In vitro and in vivo uroselectivity of B8805-033, an antagonist with high affinity at prostatic alpha1A- vs. alpha1B- and alpha1D-adrenoceptors.

We have investigated the pharmacological properties of B8805-033 [(+/-)- 1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl] amino]-2,4(1H,3H)-pyrimidinedione], a new alpha1A-adrenoceptor (AR) selective antagonist. In radioligand binding studies, B8805-033 was 150- to 1200-fold selective for alpha1A-ARs (pKi rat cerebral cortex 8.70, cloned human receptor 7.71) relative to alpha1B-ARs (pKi rat cerebral cortex 5.60, rat liver 5.39, cloned human receptor 5.16) and alpha1D-ARs (pKi cloned human receptor 5.49). B8805-033 inhibited noradrenaline (NA) induced contractions mediated by alpha1A-ARs in rat vas deferens and rabbit and human prostate (pA2 7.62-8.40) much more potently than those mediated by alpha1B-ARs in guinea pig and mouse spleen or by alpha1D-ARs in rat aorta and pulmonary artery (pA2 5.21-5.52). With the exception of a high agonist affinity at 5-HT1A receptors (pKi 9.74 in pig cortex, pD2 6.82 for contraction of rabbit basilar artery) and a moderate to low affinity at histamine H1-receptors (pA2 6.74) and beta1-ARs (pA2 5.75), B8805-033 did not interact with a number of other neurotransmitter receptors (pKi or pA2<5.0). From the i.v. doses of B8805-033 to either inhibit the urethral pressure response to NA by 50% (29 nmol/kg) or to evoke a fall in diastolic blood pressure by 25% (1.54 micromol/kg) in anaesthetized dogs, an urethral/ vascular selectivity ratio of 52 was obtained, far exceeding that found for the nearly unselective prazosin (ratio 1.8). We conclude that B8805-033 is a highly alpha1A-AR selective antagonist, which may potentially be useful as pharmacological tool to investigate alpha1-AR heterogeneity and in the treatment of benign prostatic hyperplasia.

In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor.

We have investigated the bronchodilator and anti-inflammatory properties of roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-dichloropyrid-4-yl]-benzamide), a novel, highly potent, and selective phosphodiesterase 4 (PDE4) inhibitor. Additionally, we compared the effects of roflumilast and its N-oxide, the primary metabolite in vivo, with those of the PDE4 inhibitors piclamilast, rolipram, and cilomilast. Roflumilast inhibited the ovalbumin-evoked contractions of tracheal chains prepared from sensitized guinea pigs (EC(50) = 2 x 10(-7) M) but showed no relaxant effect on tissues contracted spontaneously. In spasmogen-challenged rats and guinea pigs, intravenously administered roflumilast displayed bronchodilatory activity (ED(50) = 4.4 and 7.1 micromol/kg, respectively). Furthermore, roflumilast dose dependently attenuated allergen-induced bronchoconstriction in guinea pigs (ED(50) = 0.1 micromol/kg i.v.). Roflumilast given orally (ED(50) = 1.5 micromol/kg) showed equal potency to its N-oxide (ED(50) = 1.0 micromol/kg) but was superior to piclamilast (ED(50) = 8.3 micromol/kg), rolipram (ED(50) = 32.5 micromol/kg), and cilomilast (ED(50) = 52.2 micromol/kg) in suppressing allergen-induced early airway reactions. To assess the anti-inflammatory potential of orally administered roflumilast, antigen-induced cell infiltration, total protein, and TNFalpha concentration in bronchoalveolar lavage fluid of Brown Norway rats were determined. Roflumilast and its N-oxide equally inhibited eosinophilia (ED(50) = 2.7 and 2.5 micromol/kg, respectively), whereas the reference inhibitors displayed lower potency (ED(50) = 17-106 micromol/kg). Besides, orally administered roflumilast abrogated LPS-induced circulating TNFalpha in the rat (ED(50) = 0.3 micromol/kg), an effect shared by its N-oxide, with both molecules exhibiting 8-, 25-, and 310-fold superiority to piclamilast, rolipram, and cilomilast, respectively. These results, coupled with the in vitro effects of roflumilast on inflammatory cells, suggest that roflumilast represents a potential new drug for the treatment of asthma and chronic obstructive pulmonary disease.

