Methoctramine selectively blocks cardiac muscarinic M2 receptors in vivo

Summary The antimuscarinic effects of methoctramine (N, N- bis[6-[(2-methoxybenzyl)amino]hexyl]-1,8-octanediamine tetrahydrochloride), a polymethylene tetraamine endowed with high cardioselectivity in vitro, were assessed in two in vivo preparations. Methoctramine (300 g/kg i.v.) strongly inhibited the methacholine- and muscarine-induced bradycardia in the anaesthetized and pithed rat, respectively. The same dose of methoctramine did not significantly affect the depressor action of methacholine in the anaesthetized rat mediated by vascular M₂ receptors. Furthermore, even high doses of methoctramine (up to 1 mg/kg i. v.) did not reduce the ganglionic M₁ receptor-mediated tachycardia and pressor response to muscarine or McN-A-343 in the pithed rat. These data suggest that methoctramine while showing high affinity for cardiac M₂ receptors has rather low affinity for ganglionic M₁ and vascular M₂ receptors. This in vivo study thus provides further evidence to support the view that methoctramine is a potent and highly selective antagonist of cardiac M₂ receptors. Send offprint requests to: G. Lambrecht at the above address     

Binding characteristics and functional G protein coupling of muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes

Summary The non-selective labelled antagonist [³H]N-methyl-scopolamine ([³H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a K_D-value of 0.2–0.3 nmol/l and a receptor concentration (B_max) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M₁ (rat cerebral cortex), M₂ (rat heart), M₃ (rat submandibular gland) and M₄ (data from Dörje et al. 1991) receptors. The M₂-selective agent AF-DX 116, the group of M₂/M₄-selective compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the M3-selective HHSiD showed affinities corresponding to M₂ and/or M₄ sites. The intermediate affinity of 4-DAMP favours a mixed M₂/M₄ receptor population mainly containing M₂ receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [³⁵S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level. Send offprint requests to C. Liebmann at the above address     

Selective blockade in vivo of cardiac muscarinic M2 receptors by a polymethylene tetramine, BHC-9C

The antimuscarinic effects of BHC-9C (N,N'-bis[6-[(2-methoxybenzyl)amino]hexyl]-1,9-nonanediamine tetrahydrochloride), a member of a series of polymethylene tetraamines with unprecedented in vitro selectivity for cardiac muscarinic M2 receptors, were assessed in several in vivo test systems. BHC-9C (300 micrograms/kg i.v.) proved to be a potent antagonist at cardiac M2 receptors that mediate the decrease in heart rate in the pithed rat. In contrast, it displayed no considerable blocking activity at vascular M2 receptors subserving vasodepression and at ganglionic M1 receptors that mediate cardiovascular stimulation in the anaesthetized and pithed rat, respectively. These in vivo data are consistent with the suggestion based on in vitro experiments that BHC-9C is a highly selective antagonist of cardiac muscarinic M2 receptors.     

Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic receptor antagonist tripitramine

