Gertraud Hanft

Subclassification of α1‐adrenoceptor recognition sites by urapidil derivatives and other selective antagonists

1 The affinities of urapidil derivatives and other antagonists for α1‐adrenoceptors labelled by [3H]‐prazosin were determined on membranes of six different rat tissues. 2 Urapidil and its 5‐acetyl‐, 5‐formyl‐ and 5‐methyl‐derivative displaced [3H]‐prazosin from α1‐adrenoceptor binding sites in a concentration‐dependent manner which varied with tissue. IC50 values were lower in vas deferens, hippocampus and cerebral cortex than in heart, liver and spleen. For 5‐methyl‐urapidil, binding to two distinct sites could be demonstrated with mean K1 values of about 0.6 and 45 nM. Saturation binding studies with [3H]‐prazosin in the presence of 5‐methylurapidil indicated a competitive type of interaction between 5‐methyl‐urapidil and [3H]‐prazosin. 3 The proportion of [3H]‐prazosin binding sites with high affinity for 5‐methyl‐urapidil was 58% in vas deferens, 69% in hippocampus, 41% in cerebral cortex and 23% in myocardium. In liver and spleen virtually no high affinity sites were found. These values were in good agreement with the percentages of binding sites with high affinities for WB‐4101 and phentolamine, indicating that all these antagonists bind to the same subtype of α1 ‐recognition sites, whereas other α‐antagonists like BE 2254, yohimbine and unlabelled prazosin did not discriminate between two binding sites. 4 Preincubating membranes of the cerebral cortex with chloroethylclonidine preferentially inactivated [3H]‐prazosin binding sites with low affinity for 5‐methyl‐urapidil. 5 The antagonist potencies of 5‐methyl‐urapidil and WB‐4101 against α1‐adrenoceptor‐mediated contractile responses were higher in vas deferens than in myocardium. The α1‐mediated effects in vas deferens but not in the heart were highly susceptible to nitrendipine. 6 Using 5‐methyl‐urapidil, the existence of two distinct α1‐adrenoceptor recognition sites could be demonstrated which correspond to the proposed α1A‐ and α1B‐subtypes. Since 5‐methyl‐urapidil is one of the ligands with most selectivity between these subtypes in binding studies it may serve as a valuable tool for such investigations.

5-Methyl-urapidil discriminates between subtypes of the α1-adrenoceptor

The affinity of 5-methyl-urapidil of α1-adrenoceptors was determined from the inhibition of [3H]prazosin binding on membranes of different rat tissues. In hippocampus, vas deferens and heart 5-methyl-urapidil displaced [3H]prazosin in a biphasic manner with mean pK1 values between 9.1 and 9.4 for the high-affinity site and 7.2 to 7.8 for the low-affinity site. Only the low affinity site was found in spleen and liver. At present, 5-methyl-urapidil is the antagonist which most clearly discriminates between α1-adrenoceptor subtypes.

Radioligand binding studies of α1-adrenoceptor subtypes in rat heart

1 In order to characterize the α1‐adrenoceptor subtypes mediating positive inotropic effects of adrenaline (in the presence of propranolol) in rat right ventricular strips and the Ca2+ sources used to elicit them, we have used radioligand binding to identify the α1‐adrenoceptor subtypes present in rat heart and the α1‐adrenoceptor affinity and subtype‐selectivity of various pharmacological tools. 2 Amitryptiline, mianserin, trimipramine, oxaprotiline, clonidine, chloroethylclonidine, phenoxybenzamine, BE 2254 and 8‐OH‐DPAT competed for [3H]‐prazosin binding in rat heart, vas deferens, liver, spleen, cerebral cortex and hippocampus but none of them displayed detectable α1‐adrenoceptor subtype‐selectivity; nitrendipine did not compete for [3H]‐prazosin binding in concentrations up to ***5 μmoll−1. 3 The α1A‐adrenoceptor‐selective, 5‐methyl‐urapidil, (+)‐niguldipine, and to a lesser extent (–)‐niguldipine competed for [3H]‐prazosin binding in rat heart, vas deferens, cerebral cortex and hippocampus with shallow and biphasic curves; analysis of these curves demonstrated that rat heart contains α1A‐ and α1B‐adrenoceptors in a 20:80 ratio. 4 Treatment of rat right ventricular strips with 100 μmoll−1 chloroethylclonidine for 30 min at 30°C followed by 60 min washout reduced the number of α1‐adrenoceptors, as assessed by [3H]‐prazosin saturation experiments, by 74%. Treatment with 100 μmol 1−1 CdCl2 did not affect number or affinity of cardiac α1‐adrenoceptors and combined treatment with chloroethylclonidine and CdCl2 reduced α1‐adrenoceptor number by 90%. 5 Treatment of rat right ventricular strips with chloroethylclonidine steepened 5‐methyl‐urapidil competition curves and increased the relative contribution of α1A‐adrenoceptors from 26 to 89%. Treatment with CdCl2 did not affect 5‐methyl‐urapidil competition curves and combined treatment with chloroethylclonidine and CdCl2 increased the relative contribution of α1A‐adrenoceptors to 66%. 6 We conclude that rat heart contains α1A‐ and α1B‐adrenoceptors in a 20:80% ratio. Treatment with chloroethylclonidine reduces α1B‐adrenoceptor number by 96% but has only minor effects on α1A‐adrenoceptor density. Treatment with CdCl2 does not affect the number of either α1‐adrenoceptor subtype.