Failure of AH11110A to functionally discriminate between alpha(1)-adrenoceptor subtypes A, B and D or between alpha(1)- and alpha(2)-adrenoceptors.

The potency of the putatively alpha(1B)-adrenoceptor selective drug, 1-[biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol (AH11110A), to antagonize contraction upon stimulation of alpha(1A)-adrenoceptors in rat vas deferens and rat perfused kidney, alpha(1B)-adrenoceptors in guinea-pig spleen, mouse spleen and rabbit aorta, and alpha(1D)-adrenoceptors in rat aorta and pulmonary artery was evaluated and compared to that of a number of subtype-discriminating antagonists. N-[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]-3-methyl-4-oxo-2-phenyl-4H-1-benzopyran-8-carboxamide (Rec 15/2739) and (+/-)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805-033) were confirmed as selective for alpha(1A)-adrenoceptors, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY 7378), 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]-8-azaspiro[4.5]decane-7,9-dione (MDL 73005EF), and cystazosin were found to be selective for alpha(1D)-adrenoceptors, whereas spiperone was weakly selective for alpha(1B)-over alpha(1A)-adrenoceptors. However, from the functional affinity profile obtained for AH11110A at alpha(1A)-adrenoceptors (pA(2)=6.41 in rat vas deferens), alpha(1B)-adrenoceptors (pA(2)=5.40-6.54) and alpha(1D)-adrenoceptors (pA(2)=5.47-5.48), the affinity and presumed selectivity previously obtained for AH11110A in radioligand binding studies at native alpha(1B)- and cloned alpha(1b)-adrenoceptors (pK(i)=7.10-7.73) could not be confirmed. Additionally, AH11110A enhanced the general contractility of rat vas deferens, produced a bell-shaped dose-response curve of vasodilation in perfused rat kidney, and its antagonism in most other tissues was not simply competitive. The affinity of AH11110A for prejunctional alpha(2)-adrenoceptors in rabbit vas deferens (pA(2)=5.44) was not much lower than that displayed for alpha(1)-adrenoceptor subtypes, revealing that AH11110A, besides alpha(1)-adrenoceptors, also interacts with alpha(2)-adrenoceptors, and thus may be unsuitable for alpha-adrenoceptor subtype characterization, at least in smooth muscle containing functional studies.

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.

Multiple mechanisms of action: the pharmacological profile of budipine.

Four major components of the mechanism of action have been identified for the antiparkinsonian drug budipine up to now. 1) The primary action of budipine is an indirect dopaminergic effect as shown by facilitation of dopamine (DA) release, inhibition of monoamine oxidase type B (MAO-B) and of DA (re) up-take and stimulation of aromatic L-amino acid decarboxylase (AADC), which in sum might be responsible for enhancing the endogenous dopaminergic activity. 2) Radioligand and functional studies at the N-methyl-D-aspartate (NMDA) type glutamate receptor characterize budipine as a low-affinity, uncompetitive antagonist with fast kinetics and moderate voltage-dependency at the phencyclidine (PCP) binding site, comparable to that observed with amantadine, thereby counteracting an increased excitatory glutamatergic activity. 3) The antimuscarinic action of budipine, verified by functional and binding studies at native muscarinic M1-M3 and human recombinant m1-m5 receptor subtypes in vitro, is up to 125-fold weaker than that of biperiden and corresponds to its approximately 100-fold lower potency to cause experimentally-induced peripheral antimuscarinic effects and explains only part of its high potency, which equals biperiden, to suppress cholinergically evoked tremor. 4) An additional inhibition of striatal gamma-aminobutyric acid (GABA) release by budipine may be beneficial to suppress an increased striatal GABAergic output activity. The contribution of other observed effects to the therapeutic action of budipine, i.e. weak stimulation of noradrenaline and serotonin release, binding to brain sigma1 receptors and blockade of histamine H1 receptors, is not yet clear. By means of these multiple mechanisms, budipine might correct the imbalance of striatal output pathways by restoring DA levels in the striatum, and positively influence the secondary changes in other transmitter systems (glutamate, acetylcholine, GABA) observed in Parkinson's disease.

