Concentration-contraction Curves Research Articles

Pharmacological characterisation of the CB1 receptor antagonist activity of cannabidiol in the rat vas deferens bioassay

Cannabidiol is increasingly considered for treatment of a wide range of medical conditions. Binding studies suggest that cannabidiol binds to CB1 receptors. In the rat isolated vas deferens bioassay, a single electrical pulse causes a biphasic contraction from nerve-released ATP and noradrenaline. WIN 55,212-2 acts on prejunctional CB1 receptors to inhibit release of these transmitters. In this bioassay, we tested whether cannabidiol and SR141716 were acting as competitive antagonists of this receptor. Monophasic contractions mediated by ATP or noradrenaline in the presence of prazosin or NF449 (P2X1 inhibitor), respectively, were measured to a single electrical pulse delivered every 30 min. Following treatment with cannabidiol (10–100 μM) or SR141716 (0.003–10 μM), cumulative concentrations of WIN 55,212–2 (0.001–30 μM) were applied followed by a single electrical pulse. The WIN 55,212–2 concentration-contraction curve EC50 values were applied to global regression analysis to determine the pKB. The antagonist potency of cannabidiol at the CB1 receptor in the rat vas deferens bioassay matched the reported receptor binding affinity. Cannabidiol was a competitive antagonist of WIN 55,212–2 with pKB values of 5.90 when ATP was the effector transmitter and 5.29 when it was noradrenaline. Similarly, SR141716 was a competitive antagonist with pKB values of 8.39 for ATP and 7.67 for noradrenaline as the active transmitter. Cannabidiol's low micromolar CB1 antagonist pKB values suggest that at clinical blood levels (1–3 μM) it may act as a CB1 antagonist at prejunctional neuronal sites with more potency when ATP is the effector than for noradrenaline.

Vasoactive effect of a sheet sample of Oxytenanthera abyssinica (Poaceae) on a model of coronary arteries of pigs

Background: The cost of treatments for cardiovascular diseases, in particular for populations in developing countries such as Senegal, requires an alternative which consists in developing research into new therapeutic approaches with the use of plants from the traditional pharmacopoeia. On the other hand, many of these plants have not been the subject of scientific studies. Aims and Objectives: In our study, we set out to study the effects on the vasomotricity of a plant from an in vitro model. As specific objectives, research of the vasorelaxant effects of the hydroethanolic extract of the leaves of Oxytenanthera abyssinica, on a model of pig coronary artery. Materials and Methods: The coronary artery rings were incubated with O. abyssinica (FOA) leaf extract at different concentrations (10–4 mg/ml, 5.10–4 mg/ml, 10–3 mg/ml, 5.10–3 mg/ml, 10–2 mg/ml, 5.10–2 mg/ml, 10–1 mg/ml, and 5.10–1 mg/ml) in an isolated organ system. The device comprises a set of devices including isometric tension sensors. The vessel rings to be studied are mounted on these. They are connected to metal rods that develop isometric tension. These sensors make it possible to measure the intensity of the voltage developed which reflects the vascular reactivity. After incubation, the rings are contracted with U46619 to produce a concentration-contraction curve with and without endothelium. The measured voltages are collected and represented in the form of a curve using the GraphPad software Prism version 6.01®. The results are expressed as means ± SEM of 08 experiments. The analysis of variance test (ANOVA) was used to compare the means. P < 0.05 was considered statistically significant. Results: The results obtained in this study showed that on isolated pig coronary artery rings, contracted at U46619, the hydroethanolic extract of O. abyssinica (FOA) leaves induces vascular relaxation. This vasorelaxation is obtained at concentrations ranging from 10−4 to 5.10−1 mg/ml with a relaxation percentage of approximately 100%. This percentage of relaxation is obtained both in the presence and in the absence of functional endothelium. These results suggest that the hydroethanolic extract of leaves of O. abyssinica possesses dose-dependent vasodilator properties on isolated pig coronary artery rings with endothelium-independent character. It thus appears that the raw extract of leaves of O. abyssinica can act on two ways. Conclusion: The results of our study made it possible to demonstrate a vascular relaxing effect of the hydroethanolic extract of the leaves of O. abyssinica. This vasorelaxant effect is dose dependent and endothelium independent.

