Buspirone functionally discriminates tissues endowed with alpha1-adrenoceptor subtypes A, B, D and L.

The affinity for functional alpha1-adrenoceptor subtypes of buspirone in comparison with its close structural analogs and selective alpha1D-adrenoceptor antagonists, BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]dec ane-7,9-dione) and MDL 73005EF (8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro+ ++[4.5]decane-7,9-dione), was determined, namely at subtype A in rat vas deferens and perfused kidney, at subtype B in guinea-pig and mouse spleen, at subtype L in rabbit spleen, and at subtype D in rat aorta and pulmonary artery against noradrenaline-evoked contractions. BMY 7378 and MDL 73005EF were confirmed as 30- and 20-fold selective antagonists, respectively, for alpha1D- over both alpha1A- and alpha1B-adrenoceptors. Buspirone was a weak antagonist without intrinsic activity at alpha1A-adrenoceptors in rat vas deferens (pA2 = 6.12), at alpha1B-adrenoceptors in guinea-pig and mouse spleen (pA2 = 5.54 and 5.59) and at alpha1L-adrenoceptors in rabbit spleen (pA2 = 4.99), but caused partial vasoconstriction in rat kidney that was attenuable by the subtype D-selective adrenoceptor antagonist BMY 7378, but hardly by the subtype A-selective adrenoceptor antagonist B8805-033 ((+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-be nzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedion e), confirming the additional presence of alpha1D-adrenoceptors mediating rat renal vasoconstriction. Buspirone behaved as a partial agonist at alpha1D-adrenoceptors in rat aorta (pD2 = 6.77, intrinsic activity (i.a.)= 0.40) and pulmonary artery (pD2 = 7.16, i.a. = 0.59). With buspirone as agonist in these tissues, the pA2 values of subtype-discriminating antagonists were consistent with their alpha1D-adrenoceptor affinity determined in rat aorta against noradrenaline and with published binding data on cloned alpha1d-adrenoceptors. The results provide pharmacological evidence that (1) in functional preparations for the A subtype, like rat vas deferens and perfused kidney, for the B subtype, like guinea-pig and mouse spleen, and for the L subtype, like rabbit spleen, buspirone is a weak antagonist without intrinsic activity, but (2) behaves as a partial agonist in rat aorta and pulmonary artery as models for the D subtype and (3) detects an additional vasoconstrictor alpha1D-adrenoceptor in rat kidney. Buspirone, like its close analogs BMY 7378 and MDL 73005EF, thus might also be a useful tool for functionally discriminating alpha1D- from alpha1A-, alpha1B- and alpha1L-adrenoceptors in various tissues.     

Characterization of alpha 1 D-adrenoceptor subtype in rat myocardium, aorta and other tissues.

1. This study was done to characterize the functional role of alpha 1D-adrenoceptors in rat myocardium, aorta, spleen, vas deferens and prostate by use of the selective antagonist BMY 7378. 2. BMY 7378 inhibited [3H]-prazosin binding to aortic membranes with a potency (pKi 9.8 +/- 0.40) approximately 100 fold higher than in right ventricular membranes (pKi 7.47 +/- 0.11) and approximately 1,000 fold higher than that in plasma membranes of the prostate (pKi 6.62 +/- 0.39), vas deferens (pKi 6.67 +/- 0.15), salivary gland (pKi 6.46 +/- 0.38) and liver (6.58 +/- 0.06). 3. BMY 7378 antagonized the positive inotropic effects of phenylephrine (in the presence of 1 microM propranolol) on right ventricles (pA2 7.0 +/- 0.11), left atria (pKB 7.04 +/- 0.18) and papillary muscles (pKB 6.9 +/- 0.1) and inhibited phenylephrine-induced increase in inositol phosphates. 4. BMY 7378 was approximately 100 fold more potent as an antagonist of phenylephrine on aortic strips (pA2 9.0 +/- 0.13) than on vas deferens (pKB 7.17 +/- 0.08) and spleen (pKB 7.16 +/- 0.21); it was ineffective on the prostate. 5. Chloroethylclonidine suppressed the maximal effects of phenylephrine on spleen; 5-methylurapidil antagonized the effects of phenylephrine on aortic strips (pA2 7.98 +/- 0.08), vas deferens (pKB 8.89 +/- 0.07) and prostate (pKB 8.85 +/- 0.21). 6. BMY 7378 caused a dose (0.1-100 nmol kg-1)-dependent decrease in mean blood pressure of urethane-anaesthetized rats and its hypotensive efficacy was equal to that of hexamethonium. 7. The data suggest that alpha 1D-adrenoceptors play a significant role in rat aorta, a minor role in the heart, vas deferens and spleen and virtually no role in the prostate.     

