VASORELAXANT EFFECTS OF ATROPINE: ROLE OF NITRIC OXIDE/ ENDOTHELIUM DERIVED RELAXING FACTOR (original) (raw)
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Vasorelaxant effects of atropine in pulmonary artery rings: role of NO/ EDRF
Indian Journal of Pharmacology
This study examined the effects of atropine on isolated rat pulmonary artery rings with or without intact endothelium in the absence and presence of nitric oxide synthase inhibitors, N-omega nitro-L-arginine methyl ester (L-NAME) or N-omega nitro-L-arginine (L-NOARG) that precontracted with phenylephrine (PHE), 5-hydroxytryptamine (5-HT) or KCl. Methods: Segments of the main pulmonary artery (MPA) of male Wistar rats was carefully cut to obtain one ring of approximately three millimeter in diameter was transferred to fresh ice-cold Krebs buffer. Endothelial cells were removed and artery rings were mounted under one gram resting tension. Tension responses were recorded isometrically with Grass FTO3 transducers and were displayed on a Grass model 7 polygraph. The intact endothelium rings precontracted with PHE were confirmed with acetylcholine (ACh), which relaxed artery rings. Results: Atropine (1 nM) blocked the vasorelaxant effect of acethylcholine (1 µM) in pulmonary artery rings precontracted with PHE (100 nM). Also atropine (10 nM-5 µM) produced concentration dependent relaxations in these rings precontracted with PHE or 5-HT, but did not relax rings precontracted with KCl. The vasorelaxant effects of atropine were partially inhibited by the mechanical remove of endothelium or pretreatment rings with L-NAME or L-NOARG, although they were not statistically significant. Conclusion: The results indicate that the ability of atropine to relax pulmonary artery rings may depend on the mechanism of action of the precontracting agonist and suggest that the vasorelaxant effect of atropine is not wholly mediated by the release of NO/EDRF. EDRF L-NAME L-NOARG NO pulmonary artery the release of an endogenous dilator, such as EDRF, or by inhibiting the action of an endogenous vasoconstrictor 9. The demonstration in 1987 of the formation of NO by an enzyme in vascular endothelial cells opened up what can now be considered a new area of biological research 10,11. NO, which accounts for the biological properties of EDRF, is the endogenous stimulator of the soluble guanylate cyclase. In addition, NO is a potent vasodilator that activates guanylate cyclase resulting in the generation of cGMP, which is presumed to be the principal effector of NO-induced vasorelaxation in various tissues. NO is synthesized from the amino acid L-arginine by an enzyme, the NO synthase 12 .
British Journal of Pharmacology, 2000
A component of isoprenaline-mediated vasorelaxation in pulmonary arteries is mediated by nitric oxide (NO). We examined the eects of physiological concentrations (4400 mM) of L-arginine on isoprenaline-induced relaxation in rat pulmonary arteries, and following inhibition of L-arginine uptake with L-lysine. In addition, we examined the role of the endothelium, and whether L-arginine aected acetylcholine (ACh)-induced relaxation. 2 Isoprenaline-induced relaxation was potentiated by 400 mM L-arginine in pulmonary arteries; maximum relaxation was increased from 83+4% of initial tone to 94+4% (P50.05). L-lysine (10 mM) not only abolished the potentiation by L-arginine, but suppressed relaxation compared to control (70+4%, P50.05), even in the absence of L-arginine added to the bath. Blockade of NO synthase with 100 mM L-NMMA or removal of the endothelium inhibited isoprenaline-induced relaxation to the same extent as L-lysine, and under these conditions the presence or absence of 400 mM L-arginine made no dierence. L-lysine had no additional eect when applied in combination with L-NMMA. 3 The eect of extracellular L-arginine was concentration dependent, with an apparent EC 50 of *1±7 mM. 4 Relaxation to the membrane permeant cyclic AMP analogue CPT cyclic AMP was also potentiated by L-arginine and inhibited by L-lysine. There was however no dierence in relaxation induced by acetylcholine (ACh) in the presence of L-arginine or L-lysine, and isoprenaline-induced relaxation of mesenteric arteries was unaected by L-arginine or L-lysine. 5 These results strongly suggest that extracellular L-arginine is critically important for development of the NO-and endothelium-dependent component of cyclic AMP-induced vasorelaxation in rat pulmonary arteries, but is not required for ACh-induced relaxation. As the apparent EC 50 for this eect is in the low micromolar range it is likely to be fully activated in vivo, as plasma L-arginine is 4150 mM.
