The Involvement of Muscarinic Receptor Subtypes in the Mediation of Hypothermia,Tremor, and Salivation in Male Mice

The potency of centrally administered non-selective (atropine and N-methyl scopolamine) and putatively selective muscarinic antagonists (pirenzepine, AF-DX 116 and 4-DAMP) in inhibition of oxotremorine-induced hypothermia, tremor and salivation in male mice has been compared with their potency in vitro in three functional systems, where muscarinic effects are mediated preferentially by M₁ (i.e. superior cervical ganglion), M₂ (i.e. atrium), and M₃ (i.e. ileum) receptors. Atropine, N-methyl scopolamine and 4-DAMP potently abolished the effects of oxotremorine. Pirenzepine abolished tremor and salivation, whereas hypothermia was antagonized partially only. AF-DX 116 had but weak antagonistic effects. Atropine and N-methyl scopolamine were potent antagonists in all three in vitro test systems. High potency was also seen with 4-DAMP, in particular in the ileum preparation. Pirenzepine showed its highest potency in the ganglion preparation. AF-DX 116 was a weak and non-selective antagonist in all three in vitro preparations. Our studies indicate that the muscarinic induction of tremor and salivation may be preferentially mediated by M₃ receptors whereas both M₂ and M₃ receptors may be involved in the mediation of hypothermia. However, the overall conclusion is that compounds with higher receptor subtype selectivity are needed.     

Pharmacological characterization of muscarine receptors of PC 12 (rat phaeochromocytoma) cells

Summary The muscarinereceptors of PC12 (rat phaeochromocytoma) cells were studied in functional and binding experiments. The catecholamine stores of PC 12 cells were labelled by incubation of the cells with tritiated noradrenaline. Muscarinic agonists elicited concentration-dependent release of tritium which consisted overwhelmingly of unchanged ³H-noradrenaline. The rank order of potency was: oxotremorine > acetylcholine > muscarine = methacholine > carbachol > bethanechol. The release evoked by carbachol (0.1 mmol/l) was inhibited with high potency by the M₁-selective antagonist telenzepine (pK _i = 8.82), with intermediate potency by pirenzepine (pK _i = 7.00) and with low potency by the M₂-selective antagonist AF-DX 116 (pK _i = 5.74).The binding of 3H-N-methylscopolamine to PC 12 membranes was inhibited by various non-selective and subtype-selective muscarinic antagonists with the following rank order of potency: telenzepine = atropine > 4-DAMP > dicyclomine > pirenzepine > HHSiD > AF-DX 116. A similar rank order was obtained for the inhibition by these compounds of 3H-telenzepine binding to Mi-receptors in membranes of the cerebral cortex of the guinea pig. The Hill coefficients for inhibition of 3H-N-methylscopolamine binding (to PC 12 membranes) by pirenzepine, telenzepine and AF-DX 116 were below unity. Specific binding of both ³H-telenzepine and 3H-N-methylscopolamine to muscarine receptors of PC 12 membranes was saturable and of high affinity; the maximal number of binding sites was higher for ³H-N-methylscopolamine than for 3H-telenzepine (calculated for the active (+)enantiomer).PC 12 cells are presumably endowed with more than one subtype of muscarine receptors. The predominant receptor is an atypical receptor; it is neither a M₂- nor a M₃-receptor, and in spite of the high affinity of telenzepine for this receptor it is probably also not an M₁-receptor.     

An M3-like muscarinic autoreceptor regulates the in vivo release of acetylcholine in rat striatum

Selective muscarinic antagonists were used in an attempt to characterize the muscarinic autoreceptor modulating the release of acetylcholine in the striatum of the rat. In vivo microdialysis was applied to infuse atropine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), pirenzepine or AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro[2,3-b][1,4]benzodiazepine-6-one), leading to a dose-dependent increase in the overflow of acetylcholine, the order of potency being: atropine greater than 4-DAMP greater than pirenzepine greater than AF-DX 116. We conclude from these data that the muscarinic receptor modulating release in the striatum is of the M3 type.     

