Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker

1. The objective of the present study was to investigate the mechanism of the relaxant activity of marrubenol, a diterpenoid extracted from Marrubium vulgare. In rat aorta, marrubenol was a more potent inhibitor of the contraction evoked by 100 mM KCl (IC50: 11.8+/-0.3 microM, maximum relaxation: 93+/-0.6%) than of the contraction evoked by noradrenaline (maximum relaxation: 30+/-1.5%). 2. In fura-2-loaded aorta, marrubenol simultaneously inhibited the Ca2+ signal and the contraction evoked by 100 mM KCl, and decreased the quenching rate of fura-2 fluorescence by Mn2+. 3. Patch-clamp data obtained in aortic smooth muscle cells (A7r5) indicated that marrubenol inhibited Ba2+ inward current in a voltage-dependent manner (KD: 8+/-2 and 40+/-6 microM at holding potentials of -50 and -100 mV, respectively). 4. These results showed that marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels.     

Ethaverine, a derivative of papaverine, inhibits cardiac L-type calcium channels.

Ethaverine is a derivative of papaverine used in the treatment of peripheral vascular disease and is thought to cause vasodilation by reducing intracellular Ca2+ concentrations in vascular smooth muscle cells. We tested its effects on single, dihydropyridine-sensitive, L-type calcium channels from porcine cardiac muscle, incorporated into planar lipid bilayers. L-type calcium channels were activated by step depolarizations from a holding potential of -60 mV to a test potential of 0 mV, and unitary currents carried by 100 mM BaCl2 were recorded. Channel activity was enhanced by the presence of the dihydropyridine agonist (+)-202-791 (0.5 microM) and the activated alpha subunit of the stimulatory GTP-binding protein, Gs. We found that 0.3-30 microM ethaverine on either side of the channel caused a reduction in the channel open probability (EC50 approximately 1 microM), with the higher concentrations inhibiting channel activity almost completely. In addition, the ethaverine caused a small reduction in the unitary current amplitude of single open channels (approximately 20%). To test whether the effect of ethaverine on open probability was due to a displacement of the dihydropyridine agonist, we studied the effect of ethaverine on the binding of [3H]nitrendipine to cardiac sarcolemma and found that ethaverine inhibited [3H]nitrendipine binding with a Ki of approximately 8.5 microM. Ethaverine also inhibited the binding of [3H]diltiazem and [3H]verapamil, with Ki values of 1-2 microM. Because ethaverine is structurally related to verapamil, it is likely that ethaverine acts by binding to the verapamil binding sites on the L-type calcium channels to inhibit channel activation and dihydropyridine binding.     

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.     

Naftopidil, a new alpha-adrenoceptor blocking agent with calcium antagonistic properties: characterization of Ca2+ antagonistic effects.

The newly developed antihypertensive agent naftopidil blocks alpha 1-adrenoceptors and inhibits Ca2+ entry via potential-dependent channels in vascular muscle. The aim of our study was to detect possible Ca2+ channel blocking activity in various isolated preparations of the guinea pig heart. Prazosin and verapamil were used for reference. In papillary muscles, 10 microM of all drugs reduced the force of contraction Fc. The action potential duration and the refractory period were hardly affected by naftopidil, decreased by verapamil, and slightly increased by prazosin. In constant-flow Langendorff hearts, the drugs reduced the perfusion pressure, decreased the Fc, and slowed the spontaneous heart rate (order of potency: verapamil much greater than naftopidil greater than prazosin). In voltage-clamped ventricular cardiomyocytes, the calcium current ICa was completely inhibited by verapamil (pD2 value of 6.9) and to 53.5% by naftopidil (pD2 value of 6.4). Prazosin (10 microM) decreased ICa by little more than 10%. There were no differences in the steady-state inhibition of ICa by the two enantiomers of naftopidil. The block of ICa was clearly use dependent. Radioligand binding studies with (+)-[3H]PN 200-110. (-)-[3H]desmethoxy-verapamil, and (+)-cis-[3H]diltiazem in guinea pig skeletal muscle T-tubulus membranes demonstrated that racemic naftopidil exhibited some affinity for the three distinct drug receptor domains of the L-type Ca2+ channel. In conclusion, the present data are consistent with the hypothesis that naftopidil is a weak ligand for L-type calcium channels. It partially blocks ICa and shows no stereoselectivity.     

