Conditional involvement of muscarinic M1 receptors in vagally mediated contraction of guinea-pig bronchi

The involvement of ganglionic muscarinic M₁ receptors in vagally induced bronchoconstriction in guinea-pig airways is controversial. Therefore, we studied the effects of the M₁-selective muscarinic receptor antagonist pirenzepine on vagus nerve (VNS, preganglionic) and electrical field stimulation (EFS, postganglionic)-induced contractions of the guinea-pig main bronchus under various experimental conditions.Using identical stimulation parameters for VNS and EFS (8V, 30 Hz, 0.5 ms, 5s every min), the amplitude of the VNS-induced twitch contractions was 30.4% of the EFS-induced responses, and pirenzepine showed 2.3-fold selectivity (pIC₅₀-values 6.45 and 6.09, respectively) to inhibit vagally induced contractions. With the stimulation frequency for EFS lowered to match contraction levels obtained using VNS, pirenzepine was equipotent to inhibit both types of response at M₃ receptor-selective concentrations, suggesting that M₁ receptors are not involved. By contrast, when the stimulation episode was prolonged until plateau contraction (10–20 s), in the presence of the nicotinic antagonist hexamethomum (5 M), the M₂ receptor antagonist AQ-RA 741 (0.1 M) and the -adrenoceptor antagonist timolol (1 M), and again using matched VNS- and EFS-induced contraction levels, pirenzepine inhibited nerve stimulation-evoked responses in a biphasic manner, yielding (pIC₅₀-values of 8.12 indicative of M₁ receptor blockade) and 6.43 (indicative of M₃ receptor blockade) for the first and second phase, respectively, while postganglionic stimulation showed a purely monophasic inhibition (pIC₅₀ = 6.32).These results show that facilitatory muscarinic M₁ receptors are involved in vagally mediated contraction of guinea-pig bronchi, under conditions of elevated neurotransmission and partial nicotinic receptor blockade.     

Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea-pig isolated uterus

1. In many smooth muscle tissues a minor M₃-muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2-mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M₃-mACh receptors.
2. Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus.
3. Antagonist affinity estimates made at `protected'' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea-pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine>methoctramine. Carbachol-induced contractions were insensitive to the M₄-selective muscarinic toxin MTx-3, or PD102807 (0.1 μM) ruling out a role for M₄-mACh receptors.
4. The agonist affinity value for L-660,863, a putative `M₂-selective'' agonist of 5.44±0.30 (n=6) was significantly different from that reported in guinea-pig atria. In contrast, the pK_A value for carbachol (4.22±0.17; n=8) agrees with that reported for guinea-pig ileum.
5. Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of G_i-proteins in contraction.
6. Radioligand binding studies revealed a K_D for N-[³H]-methylscopolamine of 0.12±0.05 nM and a B_max of 147±18 fmol mg protein⁻¹. Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M₂-mACh receptors.
7. These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

     

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     

Selectivity of antimuscarinic compounds for muscarinic receptors of human brain and heart

Seven antimuscarinic compounds, used mostly for the treatment of extrapyramidal problems, were tested in vitro in radioligand binding assays for evidence of selectivity for two different pharmacological subtypes of the human muscarinic receptor, M₁, a predominant form in brain, and M₂, a predominant form in heart. Although biperiden, scopolamine, procyclidine, and benztropine showed significant selectivity in the in vitro assays, it is likely that in clinical practice biperiden would be the drug of choice to avoid any antimuscarinic effects on the heart.     

