Isolated Rabbit Mesenteric Artery Research Articles

Inhibition of TRPC6 Channels by TRPC1/C5 Channel Activity Through a Ca 2+ and Pkc-Dependent Mechanism

Vascular myocytes express two groups of functional canonical transient receptor (TRPC) channels, which we have termed TRPC1 for TRPC1-containing and TRPC3/6/7 for non-TRPC1 containing channels. These two groups have distinct properties from each other, for example, TRPC1 channels have a single unitary conductance of 2-3 pS and require PIP2 and PKC for activation, whereas TRPC3/6/7 channels have multiple conductances between 10-70 pS and are inhibited by PIP2 and PKC. The present work investigated interactions between TRPC1/C5 and TRPC6 channels activities evoked by angiotensin II (Ang II) in freshly isolated rabbit mesenteric artery myocytes.

Long-Term Inhibition of RhoA Attenuates Vascular Contractility by Enhancing Endothelial NO Production in an Intact Rabbit Mesenteric Artery

RhoA plays a critical role in regulating NO production in cultured endothelial cells. To determine its role in in situ endothelial cells, we investigated the effects of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors and a RhoA-binding domain of Rho-kinase (RB) on vascular contractility in the isolated rabbit mesenteric artery. Ex vivo treatment of the strips with 3x10(-5) mol/L simvastatin and fluvastatin for approximately 24 to 30 hours significantly attenuated the contractile response to phenylephrine and high K+ in the presence of endothelium. The addition of N(omega)-nitro-L-arginine methyl ester and the removal of endothelium abolished the attenuation of the contractile response. The cotreatment with geranylgeranyl pyrophosphate prevented the statin-induced attenuation of the contractile response, whereas geranylgeranyl transferase inhibitor mimicked the effect of simvastatin. Treatment with simvastatin enhanced the bradykinin-induced endothelium-dependent relaxation in the mesenteric artery, whereas it had no effect on the bradykinin-induced [Ca2+]i elevation in endothelial cells of the aortic valves. Introduction of RB to the strips using a cell-penetrating peptide of Tat protein (TATHA-RB) attenuated the contractile responses in a NO-dependent manner. However, a Rac1/Cdc42-binding fragment of p21-activated protein kinase, RB without Tat peptide or TATHA-protein A had no effect. The in vivo treatment of rabbit with simvastatin and TATHA-RB attenuated the contractility in a NO-dependent manner. Simvastatin and TATHA-RB significantly upregulated eNOS in the rabbit mesenteric artery. The present study provides the first evidence that RhoA plays a physiological role in suppressing NO production in in situ endothelial cells.

Different AT1 receptor subtypes at pre- and postjunctional sites: AT1A versus AT1B receptors.

Angiotensin (AT) II is known to enhance responses to electrical field stimulation (EFS) via AT1 receptors located on sympathetic nerve terminals. Differences in potency exist between AT1 receptor antagonists regarding the inhibition of the prejunctional and postjunctional AT1 receptors. It is hypothesized that prejunctional AT1 receptors might belong to the AT1B receptor subtype. Accordingly, the authors investigated whether AT1B receptor inhibition by high concentrations of PD123319 could suppress ATII-augmented noradrenergic transmission (prejunctional) in the rabbit thoracic aorta by means of a noradrenaline spillover model. Additionally, the influence of PD123319 on ATII-enhanced constrictor responses to electrical field stimulation was investigated in the isolated rabbit mesenteric artery. Furthermore, the authors investigated whether PD123319 could influence the constrictor responses (postjunctional) to ATII in both preparations. In the thoracic aorta, ATII (10 nM) caused a significant enhancement of EFS-evoked [3H]-noradrenaline release by a factor of 2.0 +/- 0.1. This reinforcement could be inhibited by PD123319 (0.1, 1, and 10 microM). The constrictor response to ATII was unaffected by PD123319. In the mesenteric artery, ATII (0.5 nM) caused a significant enhancement of constrictor responses to EFS by factors of 2.9 +/- 0.3, 2.3 +/- 0.3, and 1.6 +/- 0.1 at 1, 2, and 4 Hz, respectively. This enhancement could be attenuated by PD123319 (1 and 10 microM). The constrictor response to ATII was unaffected by PD123319. It is concluded that the prejunctional AT1 receptors belong to the AT1B subtype whereas postjunctional AT1 receptors do not.