Affinity of the miotic drug, dapiprazole, at alpha 1-adrenoceptor subtypes A, B and D.

The functional affinities of the alpha 1-adrenoceptor antagonist, dapiprazole, currently being used to reverse diagnostic pupillary dilation, were determined at subtype A in rat vas deferens, at subtype B in guinea-pig spleen and at subtype D in rat aorta and compared with various alpha 1-adrenoceptor subtype-discriminating antagonists. Dapiprazole had relatively high affinity both at rat vas deferens alpha 1A-adrenoceptors (pA2 = 7.93) and at rat aortic alpha 1D-adrenoceptors (pA2 = 8.26), whereas its affinity at guinea-pig splenic alpha 1B-adrenoceptors (pA2 = 7.13) was lower. The reference antagonists, 5-methylurapidil and the 5-methylurapidil/flesinoxan hybrid, B8805-033 ((+/-)- 1,3,5-trimethyl-6[[3[4(2(2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin -5-yl)-1-piperazinyl]propyl]-amino]2,4(1H,3H)-pyrimidinedione), were 40- and 1500-fold selective for the A subtype, whereas spiperone and BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1- piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione diHCI) were confirmed as selective for the B and D subtypes of alpha 1-adrenoceptors, respectively. Thus, in functional experiments dapiprazole seems to be moderately selective (approximately 10-fold) for the A and D over the B subtype of alpha 1-adrenoceptors; the possible therapeutic consequence of this is discussed.

Contraction of guinea-pig gallbladder: muscarinic M3 or M4 receptors?

The muscarinic receptor mediating contraction of the guinea-pig isolated gallbladder, currently being disputed to belong either to the M3 or M4 subtype, was characterized by subtype-preferring agonists and discriminating antagonists. Highly significant correlations of agonist potencies to contract the gallbladder, e.g., arecaidine propargyl ester, oxotremorine, 5-methylfurtrethonium > arecoline, arecaidine 2-butyne-1,4-diyl bisester > (R)-nipecotic acid ethyl ester > 4-[[N-(4-chlorophenyl)carbamyl]oxy]-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343), (S)-nipecotic acid ethyl ester > 4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyltrimethylammonium chloride (McN-A-343) were found with muscarinic M3 receptors mediating contraction of the guinea-pig ileum and vasodilation in rat perfused kidney. Functional affinities at guinea-pig gallbladder muscarinic receptors of antagonists known to distinguish between native or cloned muscarinic M3/m3 and M4/m4 receptors, e.g., himbacine, methoctramine, mefurtramine, tripitramine, idaverine, zamifenacin and 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyr ido(2,3-b)(1,4)benzodiazepin-6-one (AQ-RA 741), were consistent with those at guinea-pig ileal muscarinic M3 receptors but not with published data at recently defined muscarinic M4 receptors in rabbit anococcygeus muscle or at muscarinic M1 and M2 receptors in rabbit vas deferens. Antagonist affinities at guinea-pig gallbladder correlated also best with published binding data on native or cloned muscarinic M3/m3 receptors but not with those for muscarinic M4/m4 receptors. The agonist potencies and antagonist affinities suggest that smooth muscle contraction elicited by muscarinic stimuli in guinea-pig gallbladder is mediated by functional muscarinic M3 receptors.

Functional evidence for an alpha 1B-adrenoceptor mediating contraction of the mouse spleen.