Interactions of calmodulin antagonists with calcium antagonists binding sites.

Calmodulin antagonists have calcium entry blocking properties. In order to quantitatively investigate the interactions of these drugs with calcium channels, their effect on [3H]nitrendipine and [3H]d-cis-diltiazem binding to rat cerebral cortex membrane preparations was compared to their inhibitory effect on the activation of cyclic nucleotide phosphodiesterase by calmodulin. The potency of most antagonists to inhibit [3H]nitrendipine binding was correlated with their calmodulin inhibitory potency. However, bepridil (K0.5 = 280 nM), chlorpromazine (K0.5 = 3 microM), triflupromazine (K0.5 = 1.5 microM), imipramine (K0.5 = 3 microM) and propranolol (K0.5 = 14 microM) were much more active on [3H]d-cis-diltiazem binding than on either [3H]nitrendipine binding or calmodulin, suggesting that these compounds bind to higher affinity sites on the calcium antagonist target protein. Moreover, the potencies of these compounds to compete with [3H]d-cis-diltiazem and to inhibit calcium-induced contractions in depolarized smooth muscle were correlated (R = 0.76, p less than 0.02). These data suggest that low concentrations of these hydrophobic drugs which have calcium and calmodulin antagonistic properties inhibit smooth muscle contraction through calcium entry blockade, not calmodulin antagonism.     

Modulation by protease-activated receptors of the rat duodenal motility in vitro: possible mechanisms underlying the evoked contraction and relaxation

1 The present study examined effects of agonist enzymes and receptor-activating peptides for protease-activated receptors (PARs) on duodenal motility in the rat, and also investigated possible mechanisms underlying the evoked responses. 2 Thrombin at 0.03-0.1 microM and the PAR-1-activating peptide SFLLR-NH2 at 3-100 microM or TFLLR-NH2 at 10-50 microM produced a dual action, relaxation followed by contraction of the duodenal longitudinal muscle. The PAR-2-activating peptide SLIGRL-NH2 at 10-100 microM elicited only small contraction. Trypsin at 0.08 microM induced small contraction, or relaxation followed by contraction, depending on preparations. The PAR-4-activating peptide GYPGKF-NH2 at 1000 microM exhibited no effect. 3 The contractile responses of the duodenal strips to TFLLR-NH2 and to SLIGRL-NH2 were partially attenuated by the L-type calcium channel blocker nifedipine (1 microM), the protein kinase C inhibitor GF109203X (1 microM) and the tyrosine kinase inhibitor genistein (15 microM), but were resistant to indomethacin (3 microM) and tetrodotoxin (1-10 microM). 4 The relaxation of the preparations exerted by TFLLR-NH2 was unaffected by indomethacin (3 microM), propranolol (5 microM), NG-nitro-L-arginine methyl ester (100 microM) and tetrodotoxin (1-10 microM). This relaxation was resistant to either GF109203X (1 microM) or genistein (15 microM), but was, remarkably, attenuated by combined application of these two kinase inhibitors. 5 Apamin (0.1 microM), an inhibitor of calcium-activated, small-conductance potassium channels, but not charybdotoxin (0.1 microM), completely abolished the PAR-1-mediated duodenal relaxation, and significantly enhanced the PAR-1-mediated contraction. 6 These findings demonstrate that PAR-1 plays a dual role, suppression and facilitation of smooth muscle motility in the rat duodenum, while PAR-2 plays a minor excitatory role in the muscle, and that PAR-4 is not involved in the duodenal tension modulation. The results also suggest that the contractile responses to PAR-1 and PAR-2 activation are mediated, in part, by activation of L-type calcium channels, protein kinase C and tyrosine kinase, and that the relaxation response to PAR-1 activation occurs via activation of apamin-sensitive, but charybdotoxin-insensitive, potassium channels, in which both protein kinase C and tyrosine kinase might be involved synergistically.     

