Mechanisms of acetylcholine-induced vasorelaxation in high K+-stimulated rabbit renal arteries

To characterize the mechanisms of acetylcholine (ACh)-induced vasorelaxation in rabbit renal arteries precontracted with high K+ (100 mM), muscle tension and cytosolic free Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded arterial strips. In the artery with endothelium, high K+ increased both [Ca2+]i and muscle tension. Addition of ACh (10 microM) during high-K+ induced contraction significantly relaxed the muscle and induced additional increase in [Ca2+]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM). ACh increased [Ca2+]i without relaxing the muscle. In the artery without endothelium, high K+ increased both [Ca2+]i and muscle tension although ACh was ineffective, suggesting that ACh acts selectively on endothelium to increase [Ca2+]i. 4-DAMP (10 nM) or atropine (0.1 microM) abolished the ACh-induced increase in [Ca2+]i and relaxation. However, pirenzepine (0.1 microM), AF-DX 116 (1 microM) and tropicamide (1 microM) were ineffective. The ACh-induced increase of [Ca2+li and vasorelaxation was significantly reduced by 3 microM gadolinium, 10 microM lanthanum or 10 microM SKF 96365. These results suggest that, in rabbit renal artery, ACh-evoked relaxation of 100 mM K+-induced contractions is mediated by the release of endothelial NO. ACh may stimulates the M3 subtype of muscarinic receptor in the endothelial cells, resulting in the opening of the nonselective cation channels followed by an increase of [Ca2+]i and stimulation of NO synthase.     

Capsaicin-induced relaxation in rabbit coronary artery.

In the present study mechanism of inhibitory effects of capsaicin on the contractility of rabbit coronary artery were studied by measurement of isometric tension and intracellular Ca2+ concentration. Capsaicin (1 microM to 30 microM) relaxed the coronary artery pre-contracted with prostaglandin (PG) F2alpha (1 microM) in a concentration-dependent manner. The PGF2alpha-induced increase in intracellular Ca2+ concentration was also inhibited. The effects of capsaicin were readily reversed by washing capsaicin from the bath. Capsaicin-induced relaxation was not attenuated by pretreatment with capsazepine (1 microM), a blocker of vanilloid receptor or ruthenium red (1 microM), a blocker of non-selective cation channel. Previous exposure to a high concentration of capsaicin (100 microM) or repeated application of capsaicin did not eliminate the relaxation response to subsequent application of capsaicin. Increasing the external K+ concentration to 80 mM significantly attenuated the capsaicin-induced relaxation with simultaneous change in intracellular Ca2+ concentration. Pretreatment with iberiotoxin (100 nM), a blocker of Ca2+-activated K+ channel, only partially inhibited the capsaicin-induced relaxation. However, application of 4-aminopyridine (4-AP, 1 mM), a blocker of delayed rectifier K+ current significantly inhibited the capsaicin-induced relaxation with concomitant attenuation of the effect on intracellular Ca2+ concentration. These results indicate that capsaicin may have a direct relaxing effect on the smooth muscle contractility, and relaxation may be due to activation of the 4-AP-sensitive, delayed rectifier K+ channels in the rabbit coronary artery.     

Role of endothelium and nitric oxide in modulating in vitro responses of colonic arterial and venous rings to vasodilatory neuropeptides in horses

The objective of this study was to determine and compare the in vitro responses of equine large colon arterial and venous rings to vasodilatory neuropeptides; calcitonin gene-related peptide (CGRP); substance P (SP); vasoactive intestinal polypeptide (VIP); and acetylcholine (ACh), a standard nonpeptide endothelium-dependent vasodilator. Responses of vessel rings to graded concentrations (10(-11) M to 10(-5) M) of each drug were determined in endothelium-intact, denuded, and Nomega-nitro-L-arginine methyl ester (L-NAME, 10(-5) M)-treated rings that were pre-contracted with norepinephrine. Percentage maximal relaxation (PMR), defined as the % decrease from the contracted state, was determined. Because all rings did not relax at least 50%, EC50 values could not be consistently calculated. Arterial rings with intact endothelium were more sensitive to CGRP, compared with VIP and SP, and venous rings of all conditions were more sensitive to VIP than CGRP or SP. Overall, arteries had a greater PMR for ACh compared with SP and VIP. Intact and L-NAME treated arteries had a greater PMR than denuded arteries; there were no differences in PMR of intact and L-NAME treated arteries. Veins had a greater PMR for VIP than CGRP, SP, or ACh. Calcitonin gene-related peptide caused greater relaxation in intact arteries, whereas VIP causes greater relaxation in veins. Arterial relaxation was dependent upon the presence of intact endothelium. The response of veins to VIP among the conditions tested was not different, suggesting VIP has direct actions on venous smooth muscle. These neuropeptides modulate vasomotor tone via vasorelaxation in colonic arteries and veins.     