Involvement of endothelial NO in the dilator effect of VIP on rat isolated pulmonary artery
Regulatory Peptides, 2007
The endothelium and its interaction with smooth muscle play a central role in the local control of the pulmonary vasculature, and endothelial dysfunction is thought to contribute to pulmonary hypertension and chronic obstructive pulmonary disease. Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, relaxes the rat pulmonary artery, but there is controversy as to whether or not this action of VIP depends on the endothelium. The aim of this study, therefore, was to investigate the role of the endothelium and nitric oxide (NO), the major endothelium-derived relaxing factor, in the dilator action of VIP on the rat isolated pulmonary artery. Pulmonary artery preparations pre-contracted by the α 1adrenoceptor agonist L-phenylephrine were relaxed by VIP (0.003-1 μM) and acetylcholine (0.003-10 μM) in a concentration-dependent manner. Mechanical removal of the endothelium reduced the maximal response to VIP by about 50% and practically abolished the response to acetylcholine. Inhibition of NO synthesis by N ω -nitro-L-arginine methyl ester (0.5 mM) had a similar effect, abolishing the vasorelaxation caused by acetylcholine and attenuating the vasorelaxation caused by VIP by about 50%. From these data it is concluded that the relaxant action of VIP on the rat isolated pulmonary artery depends in part on the presence of the endothelium and that this part is mediated by endothelial NO.
British Journal of Pharmacology, 1997
Noradrenaline induces a meagre vasoconstriction in small muscular pulmonary arteries compared to large conduit pulmonary arteries. We have examined whether this may be partially related to dierences in the b-adrenoceptor-mediated vasorelaxation component and, in particular, b-adrenoceptor-mediated NO release. 2 Noradrenaline induced a bell-shaped concentration-response in large (1202+27 mm) and small (334+12 mm) pulmonary arteries of the rat. In large arteries tension increased to 95.6+1.8% of 75 mM KCl (KPSS; n=8) at 2 mM, above which tension declined. The response in small arteries was meagre (12+1.5% KPSS, n=9), peaking at 0.2 mM. N G-monomethyl-L-arginine (L-NMMA; 100 mM) abolished the decline in tension induced by higher concentrations of noradrenaline in large arteries, and increased maximum tension (117+3.5% KPSS, n=5, P50.05). In small arteries peak tension doubled (22.0+3.4% KPSS, n=6, P50.01), but still declined above 0.2 mM. 3 Propranolol (1 mM) abolished the decline in tension at higher concentrations of noradrenaline in both groups, but increased tension substantially more in small (37.4+3.7% KPSS, n=5, P50.001) than in large arteries (112.2+3.7% KPSS, n=9, P50.05). In the presence of L-NMMA, propranolol had no additional eect on large arteries, whereas in small arteries there was greater potentiation than for either agent alone (67.8+5.9% KPSS, n=4). 4 b-Adrenoceptor-mediated relaxation was examined in arteries constricted with prostaglandin F 2a (50 mM). In the presence of propranolol isoprenaline caused an unexpected vasoconstriction, which was abolished by phentolamine (10 mM). In the presence of phentolamine, isoprenaline caused a maximum relaxation of 43.3+2.1% (n=6) in large, and 49.0+4.5% (n=6) in small arteries. L-NMMA substantially reduced relaxation in large arteries (7.4+1.5%, n=6, P50.01), but was less eective in small arteries (26.8+5.8, n=5, P50.05). 5 Atenolol (b 1-antagonist, 5 mM) reduced relaxation to isoprenaline (large: 34.8+4.5%, n=5; small: 35.0+1.9%,n=6), but in combination with L-NMMA had no additional eect over L-NMMA alone. ICI 118551 (b 2-antagonist, 0.1 mM) reduced isoprenaline-induced relaxation more than atenolol (large: 18.0+4.6%, n=6, P50.05; small: 25.6+10.7%, n=6, P50.05). ICI 118551 in combination with L-NMMA substantially reduced relaxation (large: 4.8+2.6%, n=9; small: 6.5+3.6%, n=5). 6 Salbutamol-induced relaxation was reduced substantially by L-NMMA in large arteries (control: 34.7+6.4%, n=6; +L-NMMA: 8.3+1.3%, n=5, P50.01), but to a lesser extent in small arteries (control: 50.9+7.5%, n=6; +L-NMMA: 23.0+0.7%, n=5, P50.05). Relaxation to forskolin was also partially antagonized by L-NMMA. 7 These results suggest that the meagre vasoconstriction to noradrenaline in small pulmonary arteries is partially due to a greater b-adrenoceptor-mediated component than in large arteries. b-Mediated vasorelaxation in large arteries was largely NO-dependent, whereas in small arteries a signi®cant proportion was NO-independent. Noradrenaline stimulation was also associated with NO release that was independent of b-adrenoceptors.