Different subtypes of alpha 1A-adrenoceptor mediating contraction of rat epididymal vas deferens, rat hepatic portal vein and human prostate distinguished by the antagonist RS 17053.

1. The alpha 1-adrenoceptor subtype mediating contraction of the rat hepatic portal vein to phenylephrine was characterized by use of competitive antagonists previously shown to have selectivity between the expressed alpha 1-subtype clones. Prazosin competitively antagonized the phenylephrine contractions with a pA2 value of 9.2, as did WB 4101 (pA2 9.4), 5-methyl urapidil (pA2 8.6), indoramin (pA2 8.4) and BMY 7378 (pA2 6.5). 2. The pA2 values on the rat portal vein correlated highly with their previously published pA2 values for the alpha 1A-adrenoceptors mediating contraction of the rat epididymal vas deferens and human prostate and poorly with those for the alpha 1B- and alpha 1D-adrenoceptors mediating contraction of the rat spleen and aorta, respectively. The antagonist pA2 values on the rat portal vein correlated highly with their previously published pK1 values for the expressed alpha 1a-clone and poorly with those for the expressed alpha 1b- and alpha 1d-clones. Therefore the results show that contraction of the rat portal vein to phenylephrine is mediated by alpha 1A-adrenoceptors. 3. The novel alpha 1-adrenoceptor antagonist RS 17053 had a relatively high affinity for the alpha 1A-adrenoceptors mediating contraction of the rat epididymal vas deferens (pA2 9.5) compared with the alpha 1B-adrenoceptors in the rat spleen (pA2 7.2) or the alpha 1D-adrenoceptors in the rat aorta (pKB 7.1), in agreement with its selectivity for the expressed alpha 1a-clone. However, RS 17053 had over 100 fold lower affinity for the alpha 1A-adrenoceptors mediating contraction of the rat portal vein (pKB 7.1) and human prostate (pKB 7.1) compared with its affinity for the alpha 1A-adrenoceptors in the rat epididymal vas deferens or the expressed alpha 1a-clone. 4. The difference in affinity of RS 17053 between the rat epididymal vas deferens and rat portal vein cannot be explained by a species difference in the receptor. Therefore RS 17053 may distinguish between subtypes of the alpha 1A-adrenoceptor in the rat portal vein and human prostate compared with those in the rat epididymal vas deferens or the expressed alpha 1a-clone.     

Differential antagonism by conotoxin rho-TIA of contractions mediated by distinct alpha1-adrenoceptor subtypes in rat vas deferens, spleen and aorta