1. The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M₂-like, i.e. having antagonist affinity profiles that are qualitatively similar but quantitatively dissimilar compared to cardiac M₂ receptors. The present study sought to establish definitely the identity of these receptor subtypes by using the selective muscarinic receptor antagonist, tripitramine. Guinea-pig atria and guinea-pig trachea (postjunctional contractile response) were included for reference.
2. It was found that tripitramine antagonized methacholine-induced contractions of the guinea-pig lung strip with a pK_B value of 8.76±0.05. Both the parallel shifts of the concentration-response curves and the slope of the Schild plot being not significantly different from unity (when antagonist preincubation was for 2 h) indicated the involvement of a single population of receptors in the contractile response. From the pK_B values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Roffel et al., 1993), this single population of receptors can only be classified as M₂-like.
3. Tripitramine antagonized methacholine-induced negative chronotropic and inotropic responses in guinea-pig right and left atria with apparent pK_B values of 9.4–9.6. However, such values were only obtained when antagonist preincubation was relatively long and/or antagonist concentration relatively high (e.g. with 1 h at 100 or 300 nM but 3 h at 30 nM). It thus appears that low concentrations of tripitramine do not readily equilibrate with M₂ receptors in guinea-pig atria nor with M₂-like receptors in the guinea-pig lung strip.
4. Tripitramine increased electrical field stimulation-induced cholinergic twitch contractions in guinea-pig trachea in concentrations of 0.3–100 nM, by blocking prejunctional muscarinic inhibitory autoreceptors; with higher concentrations, twitch contractions were progressively diminished, as a result of blocking postjunctional M₃ receptors (apparent pK_B value 6.07±0.15). The pEC₂₀ value (−log concentration that increases twitch by 20% of maximum) was 8.29±0.08, which would suggest that M₄ receptors are involved in this response.
5. Oxotremorine-induced inhibition of the release of prelabelled [³H]-acetylcholine from guinea-pig trachea, under conditions where there is no auto-feedback, was blocked by tripitramine (2 h preincubation) with a pK_B value of 8.56±0.06. The slope of the corresponding Schild plot was not significantly different from unity, which together with the parallel shifts of the concentration-response curves indicated the involvement of a single muscarinic receptor subtype.
6. Since the pK_B value for tripitramine at prejunctional receptors in guinea-pig trachea is in between the affinities towards M₂ and M₄ receptors, correlation plots were constructed to compare the pK_B values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Kilbinger et al., 1995) to reported affinities at M₁–M₄ receptors. This showed rather similar distribution patterns of the data points around the line of equality in the case of M₂ and M₄ receptor subtypes. However, the correlation coefficient was markedly better for M₂ (0.9667) than for M₄ (0.5976). Since recent evidence suggests that M₄ receptors are not expressed in cholinergic nerves from guinea-pig trachea, it is concluded that prejunctional muscarinic autoinhibitory receptors in this tissue exhibit an atypical M₂ type character, with a pharmacological profile distinct from cardiac M₂ receptors.

     

Subclassification of muscarinic receptors in the heart,urinary bladder and sympathetic ganglia in the pithed rat

Summary In pithed normotensive rats muscarinic receptors were characterized heart, urinary bladder and sympathetic ganglia; the selectivity of some classical muscarinic agents for these subtypes was investigated. The potencies in decreasing heart rate, increasing bladder pressure and increasing diastolic blood pressure were measured for the following, intraarterially administered cholinergic agonists: McN-A-343 ([4-m-chlorophenylcarbamoyloxy]-2-butynyltrimethylammonium), pilocarpine, carbachol, oxotremorine, arecoline, acetyl--methylcholine and acetylcholine. The selective M₁-antagonist pirenzepine, the mixed M₁/M₂-antagonist dexetimide and the cardioselective M₂-antagonist gallamine were used as tools for identification of the receptors. All data were obtained after intravenous pretreatment with a high dose of atenolol to eliminate tachycardia induced by stimulating sympathetic ganglionic muscarinic receptors.Dexctimide strongly antagonized the bradycardia as well as the increase in bladder pressure induced by pilocarpine, carbachol, oxotremorine, arecoline, acetyl--methylcholine and acetylcholine, whereas pirenzepine was much less effective. Gallamine antagonized the bradycardia, whereas no influence was found on the bladder contraction. Pilocarpine acted as a partial agonist in reducing heart rate as well as in increasing bladder pressure, whereas McN-A-343 was almost ineffective in doses up to 1 mg/kg.The hypertensive response to pilocarpine and carbachol was less pronounced than that produced by McN-A-343. Pirenzepine and dexetimide significantly antagonized the hypertensive response to McN-A-343 and pilocarpine, whereas gallamine was much less effective. The hypertensive response induced by carbachol was totally blocked by hexamethonium. The other agonists used in this study did not produce a significant increase in diastolic blood pressure in doses that produced a maximal effect on heart rate and urinary bladder pressure.Simultaneously, intraarterially infused acetylcholine dose-dependently and reversibly decreased the pressor response to intravenously administered McN-A-343.These data suggest that muscarinic receptors in rat sympathetic ganglia belong to the M₁-subtype, whereas the muscarinic receptors in rat heart and urinary bladder represent heterogenous populations of M₂-receptors. The agonists used in this study, though, could not discriminate between these heterogenous M₂-receptors.Like McN-A-343, pilocarpine appears to be a rather selective M₁-agonist. In this study the M₁/M₂ selectivity of muscarinic agents with pronounced M₂-agonist activity could not be evaluated since M₂-receptor stimulation interferes with the hypertensive response to M₁-receptor stimulation.     