The β2-adrenoceptor agonist bronchodilators terbutaline and orciprenaline are also weak α1-adrenoceptor antagonists

Recently, the β2-adrenoceptor agonist terbutaline was shown to have α1-adrenolytic activity in mouse isolated pulmonary arteries in vitro and to lower pulmonary artery pressure in anaesthetised mice. The aim of our study was to determine the α1-adrenoceptor antagonist activity of terbutaline and its structurally close resorcinol, orciprenaline, in rat isolated small mesenteric arteries set up for myography. Their α1-adrenoceptor antagonist potency was then compared with their potency as β2-adrenoceptor agonists. Concentration-response curves to methoxamine were competitively antagonised by terbutaline (30–300 μM) or orciprenaline (30–300 μM) with a pKB of 4.70 ± 0.09 or 4.79 ± 0.17, respectively. Both terbutaline and orciprenaline fulfilled the criteria for simple, silent competitive antagonism. Terbutaline (30–300 μM) had no effect on endothelin-1 concentration-contraction curves. Our findings suggest that after oral dosing of terbutaline, the maximum plasma levels would NOT reach levels to show α1-adrenoceptor antagonist activity. In conclusion, our work has provided additional quantitative evidence that terbutaline and orciprenaline are weak competitive α1-adrenoceptor antagonists, but this additional property is probably not therapeutically important in the clinical treatment of asthma or pulmonary artery hypertension with these more potent β2-adrenoceptor agonists.

Effect of RhoA/Rho‐kinase inhibitor in Human Internal Mammary Artery and Clinical Implications

ObjectivesCoronary artery bypass grafting surgery (CABG) is the treatment of choice for severe coronary artery disease (CAD). Graft failure is a common complication after CABG. Different biological mechanisms contribute to the pathophysiology of vascular graft failure including vasospasm. RhoA/Rho‐kinase (ROCK) pathway plays an important role in cell contraction of cardiovascular disease. ROCK is a downstream effector of small GTP‐binding protein RhoA and has two isoforms, ROCK1 and ROCK2. This study aimed to investigate the inhibitory effect of ROCK inhibitor fasudil on the vasoconstriction in human internal mammary artery (IMA) from patients undergoing CABG and to explore whether the combination of fasudil and nitroglycerin (NTG) is superior than being used alone.MethodsIsolated human IMA rings (n=8 in each experimental group) taken from 58 patients undergoing CABG were studied in myograph. Cumulative concentration‐relaxation curves for fasudil (−9 to −3.5 log M) were established in IMA precontracted with KCl (30 mM) and U46619 (−8 log M). Cumulative concentration‐contraction curves for KCl (5 to 120 mM) and U46619 (−9 to −4.5 log M) were also established in two ways: (1) in three groups pretreated with the plasma concentration (−6.3 log M) and 10‐fold higher concentration of fasudil (−5.3 log M) or vehicle to estimate the depression effect of fasudil; (2) in four groups pretreated with fasudil (−6.3 log M), NTG (0.1 μM), the combination, and vehicle for 40 minutes. Western Blot was used to measure the change of the ROCK2 protein.ResultsFasudil caused similar cumulative relaxation in KCl‐ or U46619‐ contracted (p &gt; 0.05) rings. Pretreatment with −5.3 log M fasudil significantly depressed subsequent contraction by KCl (P &lt; 0.01 vs. control, P &lt; 0.05 vs. −6.3 log M) and U46619 (P &lt; 0.05 vs. control or −6.3 log M). Pretreatment with the combination of NTG and fasudil significantly depressed the cumulative contraction by KCl (P &lt; 0.01 vs. control; P &lt; 0.05 vs. −5.3 log M; P &lt; 0.01 vs. NTG). Similarly, in U46619‐induced contraction, the combination also significantly depressed the contraction (P &lt; 0.001 vs. control; P &lt; 0.05 vs. −5.3 log M fasudil; P &lt; 0.01 vs. NTG). Fasudil also caused a decrease of ROCK2 protein content (p &lt; 0.05).ConclusionsFasudil has a potent inhibitory effect on the vasoconstriction mediated by KCl and U46619 in human conduit arteries. Combination of fasudil and NTG is more effective than fasudil or NTG alone. These findings provide a new antispastic method in CABG and may be beneficial to long‐term patency of the grafts.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China [81870288 &amp; 81870227]; the Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2019XK310001 &amp; 2018TX31002]; Tianjin Science and Technology Committee[18PTZWHZ00060];Binhai New Area Health Bureau (2016BWKY007 &amp; 2018BWKZ005).