Interaction of pinaverium (a quaternary ammonium compound) with 1,4-dihydropyridine binding sites in rat ileum smooth muscle.

1. The interaction of pinaverium bromide, a quaternary ammonium compound, with binding sites for (L-type) calcium channel blockers was investigated in rat ileum smooth muscle. 2. Pinaverium inhibited [3H]-(+)-PN200-110 ([3H]-(+)-isradipine) specific binding to tissue homogenates incompletely (Ki 0.38 microM; maximal inhibition 80%). In contrast, binding to single cell preparations (obtained by collagenase treatment) and to saponin-treated homogenates was completely inhibited. These data are compatible with the view that, in untreated homogenates, 20% of [3H]-(+)-isradipine binding sites are not accessible to pinaverium because it is associated with sealed inside-out vesicles. 3. Pinaverium bromide increased the apparent KD of [3H]-(+)-isradipine binding to saponin-treated homogenates but did not significantly affect the Bmax value. Moreover, the dissociation rate constant of [3H]-(+)-isradipine binding was not changed by pinaverium. These data suggest that pinaverium interacts with the dihydropyridine binding site in a competitive manner. However, in contrast to uncharged dihydropyridine calcium antagonists, pinaverium inhibited, rather than stimulated, [3H]-diltiazem binding to rat brain membranes (at 30-37 degrees C). 4. Although Bmax values of [3H]-(+)-isradipine were similar in homogenates prepared from tissue and cells (collagenase-treated), the KD value was significantly higher in cell homogenates (166 vs 95 pM). Similarly, the Ki value of pinaverium was higher in cell preparations than in tissue homogenates (0.77 vs 0.38 microM). Thus, collagenase can significantly modify the dihydropyridine recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of an homologous series of aliphatic alcohols on neuronal and smooth muscle voltage-dependent Ca2+ channels.

The acute inhibitory actions of alcohol on K(+)-stimulated 45Ca2+ uptake into synaptosomes shows regional variation in sensitivity throughout the brain, suggesting the possibility of a selective action on a specific Ca2+ channel subtype. This was examined by comparing the effects of a homologous series of aliphatic alcohols on synaptosomal Ca2+ channels with their actions on K(+)-stimulated Ca2+ channels in guinea-pig intestinal longitudinal muscle, which have been demonstrated to be of the L-type. K(+)-stimulated contraction of and [3H]nitrendipine binding to smooth muscle were both inhibited by the alcohols at similar concentrations, with the potency increasing with chain length. In synaptosomes, however, K(+)-stimulated 45Ca2+ uptake was 5-30 times more sensitive to the inhibitory actions of alcohol than were [3H]nitrendipine and [125I]omega-conotoxin binding. These observations suggest that K(+)-stimulated 45Ca2+ uptake is mediated by a non-L non-N type channel which is more sensitive to the acute effects of alcohols. This is supported by the observation that K(+)-stimulated 45Ca2+ uptake which is insensitive to L- and N-channel antagonists was inhibited by funnel web spider venom.     

