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1 The objective was to determine the role of muscarinic receptor subtypes in mediating contraction of the human detrusor smooth muscle in vitro. 2 Contractile responses of human detrusor muscle strips to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (pirenzepine, methoctramine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), tropicamide, oxybutynin and tolterodine). Affinity estimates (pKB values) were calculated for the antagonists and correlated with values at the cloned muscarinic receptor subtypes quoted in the literature. 3 Pirenzepine, methoctramine and tropicamide drugs that have high affinities at M1, M2 and M4-receptors, respectively, all had low affinities on the human detrusor (pKB values of 6.8, 6.9 and 6.5, respectively), whilst the M3-selective antagonist 4-DAMP had a high affinity (9.5). Schild plots for all four antagonists had slopes of unity indicating an action at a single receptor. Oxybutynin and tolterodine also acted as competitive antagonists with affinity estimates of 7.6 and 8.1, respectively. 4 When the antagonist affinities obtained on the bladder were plotted against the values published for these antagonists at the cloned muscarinic receptor subtypes, the best correlations were obtained for the m3- and m5-muscarinic receptor subtypes. 5 These data suggest that direct contractile responses of the human detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M3-muscarinic receptor subtype with no contribution from the major M2-receptor population.

1. In urinary bladder, M(2)-muscarinic receptors predominate, but it is the smaller population of M(3)-receptors which mediate detrusor contraction. This study examines the M(2) : M(3) ratio and the role of M(2)-receptors in contraction of pig urinary bladder. 2. Competition experiments with [(3)H]-QNB determined the ratio of M(2) : M(3). In functional studies, affinity values (pK(B)) for 4-DAMP, darifenacin and methoctramine were calculated. Similar experiments were performed on tissues following selective M(3)-inactivation (incubation with 40 nM 4-DAMP mustard in the presence of 1 microM methoctramine to protect M(2)-receptors), precontraction with 50 mM KCl and relaxation with isoprenaline (30 microM) or forskolin (1 microM). 3. In competition binding, displacement of [(3)H]-QNB by 4-DAMP, darifenacin and methoctramine best fitted a two-site model suggesting a predominant (70 - 80%) population of M(2)-receptors. 4. On normal detrusor in vitro, 4-DAMP and methoctramine caused surmountable antagonism of responses to carbachol with pK(B) values of 9.37+/-0.07 and 6.05+/-0.05 respectively. Darifenacin caused unsurmountable antagonism, the apparent pK(B) value being 8.61+/-0.10. 5. In tissues where the M(3)-receptors had been inactivated and cyclic AMP levels elevated, 4-DAMP and darifenacin were less potent, with apparent pK(B) values of 8.72+/-0.08 and 6.74+/-0.07. In contrast, methoctramine was more potent, the apparent pK(B) value increasing significantly to 6.86+/-0.06. 6. se data suggest that the pig bladder possesses a similar muscarinic receptor population to the human bladder and that the M(3)-receptor subtype mediates contraction of the normal detrusor muscle. However an involvement of M(2)-receptors in contraction can be observed following pharmacological manipulation of the receptor population.

1. The objective of the study was to determine the role of muscarinic receptor subtypes in mediating contraction of the porcine detrusor smooth muscle in vitro. 2. Strips of pig detrusor muscle were set up in physiological salt solution and the tensions developed by the tissues were recorded. Responses to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (4-DAMP, methoctramine, darifenacin, oxybutynin, tolterodine and pirenzepine). Antagonist affinity values (pKB values) were calculated and compared with those quoted in the literature for these antagonists at each of the muscarinic receptor subtypes. 3. The M3-selective antagonists, 4-DAMP and darifenacin had high affinities (pKB values of 9.4 and 8.6, respectively). Oxybutynin, tolterodine and pirenzepine had affinities of 8.2, 8.1 and 6.8, respectively, whilst the M2-selective agent methoctramine had a relatively low affinity (pKB = 6.1). The rank order of affinities was, therefore, 4-DAMP > darifenacin > oxybutynin > tolterodine > pirenzepine > methoctramine for the pig detrusor. Correlation of the antagonist affinities obtained on the bladder with those published for these antagonists at the five muscarinic receptor subtypes identified the M3(m3)-receptor as the muscarinic subtype mediating detrusor contractile responses in vitro. 4. These data suggest that a small population of M3-muscarinic receptors must mediate direct contractile responses of the pig detrusor muscle to muscarinic receptor stimulation in vitro.

