Rabbit Vas Deferens Preparation Research Articles

ChemInform Abstract: 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

Abstract ( 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 50 s for displacement of 3 H- 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.

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.

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.

Binding of muscarinic toxins MT×1 and MT×2 from the venom of the green mamba Dendroaspis angusticeps to cloned human muscarinic cholinoceptors

Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Previous competition binding studies indicate that the MTxs may be selective for the M, subtype of muscarinic acetylcholine receptors. The present work was undertaken in order to clarify the muscarinic subtype specificity and functional effects of MTx1 and MTx2. Binding interactions were determined using 3 H- N-methyl scopolamine (NMS) and cloned human muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary functional studies were performed on rabbit vas deferens preparations, which contain M 1 cholinoceptors. MTx1 and MTx2 inhibited 3H-NMS binding to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for ml receptors: K for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and K i for MTx2 were 364 nM at m1 and 1.2μM at m3 receptors. At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit vas deferens preparations, MTx1 and MTx2 at concentrations above 50 nM behaved in a similar way to the relatively selective M 1-agonists M cN-A-343 and CPCP (4-[ N-(4-chlorophenyl)carba-moyloxy]-4-pent-2-ynyl-trimethylammonium iodide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 receptors, but they also reveal a slightly weaker effect at m3 receptors. The interaction at m1 receptors apears to be essentially irreversible, implying that the toxins could be useful tools in studies of the functional role of m1 muscarinic receptors.

A pharmacological profile of the novel, peripherally‐selective k‐opioid receptor agonist, EMD 61753

1. The pharmacological properties of the novel diarylacetamide kappa-opioid receptor agonist, EMD 61753, have been compared with those of ICI 197067 (a centrally-acting kappa agonist) and ICI 204448 (a peripherally-selective kappa agonist). 2. EMD 61753 binds with high affinity (IC50 5.6 nM) and selectivity (kappa:mu:delta:sigma binding ratio 1:536:125: > 1,786) to kappa-opioid receptors and is a full and potent (IC50 54.5 nM) agonist in an in vitro assay for kappa-opioid receptors (rabbit vas deferens preparation). 3. Systemically-applied [14C]-EMD 61753 is found in high concentrations in the lungs, liver, adrenal glands and kidneys. Considerably less radioactivity is detected in the whole brain, and this radioactivity is concentrated in the region of the cerebral ventricles in the choroid plexuses. EMD 61753 penetrates only poorly into the CNS. 4. EMD 61753 was weakly effective in pharmacological tests of central activity. This compound reversed haloperidolol-induced DOPA accumulation in the nucleus accumbens of the rat only at a dose of 30 mg kg-1, s.c., (doses of 0.1, 1.0 and 10 mg kg-1, s.c., and 1.0, 10 and 100 mg kg-1, p.o., were inactive). Hexobarbitone-induced sleeping in mice was prolonged by EMD 61753 at threshold doses of 10 mg kg-1, s.c., and 100 mg kg-1, p.o., whereas the motor performance of rats in the rotarod test was impaired by EMD 61753 with an ID50 value of 453 mg kg-1, s.c. 5. EMD 61753 produced dose-dependent, naloxone-reversible antinociception in the mouse formalin test (1st phase ID50 1.9 mg kg-1, s.c., and 10.4 mg kg-1, p.o.; 2nd phase ID50 0.26 mg kg-1, s.c., and 3.5 mg kg-1, p.o.) and rodent abdominal constriction test (ID50 mouse 1.75 mg kg-1, s.c., and 8.4 mg kg-1, p.o.; ID50 rat 3.2 mg kg-1, s.c., and 250 mg kg-1, p.o.). EMD 61753 was inactive, or only weakly effective, in the rat pressure test under normalgesic conditions. After the induction of hyperalgesia with carrageenin, however, this compound elicited potent, dose-dependent (ID50 0.08 mg kg-1, s.c., and 6.9 mg kg-1, p.o., after remedial application, and 0.2 mg kg-1, s.c., and 3.1 mg kg-1, p.o., after prophylactic application) and naloxone-reversible antinociception. The antinociceptive action of systemically-applied (50 mg kg-1, p.o.) EMD 61753 in the hyperalgesic pressure test was completely inhibited by injection of the K-opioid antagonist norbinaltorphimine (100 Lg) into the inflamed tissue, a result which indicates that this opioid effect is mediated peripherally.6. Cutaneous plasma protein extravasation produced by antidromic electrical stimulation of the rat saphenous nerve was dose-dependently inhibited by systemically-applied EMD 61753 (ID13 values 3.7 mg kg-1, s.c., and 35.8 mg kg-1, p.o.), and this effect was completely antagonized by intraplantar application of norbinaltorphimine (50 microg). Extravasation elicited by the intraplantar application of substance P (10 microg) was not influenced by the administration of EMD 61753.7. EMD 61753 produced dose-dependent diuresis in non-hydrated rats at doses of and above 1.0 mg kg-1, s.c., and 10 mg kg-1, p.o., and in saline-loaded rats at doses of and above 10 mg kg-1, s.c.,and 30mgkg-1, p.o.8. The prostaglandin-mediated fall in mean arterial blood pressure elicited in anaesthetized rats by i.v.application of arachidonic acid was not inhibited by prior treatment with EMD 61753 (10mg kg-1,p.o.). Thus, a blockade of prostaglandin synthesis via inhibition of cyclo-oxygenase activity does not contribute to the in vivo effects of EMD 61753 and its metabolites.9 The present experiments therefore indicate that EMD 61753 is a potent, selective and orally-effective full ic-opioid receptor agonist which has a limited ability to penetrate the blood-brain barrier and elicit centrally-mediated sedation, putative aversion, diuresis, and antinociception. The inhibitory actions of systemically-applied EMD 61753 against hyperalgesic pressure nociception and neurogenic inflammation are mediated peripherally, probably by opioid receptors on the endings of sensory nerve fibres.

