Rabbit Vas Deferens Research Articles

Affinity profile at α 1- and α 2-adrenoceptor subtypes and in vitro cardiovascular actions of (+)-boldine

The present study examines the functional and binding affinities of the aporphine alkaloid, (+)-boldine, at different α 1- and α 2-adrenoceptor subtypes, namely, α 1A (rat vas deferens and kidney) and its L-like state (rabbit spleen), α 1B (guinea pig spleen, mouse spleen and rabbit aorta), α 1D (rat aorta and pulmonary artery), at possible subtypes of prejunctional α 2-adrenoceptors in rat and rabbit vas deferens and rat atrium, α 2D in guinea pig ileum, cloned human α 1-adrenoceptor subtypes A, B and D and α 2-adrenoceptor subtypes A, B and C as well as rat α 2D-adrenoceptors. Additionally, we investigated its Ca 2+ channel antagonism in vascular and cardiac preparations. (+)-Boldine had higher affinity at α 1-adrenoceptor subtype A (p A 2=7.46, p K i=7.21) compared with its L-like state (p A 2=5.63) or subtype B (p A 2=5.98– 6.12, p K i=5.79) and subtype D (p A 2=6.18–6.37, p K i=6.09). Its affinities at α 2-adrenoceptors in rat and rabbit vas deferens and rat atrium (p A 2=6.02, 6.36, 6.06, respectively) were identical, but lower at guinea pig ileum α 2D-adrenoceptors (p A 2=4.38). (+)-Boldine displayed nearly undistinguishable affinity at cloned human α 2-adrenoceptor subtypes A, B and C (p K i=6.26, 5.79 and 6.35, respectively), whereas its affinity at rat α 2D-adrenoceptors was low (p K i=4.70). In perfused rat kidney, (+)-boldine inhibited K +-evoked vasoconstriction at doses 70-fold higher than diltiazem. In guinea pig Langendorff heart, (+)-boldine (10 −5–2×10 −4 M) was equieffective in increasing coronary flow and in depressing cardiac force, while lower concentrations already depressed heart rate. In papillary muscles from guinea pig, (+)-boldine (10 −6–10 −5 M) mainly prolonged the duration of action potential at levels >30% of repolarization. These data reveal that (+)-boldine, except for its moderate selectivity (15 to 25-fold) for α 1A-adrenoceptors, does not discriminate between the α 1-adrenoceptor subtypes B and D and α 2-adrenoceptor subtypes A, B and C, at which the drug consistently displays micromolar affinity. In vascular and cardiac preparations, (+)-boldine, although being at least 50-fold weaker than diltiazem, shows Ca 2+ channel antagonistic properties but no specificity for coronary dilatation relative to cardiodepression.

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors.

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M1 receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [3H]N-methylscopolamine binding to cloned muscarinic M1 and M4 receptors, and reduced binding to M5 subtype with lower affinity, while they reversibly inhibited the binding of [3H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M1 receptors at a site distinct from the classical one, and to have affinity for some alpha-adrenoceptors.

CJ-15,208, a novel kappa opioid receptor antagonist from a fungus, Ctenomyces serratus ATCC15502.

A novel kappa opioid receptor binding inhibitor CJ-15,208 (I) was isolated from the fermentation broth of a fungus, Ctenomyces serratus ATCC15502. The structure of I was determined to be a cyclic tetrapeptide consisting of one tryptophan, one D-proline, and two L-phenylalanine. Compound I was a selective binding inhibitor for the kappa opioid receptor: 47 nM (IC50) for kappa, 260 nM for mu, and 2,600 nM for delta. In the electrically-stimulated twitch response assay of rabbit vas deferens I recovered the suppression by a kappa agonist asimadoline with an ED50 of 1.3 microM, indicating that it is a kappa antagonist.

