Angus M Shaw

β1-, β2- and atypical β-adrenoceptor-mediated relaxation in rat isolated aorta

β‐adrenoceptor‐mediated relaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre‐constricted with a sub‐maximal concentration of noradrenaline (1 μM) and relaxant responses to cumulative concentrations of β‐adrenoceptor agonists obtained. The concentration‐response curve (CRC) to isoprenaline was shifted to the right by propranolol (0.3 μM) with a steepening of the slope. Estimation of the magnitude of the shift from EC50 values gave a pA2 of 7.6. Selective β1‐ and β2‐adrenoceptor antagonists, CGP 20712A (0.1 μM) and ICI 118551 (0.1 μM), respectively, produced 4 and 14 fold shifts of the isoprenaline CRC. Atypical β‐adrenoceptor agonists also produced concentration‐dependent relaxation of aortic rings. The order of potency of the β‐adrenoceptor agonists was (−log EC50): isoprenaline (6.25)>cyanopindolol (5.59)>isoprenaline+propranolol (5.11)>CGP 12177A (4.40)>ZD 2079 (4.24)>ZM 215001 (4.07)>BRL 37344 (3.89). Relaxation to CGP 12177A and ZM 215001 was unaffected by propranolol (0.3 μM). SR 59230A (1 μM) and cyanopindolol (1 μM), β3‐adrenoceptor antagonists, had no effect on the isoprenaline (in the presence of propranolol) or CGP 12177A CRCs. Bupranolol and CGP 20712A, at μM concentrations (β4‐adrenceptor antagonists), inhibited responses to isoprenaline (in the presence of propranolol) and CGP 12177A. In conclusion, atypical β‐adrenoceptors co‐exist with β1‐ and β2‐adrenoceptors in rat aorta. Although non‐conventional partial agonists and selective β3‐adrenoceptor agonist cause relaxation, the vascular atypical β‐adrenoceptor does not appear to correspond to the β3‐adrenoceptor. There are, however, similarities with the putative β4‐adrenoceptor.

Evidence against β3‐adrenoceptors or low affinity state of β1‐adrenoceptors mediating relaxation in rat isolated aorta

The presence of β3‐adrenoceptors and the low affinity state of the β1‐adrenoceptor (formerly ‘putative β4‐adrenoceptor’) was investigated in ring preparations of rat isolated aorta preconstricted with phenylephrine or prostaglandin F2α (PGF2α). Relaxant responses to isoprenaline, selective β3‐adrenoceptor agonists (BRL 37344, SR 58611A, CL 316243) and non‐conventional partial agonists (CGP 12177A, cyanopindolol, pindolol) were obtained. In phenylephrine‐constricted, but not PGF2α‐constricted rings, relaxations to isoprenaline showed a propranolol‐resistant component. In phenylephrine‐constricted rings, relaxations to BRL 37344 (pEC50, 4.64) and SR 58611A (pEC50, 4.94) were not antagonized by the selective β3‐adrenoceptor antagonist SR 59230A (1 μM). CL 316243 (100 μM) failed to produce relaxation. In PGF2α‐constricted rings only SR 58611A produced relaxation, which was not affected by SR 59230A (3 μM). Non‐conventional partial agonists produced relaxation in phenylephrine‐constricted but not PGF2α‐constricted rings. The relaxation to CGP 12177A was unaffected by SR 59230A (1 μM) or by CGP 20712A (10 μM), reported to block the low affinity state of the β1‐adrenoceptor. β‐adrenoceptor antagonists also produced relaxation in phenylephrine‐constricted rings with an order of potency of (pEC50 values): bupranolol (5.5)∼38;SR 59230A (5.47)∼38;cyanopindolol (5.47)>pindolol (5.30)>alprenolol (5.10)>propranolol (4.83)>ICI 118551 (4.60)>CGP 12177A (4.38)∼38;CGP 20712A (4.35). Bupranolol (100 μM), alprenolol (30 μM), propranolol (100 μM) and SR 59230A (10 μM) produced no relaxation in PGF2α‐constricted rings. These results provide no evidence for the presence of functional β3‐adrenoceptors or the low affinity state of the β1‐adrenoceptor in rat aorta.

Role of endothelium/nitric oxide in atypical β-adrenoceptor-mediated relaxation in rat isolated aorta

The role of endothelium in the modulation of classical and atypical beta-adrenoceptor-mediated vasorelaxation was investigated in ring preparations of rat isolated thoracic aorta. Rings were pre-constricted with a sub-maximal concentration of noradrenaline (1 microM) and relaxant responses to cumulative concentrations of beta-adrenoceptor agonists obtained. Endothelium removal or pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) or 1H-[1,2,4] oxadiazolol[4,3,-a] quinoxalin-1-one (ODQ, 10 microM) significantly reduced the relaxant effects of isoprenaline, but had less effect on relaxant responses to the atypical beta-adrenoceptor agonist, (+/-)-4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one hydrochloride (CGP 12177A). Sodium nitroprusside (3 nM) shifted the isoprenaline concentration-response curve to the left and restored the attenuated responses in the presence of L-NAME back to control levels. Sodium nitroprusside had little effect on the CGP 12177A concentration-response curve. The results show that the endothelium/nitric oxide (NO) pathway modulates beta-adrenoceptor-mediated vasorelaxation in rat aorta and that classical beta-adrenoceptors are modulated to a greater extent than atypical beta-adrenoceptors.

