1 The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9-0-methylboldine) and glaucine (2,9-0-dimethylboldine) on 5 These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectivity of action for the aIA-adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2-hydroxy function, induces a significant increase in aCIA-subtype selectivity and affinity.

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.

Ryanodine (10 microM), thapsigargin (1 microM) and cyclopiazonic acid (10 microM) produced a slow, sustained contractile response in rat aorta that only can be observed in Ca2+-containing solution. In Ca2+-free medium, no response to the drugs was obtained, which suggests that the contraction elicited in presence of Ca2+ is mainly due to the contribution of extracellular influx. This Ca2+ entry does not depend on the opening of dihydropyridine-dependent Ca2+-channels for nimodipine does not affect this. Noradrenaline (1 microM) induced a biphasic response in Ca2+-free medium that was mediated by two different Ca2+ compartments, one of which is common to caffeine (10 mM), and is also depleted by ryanodine (10 microM), thapsigargin (1 microM) and cyclopiazonic acid (10 microM). This compartment loses its Ca2+ content after long exposure (65 min) to Ca2+-free EDTA-containing solution and its refilling was also affected by the three agents tested. The other compartment depleted by noradrenaline, but not by caffeine, was also insensitive to ryanodine, thapsigargin and cyclopiazonic acid, and did not lose its Ca2+ after 65 min in Ca2+-free medium. Contractions induced by noradrenaline (1 microM) or caffeine (10 mM) in Ca2+-free medium were not affected by ryanodine, thapsigargin and cyclopiazonic acid when these agents were added 1 min before or during the response to each agonist. After depletion of internal Ca2+ stores sensitive to noradrenaline, an increase in the resting tone (IRT) of rat aorta was observed when Ca2+ was added again in absence of the agonist. This IRT was not affected by treatment with ryanodine, thapsigargin and cyclopiazonic acid, and represents a Ca2+ entry pathway dependent on the depletion of the noradrenaline-sensitive Ca2+ compartment. In conclusion, we can differentiate two Ca2+ entry pathways in rat aorta that depend on the previous depletion of two internal Ca2+ compartments: One corresponds to the classic capacitative Ca2+ entry model and is promoted by depletion of the internal pool sensitive to noradreanline, caffeine, ryanodine, thapsigargin and cyclopiazonic acid, the other is dependent only on depletion of an alpha1-adrenoceptor-sensitive Ca2+ pool.

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Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.