In vitro characterization of tripitramine, a polymethylene tetraamine displaying high selectivity and affinity for muscarinic M2 receptors.

1. The antimuscarinic effects of tripitramine were investigated in vitro in isolated driven left (force) and spontaneously beating right (force and rate) atria as well as in the ileum of guinea-pig and rat and in the trachea and lung strip of guinea-pig and compared with the effects of methoctramine. 2. Tripitramine was a potent competitive antagonist of muscarinic M2 receptors in right and left atria. The pA2 values ranged from 9.14 to 9.85. However, in the guinea-pig and rat left atria but not in guinea-pig right atria, tripitramine at lower concentrations (3-10 nM) produced a less than proportional displacement to the right of agonist-induced responses owing to the presence of a possible saturable removal process. 3. Tripitramine was about three orders of magnitude less potent in ileal and tracheal than in atrial preparations (pA2 values ranging from 6.34 to 6.81) which makes it more potent and more selective than methoctramine. 4. Another intriguing finding was the observation that the pA2 value of 7.91 observed for tripitramine in guinea-pig lung does not correlate with that found at both muscarinic M2 and M3 receptor subtypes, which clearly indicates that the contraction of guinea-pig lung strip is not mediated by these muscarinic receptor subtypes. 5. A combination of tripitramine with atropine resulted in addition of the dose-ratios for left atria as required for two antagonists interacting competitively with the same receptor site, whereas the same combination gave a supra-additive antagonism on guinea-pig ileum which suggests that tripitramine interacts with a second interdependent site. 6. Tripitramine was more specific than methoctramine since, in addition to muscarinic receptors, it inhibited only frog rectus abdominis muscular (pIC50 value of 6.14) and rat duodenum neuronal (pIC50 value of 4.87) nicotinic receptors among receptor systems investigated, namely alpha 1-, alpha 2-, and beta 1-adrenoceptors, H1- and H2-histamine receptors, and muscular and neuronal nicotinic receptors.     

Muscarinic receptor subtypes controlling the cationic current in guinea-pig ileal smooth muscle

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells.
2. Ascending concentrations of carbachol (3–300 μM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (G_max) of 27.4±1.4 nS and a mean −log EC₅₀ of 5.12±0.03 (mean±s.e.mean) (n=114).
3. Muscarinic antagonists with higher affinity for the M₂ receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA₂ values of 8.1, 8.0 and 9.1, respectively.
4. All M₃ selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC₅₀ for carbachol. At higher concentrations M₃ antagonists shifted the agonist curve to the right, increasing the EC₅₀, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in G_max (M₃ effect) and significant increase in the EC₅₀ (M₂ effect) in the same concentration range.
5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPγS to the pipette (without application of carbachol) was unaffected.
6. The results support the hypothesis that carbachol activates M₂ muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M₃ receptors. As an increasing fraction of M₃ receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M₂ receptors are disabled.

     

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation

Summary The agonistic potencies of (±)muscarine, (±)cis - 2 - methyl - 5 - [(dimethylamino)methyl] - 1,3 -oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M₁ and M₂ receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M₂ receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, “true” M₁ and M₂ potencies were determined after selective blockade of M₁ and M₂ receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M₁ and M₂ receptors: (+)cis-oxathiolane > (±)cis-oxathiolane > (±)muscarine > (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (−) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (±)muscarine showed similar potencies at M₁ and M₂ receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3–7 fold) for M₁ receptors indicating the potential usefulness of these compounds in the development of selective M₁ receptor agonists. Send offprint requests to F. Cantalamessa at the above address     

Central muscarinic receptor subtypes involved in pilocarpine-induced salivation, hypertension and water intake

Background and purpose:

Recent evidence has suggested that pilocarpine (ACh receptor agonist) injected peripherally may act centrally producing salivation and hypertension. In this study, we investigated the effects of specific M₁ (pirenzepine), M₂/M₄ (methoctramine), M₁/M₃ (4-DAMP) and M₄ (tropicamide) muscarinic receptor subtype antagonists injected into the lateral cerebral ventricle (LV) on salivation, water intake and pressor responses to peripheral pilocarpine.