ICI 204448: a κP‐opioid agonist with limited access to the CNS

1. A number of compounds were evaluated in an attempt to identify a kappa-opioid receptor agonist with limited access to the central nervous system. 2. Quaternary derivatives of the kappa-opioid agonists tifluadom, U-50488H and ethylketocyclazocine were essentially devoid of opioid activity in a range of isolated tissue preparations. 3. A novel compound - ICI 204448 - is described which produced a potent and naloxone-reversible inhibition of electrically-evoked contraction of the guinea-pig ileum, mouse vas deferens and rabbit vas deferens preparations. ICI 204448 was shown to displace the binding of the kappa-opioid ligand [3H]-bremazocine from guinea-pig cerebellum membranes. 4. Ex vivo binding studies in mice showed ICI 204448 to be well absorbed following subcutaneous administration. The brain levels achieved by ICI 20448 were substantially lower than those produced by kappa-agonists such as U-50488H and tifluadom. 5. A good correlation was found for a range of opioids between lipophilicity and degree of CNS penetration.

Selectivity for opioid receptor subtypes of enkephalin analogues in isolated smooth muscle and in the analgesic effect in mice.

Selectivity for opioid receptor subtypes of enkephalin analogues (KK-1, -2, -3 and -4) of Tyr moiety on the N-terminal, and Phe-ol group on the C-terminal, connected with the methylene group (n = 1-4) were examined in isolated smooth muscle preparations and in the analgesic effect in mice. In the longitudinal muscle preparations of guinea pig ileum (GPI), morphine, U-50488H and all the enkephalin analogues inhibited electrically evoked contractions, and the inhibitory effects of morphine, KK-1, KK-2 and KK-3 were antagonized by naloxone with relatively high pA2 values, while that of U-50488H and KK-3 were preferentially antagonized by norbinaltorphimine. In the rabbit vas deferens preparations (RVD), on the other hand, U-50488H, KK-3 and KK-4 showed weak inhibitory effects and the inhibition of U-50488H and KK-3 were antagonized by norbinaltorphimine. By intracerebroventricularly (i.c.v.) injection, enkephalin analogues produced analgesia in the acetic acid (AcOH) writhing test, and the effect of KK-1 and KK-2 as well as morphine was antagonized by 1 mg/kg naloxone, while those of U-50488H and KK-3 were sensitive to 1 mg/kg Mr2266. In conclusion, enkephalin analogues with a short methylene chain between the functional groups, KK-1 and KK-2, mainly exert their effect through opioid mu-receptors, while those of longer chain, KK-3 and KK-4, act through kappa-receptors preferentially, and KK-3 is situated in the alternating point of the selectivity for mu- and kappa-receptors.

Muscarinic receptor differentiation

The plant alkaloid brucine is an analogue of strychnine and is known to be an allosteric modulator at cloned M1 muscarinic receptors. The functional effects of brucine were examined on the M1 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 M1 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 M1 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 α1-adrenoceptor ligand prazosin from its specific binding sites in membranes made from rat cerebral cortex and rat vas deferens. The apparent Ki values were 150 and 3.4 μM in the cortical and vas deferens membranes, respectively. The positive allosterism found with brucine at cloned M1 receptors seems to be mirrored at native M1 receptors. However, the unexpected blocking effects at α1-adrenoceptors indicates that more selective ligands than brucine are required as starting points for the design of specific enhancers of the activity of M1 receptors with therapeutic potential.

ChemInform Abstract: Synthesis and Biological Activities of Dynorphin A Analogues with Opioid Antagonist Properties.

