Allan MacDonald

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.

α1-Adrenoceptor subtypes involved in vasoconstrictor responses to exogenous and neurally released noradrenaline in rat femoral resistance arteries

The α1‐adrenoceptor subtypes involved in responses to exogenous and neurally released noradrenaline in rat femoral resistance arteries were characterised using a small vessel myograph, with antagonists prazosin (nonsubtype selective), 5‐methyl‐urapidil (α1A‐selective), BMY 7378 (α1D‐selective) and the alkylating agent chloroethylclonidine (preferential for α1B‐). Prazosin and 5‐methyl‐urapidil produced rightward shifts of the exogenous noradrenaline concentration – response curve (CRC) with pA2 values of 9.2 and 9.1 respectively, in agreement with the presence of α1A‐adrenoceptors. BMY 7378 (1 μM) shifted the noradrenaline CRC with an apparent pKB of 6.7, in agreement with the presence of α1A‐, but not α1D‐, adrenoceptors. Chloroethylclonidine at 1 μM had no effect and at 10 μM produced only a small reduction (c. 20%) in the maximum response to noradrenaline, indicating little, if any, contribution from α1B‐adrenoceptors. Responses of the rat femoral resistance arteries to electrical field stimulation (EFS) at 5–30 Hz for 10 s and 0.05 ms pulse width were principally due to α1‐adrenoceptor stimulation. Prazosin and 5‐methyl‐urapidil inhibited EFS‐mediated responses with pIC50s of 9.3 and 8.2, respectively, consistent with the α1A‐adrenoceptor being the predominant subtype. Responses to EFS at 10–30 Hz were relatively insensitive to BMY 7378 (pIC50, 6.5–6.7), while responses to 5 Hz were inhibited with a significantly higher pIC50 of 8.02, suggesting the contribution of α1D‐adrenoceptors. Chloroethylclonidine had no effect on responses to EFS, ruling out the contribution of an α1B‐subtype. In the presence of cocaine, the predominant subtype involved in responses to EFS was the α1A‐adrenoceptor, with a contribution from α1D‐adrenoceptors at low frequency, as seen in the absence of cocaine. However, there was also a significant increase in the sensitivity to BMY 7378 at higher frequencies, suggesting that a further small α1D‐adrenoceptor component may be uncovered in the presence of cocaine. The present study has shown a predominant role of the α1A‐adrenoceptor in contractions due to exogenous noradrenaline and to neurally released noradrenaline in rat femoral resistance arteries. α1D‐Adrenoceptors are not involved in responses to exogenous noradrenaline but appear to be activated by neurally released noradrenaline at a low frequency of stimulation and at higher frequencies in the presence of neuronal‐uptake blockade.

Functional characterization of α1‐adrenoceptor subtypes in human skeletal muscle resistance arteries

α1‐adrenoceptor subtypes in human skeletal muscle resistance arteries were characterized using agonists noradrenaline (non‐selective) and A61603 (α1A‐selective), the antagonists prazosin (non‐selective), 5‐methyl‐urapidil (α1A‐selective) and BMY7378 (α1D‐selective) and the alkylating agent chloroethylclonidine (preferential for α1B). Small arteries were obtained from the non‐ischaemic skeletal muscle of limbs amputated for critical limb ischaemia and isometric tension recorded using wire myography. Prazosin antagonized responses to noradrenaline with a pA2 value of 9.18, consistent with the presence of α1‐adrenoceptors, although the Schild slope (1.32) was significantly different from unity. 5‐Methyl‐urapidil competitively antagonized responses to noradrenaline with a pKB value of 8.48 and a Schild slope of 0.99, consistent with the presence of α1A‐adrenoceptors. In the presence of 300 nM 5‐methyl‐urapidil, noradrenaline exhibited biphasic concentration response curves, indicating the presence of a minor population of a 5‐methyl‐urapidil‐resistant subtype. Contractile responses to noradrenaline were not affected by 1 μM chloroethylclonidine suggesting the absence of α1B‐adrenoceptors. Maximum responses to noradrenaline and A61603 were reduced to a similar extent by 10 μM chloroethylclonidine, suggesting an effect of chloroethylclonidine at α1A‐adrenoceptors at the higher concentration. BMY7378 (10 and 100 nM) had no effect on responses to noradrenaline. BMY7378 (1 μM) poorly shifted the potency of noradrenaline giving a pA2 of 6.52. These results rule out the presence of the α1D‐subtype. These results show that contractile responses to noradrenaline in human skeletal muscle resistance arteries are predominantly mediated by the α1A‐adrenoceptor subtype with a minor population of an unknown α1‐adrenoceptor subtype.

