Activation of nitric oxide synthase by beta 2-adrenoceptors in human umbilical vein endothelium in vitro.

1. Some animal studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of beta2-adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibits this response. 2. The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial beta-adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine-3',5'-monophosphate (cyclic AMP). 3. Vasorelaxant responses were determined in umbilical vein rings to the nonselective beta-adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2alpha. 4. NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]-L-arginine to [3H]-L-citrulline, and adenylyl cyclase activity by the conversion of [alpha-32P]-ATP to [32P]-cyclic AMP. 5. Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by beta2- (but not beta1-) adrenergic blockage, and by endothelium removal or 1 mM L-NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L-NMMA, with a reduction in Emax from 90.0+/-9.3% to 50.5+/-9.9% (P<0.05). 6. Isoprenaline 1 microM increased NOS activity in HUVEC (34.0+/-5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration-dependent manner; this response was inhibited by beta2 (but not beta1-) adrenergic blockade. Forskolin 1 microM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 microM. The increase in L-arginine to L-citrulline conversion observed with each agent was abolished by coincubation with NOS inhibitors. 7. These results indicate that endothelial beta2-adrenergic stimulation and cyclic AMP elevation activate the L-arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.

Potentiation of cyclic AMP-mediated vasorelaxation by phenylephrine in pulmonary arteries of the rat.

Alpha1-adrenoceptor agonists may potentiate relaxation to beta-adrenoceptor agonists, although the mechanisms are unclear. We compared relaxations induced by beta-adrenoceptor agonists and cyclic AMP-dependent vasodilators in rat pulmonary arteries constricted with prostaglandin F2alpha (PGF2alpha) or the alpha1-adrenoceptor agonist phenylephrine (PE). In addition, we examined whether differences were related to cyclic AMP- or nitric oxide (NO) and cyclic GMP-dependent pathways. Isoprenaline-induced relaxation was substantially potentiated in arteries constricted with PE compared with PGF2alpha. Methoxamine was similar to PE, whereas there was no difference between PGF2alpha and 30 mM KCl. The potentiation was primarily due to a marked increase in the NO-independent component of relaxation, from 9.1+/-1.7% for PGF2alpha to 55.1+/-4.4% for PE. NO-dependent relaxation was also enhanced, but to a lesser extent (50%). Relaxation to salbutamol was almost entirely NO-dependent in both groups, and was potentiated approximately 50% by PE. Relaxation to forskolin (activator of adenylate cyclase) was also enhanced in PE constricted arteries. Part of this relaxation was NO-dependent, but the major effect of PE was to increase the NO-independent component. Propranolol diminished but did not abolish the potentiation. There was no difference in response to CPT cyclic AMP (membrane permeant analogue) between PE and PGF2alpha, suggesting that mechanisms distal to the production of cyclic AMP were unchanged. Relaxation to sodium nitroprusside (SNP) was the same for PE and PGF2alpha, although relaxation to acetylcholine (ACh) was slightly depressed. This implies that potentiation by PE does not involve the cyclic GMP pathway directly. Mesenteric arteries constricted with PE did not show potentiation of isoprenaline-induced relaxation compared to those constricted with PGF2alpha, suggesting that this effect may be specific to the pulmonary circulation. These results clearly show that PE potentiates both the NO-independent and -dependent components of cyclic AMP-mediated relaxation in pulmonary arteries of the rat, although the effect on the former is more profound. We suggest that potentiation of both components is largely due to direct activation of adenylate cyclase via alpha1-adrenoceptors, within the smooth muscle and endothelial cells respectively.

Membrane potential-dependent and -independent vasodilation in small pulmonary arteries from chronically hypoxic rats.

Chronic hypoxia is associated with altered pulmonary vasoreactivity, and it has been suggested that an increased response to voltage-dependent vasodilators may relate to enhanced Ca++ entry via voltage-dependent channels, secondary to depolarization. Few studies have been performed on small pulmonary arteries, and it is unknown whether they are depolarized after chronic hypoxia. We examined the resting membrane potential, and the actions of voltage-dependent (verapamil, levcromakalim) and -independent (isoproterenol, forskolin, papaverine) vasodilators in small ( approximately 300 microm internal diameter) pulmonary arteries from chronically hypoxic rats. The resting membrane potential was more positive in arteries after chronic hypoxia (control: -60 +/- 0.5 mV; hypoxic: -54.4 +/- 1.1 mV; P < .01), and this was reflected by a shift to the left of the response curves for K+ and 4-aminopyridine. In arteries constricted with prostaglandin F2alpha the response to verapamil and levcromakalim was increased after chronic hypoxia, although maximum prostaglandin F2alpha-induced tension was unchanged, which implies a reduction in voltage-independent constrictor mechanisms. Although vasorelaxation to isoproterenol was depressed in arteries from hypoxic rats, forskolin-induced relaxation was enhanced substantially, and because the response to the phosphodiesterase inhibitor papaverine was unchanged, we suggest that this reflects an up-regulation of adenylate cyclase. In conclusion, chronic hypoxia resulted in a significant depolarization in small pulmonary arteries, but this may explain only partly the increased efficacy of voltage-dependent vasodilators. Whether the reduction in voltage-independent constrictor mechanisms is related to the apparent up-regulation of adenylate cyclase remains to be elucidated.

Noradrenaline, beta-adrenoceptor mediated vasorelaxation and nitric oxide in large and small pulmonary arteries of the rat.