Characterisation of the response of equine digital arteries and veins to substance P

Substance P (SP), a potent vasodilator, has been detected in equine digital sensory-motor nerves. The aim of the study was to characterise the functional responses of equine digital blood vessels to exogenous SP. Pre-constricted equine digital arteries (EDA) and veins (EDV) vasodilated in a biphasic, endothelium- and concentration-dependent manner to SP. A nitric oxide (NO) synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; 300 microm) inhibited both phases of the relaxation response curve of EDAs to SP by >70%. In EDVs, the first relaxant phase to SP was largely L-NAME-resistant, whereas the second phase was inhibited by 60%. Both L-NAME and a cyclo-oxygenase inhibitor (ibuprofen; 10 microm) were required to inhibit EDV relaxation to SP by > or =80%. Experiments determining the receptor mediated responses to physiological concentrations of SP (1 nm) revealed that the relaxant responses of both EDA and EDV were inhibited by a neurokinin-1 (NK1) receptor antagonist (CP-96 345; 10 nm). In conclusion, SP is an endothelium-dependent vasodilator of both EDA and EDV. NO is the predominant pathway activated in EDA, whereas both prostacyclin and NO pathways are involved in EDVs. NK1 receptors appear to mediate responses to low concentrations of SP.     

Effects of substance P on nicotine-induced intracellular Ca2+ dynamics in bovine adrenal chromaffin cells

Substance P (SP) is colocalized with ACh in splanchnic nerves that innervate into adrenal medulla and the peptide has been shown to inhibit nicotinic agonists-induced catecholamine secretion. To elucidate the effects of SP on cytosolic Ca2+ dynamics, the present study was conducted using fura-2-loaded isolated bovine adrenal chromaffin cells. Stimulation of the cells with nicotine (10-100 microM) produced a rapid rise of cytosolic Ca2+ concentration ([Ca2+]i), the peak level of which increased in a dose-dependent manner, followed by a gradual decay. In the presence of 10 microM SP, the dose-response relationship of the peak levels shifted downward. Quantitative analyses implied that SP inhibits the nicotine-induced Ca2+ influx in a noncompetitive manner. Nicotinic acetylcholine receptor is composed of two major functional domains: an agonist-binding site and an ionophore or channel domain. Agonist binding activates ionophore/channel domain and causes mainly Na+ influx. This Na+ influx depolarizes the cell and activates voltage-dependent Ca2+ channels. Based on this fact, the present results indicate that SP dose not block nicotine binding sites but interferes with other sites of nicotinic receptor/channel molecule, most probably a channel domain. It was suggested that SP colocalized with ACh in splanchnic nerves functions as a physiological modulator of catecholamine secretion by non-competitively suppressing ACh-induced cytosolic Ca2+ dynamics in bovine adrenal chromaffin cells.     

Palmar digital vessel relaxation in healthy horses and in horses given carbohydrate

OBJECTIVE: To compare in vitro smooth muscle relaxation of palmar digital vessels from healthy horses with those from horses in the prodromal stage of experimentally (carbohydrate) induced laminitis. ANIMALS: 16 adult horses. PROCEDURE: Segments of palmar digital vessels were obtained from 5 healthy horses and 6 horses given carbohydrate. Vascular rings from the palmar digital artery and vein were suspended in individual organ baths containing buffer solution and indomethacin; isometric tension was recorded, and contraction and relaxation were compared. Smooth muscle contraction in response to cumulative addition of phenylephrine was recorded in the absence and presence of 1 microM NG-nitro-L-arginine methyl ester (L -NAME). After wash out, vascular rings were preconstricted with phenylephrine (0.3 microM), and cumulative endothelium-dependent (acetylcholine-induced) and independent (nitroprusside-induced) smooth muscle relaxations were recorded in the absence or presence of L -NAME. RESULTS: Phenylephrine increased vascular smooth muscle tone in ring preparations of palmar digital arteries and veins. Addition of acetylcholine or nitroprusside induced relaxation of palmar digital artery and vein ring preparations. Use of L-NAME (1 microM) significantly reduced maximal relaxation induced by acetylcholine, but not by nitroprusside. Maximal relaxation induced by acetylcholine, but not by nitroprusside, was reduced in vascular rings prepared from carbohydrate-overloaded horses. CONCLUSION AND CLINICAL RELEVANCE: Reduced endothelium-dependent relaxation of palmar digital vessels may have a role in the pathophysiology of acute laminitis after carbohydrate overload in horses.     