Contributions of α1‐adrenoceptors, α2‐adrenoceptors and P2x‐purinoceptors to neurotransmission in several rabbit isolated blood vessels: role of neuronal uptake and autofeedback

1 . The roles of autofeedback and neuronal uptake in neurotransmission produced by electrical field stimulation in several rabbit isolated blood vessels were examined. 2 . Blocking drugs were used to separate the possible purinergic and noradrenergic contributions to the end organ response: prazosin, antagonist at postjunctional α1‐adrenoceptors; rauwolscine and yohimbine, antagonists at pre‐ and postjunctional α2‐adrenoceptors; α,β‐methylene ATP, desensitizing agent at postjunctional P2x‐purinoceptors. In addition to desensitizing postjunctional P2x‐purinoceptors, α,β‐methylene ATP potentiated the noradrenergic component of the nerve‐induced responses. 3 . In the presence of an intact neuronal uptake mechanism, the vessels showed different contributions of purinergic (via P2x‐purinoceptors) and noradrenergic (via α1‐adrenoceptors and α2‐adrenoceptors) components to the end organ response to nerve stimulation: saphenous artery (approximately equal contributions from P2x‐purinoceptors and α1‐adrenoceptors), ileocolic artery (mainly P2x‐purinoceptors with a smaller contribution from α1‐adrenoceptors), plantaris vein (mainly α1‐adrenoceptors with a small contribution from α2‐adrenoceptors and P2x‐purinoceptors) and saphenous vein (α1‐adrenoceptors). 4 . The presence of α2‐adrenoceptor‐mediated autofeedback could be demonstrated for both purinergic and noradrenergic components of the nerve‐induced responses in the artery preparations. In the veins, potentiation of nerve‐induced responses by α2‐adrenoceptor antagonists could not be studied due to blockade of postjunctional α2‐adrenoceptor‐mediated vasoconstriction. 5 . Blockade of neuronal uptake with cocaine potentiated the noradrenergic component of the nerve‐induced responses. Both α1‐adrenoceptor‐ and α2‐adrenoceptor‐mediated components were potentiated, with a relatively greater potentiation of the α2‐adrenoceptor‐mediated component. In the case of saphenous vein an α2‐adrenoceptor‐mediated component which was previously absent was uncovered. 6 . Blockade of neuronal uptake with cocaine had no effect or reduced the purinergic component of responses, the latter effect presumably due to enhanced α2‐adrenoceptor‐mediated autofeedback. 7 . In the presence of cocaine, nerve‐induced responses in the saphenous vein were biphasic. Rauwolscine potentiated the first phase and inhibited the second phase thus demonstrating effects of pre‐ and postjunctional α2‐adrenoceptor‐mediated activation in the same preparation. 8 . In conclusion, neuronal uptake and autofeedback processes play important and complex interacting parts in determining the relative contributions of α1 and α2‐adrenoceptors and P2x‐purinoceptors in the end organ response to neurotransmission in blood vessels.

Adrenoceptors mediating relaxation to catecholamines in rat isolated jejunum.

1 The characteristics of adrenoceptors mediating relaxation to catecholamines in rat isolated jejunum were investigated. 2 Catecholamines and BRL 37344 produced relaxation of the KCl‐contracted strips with an order of potency of isoprenaline (1.0) > BRL 37344 (0.63) > noradrenaline (0.1) > adrenaline (0.04). 3 In the presence of both prazosin (1 μm) and propranolol (1 μm) only small dextral shifts of the concentration‐response curves to agonists were observed and an order of potency of BRL 37344 (2.5) > isoprenaline (1.0) > noradrenaline (0.2) > adrenaline (0.1) was obtained. 4 In the presence of prazosin (1 μm) and propranolol (1 μm), cyanopindolol (0.1 − 10 μm) produced a concentration‐dependent rightward shift of the concentration‐response curve to adrenaline with a Schild slope not significantly different from unity and a mean pA2 value of 7.01. 5 The resistance of relaxant responses to propranolol, the relatively high potency of BRL 37344 compared to catecholamines and the competitive antagonism of relaxant responses to adrenaline by cyanopindolol suggest that β‐adrenoceptors in rat small intestine are mainly atypical in nature.

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.

Increased α1- and α2-adrenoceptor-mediated contractile responses of human skeletal muscle resistance arteries in chronic limb ischemia

Objective: Recently, we have shown augmented contractile responses of skeletal muscle resistance arteries to noradrenaline in patients with critical limb ischemia. We investigated whether this increased sensitivity in skeletal muscle resistance arteries is due to either α1- or α2-adrenoceptor-mediated responses or both. Methods: Skeletal muscle resistance arteries were isolated from the proximal (non-ischemic) and distal (ischemic) parts of limbs amputated for critical limb ischemia and mounted on a small vessel wire myograph. Cumulative concentration response curves of the vessel segments to noradrenaline, phenylephrine and brimonidine were obtained in the presence or the absence of the selective antagonists, prazosin and RS79948. Results: Noradrenaline and phenylephrine produced almost equal maximal contractile responses. Brimonidine responses were smaller and were almost abolished by 0.1 μM RS 79948 while those of phenylephrine and noradrenaline were not affected. Prazosin reduced the maximum responses to brimonidine, shifted the concentration response curves of noradrenaline and phenylephrine rightwards giving p K B values of 9.86 and 9.33, respectively. Maximum responses produced by all three agonists in distal vessels were significantly higher than those obtained in proximal vessels. Conclusions: Noradrenaline contractile responses in skeletal muscle resistance arteries are predominantly mediated by α1-adrenoceptors. Both α1- and α2-adrenoceptor-mediated responses are increased in the arteries from ischemic regions that may aggravate the decreased blood flow to the limbs due to arterial occlusion.