Long-Acting Fentanyl Analogues: Synthesis and Pharmacology of N-(1-Phenylpyrazolyl)- N-(1-phenylalkyl-4-piperidyl)propanamides

The synthesis of new fentanyl analogues in which the benzene ring of the propioanilido group has been replaced by phenylpyrazole is described. Antinociceptive activity was evaluated using the writhing and hot plate tests in mice. Two compounds, 3b and 3d , showed interesting analgesic properties, being more potent than morphine and less than fentanyl but with longer duration of action. These compounds inhibited the electrically evoked muscle contraction of guinea pig ileum and mouse vas deferens but not that of rabbit vas deferens and the effects could be reversed by antagonists (naloxone and/or CTOP), thus indicating that the compounds acted as μ agonists. Finally, the binding data confirmed that the compounds had high affinity and selectivity for the μ receptor.

Analysis of the muscarinic receptor subtype mediating inhibition of the neurogenic contractions in rabbit isolated vas deferens by a series of polymethylene tetra-amines.

The pharmacological characteristics of the presynaptic muscarinic receptor subtype, which mediates inhibition of the neurogenic contractions in the prostatic portion of rabbit vas deferens, have been investigated by using a series of polymethylene tetra-amines, which were selected for their ability to differentiate among muscarinic receptor subtypes. It was found that all tetra-amines antagonized McN-A-343-induced inhibition in electrically stimulated rabbit vas deferens in a competitive manner and with affinity values (pA:(2)) ranging between 6.27+/-0.09 (spirotramine) and 8.51+/-0.02 (AM170). Competition radioligand binding studies, using native muscarinic receptors from rat tissues (M(1), cortex; M(2), heart; M(3), submaxillary gland) or from NG 108-15 cells (M(4)) and human cloned muscarinic M(1)-M(4) receptors expressed in CHO-K1 cells, were undertaken with the same tetra-amines employed in functional assays. All antagonists indicated a one-site fit. The affinity estimates (pK:(i)) of tetra-amines calculated in binding assays using native receptors were similar to those obtained using cloned receptors. Among these compounds some displayed selectivity between muscarinic receptor subtypes, indicating that they may be valuable tools in receptor characterization. Spirotramine was selective for M(1) receptors versus all other subtypes (pK:(i) native: M(1), 7.32+/-0.10; M(2), 6.50+/-0.11; M(3), 6.02+/-0.13; M(4), 6.28+/-0.16; pK:(i) cloned: M(1), 7.69+/-0.08; M(2), 6.22+/-0.14; M(3), 6.11+/-0.16; 6.35+/-0.11) whereas CC8 is highly selective for M(2) receptors versus the other subtypes (pK:(i) native: M(1), 7.50+/-0.04; M(2), 9.01+/-0.12; M(3), 6.70+/-0.08; M(4), 7.56+/-0.04; pK:(i) cloned: M(1), 7.90+/-0.20; M(2), 9.04+/-0.08; M(3), 6.40+/-0.07; M(4), 7.40+/-0.04). Furthermore, particularly relevant for this investigation were tetra-amines dipitramine and AM172 for their ability to significantly differentiate M(1) and M(4) receptors. The apparent affinity values (pA:(2)) obtained for tetra-amines in functional studies using the prostatic portion of rabbit vas deferens correlated most closely with the values (pK:(i)) obtained at either native or human recombinant muscarinic M(4) receptors. This supports the view that the muscarinic receptor mediating inhibition of neurogenic contractions of rabbit vas deferens may not belong to the M(1) type but rather appears to be of the M(4) subtype.

Failure of AH11110A to functionally discriminate between α 1-adrenoceptor subtypes A, B and D or between α 1- and α 2-adrenoceptors

The potency of the putatively α 1B-adrenoceptor selective drug, 1-[biphenyl-2-yloxy]-4-imino-4-piperidin-1-yl-butan-2-ol (AH11110A), to antagonize contraction upon stimulation of α 1A-adrenoceptors in rat vas deferens and rat perfused kidney, α 1B-adrenoceptors in guinea-pig spleen, mouse spleen and rabbit aorta, and α 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-4 H-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(1 H,3 H)-pyrimidinedione (B8805-033) were confirmed as selective for α 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 α 1D-adrenoceptors, whereas spiperone was weakly selective for α 1B-over α 1A-adrenoceptors. However, from the functional affinity profile obtained for AH11110A at α 1A-adrenoceptors (p A 2=6.41 in rat vas deferens), α 1B-adrenoceptors (p A 2=5.40–6.54) and α 1D-adrenoceptors (p A 2=5.47–5.48), the affinity and presumed selectivity previously obtained for AH11110A in radioligand binding studies at native α 1B- and cloned α 1b-adrenoceptors (p K 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 α 2-adrenoceptors in rabbit vas deferens (p A 2=5.44) was not much lower than that displayed for α 1-adrenoceptor subtypes, revealing that AH11110A, besides α 1-adrenoceptors, also interacts with α 2-adrenoceptors, and thus may be unsuitable for α-adrenoceptor subtype characterization, at least in smooth muscle containing functional studies.