Blockade of spatial learning by the M1 muscarinic antagonist pirenzepine

Two experiments were conducted to determine the effects of the M₁ muscarinic receptor antagonist pirenzepine on place navigation in a water maze. In the first experiment rats were required to learn the location of a hidden platform following intracerebroventricular injections of equimolar doses of pirenzepine or scopolamine methylbromide. Both drugs dose-dependently impaired spatial learning according to both escape latency data and transfer test analysis. Pirenzepine was approximately 3 times less potent than scopolamine, a potency ratio which suggests M₁ receptor mediation of the impairment. In the second experiment pirenzepine (192.3 g/rat ICV) was injected prior to training on a simultaneous place dicrimination task in the water maze. Impairments of choice accuracy were found with a dose of 20 g/rat in the absence of any marked increases in either errors of omission or choice latency. These data suggest that M₁ receptor blockade impairs processes which are involved in spatial learning.     

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     

Characterization of postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle

The present study was designed to characterize the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle by means of a series of muscarinic agonists and subtype-preferring key muscarinic antagonists. Cumulative addition of muscarinic agonists elicited concentration-dependent contractions with the following rank order of potency (pD₂ values): (+)-muscarine (6.36) ≥ oxotremorine M (6.21) ≥ arecaidine propargyl ester (APE) (6.18) > carbachol (5.68)=(±)-methacholine (5.65) > 4-(4-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium chloride (4-Cl-McN-A-343) (4.28) > 4-(3-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) (3.89). (+)-Muscarine, oxotremorine M, carbachol and (±)-methacholine behaved as full agonists, whereas APE, 4-Cl-McN-A-343 and McN-A-343 displayed partial agonism. The contractile responses of the rat anococcygeus muscle to (±)-methacholine were competitively antagonized by pirenzepine (pA₂=6.92), 11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl] 5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one (AQ-RA 741; pA₂=6.75), himbacine (pA₂=7.11), (±)-p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD; pA₂=7.68) and the (R)- and (S)-enantiomers of hexahydro-difenidol [(R)-HHD: pA₂=8.52; (S)-HHD: pA₂=6.06]. A comparison of the pA₂ values derived from studies of contraction in rat anococcygeus muscle with literature binding (pK_i values) and functional affinities (pA₂ values) obtained at native M₁-M₄ receptors strongly suggests that the postjunctional muscarinic receptors mediating contraction in rat anococcygeus muscle are of the M₃ subtype. Received: 18 April / Accepted: 18 July 1997     

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction

In gallbladder smooth muscle, carbachol interacts with M₃ receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [³H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates and contraction were measured at various times (0–90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 g/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [³²P]ADP-ribosylation of a –40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates. The time course of [³H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [³H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [³H]inositol trisphosphates and on contraction were abolished by atropine. Pretreatment with pertussis toxin resulted in ADP-ribosylation of a 40/41 kDa protein from gallbladder cell membranes but did not affect the concentration-response or time course of carbachol-induced contraction. These results indicate that carbachol-induced contraction of gallbladder smooth muscle cells is accompanied by the activation of inositol phosphate turnover and does not involve a pertussis toxin-sensitive G-protein.This article is based in part on the doctoral thesis of Burkhard Mackensen at the Faculty of Medicine, University of Hamburg, Germany. Some of the results were presented at the meeting of the American Gastroenterological Association (AGA) in San Francisco 1992 (von Schrenck et al. 1992) Correspondence to: T. von Schrenck at the above address     

Pharmacological characterization of muscarinic receptors in the uterus of oestrogen-primed and pregnant rats