1 The present study has investigated the alpha1-adrenoceptor subtype mediating contraction of the rat isolated prostatic vas deferens and the possible effector mechanisms involved in this response by use of functional experiments. 2 Contractions to noradrenaline in the rat isolated prostatic vas deferens were antagonized by prazosin (9.4, 1.04+/-0.19, pA2 and Schild plot slope), 5-methyl urapidil (8.9, 1.10+/-0.13), BMY 7378 (6.4, 1.53+/-0.07) and RS 17053 (8.3, 1.13+/-0.18). These affinities are consistent with the response being mediated by the alpha1A-adrenoceptor subtype. 3 The contraction to noradrenaline at 37 degrees C consisted of an initial phasic response, composed of many rhythmic contractile spikes and a more slowly developing tonic contraction. When the temperature was lowered to 25 degrees C the phasic contraction became a smooth single response which was increased in magnitude. 4 In Ca2+-free Krebs solution the tonic contraction to noradrenaline (10(-4) M) was abolished, suggesting that this response was dependent on influx of extracellular Ca2+. After 2 min in Ca2+-free Krebs solution at 37 degrees C and 25 degrees C the phasic response to noradrenaline (10(-4) M) was 38+/-2% and 91+/-4%, respectively, compared with the phasic contraction to noradrenaline (10(-4) M in normal Krebs solution) and after 30 min it was abolished at 37 degrees C and was 7+/-1% at 25 degrees C. Ryanodine abolished the noradrenaline response in Ca2+-free Krebs solution for 2 min at 25 degrees C, while cyclopiazonic acid reduced it to 36+/-2%. 5 In normal Krebs solution at 25 degrees C the protein kinase C inhibitor calphostin C reduced the tonic contraction to noradrenaline (10(-5) M) from 36+/-8% to 14+/-3% compared with the phasic contraction to noradrenaline (10(-4) M). The DAG kinase inhibitor R 59022 increased the contraction following the initial phasic response to a maximum of 107+/-17% after 35 s, before dropping down to a well maintained contraction which was still greater in magnitude compared with the control. Nifedipine (3x10(-7) M) reduced the tonic contraction from 49+/-6% to 7+/-1% but did not reduce the phasic response. Ryanodine (10(-4) M) reduced the phasic contraction from 50+/-2% to 7+/-1% and the tonic response from 47+/-5% to 27+/-5%. 6 The phorbol ester phorbol-12,13-dibutyrate at 25 degrees C produced a transient contraction of the rat prostatic vas deferens, maximum response (10(-5) M) 48+/-4%, compared with the maximum tonic response to noradrenaline. The contraction to PDBu (10(-5) M) was reduced to 23+/-2% by calphostin C (10(-6) M) and to 15+/-1% by nifedipine (3x10(-7) M) and was abolished after 2 min in Ca2+-free Krebs solution. 7 In conclusion, the alpha1A-adrenoceptor mediated contraction to noradrenaline of the rat prostatic vas deferens appears to consist of an initial phasic component due to the release of intracellular Ca2+ from ryanodine-sensitive stores. These stores are depleted in the absence of extracellular Ca2+ and this depletion is slower at 25 degrees C than at 37 degrees C. The phasic contraction is followed by a tonic contraction involving activation of protein kinase C by diacylglycerol and influx of Ca2+ through nifedipine-sensitive channels.

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.

1. The predominant alpha 1-adrenoceptor mediating contractions of the human vas deferens has been characterised in vitro by use of subtype selective antagonists. 2. Responses of human epididymal vas deferens were obtained to phenylephrine in the presence of amine uptake inhibitors and propranolol. The effects of the alpha 1-adrenoceptor antagonists, 5-methylurapidil, oxymetazoline, WB4101, prazosin and chloroethylclonidine were examined and also the L-type calcium channel blocker, nifedipine. 3. 5-Methylurapidil, WB4101, oxymetazoline and prazosin acted as competitive antagonists of the responses to phenylephrine, yielding pA2 values of 8.8, 9.2, 7.7 and 8.8 respectively. All four antagonists produced Schild plots with slopes similar to unity and maximum responses to phenylephrine were not altered in the presence of any of the antagonists. 4. Tamsulosin (1 nM) caused rightward shifts of phenylephrine concentration-response curves yielding an apparent pKB value of 10.0. However, maximum responses were also reduced by 51% with this concentration of antagonist. 5. Incubation of tissues with chloroethylclonidine (100 microM for 40 min) failed to alter responses significantly but the presence of nifedipine (1 microM) reduced maximum responses to phenylephrine by 32%. 6. The high affinity of 5-methylurapidil, oxymetazoline and WB4101, together with the failure of chloroethylclonidine to antagonize responses, indicate that the predominant alpha 1-adrenoceptor mediating contraction of the human vas deferens has the characteristics previously described for the pharmacologically-defined alpha 1A-adrenoceptor. The data are also consistent with those described for the cloned alpha 1c-adrenoceptor subtype thereby supporting the hypothesis that the two receptors are identical. The human vas deferens therefore represents a readily accessible preparation for functional studies of the human alpha 1A-adrenoceptor.