alpha 1-Adrenoceptor agonists ((-)-adrenaline = (-)-noradrenaline > > L-phenylephrine > methoxamine > (-)-(4a R, 10a R)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2 H-naphth[2,3-b]-1,4-oxazine (SDZ NVI 085) > cirazoline) evoked contraction of isolated mouse spleen strips, whereas oxymetazoline and indanidine were nearly inactive. Splenic contractions elicited by (-)-noradrenaline were inhibited by chloroethylclonidine (3 x 10(-6) - 6 x 10(-5) M) and partially attenuated by SZL-49 (10(-7) -10(-6) M), but remained resistant to (+/-)-isradipine (10(-9) -10(-7) M). The contractions were competitively antagonized by low concentrations of the alpha 1B-adrenoceptor-selective antagonist, spiperone (pA2 = 8.29), but by relatively high concentrations of the alpha 1A-adrenoceptor-selective receptor antagonists, tamsulosin (pA2 = 8.62), 5-methyl-urapidil (pA2 = 7.03), (+)-niguldipine (pA2 = 6.26) and the alpha 1D-adrenoceptor-selective antagonist, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro-[4.5]dec ane-7, 9-dione (BMY 7378) (pA2 = 6.76). Functional antagonist affinities at mouse spleen alpha 1-adrenoceptors were consistent with those at guinea-pig splenic alpha 1B-adrenoceptors, but not with those of either rat vas deferens alpha 1A- or rat aortic alpha 1D-adrenoceptors. Antagonist affinities at mouse spleen alpha 1-adrenoceptors correlated also best with published antagonist data on cloned and expressed alpha 1b-adrenoceptors but less well with those for either alpha 1a- or alpha 1d-adrenoceptors. The results provide pharmacological evidence that the alpha 1-adrenoceptor mediating smooth muscle contraction of mouse spleen is the B subtype.

Characterization of vascular P2 purinoceptors in the rat isolated perfused kidney.

In isolated, constant-pressure perfused rat kidneys at basal vascular tone, injected P2 purinoceptor agonists evoked vasoconstriction (alpha, beta-methylene ATP > beta, gamma-methylene ATP > ATP-gamma-S > 2-methylthio ATP > ATP > ADP = UTP). In kidneys with raised tone, the nucleotides produced vasodilatation at low doses (2-methylthio ATP > ADP = ATP = ATP-gamma-S > UTP; alpha, beta-methylene ATP and beta, gamma-methylene ATP, inactive), and constriction at high doses (alpha, beta-methylene ATP > beta, gamma-methylene ATP > ATP-gamma-S > 2-methylthio ATP > ADP = ATP > UTP). Removal of the endothelium abolished the dilator responses to the agonists. NG-Nitro-L-arginine methylester (L-NAME, 5 x 10(-5) M) abolished vasorelaxation in response to 2-methylthio ATP, a response which could be restored by additional L-arginine (3 x 10(-3) M). Both vasodilatation and constriction due to the nucleotides remained unaffected by indomethacin (3 x 10(-6) M), S-(p-nitrobenzyl)-6-thioinosine (3 x 10(-5) M) and 8-phenyltheophylline (3 x 10(-6) M). Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 1-3 x 10(-6) M), inhibited vasoconstriction caused by alpha, beta-methylene ATP, 2-methylthio ATP and UTP, but not by ATP. Suramin (3 x 10(-5) M) caused a rightward shift of the dose-response curves for constriction caused by alpha, beta-methylene ATP (27-fold) and 2-methylthio ATP (5-fold), whereas the ATP curve was shifted to the left (20-fold). With Evans blue (10(-5) M), vasodilatation due to the nucleotides was abolished and the dose-response curves for vasoconstriction caused by ATP and UTP were shifted left more than 100-fold, the effect to both could not be antagonized by PPADS (3 x 10(-6) M). These results suggest: (1) the different rank orders of P2 purinoceptor agonist potencies for constrictor and dilator responses in perfused rat kidney are consistent with mediation via P2x and P2Y purinoceptors, respectively; (2) P2X purinoceptors, selectively sensitive to blockade by PPADS, are located on vascular smooth muscle; (3) endothelial P2Y purinoceptor stimulation results in vasodilatation involving NO synthesis but not release of prostanoids; (4) Evans blue, which appears to combine selective P2Y purinoceptor blockade and strong inhibition of ecto-nucleotidases, potentiates vasoconstriction in response to the degradable nucleotides, ATP, 2-methylthio ATP and UTP; (5) additionally, Evans blue unmasks a PPADS-insensitive P2U purinoceptor where the nearly equipotent nucleotides, ATP and UTP, can produce vasoconstriction.

In functional experiments, risperidone is selective, not for the B, but for the A subtype of alpha 1-adrenoceptors.

