UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine]-mediated vasodilator responses were studied on wire myograph-mounted mouse aorta to determine the cells involved, mechanisms of action, and subtypes of ␣ 2 -adrenoceptors. In the presence of induced tone, UK-14,304 produced concentration-related vasodilatation that was abolished by rauwolscine, N -nitro-L-arginine methyl ester (L-NAME), or endothelium removal, indicating that endothelial ␣ 2 -adrenoceptors can release nitric oxide. In the ␣ 2A -adrenoceptor knockout mouse and the D79N mouse, a functional knockout of the ␣ 2A -adrenoceptor, these relaxant effects of UK-14,304 were lost, indicating the involvement of the ␣ 2A -adrenoceptor. UK-14,304 could also contract aorta: a small contraction occurred at high concentrations, was enhanced by L-NAME, and was absent in the ␣ 1D -adrenoceptor knockout mouse, indicating activation of the ␣ 1D -adrenoceptor. There was no evidence for a contractile ␣ 2 -adrenoceptor-mediated response. A fluorescent ligand, quinazoline piperazine bodipy, antagonized the relaxant action of UK-14,304. This compound could be visualized on aortic endothelial cells, and its binding could be prevented by rauwolscine, providing direct evidence for the presence of ␣ 2 -adrenoceptors on the endothelium. Norepinephrine reduced tone in the ␣ 1D -adrenoceptor knockout and controls, an effect blocked by rauwolscine and L-NAME but not by prazosin. This suggests that norepinephrine activates endothelial ␣ 2 -adrenoceptors. In conclusion, the endothelium of mouse aorta has an ␣ 2A -adrenoceptor that responds to norepinephrine; promotes the release of nitric oxide, causing smooth muscle relaxation; and that can be directly visualized. Knockout or genetic malfunction of this receptor should increase arterial stiffness, exacerbated by raised catecholamines, and contribute to heart failure.All three ␣ 2 -adrenoceptors have distinct, yet poorly defined, roles in the control of the vascular system. The limited selectivity of agonists and antagonists has therefore prompted the use of transgenic mouse models. The subtypes are ␣ 2A , ␣ 2B , and ␣ 2C : the mouse ortholog of the human ␣ 2A -adrenoceptor is sometimes called the ␣ 2D -or ␣ 2A/D -adrenoceptor; we use the generic term ␣ 2A -adrenoceptor (Alexander et al., 2004). They have two direct pharmacological effects on blood vessels that can modify vascular tone: a direct vasopressor action (for review, see Wilson et al., 1991;Guimaraes and Moura, 2001) and vasodilatation via endothelium-derived relaxant factors (Cocks and Angus, 1983;Vanhoutte, 2001). They also reduce sympathetic traffic centrally and inhibit transmitter release from sympathetic postganglionic nerves (Starke, 2001), although this is not well established as a physiological phenomenon in blood vessels. Available pharmacological data and knockout studies, although not definitive, present evidence for, at least, ␣ 2A -, ␣ 2B -, and ␣ 2C -adrenoceptors for vasoconstriction, ␣ 2A -and ␣ 2C -adrenoceptors for sympatho-...
1 The postjunctional x-adrenoceptors mediating contractions in the isolated vascular bed of the perfused rat tail have been investigated, in the presence and absence of an increase in perfusion pressure by arginine vasopressin (AVP). 2 In the absence of AVP, bolus doses of noradrenaline (NA) and phenylephrine produced pressor responses of similar time course, while UK-14,304 was practically inactive. Responses to noradrenaline were inhibited more by 0.05 yM prazosin than by 1 yM rauwolscine, suggesting the presence of ax-adrenoceptors.3 Following a sustained elevation in perfusion pressure by AVP, both UK-14,304 and NA (the latter in the presence of 0.05pM prazosin to inhibit a1-adrenoceptors) elicited dose-dependent pressor responses. The maximum response to UK-14,304 under these conditions was approximately 30% of the maximum response to NA in the absence of prazosin and AVP. Responses to phenylephrine were not affected by the AVP-induced increase in vascular tone. 4 In the presence of AVP, pressor responses to UK-14,304 were resistant to 0.05pM prazosin and susceptible to antagonism by 1 yM rauwolscine (-log Kb 7.65 + 0.15). Similarly, responses to NA in the presence of 0.05 pM prazosin and AVP were inhibited by 1 ,M rauwolscine. This represents the first demonstration of prazosin-resistant, rauwolscine-sensitive a2-adrenoceptor-mediated responses in the vasculature of the rat tail. 5These results suggest that in isolated vascular preparations, functional populations of postjunctional LX2-adrenoceptors may be 'uncovexed' by the presence of AVP.