Benign prostatic enlargement is a common cause of bladder outlet obstruction. Recent work has demonstrated the important role played by the sympathetic nervous system in the control of prostatic muscle tone. Although isometric muscle strip studies and clinical trials have highlighted the influence of alpha-1 adrenoceptors, radioisotope ligand binding studies have demonstrated a relatively increased density of alpha-2 adrenoceptors in the muscle within prostatic tissue, the significance of which is as yet unexplained. Forty patients entered a study using pharmacological muscle strip experiments, radioligand binding assays and receptor autoradiography. Pharmacological data from these studies confirmed that contraction of prostatic muscle is mediated predominantly by alpha-1 adrenoceptor stimulation, with no evidence of significant alpha-2 adrenoceptor or cholinergic mediated effects. Radioligand binding studies confirmed that there is a higher concentration of alpha-1 binding sites as contrasted to alpha-2 within normal prostate, but that this relationship approaches equity in adenomatous prostate. Autoradiographic localisation demonstrated that alpha-1 adrenoceptor binding is predominant within prostatic stroma with only a small component of alpha-2 adrenoceptors in this compartment. This comprehensive study supports the suggestion that prostatic muscular contraction is controlled by the influence of the sympathetic nervous system acting via alpha-1 adrenoceptors. These findings support the therapeutic use of specific alpha-1 adrenoceptor blockade in the management of benign prostatic hyperplasia.

Authors from Sheffield and the USA investigated the role of neurokinin receptors in mediating contraction of detrusor muscle strips from human and pig. They found that NK-2 receptors subtypes mediate neurokinin A-induced contraction of the detrusor muscle, and that the NK-3 receptor appears not to have such a role. Another study, from a variety of European and American centres, determined the expression and plasma membrane location of isoforms of Na,K-ATPase in the rat ventral prostate; they were localised in the lateral and basolateral plasma membrane domain prostatic epithelial cells, and the authors draw conclusions about the significance of these findings. Finally authors from Yale University investigated the effects of castration on the expressions of endothelins, endothelin receptors and converting enzyme-1 in the rat seminal vesicle. Their findings led them to suggest that testosterone has a regulatory role in the expression of the endothelin receptor system in the rat seminal vesicle. To investigate the role of neurokinin (NK)-2 and -3 receptors in mediating the contraction of detrusor muscle strips from human and pig, to determine whether the pig is a good model for the study of tachykinin receptors in the human bladder, as the biological actions of tachykinins, e.g. substance P and NKA are mediated via three distinct receptor subtypes, NK-1, -2 and -3. Strips of detrusor muscle were obtained from the bladder dome and neck of female pigs and from patients undergoing cystectomy. Cumulative concentration-response curves to NKA were obtained in the absence and presence of either the NK-2 receptor-selective antagonist SR48968 or the NK-3 receptor-selective antagonist SB223412. NKA produced concentration-dependent contractions in the human and pig detrusor muscle; the curves were shifted to the right by SR48968, with high affinity (pKB 8.9, 8.3 and 8.0 in the human, pig dome and pig neck, respectively), whereas SB223412 had a minimal effect (pKB 5.8, 5.8 and 6.3, respectively). These data confirm that the NK-2 receptor subtype mediates NKA-induced contraction of the human and pig detrusor muscle. The NK-3 receptor appears to have no role in detrusor contraction of either species. The results also provide evidence that the NK-2 receptor in human and pig are the same, and the latter may be an appropriate species to study tachykinin-induced contractions in human bladder. [ABSTRACT FROM AUTHOR]

Summary 1 The objective was to determine the role of muscarinic receptor subtypes in mediating contraction of the human detrusor smooth muscle in vitro . 2 Contractile responses of human detrusor muscle strips to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (pirenzepine, methoctramine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), tropicamide, oxybutynin and tolterodine). Affinity estimates (pK B values) were calculated for the antagonists and correlated with values at the cloned muscarinic receptor subtypes quoted in the literature. 3 Pirenzepine, methoctramine and tropicamide drugs that have high affinities at M1 , M2 and M4 -receptors, respectively, all had low affinities on the human detrusor (pK B values of 6.8, 6.9 and 6.5, respectively), whilst the M3 -selective antagonist 4-DAMP had a high affinity (9.5). Schild plots for all four antagonists had slopes of unity indicating an action at a single receptor. Oxybutynin and tolterodine also acted as competitive antagonists with affinity estimates of 7.6 and 8.1, respectively. 4 When the antagonist affinities obtained on the bladder were plotted against the values published for these antagonists at the cloned muscarinic receptor subtypes, the best correlations were obtained for the m3- and m5-muscarinic receptor subtypes. 5 These data suggest that direct contractile responses of the human detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M3 -muscarinic receptor subtype with no contribution from the major M2 -receptor population. [ABSTRACT FROM AUTHOR]