α1B- but not α1A-adrenoceptors mediate inositol phosphate generation

SummaryWe used novel highly subtype-selective antagonists to study whether α1A- and/or α1B-adrenoceptors mediate the stimulation of inositol phosphate generation by noradrenaline in rat cerebral cortex. Phentolamine (10 μM) and prazosin (100 nM) completely abolished the stimulated inositol phosphate generation. The α1A-selective antagonists 5-methyl-urapidil (100 nM) and (+)− and (−)-niguldipine (10 nM) caused only weak inhibition or none at all although these concentrations occupied α1A-adrenoceptors almost completely. In contrast, pretreatment with the irreversible α1B-selective chloroethylclonidine reduced the noradrenaline-stimulated inositol phosphate generation by 76 ± 8%. These data demonstrate that α1B-adrenoceptors couple to inositol phosphate generation; the signal transduction system of α1A-adrenoceptors remains unclear.

Functional studies on α1‐adrenoceptor subtypes mediating inotropic effects in rat right ventricle

1 We have studied the α1‐adrenoceptor subtypes mediating inotropic effects of adrenaline in rat right ventricle and the Ca2+ sources used to elicit these effects. (α1A‐Adrenoceptor‐mediated contractile effects in rat vas deferens were studied for comparison in some cases. 2 Treatment with chloroethylclonidine did not affect the maximal β‐adrenoceptor‐mediated inotropic effects in rat right ventricle or the maximal α1A‐adrenoceptor‐mediated contractile effects in rat vas deferens; it did not alter the potency of isoprenaline in the ventricle and reduced the potency of the α‐adrenoceptor antagonists in vas deferens only slightly. Treatment of right ventricular strips with CdCl2 markedly reduced resting tension and enhanced maximal inotropic effects of isoprenaline but did not affect its potency. 3 Inactivation of cardiac α1B‐adrenoceptors by treatment with chloroethylclonidine slightly enhanced the maximal inotropic effects of the. full agonist, adrenaline and of several partial agonists. 4 Schild analysis of inhibition experiments with the α1A‐adrenoceptor‐selective antagonists, 5‐methylurapidil and (±)‐tamsulosin, demonstrated that adrenaline causes its inotropic effects mainly via the α1B‐adrenoceptor subtype. Schild analysis of 5‐methyl‐urapidil inhibition experiments in chloroethylclonidine‐treated ventricles indicated that only α1A‐adrenoceptors mediate the inotropic effects of adrenaline following inactivation of the α1B‐adrenoceptors. 5 In control ventricles the organic Ca2+ entry blocker, nitrendipine and treatment with the inorganic Ca2+ entry blocker, CdCl2 did not reduce inotropic effects of adrenaline whereas ryanodine treatment inhibited them. In contrast, nitrendipine and CdCl2 treatment had major inhibitory effects in chloroethylclonidine‐treated but lacked inhibitory effects in phenoxybenzamine‐treated ventricular strips. 6 We conclude that inotropic effects of adrenaline in rat heart are mediated mainly by α1B‐adrenoceptors via release of Ca2+ from an intracellular pool. Following inactivation of α1B‐adrenoceptors by chloroethylclonidine treatment, α1A‐adrenoceptors can fully compensate and mediate inotropic effects by promoting influx of extracellular Ca2+ at least partly via voltage‐operated channels. Therefore, we speculate that α1B‐adrenoceptors exert a tonic inhibitory effect on α1A‐adrenoceptors.