Release of a soluble ATPase from the rabbit isolated vas deferens during nerve stimulation.

The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated. Superfusate collected during EFS rapidly metabolised exogenous ATP (100 microM) and 50% was broken down in 5.67+/-0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 40 microM and V(max) of 20.3 nmol ATP metabolized min(-1) ml(-1) superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80 degrees C for 10 min. ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC(50)=25 microM (95% confidence limits=22-27 microM), Hill slope=-1.06+/-0.04). When EFS was applied three times at 30 min intervals, ATP metabolism was 20-30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency-dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz. In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea-pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species-dependent.

Effects of brucine, a plant alkaloid, on M 1 muscarinic receptors and α 1-adrenoceptors in the rabbit vas deferens preparation

The plant alkaloid brucine is an analogue of strychnine and is known to be an allosteric modulator at cloned M 1 muscarinic receptors. The functional effects of brucine were examined on the M 1 muscarinic receptors in the rabbit isolated vas deferens preparation. Brucine (10–100 μM) enhanced the effects of the muscarinic agonist McN-A-343 at presynaptic M 1 muscarinic receptors in the rabbit isolated vas deferens preparation, but only when brucine was added prior to McN-A-343. This effect is indicative of a positive allosteric action. It was poorly reversed on washing. Brucine did not affect the responses to the mamba venom muscarinic toxins MT2 and MT4, which are also allosteric activators in this preparation. Brucine (10–100 μM) caused a significant decrease in the twitch response to electrical stimulation in the rabbit vas deferens preparation, which was not antagonised by 100 nM pirenzepine (an M 1 muscarinic antagonist). Brucine and MT4, but not MT2, caused significant decreases ( p<0.05) in the responses to noradrenaline in the rabbit vas deferens preparation. Responses to ATP and KCl were not affected. In radioligand binding assays, brucine displaced the α 1-adrenoceptor ligand prazosin from its specific binding sites in membranes made from rat cerebral cortex and rat vas deferens. The apparent K i values were 150 and 3.4 μM in the cortical and vas deferens membranes, respectively. The positive allosterism found with brucine at cloned M 1 receptors seems to be mirrored at native M 1 receptors. However, the unexpected blocking effects at α 1-adrenoceptors indicates that more selective ligands than brucine are required as starting points for the design of specific enhancers of the activity of M 1 receptors with therapeutic potential.

New analogues of oxotremorine and oxotremorine-M: Estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M 1), guinea pig left atrium (M 2), guinea pig ileum and urinary bladder (M 3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist.

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pKi of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 microM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system.

Pharmacology of [Tyr1]nociceptin analogs: receptor binding and bioassay studies.