Characterization of muscarinic receptor subtype mediating contraction and relaxation in equine coronary artery in vitro

In coronary arterial rings isolated from horses, 10-^-8-10^-6 mol/l acetylcholine (ACh) induced concentration-dependent contractions which were potentiated by the removal of endothelium and by pretreatment with I,-nitro-arginine (LNAG) or methylene blue (MB). Relatively lower concentrations of Ach 10-¹⁴-10-⁸ mol/l) induced relaxation when the coronary rings were contracted by phenylephrine (PE). ACh-induced contractions in the coronary rings without endothelium were competitively inhibited by each muscarinic subtype selective antagonist in the following order of potency: 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine ≥ parafluoro-hexahydrosiladiphenidol (pFHHSiD) > methoctramine. ACh-induced relaxation in the rings with endothelium was inhibited by LNAG or MB, and by each selective antagonist in the following order of potency: 4-DAMP < pFHHSiD ≥ pirenzepine ≥ methoctramine. These results suggest that the ACh-induced contraction and relaxation in equine coronary arteries are mediated mainly by an M₃-receptor located on the smooth muscle cells and endothelial cells, respectively, and that the stimulation of the M₃-receptor on the endothelial cells liberates nitric oxide.     

Characterization of agonist-induced endothelium-dependent vasodilatory responses in the vascular bed of the equine digit

The role of endothelium-derived relaxing factors was studied in the regulation of vascular responses in the Krebs perfused equine isolated digit. Perfusion pressure was recorded in response to bolus doses of 5-hydroxytryptamine (6 nmol) alone or co-administered with carbachol (CCh; 0.2 micromol), bradykinin (BK; 0.2 nmol), substance P (SP; 0.2 nmol) or sodium nitroprusside (SNP; 0.2 micromol). N(omega)-Nitro-L-Arginine methyl ester hydrochloride (L-NAME; 300 microm) caused partial but significant inhibition of CCh-induced vasodilatory response, whereas BK and SP-induced responses were resistant to L-NAME. High potassium (K(+), 30 mm) and the cytochrome P-450 (CYP) epoxygenase inhibitor, clotrimazole (10 microm) plus L-NAME (100 microm), completely abolished the CCh, BK and SP-induced vasodilatory responses, whereas the response to SNP was unaffected. In contrast, the L-NAME-resistant proportion of CCh, BK and SP-induced vasodilatory response was not inhibited by the highly selective CYP2C9 inhibitor, sulphaphenazole (10 microm). The cyclo-oxygenase inhibitor, ibuprofen (10 microm) did not affect the CCh, BK and SP-induced responses. These data demonstrate that CCh, BK and SP-induced relaxation in the equine digit involve a combination of the NO and endothelium-derived hyperpolarizing factor (EDHF) pathways. These results do not support the evidence for the involvement of CYP-derived epoxyeicosatrienoic acids and the exact nature of EDHF in the equine digit remains to be established.     

Nitrergic inhibition of tachykininergic neuro-muscular transmission via cyclic GMP in the hamster ileum