Experimental approach:

Male Holtzman rats with stainless steel cannulae implanted in the LV were used. Salivation was measured in rats anaesthetized with ketamine (100 mg per kg body weight) and arterial pressure was recorded in unanaesthetized rats.

Key results:

Salivation induced by i.p. pilocarpine (4 μmol per kg body weight) was reduced only by 4-DAMP (25–250 nmol) injected into the LV, not by pirenzepine, methoctramine or tropicamide at the dose of 500 nmol. Pirenzepine (0.1 and 1 nmol) and 4-DAMP (5 and 10 nmol) injected into the LV reduced i.p. pilocarpine-induced water intake, whereas metoctramine (50 nmol) produced nonspecific effects on ingestive behaviours. Injection of pirenzepine (100 nmol) or 4-DAMP (25 and 50 nmol) into the LV reduced i.v. pilocarpine-induced pressor responses. Tropicamide (500 nmol) injected into the LV had no effect on pilocarpine-induced salivation, pressor responses or water intake.

Conclusions and implications:

The results suggest that central M₃ receptors are involved in peripheral pilocarpine-induced salivation and M₁ receptors in water intake and pressor responses. The involvement of M₃ receptors in water intake and pressor responses is not clear because 4-DAMP blocks both M₁ and M₃ receptors.     

In vivo modulation of histamine release by autoreceptors and muscarinic acetylcholine receptors in the rat anterior hypothalamus

The modulation of histamine release by histamine and muscarinic acetylcholine receptors was investigated by using the push-pull technique. The anterior hypothalamic area of the conscious, freely moving rat was superfused through the push-pull cannula with CSF or with CSF containing drugs and the release of endogenous histamine was determined in the superfusate.Hypothalamic superfusion with tetrodotoxin (10 mol/1) led to a pronounced and sustained decrease in the histamine release rate. Superfusion with compound 48/80 (100 mg/1) was ineffective. Hypothalamic superfusion with the H₃ agonist (R)--methylhistamine inhibited, while superfusion with the H₃ antagonist thioperamide enhanced the release of histamine. The release of histamine was inhibited on hypothalamic superfusion with the muscarinic receptor agonists carbachol or oxotremorine. Histamine release was enhanced by atropine, and this release-enhancing effect was abolished by oxotremorine. The selective M₁ antagonist pirenzepine (100 mol/I) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10 ol/1), which blocks M₁ and M₃ receptors, also enhanced the release rate of histamine. On the other hand, 50 and 100 moI/I methoctramine (M₂ receptor antagonist) 10 and 100 moI/l p-fluoro-hexahydro-siladifenidol (p-F-HHSiD, a M₃ receptor antagonist) were ineffective.It is concluded that histamine released in the hypothalamus originates predominantly from neurons. The release of histamine is modulated by H₃ autoreceptors. The histamine release is also modulated by cholinergic neurons which modify histamine release from histaminergic neurons by stimulating M₁ muscarinic acetylcholine heteroreceptors probably located on histaminergic neurons.Supported by the Fonds zur Förderung der wissenschaftlichen Forschung Correspondence to: H. Prast at the above address     

Evidence that SK & F 104078 does not differentiate between pre- and postjunctional α2-adrenoceptors

Summary We have investigated the proposed postjunctional selectivity of the a2-adrenoceptor antagonist SK & F 104078 employing the pithed rat, rat isolated atrium and human saphenous vein preparations. In the pithed rat, SK & F 104078 (5 mg kg^–1) produced a 20-fold shift in the prejunctional ID₅₀ (concentration of agonist producing 50% inhibition of the cardioacceleration to a single stimulus) and a 3-fold shift in the postjunctional ED₅₀ (concentration producing 50% of maximum rise in diastolic blood pressure) of the ₂-adrenoceptor agonist xylazine. However, the ₂-adrenoceptor antagonist yohimbine also showed apparent selectivity for prejunctional receptors in the pithed rat. In the rat isolated atrium, yohimbine was approximately 10 times more potent than SK & F 104078 at enhancing the stimulation-evoked release of tritium in tissues preincubated with (³H)-noradrenaline. In the human saphenous vein, yohimbine and SK & F 104078 had pA₂ values of 7.40 and 6.33, respectively, against contractions to noradrenaline, so that yohimbine was again approximately ten times more potent than SK & F 104078. In conclusion, SK & F 104078 behaved like yohimbine in its relative potencies at pre- and postjunctional ₂-adrenoceptors both in vivo and in vitro, so that we fail to find any selectivity of SK & F 104078 for postjunctional ₂-adrenoceptors.Send offprint requests to J. R. Docherty at the above address     