1 Cutaneous resistance arteries (c.r.a.) (internal diameter=240.94+5.42 mm, n=67/25 (number arteries/number animals)) from New Zealand white rabbits were mounted in wire myographs and a normalization procedure followed. 2 Cumulative concentration-response curves (CCRCs) were constructed for the a-adrenoceptor agonists noradrenaline (NA), (R)A61603 and phenylephrine (PE) in the presence of cocaine (3 mM), propranolol (1 mM) and corticosterone (10 mM). The eects of competitive a 1 -adrenoceptor antagonists, prazosin, WB4101, 5-methyl-urapidil, HV723, BMY7378 and the irreversible a 1B selective compound chloroethylclonidine (CEC) were examined versus the potency and maximum response of the c.r.a.s to noradrenaline. 3 The high potency of A-61603 relative to PE has been shown to dierentiate both functional and binding site a 1A -or a 1B -adrenoceptors from a 1D -adrenoceptors: A-61603 was 944 times more potent than phenylephrine (at EC 50 ) suggesting the presence of a functional a 1A or a 1B as opposed to an a 1D -subtype. 4 Exposure to chloroethylclonidine (CEC; 100 mM) decreased the maximum response to noradrenaline but did not signi®cantly change noradrenaline sensitivity indicating that a substantial part of noradrenaline-induced vasoconstriction in rabbit cutaneous arteries is CEC-insensitive. 5 The potencies of prazosin (pA 2 =9.14) and WB4101 (pA 2 =9.30) indicate the involvement of prazosin-sensitive functional a 1 -adrenoceptors. The slopes of corresponding Schild plots for prazosin and WB4101 did not include negative unity which implies the possible involvement of more than one functional a 1 -adrenoceptor subtype in noradrenaline-induced vasoconstriction in rabbit cutaneous resistance arteries. In contrast to this, in the case of 5-methyl-urapidil and HV723, the Schild plot slope parameters were not signi®cantly dierent from negative unity over the range of concentrations used; the low pA 2 value for 5-methylurapidil (7.27) suggests the non-involvement of an a 1A -or an a 1D -adrenoceptor; the low pA 2 value for HV723 (8.47) was similar to that against responses postulated as a 1L . 6 We conclude that rabbit cutaneous resistance arteries express a prazosin-sensitive functional a 1 -adrenoceptor resembling the a 1B and another low anity site for prazosin which on the basis of the functional antagonism produced by HV723 most closely resembles the a 1L -adrenoceptor; the low pA2 value for HV723 (8.47) is similar to that against responses postulated as a 1L .
1 a 1 -Adrenoceptor (AR) subtypes in mouse carotid arteries were characterised using a combination of agonist/antagonist pharmacology and knockout (KO) mice. 2 Phenylephrine (PE) was most potent in the a 1B -KO (pEC 50 ¼ 6.970.2) followed by control (pEC 50 ¼ 6.370.06) and a 1D -KO (pEC 50 ¼ 5.570.07). Both N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulphonamide hydrobromide (A-61603) and 5-hydroxytryptamine (5-HT) were more potent in the a 1D -KO (pEC 50 ¼ 7.470.27 and 7.470.05, respectively) than the control (pEC 50 ¼ 6.970.09 and 6.970.08, respectively) and equipotent with the control in the a 1B -KO (pEC 50 ¼ 6.770.07 and 6.870.04). Maximum responses to PE and A-61603 were reduced in the a 1D -KO compared to control; there was no difference in maximum responses to 5-HT. 3 In control arteries, prazosin and 5-methylurapidil acted competitively with pA 2 of 9.6 and 7.5, respectively. BMY7378 produced antagonism only at the highest concentration used (100 nM; pK B 8.3). 4 Prazosin, 5-methylurapidil and BMY7378 acted competitively in a 1B -KO carotid arteries with pA 2 of 10.3, 7.6 and 9.6, respectively. 5 In the a 1D -KO, against PE, 5-methylurapidil produced a pA 2 of 8.1. pK B values were calculated for prazosin (10.6) and BMY7378 (7.0). Against A-61603, 5-methylurapidil had a pA 2 of 8.5, prazosin 8.6, while BMY7378 had no effect. 6 In conclusion, the a 1B -KO mediates contraction solely through a 1D -ARs and the a 1D -KO through a 1A -ARs. Extrapolating back to the control from the knockout data suggests that all three subtypes could be involved in the responses, but we propose that the a 1D -AR causes the contractile response and that the role of the a 1B -AR is mainly regulatory.
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