The ability of the conotoxin rho-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha1A-adrenoceptors in rat vas deferens, alpha1B-adrenoceptors in rat spleen and alpha1D-adrenoceptors in rat aorta, and to inhibit the binding of [125I]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha1-adrenoceptors was investigated. rho-TIA (100 nM to 1 microM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA2 approximately 7.2, n=4). This suggests that rho-TIA is a competitive antagonist of alpha1A- and alpha1D-adrenoceptors with no selectivity between these subtypes. Incubation of rho-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that rho-TIA is a non-competitive antagonist at alpha1B-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of rho-TIA in inhibiting contractions was examined with similar occupancies (approximately 25%) at each subtype. Its potency (pIC50) was 12 times higher in spleen (8.3+/-0.1, n=4) than in vas deferens (7.2+/-0.1, n=4) or aorta (7.2+/-0.1, n=4). In radioligand binding assays, rho-TIA decreased the number of binding sites (B(max)) in membranes from HEK293 cells expressing the rat alpha1B-adrenoceptors without affecting affinity (K(D)). In contrast, in HEK293 cells expressing rat alpha1A- or alpha1D-adrenoceptors, rho-TIA decreased the K(D) without affecting the B(max). It is concluded that rho-TIA will be useful for distinguishing the role of particular alpha1-adrenoceptor subtypes in native tissues.     

Effects of castration on alpha 1-adrenoceptor subtypes in the rat aorta

The expression of alpha 1-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the alpha 1-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the alpha 1B/alpha 1D-adrenoceptor alkylating agent chloroethylclonidine resulted in approximately 1600-fold rightward shift in the concentration-response curves to noradrenaline. The pA2 values found for WB 4101, benoxathian (alpha 1A-selective) and BMY 7378 (alpha 1D-selective) indicate that alpha 1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pKB estimated through the lowest effective concentrations of the alpha 1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pKB estimated in aorta from control rats is consistent with the interaction with alpha 1D-adrenoceptors (7.58 +/- 0.06), while that calculated in organs from control rats is consistent with alpha 1A-adrenoceptors (8.76 +/- 0.09). These results suggest that castration induces plasticity in the alpha 1-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.     

Investigation of the subtypes of alpha1-adrenoceptor mediating contractions of rat vas deferens

1 The subtypes of alpha1-adrenoceptor mediating contractions of rat vas deferens to endogenous and exogenous noradrenaline and to the exogenous agonists methoxamine, phenylephrine and A61603 have been examined. 2 The effects of antagonists on the shape of concentration-response curves, both tonic and phasic, to the four agonists were analysed. Prazosin produced parallel shifts in all cases. Particularly for RS 17053 against noradrenaline, there was some evidence for a resistant component of the agonist response. High concentrations of RS 17053 (1-10 microM) virtually abolished tonic contractions but phasic contractions were resistant. 3 A series of nine antagonists (the above and WB4101, benoxathian, phentolamine, BMY 7378, HV 723, spiperone) were investigated against contractions to noradrenaline. The correlation with the potency of the series of alpha1-adrenoceptor antagonists against contractions to noradrenaline was significant only for the alpha1A-adrenoceptor ligand binding site (r=0.88, n=9, P<0.01). 4 In epididymal portions (nifedipine 10 microM), the isometric contraction to a single electrical pulse is alpha1-adrenoceptor mediated. The correlation with ligand binding sites for 11 antagonists (the above plus ARC 239 and (+)-niguldipine) was significant only for the alpha1D-adrenoceptor subtype (r=0.65, n=11, P<0.05). 5 In conclusion, tonic contractions of rat vas deferens produced by exogenous agonists are mediated predominantly by alpha1A-adrenoceptors, although a second subtype of receptor may additionally be involved in phasic contractions. Nerve-stimulation evoked alpha1-adrenoceptor mediated contractions seem to predominantly involve non-alpha1A-adrenoceptors, and the receptor involved resembles the alpha1D-receptor.     

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of alpha1-adrenoceptor subtypes in rabbit aorta and arteries: regional difference and co-existence.