Two series of nociceptin (NC)-related peptides with or without replacement of the N-terminal Phe by Tyr have been investigated in an attempt to obtain compounds that interact with the NC receptor (ORL1) and classic opioid receptors. When tested for their ability to displace [3H]NCNH2 ([3H]nociceptin amide; ORL1 sites) or the selective opioid receptor ligands [3H]DAMGO (mu), [3H]deltorphin II (delta) and [3H]U69593 (kappa) from their respective binding sites in guinea-pig brain membranes, [Tyr1]NCNH2 and [Tyr1]NC(1-13)NH2 showed high affinities (Ki 2nM and 5 nM, respectively) for ORL1 and approximately tenfold lower potency for mu (32nM and 44nM) and kappa sites (42 nM and 48 nM). They also interacted, but with low potency (Ki 410 nM and 310 nM) with delta sites. Shorter fragments as [Tyr1]NC(1-9)NH2 and [Tyr1]NC(1-5)NH2 were found to be inactive on ORL1, delta and kappa sites, and extremely weak (Ki 2224 nM and 4228 nM, respectively) on mu. Results of bioassays performed on the guinea-pig ileum (ORL1 and mu receptors), mouse vas deferens (ORL1 and delta receptors), and rabbit vas deferens (kappa receptor) confirmed (at least partially) the data of the binding by showing that [Tyr1]NC analogs interact with functional ORL1 as well as with classic opioid receptors. [Tyr1]NCNH2 and [Tyr1]NC(1-13)NH2 behaved as mixed ORL1/opioid receptor agonists showing similar affinities as the control NC sequence while [Tyr1]NC(1-9)NH2 and [Tyr1]NC(1-5)NH2 were inactive on ORL1 receptors but maintained some activities on opioid receptors: their effects were prevented by naloxone. The results of this study indicate that the replacement of Phe1 by Tyr in NC leads to compounds which bind both the ORL1 and mu/kappa receptors and may represent new promising agents for use in peripheral organs.

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M

Two subseries of nonquaternized (5a–10a) and quaternized derivatives (5b–10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: Ml rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/Ml-antagonist (pD2=5.85; pA2=4.76, respectively). In in vivo tests nonquaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Species-dependent effects of adenosine receptor agonists on contractile responses of vas deferens to ATP.

1. Experiments were carried out to examine the postjunctional actions of adenosine receptor agonists on the smooth muscle of the vas deferens of the guinea-pig and rabbit. 2. Although they produced neither contraction nor relaxation by themselves, adenosine analogues enhanced contractions of the guinea-pig vas deferens induced by 10 microm ATP. The rank order of potency was N6-cyclopentyladenosine (CPA) > 5'-N-ethylcarboxamidoadenosine (NECA) > adenosine > CGS 21680. Dose-response curves for NECA were shifted to the right by the nonselective adenosine receptor antagonist 8(p-sulphophenyl)theophylline (8-SPT; 100 microM) and by the selective A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 1 mM). 3. In the rabbit vas deferens, contractions induced by ATP (1 mM) were inhibited rather than facilitated by NECA. Neither CPA, R(-)-N6-(2-phenyl isopropyl)-adenosine (R-PIA) nor CGS 21680 had any effect. 4. The results indicate that the smooth muscle of the guinea-pig vas deferens expresses facilitatory adenosine A1 receptors but not adenosine A2 receptors. In contrast, in rabbit there are postjunctional inhibitory adenosine A2A receptors but not adenosine A1 receptors.

Subtypes of muscarinic receptors in rat duodenum A comparison with rabbit vas deferens, rat atria, guinea-pig ileum and gallbladder by using imperialine

The specific binding of [ 3H]QNB to rat duodenum smooth muscle membranes was a saturable process and Scatchard transformation of the saturation curves indicated a linear plot n H = 1.017 ± 0.071 . The K D and B max values were 0.168 ± 0.025 nM and 46.7 ± 8.6 fmol/mg protein, respectively. Analyses of competition curves using pirenzepine and guanylpirenzepine indicated more than one class of binding site. A minor population of muscarinic binding sites showed high affinity (M 1) for both pirenzepine 19.3 ± 1.2%; pK i = 8.29 ± 0.36 and guanylpirenzepine 29.4 ± 2.0%; pK i = 7.28 ± 0.11 . The antagonistic affinity values of pirenzepine and guanylpirenzepine for the remaining low affinity binding sites, and that of methoctramine indicated the presence of both M 2 and M 3 subtypes. McN-A-343 produced relaxations in rat duodenum and inhibited twitch contractions of rabbit vas deferens induced by electrical stimulation in a concentration dependent manner. Carbachol (Cch) exerted concentration-dependent negative inotropic effect in rat atria and contractile effects in guinea-pig gallbladder and ileum longitudinal muscle-myenteric plexus preparation. Imperaline displaced the concentration-response curves to McN-A-343 and Cch to the right in parallel, without affecting the maximum responses in all tissues studied. The rank order of the pA 2 values was rabbit vas deferens > rat atria > guinea-pig gallbladder = guinea-pig ileum > rat duodenum. The presynaptic muscarinic receptors at the rat duodenum and rabbit vas deferens were concluded to be of M 1 and M 4 subtypes, respectively.