Urethane,but not pentobarbitone,attenuates presynaptic receptor function in rats: a contribution to the choice of anaesthetic

Background and purpose:

We examined whether cannabinoid CB₁ and histamine H₃ receptors resemble α₂-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats.

Experimental approach:

Effects of the cannabinoid agonist CP-55,940 and the H₃ receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with ³H-noradrenaline.

Key results:

The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB₁ or H₃ receptors and α₂ adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked ³H-noradrenaline release.

Conclusions and implications:

Urethane, but not pentobarbitone, abolished the H₃ receptor-mediated vascular response in pithed rats and attenuated the CB₁ receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB₁, H₃ and α₂ receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.     

Loss of selectivity of so-called selective α1-adrenoceptor agonists after phenoxybenzamine

Summary This study examined the nature of -adrenoceptor subtype involved in pressor responses to so-called selective ₁-adrenoceptor agonists after treatment with phenoxybenzamine in vivo. The influence of prazosin (0.1 mg/kg) and of yohimbine (1 mg/kg) on the dose-response curves for cirazoline in the pithed rat, and for phenylephrine in the anaesthetized dog were compared, after various doses of phenoxybenzamine.In the pithed rat, after 0.05 mg/kg phenoxybenzamine, prazosin caused a displacement of the dose-response curve of cirazoline to the right which was much larger than that caused by yohimbine; after 0.3 mg/kg phenoxybenzamine, prazosin and yohimbine caused about equal displacements; after 1 mg/kg phenoxybenzamine, yohimbine caused a marked displacement, while prazosin was without effect.In the anaesthetized dog, after 1 mg/kg phenoxybenzamine, prazosin and yohimbine produced about equal rightward shifts of the dose-response curve for phenylephrine. However, after 3 mg/kg phenoxybenzamine the rightward shift of the dose-response curve for phenylephrine was much larger after yohimbine than after prazosin. In the anaesthetized dog, verapamil (1 mg/kg) caused a small and parallel rightward shift of the dose-response curve for phenylephrine before phenoxybenzamine and a large and nonparallel one after phenoxybenzamine (3 mg/kg); the effect of verapamil on responses to the selective ₂-adrenoceptor agonist UK-14,304 (before and after phenoxybenzamine) were similar to those on responses to phenylephrine after phenoxybenzamine.It is concluded that after 1 mg/kg phenoxybenzamine in the pithed rat or after 3 mg/kg phenoxybenzamine in the anaesthetized dog, the responses to the so-called selective ₁-adrenoceptor agonists cirazoline and phenylephrine, respectively, are predominantly or totally ₂-adrenoceptor-mediated. This explains why, after inactivation of ₁-adrenoceptors by phenoxybenzamine, the so-called selective ₁- and ₂-adrenoceptor agonists are equally antagonized by calcium entry blockers.This work was supported by grants from INIC (Instituto Nacional de Investigação Cientifica): FmP₁ and FMP₃ Send offprint requests to S. Guimarães at the above address     

Brain M3 muscarinic receptors are involved in the ACTH-induced reversal of hemorrhagic shock

Summary In an experimental model of bleeding-induced hemorrhagic shock causing the death of all saline-treated rats within 30 min, the intravenous injection of ACTH-(1–24) at the dose of 160 g/kg induced a sustained reversal of the shock condition, with almost complete recovery of blood pressure, pulse amplitude, respiratory rate, heart rate, and 100% survival, at least for the 2 h of observation. This effect of ACTH-(1–24) was prevented by the intracerebroventricular injection of 4-DAMP (a highly selective antagonist for M₁ and M3 muscarinic receptors), but unaffected by the intracerebroventricular injection of pirenzepine (a highly selective antagonist for M₁ muscarinic receptors). These data indicate that an essential step in the complex mechanism of the ACTH-induced shock reversal may be the activation of brain M₃ muscarinic receptors. Send offprint requests to S. Guarini at the above address     