The present study was designed to explore the inhibitory mechanism by nitric oxide (NO) of the tachykininergic neuro-muscular transmissions in the hamster ileum. In the presence of guanethidine (1 μM), atropine (0.5 μM), nifedipine (0.1 μM) and apamin (100 nM), electrical field stimuli (EFS; 0.5 ms duration, 15 V) evoked non-adrenergic, non-cholinergic excitatory junction potentials (EJPs) in circular smooth muscle cells. The EJPs were markedly inhibited by the tachykinin NK1 receptor antagonists [D-Pro(4), D-Trp(7,9)]-SP(4-11) (3 μM). Both the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 200 μM) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 μM), did not affect on the resting membrane potentials, but enhanced the tachykininergic EJPs. In the presence of L-NAME (200 μM), exogenously applied NO (10 μM) and the membrane permeable analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, 3 mM), significantly inhibited the tachykininergic EJPs. Application of EFS (0.5 msec duration, 15 V) with trains of 20 pulses at 20 Hz increased amount of released substance P (SP). The release of SP was further increased by the treatment of L-NAME or ODQ, but markedly reduced by exogenously applied NO and 8-Br-cGMP. These results suggest that the endogenous NO may inhibit the tachykininergic neuro-muscular transmissions by the decrease of SP release from the tachykininergic neurons, possibly through a guanylate cyclase-cGMP-dependent mechanism in the hamster ileum.     

Endothelium-dependent relaxation of canine uterine artery in response to acetylcholine: the possible involvement of alternative pathways

The effect of acetylcholine on the isolated, pre-contracted, uterine artery of non-pregnant dog was investigated. Acetylcholine-induced concentration-dependent relaxation of isolated canine uterine artery with endothelium (pEC50 = 6.48 +/-0.01, n = 37) and was without effect on arterial segments denuded of endothelium. Indomethacin, 4-aminopyridine (10-5 m) and pre-contraction with K+-rich Krebs-Ringer bicarbonate solution had no effect on acetylcholine-induced relaxation. NG-nitro-l-arginine (l-NOARG) (10-5 m) inhibited relaxation evoked by acetylcholine. Indomethacin applied with l-NOARG led to further inhibition of acetylcholine-induced relaxation. In the presence of both l-NOARG and indomethacin, 4-aminopiridine did not provoke further inhibition of acetylcholine-induced relaxation of canine uterine artery. It is concluded that the acetylcholine-induced relaxation of canine uterine artery is probably mediated by endothelial production of nitric oxide (NO). However, if NO-synthase is inhibited, acetylcholine-induced vasorelaxation may be, in part, mediated through activation of cyclooxygenase pathway.     

Anticholinergic effects of artemisinin, an antimalarial drug, in isolated guinea pig heart preparations

Concern has been growing about the cardiac toxicity of antimalarial drugs. Artemisinin, a unique type of antimalarial drug originating from a Chinese medicinal plant, has minimal adverse effects, but it has been reported to inhibit delayed rectifier potassium current, a voltage-gated potassium current. However, no studies have been published concerning the effect of artemisinin on ligand-gated potassium currents. Therefore, in the present study, we examined the influence of artemisinin on the acetylcholine receptor-operated potassium current (IK.ACh), a ligand-gated potassium current, in guinea pig atrial myocytes using a patch clamp technique. Artemisinin (1 to 300 microM) inhibited I(K.ACh) induced by extracellular application of both carbachol (1 microM) and adenosine (10 microM) and that induced by intracellular loading of GTPgammaS (100 microM) in a concentration-dependent manner. Artemisinin inhibited carbachol-induced, adenosine-induced, and GTPgammaS-activated IK.ACh within almost the same concentration range. In left atria, artemisinin (1 to 100 microM) partially reversed the shortening of action potential duration induced by carbachol in a concentration-dependent manner. Carbachol-induced negative inotropic action in left atria was also inhibited by artemisinin (10 to 300 microM). In conclusion, we suggest that the anticholinergic action of artemisinin is mediated through inhibition of IK.ACh via inhibition of the muscarinic potassium channel and/or associated GTP-binding proteins.     

Effects of high-K+, Na+-deficient solution on contractility of the smooth muscles of the bovine trachea

A high-K+, Na+-deficient (I-154 K+) solution induced contraction followed by gradual relaxation of the smooth muscles of the bovine trachea, while hyperosmotic addition of 65 mM KCl induced a large sustained contraction. Exposure of the muscle to the I-154 K+ solution induced an increase in the ratio of cellular water content and a sustained increase in oxidized flavoprotein fluorescence or reduced pyridine nucleotide fluorescence. The I-154 K+ solution also induced a sustained increase in [Ca2+]i level. Decreases in developed tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl but not pyruvate. Substitution of KI for KCl in the I-154 K+ solution produced a greater inhibition of contraction, while substitution with K-propionate produced no inhibition of contraction. Moreover, decreases in developed tension and increases in cellular water content were both prevented by addition of 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), but not 10 microM bumetanide or 1 mM acetazolamide. In conclusion, I-154 K+ solution induced-relaxation in the bovine trachea may be due to swelling of smooth muscle cells and the mechanism of swelling is probably involved in DIDS-sensitive anion movement.     