1. Radioligand binding and contractility studies were undertaken to determine the subtype/s of muscarinic receptors present in uteri of oestrogen-treated and late pregnant rats.
2. Competition binding studies with uterine membrane preparations and [³H]-QNB (quinuclidinyl benzilate) provided negative log dissociation constants (pK_i) for each antagonist as follows; oestrogen-treated – atropine (7.98)⩾himbacine (7.83)>methoctramine (7.52)⩾hexahydrosiladiphenidol (HHSiD; 7.32)⩾5,11-dihydro-11-[[[2-[2 - [(dipropylamino)methyl] - 1piperidinyl]ethyl]amino] - carbonyl] - 6H-pyrido- [2,3 - b][1,4] - benzodiazepin - 6-one (AF - DX 384; 7.10)>11 - [[2 - [(diethylamino)methyl]-1-piperidinyl]- acetyl]5,11-dihydro-6H-pyridol]2,3,-b][1,4]benzodiazepin-6-one (AF-DX 116, 6.77)>pirenzepine (6.17); late pregnant – atropine (8.05)⩾methoctramine (7.95)⩾himbacine (7.71)⩾HHSiD (7.52)⩾AF-DX 384 (7.34)>AF-DX 116 (6.72)>pirenzepine (6.18).
3. The potency of carbachol in causing uterine contraction was similar in preparations from pregnant and non-pregnant animals (pD₂=5.57 and 5.46, respectively). Each muscarinic antagonist caused parallel, rightward shifts of carbachol concentration-response curves. The pA₂ estimates were: oestrogen-treated – atropine (9.42)>himbacine (8.73)⩾HHSiD (8.68)⩾methoctramine (8.49)⩾AF-DX 384 (7.91)⩾AF-DX 116 (7.36)⩾pirenzepine (7.26); late pregnant – atropine (9.48)>himbacine (8.37)⩾HHSiD (8.22)⩾methoctramine (8.01)⩾AF-DX 116 (7.73)⩾AF-DX 384 (7.44)⩾pirenzepine (6.92).
4. The relative pK_i estimates for antagonists obtained in membrane preparations from oestrogen-treated rats suggest the presence of muscarinic M₂ subtypes. In functional studies pA₂ values indicated the additional presence of muscarinic M₃ receptor or, possibly an atypical receptor subtype. The similarity between pK_i and pA₂ estimates obtained in uteri from oestrogen-treated and pregnant animals, respectively, indicates that pregnancy does not affect myometrial muscarinic receptors in the rat.

     

Characterization of muscarinic receptors mediating release of epithelial derived relaxant factor (EpDRF) in guinea-pig isolated trachea

Summary Muscarinic receptors mediating the release of epithelial derived relaxant factor (EpDRF) have been studied by using both contractions of the guinea-pig tracheal strip (with epithelium intact or denuded) or a coaxial bioassay assembly (rat anococcygeus-recipient; guinea-pig trachea-donor tissue). Indomethacin (1 M/l) and physostigmine (0.1 M/l) were both present throughout the study.In the tracheal strip studies, the potencies and maximal effects of all agonists studied (acetylcholine, arecoline, bethanechol, carbachol, (+)cis-dioxolane, ethoxyethyltrimethylammonium, L-660,863, (±)methacholine and OXA-22) were not affected or were only slightly (but significantly) reduced by removal of the epithelium. The -log K_B for the muscarinic antagonists, atropine, pirenzepine, methoctramine and 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) were also not affected and the -log K_B values were consistent with M₃ muscarinic receptor function. However, the -log K_B value of para-fluoro-hexahydro-siladifendol (p-F-HHSiD) was significantly (P < 0.05) increased upon epithelial denudation (epithelium intact, 7.1; epithelium removed, 7.6). The coaxial bioassay assembly provided more convincing evidence for release of EpDRF in that all muscarinic agonists studied caused relaxations of a precontracted anococcygeus tissue. These relaxations were observed only in the presence of a tracheal tube possessing an intact epithelium. The rank order of potencies for agonists at receptors mediating EpDRF dependent relaxation were similar to those estimated at receptors causing contraction. These data suggested that a substantial receptor reserve was associated with the receptors mediating both EpDRF release and contraction. The affinities of the muscarinic antagonists (atropine, pirenzepine, methoctramine, p-F-HHSiD, 4-DAMP and gallamine) indicated that M₃ receptors also mediated EpDRF release.It is concluded that EpDRF release in guinea-pig trachea is a general property of muscarinic agonists and that this process is mediated, like the contractile response, by M₃ receptors. Send offprint requests to R. M. Eglen at the above address     