Pharmacological analysis of the interaction of antimuscarinic drugs at M<Subscript>2</Subscript> and M<Subscript>3</Subscript> muscarinic receptors in vivo using the pithed rat assay

Muscarinic receptor antagonists form the mainstay of the therapeutic options for airway, bladder, and gastrointestinal smooth muscle disorders. Both M₂ and M₃ muscarinic receptors are involved in mediating smooth muscle contractility, although the relative functional contribution of each subtype, especially in the disease state, is unclear. Because the potency and selectivity of compounds for a given receptor in an in vivo setting can be dissimilar to that observed in an in vitro system, we developed an in vivo assay to simultaneously determine the absolute potency and selectivity of muscarinic receptor antagonists at M₂ and M₃ receptors using the pithed rat. Methacholine (MCh)-induced bradycardia and depressor responses were used as surrogate functional endpoints for M₂ and M₃ receptor activation, respectively. The influence of the muscarinic antagonists, tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, or tiotropium on the MCh-induced responses were studied. The estimated DR₁₀ values (dose producing a tenfold shift in the MCh curve) of tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, and tiotropium for the M₂ muscarinic receptor-mediated bradycardia were 0.22, 1.18, ∼2.6, 0.025, 0.40, and 0.0026 mg/kg, respectively, and 0.14, 0.18, 0.11, 3.0, 0.18, and 0.0017 mg/kg, respectively, for the M₃ muscarinic receptor-mediated depressor response. In a separate set of experiments, a single intravenous dose of tiotropium was administered before a MCh curve at 1, 3, 6, or 9 h to determine if tiotropium exhibited time-dependent selectivity for the M₃ receptor as has been reported from in vitro studies. The results indicate a slight preference of tiotropium for the M₃ receptor at later time points. The pithed rat assay may serve useful for elucidating the functional contribution of M₂ and M₃ receptors to the in vivo pharmacological effects of antagonists in disease animal models.     

Characterization of the muscarine receptor type on paracrine cells activated by McN-A-343 in the mouse isolated stomach

Summary An attempt was made to characterize the muscarine receptor type(s) involved in acid secretion in the mouse isolated stomach when stimulated by the muscarinic agonist McN-A-343. A series of 8 muscarinic antagonists was used with preference for Mr receptors (telenzepine and pirenzepine), M₁ and M₂ receptors (secoverine), M₂ receptors (AF-DX 116 and himbacine) and M₁ and M₃ receptors (p-F-HHSiD and HHSiD). BTM-1086 was used as a high affinity antagonist at the M₁ receptor however with little selectivity.Receptor type preferences were determined in binding experiments with [³H]telenzepine in cortical membranes (M₁) and [³H]N-methylscopolamine in atrial (M₂) or salivary gland (M₃) membranes, derived from guinea pigs. No antagonist with M₃ preference could be identified in the binding studies.A fixed antagonist concentration of 1 mol/l was used to antagonize acid secretion stimulated by 10 mo1/l McN-A-343. By plotting the percentage inhibition obtained in the functional test against the Ki values determined in binding experiments for each antagonist at M₁, M₂ and M₃ binding sites, an affinity-inhibition curve could only be constructed when based on the antagonist affinities to the Mr receptor. No statistically significant fit was found using antagonist affinities to the M₂ or M₃ receptor.Thus, in accordance with the presumed Mr selectivity of the agonist McN-A-343, the rank order of potencies of different antagonists point to the M₁ nature of the muscarine receptor which stimulates acid secretion in the mouse isolated stomach upon activation by McN-A-343. Though M₂ receptors were completely ruled out, M₃ receptors may still contribute to some extent to the acid stimulating effect of McN-A-343 in this tissue. Send offprint request to W. Kromer at the above address     

AE9C90CB: a novel, bladder-selective muscarinic receptor antagonist for the treatment of overactive bladder

Background and purpose:

AE9C90CB (N- [(1R, 5S, 6R)-3-azabicyclo [3.1.0] hex-6-ylmethyl]-2-hydroxy-N-methyl-2, 2-diphenylacetamide), a novel muscarinic receptor antagonist, was synthesized for the treatment of overactive bladder. Here we describe the in vitro and in vivo profiles of AE9C90CB for action in bladder over salivary gland and compare it with four agents already in clinical use (tolterodine, oxybutynin, solifenacin and darifenacin).