British Journal of Pharmacology, 1992
on vasodilatation induced by ATP, substance P, 5-hydroxytryptamine (5-HT), bradykinin and sodium nitroprusside (SNP) were examined in the guinea-pig coronary bed, by use of a Langendorff technique. The effects of these inhibitors of nitric oxide synthesis were assessed on their ability to inhibit both the amplitude and the area of the vasodilator response. 2 The vasodilator responses evoked by low doses of 5-HT (5 x 10`-'°5 x 10 mol) were almost abolished by L-NAME and L-NOARG (both at 10-5, 3 x 10-5 and 1O-4M), although L-NOARG (3 x 10-s M) was significantly less potent than L-NAME (3 x 10-5 M) as an inhibitor of vasodilator responses to 5-HT (5 x 10-8 mol). 3 The vasodilator responses evoked by substance P (5 x 102-5 X I0-mol) were reduced in the presence of L-NAME and L-NOARG (both at 10-5 and 3 x 10-5 M). The response to substance P was almost abolished by L-NAME and L-NOARG (both at 10-4 M). 4 The amplitude of the vasodilator responses to ATP (5 x 10-" and 5 x 10-9-5 x 10-mol) was little affected by either L-NAME or L-NOARG (both at 10-5, 3 x 10-5 and 10-4 M). However, the area of the response to ATP (5 x 10-`o-5 x 10-7 mol) was inhibited by L-NAME (10-5, 3 x 10-5 and 10-4M) and to a lesser extent by L-NOARG (10-5 and 10-M). 5 The amplitude and area of the vasodilator responses to bradykinin (5 x 10-12-5 x 10-11 mol) were reduced, but not abolished, by L-NOARG and L-NAME. 6 Neither the amplitude nor area of the responses to sodium nitroprusside (5 x 10-'°-5 x 10-7 mol) were inhibited by either L-NAME or L-NOARG (both at 10-5 and 3 x 10-5 M). 7 It is concluded that in the guinea-pig coronary vasculature, the vasodilatation evoked by substance P and low doses of 5-HT is mediated almost exclusively via nitric oxide, whereas the vasodilatations evoked by ATP and bradykinin appear to involve other mechanisms in addition to the release of nitric oxide. L-NAME was a more effective agent than L-NOARG in inhibiting the vasodilator actions of 5-HT and ATP in this preparation.
British Journal of Pharmacology, 2000
The NO-dependent component of cyclic AMP-induced vasorelaxation in rat pulmonary arteries is critically dependent on extracellular L-arginine but independent of endothelial cell intracellular [Ca 2+ ]. We examined whether L-arginine uptake was also essential for NO production induced by passive stretch or isometric tension, processes also reported to be Ca 2+-independent. 2 The passive length-tension curve was depressed by physiological concentrations of L-arginine (400 mM; P50.05). Inhibition of the y + transporter with 10 mM L-lysine, NO synthase with L-NAME (100 mM), or protein tyrosine kinase with erbstatin A (30 mM) caused identical upward shifts (P50.001), alone or in combination. Tyrphostin 23 was similar to erbstatin A, whilst the inactive analogue tyrphostin A1 and genistein were without eect. 3 L-arginine (400 mM) shifted the PGF 2a concentration-response curve under isometric conditions to the right (P50.05), whereas L-NAME or L-lysine caused a leftward shift (P50.001). Tyrphostin 23 (30 mM) more than reversed the L-arginine-induced suppression of PGF 2a-induced tension; subsequent addition of L-NAME had no eect. The L-lysine-sensitive component of CPT cyclic AMP-induced vasorelaxation was abolished by erbstatin A. 4 ACh-induced vasorelaxation was *80% inhibited by L-NAME, but was not aected by L-lysine or 400 mM L-arginine. Erbstatin A reduced the vasorelaxation by only *25%. 5 We conclude that activation of NO production by stretch, isometric tension, or cyclic AMP in rat pulmonary arteries is critically dependent on the presence and uptake of physiological concentrations of extracellular L-arginine, and protein tyrosine kinase activity. This directly contrasts with ACh-induced vasorelaxation, which was independent of extracellular L-arginine, and relatively unaected by tyrosine kinase inhibition.