The relative potencies of cholinomimetics and muscarinic antagonists on the rat iris in vivo: effects of pH on potency of pirenzepine and telenzepine

Summary The effects of cholinomimetics and muscarinic antagonists were compared following topical administration to the eyes of anaesthetized rats. For tests with cholinomimetics, clonidine (0.3 mg/kg) was used to induce mydriasis via central inhibition of parasympathetic tone. Full, dose-dependent miosis was induced by acetylcholinesterase inhibitors [physostigmine > neostigmine > tetrahydroaminoacridine (THA)] and by membrane channel blockers (4-aminopyridine > 3,4-diaminopyridine). Oxotremorine was the most potent direct agonist tested [oxotremorine > arecaidine propargylester (APE) > arecolne > carbachol > ethoxyethyltrimethyl-ammonium iodide (EOE) > RS 86]. Some putative M1 selective agonists were weakly active or behaved as partial agonists (pilocarpine > AH6405 > Mc-A-343 > isoarecoline). Of the antagonists, compared in non-clonidine treated rats, scopolamine hydrochloride was the most potent. Of the receptor selective antagonists the M₂ (ileal) selective compounds hexahydrosiladifenidol and 4-DAMP were more potent than either M₁ selective (pirenzepine, telenzepine) or M₂ (atrial) selective (AF DX 116) drugs. These data tentatively suggest the involvement of an M₂ (ileal) type muscarinic receptor. Potency was lower for quaternary structures, probably due to impaired corneal penetration. The potency of pirenzepine and telenzepine was increased 60-fold at low pH following topical administration. Acid induced corneal damage does not appear to account for this potency shift as the effects of scopolamine and several agonists (oxotremorine, pilocarpine and McN-A-343) were not substantially altered by acid media. For pirenzepine the potency shift appears to be related to protonation of the second amino group (N1) in the piperazine tail (pK _a = 2.05). Intraocular injections suggest that diprotonation facilitates penetration through the cornea. This anomalous behaviour of pirenzepine may contribute to its potency in gastric acid inhibition where the acid environment of the stomach would favour the diprotonated state and therefore penetration through the epithelium. Send offprint requests to J. J. Hagan at the above address     

Two types of neuronal muscarine receptors modulating acetylcholine release from guinea-pig myenteric plexus

Summary Longitudinal muscle strips of the guinea-pig ileum were incubated with [³H]choline and the effects of muscarinic agonists on smooth muscle contraction and on spontaneous and electrically-evoked outflow of tritium were studied. Muscarine and pilocarpine concentration-dependently increased both muscle contraction and spontaneous outflow of [³H]ACh, and inhibited the electrically-evoked outflow of [³H]ACh. The increase in spontaneous outflow was prevented by tetrodotoxin and scopolamine, but not by hexamethonium. Oxotremorine (1–100 M) did not increase the spontaneous outflow of tritium.Pirenzepine in concentrations of 10 and 100 nM hardly affected the muscle contractions induced by pilocarpine, but significantly antagonized the pilocarpine-evoked increases in [³H]ACh outflow. Likewise, pirenzepine (100 nM) antagonized more effectively the enhancement by muscarine of spontaneous outflow than the inhibitory effect of muscarine on the electrically-evoked release of [³H]ACh. Scopolamine (1 and 10 nM) antagonized to a similar extent the effects of pilocarpine on spontaneous outflow of [³H]ACh and on muscle contraction.The results suggest that the cholinergic nerves of the myenteric plexus are endowed with excitatory (ganglionic) and inhibitory (prejunctional) muscarine receptors which modulate the release of ACh and which differ in their affinities to pirenzepine.     

Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle

1. The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M₃ receptors in rabbit urinary bladder smooth muscle.
2. (+)-Cis-dioxolane induced concentration-dependent contractions of the iris sphincter muscle (pEC₅₀=6.41±0.10, E_max=181±17 mg, n=38) and urinary bladder smooth muscle (pEC₅₀=6.97±0.04, E_max=4.28±0.25 g, n=54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK_B values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30±0.07 and 9.40±0.04), AQ-RA 741 (6.35±0.04 and 6.88±0.03), darifenacin (9.56±0.05 and 9.12±0.05), methoctramine (5.75±0.07 and 5.81+0.06), oxybutynin (8.10±0.09 and 8.59±0.06), pirenzepine (6.79±0.05 and 6.89±0.04), secoverine (7.54±0.05 and 7.66±0.05), p-F-HHSiD (7.55±0.09 and 7.50±0.05) and zamifenacin (8.69±0.10 and 8.36±0.06). A significant correlation between the pK_B values in the bladder and the pK_B values in the iris was obtained.
3. In both tissues, the pK_B values correlated most favorably with pK_i values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors.
4. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically-defined muscarinic M₃ receptor.

     

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     

Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK₁, NK₂ and NK₃ receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle.
2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK₁ and NK₃ receptor-selective agonists.
3. Among the natural tachykinins neurokinin B (EC₅₀=3.2 nM; 95% c.l.=2.0–5.1; n=4) was the most potent, being about 40 and 25 fold more potent than substance P (EC₅₀=121.6 nM; 95% c.l.=94–157; P<0.01; n=4) and neurokinin A (EC₅₀=83.4 nM; 95% c.l.=62–112; P<0.01; n=4), respectively. Among the synthetic analogues the NK₃ receptor-selective agonist senktide (EC₅₀=1.1 nM; 95% c.l.=0.7–1.8; n=8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar⁹]substance P sulfone (NK₁ receptor-selective) (EC₅₀=130.4 nM; 95% c.l.=99–172; P<0.01; n=8), [βAla⁸]NKA (4–10) (NK₂ receptor-selective) (EC₅₀=120.1 nM; 95% c.l.=95–151; P<0.01; n=8) and septide (NK₁ receptor-selective) (EC₅₀=22.6 nM; 95% c.l.=18–28; P<0.01; n=8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar E_max, averaging about 50% of that produced by KCl (80 mM).
4. Atropine (1 μM) did not affect the responses to either NK₁ or NK₂ receptor-selective agonists, whereas it reduced the E_max of senktide by about 50%, without affecting its potency (EC₅₀). Tetrodotoxin (1 μM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK₁ and NK₂ receptor-selective antagonists GR 82334 and MEN 11420 (1 μM each), respectively.
5. GR 82334 (1 μM) blocked with apparent competitive kinetics septide- (apparent pK_B=7.46±0.10; n=5) and [Sar⁹]substance P sulfone- (apparent pK_B=6.80±0.04; n=4) induced contractions. MEN 11420 (30–300 nM), a novel potent NK₂ receptor antagonist, potently antagonized [βAla⁸]NKA (4–10), with competitive kinetics (pK_B=8.25±0.08; n=12: Schild plot slope=−0.90; 95% c.l.=−1.4; −0.35). The NK₃ receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (E_max inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration–response curve to methacholine (0.1–300 μM).
6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK₁, NK₂ and NK₃ receptors. The responses obtained by activating NK₁ and NK₂ receptors are atropine-resistant. The contraction obtained by stimulating NK₃ receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK₁/NK₂ receptors. The role of the NK₃ receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

     

Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris

Summary The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with ¹⁴C-choline. The stimulation-evoked ¹⁴C-overflow was doubled in the presence of atropine 0.1 mol/l but unaffected by the agonist (±)-methacholine (50 mol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mol/l). peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M₂-selective antagonists methoctramine (10 mol/l) and gallamine (100 µmol/l). increased both the ¹⁴Goverflow and the peak contractions evoked by field stimulation. In contrast, the M₃-selective antagonist hexahydrosiladifenidol (0.1–10 mol/l) failed to affect the evoked ¹⁴C-release but concentration-dependently (1–10 mol/l) reduced the iris contractions. Pirenzepine (10 mol/l) enhanced the evoked ¹⁴C-overflow and inhibited the peak contractions (0.1–10 mol/l; maximal effect at 10 mol/l). The low potency of the antagonist at both receptor sites indicates that an M₁ muscarine receptor is not involved. The results are consistent with the idea of M₂ muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M₃-like receptors inducing the contraction of the sphincter muscle. Send offprint requests to I. T. Bognar at the above address     