The potency of the antipsychotic drug, risperidone, to antagonize alpha 1A-adrenoceptor-mediated contraction in rat vas deferens and vasoconstriction in rat perfused kidney, and alpha 1B-adrenoceptor-mediated contractions in spleen from guinea-pig and mouse was evaluated and compared to that of alpha 1-adrenoceptor subtype-discriminating antagonists. Prazosin was found to be unselective; 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 5-methyl-urapidil, indoramin and (+)-niguldipine were confirmed as selective for the alpha 1A-adrenoceptor, whereas spiperone was weakly alpha 1B-selective. Risperidone was equipotent to prazosin at alpha 1A-adrenoceptors in rat vas deferens and kidney. However, at guinea-pig and mouse splenic alpha 1B-adrenoceptors, the affinity values of risperidone were 10-fold lower than those of prazosin. Thus, in functional experiments the presumed high selectivity of risperidone for the B subtype of alpha 1-adrenoceptors could not be confirmed, the drug instead appears to be moderately selective (10-fold) for the A subtype.

Pathways involved in muscarinic M1 and M2 receptor stimulation in rabbit vas deferens.

Pretreatment of the field-stimulated rabbit isolated vas deferens for 30 min with LiCl (2 x 10(-2) and 4 x 10(-2) M) attenuated the inhibition of neurogenic twitch contractions due to muscarinic M1 receptor stimulation by 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343), and enhanced the muscarinic M2 receptor-mediated potentiation of contractions evoked by carbachol. When the tissues were preincubated for 5 min with the adenylate cyclase activator, forskolin (3 x 10(-8)-3 x 10(-7) M), the response to carbachol was attenuated whereas that to 4-Cl-McN-A-343 remained unchanged. 1,9-Dideoxy-forskolin (3 x 10(-7) and 10(-6) M), which fails to activate cyclase, did not abolish the carbachol effect. In addition, desensitization of the response to 4-Cl-McN-A-343 but not to carbachol occurred in preparations incubated for 90 min with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA, 3 x 10(-8)-3 x 10(-7) M), whereas its inactive 4 alpha-stereoisomer (4 alpha-PMA, 3 x 10(-7) M) was without effect. In unstimulated preparations, LiCl, forskolin and PMA did not impair contractions due to exogenous ATP (10(-3) M). These findings are consistent with the hypothesis that, in rabbit vas deferens, inhibitory muscarinic M1 receptors stimulate LiCl-sensitive phosphatidylinositol turnover (IP3 pathway) involving protein kinase C, whilst excitatory muscarinic M2 receptors are coupled to inhibition of adenylate cyclase, resulting in reduced levels of cyclic AMP.

Characterization of the alpha 1-adrenoceptor subtype mediating contraction of guinea-pig spleen.

A series of alpha 1-adrenoceptor agonists evoked concentration-dependent contraction of isolated guinea-pig spleen strips ((-)-adrenaline > (-)-noradrenaline >> L-phenylephrine > (-)-(4aR, 10aR)-3, 4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2H-naphth [2,3-b]-1,4-oxazine (SDZ NVI 085) > cirazoline), whereas indanidine, methoxamine, oxymetazoline and UK-14.304 were ineffective. (-)-Noradrenaline-induced contractions were inhibited by chloroethylclonidine (3 x 10(-6)-6 x 10(-5) M) and partially attenuated by SZL-49 (10(-7)-10(-6) M), but remained resistant to (+/-)-isradipine (10(-9)-10(-7) M). The contractions of the splenic strips were competitively antagonized by low concentrations of the alpha 1B-adrenoceptor-selective antagonist, spiperone (pA2 8.05), but by relatively high concentrations of the alpha 1A-adrenoceptor-selective antagonists, (+)-niguldipine (pA2 6.32) and 5-methyl-urapidil (pA2 6.95). The affinities of subtype-selective antagonists determined at guinea-pig spleen alpha-adrenoceptors significantly correlated with pKi values at rat liver alpha 1B binding sites (r = 0.96) and pA2 values at putative alpha 1B-adrenoceptors in rat aorta (r = 0.95), but differed from pKi values at rat cortical alpha 1A binding sites and pA2 values at alpha 1A-adrenoceptors in rat vas deferens. Also no correlation was obtained between antagonist affinities at guinea-pig spleen alpha-adrenoceptors and alpha 1C binding sites in rabbit liver. Thus, from the (1) potencies of agonists, (2) affinities of subtype-selective antagonists and (3) differential sensitivity of the contractions to alpha 1-adrenoceptor inactivating agents and their resistance to Ca2+ channel blockade, the alpha 1-adrenoceptor mediating smooth muscle contraction of guinea-pig spleen can be best characterized as being of the B subtype.