Comparison of the in vitro pharmacological profiles of long-acting muscarinic antagonists in human bronchus

Background and purposeLong-acting muscarinic antagonists (LAMAs) have been recommended for the treatment of chronic obstructive pulmonary disease and (more recently) asthma. However, the in vitro pharmacological profiles of the four LAMAs currently marketed (tiotropium, umeclidinium, aclidinium and glycopyrronium) have not yet been compared (relative to ipratropium) by using the same experimental approach. Experimental approachWith a total of 560 human bronchial rings, we investigated the antagonists' potency, onset and duration of action for inhibition of the contractile response evoked by electrical field stimulation. We also evaluated the antagonists' potency for inhibiting cumulative concentration-contraction curves for acetylcholine and carbachol. Key resultsThe onset and duration of action were concentration-dependent. At submaximal, equipotent concentrations, the antagonists' onsets of action were within the same order of magnitude. However, the durations of action differed markedly. After washout, ipratropium's inhibitory activity decreased rapidly (within 30–90 min) but those of tiotropium and umeclidinium remained stable (at above 70%) for at least 9 h. Aclidinium and glycopyrronium displayed less stable inhibitory effects, with a progressive loss of inhibition at submaximal concentrations. In contrast to ipratropium, all the LAMAs behaved as insurmountable antagonists by decreasing the maximum responses to both acetylcholine and carbachol. Conclusions and implicationsThe observed differences in the LAMAs' in vitro pharmacological profiles in the human bronchus provide a compelling pharmacological rationale for the differences in the drugs' respective recommended daily doses and frequencies of administration.

Functional estimation of endothelin-1 receptor antagonism by bosentan, macitentan and ambrisentan in human pulmonary and radial arteries in vitro

BackgroundEndothelin receptor antagonists are approved for pulmonary arterial hypertension. Development of selective ETA-receptor antagonists over mixed or dual receptor antagonists has depended on a range of receptor binding assays, second messenger assays and functional blood vessel assays. This study compared the 3 clinically-approved endothelin receptor antagonists in assays of human isolated pulmonary and radial arteries in vitro. MethodsHuman isolated pulmonary (i.d. 5.5mm) and human radial (i.d. 3.23mm) artery ring segments were mounted in organ baths for isometric force measurement. Single concentration-contraction curves to endothelin-1 were constructed in the absence or presence of bosentan (1–10µM), macitentan (0.03–0.3µM) or ambrisentan (0.1–1µM). ResultsAll 3 endothelin antagonists caused competitive rightward shifts in the endothelin-1 concentration-response curves in both arteries. The Clark plot and analysis gave the following pKB values: bosentan, pulmonary artery 6.28±0.13 and radial artery 6.04±0.10; macitentan, pulmonary artery 8.02±0.13 and radial artery 7.49±0.08; and ambrisentan, pulmonary artery 7.38±0.13 and radial artery 6.96±0.10. ConclusionsNoting the maximum plasma levels attained from recommended oral doses of each antagonist in volunteers, the pKB findings here show that there would be significant antagonism of endothelin-1 contraction in the pulmonary and radial arteries at therapeutic plasma levels. This functional assay confirms in human tissue that much higher plasma concentrations of endothelin-1 receptor antagonists are required to be effective than those predicted from binding or other biochemical assays.

Novel α1-adrenoceptor antagonism by the fluroquinolone antibiotic trovafloxacin

Trovafloxacin, a fluroquinolone antibiotic, was recently found to be an inhibitor of pannexin-1 channels through which ATP is released as “find-me” signals in apoptotic Jurkat cells. Our interest in the role of pannexin-1 channels in α1-adrenoceptor-mediated vasoconstriction led us to the novel finding reported here. Concentration-response curves to methoxamine and phenylephrine were competitively antagonised by trovafloxacin (1–30µM) with a pKB of 5.54 and 5.32, respectively, in rat mesenteric small arteries isolated for myography. In comparison, prazosin (1–10nM) antagonised methoxamine concentration-response curves with a pKB of 9.76. Trovafloxacin (1–30µM) had no effect on either the thromboxane mimetic (U46619) or endothelin-1 concentration-contraction curves. Interestingly, the concentration range is similar for trovafloxacin antagonising the 3 distinct pharmacological targets: (i) fourth generation fluroquinolone antibiotic, (ii) pannexin-1 channel inhibitor in apoptotic cells, and now (iii) as an α1-adrenoceptor antagonist. When trovafloxacin was in use clinically, CNS side effects of dizziness, flushing and headache consistent with α1-adrenoceptor antagonism were common. We conclude that trovafloxacin with its quinolone moiety is a weak α1-adrenoceptor competitive antagonist in comparison with prazosin.

Association of rat thoracic aorta dilatation by astragaloside IV with the generation of endothelium-derived hyperpolarizing factors and nitric oxide, and the blockade of Ca2+ channels.