Possible involvement of K+ channel opening to the interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.

We have previously shown that interleukin-1 beta relaxes vascular smooth muscle by the NO-dependent and independent mechanisms (Takizawa et al.: Eur. J. Pharmacol. 330: 143-150, 1997). In this study, we investigated the mechanism of NO-independent relaxation. Treatment of the rat aorta with interleukin-1 beta for 24 hr inhibited the high-K+ induced contraction by decreasing cytosolic Ca2+ level ([Ca2+]i). The relationship between [Ca2+]i and tension in intact muscle and the pCa-tension curves in permeabilized muscle suggested that Ca2+ sensitivity of contractile element was not changed after the interleukin-1 beta-treatment. After a treatment with interleukin-1 beta for 24 hr, contractile effects of phenylephrine (1 microM-10 microM) were markedly inhibited in the presence of L-NMMA (100 microM) applied to inhibit NO synthesis. A blocker of ATP-sensitive K+ channel, glibenclamide (1 microM), partially recovered the interleukin-1 beta-induced inhibition. In contrast, a blocker of Ca(2+)-activated K+ channel, charybdotoxin (0.1 microM), was ineffective. These results suggest that membrane hyperpolarization due to activation of ATP-sensitive K+ channels may partly be responsible for the NO-independent mechanism of interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.     

Femtomolar bradykinin-induced relaxation of isolated bovine coronary arteries, mediated by endothelium-derived nitric oxide

We reported previously that bradykinin induces endothelium-dependent relaxation at nanomolar (n M ) concentrations in isolated bovine coronary arteries with an intact endothelium. Recently we have found that in the presence of 10 μ m indomethacin, femtomolar (f M ) concentrations of bradykinin induce endothelium-dependent relaxation in some bovine coronary arteries (≈ 10% of the coronary arteries examined). The present study was designed to characterize the relaxation induced by f M bradykinin. Relaxation was completely abolished by repeated application of f M bradykinin, by 100 μ m N^ω- nitro- l - arginine methyl ester and by 10 μ m methylene blue. Relaxation induced by n M bradykinin was partly affected by these treatments. Relaxation induced by both concentrations of bradykinin was inhibited by a B₂-kinin receptor antagonist, [Thi^5,8, D-Phe⁷]-bradykinin, in a concentration-dependent manner, but not by a B₁-kinin receptor antagonist, des-Arg⁹, [Leu⁸]-bradykinin. In the presence of 10 μ m captopril, an angiotensin-converting enzyme (ACE) inhibitor, all coronary arteries examined in this experiment showed endothelium-dependent relaxation to f M bradykinin. These results show that some bovine coronary arteries relax in response to f M bradykinin, and this response is mediated predominantly by the release of nitric oxide via stimulation of endothelial B₂-kinin receptors. The relaxation may be dependent on ACE activity.     

Benzodiazepines inhibit the acetylcholine receptor-operated potassium current (IK.ACh) by different mechanisms in guinea-pig atrial myocytes

The anticholinergic effects of 7 benzodiazepines, bromazepam, camazepam, chlordiazepoxide, diazepam, lorazepam, medazepam and triazolam, were compared by examining their inhibitory effects on the acetylcholine receptor-operated potassium current (I(K).(ACh)) in guinea-pig atrial myocytes. All of these benzodiazepines (0.3-300 μM) inhibited carbachol (1 μM)-induced I(K).(ACh) in a concentration-dependent manner. The ascending order of IC(50) values for carbachol-induced I(K).(ACh) was as follows; medazepam, diazepam, camazepam, triazolam, bromazepam, lorazepam and chlordiazepoxide (>300 μM). The compounds, except for bromazepam, also inhibited I(K).(ACh) activated by an intracellular loading of 100 μM guanosine 5'-[γ-thio]triphosphate (GTPγS) in a concentration-dependent manner. The ascending order of IC(50) values for GTPγS-activated I(K).(ACh) was as follows; medazepam, diazepam, camazepam, lorazepam, triazolam chlordiazepoxide (>300 μM) and bromazepam (>300 μM). To clarify the molecular mechanism of the inhibition, IC(50) ratio, the ratio of IC(50) for GTPγS-activated I(K).(ACh) to carbachol-induced I(K).(ACh), was calculated. The IC(50) ratio for camazepam, diazepam, lorazepam, medazepam and triazolam was close to unity, while it for chlordiazepoxide could not be calculated. These compounds would act on the GTP binding protein and/or potassium channel to achieve the anticholinergic effects in atrial myocytes. In contrast, since the IC(50) ratio for bromazepam is presumably much higher than unity judging from the IC(50) values (104.0 ± 30.0 μM for carbachol-induced I(K).(ACh) and >300 μM for GTPγS-activated I(K).(ACh), it would act on the muscarinic receptor. In summary, benzodiazepines had the anticholinergic effects on atrial myocytes through inhibiting I(K).(ACh) by different molecular mechanisms.     

