have been known to be effective for the treatment of exertional angina and essential hypertension. Selective and nonselective voltage-dependent calcium channel blockers also have inhibitory action on the acute and tonic pain behaviors resulting from thermal stimulation, subcutaneous formalin injection and nerve injury. This study was undertaken to investigate the effects of iontophoretically applied Ca++ and its antagonists on the responses of WDR (wide dynamic range) cells to sensory inputs. The responses of WDR cells to graded electrical stimulation of the afferent nerve and also to thermal stimulation of the receptive field were recorded before and after iontophoretical application of Ca++, EGTA, Mn++, verapamil, omega-conotoxin GVIA, omega-conotoxin MVIIC and omega-agatoxin IVA. Also studied were the effects of a few calcium antagonists on the C-fiber responses of WDR cells sensitized by subcutaneous injection of mustard oil (10%). Calcium ions and calcium channel antagonists (Mn++, verapamil, omega-conotoxin GVIA & omega-agatoxin IVA) current-dependently suppressed the C-fiber responses of WDR cells without any significant effects on the A-fiber responses. But omega-conotoxin MVIIC did not have any inhibitory actions on the responses of WDR cell to A-fiber, C-fiber and thermal stimulation. Iontophoretically applied EGTA augmented the WDR cell responses to C-fiber and thermal stimulations while spinal application of EGTA for about 20 ~ 30 min strongly inhibited the C-fiber responses. The augmenting and the inhibitory actions of EGTA were blocked by calcium ions. The WDR cell responses to thermal stimulation of the receptive field were reduced by imtophoretical application of Ca++, verapamil, omega -agatoxin IVA, and omega-conotoxin GVIA but not by omega-conotoxin MVIIC. The responses of WDR cells to C-fiber stimulation were augmented after subcutaneous injection of mustard oil (10%, 0.15 ml) into the receptive field and these sensitized C-fiber responses were strongly suppressed by iontophoretically applied Ca++, verapamil, omega-conotoxin GVIA and omega-agatoxin IVA. These experimental findings suggest that in the rat spinal cord, L-, N-, and P-type, but not Q-type, voltage-sensitive calcium channels are implicated in the calcium antagonist-induced inhibition of the normal and the sensitized responses of WDR cells to C-fiber and thermal stimulation, and that the suppressive effect of calcium and augmenting action of EGTA on WDR cell responses are due to changes in excitability of the cell.