The aim of the present study was to elucidate the roles of endothelium-derived hyperpolarizing factors (EDHFs) and nitric oxide (NO) in mediating the vasodilatation response to astragaloside IV and the effects of astragaloside IV on voltage-dependent Ca2+ channels and receptor-operated Ca2+ channels in rat thoracic aortic rings precontracted with potassium chloride (KCl; 60 mM) or phenylephrine (PHE; 1 µM). The results showed that astragaloside IV (1×10−4-3×10−1 g/l) concentration-dependently relaxed the contraction induced by KCl (10–90 mM) or PHE (1×10−9-3×10−5 µM) and inhibited concentration-contraction curves for the two vasoconstrictors in the aortic rings. Preincubation with Nω-nitro-L-arginine methyl ester (L-NAME, 100 µM) significantly attenuated astragaloside IV-induced relaxation in the endothelium-intact and -denuded arterial rings precontracted with PHE. Astragaloside IV, following preincubation with L-NAME (100 µM) plus indomethacin (10 µM), exerted vasodilatation, which was depressed by tetraethtylamine (1 mM) and propargylglycine (100 µM), but not by carbenoxolone (10 µM), catalase (500 U/ml) or proadifen hydrochloride (10 µM). The action mode of astragaloside IV was evident in comparison to nifedipine. Inhibition of PHE-induced contraction by astragaloside IV (100 mg/l) was more potent compared to inhibition of KCl-induced contraction, while inhibition of KCl-induced contraction by nifedipine (100 mg/l) was more potent compared to inhibition of PHE-induced contraction by nifedipine (100 mg/l). In addition, the combination of astragaloside IV and nifedipine exhibited synergistic and additive inhibitory effects on contraction evoked by KCl, which was similar to PHE. In conclusion, astragaloside IV, as a Ca2+ antagonist, relaxes the vessels through the blockade of superior receptor-operated Ca2+ and inferior voltage-dependent Ca2+ channels, which modulate NO from vascular endothelial cells and vascular smooth muscle cells, and EDHFs including K+ and hydrogen sulfide.

Elastic and Muscular Arteries Differ in Structure, Basal NO Production and Voltage-Gated Ca(2+)-Channels.

In the last decades, the search for mechanisms underlying progressive arterial stiffening and for interventions to avoid or reverse this process has gained much attention. In general, arterial stiffening displays regional variation and is, for example, during aging more prominent in elastic than in muscular arteries. We hypothesize that besides passive also active regulators of arterial compliance [i.e., endothelial and vascular smooth muscle cell (VSMC) function] differ between these arteries. Hence, it is conceivable that these vessel types will display different time frames of stiffening. To investigate this hypothesis segments of muscular arteries such as femoral and mesenteric arteries and elastic arteries such as the aorta and carotid artery were isolated from female C57Bl6 mice (5–6 months of age, n = 8). Both microscopy and passive stretching of the segments in a myograph confirmed that passive mechanical properties (elastin, collagen) of elastic and muscular arteries were significantly different. Endothelial function, more specifically basal nitric oxide (NO) efficacy, and VSMC function, more specifically L-type voltage-gated Ca2+ channel (VGCC)-mediated contractions, were determined by α1-adrenoceptor stimulation with phenylephrine (PE) and by gradual depolarization with elevated extracellular K+ in the absence and presence of eNOS inhibition with L-NAME. PE-mediated isometric contractions significantly increased after inhibition of NO release with L-NAME in elastic, but not in muscular vessel segments. This high basal eNOS activity in elastic vessels was also responsible for shifts of K+ concentration-contraction curves to higher external K+. VGCC-mediated contractions were similarly affected by depolarization with elevated K+ in muscular artery segments or in elastic artery segments in the absence of basal NO. However, K+-induced contractions were inhibited by the VGCC blocker diltiazem with significantly higher sensitivity in the muscular arteries, suggestive of different populations of VGCC isoforms in both vessel types. The results from the present study demonstrate that, besides passive arterial wall components, also active functional components contribute to the heterogeneity of arterial compliance along the vascular tree. This crucially facilitates the search for (patho) physiological mechanisms and potential therapeutic targets to treat or reverse large artery stiffening as occurring in aging-induced arterial stiffening.

β-adrenergic antagonists influence abdominal aorta contractility by mechanisms not involving β-adrenergic receptors.