Effects of GR-89696 and the novel peripherally selective OP2 agonists, EMD-61569 and EMD-61747, against focal cerebral ischemia in the rat.

We examined whether increases in blood-brain barrier (BBB) permeability occurring after stroke can be exploited to apply protective substances selectively to ischemic tissue. To do this, the actions of the peripherally selective OP2 agonists, EMD-61569 and EMD-61747, have been compared with those of the centrally acting OP2 agonist, GR-89696, in the rat permanent focal ischemia model. EMD-61569, EMD-61747 and GR-89696 all bound with high affinity to OP2 receptors and were potent agonists in the rabbit vas deferens functional assay. These substances also potently inhibited electrically-induced overflow of dopamine from slices of rat nucleus accumbens. EMD-61747 and EMD-61569 penetrate poorly into the CNS under normal conditions and reverse haloperidol-induced L-DOPA accumulation in the nucleus accumbens of the rat only at high doses, in contrast to GR-89696. Permanent unilateral occlusion of the middle cerebral artery (MCAO) was associated with a disruption of the BBB and an increase in the concentration of EMD-61747 in the area of the infarct. GR-89696 at a dose of 0.1 mg/kg s.c. produced a reduction in infarct volume by 38% after MCAO, EMD-61569 and EMD-61747 had no influence on swelling and ischemic damage. We conclude that EMD-61747 and EMD-61569 are potent OP2 agonists, which usually have a limited ability to penetrate the BBB. The change in the properties of the BBB in ischemic tissue was not sufficient to elicit neuroprotection, since both EMD-61747 and EMD-61569 were inactive in the focal ischemia model. Conversely, GR-89696 had a robust protective action, and probably powerful OP2-typical side effects as a consequence of its unrestricted central activity.

Synthesis and pharmacology of the putative novel muscarinic agonist ( S)-4-F-MePyMcN [( S)-4-(pyrrolidino)-1-methyl-2-butynyl-N-(4-fluorophenyl) carbamate oxalate

( S)-4-F-MePyMcN [( S)-4-(pyrrolidino)-1-methyl-2-butynyl-N-(4-fluorophenyl) carbamate oxalate] has been suggested to be a selective agonist at the M 1 subtype of muscarinic receptor [Lambrecht G. et al., Life Sci. 56 (1995) 815–822]. We synthesized the compound and tested its selectivity for different muscarinic receptors with binding experiments using rat cerebral cortex synaptosomal membranes and cloned human m1 to m5 receptors and by functional experiments on rabbit vas deferens preparations. There was little difference in affinity for the compound at the different cloned muscarinic receptors (IC 50s for displacement of 3H- N-methylscopolamine were 0.7-1.0 μM). On rabbit vas deferens preparations, ( S)-4-F-MePyMcN did reduce twitch responses to electrical stimulation like the known M 1 agonist McN-A-343, but unlike McN-A-343 the compound reduced postsynaptic sensitivity to noradrenaline, ATP and KCl. Because of these additional actions, ( S)-4-F-MePyMcN may not be suitable as a tool to study M 1 muscarinic receptors selectively.

M2 receptor binding of the selective antagonist AF-DX 384: possible involvement of the common allosteric site.

