The mRNA levels for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The alpha1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), alpha1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both alpha1A- and alpha1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the alpha1B-subtype were a minority in all vessels (1.7-11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to alpha1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 micromol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro (4,5) decane-7-dionedihydrochloride} (BMY-7378, an alpha1D-adrenoceptor antagonist) confirm the relevant role of alpha1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an alpha1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of alpha1A- and alpha1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of alpha1A/alpha1L-adrenoceptors with minor participation of the alpha1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the alpha1L-adrenoceptor could be a functional isoform of the alpha1A-subtype.

1 The role of a constitutively active population of a 1D -adrenoceptors was analysed in arteries obtained from spontaneously hypertensive rats (SHR) and controls (WKY) divided into three groups: young prehypertensive, adult hypertensive, and adult animals chronically treated with captopril (50 mg kg 71 per day orally) in order to prevent the hypertensive state. 2 In adult SHR, a signi®cant increase in BMY 7378 potency (not in prazosin potency) was observed in aorta, mesenteric artery, and the ®rst and second branches of the small mesenteric arteries with respect to WKY rats. This di erence was not observed in iliac and tail arteries, which suggests an increased functional role of a 1D -adrenoceptors only in some vessels of SHR. 3 The increase in the resting tone (IRT) observed in absence of agonist, inhibited by BMY 7378, that represents the constitutively active population of a 1D -adrenoceptors, was also signi®cantly greater in aorta and mesenteric artery from adult SHR. 4 In young and captopril treated adult animals, no di erences between strains with respect to BMY 7378 potency, or IRT were observed. 5 The increase in the functional role of a 1D -adrenoceptors and their constitutive activity observed in hypertension is prevented by captopril treatment. The pathological consequence of this change is the slower rate of recovery of the basal tone after removal of an adrenergic stimulus, observed in vessels from hypertensive animals that had shown an increase in the functionality of constitutively active a 1D -adrenoceptors. This change was not observed in prehypertensive or captopril treated animals. IRT, increase in the resting tone; NA, noradrenaline; SHR, spontaneously hypertensive rats; SMA-1 and SMA-2, ®rst and second small mesenteric arterial branches; WKY, Wistar Kyoto rats

1 The a 1 -adrenergic responses of rat aorta and tail artery have been analysed measuring the contractility and the inositol phosphate (IP) formation induced by noradrenaline. Three antagonists, prazosin, 5-methylurapidil (a 1A selective) and BMY 7378 (a 1D selective) have been used in di erent experimental procedures. 2 Noradrenaline possesses a greater potency inducing contraction and IP accumulation in aorta (pEC 50 -contraction=7.32+0.04; pEC 50 -IPs=6.03+0.08) than in the tail artery (pEC 50 -contraction=5.71+0.07; pEC 50 -IPs=5.51+0.10). Although the maximum contraction was similar in both tissues (E max -tail=619.1+55.6 mg; E max -aorta-698.2+40.8 mg), there were marked di erences in the ability of these tissues to generate intracellular second messengers the tail artery being more e cient (E max -tail=1060+147%; E max -aorta=108.1+16.9%).3 Concentration response curves of noradrenaline in presence of antagonist together with concentration inhibition curves for antagonists added before (CICb) or after (CICa) noradrenaline-induced maximal response in Ca 2+ -containing or Ca 2+ -free medium have been performed. A comparative analysis of the di erent procedures as well as the mathematical approaches used in each case to calculate the antagonist potencies, were completed. 4 The CICa was the simplest method to characterize the predominant a 1 -adrenoceptor subtype involved in the functional response of a tissue. 5 In aorta, where constitutively active a 1D -adrenoeptors are present, the use of di erent experimental procedures evidenced a complex equilibrium between a 1D -and a 1A -adrenoceptor subtypes. 6 The appropriate management of LiCl in IP accumulation studies allowed us to reproduce the di erent experimental procedures performed in contractile experiments giving more technical possibilities to this methodology.