β-adrenergic receptors (β-AR) are widely distributed in the cardiovascular system, where they considerably contribute to the control of its functions. β-blockers are commonly used in the treatment of disorders of the circulatory system. They act primarily by inhibiting cardiac β-receptors. However, there are also reports of pleiotropic action of β-blockers as well as of new compounds created to study β3 adrenergic receptors. The study aimed to investigate additional mechanisms of action of β-AR inhibitors in the rabbit abdominal aorta with emphasis on their action on α-adrenergic receptors and calcium influx. Responses to propranolol, betaxolol, metoprolol and SR59230A were evaluated in phenylephrine and PGF(2alpha) precontracted aortic rings. The effect of propranolol on the phenylephrine concentration-contraction curve was examined. Propranolol (≥ 10 μM) and SR59230A (≥ 0.1 μM) induced relaxations in phenylephrine-precontracted rings, while betaxolol and metoprolol had little effect. The β-AR inhibitors produced further contraction of tissues preincubated with PGF(2alpha), excluding SR59230A, which after initial contraction, elicited marked relaxation at a concentration above 1 ĕM. 100 μM of propranolol caused a significant rightward shift of the concentration-contraction curve to phenylephrine with no reduction in the maximum response. Incubation of aortic rings in phentolamine reduced the maximal contraction to propranolol; verapamil pretreatment by contrast enhanced contractile response. In conclusion, SR59230A and propranolol most probably act as α1-AR competitive antagonists in the presence of phenylephrine in rabbit abdominal aortic rings. After α-ARs blockade, propranolol exerts a weak relaxing activity connected with Ca2+ channel inactivation. SR59230A at a high concentration acts on the rabbit aorta by an additional mechanism needing further investigation.

Paeonol induces vasodilatation in rat mesenteric artery via inhibiting extracellular Ca2+ influx and intracellular Ca2+ release

To investigate the vasodilative effect of paeonol in rat mesenteric artery and the mechanisms responsible for it. Rats were anaesthetized and sacrificed. The superior mesenteric artery was removed, dissected free of adherent tissue and cut into 2.0 mm long cylindrical segments. Isometric tension of artery rings was recorded by a myograph system in vitro. Concentration-relaxation curves of paeonol (17.8 μ mol/L to 3.16 mmol/L) were recorded on artery rings precontracted by potassium chloride (KCl) and concentration-contraction curves of KCl, 5-hydroxytryptamine (5-HT), noradrenaline (NA) or calcium chloride (CaCl2) were recorded in the presence of paeonol (10(-4.5), 10(-3.8), 10(-3.5) mol/L) respectively. And also, concentration-relaxation curves of paeonol were recorded in the presence of different potassium channel inhibitors and propranolol on rings precontracted with KCl respectively. To investigate the role of intracellular Ca(2+) release from Ca(2+) store, the contraction induced by NA (100 μ mol/L) and CaCl2 (2 mmol/L) in Ca(2+) free medium was observed in the presence of paeonol respectively. Paeonol relaxed artery rings precontracted by KCl in a concentration-dependent manner and the vasodilatation effect was not affected by endothelium denudation. Paeonol significant decreased the maximum contractions (Emax) induced by KCl, CaCl2, NA and 5-HT, as well as Emax induced by NA and CaCl2 in Ca(2+) -free medium, suggesting that paeonol dilated the artery via inhibiting the extracellular Ca(2+) influx mediated by voltage-dependent calcium channel, and receptor-mediated Ca(2+)-influx and release. Moreover, none of glibenclamide, tetraethylammonium, barium chlorded and propranolol affected the paeonol-induced vasodilatation, indicating that the vasodilatation was not contributed to ATP sensitive potassium channel, calcium-activated potassium channel, inwardly rectifying potassium channel, and β-adrenoceptor. Paeonol induces non-endothelium dependent-vasodilatation in rat mesenteric artery via inhibiting voltage-dependent calcium channel-mediated extracellular Ca(2+) influx and receptor-mediated Ca(2+) influx and release.

Vasorelaxant Alkaloids from Spirospermum penduliflorum (Menispermaceae), a Plant Used to Treat Hypertension in Malagasy Traditional Medicine

Spirospermum penduliflorum Thouars (Menispermaceae) is widely used on the eastern coast of Madagascar to treat hypertension. The aim of the present study was to analyse the vasorelaxant properties of different leaf extracts. The activity of the n-hexane, dichloromethane and methanolic extracts was tested on phenylephrine-contracted aorta. The dichloromethane extract was shown to be the most effective. Further fractionation of this extract led to the isolation of an active fraction relaxing phenylephrine-contracted aorta with an IC50 of 0.18 μg/mL {log IC50 (μg/mL) −0.74 ± 0.03} but was much less effective on KCl induced contractions. Bioassay-guided fractionation of this fraction led to the isolation of two aporphinoid alkaloids, neolitsine and dicentrine, which at concentrations of 0.1 μM and 1 μM displaced to the right the phenylephrine concentration-contraction curve. Our results show that Spirospermum penduliflorum extracts possess vasorelaxant activity in vitro that could be related to the presence of dicentrine in the extracts having an α1 antagonist activity. This finding is not in accord with the previous studies by Rasoanaivo et al where no alkaloids were detected in the leaves of Spirospermum penduliflorum.