Pharmacologic characterization of S-1255, a highly potent and orally active endothelin A receptor antagonist.

The pharmacologic properties of a novel nonpeptide endothelin (ET) receptor antagonist, S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6-isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid), was studied. [3H]S-1255 specifically bound to porcine aortic smooth muscle membranes expressing only ET(A) receptors with a Kd value of 0.39 nM. [3H]S-1255 binding was potently inhibited by ET-1 and selective ET(A) or ET(A)/ET(B) receptor antagonists, such as L-749329, SB209670, bosentan, and BQ-123, but the inhibitory effect of ET-3 and the selective ET(B) receptor antagonist, BQ-788, on the binding was weak. These inhibitory effects on [3H]S-1255 binding correlated well with those on [125I]ET-1 binding. S-1255 inhibited ET(A) receptor- and ET(B) receptor-mediated contractions in isolated rabbit femoral and pulmonary arteries with pA2 values of 8.8 and 6.3, respectively. The pA2 value of S-1255 for ET(B) receptor-mediated relaxation in isolated rabbit mesenteric artery was 7.4. Oral administration of S-1255 (0.3-10 mg/kg) caused dose-dependent inhibition of the pressor response to exogenous ET-1 (0.1 nmol/kg) in conscious normotensive rats, which was similar to that produced by intravenous administration (1 and 3 mg/kg). S-1255 (10 and 30 mg/kg, p.o.) significantly reduced blood pressure in deoxycorticosterone acetate-salt hypertensive rats from 6 h after administration, and the hypotensive effects were sustained up to 24-48 h. These results suggest that S-1255 is a highly potent and orally active ET(A) receptor antagonist.

Endothelium-independent vascular relaxation mediating ETB receptor in rabbit mesenteric arteries.

Vascular response mediating endothelin (ET)B receptor was studied using isolated rabbit mesenteric arteries. ET-1 (0.1-30 nM) caused a concentration-dependent contraction, whereas ET-3 >100 nM caused only weak contraction. Up to 1 microM of sarafotoxin S6c showed no contraction. In arteries precontracted with phenylephrine, ET-3 (0. 03-1 nM) caused a concentration-dependent relaxation, which was not affected by endothelium denudation. The ET-3-induced relaxation was antagonized by BQ-788 and PD-142893 but not by BQ-123 in the endothelium-denuded arteries. Treatment with indomethacin but not with NG-nitro-L-arginine methyl ester completely inhibited the relaxation. ET-3 stimulated the release of 6-keto-PGF1alpha and PGE2 from the endothelium-denuded arteries. ET-3 also significantly increased cAMP content but not cGMP content in the arteries. Radioligand-binding studies using serial sections of the artery revealed the expression of not only ETA but also ETB receptors in the smooth muscle layer of the arteries. These results suggest that ET-3 activates ETB receptor in smooth muscle cells of rabbit mesenteric artery, producing vasodilator prostaglandins from arachidonic acid probably via a catalysis of cyclooxygenase, which accumulates cAMP in subendothelial tissues and produces relaxations.