Involvement of muscarinic M2 and M3, but not of M1 and M4 receptors in vagally stimulated contractions of rabbit bronchus/trachea.

The inhibition of preganglionic and postganglionic contractions of the rabbit isolated bronchus/trachea by antagonists with selectivity for different muscarinic receptor subtypes was compared with their affinities at M1, M2, M3 and M4 receptors. Neither M1/M3 receptor-unselective antagonists (atropine, hexahydro-siladifenidol, thiazinamium, p-fluoro-hexahydro-sila-difenidol) nor antagonists with selectivity for the M1 over M3 subtype ((+)-biperiden, UH-AH 37, telenzepine, o-methoxy-sila-hexocyclium, pirenzepine) consistently showed a preferential inhibition of the response to preganglionic over postganglionic stimulation. Potencies for inhibition of contraction to preganglionic stimulation by antagonists discriminating more than threefold both between M1 and M3, and between M3 and M2 receptors (hexocyclium, (+)-biperiden, UH-AH 37, telenzepine, o-methoxy-silahexocyclium, p-fluoro-hexahydro-sila-difenidol, pirenzepine) are most consistent with affinities at smooth muscle M3 receptors as determined on methacholine-contracted rabbit trachea. Antagonists with a 10-fold higher affinity at M2 over M3 receptors enhanced contractions to field stimulation (AQ-RA 741 = AF-DX 384 = idaverine > himbacine = imperialine = AF-DX 116 = methoctramine >> gallamine), whereas antagonists with a selectivity profile of M4 > or = M3 > M2 (hexahydro-sila-difenidol, pirenzepine, dicyclomine) failed to increase the contractions. Secoverine (selectivity profile M4 > M2 > M3) enhanced contractions at concentrations consistent with its M2 receptor affinity. These results (1) exclude a ganglionic M1 receptor modulating excitatory vagal neurotransmission but (2) suggest the presence of an inhibitory prejunctional M2 rather than an M4 receptor and (3) identify a postjunctional smooth muscle M3 receptor in rabbit bronchus/trachea.

Characterization of muscarinic receptors mediating vasodilation in rat perfused kidney.

The muscarinic receptor mediating vasodilation of resistance vessels in the rat isolated, constant-pressure perfused kidney (preconstriction by 10(-7) M cirazoline) was characterized by subtype-preferring agonists and selective antagonists. The agonists produced vasodilation with the following rank order of potency: arecaidine propargyl ester (APE) > 5-methylfurtrethonium = methacholine = oxotremorine > (S)-aceclidine > arecaidine 2-butyne-1,4-diyl bisester > 4-Cl-McN-A-343 = (R)-nipecotic acid ethyl ester = N-ethyl-guvacine propargyl ester approximately (R)-aceclidine = (S)-nipecotic acid ethyl ester > McN-A-343. Agonist-induced vasodilation disappeared after destruction of the endothelium with detergent. Highly significant correlations of agonist potencies for vasodilation were found between rat kidney and guinea-pig ileum submucosal arterioles as well as agonist potencies at smooth muscle muscarinic M3 receptors of the guinea-pig ileum. The rank order of antagonist potencies (4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > (R)-hexahydro-difenidol approximately hexahydro-sila-difenidol > pirenzepine approximately p-fluoro-hexahydro-sila-difenidol approximately himbacine approximately AF-DX 384 approximately AQ-RA 741 > (S)-hexahydro-difenidol) to attenuate vasodilation to APE in rat kidney, correlated significantly with affinities at M3 receptors in submucosal arterioles and in smooth muscle of the guinea-pig ileum, but differed from those at M1 and M2 receptors in rabbit vas deferens. The agonist and antagonist potencies suggest that vasodilation elicited by muscarinic stimuli in endothelium-intact rat renal vasculature is mediated by functional muscarinic M3 receptors.

The adrenoceptor agonist, SDZ NVI 085, discriminates between alpha 1A- and alpha 1B-adrenoceptor subtypes in vas deferens, kidney and aorta of the rat.