Demonstration of α1A-and α1B-adrenoceptor binding sites in human brain tissue

Abstract Radioligand binding studies suggest that α 1 -adrenoceptor recognition sites are heterogeneous. Several adrenergic agents discriminate between two adrenoceptor binding sites designated α 1 A and α 1B . In the present study we demonstrate for the first time that these two subtypes exist in the human brain. 5-Methyl-urapidil and (+)-niguldipine, which have previously been shown to be α 1A -selective, inhibited [ 3 H]prazonin binding to cortical membranes in a biphasic manner. The irreversible α 1B -ligand, chloroethylclonidine, preferentially elimanated the binding sites with low affinity for (+)-niguldipine. In contrast, BE 2254 and unlabelled prazosin displaced the radioligand ina monophasic manner. The IC 50 values for prazosin were not affected by pretreatment of the membranes with chloroethylclonidine. Our data on human brain membranes are in excellent agreement with recent findings in rat tissues and suggest that the α 1 -adrenoceptor subtypes in human brain are similar to those in rat tissues.

Effect of chronic lithium administration on adrenoceptor binding and adrenoceptor regulation in rat cerebral cortex

SummaryLithium (Li) at a concentration, which exerts prophylactic effects in affective disorders is known to alter noradrenaline turnover and the β-adrenoceptor-dependent cAMP accumulation. In the present study the action of chronic Li administration (at least 5 weeks) on agonist and antagonist binding to adrenoceptors and on the regulation of adrenoceptors was investigated in rat cerebral cortex. Li treatment caused a small but significant decrease in the number of β-adrenoceptor binding sites by 10% (3H-dihydroalprenolol binding) leaving the number of α1- and α2-adrenoceptor binding sites (3H-prazosin and 3H-rauwolscine, respectively) unchanged. The affinity of the radioligands as well as the affinity of agonists to these binding sites were not altered. The up-regulation of β-adrenoceptor binding sites produced by repeated reserpine injections was inhibited by 32% in rats treated concomitantly with Li, although the noradrenaline depleting effect of reserpine was not impaired. In contrast, Li treatment had no effect on the up-regulation of β-adrenoceptor binding induced by 6-OH-dopamine, nor did it alter the β-adrenoceptor down-regulation following chronic administration of desipramine. The up-regulation of α1-adrenoceptor binding sites caused by reserpine or 6-OH-dopamine also remained unaffected by Li. It is concluded that chronic Li has limited effects on cortical adrenoceptors and their regulation. The inhibition of β-adrenoceptor up-regulation caused by reserpine may reflect an action of Li on non-adrenergic systems rather than a general “stabilizing” effect on adrenoceptors proposed previously.

α1-Adrenoceptors of rat myocardium: Comparison of agonist binding and positive inotropic response

SummaryBinding of agonists to al-adrenoceptors labelled by 3H-prazosin was investigated in membranes of rat myocardium and compared to the inotropic response elicited by α1-adrenoceptor activation on isolated right ventricles.1.At 30°C the full agonists, adrenaline and phenylephrine, displaced 3H-prazosin with a shallow inhibition curve. The data are compatible with the assumption that 32% of the binding sites were in a state of high affinity for the agonist adrenaline (KI 85 nmol/l) and 68% in a low affinity state (KI I738 nmol/l). GTP transformed all binding sites into the low affinity form suggesting that at least some of the cardiac α1-adrenoceptors are coupled to N-proteins.2.At 0°C most of the binding sites (86%) were in a state of high affinity for agonists (KI for adrenaline: 91 nmol/l).3.For several partial agonists and antagonists (cirazoline, methoxamine, indanidine (Sgd 101-75), oxymetazoline and phentolamine) no such distinct temperature- and GTP-shifts could be demonstrated suggesting a different kind of interaction with al-binding sites.4.When temperature was changed during incubation with adrenaline, a rise of temperature (from 0°C to 30°C) converted high affinity sites into the low affinity form, whereas a decrease in temperature (from 30°C to 0°C) failed to induce the high affinity state for agonists. Short term incubation (0.5 min) with adrenaline at 30°C resulted in significantly lower IC50 values as compared to equilibrium conditions at the same temperature.5.Occupancy of al-adrenoceptor binding sites with adrenaline at O°C but not at 30°C rather closely paralleled the concentration-response curve for the α1-mediated increase in ontractile force.6.Irreversible blockade of α1-adrenoceptors with phenoxybenzamine decreased the maximum inotropic response but only slightly affected pD2 values for the α1-stimulant effect of adrenaline. Our binding experiments suggest that myocardial α1-adrenoceptors can exist in states of different affinity for agonists. Some agonists like adrenaline and phenylephrine seem to induce a temperature-dependent change in the conformation of the receptor which may represent a rapid form of desensitization. Since no appreciable receptor reserve was detected it is hypothesized that the high affinity state which can be measured at 0°C under equilibrium conditions may be relevant to the initiation of the functional response.