Bank, She eld, ST10 2TN and 4 8 Palace Garden, Royston, Hertfordshire, S68 5AD1 It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the a 1A -adrenoceptor-mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. 2 The present study describes the a 1 -adrenoceptor (a 1 -AR) subtype selectivities of two novel a 1 -AR antagonists, Ro 70-0004 (aka RS-100975) and a structurally-related compound RS-100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second-messenger studies in intact CHO-K1 cells expressing human cloned a 1A -, a 1B -and a 1D -AR showed nanomolar a nity and signi®cant a 1A -AR subtype selectivity for both Ro 70-0004 (pK i 8.9: 60 and 50 fold selectivity) and RS-100329 (pK i 9.6: 126 and 50 fold selectivity) over the a 1B -and a 1D -AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. 3 Noradrenaline-induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70-0004 (pA 2 8.8 and 8.9), RS-100329 (pA 2 9.2 and 9.2), tamsulosin (pA 2 10.4 and 9.8) and prazosin (pA 2 8.7 and 8.3 respectively). A nity estimates for tamsulosin and prazosin in antagonizing a 1 -AR-mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70-0004 and RS-100329 were approximately 100 fold less potent (pA 2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). 4 The a 1A -AR subtype selectivity of Ro 70-0004 and RS-100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a`uroselective' agent for the treatment of symptoms associated with benign prostatic hyperplasia.

1 The present study characterizes and classifies ax-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned al-adrenoceptor subtypes, native aIA-adrenoceptors in submaxillary gland of rat, and MIA-adrenoceptors in several other tissues of rat).2 Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating al-adrenoceptor heterogeneity. The al-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this aI-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 JLM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil.3 The following antagonists interacted competitively with noradrenaline at the al-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3)>prazosin (9.5)--HV 723 (9.3) -5-methyl-urapidil (9.2)>phentolamine (8.6)>spiperone (pA2 = 8.1)toxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)-and R(-)-niguldipine, the S(+ )-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with a,-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB =9.0). Thus, the results with antagonists define the aI-adrenoceptor as an cXIA-adrenoceptor. 4 An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the aIA-adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1)t adrenaline (2)>noradrenaline (5) 6 The present study demonstrates that the MlA-adrenoceptor is the predominant a-adrenoceptor subtype mediating vasoconstrictor responses to exogenously administered noradrenaline in the isolated perfused kidney of rat. More importantly, OIA-adrenoceptors mediating vasoconstrictor responses to noradrenaline exhibited a pharmacological equivalency to the cloned bovine xic-adrenoceptor. Thus, definitive functional pharmacological data are provided for equating the two receptors and support results derived recently from molecular and radioligand binding studies.

1 Experiments were undertaken to characterize the ml-adrenoceptor subtype mediating vasoconstrictor responses to periarterial noradrenergic nerve stimulation (PNS) in the isolated perfused kidney of the rat.2 Vasoconstrictor responses to nerve stimulation were inhibited by prazosin (10 nM), 5-methyl-urapidil (30 nM), and nitrendipine (1 tM) but were resistant to inhibition by chloroethylclonidine (1001M). 3 5-Methyl-urapidil (30 nM), chloroethylclonidine (100 gM) and nitrendipine (1 pLM) did not inhibit the neuronal release of tritium from nerves loaded with [3H]-noradrenaline. 4 The results suggest that renovascular xA-adrenoceptors receive a noradrenergic innervation and that the innervated receptors are coupled to dihydropyridine-sensitive calcium channels.

Novel arylpiperazines were identified as alpha 1-adrenoceptor (AR) subtype-selective antagonists by functional in vitro screening. 3-[4-(ortho-Substituted phenyl)piperazin-1-yl]propylamines were derivatized with N,N-dimethyl anthranilamides, nicotinamides, as well as carboxamides of quinoline, 1,8-naphthyridine, pyrazolo[3,4-b]pyridine, isoxazolo[3,4-b]pyridine, imidazo[4,5-b]pyridine, and pyrazolo[1,5-a]pyrimidines. Strips of rabbit bladder neck were employed as a predictive assay for antagonism in the human lower tract. Rings of rat aorta were used as a "negative screen" for the test antagonists. Binding to alpha 1-ARs was relatively sensitive to size and electronic features of the arylpiperazine portion of the antagonists and permissive to these features on the heteroaryl carboxamide side. These structure-affinity findings were exploited to produce nicotinamides (e.g. 13ii and 25x) and pyrazolo[3,4-b]pyridines (e.g. 37f and 37y) ligands with nanomolar affinity at the alpha 1-AR subtype prevalent in the human lower urinary tract(pA2 values: 8.8, 10.7, 9.3, and 9.9, respectively) and displaying 2-3 orders of magnitude selectivity over the alpha 1D-AR.

1 The a 1 -adrenoceptor population mediating contractile responses to noradrenaline (NA) in smooth muscles of the bladder neck from rabbit (RBN) has been characterized by use of quantitative receptor pharmacology. 2 Experiments with several`key' a 1 -adrenoceptor antagonists of varying subtype selectivities (RS-17053, BMY 7378, indoramin, 5-methylurapidil, prazosin, REC 15/2739, SNAP 5089, terazosin, WB 4101, tamsulosin, (+)-cyclazosin and RS-100329) were conducted. Schild regression analyses yielded a nity (mean pK b ) estimates of 7. 1, 6.2, 8.6, 8.6, 8.4, 9.3, 7.0, 7.4, 8.9, 10.0, 7.1 and 9.3, respectively, although deviations from unit Schild regression slope question the robustness of data for RS-17053 and SNAP 5089. 3 The nature of antagonism by these agents and the pro®le of a nity determinations generated together suggest that a single a 1 -adrenoceptor subtype mediates contractile responses of RBN to NA. Additional studies with phenylephrine indicated also an agonist-independence of this pro®le. Pharmacologically, this pro®le was reminiscent of that described as`a 1L '-adrenoceptor, which has been shown to mediate contractions of several tissues including lower urinary tract (LUT) tissues of man. Furthermore, a similarity was noticed between the`a 1L '-adrenoceptor described here in RBN and the rabbit and human cloned a 1a -adrenoceptor (based on data from both whole cell radioligand binding at 378C and [ 3 H]-inositol phosphates accumulation assays), characterizations of which have been published elsewhere. 4 In conclusion, the RBN appears to provide a predictive pharmacological assay for the study of NAinduced smooth muscle contraction in LUT tissues of man.