This study was done to determine the alpha1-adrenoceptor subtypes and to characterize the functional role of alpha1D-adrenoceptors in the following rabbit arteries: thoracic and abdominal aorta, mesenteric, renal and iliac arteries. In all arteries, selective alpha1D-adrenoceptor antagonist BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspirol(4,5) decane-7,9-dione dihydrochloride) dose dependently shifted the concentration-response curves for norepinephrine to the right. Schild plots of the results obtained from the inhibition by BMY 7378 for norepinephrine yielded a straight line with a slope of unity in thoracic (pA2 6.54+/-0.02) and abdominal (pA2 6.73+/-0.03) aorta. Slopes of Schild plots obtained from the inhibition by BMY 7378 for norepinephrine were significantly different from unity in mesenteric, renal and iliac arteries. Slopes of Schild plots for BMY 7378 were not different from unity in chloroethylclonidine-treated thoracic (pA2 6.49+/-0.14) and abdominal (pA2 6.61+/-0.11) aorta. Slopes of Schild plots for BMY 7378 were significantly different from unity in chloroethylclonidine-treated mesenteric, renal and iliac arteries. On the other hand, in Ca2+-free physiological saline solution (Ca2+-free PSS) slopes obtained from Schild plots for BMY 7378 were not different from unity in thoracic (pA2 6.41+/-0.09) and abdominal (pA2 6.28+/-0.07) aorta and mesenteric (pA2 6.55+/-0.06), renal (pA2 6.24+/-0.10) and iliac (pA2 6.64+/-0.13) arteries. BMY 7378 inhibited [3H]prazosin binding to thoracic (pKi 6.44+/-0.08) and abdominal (pKi 6.59+/-0.02) aorta with low potency, and mesenteric (pKi High 8.66+/-0.28, pKi Low 6.34+/-0.14), renal (pKi High 8.71+/-0.33, pKi Low 6.45+/-0.03) and iliac artery (pKi High 8.60+/-0.24, pKi Low 6.56+/-0.13). These results suggest that alpha1D-adrenoceptors play a significant role for contractile responses in renal and iliac artery, but play virtually no role in thoracic and abdominal aorta and that an alpha1-adrenoceptor subtype, which is pharmacologically distinguishable from the alpha1A-, alpha1B- and alpha1D-adrenoceptor subtype, may co-exist in mesenteric artery.     

The alpha (1D)-adrenoceptor antagonist BMY 7378 is also an alpha (2C)-adrenoceptor antagonist

1 We have investigated the actions of the alpha(1D)-adrenoceptor selective antagonist BMY 7378 in comparison with yohimbine at alpha(1)- and alpha(2)-adrenoceptors. 2 In rat aorta (alpha(1D)-adrenoceptor), BMY 7378 (pA(2) of 8.67) was about 100 times more potent than yohimbine (pA(2) of 6.62) at antagonizing the contractile response to noradrenaline. 3 In human saphenous vein (alpha(2C)-adrenoceptor), BMY 7378 (pA(2) of 6.48) was approximately 10 times less potent than yohimbine (pA(2) of 7.56) at antagonizing the contractile response to noradrenaline. 4 In prostatic portions of rat vas deferens, BMY 7378 (10 mum) did not significantly affect the concentration-dependent inhibition of single pulse nerve stimulation-evoked contractions by xylazine (an action at prejunctional alpha(2D)-adrenoceptors). 5 In ligand-binding studies, BMY 7378 showed 10-fold selectivity for alpha(2C)-adrenoceptors (pK(i) of 6.54) over other alpha(2)-adrenoceptors. 6 It is concluded that BMY 7378, in addition to alpha(1D)-adrenoceptor selectivity in terms of alpha(1)-adrenoceptors, shows selectivity for alpha(2C)-adrenoceptors in terms of alpha(2)-adrenoceptors.     

Existence of functional alpha1A- and alpha1D- but no alpha1B-adrenoceptor subtypes in rat common carotid arteries

Using the cannula inserting method, vasoconstrictor responses to alpha1-adrenoceptor agonists (noradrenaline [NA], phenylephrine [PE] and methoxamine [ME]) and effects of alpha1-adrenoceptor antagonists (WB4101, chloroethylclonidine [CEC] and BMY7378) were investigated in isolated and perfused rat common carotid arteries. The rank order of agonist potency and efficacy was NA = PE > ME. Either WB4101 or BYM7378 inhibited NA- and PE-induced constrictions in a dose-related manner. CEC did not inhibit the NA- and PE-induced responses. The ME-induced responses were also significantly blocked by either WB4101 or BMY7378. From these results, it is concluded that there are functional alpha1A- and alpha1D-adrenoceptor subtypes in rat common carotid arteries, but no functional alpha1B subtype.     