α1‐Adrenoceptor antagonist properties of CGP 12177A and other β‐adrenoceptor ligands: evidence against β3‐ or atypical β‐adrenoceptors in rat aorta

The α1‐adrenoceptor antagonist properties of the β‐adrenoceptor nonconventional partial agonist, CGP 12177A, was investigated in functional assays in rat aorta and in radioligand binding assays in rat cerebral cortical membranes. In addition, binding affinities of other β‐adrenoceptor ligands were measured to investigate any correlation between α1‐adrenoceptor affinity and relaxant potency in phenylephrine‐constricted rings. In functional studies, CGP 12177A produced parallel rightward shifts of the phenylephrine CRC with no reduction in the maximum responses. Schild regression analysis gave a straight line with a slope of 0.95 (95% CL: 0.87–1.04), suggesting reversible competitive antagonism, and gave a pKB value of 5.26. In contrast, CGP 12177A (300 μM) had no effect on contraction induced by the thromboxane‐mimetic, U46619. In binding studies, CGP 12177A competed monophasically with [3H]prazosin binding (Hill slope, 0.95, 95% CL: 0.76–1.13), giving a pKi value of 5.48, in good agreement with the pKB from functional studies. Competition experiments with various other β‐adrenoceptor ligands showed that they all displaced [3H]prazosin in a manner consistent with one‐site competition. pKi values were as follows: SR 59230A, 6.25; cyanopindolol, 6.33; bupranolol, 6.35; alprenolol, 5.90; propranolol, 5.80; BRL 37344, 5.50; ICI 118551, 5.55; CGP 20712A, 5.26. The pKi values correlated well with the pEC50 values for relaxation of phenylephrine‐constricted rat aorta obtained previously (r2=0.984, P<0.0001). In conclusion, relaxant effects of CGP 12177A and other β‐adrenoceptor ligands in phenylephrine‐constricted rat aorta can be attributed to α1‐adrenoceptor blockade and are unrelated to effects at β3‐adrenoceptors or atypical β‐adrenoceptors.

Mechanisms of U46619- and 5-HT-induced contraction of bovine pulmonary arteries: role of chloride ions

Thromboxane A2 and 5‐hydroxytryptamine (5‐HT) are implicated in pulmonary hypertension. The involvement of chloride, voltage‐operated calcium channels (VOCCs), store‐operated calcium channels (SOCCs) and the Rho kinase in the contractile response of bovine pulmonary arteries (BPA) to the thromboxane A2 mimetic U46619 and 5‐HT was investigated.

Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium

Background and purpose:  Thromboxane A2 and endothelial dysfunction are implicated in the development of pulmonary hypertension. The receptor‐transduction pathway for U46619 (9,11‐dideoxy‐9α, 11α‐methanoepoxy prostaglandin F2α)‐induced contraction was examined in endothelium‐intact (E+) and denuded (E−) rat pulmonary artery rings.

Relaxation to bradykinin in bovine pulmonary supernumerary arteries can be mediated by both a nitric oxide-dependent and -independent mechanism.

The aim of the present study was to determine the relative contribution of prostanoids, nitric oxide and K+ channels in the bradykinin‐induced relaxation of bovine pulmonary supernumerary arteries. In endothelium‐intact, but not denuded rings, bradykinin produced a concentration‐dependent relaxation (pEC50, 9.6±0.1), which was unaffected by the cyclo‐oxygenase inhibitor indomethacin. The nitric oxide scavenger hydroxocobalamin (200 μM, pEC50, 8.5±0.2) and the nitric oxide synthase inhibitor L‐NAME (100 μM, pEC50, 8.9±0.1) and the combination of L‐NAME and hydroxocobalamin (pEC50, 8.1±0.2) produced rightward shifts in the bradykinin concentration response curve. The guanylyl cyclase inhibitor ODQ (10 μM, pEC50, 9.6±0.4) did not affect the response to bradykinin. Elevating the extracellular [K+] to 30 mM did not affect the response to bradykinin but abolished the response when ODQ or L‐NAME was present. The K+ channel blocker apamin (100 nM), combined with charybdotoxin (100 nM), produced a small reduction in the maximum response to bradykinin but they abolished the response to bradykinin when ODQ, L‐NAME or hydroxocobalamin were present. Apamin (100 nM) combined with iberiotoxin (100 nM) also reduced the response to bradykinin in the presence of hydroxocobalamin or L‐NAME. The concentration response curve for sodium nitroprusside‐induced relaxation was abolished by ODQ (10 μM) and shifted to the right by apamin and charybdotoxin. These studies suggest that in bovine pulmonary supernumerary arteries bradykinin can stimulate the formation of nitric oxide and activate an EDHF‐like mechanism and that either of these pathways alone can mediate the bradykinin‐induced relaxation. In addition nitric oxide, acting through guanylyl cyclase, can activate an apamin/charbydotoxin‐sensitive K+ channel in this tissue.

Mechanisms involved in the regulation of bovine pulmonary vascular tone by the 5-HT1B receptor

Background and purpose:  5‐HT1B receptors may have a role in pulmonary hypertension. Their relationship with the activity of BKCa, a T‐type voltage‐operated calcium channel (VOCC) and cyclic nucleotide‐mediated relaxation was examined.