The potency of the alpha 1-adrenoceptor agonist (-)-(4aR, 10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2H -naphth [2,3-b]-1,4-oxazine (SDZ NVI 085) was investigated both in isolated vas deferens and perfused kidney of the rat, two tissues with alpha 1A-adrenoceptor subtype characteristics, and in the rat thoracic aorta, in which the contribution of different alpha 1-adrenoceptor subtypes mediating contraction is controversial. In vas deferens and kidney, SDZ NVI 085 evoked smooth muscle contraction and vascular constriction and was of similar potency to L-phenylephrine. Contractions of vas deferens in response to (-)-noradrenaline and SDZ NVI 085 were resistant to chloroethylclonidine treatment (3 x 10(-5) M), sensitive to (+/-)-isradipine (10(-8) M) and competitively antagonized by 5-methyl-urapidil (pA2 = 9.04 and 8.82, respectively). The potencies of a number of alpha 1A-/alpha 1B-adrenoceptor-discriminating antagonists to reverse renal vasoconstriction due to either (-)-noradrenaline or SDZ NVI 085, and their affinities in vas deferens correlated significantly with their pKi values at alpha 1A binding sites in rat cortex. In rat aorta, SDZ NVI 085 up to 5 x 10(-4) M failed to evoke contraction. The affinities of subtype-selective antagonists determined in aorta correlated significantly with the pKi values at alpha 1B binding sites but differed from pKi values at alpha 1A sites in rat cortex. Thus, the contractile alpha 1-adrenoceptor in rat aorta can be best characterized as B subtype. SDZ NVI 085 might be a selective alpha 1A-adrenoceptor agonist and thus be used as a new tool either to detect (rat vas deferens and kidney) or exclude (rat aorta) a contribution of alpha 1A-adrenoceptors functionally involved in smooth muscle contraction.

External ATP antagonizes the effect of potassium channel openers in guinea-pig ventricular papillary muscle.

Right ventricular papillary muscles of the guinea-pig heart were electrically stimulated. Cromakalim 10-100 microM and Ro 31-6930 3 microM depressed the contractile force and shortened the duration of action potentials. Glibenclamide 0.3-3 microM, ATP 100 microM and alpha, beta-methylene ATP (alpha, beta-meATP) 30 microM antagonized these effects. Suramin 300 microM failed to reverse the alpha, beta-meATP-evoked antagonism of the action of cromakalim. It is concluded that both intra- and extracellular ATP may interfere with potassium channel openers and that extracellular ATP does not act via the known P2-purinoceptor subtypes.

Affinity profiles of pizotifen, ketotifen and other tricyclic antimuscarinics at muscarinic receptor subtypes M1, M2 and M3.

The affinity of pizotifen, ketotifen and other tricyclic antimuscarinic drugs for different muscarinic receptor subtypes was investigated in vitro in functional experiments with field-stimulated vas deferens of the rabbit (M1 and M2 receptors) and with ileum and trachea of the guinea-pig (M3 receptors). All compounds were competitive antagonists in the three tissues. Like the close analogue cyproheptadine (pA2 = 7.99-8.08), pizotifen (pA2 = 7.23-7.81) and ketotifen (pA2 = 6.34-6.99) were devoid of selectivity for the receptor subtypes studied. Thiazinamium, although exhibiting high affinity for muscarinic receptors (pA2 = 7.83-8.51), was found to be non-selective. In contrast, the novel pirenzepine analogue nuvenzepine was selective for M1 receptors (pA2 = 6.63-7.74). The lack of selectivity of cyproheptadine, pizotifen and ketotifen is reflected in the chemical structures of these drugs. All three antagonists are composed of a very similar tricyclic ring system linked to a 1-methyl-4-piperidylene ring. The finding that thiazinamium, pizotifen and cyproheptadine were potent muscarinic antagonists and possessed non-selective affinity characteristics may have therapeutic implications.

Is field (vagal) stimulation of gastric acid secretion mediated by M1 or non-M1 muscarinic receptors? A methodical problem exemplified in the mouse stomach in vitro.