O-332 - Mechanism of endothelin-3-induced relaxation in isolated rabbit mesenteric arteries

O-332 - Mechanism of endothelin-3-induced relaxation in isolated rabbit mesenteric arteries

Effects of propofol on isolated rabbit mesenteric arteries and veins

We have investigated the effect of propofol on isolated rabbit mesenteric arteries and veins. Isometric tension was measured in rings of arteries (with or without endothelium) or veins in organ chambers. The preparation was stimulated with noradrenaline 10(-6) mol litre-1, K+ 50 mmol litre-1 and caffeine 20 mmol litre-1 in the presence or absence of propofol. Propofol potentiated noradrenaline-induced contractions at lower concentrations (3 x 10(-5) mol litre-1) and attenuated them at greater concentrations (10(-4) and 3 x 10(-4) mol litre-1) in arteries with endothelium. Propofol inhibited noradrenaline-induced contractions in arteries without endothelium. In contrast, propofol produced venodilatation in a concentration-dependent manner (10(-5) to 3 x 10(-4) mol litre-1) of significantly greater magnitude than that in arteries. Propofol inhibited K+-induced contraction of both arteries and veins. It decreased the relaxation induced by acetylcholine (3 x 10(-8), 10(-7) and 3 x 10(-7) mol litre-1) of noradrenaline-induced contractions of arteries. Propofol did not affect caffeine-induced contractions after pretreatment with increased Ca2+. We conclude that propofol has a more potent vasodilator effect on veins than on arteries. Vasoconstriction induced by propofol may be associated with inhibition of endothelium-derived relaxing factor, whereas vasodilatation induced by propofol may be associated with block of voltage-gated influxes of extracellular Ca2+.

Effect of sevoflurane on the vascular reactivity of rabbit mesenteric artery

Sevoflurane is well known to cause depression of cardiovascular function, but detailed information on its actions on the contractility and reactivity of blood vessels is lacking. We have assessed therefore the direct effect of this anaesthetic on the functional reactivity of isolated rabbit mesenteric artery ring preparations. We found that contractions of endothelium intact rings induced by noradrenaline and phenylephrine were significantly attenuated by 4% sevoflurane; the observation that the maximal tension generation decreased without a significant reduction in pD2 is consistent with the view that receptor dysfunction was not involved. The effect of sevoflurane was not affected by NG-monomethyl-L-arginine. Sevoflurane 4% also produced attenuation of noradrenaline-induced contractions of endothelium denuded ring preparations. The contractions of endothelium denuded ring preparations produced by noradrenaline in Ca(2+)-free media in the presence of K+ were not affected by 4% sevoflurane, but sevoflurane depressed external Ca(2+)-dependent contractions. When vasodilators (acetylcholine and nitroglycerin) were added to the bathing media in the presence of 2% sevoflurane, the endothelium-dependent relaxation produced by acetylcholine, but not the endothelium-independent relaxation produced by nitroglycerin, was attenuated; superoxide dismutase inhibited the effect of sevoflurane on endothelium-dependent relaxation. These results are consistent with the view that sevoflurane inhibits alpha adrenoceptor-mediated contractions of isolated rabbit mesenteric artery ring preparations; this effect may be caused by reduced Ca2+ influx, as estimated from the effect on external Ca(2+)-dependent contractions, but is unlikely to be caused by reduced Ca2+ release from the sarcoplasmic reticulum of vascular smooth muscle, as estimated from noradrenaline-induced contractions in Ca(2+)-free bathing media.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor-induced rapid relaxation of the isolated rabbit mesenteric artery

Epidermal growth factor (EGF) (10–100 ng ml −1) induced a rapidly developing relaxation of precontracted rabbit mesenteric artery rings within 30 min of exposure. Indomethacin or protein synthesis inhibitors prevented or acutely reversed the effect of EGF on the preparation and an erbstatin analogue significantly reduced it. It is concluded that the EGF-mediated relaxation may be induced by the rapid production of prostanoids via a cascade of biochemical events initiated by the tyrosine-kinase activity of the receptors for EGF. One or more proteins rapidly produced and rapidly depleted appear to be involved in the activation of arachidonate metabolism.

Selective in vivo antagonism of pinacidil-induced hypotension by the guanidine U37883A in anesthetized rats.

