Alpha-1 adrenoceptors: evaluation of receptor subtype-binding kinetics in intact arterial tissues and comparison with membrane binding

1. The binding kinetics of [(3)H]-prazosin were measured using intact segments of rat tail artery (RTA) and thoracic aorta (RAO), and the data were compared with those obtained using a conventional membrane ligand-binding method. 2. In intact RTA and RAO segments, [(3)H]-prazosin bound reversibly in a time-dependent and receptor-specific manner at 4°C to alpha-1 adrenoceptors (ARs) of the plasma membrane, with affinities (pK(D): 9.5 in RTA; 9.9 in RAO) that were in agreement with values estimated by a conventional membrane ligand-binding method. However, nonspecific binding was considerably higher in RAO than RTA, failing to detect clearly the specific binding at high concentrations (>300 pM) of [(3)H]-prazosin in binding experiments with RAO segments and membranes. 3. The abundance of receptor in the RTA and RAO (B(max) mg(−1) of total tissue protein), estimated using the tissue segment-binding approach (527±14 fmol mg(−1) for RTA; 138±4 fmol mg(−1) for RAO), was about 25-fold higher than values estimated using a conventional membrane-binding method (22±5 fmol mg(−1) for RTA; 5±1 fmol mg(−1) for RAO). 4. Binding competition experiments using intact tissue segments or membranes derived from RTA tissue yielded comparable data, indicating a coexistence of alpha-1A AR (high affinity for prazosin, KMD-3213 and WB4101 and low affinity for BMY 7378) and alpha-1B AR (high affinity for prazosin but low affinity for KMD-3213, WB4101 and BMY 7378). 5. In RAO tissue, careful evaluation of the tissue segment-binding assay revealed the coexpression of alpha-1B AR (high affinity for prazosin, but low affinity for KMD-3213 and BMY 7378) and alpha-1D AR (high affinity for prazosin and BMY 7378, but low affinity for KMD-3213), whereas the membrane-binding approach failed to detect these receptor subtypes with certainty. 6. The present study indicates that previous estimates of alpha-1 AR density and alpha-1 AR subtypes obtained by a conventional membrane-binding approach, as opposed to our improved tissue segment-binding assay, may have substantially underestimated the abundance of receptors present in arterial tissues, and may have failed to identify accurately the presence of receptor subtypes. Advantages and disadvantages of the tissue segment-binding approach are discussed

Alpha-1D adrenoceptors are involved in reserpine-induced supersensitivity of rat tail artery

1. We examined reserpine-induced chemical denervation supersensitivity with special reference to alpha-1 adrenoceptor (AR) subtypes. 2. Chronic treatment with reserpine for 2 weeks depleted noradrenaline in the tail artery and spleen of rats. Noradrenaline in the thoracic aorta was negligible before and after reserpine treatment. 3. The treatment with reserpine produced supersensitivity in the contractile responses of the rat tail artery to phenylephrine, 5-HT and KCl, resulting in leftward shift of concentration–response curves (11.6-, 2.5- and 1.1-fold at EC(50) value, respectively). These results suggest a predominant sensitization of the alpha-1 AR-mediated response by reserpine treatment. 4. BMY 7378 at a concentration (30 nM) specific for blocking the alpha-1D AR subtype, but not KMD-3213 at a concentration (10 nM) selective for blocking the alpha-1A AR subtype, inhibited the supersensitivity of the phenylephrine-induced response in the reserpine-treated artery. On the other hand, the response to phenylephrine in reserpine-untreated artery was selectively inhibited by the same concentration of KMD-3213, but not by BMY 7378. Prazosin, a subtype-nonselective antagonist, blocked the responses to phenylephrine with the same potency, regardless of reserpine treatment. 5. In the thoracic aorta and spleen, no supersensitivity was produced in the responses to phenylephrine by reserpine treatment. 6. In a tissue segment-binding study using [(3)H]-prazosin, the total density and affinity of alpha-1 ARs in the rat tail artery were not changed by treatment with reserpine. However, alpha-1D AR with high affinity for BMY 7378 was significantly detected in reserpine-treated tail artery, in contrast to untreated artery. Decreases in alpha-1A AR with high affinity for KMD-3213 and alpha-1B AR with low affinities for KMD-3213 and BMY 7378 were also estimated in reserpine-treated tail artery. 7. Alpha-1D AR mRNA in rat tail artery increased to three-folds by reserpine treatment, whereas the levels of alpha-1A and 1B mRNAs were not significantly changed. 8. The present results suggest that chronic treatment with reserpine affects the expression of alpha-1 AR subtypes of rat tail artery and that the induction of alpha-1D ARs with high affinity for catecholamines is in part associated with reserpine-induced supersensitivity