Alterations of Endothelium-Dependent Digital Vascular Responses in Horses Given Low-Dose Endotoxin

Low doses of endotoxin cause vasoconstriction and hypoperfusion of the digit, small intestine, and cecum in horses. To determine the potential cause of these vascular alterations, in vitro vascular responses of palmar digital arteries and veins were determined in 8 horses after intravenous (IV) infusion of 1 L 0.9% NaCl (control) and 0.1 μg/kg Escherichia coli 055:B5 endotoxin in 1 L of 0.9% NaCl (endotoxin-treated). Vessels were surgically removed under general anesthesia, cut into 4-mm vascular rings, suspended in tissue baths, and attached to force displacement transducers for measurement of vascular tension. Cumulative concentration response curves to acetylcholine, bradykinin, nitroprusside, norepinephrine, 5-hydroxytryptamine (serotonin), and endothelin were determined. Maximal relaxation or contraction and the concentrations needed to produce 50% maximal relaxation or contraction were determined. Palmar digital arteries from endotoxin-treated horses relaxed significantly less in response to acetylcholine and bradykinin (endothelium-dependent), but not to nitroprusside (endothelium-independent) when compared with arteries from control horses. Digital arteries from endotoxin-treated horses also contracted significantly more with norepinephrine but less with serotonin. Digital veins responded less than digital arteries. In another study, vascular reactivity experiments documented that acetylcholine and bradykinin were endothelium-dependent vasodilators (endothelium-denuded vessels relaxed less than control vessels) in palmar digital vessels. Additionally, maximal relaxations for both vasodilators were significantly inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide antagonist, suggesting that acetylcholine and bradykinin cause relaxation through the nitric oxide pathway. The data from these studies indicate that low dose endotoxin impairs endothelium-dependent relaxation and augments adrenergic contraction of palmar digital arteries in horses.     

The relaxant effect of vasoactive intestinal polypeptide in the isolated canine uterine artery: the role of endothelium

The purpose of this study was to examine the effect of vasoactive intestinal polypeptide (VIP) on the uterine artery obtained from non-pregnant dogs. VIP (3 x 10(-9)-3 x 10(-7) M) induced concentration-dependent relaxation in canine uterine arteries with intact endothelium, pre-contracted with 10(-5) M phenylephrine (pEC(50) = 7.52 +/- 0.02, maximal response was 82.19 +/- 2.15%, n = 36). The administration of the cyclooxygenase inhibitor indomethacin (10(-5) M) or 4-aminopyridine (4-AP), a blocker of potassium channels (10(-5) M), did not modify the relaxation induced by VIP. Contrary to this, N(G)-nitro-L-arginine (L-NOARG) (10(-5) M) inhibited relaxation is evoked by VIP. Indomethacin applied with L-NOARG did not provoke further inhibition of VIP-induced relaxation. In the presence of both L-NOARG and L-NOARG + indomethacin, 4-AP led to the further inhibition of VIP-induced relaxation of canine uterine artery. It is concluded that VIP induces endothelium-dependent relaxation of uterine arteries of non-pregnant dogs, which can be entirely explained by the production of nitric oxide (NO) from the endothelial cells. We proposed that when NO synthesis is inhibited, VIP induces further relaxation, independent of the edothelium-derived relaxing factors, probably through activation of K(+) channels.     