The pyridylcyanoguanidine pinacidil exerts its hypotensive effect by opening ATP-sensitive potassium channels (K+ATP) in vascular smooth muscle. Direct glyburide-like antagonism of pinacidil-induced vasorelaxation by the guanidine U37883A (4-morpholinecarboximidine-N-1-adamantyl-N'- cyclohexylhydrochloride) has recently been demonstrated in isolated rabbit mesenteric artery. We herein report the first detailed in vivo cardiovascular interaction between U37883A and pinacidil in an anesthetized rat model. U37883A, administered from 0.1 to 3.0 mg/kg i.v. 10 min subsequent to pinacidil, immediately and dose-dependently reversed pinacidil's steady-state hypotensive effects by 29-100% (ED50 = 0.4 mg/kg), while reversal of pinacidil's tachycardiac effects from 10 to 79% was evident with 0.1-1.0 mg/kg i.v. U37883A (ED50 = 0.5 mg/kg). In contrast to these effects, pretreatment with 0.3-3.0 mg/kg i.v. U37883A only moderately inhibited the acute pinacidil-induced hypotension by 6-58%. Because U37883A's separate bradycardiac effects lowered basal heart rate, U37883A pretreatment precipitated a paradoxical 15-51% augmentation of sustained pinacidil-induced tachycardia, although absolute heart rates were below those seen with pinacidil alone. Qualitatively similar K+ATP blocking effects by U37883A were also observed in rats treated with the K+ATP openers (PCOs) cromakalim (BRL 34915), RPS 49365 and minoxidil. However, U37883A-treated rats remained responsive to the hypotensive action of both i.v. sodium nitroprusside and isoproterenol and buccal nifedipine. This study corroborates prior in vitro and in vivo findings and establishes that the guanidine U37883A is an effective and relatively selective blocker of PCO-induced vasodilation in the anesthetized rat. U37883A also appears more effective at closing basally and pinacidil-opened K+ATP than preventing K+ATP opening by pinacidil in vivo.

A sensitive in vitro functional assay to detect K+ — channel-dependent vasodilators

This study describes a sensitive in vitro relaxation assay using isolated rabbit mesenteric artery to detect the activity of a vasodilator as a K+-channel activator. Thus, comparison of several known K+-channel activators was made with other vasodilators known to work via various cellular mechanisms. The vasodilators used were minoxidil sulfate (MNXS; 5 μM), BRL-34915 (cromakalim, 0.1 μM), nicorandil (10 μM), pinacidil (1 μM), diazoxide (100 μM), sodium nitroprusside (10 μM), forskolin (1 μM), D600 (0.5 and 10 μM), hydralazine (10 μM), and viprostal (PGE1 analog, 5 μM). The concentrations chosen were equipotent to produce greater than 80% relaxation of the maximal norepinephrine (NE) (5 μM) contraction. At these concentrations, MNXS, cromakalim, pinacidil, nicorandil, and diazoxide were found to be ineffective in producing relaxation of 80 mM K+-contractions. Subsequently, pretreatment of tissues with 20 mM K+ before NE contraction was found to attenuate relaxation significantly by these agents, but had no effect on the relaxations by forskolin or D600. These initial criteria helped to establish cromakalim, pinacidil, nicorandil, and diazoxide as compounds acting similarly to MNXS as K+-channel-dependent. In another set of experiments, the effects of tetraethylammonium (TEA) (10 mM), Ba2+ (0.5 mM), and glyburide (1 μM) as K+-channel blockers were examined. Again it was found that these blockers had the most inhibitory effect on the class of compounds identified as K+ channel activators. Additionally, it was found that these K+-channel activators were without any significant effect on the NE-sensitive intracellular Ca2+ release as studied by contraction in a Ca2+-free solution. Thus, this series of functional criteria clearly show that the profile of these K+-channel activators is distinctly different from the vasodilators working via other mechanisms such as cyclic AMP (cAMP) (forskolin), cyclic GMP (cCMP) (nitroprusside), and Ca2+ antagonists (D600). It is suggested that appropriately defined, systematic functional studies, such as the one described here, can provide a sensitive and reproducible vascular model to discover and delineate the role of pharmacologically relevant mechanisms for vasodilation.