介导大鼠肠系膜动脉血管床收缩反应的α1—肾上腺素受体亚型特征

目的：研究去甲肾上腺素（ＮＥ）介导大鼠肠系膜血管床（ＭＶＢ）收缩的α在－肾上腺素受体（α１－ＡＲ）亚型,方法：用灌流大鼠ＭＶＢ标本收缩功能实验和克隆细胞放射配体结合实验测定α１－ＡＲ亚型选择性拮抗剂ｐＡ２和ｐＫｉ,并作相关分析。结果：α１Ａ－ＡＲ选择性拮抗剂ＲＳ－１７０５３,ＷＢ４１０１,５－ＭＵ及α１Ｄ－ＡＲ选择性拮抗剂ＢＭＹ７３７８的ｐＡ２分别为８．９８±０．２８,９．１６±０．２０,８．６９     

Alpha(1D)-adrenoceptors mediate nerve and agonist-evoked contractions in mouse vas deferens: evidence obtained from knockout technology

1 It has been demonstrated that nerve-evoked contractions of the rat vas deferens involve alpha(1D)-adrenoceptors. Definitive evidence for a similar alpha(1D)-adrenoceptor-mediated response in mouse vas deferens has been more difficult to obtain. In this study, we have used alpha(1D)-adrenoceptor knockout (alpha(1D)-KO) mice to aid in the pharmacological characterization. 2 Mouse whole vas deferens was stimulated with a single pulse every 5 min. Once a stable response had been obtained, vehicle or antagonist was administered cumulatively at 5-min intervals and a response to stimulation obtained 5 min later. Cumulative concentration-response curves were also obtained for noradrenaline. 3 In vas deferens from alpha(1D)-KO mice, the contractile response to low concentrations of noradrenaline and the contractile response to a single stimulus were significantly reduced as compared to wild type (WT). 4 The alpha(1D)-adrenoceptor selective antagonist, BMY 7378, produced a concentration-dependent inhibition of single pulse-evoked contractions of vas deferens from WT and alpha(1D)-KO mice. BMY 7378 was significantly less potent in inhibiting stimulation-evoked contractions in vas deferens from alpha(1D)-KO mice. 5 It is concluded that alpha(1D)-adrenoceptors mediate a component of nerve- and agonist-evoked contractions of the vas deferens of WT mice.     

Analysis of alpha 1L-adrenoceptor pharmacology in rat small mesenteric artery.

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 alpha-adrenoceptor antagonist, zolertine, inhibits alpha1D- and alpha1A-adrenoceptor-mediated vasoconstriction in vitro