Experimental approach:

Radioligand binding assay and isolated tissue-based functional assay were used to evaluate affinity, potency, and receptor subtype selectivity of compounds. Inhibition of carbachol-induced increase in intravesicular pressure and salivary secretion were measured in anaesthetized rabbits to assess the functional selectivity.

Key results:

In vitro radioligand binding study using human recombinant muscarinic receptors showed that AE9C90CB had greater affinity for M₃ muscarinic receptors with pKi of 9.90 ± 0.11 and was 20-fold more selective for M₃ than for M₂ muscarinic receptors. AE9C90CB exhibited an unsurmountable antagonism on rat bladder strips (pK_B, 9.13 ± 0.12). In anaesthetized rabbits after intravenous administration, AE9C90CB dose dependently inhibited carbachol-induced increase in intravesicular pressure and salivary secretion, and exhibited functional selectivity for urinary bladder over salivary gland which was ninefold better than that of oxybutynin.

Conclusions and implications:

We have identified AE9C90CB, a compound exhibiting moderate selectivity for M₃ over M₂ receptors but greater selectivity for urinary bladder over salivary gland than oxybutynin, tolterodine, solifenacin and darifenacin. Therefore, AE9C90CB may be a promising compound for the treatment of overactive bladder with reduced potential to cause dry mouth than currently available antimuscarinic drugs.     

(−)Tertatolol is a potent antagonist at pre- and postsynaptic serotonin 5-HT1A receptors in the rat brain

Summary The potential 5-HT_1A antagonist properties of the ß-antagonist tertatolol were assessed using biochemical and electrophysiological assays in the rat. (±) Tertatolol bound with high affinity (K_i = 38 nM) to 5-HT_1A sites labelled by [³H]8-OH-DPAT in hippocampal membranes. The (–)stereoisomer (K_i = 18 nM) was about 50-fold more potent than the (+)stereoisomer (K_i = 864 nM) to inhibit the specific binding of [³H]-8-OHDPAT. As expected of a 5-HT_1A antagonist, (–)tertatolol prevented in a concentration-dependent manner (K_i = 24 nM) the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in rat hippocampal homogenates. Furthermore in vivo pretreatment with (–)tertatolol (5 mg/kg s.c.) significantly reduced the inhibitory influence of 8-OH-DPAT (0.3 mg/ kg s.c.) on the accumulation of 5-hydroxytryptophan in various brain areas after the blockade of aromatic L-amino acid decarboxylase by NSD-1015 (100 mg/kg i.p.). In vitro (in brainstem slices; K_i 50 nM) and in vivo (in chloral hydrate anaesthetized rats; ID₅₀ 0.40 mg/kg i.v.), (–)tertatolol prevented the inhibitory effects of the 5-HT_1A receptor agonists 8-OH-DPAT, ipsapirone and lesopitron on the firing rate of serotoninergic neurones within the dorsal raphe nucleus. In about 25% of these neurones, the basal firing rate was significantly increased by (–)tertatolol (up to +47% in vitro, and +30% in vivo). These data indicate that (-)tertatolol is a potent competitive antagonist at both pre (in the dorsal raphe nucleus) - and post (in the hippocampus) - synaptic 5-HT_1A receptors in the rat brain.Abbreviations ACSF artificial cerebrospinal fluid - BMY-7378 8-[2-[4(2-methoxy-phenyl)-1-piperazinyl]ethyl]-8-azaspiro-[4,5]-decane-7,9dione - DRN dorsal raphe nucleus - 5-HIAA 5-hydroxyindole acetic acid - 5-HT 5-hydroxytryptamine (serotonin) - 5-HTP 5-hydroxytryptophan - MDL 73005 EF 8-[2-[(2,3-dihydro-1,4-benzodioxin-2-yl)-methylamino]ethyl]-8-azaspiro-[4,5]-decane-7,9-dione, methyl sulfonate - NAN-190 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine - 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin - (S)-UH-301 (S)-5-fluoro-8-hydroxy-2-(dipropylamino) tetralin - WAY-100,135 N-tert-butyl-3,4-(2-methoxyphenyl)-piperazin-1-yl-2-phenylpropanamide Correspondence to T. Jolas at the above address     