Effects of manganese tetrakis (4‐N‐methylpyridyl) porphyrin (MnTMPyP) on dexmedetomidine‐induced contractions in the aorta of normal and septic rats

Study ObjectiveThe aim of the present study was to investigate the effects of peroxynitrite scavenging on dexmedetomidine‐induced contractions of the rat aorta after acute in vivo exposure to lipopolysaccharide. (LPS)MethodologyMale SD rats (eight to ten weeks old) were injected intraperitoneally with (1mg/kg) of LPS or normal saline. Two hours later, their aortas were harvested. Aortic rings were suspended for isometric tension recording in organ chambers and incubated with vehicle, L‐NAME (100 uM), indomethacin (10 uM), 1400W (10uM), and the peroxynitrite decomposition catalyst manganese tetrakis (4‐N‐methylpyridyl) porphyrin (MnTMPyP) (10uM). Concentration‐contraction curves to dexmedetomidine (10nM to 10uM) were obtained. Plasma cytokine concentrations were measured before and after the administration of LPS. The expression of iNOS and 3‐nitrotyrosine were measured using immunofluorescence and Western blotting.ResultsLPS treatment attenuated dexmedetomidine‐induced contractions after acute in vivo exposure to LPS and in the presence of MnTMPyP and 1400W, in aortic rings without endothelium. TNF‐á and IL‐6 were increased after 2 hours of LPS.ConclusionThese data indicate that LPS attenuates the contractile responses to dexmedetomidine and ex vivo treatment with MnTMPyP and 1400W. Circulating endotoxin induces the release of the cytokines TNF‐á and IL‐6 after two hours.

Chiral recognition of doxazosin enantiomers in 3 targets for therapy as well as adverse drug reactions in animal experiments

Doxazosin used in benign prostatic hyperplasia has the side effects of causing hypotension and the risk of heart failure. The 3 targets of α(1A)-adrenoceptors (in the prostate), α(1D)-adrenoceptors (in the aorta), and an unknown mechanism (in the heart) are involved, respectively. We hypothesized that there is a chiral recognition of doxazosin enantiomers in the 3 targets. Using isolated rat aorta (α(1D)-adrenoceptors) and rabbit prostate (α(1A)-adrenoceptors), we examined pA(2) and pK(B) values of doxazosin enantiomers. We observed chronotropic and inotropic effects of doxazosin enantiomers in isolated rat and rabbit heart tissues. (-)Doxazosin and (+)doxazosin produced a shift to the right of concentration-contraction curves for noradrenalin (aorta) and phenylephrine (prostate smooth muscle). The pA(2) value of (-)doxazosin (8.625± 0.053) was smaller than (+)doxazosin (9.503± 0.051) in rat aorta, but their pK(B) values in rabbit prostate were the same. In rat and rabbit heart tissues, (+)doxazosin (3-30µmol·L(-1)) significantly decreased atrial rate, and produced negative inotropic effects; however, (-)doxazosin did not affect the atrial rate, and produced positive inotropic effects in the atria. Thus, the chiral carbon atom of doxazosin does not affect its activity at the therapeutic target of α(1A)-adrenoceptors in the prostate, but significantly changes its blocking activity against α(1D)-adrenoceptors in the aorta, and produces opposite inotropic effects in the atria via an α(1)-adrenoceptor-independent mechanism.

Secondhand cigarette smoke exposure causes upregulation of cerebrovascular 5‐HT1B receptors via the Raf/ERK/MAPK pathway in rats

Cigarette smoke exposure increases the risk of stroke. Upregulation of 5-hydroxytryptamine 1B (5-HT(1) (B) ) receptors is associated with the pathogenesis of cerebral ischaemia. This study examined the hypothesis that the expression of 5-HT(1) (B) receptors is altered in brain vessels after secondhand smoke (SHS) exposure. Rats were exposed to SHS in vivo for 200 min daily for 8 weeks. The contractile responses of isolated cerebral arteries were studies by a sensitive myograph. The mRNA and protein expression for 5-HT(1) (B) receptors were examined by real-time PCR, Western blot and immunofluorescence respectively. In addition, the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway was evaluated. The results showed that SHS exposure shifted the 5-HT(1) (B) receptor-mediated concentration-contraction curve towards the left with a markedly increased maximal contraction. Furthermore, there were significant elevations in mRNA level and protein expression of 5-HT(1) (B) receptors in SHS-exposed rats. Immunostaining revealed that the 5-HT(1) (B) receptors were localized to the smooth muscle cells of cerebral arteries. SHS was also found to induce the phosphorylation of Raf-1 and ERK1/2 proteins. The administration of a Raf-1 inhibitor GW5074 attenuated the 5-HT(1) (B) receptor upregulation. Secondhand smoke exposure upregulates cerebrovascular 5-HT(1) (B) receptors in rats. The receptor upregulation is associated with Raf/ERK/MAPK activation.