Purine-mediated relaxation and constriction of isolated rabbit mesenteric artery are not endothelium-dependent

The distribution of P1- and P2-purinoceptors in isolated transverse ring preparations of rabbit mesenteric artery was studied by comparing responses to ATP, alpha, beta-methylene ATP and adenosine in the presence and absence of endothelium. At resting tone in the presence of endothelium, ATP, alpha, beta-methylene ATP and acetylcholine, but not adenosine, produced transient concentration-dependent contractions. Alpha, beta-Methylene ATP was 5000 times more potent than ATP in this respect. Contractions to ATP or alpha, beta-methylene ATP in intact preparations were not significantly different from preparations without endothelium, but contractions to acetylcholine were significantly enhanced in the absence of endothelium. In the presence of endothelium, after desensitisation of the preparations to alpha, beta-methylene ATP, contractions to ATP, but not those to noradrenaline, were either greatly reduced or abolished. In preparations with tone raised by noradrenaline, acetylcholine, ATP and adenosine each produced relaxation in the presence of endothelium; however, in the absence of the endothelium, relaxations to acetylcholine were abolished, while those to ATP and adenosine were not significantly altered. 8-Phenyltheophylline significantly antagonised relaxation to adenosine in the presence of endothelium, whereas relaxation to ATP was unaffected. In raised tone preparations, with and without endothelium, alpha, beta-methylene ATP caused further contraction. However, in raised tone preparations with or without endothelium, that had been desensitised to alpha, beta-methylene ATP before elevation of muscle tone, ATP still caused relaxation, whereas alpha, beta-methylene ATP had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dithiothreitol on the isolated rabbit mesenteric artery.

Rabbit mesenteric artery strips exposed to 10(-3) M dithiothreitol (DTT) were contracted with a series of concentrations of histamine and 2-pyridylethylamine (PEA). DTT exposure increased the sensitivity to histamine 100-fold but increased the sensitivity to PEA only 4-fold. DTT did not reduce dimaprit-induced relaxations, but reduced histamine-induced relaxations. Following a high concentration of histamine (10(-3) M), DTT itself produced a sustained, slowly developing contraction (29 +/- 6.8% of the maximal contraction) relaxed by 7 X 10(-6) M mepyramine but not by 10(-6) M phentolamine. Metiamide (3 x 10(-5) M) potentiated DTT-induced contractions (29 +/- 6.8 before, 57 +/- 7.5% after metiamide, as a percent of maximal contraction). Changing the bathing fluid and repeating DTT exposure slowly relaxed previously contracted strips. DTT did not prevent the increase in sensitivity of relaxant histamine receptors on exposure to cold. We conclude that DTT, in addition to potentiating histamine H1-receptor responses, releases histamine presumably from non-mast cell pools when they are loaded with a high concentration of exogenous histamine.

Demonstration of specific vascular dopamine receptors mediating vasodilation on the isolated rabbit mesenteric artery.

In the isolated rabbit mesenteric artery, pretreated with phenoxybenzamine (10−5 M) and contracted with prostaglandin F2α (5×10−7–5×10−6 M) the relaxing effects of dopamine were analyzed.

O-108) - Relaxation of isolated rabbit mesenteric artery by N4-substituted quinazoline derivatives

O-108) - Relaxation of isolated rabbit mesenteric artery by N4-substituted quinazoline derivatives

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F 2α

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F 2α (PGF 2α) dopamine (10 −6M to 3×10 −4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10 −7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10 −5 and 10 −4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries in vitro . They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.

Effects of temperature on the responses to noradrenaline, isoprenaline and histamine on isolated rabbit mesenteric artery.

On the rabbit mesenteric artery, the elevation of temperature from 25 degrees up to 42 degrees diminished the affinity of histamine, whereas that of noradrenaline to the alpha-adrenoceptors remained unchanged. The presence of beta-adrenoceptors could not be demonstrated.