Characterization of 5-HT receptors mediating contraction and relaxation of the longitudinal muscle of guinea-pig distal colon in vitro

A range of agonists and antagonists were used to characterize the receptors through which 5-hydroxytryptamine (5-HT) contracts and relaxes the longitudinal muscle of segments of guinea-pig distal colon, in vitro. 5-HT contracted the longitudinal muscle over the concentration range 10^–9 to 10^–4 mol/l. The 5-HT3 receptor agonist, 2-methyl-5-HT, produced concentration dependent contractions over the range 10^–6 to 10^–4 mol/l. 5-methoxytryptamine, an agonist at 5-HT4 receptors, caused contractions over a concentration range of 10^–8 to 10^–4 mol/l. The 5-HT4 antagonist, SDZ 205-557 (5 × 10^–7 mol/l) substantially suppressed the responses to low concentrations of 5-HT and to 5-methoxytryptamine, but had no effect on the responses to higher concentrations of 5-HT. In contrast, the 5-HT3 antagonist, granisetron (10^–6 mol/l), blocked the effect of 2-methyl-5-HT and substantially depressed responses to high concentrations of 5-HT, but had no effect on lower concentrations of 5-HT. Granisetron produced a small reduction in the response to 5-methoxytryptamine. Tetrodotoxin (TTX) (3 × 10^–7 mol/l) almost abolished the response to 5-methoxytryptamine and markedly suppressed the response to 2-methyl-5-HT, but the responses to 5-HT were only partially reduced. The 5-HT, antagonist, methiothepin 10^–6 mol/l. depressed the response to 5-HT 10^–7 to 10^–4 mol/l. and blocked its TTX insensitive component. The 5-HT₂ antagonist, ketanserin, in concentrations up to 10^–5 mol/l, had no effect on the contractions evoked by 5-HT.The response to 5-HT was substantially depressed by hyoscine (3 × 10^–6 mol/l. The tachykinin antagonist, spantide 10^–5 mol/l. depressed the response to 5-HT but to a lesser extent than hyoscine. Spantide and hyoscine combined completely blocked the contractile responses to 5-HT Responses to 2-methyl-5-HT were partially suppressed by hyoscine (3 x 10^–6 mol/l. and spantide (10^–5 mol/l) and completely blocked when both byoscine and spantide were present. Contractions evoked by 5-methoxytryptamine were partially blocked by hyoscine (3 × 10^–6 mol/l) and were unaffected by spantide (10^–5 mol/l), but a combination of hyoscine and spantide completely blocked such responses.When the excitatory transmission was blocked with hyoscine (3 × 10^–6 mol/l) and spantide 10^–5 mol/l) and the tone of the muscle raised, an inhibitory response to 5-HT was revealed that had a threshold concentration between 10^–7 mol/l) and 3 × 10^–7 mol/l, and a maximum effect at 10^–4 mol/l. It was blocked by TTX (3 × 10^–7 mol/l) and granisetron 10^–6 mol/l. while N-nitro-l-arginine (NOLA) (10^–4 mol/l) and SDZ 205-557 (5 × 10^–7 mol/l) had no effect. Apamin A 10^–6 mol/l. partially suppressed this response.It is concluded that 5-HT3, 5-HT4 and 5-HT₁-like receptors mediate contraction of the longitudinal muscle of the distal colon. The 5-HT3 and 5-HT4 receptors are located on the excitatory motor neurons innervating the longitudinal muscle and the 5-HT₁-like receptor is located on the muscle. 5-HT3 receptors are also found on inhibitory neurons to the muscle. Correspondence to: D. J. Woollard at the above address     