Vasorelaxing effect in rat thoracic aorta caused by denudatin B, isolated from the Chinese herb, magnolia fargesii

Denudatin B is an antiplatelet agent isolated from the flower buds of Magnolia fargesii. We studied the effects of denudatin B on the vasoconstriction of rat thoracic aorta induced by high potassium (K+) solution, norepinephrine (NE) and caffeine, and to elucidate its mode of action. The contraction of rat aorta caused by high K+ (60 mM) and cumulative concentrations of CaCl2 (0.03-3 mM) was inhibited concentration dependently by denudatin B with an IC50 of 21.2 micrograms/ml. NE (3 microM)-induced phasic and tonic contractions of rat aorta were inhibited by pretreatment with denudatin B (10-100 micrograms/ml). The relaxing action of denudatin B persisted in denuded aorta, in Ca2(+)-free and EGTA (2 mM)-containing medium. The vasorelaxing effects were not affected by indomethacin (20 microM), hemoglobin (10 microM) or methylene blue (50 microM) and were not accompanied by PGI2 formation. In quin-2/AM-loaded cultured rat vascular smooth muscle cells, denudatin B (100 micrograms/ml) inhibited the increase of intracellular calcium caused by NE (3 microM) in the presence or absence of extracellular calcium. Denudatin B did not affect the caffeine (10 mM)-induced contraction and the increase in intracellular calcium. Denudatin B (100 micrograms/ml) increased the cGMP, but not the cAMP level in intact and denuded aorta. The 45Ca2+ influx induced in rat aorta by high K+ (60 mM) or NE (3 microM) was markedly inhibited by denudatin B in a concentration-dependent manner. These results indicate that denudatin B relaxed vascular smooth muscle by inhibiting the Ca2+ influx through voltage-gated and receptor-operated Ca2+ channels; its effect to increase cGMP may enhance the vasorelaxation.     

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1. The effects of norbormide on the contractility of endothelium-deprived rat, guinea-pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2. In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5-50 microM) induced a concentration-dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10-800 microM) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3. In resting rat and guinea-pig aortae, guinea-pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10-100 microM) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2(+)-free medium. Norbormide (up to 100 microM) was ineffective in phenylephrine-contracted guinea-pig and rat aorta. 4. In A7r5 cells, a cell line from rat aorta, norbormide prevented high K(+)- but not 5-hydroxytryptamine-induced intracellular calcium transients. 5. These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.     

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.     

Lack of involvement of endothelin-1 in angiotensin II-induced contraction of the isolated rat tail artery

1. The contribution of endothelin-1 (ET-1) to angiotensin II (Ang II)-mediated contraction of the isolated rat tail artery was assessed with measurements of tension, and cytosolic calcium ([Ca(2+)](i)). The distribution of the AT(1) receptor was studied with RT - PCR and immunohistochemistry. 2. Ang II induced an endothelium-independent contraction (pEC(50) 7.95+/-0.06 and E(max): 0.46 g+/-0.05 with endothelium vs 7.81+/-0.02 and 0.41 g+/-0.07 without endothelium; P>0.05). Ang II (0.003 - 0.3 microM)-induced a non-sustained contraction of endothelium-intact preparations which was not antagonized by BQ-123 (1 microM), but was inhibited by losartan (10 nM). In addition, the maximal contraction induced by ET-1 (0.1 microM) could be further increased by the addition of 0.1 microM Ang II. 3. Ang II (0.001 - 0.3 microM) elevated [Ca(2+)](i) in single vascular smooth muscle cells (VSMCs) in a dose-dependent manner (pEC(50) 9.12+/-0.26) and the Ang II-induced increases in [Ca(2+)](i) were not affected by a Ca(2+)-free solution, but were abolished by pretreatment with caffeine (5 mM). Ang II did not increase [Ca(2+)](i) in endothelial cells. ET-1 (0.1 microM) increased [Ca(2+)](i) in single VSMCs in a normal Ca(2+) containing physiological saline solution (PSS), but not in a Ca(2+)-free solution. 4. Ang II-induced contraction was insensitive to inhibition by nifedipine (0.1 microM), an antagonist of L-type voltage-gated Ca(2+) channels, and SK&F96365 (10 microM), which blocks non-selective cation channels, whereas that to ET-1 was inhibited by SK&F69365. 5. RT - PCR data indicate the expression of AT(1A) and AT(1B) on both VSMCs and endothelial cells, but immunohistochemical evidence illustrates that the AT(1) is located primarily on VSMCs. 6. These results indicate that endothelium-derived ET-1 is not involved in the Ang II-mediated vasoconstriction of the rat tail artery and that Ang II- and ET-1-mediated VSM contractions utilize distinct pathways.     