Vasodilation effect of 2-benzyl-5-hydroxy-6-methoxy-3, 4-dihydroisoquinolin-1-one

A 2-Benzyl-5-hydroxy-6-methoxy-3, 4-dihydroisoquinolin-1-one (ZC2) is a newly synthesized isoquinolinone compound. Its effect on vasodilation was evaluated in the present study. Isometric tension of rat artery rings was recorded by a sensitive myography system in vitro. The results showed that ZC2 relaxed rat mesenteric arteries pre-contracted by KCl, phenylephrine and 9, 11- dideoxy- 11α, 9α-epoxymethano-prostaglandin F2α (U46619), and abdominal aorta pre-contracted by KCl in a concentration-dependent manner. The ZC2-induced vasodilation was not affected by an endothelium denudation. ZC2 rightwards shifted the concentration-contraction curves, induced by KCl, phenylephrine, and 5-hydroxytryptamine (5-HT) in a non-parallel manner, which suggests that the vasodilation effects are most likely via voltage-dependent calcium channel (VDCC) and receptor-operated calcium channel (ROCC). Moreover, in Ca(2+)-free medium, ZC2 concentration-dependently depressed the vasoconstrictions induced by phenylephrine and CaCl(2), and decreased a contractile response induced by caffeine, which indicates a role of extracellular Ca(2+) influx inhibition through VDCC and ROCC, and intracellular Ca(2+) release from Ca(2+) store via the ryanodine receptors. Glibenclamide did not affect the vasodilation induced by ZC2, suggesting that ATP sensitive potassium channel is not involved in the vasodilation. The results indicate that ZC2 induces vasodilation by inhibiting the VDCC and ROCC, and receptormediated Ca(2+) influx and release. The inhibition of intracellular Ca(2+) release may be mediated via the ryanodine receptors.

Action of calcium antagonists and agonists on isolated human thoracic arteries used for coronary artery bypass grafting

BackgroundThe goal of this study was to investigate the modulation of the contraction-relaxation effects in isolated human thoracic artery samples of three calcium-channel antagonists, amlodipine (CAS [88150-42-9]), cerebrocrast (CAS [118790-71-9]) and diltiazem (CAS [42399-41-7]), and two calcium-channel agonists, CGP 28392 (CAS [89289-93-0]) and benzimidazole derivative. To estimate the endothelial function of the artery samples, carbachol, an agonist of muscarinic receptors, was used. MethodsThe experiments were conducted on isolated human thoracic artery samples, and their isometric contractions were recorded using an i-FOT10 force transducer. Cumulative concentration-contraction curves of the tested agents (10−7 to 10−4M) were established. ResultsCarbachol at concentrations of 10−7 to 10−6M did not cause any relaxation of artery rings precontracted by 10−4M phenylephrine, and at concentrations of 10−5 and 10−4M, isometric contractions increased by 7% and 20%, respectively. In response to amlodipine, cerebrocrast and CGP28392, the contraction of artery samples increased significantly, whereas diltiazem and benzimidazole derivative caused their relaxation by ~55%. ConclusionThe obtained data indicate that the endothelium of the thoracic artery, which is used for coronary artery bypass grafting, is damaged by and may have some influence on the inadequate response of 1,4-dihydropyrine type calcium-channel antagonists.

Muscarinic receptor subtypes involved in regulation of colonic motility in mice: Functional studies using muscarinic receptor-deficient mice