Selective blockade of muscarinic M2 receptors in vivo by the new antagonist tripitramine

The antimuscarinic effects of tripitramine (1, 1, 24--tris [[5, 11-dihydro-6-oxo-6H-pyrido [2, 3-b][1, 4]-benzodiazepin-11-yl)(carbonyl] methyl]-8, 17-dimethyl-1, 8, 17, 24-tetraazatetracosane tetraoxalate), a member of a series of polymethylene tetraamines with in vitro cardioselectivity, were assessed in two in vivo preparations: anaesthetized and pithed rats. The well-known M₂ selective antagonist methoctramine was used in a comparative study. Tripitramine (0.0202 mol/kg i.v.) proved to be a potent antagonist at cardiac M₂ receptors that mediate the decrease in heart rate in the pithed rat; the same dose of this antagonist in the anaesthetized rat did not significantly affect the depressor action of methacholine mediated by vascular M₃ receptors. In the pithed rat, this dose did not affect the ganglionic M₁ receptor-mediated tachycardia and pressor response to muscarme or McN-A-343. These in vivo data are consistent with the in vitro findings and confirm that tripitramine is a more potent and selective muscarinic M₂ receptor antagonist than methoctramine.     

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.     

A1 adenosine receptors and muscarinic cholinoceptors in myocardial ischemia

The regulation of cardiac A₁ adenosine receptors and M₂ muscarinic cholinoceptors was investigated in ischemic rat hearts. Ischemia was induced in isolated, perfused hearts either by stop (stop-flow) or by reduction (low-flow) of perfusion flow. Receptor densities and affinities were determined by radioligand binding. The mRNA concentrations of the receptors and of control messages were measured by quantitative polymerase chain reactions (PCR). Second messenger coupling of the receptors was evaluated by measuring their inhibition of adenylate cyclase activity.Up to 60 min of stop-flow ischemia and 6 h of lowflow ischemia, cardiac A₁ adenosine receptor density and affinity, and adenosine receptor-mediated inhibition of adenylate cyclase, did not change significantly, compared to non-ischemic hearts. Receptor down-regulation, however, could be induced by perfusion with the A₁ receptor agonist R-phenyl-isopropyl-adenosine (R-PIA) during normal flow. After 6 h of perfusion with R-PIA (0.1 mol/l), A₁ adenosine receptor density was reduced. Agonist-induced receptor down-regulation was not found after perfusion with R-PIA in low-flow ischemia. The density and the affinity of muscarinic cholinoceptors were not affected during stop-flow ischemia up to 1 h either, whereas the density was down-regulated to 75% of controls (P<0.05) after 6 h of low-flow ischemia. This intervention also reduced inhibition of adenylate cyclase via muscarinic cholinoceptors. In non-ischemic hearts, perfusion with carbachol (10 µmol/l) suppressed receptor densities to 72% of control values.No significant changes in the concentration of A₁ adenosine receptor or M₂ cholinoceptor mRNAs occurred during normal flow, stop-flow and low-flow ischemia. Likewise, agonist stimulation with R-PIA or carbachol during normal flow did not change the respective receptor mRNA concentrations significantly. Conclusion: Although a down-regulation of A₁ adenosine receptor density was demonstrated after receptor agonist perfusion with normal flow, adenosine did not affect the density or functional activity of cardiac A₁ adenosine receptors in the ischemic myocardium. In contrast, muscarinic cholinoceptor density and function was down-regulated after prolonged ischemia. The lack of an agonist-induced down-regulation of A₁ adenosine receptors in the presence of decreasing activity of m-cholinoceptors suggests a growing importance of the adenosine system in myocardial ischemia.