Comparative studies of AJG049, a novel Ca2+ channel antagonist, on voltage-dependent L-type Ca2+ currents in intestinal and vascular smooth muscle

BACKGROUND AND PURPOSE: Antagonists of Ca2+ channels reduce contraction of intestinal smooth muscle but also affect vascular smooth muscle. We have therefore examined the effects of AJG049, a newly synthesized antagonist for regulation of gut motility, on voltage-dependent L-type Ca2+ channels, in vascular and intestinal smooth muscle, comparing AJG049 with two other Ca2+ channel antagonists, verapamil and diltiazem. EXPERIMENTAL APPROACH: Affinities of AJG049 for various types of voltage-dependent Ca2+ channels were examined by binding studies. Effects of AJG049 on voltage-dependent inward Ca2+ (or Ba2+) currents (ICa or IBa) in dispersed smooth muscle cells from guinea-pig ileum, colon and mesenteric artery were measured using conventional whole-cell configurations. KEY RESULTS: In binding studies, AJG049 showed a high affinity for the diltiazem-binding site of L-type Ca2+ channels. In whole-cell configuration, AJG049 suppressed ICa in ileal myocytes, with concentration-, voltage-and use-dependencies. AJG049 shifted the steady-state inactivation curve of ICa to the left. The order of potency to inhibit ICa in ileal myocytes was AJG049>verapamil>diltiazem. AJG049 also suppressed IBa in guinea-pig mesenteric arterial myocytes, showing concentration- and voltage-dependencies and the potency order for this action was also AJG049>verapamil>diltiazem. For the relative ratio of Ki values between ileal and mesenteric arterial myocytes, the order was AJG049>diltiazem>verapamil. CONCLUSIONS AND IMPLICATIONS: These results show that AJG049 inhibits L-type Ca2+ channels mainly through the diltiazem-binding site(s). From our results, AJG049 showed a little selectivity for these Ca2+ channels in intestinal smooth muscle.     

Selegiline, an MAO-B inhibitor, attenuates airway smooth muscle contraction in the rat trachea

Selegiline is widely used for Parkinson's disease and sometimes for Alzheimer's disease. It is reported to affect intracellular Ca(2+) concentration. Since intracellular Ca(2+) is partly regulated by phosphatidylinositol (PI) response and is important for smooth muscle contraction, selegiline may affect airway smooth muscle tension. We examined the effects of selegiline on acetylcholine (ACh)- and KCl-induced contractile and PI responses in rat trachea. The trachea was cut into 3-mm-wide ring segments or 1-mm-wide slices. ACh (3 microM, 50% effective dose) or KCl (40 mM) was added, and ring relaxation was induced by the addition of selegiline. Tracheal slices were incubated with [(3)H]myo-inositol and 3 microM ACh in the presence of selegiline, and [(3)H]inositol monophosphate (IP(1)) was measured. Selegiline dose-dependently attenuated ACh- and KCl-induced tracheal ring contractions. Fifty-percent inhibitory doses (ID50) of selegiline against ACh- and KCl-induced contraction were 120 +/- 30 microM and 80 +/- 20 microM, respectively. Basal and ACh-induced IP(1) accumulation were 2.20 +/- 0.20 Bq and 7.88 +/- 0.23 Bq, respectively, and selegiline at a dose of 1000 microM attenuated ACh-induced IP(1) accumulation (5.44 +/- 0.30 Bq). These results suggest that selegiline inhibits contractile responses through the inhibition of voltage-operated Ca(2+) channels and the PI response.     