1. Highly controversial claims have been put forward in the literature as to the involvement of either M1 or non-M1 muscarinic receptors in the field (vagal) stimulation of gastric acid secretion. This mini-review considers three available sets of data obtained in the mouse isolated, lumen-perfused stomach. 2. While pA2 values seemed to favour non-M1 receptors, a comparison between M1 selective versus non-selective antagonists on the one hand and field stimulation versus bethanechol stimulation on the other clearly pointed to M1 receptors being involved. 3. In the present review we discuss a novel experimental approach supporting the latter concept of M1 receptors. This novel approach provides a simple though provocative way to deal with the particular difficulties in determining pA2 values in the acid-secreting gastric mucosa. It is based on the rank order in the effects of different antagonists relative to their receptor type-dependent affinities, when employed at a fixed concentration, rather than on their pA2 values.

Vasodilatation elicited by 5-HT1A receptor agonists in constant-pressure-perfused rat kidney is mediated by blockade of alpha 1A-adrenoceptors.

The vasodilator mechanism of the putative serotonin1A (5-HT) receptor agonists, urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was investigated in constant-pressure perfused rat kidneys. The compounds (10(-12)-10(-7) mol bolus injection) neither enhanced basal flow nor evoked vasodilatation in kidneys preconstricted by 27 mM KCl, 1.5 mM BaCl2 or 10(-6) M prostaglandin (PG)F2 alpha, but evoked a dose-dependent, reversible and spiroxatrine-resistant increase in vasodilatation of organs preconstricted by 6 x 10(-7) M noradrenaline. 5-Carboxamidotryptamine and sumatriptan did not reverse the vasoconstriction induced by all stimuli or that induced by noradrenaline in the presence of 5-HT2 plus 5-HT3 receptor blockade. No correlation for the vasorelaxant drugs was found between their -log ED50 in rat kidney and pKi values at 5-HT1A binding sites in pig cortex as determined in radioligand experiments. The relaxation in rat kidney induced by 5-HT1A receptor agonists and alpha 1A-adrenoceptor-selective antagonists (WB 4101 and (+)-niguldipine) was significantly correlated with pKi values at alpha 1A binding sites in rat cortex and the pA2 values derived from contraction studies for competitive antagonism at alpha 1-adrenoceptors in prostatic portions of the rat vas deferens, but differed from pKi values for alpha 1B binding sites in rat cortex. Thus, the vasodilator effect of the 5-HT1A receptor agonists urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-OH-DPAT in the noradrenaline-perfused rat kidney appears to be mediated by their concomitant alpha 1A-adrenoceptor blockade. No evidence for a vasodilator effect mediated through 5-HT1A receptors was found under our experimental conditions.

Telenzepine inhibits electrically-stimulated, acetylcholine plus histamine-mediated acid secretion in the mouse isolated stomach by blockade of M1 muscarine receptors.

1. The muscarine receptor mediating electrically-stimulated acid secretion in the mouse isolated stomach was characterized using a variety of muscarine receptor antagonists confirming the M1 nature of the antagonist effect of telenzepine. 2. Field stimulation (7 V, 10 Hz, 0.5 ms) resulted in a plateau acid secretion over at least 90 min which was completely blocked by either 1 mumol/l TTX or H2 receptor antagonists (100 mumol/l cimetidine or 10 mumol/l lupitidine). Ranitidine, which is known to potently inhibit mucosal acetylcholine esterase, was ineffective. Compound 48/80 at 100 mumol/l, which depletes mucosal histamine stores, initially mimicked electrical stimulation but subsequently prevented it from inducing acid secretion. 3. 10 muscarine receptor antagonists with differing relative affinities for M1, M2 and M3 receptors were introduced at 1 mumol/l to inhibit electrically-stimulated acid secretion. The percentages inhibition were plotted against binding affinities of the antagonists at either M1, M2 or M3 binding sites. A statistically significant correlation between functional and binding data was detected only when based on M1 affinities. 4. It is concluded that field stimulation, which probably mimicks vagal drive, results in muscarinic M1 receptor activation on paracrine cells to release histamine. Histamine then stimulates parietal cells to secrete acid. Hence, according to the present and our previous data, telenzepine inhibits acid secretion under these conditions by blocking M1 receptors at least partially located on histamine-releasing paracrine cells.