Equine coronary artery responds to 5-hydroxytryptamine with relaxation in vitro

Isolated equine coronary arteries responded to 5-hydroxytryptamine (5-HT) with relaxations in both endothelium-dependent and endothelium-independent mechanisms. Experiments were designed to characterize the 5-HT receptor sub type mediating these relaxations. Both 5-HT and alpha-methyl-5-HT (α-Me-5- HT; 5-HT₂ agonist) produced concentration-dependent relaxations in equine coronary arteries precontracted with a thromboxane A₂ derivative (0N011113). The degree of the maximal relaxation induced by α t-Me-5-HT was about one-half of that induced by 5-HT. In the coronary arteries without endothelium, α -Me-5-HT produced no relaxation, but 5-HT caused relaxation, which was inhibited by a 5-HT₁ antagonist (methysergide, mianserin and methiothepin), but was inhibited neither by ketanserin (5-HT₂ antagonist) nor by MDL72222 (5-HT₃ antagonist). In the coronary arteries with endothelium, however, the relaxation induced by α -Me-5-HT was inhibited by ketanserin, L-nitro-arginine (NO synthase inhibitor) and methylene blue (soluble guanylate cyclase inhibitor). These results suggest that the relaxation induced by 5-HT in equine coronary arteries depends mainly on the stimulation of both 5-HTi receptor subtype on smooth muscle cells directly, and 5-HT₂ receptor subtype on endothelial cells indirectly by liberating endothe-lium-derived NO.     

Tamoxifen induces vasorelaxation via inhibition of mitogen-activated protein kinase in rat aortic smooth muscle

The contribution of the mitogen-activated protein kinase (MAPK) pathway to the relaxation induced by tamoxifen, a synthetic non-steroidal anti-estrogen, was examined in rat vascular smooth muscle. Tamoxifen (0.1-300 microM) inhibited the contraction induced by endothelin-1 (ET-1, 3 nM) in aortic smooth muscle in a concentration-dependent manner. The inhibitory effect of tamoxifen was not attenuated by 10 microM ICI 182,780, a selective antagonist of estrogen receptors. In the Ca(2+) channel inhibitor verapamil (1 microM)-pretreated strips, tamoxifen also inhibited the contraction induced by ET-1. Both PD098059 and SB203580, inhibitors of MAPK/extracellular signal-regulated kinase (ERK) kinase and p38 MAPK, respectively, inhibited ET-1-induced contraction in aortic smooth muscle. In Western blot analysis with anti-phosphorylated MAPK antibodies, ET-1 (3 nM) enhanced activities of both ERK1/2 and p38 MAPK in aortic muscle strips, which were not attenuated by the treatment with 4 mM EGTA. Tamoxifen (100 microM) inhibited the activities of ERK1/2 and p38 MAPK induced by ET-1 without significant changes in the expression of these kinases. These results suggest that tamoxifen induces relaxation of rat vascular smooth muscle, and that this is, at least in part, mediated by the inhibition of the Ca(2+)-independent MAPK pathway.     

Cdc42 contributes to phorbol ester-induced Ca2+-independent contraction of pulmonary artery smooth muscle

We determined the contribution of the Rho family of low molecular GTP-binding proteins to phorbol ester-induced contraction in swine pulmonary artery smooth muscle. In Ca2+-free medium containing 1 mM EGTA, 12-deoxyphorbol 13-isobutyrate (DPB, 1 microM), a protein kinase C (PKC) activator, elicited sustained contractions, which were not inhibited by treatment with verapamil, a voltage-dependent Ca2+ channel antagonist, and Y27632, a Rho-associated kinase inhibitor. Immunoblot analysis showed three PKC isoforms (alpha, epsilon, and zeta) and two Rho GTPases (RhoA and Cdc42) in both cytosolic and the membrane fractions from quiescent strips. DPB (1 microM) significantly induced PKCalpha and epsilon to translocate from the cytosolic to the membrane fraction in Ca2+-free medium. DPB also elicited the translocation of Cdc42, but not RhoA to the membrane fraction. Similarly, in the experiment for measurement of Rho GTPase activity by pull-down assay, DPB (1 microM) significantly increased the activity of Cdc42 in Ca2+-free medium. Norepinephrine (NE, 10 microM) stimulated the redistribution of RhoA from the cytosolic to the membrane fraction in swine pulmonary artery smooth muscle. In contrast, NE did not alter the subcellular distributions of Cdc42 and the PKC isoforms. These results indicate that phorbol ester evokes PKC-mediated Ca2+-independent contraction via a Rho GTPase pathway, especially Cdc42, in smooth muscle from swine pulmonary arteries.