Search for an endogenous cannabinoid-mediated effect in the sympathetic nervous system

Activation of CB₁ cannabinoid receptors by exogenous agonists causes presynaptic inhibition of neurotransmitter release from axon terminals. In the central nervous system, presynaptic CB₁ receptors can also be activated by endogenous cannabinoids (endocannabinoids) released from postsynaptic neurons. Except in the vas deferens, there is no indication of endocannabinoid-mediated presynaptic inhibition in the sympathetic nervous system. The aim of the present study was to search for such inhibition in pithed rats. Artificial sympathetic tone was established by continuous electrical stimulation of preganglionic sympathetic axons. The CB₁ cannabinoid receptor antagonist rimonabant (0.5 and 2 mg kg^–1 i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration. Since activation of G_q/11 protein-coupled receptors enhances endocannabinoid synthesis in the central nervous system, we attempted to stimulate endocannabinoid production by infusion of arginine vasopressin and phenylephrine (both activate G_q/11 protein-coupled receptors). Rimonabant (2 mg kg^–1 i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration during infusion of phenylephrine or vasopressin. In the final series of experiments we verified that an exogenous cannabinoid agonist produces sympathoinhibition. The synthetic CB₁/CB₂ receptor agonist WIN55212-2 (0.1 and 1 mg kg^–1 i.v.) markedly lowered blood pressure and plasma noradrenaline concentration in pithed rats with electrically stimulated sympathetic outflow. In contrast, in pithed rats with a pressor infusion of noradrenaline, WIN55212-2 did not change blood pressure or heart rate. The results verify that activation of peripheral presynaptic CB₁ receptors inhibits noradrenaline release from sympathetic nerve terminals. The lack of effect of the CB₁ receptor antagonist rimonabant indicates that, even under conditions favouring endocannabinoid synthesis, endocannabinoid-mediated presynaptic inhibition is not operating in the sympathetic nervous system of the pithed rat.     

Effects of selective A1 and A2 adenosine receptor agonists on cardiovascular tissues

Summary We investigated the negative chronotropic and vasodilating properties of new selective A₁ and A₂ adenosine agonists such as 2-chloro-N⁶-cyclopentyladenosine (CCPA) and 2-hexynyl-5-N-ethyl-carboxamidoadenosine (2-hexynyl-NECA) as compared with reference adenosine analogues. The potency of these compounds on heart rate was assessed in the rat atrial preparation and their activity on the vascular tone was determined in both rat aorta and bovine coronary artery. CCPA was found to be the most potent At agonist of those currently available in producing negative chronotropic effects (EC₅₀ = 8.2 nM). The A1 antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) blocked CCPA activity in a dose-dependent manner. There was also a significant correlation between its biological effect and the affinity for A₁ receptors as measured in the rat brain by [³H]-N⁶-cyclohexyladenosine (³[H]-CHA) binding. The A₂ selective agonist 2-hexynyl-NECA showed vasodilating properties comparable with those observed with the reference compounds, CGS 21680 and NECA. EC₅₀ values were 596 and 569 nM in rat aorta and bovine coronary artery, respectively. Moreover, the rank order of potency was similar in the two vascular districts examined, suggesting that the rat aorta is a useful model for studying the effects of adenosine derivatives on vascular tone. In addition, the potency of the compounds in inducing vasodilation was found to be correlated with their affinity for A₂ receptors as measured in the rat striatum by ³[H]-CGS 21680 binding.These data further support that A₁ receptors are involved in depressing cardiac activity and A₂ receptors in inducing vasorelaxation.Correspondence to A. Conti at the above address