1. The antagonist effect of zolertine (4-phenyl-1-[2-(5-tetrazolyl)ethyl]piperazine trihydrochloride), on vascular contraction elicited by noradrenaline in aorta, carotid (alpha1D-adrenoceptors), mesenteric (alpha1A/D-adrenoceptors) and caudal arteries (alpha1A-adrenoceptors) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats and rabbit aorta (alpha1B-adrenoceptors), was investigated in endothelium-denuded arterial rings. 2. The selective alpha1D-adrenoceptor agonist, noradrenaline, elicited concentration-dependent contractions in all arterial rings from both species. Noradrenaline selectivity was: carotid = aorta > mesenteric = rabbit aorta > caudal arteries. 3. The contractile responses induced by noradrenaline were competitively antagonized by zolertine in rat carotid and aorta arteries, yielding pA2 values of WKY, 7.48 +/- 0.18; SHR, 7.43 +/- 0.13 and WKY, 7.57 +/- 0.24; SHR, 7.40 +/- 0.08, respectively. Zolertine was a non-competitive antagonist in some blood vessels as Schild plot slopes were lower than unity. The pKb estimates for zolertine were WKY, 6.98 +/- 0.16; SHR, 6.81 +/- 0.18 in the mesenteric artery, WKY, 5.73 +/- 0.11; SHR, 5.87 +/- 0.25 in the caudal artery and 6.65 +/- 0.09 in rabbit aorta. 4. Competition binding experiments using the alpha1-adrenoceptor antagonist [3H]prazosin showed a zolertine pKi of 6.81 +/- 0.02 in rat liver (alpha1B-adrenoceptors) and 6.35 +/- 0.04 in rabbit liver (alpha1A-adrenoceptors) membranes. 5. Zolertine showed higher affinity for alpha1D-adrenoceptors compared to alpha1A-adrenoceptors, while it had an intermediate affinity for alpha1B-adrenoceptors. The ability of the alpha1-adrenoceptor antagonist zolertine to block alpha1D-adrenoceptor-mediated constriction in different vessels of WKY and SHR rats may explain its antihypertensive efficacy despite its low order of potency.     

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means.     

Effects of castration and of testosterone replacement on alpha(1)-adrenoceptor subtypes in the rat vas deferens

The contractions of the rat vas deferens in response to noradrenaline are mediated through alpha(1A)-adrenoceptors. We observed participation of alpha(1B)-adrenoceptors in these contractions after castration. We now investigated the time course of this plasticity and the effects of testosterone by determining the actions of competitive antagonists on noradrenaline-induced contractions after 7, 14, 21 and 30 days of castration. BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride) antagonised noradrenaline-induced contractions in control and castrated rats with low pA(2) values (approximately = 6.8). In control vas deferens, WB 4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) had a slope in the Schild plot no different from 1.0, while slopes lower than 1.0 (approximately 0.6) were observed for vas deferens from castrated rats. Chloroethylclonidine was ineffective in the control vas while it inhibited noradrenaline-induced contractions in vasa from castrated rats and converted the complex antagonism by WB 4101 into simple competitive antagonism. Treatment of castrated rats with testosterone prevented the effects of castration. The results suggest that alpha(1B)-adrenoceptors are detectable in vas deferens from at least the 7th through the 30th day after castration and that testosterone prevents this plasticity.     

Differential response to chloroethylclonidine in blood vessels of normotensive and spontaneously hypertensive rats: role of alpha 1D- and alpha 1A-adrenoceptors in contraction

The effects of chloroethylclonidine on alpha(1)-adrenoceptor-mediated contraction in endothelium-denuded caudal arteries and aorta from normotensive Wistar and Wistar Kyoto (WKY), and from spontaneously hypertensive (SHR) rats were evaluated. Chloroethylclonidine elicited concentration-dependent contractions. Maximal contraction was similar in caudal arteries among strains ( approximately 40% of noradrenaline effect). However, chloroethylclonidine elicited a higher contraction in aorta from SHR than from normotensive rats. In Wistar aorta chloroethylclonidine produced the smallest contractile response. In SHR aorta, BMY 7378 and 5-methylurapidil blocked chloroethylclonidine-elicited contraction, while (+)-cyclazocine did not inhibit it; while in caudal arteries, 5-methylurapidil blocked chloroethylclonidine action; the other antagonists had no effect. In chloroethylclonidine-treated aorta noradrenaline elicited biphasic contraction-response curves, indicating a high affinity (pD(2), 8.5 - 7.5) chloroethylclonidine-sensitive component and a low affinity (pD(2), 6.3 - 5.2) chloroethylclonidine-insensitive component. The high affinity component was blocked by chloroethylclonidine; while in caudal arteries noradrenaline elicited monophasic contraction-response curves with pD(2) values (6.5 - 5.7) similar to the low affinity component in aorta. Chloroethylclonidine inhibition of noradrenaline response was greater in aorta than in caudal arteries. Chloroethylclonidine increased the EC(50) values of noradrenaline approximately 1000 fold in aorta and approximately 10 fold in caudal arteries. In SHR aorta BMY 7378 protected alpha(1D)-adrenoceptors and in caudal arteries 5-methylurapidil protected alpha(1A)-adrenoceptors from chloroethylclonidine alkylation, allowing noradrenaline to elicit contraction. These results show marked strain-dependent differences in the ability of chloroethylclonidine to contract aorta; moreover, chloroethylclonidine stimulates alpha(1D)-adrenoceptors in aorta and alpha(1A)-adrenoceptors in caudal arteries. The higher contraction observed in aorta from SHR and WKY suggests an augmented number of alpha(1D)-adrenoceptors in these strains.     