. Modulatory role of a constitutively active population of ␣1D-adrenoceptors in conductance arteries. Am J Physiol Heart Circ Physiol 282: H475-H481, 2002. First published October 18, 2001 10.1152/ajpheart.00411.2001.-A constitutively active population of ␣1D-adrenoceptors in iliac and proximal, distal, and small mesenteric rat arteries was studied. The increase in resting tone (IRT) that evidences it was observed only in iliac and proximal mesenteric and was inhibited by prazosin (pIC50 ϭ 9.57), 5-methylurapidil (pIC50 ϭ 7.61), and BMY 7378 (pIC50 ϭ 8.77). Chloroethylchlonidine (100 mol/l) did not affect IRT, but when added before the other antagonists it blocked their effect. The potency shown by BMY 7378 confirms the ␣1D-subtype as responsible for IRT. BMY 7378 displayed greater inhibition of adrenergic responses in iliac (pIC50 ϭ 7.57 Ϯ 0.11) and proximal mesenteric arteries (pIC50 ϭ 8.05 Ϯ 0.2) than in distal (pIC50 ϭ 6.94 Ϯ 0.13) or small mesenteric arteries (pIC50 ϭ 6.30 Ϯ 0.14), which confirms the functional role of the ␣1D-adrenoceptor in iliac and proximal mesenteric arteries. This subtype prevents abrupt changes in iliac and proximal mesenteric artery caliber when the agonist disappears, and this modulatory role is evidenced by the slower decay in the response to norepinephrine after removal. conductance vessels; resistance vessels; constitutive activity; ␣1D-adrenoceptors IT HAS BEEN CLEARLY SHOWN that activation of ␣ 1 -adrenoceptors mediates vasoconstriction, and considerable progress has been made toward elucidating the molecular structures and signal transduction mechanisms of these adrenoceptors. Molecular cloning studies have identified three ␣ 1 -adrenoceptor cDNAs (␣ 1a , ␣ 1b , and ␣ 1d ), and their primary structure corresponds to the model of the superfamily of G protein-coupled receptors. Moreover, three distinct ␣ 1 -adrenoceptor subtypes (␣ 1A , ␣ 1B , and ␣ 1D ) that correlate well with the cloned ␣ 1 -adrenoceptors have been identified pharmacologically in functional and binding experiments (8, 9). These three subtypes display high, subnanomolar affinities for prazosin. Furthermore, functional studies have provided evidence of the existence of an additional ␣ 1L -adrenoceptor subtype displaying low affinity for prazosin and some other ␣ 1 -adrenoceptor antagonists (3,4,23,33). This ␣ 1L -adrenoceptor has no molecular correlate. However, the physiological role of each ␣ 1 -adrenoceptor in the vascular smooth muscle is unclear, and the expression of an ␣ 1 -adrenoceptor subtype in a vessel is not always related to a functional role of this subtype in the contractile tone of the vessel (10,12,16,17,29,33).In addition to the complex functionality of the different subtypes of ␣ 1 -adrenoceptors, we have shown in previous studies (7,24,26) the existence of a population of constitutively active ␣ 1D -adrenoceptors in the rat aorta but not in the tail artery. The existence of this active conformation has been revealed mainly in artificial models such as receptor mutants or systems that show ...

In the present study, we analyzed changes in intracellular Ca 2ϩ levels and inositol phosphate accumulation related to a population of ␣ 1D -adrenoceptors in rat aorta resembling constitutively active receptors. after store depletion induced only a sustained increase in [Ca 2ϩ ] i without inositol phosphate accumulation nor contractile response. Taken together these results suggest that in the aorta, Ca 2ϩ entry is required for the recovery of cytosolic calcium levels and the display of the membrane signals related to the constitutive activity of ␣ 1D -adrenoceptors, i.e., inositol phosphate formation and Ca 2ϩ entry through L-type channels, which maintains a contractile response once the agonist has been removed.