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1. To illuminate the controversy on alpha 1A- or alpha 1L-adrenoceptor involvement in noradrenaline-mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype-selective alpha 1-adrenoceptor agonists and antagonists under different experimental conditions. 2. The agonist potency order in rat SMA was: A61603 >> SKF89748-A > cirazoline > noradrenaline > ST-587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29-8.80) indicating the involvement of alpha 1L-rather than alpha 1A-adrenoceptors. 3. The putative alpha 1L-adrenoceptor antagonist JTH-601, but not the alpha 1B-adrenoceptor antagonist chloroethylclonidine (10 microM) antagonized noradrenaline-induced contractions of SMA. The potency of the selective alpha 1D-adrenoceptor antagonist BMY 7378 against noradrenaline (pA2 = 6.16 +/- 0.13) and of the selective alpha 1A-adrenoceptor antagonist RS-17053 against noradrenaline (pKB = 8.35 +/- 0.10) and against the selective alpha 1A-adrenoceptor agonist A-61603 (pKB = 8.40 +/- 0.09) were too low to account for alpha 1D- and alpha 1A-adrenoceptor involvement. 4. The potency of RS-17053 (pKB/pA2's = 7.72-8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619. 5. Selective protection of a putative alpha 1A-adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS-17053 (pA2 = 8.25 +/- 0.06 against A61603). 6. Combined concentration-ratio analysis demonstrated that tamsulosin, which does not discriminate between alpha 1A- and alpha 1L-adrenoceptors, and RS-17053 competed for binding at the same site in the SMA. 7. In summary, data obtained in our experiments in rat SMA indicate that the alpha 1-adrenoceptor mediating noradrenaline-induced contraction displays a distinct alpha 1L-adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that alpha 1L-adrenoceptors represent an affinity state of the alpha 1A-adrenoceptor in functional assays. Furthermore, there is no co-existing alpha 1A-adrenoceptor in the SMA.

The aim of this study was to evaluate whether the operational model of agonism can yield independent estimates of agonist affinity (pK(A)) and efficacy (log tau) when Furchgott's method of irreversible receptor inactivation is employed. For this purpose, the interaction between noradrenaline and phenoxybenzamine was studied in rat small mesenteric artery using a paired-curve design. Phenoxybenzamine pretreatment produced a significant rightward shift and depression of the upper asymptote of the noradrenaline concentration-effect (E/[A]) curve. Although the operational model of agonism appeared to provide an adequate fit of the individual E/[A] curves, a highly significant correlation was found between the estimates of pK(A )and log tau (r = -0.80, p < 0.0001), inconsistent with the assumption that affinity and efficacy are independent parameters (best line fit: pK(A) = -0.96 x log tau + 6.75). The pK(A) and log tau estimates were not correlated with either the pEC50s of the control curves or upper asymptotes of the phenoxybenzamine-treated curves. Simulations showed that the correlation between affinity and efficacy can be explained by the effect on the outcome of the analysis of random errors in the response measurements. Therefore, although in theory the operational model of agonism should provide independent estimates of agonist affinity and efficacy, this is unlikely to be the case with experimental data.

We have developed a fitting procedure, based on nonlinear mixed effect modelling and original work by Richards (1959, J Exp Botany 10, 290-300), to describe the degree of asymmetry of concentration-effect E/[A] curves and analysed the shape of E/[A] curves obtained with alpha1-adrenoceptor agonists in rat aorta. The four-parameter Richards model provided a significantly better fit of the data than the standard logistic/Hill model for all ligands investigated, which implies that E/[A] curves were asymmetrical. With the exception of ST 587, the asymmetry parameter (delta) tended toward zero and the Richards model could be replaced without significant loss of goodness-of-fit by the three-parameter, asymmetrical Gompertz model. The alpha1-adrenoceptor antagonist, prazosin (10 nM), had no effect on the asymmetry of the noradrenaline E/[A] curve but significantly increased the slope at the point of inflection. In contrast, pretreatment with the irreversible antagonist, phenoxybenzamine (60 nM), produced a shift of the delta estimate for noradrenaline from zero to unity, indicating a change from an asymmetrical to a symmetrical curve. Therefore, detailed statistical analysis of E/[A] curve asymmetry demonstrates that alpha1-adrenoceptors in rat aorta do not operate as a homogenous one-receptor-one-transducer system. This conclusion could not have been reached by either an analysis with the standard logistic/Hill model or visual inspection of experimental data. Overall, the curve-fitting analysis developed in this study provides a quantitative and sensitive measure of asymmetry and a novel method for the objective discrimination of agonist action on the basis of curve shape. The method is generally applicable to other pharmacological assays and provides a new tool in receptor classification studies.

1. Previously, we reported that noradrenaline (NA), in addition to its alpha 1-adrenoceptor-mediated contractile effect, may relax the rat small mesenteric artery (SMA) in order to account for steep Schild plots obtained with compounds classified as alpha 1-adrenoceptor antagonists. In this study, a relaxant action of NA has been exposed in the rat isolated, endothelium-denuded SMA precontracted by the thromboxane A2-mimetic, U46619. 2. NA, but not the selective alpha 2-adrenoceptor agonist, UK14304, produced concentration-dependent contraction of the SMA (pEC50 = 5.7 +/- 0.1). After precontraction with 0.1 microM U46619, 10 nM-30 microM NA produced a further contraction (pEC50 = 6.1 +/- 0.2), while higher concentrations of NA produced small, but significant, relaxant responses. 3. In the presence of 1 microM prazosin, 0.1-30 microM NA produced concentration dependent relaxation (pIC50 = 5.9 +/- 0.1) after precontraction with 0.1 microM U46619. The NA relaxation concentration-effect curve was completely inhibited by 1 microM of the beta 1/beta 2-adrenoceptor antagonist, timolol. However, when the concentration of prazosin was increased by 10 fold (10 microM), NA once again produced concentration-dependent relaxation (pIC50 = 4.5 +/- 0.2). This relaxation concentration-effect curve was not blocked by a 10 fold higher concentration of timolol (10 microM), nor by the presence of idazoxan (10 microM), cyanopindolol (10 microM), NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), indomethacin (10 microM) or sulpiride (1 microM). However, haloperidol (10 microM) and (+/-)-SCH-23390 (10 nM) produced significant inhibition of the relaxation, suggesting the involvement of dopamine D1 receptors. 4. Dopamine also produced concentration-dependent relaxation following U46619 precontraction (pIC50 = 5.4 +/- 0.1) which was significantly inhibited by haloperidol and (+)-SCH-23390. Pretreatment with 10 microM phenoxybenzamine for 60 min produced a significant inhibition of the dopamine and NA relaxation curves and application of the operational model of agonism yielded estimates of the affinity (pKA = 5.3 +/- 0.2 and 4.4 +/- 0.2) and efficacy (log gamma = 0.06 +/- 0.11 and 0.01 +/- 0.10) for dopamine and NA, respectively, at D1 receptors. 5. HV723 (0.1 and 1 microM), a ligand that yielded a Schild plot slope parameter of unity as an antagonist of NA in the contractile assay, produced concentration-dependent inhibition of the NA-mediated relaxation (pA2 approximately 8). 6. The results of this study indicate that NA can activate D1 receptors mediating relaxation in the rat SMA at concentrations which were encountered in our previous receptor classification experiments using competitive alpha 1-adrenoceptor antagonists.