Emodin increases Ca(2+) influx through L-type Ca(2+) channel in guinea pig gallbladder smooth muscle

Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca(2+) concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca(2+)](i) was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca(2+)](i) in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 microM (at +10 mV, 10 microM, 45.1+/-5.2% compared to control, EC(50) =3.11 microM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca(2+) influx through L-type calcium channel via PKC pathway.     

Nerve evoked P2X receptor contractions of rat mesenteric arteries; dependence on vessel size and lack of role of L-type calcium channels and calcium induced calcium release

1. Contractile responses to short trains of nerve stimulation have been characterized in small, medium and large arteries from the rat mesenteric circulation (5th - 6th, 2nd - 3rd and 1st order, respectively). In addition, sources of calcium for smooth muscle contraction have been investigated. 2. Nerve stimulation (10 pulses at 10 Hz) evoked reproducible contractions. The P2 receptor antagonist suramin (100 microM) reduced constrictions by 65.3+/-7.4, 82.7+/-3.3 and 3.1+/-6.1% in small, medium and large arteries respectively. The alpha-adrenoceptor antagonist prazosin (0.1 microM) reduced responses by 32.6+/-2.6, 27.0+/-1.5 and 97.0+/-1.9% respectively. 3. The L-type calcium channel antagonist nifedipine (1 microM) reduced nerve-evoked contractions by 2.8+/-3.3, 10.0+/-3.7 and 13.5+/-2.7% in small, medium and large arteries respectively. When the adrenergic component of contraction was blocked by prazosin (0.1 microM) nifedipine reduced responses by 4.6+/-7.9, 14.3+/-2.0 and 3.0+/-1.9% respectively. Contractile responses to exogenous alpha,beta-meATP were unaffected by the depletion of calcium stores with cyclopiazonic acid (30 microM). This indicates that mobilization of calcium from internal stores is not required for P2X receptor mediated smooth muscle contraction. We conclude that for neurogenic responses, the P2X receptor mediated component of constriction dominates in small mesenteric arteries (3rd -- 6th order) while in large arteries (1st order) noradrenaline mediates contraction. For P2X receptor mediated responses all the calcium required for smooth muscle contraction enters the cell directly through P2X receptor channels.     

Calcium antagonism by KB-2796, a new diphenylpiperazine analogue, in dog vascular smooth muscle.

The effects of KB-2796, a new diphenylpiperazine analogue, on [3H]nitrendipine ([3H]NTD) binding, KCl-induced contraction and 45Ca influx has been examined in dog vascular smooth muscle, and compared with those of other diphenylpiperazines. In the binding study, [3H]NTD was found to bind with a high affinity to a single class of sites on aortic membranes (Kd = 0.41 nM and Bmax = 31 fmol (mg protein)-1). KB-2796 inhibited specific [3H]NTD binding in a concentration-dependent manner, with a Ki value of 0.34 microM. The other diphenylpiperazine derivatives such as flunarizine and cinnarizine also inhibited binding in the same manner. Also, in the contraction study, all the diphenylpiperazines antagonized the 50 mM KCl-induced contraction of isolated mesenteric arteries concentration-dependently. The IC50 values of the compounds for KCl-induced contraction correlated strongly with the respective Ki values obtained in the [3H]NTD binding study. In the 45Ca influx study, KB-2796 also effectively inhibited KCl-induced 45Ca influx in mesenteric arteries, with an IC50 value of 0.14 microM. This was close to the IC50 value found in the KCl-induced contraction study. These findings suggest that calcium antagonism by KB-2796 is responsible for its vasorelaxing action in vascular smooth muscle.     