Although muscarinic M(2) and M(3) receptors are known to be important for regulation of gastric and small intestinal motility, muscarinic receptor subtypes regulating colonic function remain to be investigated. The aim of this study was to characterize muscarinic receptors involved in regulation of colonic contractility. M(2) and/or M(3) receptor knockout (KO) and wild-type mice were used in in vivo (defecation, colonic propulsion) and in vitro (contraction) experiments. Amount of feces was significantly decreased in M(3)R-KO and M(2)/M(3)R-KO mice but not in M(2)R-KO mice. Ranking of colonic propulsion was wild-type=M(2)R-KO>M(3)R-KO>M(2)/M(3)R-KO. In vitro, the amplitude of migrating motor complexes in M(2)R-KO, M(3)R-KO and M(2)/M(3)R-KO mice was significantly lower than that in wild-type mice. Carbachol caused concentration-dependent contraction of the proximal colon and distal colon from wild-type mice. In M(2)R-KO mice, the concentration-contraction curves shifted to the right and downward. In contrast, carbachol caused non-sustained contraction and relaxation in M(3)R-KO mice depending on its concentration. Carbachol did not cause contraction but instead caused relaxation of colonic strips from M(2)/M(3)R-KO mice. 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (McN-A-343) caused a non-sustained contraction of colonic strips from wild-type mice, and this contraction was changed to a sustained contraction by tetrodotoxin, pirenzepine and L-nitroarginine methylester (L-NAME). In the colon of M(2)/M(3)R-KO mice, McN-A-343 caused only relaxation, which was decreased by tetrodotoxin, pirenzepine and L-NAME. In conclusion, M(1), M(2) and M(3) receptors regulate colonic motility of the mouse. M(2) and M(3) receptors mediate cholinergic contraction, but M(1) receptors on inhibitory nitrergic nerves counteract muscarinic contraction.

Reactivity of the Human Internal Mammary Artery and the Gastroepiploic Artery to Constrictor Substances.

The internal mammary artery (IMA) and the gastroepiploic artery (GEA) are frequently used to construct coronary artery bypass grafts. In order to determine and to compare their contractile properties, we studied the effects of constricting agents which are liberated or infused during coronary bypass surgery. IMA and GEA were arranged as ring segments and suspended in organ baths at optimal stretch using a normalization procedure. Concentration-contraction curves to angiotensin II, norepinephrine, U-46619, endothelin-1, leukotriene C(4) and KCl were performed. The sensitivity (pD(2)) of GEA and IMA to all the agents did not differ. However, GEA developed stronger maximal contraction force than IMA to angiotensin II (P < 0.001), norepinephrine (P < 0.05), U-46619 (P = 0.06), endothelin-1 (P < 0.01), and KCl (P < 0.01), whereas leukotriene C(4) did not induce a significant contraction on GEA and IMA. The higher contractility of GEA may explain that it is more prone to spasm than IMA in the clinical setting. This study reinforces IMA as a first-choice conduit for coronary artery bypass and emphasizes the need for antispastic drugs particularly when GEA is used as bypass vessel.http://link.springer-ny.com/link/service/journals/00547/bibs/8n4p187.html</hea

Inhibition of Endothelin-1 and Hypoxia-induced Pulmonary Pressor Responses in the Rat by a Novel Selective Endothelin-A Receptor Antagonist, Di-n-butylaminocarbamyl-l-leucyl-d-tryptophanyl-d-4-chloro-Phe

Pulmonary hypertension is a kind of disease associated with a very high rate of mortality, and there are not many effective drugs for the treatment. Today, endothelin (ET)-1 receptor antagonists were proved to be effective in the treatment of pulmonary hypertension. Aiming at developing new endothelin-A receptor (ETA) antagonist for treatment of pulmonary hypertension, di-n-butylaminocarbamyl-L-leucyl-D-tryptophanyl-D-4-chloro-Phe, named GF063, was synthesized at base of selective ETA receptor antagonist BQ485 and selected for the further pharmacological characterization. The preliminary pharmacodynamics of GF063 was evaluated by radioligand receptor binding assay and test of antivasoconstriction effects in vitro and in vivo. The integrative pharmacodynamics was evaluated in hypoxia-induced rat pulmonary hypertension. In vitro, GF063 bound to ETA receptor with 100,000-fold higher affinity than to ETB receptor. GF063 concentration dependently inhibited contraction of isolated rat aortic ring induced by ET-1 and shifted the cumulative concentration-contraction response curve to right with no change in the maximal response. In vivo, GF063 inhibited the increase of mean systemic arterial pressure induced by ET-1 in anesthetized rat. In hypoxia-induced rat pulmonary hypertension model, pretreatment with GF063 (40 mg/kg, s.c.) significantly decreased pulmonary artery pressure and right ventricular hypertrophy, also significantly inhibited the increase of ET-1 level in lung, improved hemodynamics, and alleviated the wall thickness of pulmonary vessels. This study indicated that GF063, as a selective ETA receptor antagonist, could inhibit vasoconstriction effects in vivo and in vitro, could prevent pulmonary hypertension induced by hypoxia, and may have great potential to be developed as a new drug of antipulmonary hypertension.