Functional evidence of alpha1D-adrenoceptors in the vasculature of young and adult spontaneously hypertensive rats.

The role of alpha1D-adrenoceptors in the vasculature of spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY), of different ages was assessed in pithed rats by the use of the selective alpha1D-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-8-azaspiro [4.5]decane-7,9-dione dihydrochloride). BMY 7378 displaced the pressor effect of phenylephrine in young pre-hypertensive pithed SHR rats, but produced no effect in young WKY rats (dose ratio of 3.4 and 1.6, respectively), while in adult rats BMY 7378 produced a greater shift in the phenylephrine response curve than in younger animals (dose ratio of 3.2 and 6.2 in WKY and SHR, respectively). The presence of alpha1D-adrenoceptors in the vasculature of pre-hypertensive rats, suggests its role in the pathogenesis/maintenance of increased blood pressure.     

Sympathectomy reveals alpha 1A- and alpha 1D-adrenoceptor components to contractions to noradrenaline in rat vas deferens

We have previously demonstrated that contractions of rat vas deferens to exogenous noradrenaline involve predominantly alpha(1A)-adrenoceptors, but that contractions to endogenous noradrenaline involve predominantly alpha(1D)-adrenoceptors. In this study, we have examined the effects of sympathectomy on the subtypes of alpha(1)-adrenoceptor in rat vas deferens in radioligand binding and functional studies. In vehicle-treated tissues, antagonist displacement of [(3)H]prazosin binding to alpha(1)-adrenoceptors was consistent with a single population of alpha(1)-adrenoceptors. Binding affinities for a range of alpha(1)-adrenoceptor antagonists were expressed as pK(i) values and correlated with known affinities for alpha(1)-adrenoceptor subtypes. The correlation was significant only with alpha(1A)-adrenoceptors. In tissues from rats sympathectomised with 6-hydroxy-dopamine (2 x 100 mg kg(-1) i.p.), binding affinity for the alpha(1D)-adrenoceptor antagonist BMY 7378 fitted best with a two-site model. In functional studies, the potency of noradrenaline at producing total (phasic plus tonic) but not tonic contractions was increased in tissues from sympathectomised rats. Results obtained from sympathectomised rats suggest that phasic contractions are mainly alpha(1D)-adrenoceptor mediated, whereas tonic contractions are mainly alpha(1A)-adrenoceptor mediated, based on the effects of BMY 7378 and the alpha(1A)-adrenoceptor antagonist RS 100329. It is concluded that the predominant alpha(1)-adrenoceptor in vehicle-treated rat vas deferens is the alpha(1A)-adrenoceptor, both in terms of ligand binding and contractions to exogenous agonists. The alpha(1D)-adrenoceptor is only detectable by ligand binding following chemical sympathectomy, but is involved in noradrenaline-evoked contractions, particularly phasic contractions, of rat vas deferens.     

Characterisation of α1B-adrenoceptors linked to inositol phosphate formation and calcium mobilisation in human astrocytoma U373 MG cells

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.