The hypothesis was tested that M2-selective antagonists partially utilize the allosteric site of muscarinic M2 receptors. The interactions of the allosteric agent W84 (hexane-1, 6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) were studied with the M2/M4-selective AF-DX 384 [(+/-)-5, 11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1-piperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the nonselective N-methylscopolamine (NMS), and a number of other muscarinic antagonists. In isolated paced guinea pig atria, the antagonistic effect of W84 against oxotremorine- and arecaidine propargyl ester-induced negative inotropic actions reached a limiting value at higher W84 concentrations, revealing negative cooperativity (factors of cooperativity alpha = 311 and alpha = 495, respectively). The antagonistic potency of W84 in this M2 receptor model (W84 binding constant KA approximately 160 nM) was higher than at M1/M4-like receptors of rabbit vas deferens (KB approximately 800 nM) and at M3 receptors of guinea pig ileum (KB approximately 4,000 nM). In paced atria, combinations of W84 with muscarinic antagonists yielded more-than-additive antagonistic effects against oxotremorine in case of conventional antagonists such as NMS (alpha = 18) but less-than-additive effects with the M2-preferring AF-DX 384 (alpha = 444). In guinea pig heart homogenates, the equilibrium binding of [3H]NMS was only partially inhibited by W84 (alpha = 2.4), whereas [3H]AF-DX 384 binding could be suppressed completely (alpha = 194). The difference in cooperativity reflects that W84 inhibits [3H]NMS dissociation with a approximately 40-fold higher potency (ECdiss = 900 nM) than [3H]AF-DX 384 dissociation (ECdiss = 33,300 nM). [3H]NMS dissociation also could be retarded by AF-DX 384 (ECdiss = 22,000 nM), probably via an interaction with the site used by W84. The results suggest that the binding domain of AF-DX 384 partially overlaps with the common allosteric site of the M2 receptor protein.

Erinacine E as a kappa opioid receptor agonist and its new analogs from a basidiomycete, Hericium ramosum.

A kappa opioid receptor binding inhibitor was isolated from the fermentation broth of a basidiomycete, Hericium ramosum CL24240 and identified as erinacine E (1). Three analogs of 1 were produced by fermentation in other media and by microbial biotransformation. Of these compounds, 1 was shown to be the most potent binding inhibitor. Preliminary SAR studies of these compounds indicated that all functional groups and side chains were required for the activity. Compound 1 was a highly-selective binding inhibitor for the kappa opioid receptor: 0.8 microM (IC50) for kappa, >200 microM for mu, and >200 microM for delta opioid receptor. Compound 1 suppressed electrically-stimulated twitch responses of rabbit vas deferens with an ED50 of 14 microM. The suppression was recovered by adding a selective kappa opioid receptor antagonist nor-binaltorphimine, indicating that 1 is a kappa opioid receptor agonist.

IN VITRO CHARACTERIZATION OF A NOVEL, POTENT AND SELECTIVE M 3 ANTAGONIST

The pharmacological profile of the competitive muscarinic antagonist (2S, 3′R) 3-quinuclidinyl tropate, abbreviated (−)−2a, was evaluated on rabbit vas deferens (M 1/M 4-like; pA 2=9.10), guinea-pig left atrium (M 2; pA 2=9.30), guinea-pig ileum (M 3; pA 2=10.33) and guinea-pig uterus (M 4 putative; pA 2=9.70) muscarinic receptors and on the five subtypes of muscarinic receptors expressed individually in CHO-K1 cells. The drug shows an affinity for the M 3 receptor subtype at least 10-fold higher than 4-DAMP, p-HHSiD and zamifenacin, used as reference drugs. These results suggest (−)−2a as a novel, potent and selective M 3 antagonist that may have therapeutic potential in the treatment of conditions associated with increased smooth muscle contractility.

Contraction of guinea-pig gallbladder: muscarinic M 3 or M 4 receptors?

The muscarinic receptor mediating contraction of the guinea-pig isolated gallbladder, currently being disputed to belong either to the M 3 or M 4 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 M 3 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 M 3/m3 and M 4/m4 receptors, e.g., himbacine, methoctramine, mefurtramine, tripitramine, idaverine, zamifenacin and 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one (AQ-RA 741), were consistent with those at guinea-pig ileal muscarinic M 3 receptors but not with published data at recently defined muscarinic M 4 receptors in rabbit anococcygeus muscle or at muscarinic M 1 and M 2 receptors in rabbit vas deferens. Antagonist affinities at guinea-pig gallbladder correlated also best with published binding data on native or cloned muscarinic M 3/m3 receptors but not with those for muscarinic M 4/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 M 3 receptors.