1. 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 differences in the beta-adrenoceptor-mediated vasorelaxation component and, in particular, beta-adrenoceptor-mediated NO release. 2. Noradrenaline induced a bell-shaped concentration-response in large (1202+/-27 microm) and small (334+/-12 microm) pulmonary arteries of the rat. In large arteries tension increased to 95.6+/-1.8% of 75 mM KCl (KPSS; n=8) at 2 microM, above which tension declined. The response in small arteries was meagre (12+/-1.5% KPSS, n=9), peaking at 0.2 microM. N(G)-monomethyl-L-arginine (L-NMMA; 100 microM) abolished the decline in tension induced by higher concentrations of noradrenaline in large arteries, and increased maximum tension (117+/-3.5% KPSS, n=5, P<0.05). In small arteries peak tension doubled (22.0+/-3.4% KPSS, n=6, P<0.01), but still declined above 0.2 microM. 3. Propranolol (1 microM) 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, P<0.001) than in large arteries (112.2+/-3.7% KPSS, n=9, P<0.05). In the presence of L-NMMA, propranolol had no additional effect on large arteries, whereas in small arteries there was greater potentiation than for either agent alone (67.8+/-5.9% KPSS, n=4). 4. Beta-adrenoceptor-mediated relaxation was examined in arteries constricted with prostaglandin F2alpha (50 microM). In the presence of propranolol isoprenaline caused an unexpected vasoconstriction, which was abolished by phentolamine (10 microM). 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, P<0.01), but was less effective in small arteries (26.8+/-5.8, n=5, P<0.05). 5. Atenolol (beta1-antagonist, 5 microM) 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 effect over L-NMMA alone. ICI 118551 (beta2-antagonist, 0.1 microM) reduced isoprenaline-induced relaxation more than atenolol (large: 18.0+/-4.6%, n=6, P<0.05; small: 25.6+/-10.7%, n=6, P<0.05). 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, P<0.01), but to a lesser extent in small arteries (control: 50.9+/-7.5%, n=6; +L-NMMA: 23.0+/-0.7%, n=5, P<0.05). 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 beta-adrenoceptor-mediated component than in large arteries. Beta-mediated vasorelaxation in large arteries was largely NO-dependent, whereas in small arteries a significant proportion was NO-independent. Noradrenaline stimulation was also associated with NO release that was independent of beta-adrenoceptors.

Ligustrazine-induced endothelium-dependent relaxation in pulmonary arteries via an NO-mediated and exogenous L-arginine-dependent mechanism.

1. Ligustrazine (tetramethylpyrazine, TMP) is a vasodilator that has been reported to have pulmonary selective properties in vivo, but not in vitro. Although TMP is generally described as being endothelium-independent, we provide evidence here that TMP may have an endothelium-dependent and nitric oxide (NO)-mediated mechanism in pulmonary arteries that could predominate at concentrations used therapeutically in China. 2. The study was performed on isolated pulmonary (1-2 mm i.d.), intrapulmonary (200-850 microns) and mesenteric (200-400 microns) arteries of the rat using a Mulvaney-Halpen small vessel myograph, following preconstriction with phenylephrine (PE, 10 microM), prostaglandin F2 alpha (PGF2 alpha, 100 microM), or 75 mM K+ (KPSS, equimolar substitution for Na+). Values are shown as mean +/- s.e.mean, or for EC50S as mean [+/-95% confidence limits]. 3. TMP caused a concentration-dependent relaxation against all three agonists in both large (1.56 +/- 0.04 mm) and small (399 +/- 20 microM) pulmonary arteries; it was more potent in small compared to large arteries constricted with PE or PGF2 alpha (P < 0.05), but not those constricted with KPSS. The NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA, 100 microM) caused a significant shift to the right of these relationships, such that the EC50 for TMP in large pulmonary arteries constricted with PE increased from 522 [+130, -104] microM (n = 12) to 1828 [+395, -325] microM (n = 6, P < 0.01). Both removal of the endothelium and methylene blue (10 microM) had similar effects. 4. L-Arginine substantially reduced the EC50 for TMP in pulmonary arteries; in the presence of 400 microM L-arginine the EC50 for TMP in large arteries constricted with PE was 14.7 [+21.0, -8.6] microM, (n = 6, P < 0.001), and with 10 microM L-arginine 96.7 [+45.1, -30.7] microM, (n = 6, P < 0.001). Similar effects were seen in small arteries. L-Arginine had no effect in the absence of an endothelium. D-Arginine was ineffective, and inhibition of L-arginine uptake with L-lysine blocked the action of L-arginine. L-Arginine (400 microM) had no significant effect on TMP-induced relaxation in mesenteric arteries (n = 5). 5. L-Arginine itself caused a concentration-dependent relaxation in intrapulmonary arteries (639 +/- 34 microM) constricted with PE, reaching a maximum relaxation around 100-400 microM (42.4 +/- 3.0%, n = 16), but this was independent of the endothelium. TMP (10 and 100 microM) significantly enhanced the relaxation to L-arginine, with a maximum relaxation in the presence of 100 microM TMP of 81.7 +/- 6.2% (n = 5, P < 0.01), but the effect of TMP was entirely dependent on the endothelium. A similar effect was observed in PGF2 alpha-constricted pulmonary arteries. 6. These results show that TMP stimulates NO production at low concentrations in pulmonary arteries, via an apparently novel endothelium-resident mechanism that is dependent on exogenous L-arginine. Normal plasma L-arginine levels of around 150 microM would allow this mechanism to be maximally activated. As mesenteric arteries do not seem to express the mechanism to any significant extent, at low concentrations TMP would be effectively selective to the pulmonary vasculature, and may thus have potential as a therapeutic agent in pulmonary vascular disease.