Direct effects of hydrogen peroxide on airway smooth muscle tone: roles of Ca2+ influx and Rho-kinase

Reactive oxidant species are implicated in the chronic airway inflammation related to asthma and chronic obstructive pulmonary disease. This study was designed to determine mechanisms underlying contraction induced by hydrogen peroxide (H(2)O(2)), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca(2+) concentrations ([Ca(2+)](i)), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H(2)O(2) (0.01-1 mM) caused contraction with an augmentation of [Ca(2+)](i) in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca(2+) concentrations. The contractile force and [Ca(2+)](i) by H(2)O(2) (1 mM) were approximately half of those in response to 1 microM methacholine. However, contraction by H(2)O(2) was not generated under the condition that extracellular Ca(2+) concentrations were less than 0.15 mM. Verapamil (10 microM), an inhibitor of voltage-operated Ca(2+) channels, partially but significantly inhibited the H(2)O(2)-induced contraction. In contrast, SKF-96365 (1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride) (100 microM), a non-selective inhibitor of Ca(2+) channels, completely abolished both the contraction and the increase in [Ca(2+)](i) elicited by H(2)O(2). Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03-10 microM), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H(2)O(2)-induced contraction. In conclusion, both the Ca(2+) influx from the extracellular side and the Ca(2+) sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H(2)O(2). An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress.     

Effects of besipirdine at the voltage-dependent sodium channel.

1. Besipirdine (HP 749) is a compound undergoing clinical trials for efficacy in treating Alzheimer's disease. Among other pharmacological effects, besipirdine inhibits voltage-dependent sodium and potassium channels. This paper presents a pharmacological study of the interaction of besipirdine with voltage-dependent sodium channels. 2. Besipirdine inhibited [3H]-batrachotoxin binding (IC50 = 5.5 +/- 0.2 microM) in a rat brain vesicular preparation and concentration-dependently inhibited veratridine (25 microM)-stimulated increases in intracellular free sodium ([Na+]i) and calcium ([Ca2+]i) in primary cultured cortical neurones of rat. 3. Besipirdine (30-100 microM) concentration-dependently inhibited (up to 100%) veratridine-stimulated release of [3H]-noradrenaline (NA) from rat cortical slices. 4. When examined in greater detail, besipirdine was found to inhibit [3H]-batrachotoxin binding in vesicular membranes competitively. However, when examined in rat brain synaptosomes, we found that the antagonism by besipirdine was not competitive; that is, the maximal stimulation of [Ca2+]i induced by veratridine decreased with increasing concentrations of besipirdine. 5. These results show that besipirdine is an inhibitor of voltage-sensitive sodium channels and appears to bind to a site close to the batrachotoxin/veratridine binding site.     

Kavain attenuates vascular contractility through inhibition of calcium channels

Kavain is a biologically active compound from the Oceanic plant Piper methysticum (kava). Traditional medicinal uses of the kava root are many. Kava is increasingly being utilized by Western societies for its anxiolytic effects. Recent reports indicate that kavain blocks ion channels in neural tissue, relaxes precontracted ileum, and relaxes precontracted airway. Thus, we investigated the potential ability of this plant-derived compound to alter vascular smooth muscle function. Thoracic aortae were isolated from Sprague-Dawley rat and cut into 4 mm rings. Rings were placed in tissue baths and suspended from force-displacement transducers for the measurement of isometric tension. In a dose-dependent manner, kavain (10(-6) M to 10(-3) M) was found to relax aortic rings precontracted with phenylephrine (PE). This response was not dependent on functional endothelium. In addition, kavain pretreatment (3 x 10(-5) M or 3 x 10(-4) M) attenuated vascular smooth muscle contraction evoked by PE. However, kavain failed to attenuate PE-mediated contraction in calcium (Ca(++))-free buffer, indicating that intracellular signaling processes were likely not affected. Also, kavain did attenuate the contraction elicited by administration of Ca(++) to depolarized tissue. Interestingly, in rings pre-treated with the selective L-type Ca(++) channel blocker nifedipine, kavain-mediated relaxation was inhibited. Lastly, in rings selectively contracted with an L-type calcium channel activator, kavain elicited dose-dependent (and ultimately complete) relaxation. These data strongly suggest that kavain impairs vascular smooth muscle contraction, likely through inhibition of Ca(++) channels.