Dynorphin A, which displays a wide variety of physiological effects, binds to opioid receptors preferentially at the kappa receptor type. kappa-selective antagonists would be very useful as pharmacological and biochemical probes to study and better understand the action of dynorphin A at its preferred receptor. However, the development of such molecules has been elusive, and very few are known at this time. Taking these features into account, we have synthesized by the solid-phase procedure several analogues of dynorphin A containing various D-amino acid substitutions. The binding properties of the peptides have been examined at three main opioid binding sites (mu, delta, and kappa) and their kappa selectivity determined. Their biological activities have been tested in three specific pharmacological assays for agonist and/or antagonist properties. Introduction of D-Trp substitution leads to analogues, in particular [D- Trp2,8,D-Pro10]-, [D-Trp5,8,D-Pro10]-, and [D-Trp2,4,8,D-Pro10]dynorphin(1-11), showing antagonist properties in the isolated rabbit vas deferens preparation, a kappa specific bioassay. The antagonism against dynorphin A is weak, as indicated by the observed Ke values (433, 199, and 293 nM, respectively), and not very selective (kappa vs. mu). Such peptide analogues derived from the endogenous ligand and endowed with antagonist properties are the first ones reported to date and could open a promising way in designing more potent and selective kappa opioid antagonists.

Synthesis and biological activities of dynorphin A analogues with opioid antagonist properties

Dynorphin A, which displays a wide variety of physiological effects, binds to opioid receptors preferentially at the kappa receptor type. kappa-selective antagonists would be very useful as pharmacological and biochemical probes to study and better understand the action of dynorphin A at its preferred receptor. However, the development of such molecules has been elusive, and very few are known at this time. Taking these features into account, we have synthesized by the solid-phase procedure several analogues of dynorphin A containing various D-amino acid substitutions. The binding properties of the peptides have been examined at three main opioid binding sites (mu, delta, and kappa) and their kappa selectivity determined. Their biological activities have been tested in three specific pharmacological assays for agonist and/or antagonist properties. Introduction of D-Trp substitution leads to analogues, in particular [D- Trp2,8,D-Pro10]-, [D-Trp5,8,D-Pro10]-, and [D-Trp2,4,8,D-Pro10]dynorphin(1-11), showing antagonist properties in the isolated rabbit vas deferens preparation, a kappa specific bioassay. The antagonism against dynorphin A is weak, as indicated by the observed Ke values (433, 199, and 293 nM, respectively), and not very selective (kappa vs. mu). Such peptide analogues derived from the endogenous ligand and endowed with antagonist properties are the first ones reported to date and could open a promising way in designing more potent and selective kappa opioid antagonists.

Purification and characterization of an opioid antagonist from a peptic digest of bovine .KAPPA.-casein.

A chloroform/methanol extract of a peptic digest of bovine K-casein had binding activity to opioid receptors of rat brain. The active peptide was isolated by reverse-phase liquid chromatography on ODS and CN columns. The structure of the peptide was Ser-Arg-Tyr-Pro-Ser-Tyr-OCH3, which corresponds to the methyl ester of the 33 - 38th residues of κ-casein. The peptide was esterified during the extraction process, and the structure was confirmed by a chemical synthesis. The IC50 value of the peptide in a radioreceptor assay in the presence of 1 HM [3H]naloxone was 15 μM. The peptide did not have opioid agonistic activities in any of the assay systems tested. In the guinea pig ileum and rabbit vas deferens preparations, however, the peptide antagonized with morphiceptin and dynorphin A-(1-13), respectively. The peptide did not antagonize with [Leu]enkephalin in the mouse vas deferens preparations. Therefore, the peptide was an antagonist selective for the μ- and κ-types of opioid receptors. The peptide was named casoxin.

Profile of activity of κ receptor agonists in the rabbit vas deferens

The purpose of this study was to investigate further the κ opioid receptor selectivity of the field-stimulated isolated rabbit vas deferens preparation and to study the profile of a series of κ agonists in this tissue. Agonists acting at μ, δ and σ receptors were without detectable effect in the rabbit vas deferens. But a number of κ agonists, including bremazocine, tifluadom, ethylketocyclazocine, ketocyclazocine, U-50,488 and Win 42,610 all depressed contractions, producing parallel dose-response curves. Mr 2034 generally produced a shallower dose-response curve and achieved a lower maximum effect, thus acting like a partial agonist. The effect of ethylketocyclazocine was not reduced by the irreversible μ antagonist, β-funaltrexamine, confirming that it is not acting via μ receptors. Another group of drugs, including nalorphine, butorphanol and proxorphan, which produce an agonist action via κ receptors in the guinea-pig ileum and mouse vas deferens, were antagonists in the rabbit vas deferens, suggesting that this tissue will only respond to high efficacy κ agonists.

The conversion of met-enkephalin-Arg 6, Phe 7 to met-enkephalin in rabbit ear artery

We studied the opioid agonist activities of met-enkephalin-Arg 6, Phe 7 heptapeptide (ME-A,P) and its D-Ala 2-analogue in several isolated organs including rabbit ear artery (ART), guinea-pig ileum (GPI) mouse-, rat and rabbit vas deferens preparation. Both heptapeptides followed the in vitro activity pattern of met-enkephalin (ME). There was a significant parallelism in the sensitivity of ART and mouse vas deferens to opioid peptides. In ART, ME-A,P must be converted by a captopril sensitive enzyme to exert opioid agonist action. Incubation of heptapeptide with membranes prepared from rabbit ear artery revealed the captopril-sensitive enzymic conversion of ME-AP to ME as detected by high voltage paper electrophoresis.