Aims: The aim of this study was to investigate whether β-adrenoceptor (β-AR) overstimulation induced by in vivo treatment with isoproterenol (ISO) alters vascular reactivity and nitric oxide (NO) production and signal-ing in pulmonary arteries. Main methods: Vehicle or ISO (0.3 mg kg −1 day −1) was administered daily to male Wistar rats. After 7 days, the jugular vein was cannulated to assess right ventricular (RV) systolic pressure (SP) and end diastolic pressure (EDP). The extralobar pulmonary arteries were isolated to evaluate the relaxation responses, protein expression (Western blot), NO production (diaminofluorescein-2 fluorescence), and cyclic guanosine 3′,5′-monophosphate (cGMP) levels (enzyme immunoassay kit). Key findings: ISO treatment induced RV hypertrophy; however, no differences in RV-SP and EDP were observed. The pulmonary arteries from the ISO-treated group showed enhanced relaxation to acetylcholine that was abolished by the NO synthase (NOS) inhibitor N ω-nitro-L-arginine methyl ester (L-NAME); whereas relaxation elicited by sodium nitroprusside, ISO, metaproterenol, mirabegron, or KCl was not affected by ISO treatment. ISO-treated rats displayed enhanced endothelial NOS (eNOS) and vasodilator-stimulated phosphoprotein (VASP) expression in the pulmonary arteries, while phosphodiesterase-5 protein expression decreased. ISO treatment increased NO and cGMP levels and did not induce eNOS uncoupling. Significance: The present data indicate that β-AR overactivation enhances the endothelium-dependent relaxation of pulmonary arteries. This effect was linked to an increase in eNOS-derived NO production, cGMP formation and VASP content and to a decrease in phosphodiesterase-5 expression. Therefore, elevated NO bioactivity through cGMP/VASP signaling could represent a protective mechanism of β-AR overactivation on pulmonary circulation.
Cardiovascular Research, 2007
Aims b-adrenoceptor (b-AR)-mediated relaxation was characterized in pulmonary arteries from normoxic and hypoxic (as model of pulmonary hypertension) mice. The endothelial NO synthase (eNOS) pathway was especially investigated because of its potential vasculoprotective effects. Methods Pulmonary arteries from control or hypoxic (0.5 atm for 21 days) wild-type or eNOS 2/2 mice were used for pharmacological characterization of b-AR-mediated relaxation in myograph, and for immunohistochemistry using anti-b-AR antibodies. Results In pulmonary arteries from normoxic mice, isoproterenol (b-AR agonist) and procaterol (selective b 2 -AR agonist) elicited relaxation, while cyanopindolol and CL316243 (b 3 -AR agonists) were ineffective. The effect of isoproterenol was antagonized by CGP20712A and ICI118551 (b 1 -or b 2 -AR antagonists, respectively) and also partially inhibited by N v -nitro-L-arginine methylester (L-NAME, a NOS inhibitor), endothelium denudation, or eNOS gene deletion. Relaxation to procaterol was abolished by L-NAME or endothelium removal. In eNOS 2/2 mice, procaterol-induced relaxation was decreased but was insensitive to L-NAME, this residual effect involving other endothelium-dependent relaxant factors as compensatory mechanisms. Immunostaining for b 2 -AR was observed in the endothelial layer, but not the medial layer of pulmonary arteries. Pulmonary arteries from hypoxic mice exhibited decreased endothelial NO-dependent relaxation to acetylcholine. However, in these arteries, relaxation to procaterol was either unaffected (extralobar segments) or even increased (intralobar segments) and was still abolished by L-NAME or endothelium removal. Conclusion b 1 -and b 2 -AR, but not b 3 -AR, mediate relaxation of mice pulmonary arteries. The b 2 -AR component is dependent on eNOS activity and is preserved following chronic hypoxia. These data highlight the role of the b 2 -AR as a pharmacological target to induce/restore endothelial NO-dependent protective effects in pulmonary circulation.
Influence of endothelial nitric oxide on neurogenic contraction of human pulmonary arteries
European Respiratory Journal, 1995
c co on nt tr ra ac ct ti io on n o of f h hu um ma an n p pu ul lm mo on na ar ry y a ar rt te er ri ie es s ABSTRACT: The present study was designed to investigate the contribution of the endothelium and that of the L-arginine pathway on the contractile responses of isolated human pulmonary arteries to electrical field stimulation (EFS) and noradrenaline.