Acetylcholinesterase histochemistry and functional characterization of the muscarinic receptor mediating the contraction of the bovine oesophageal groove

1 By using acetylcholinesterase (AChE) histochemistry and invitro isometric techniques, we have studied the presence and distribution of AChE-positive nerves, as well as the effects of muscarinic cholinoceptor agonists and selective antagonists, in the bovine oesophageal groove. 2 AChE-positive nerves and cells were distributed widely on the oesophageal groove floor. These fibres originated from adventitial ganglia containing bodies with high AChE activity and were shown grouped as large adventitial nerve bundles. 3 Both in the presence and absence of physostigmine, acetylcholine (ACh) induced concentration dependent contractions of bovine oesophageal groove strips. The rank order of the pD₂ values for muscarinic agonists was: oxotremorine-M (7.37) = carbachol (7.14) > acetylcholine plus physostigmine (6.46) > bethanechol (5.42) > McN-A-343 (4.45) > acetylcholine (4.06). 4 Hexamethonium (10^?6–10^?4M ), a nicotinic receptor blocker, did not affect the carbachol concentration–response curve, which was significantly inhibited by the muscarinic antagonist, atropine (10^?9–10^?8M ). 5 The preferential muscarinic antagonists pirenzepine (M₁), 11-(2(-(diethyl-amino)methyl)-1-piperidinylacetyl)-5,-11-dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepine-6-one (AF-DX 116) and methoctramine (M₂), 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) and p-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) (M₃) and tropicamide (M₄) evoked rightwards displacements in a parallel manner of the carbachol control curve, and there was no decrease of the maximum response with the highest concentration of antagonist utilized. The muscarinic antagonist affinities, expressed in terms of pA₂ values, were: atropine (9.51) = 4-DAMP (9.32) > p-F-HHSiD (7.78) > tropicamide (7.40) > pirenzepine (6.91) = AF-DX 116 (6.88) = methoctramine (6.71). This muscarinic antagonist profile suggests that an M₃ receptor is involved in the carbachol induced contraction. 6 The present results suggest that a rich network of AChE- positive fibres is present in the oesophageal groove floor, where they form a nerve trunk and thinner branches accompanying blood vessels and sometimes around ganglia. The muscarinic cholinergic contraction of the bovine oesohageal groove seems to be mediated via activation of an M₃ postsynaptic muscarinic receptor.     

AF-DX 116 differentiates between prejunctional muscarine receptors located on noradrenergic and cholinergic nerves

Summary Prejunctional affinity constants of the cardioselective muscarine receptor antagonist AF-DX 116 (11-[(2[(diethyl-amino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro6 H-pyrido [2,3-b] [1,4] benzodiazepine-6-one) were determined for muscarine autoreceptors on cholinergic nerves of the guinea-pig ileum and for heteroreceptors on noradrenergic nerves of the rat heart and guinea-pig iris. AF-DX 116 antagonized with low affinity the muscarinic inhibition induced by arecaidine propargyl ester of the stimulation-evoked [³H]acetylcholine overflow (pA₂ 6.74) from the guinea-pig ileum. In contrast, AF-DX 116 was more potent in antagonizing the methacholine-induced inhibition of the stimulation-evoked [³H]noradrenaline overflow from rat heart (pA₂ 7.29) or guinea-pig iris (pA₂ 7.57). The data confirm previously reported differences between prejunctional muscarine heteroreceptors in the rat heart which belong to the cardiac subtype (M₂ or M₂) and autoreceptors in the guinea-pig ileum that cannot be distinguished from the ileal subtype (M₂) or (M₃). Send offprint requests to H. Fuder at the above address     

Characterization of presynaptic vascular muscarinic receptors inhibiting endogenous noradrenaline overflow in the portal vein of the freely moving rat.

1. In the portal vein of permanently cannulated, freely moving, unanaesthetized rats, methacholine (MCh) is able to inhibit the electrically-evoked endogenous noradrenaline (NA) overflow. This inhibition is mediated by presynaptic inhibitory muscarinic heteroreceptors. 2. By use of pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) and AF-DX 116 as M1-, M3-, and M2-selective antagonists respectively, the MCh (0.1 microM)-induced inhibition of the electrically-evoked NA overflow could be reversed to the control stimulation value dose-dependently. 3. The potency order of the antagonists was: 4-DAMP greater than AF-DX 116 greater than pirenzepine, pIC50 values being 8.50, 7.96 and 7.01, respectively. 4. From these results it was concluded that the inhibitory presynaptic heteroreceptors in the portal vein of conscious unrestrained rats are of the cardiac M2-subtype.     

M3-muscarinic receptor mediates prejunctional inhibition of noradrenaline release and the relaxation in cat femoral artery.

The aim of the present study was to analyse the muscarinic receptors involved in the vasodilation elicited by acetylcholine (ACh) and the carbachol inhibition of electrically-evoked [3H]noradrenaline (NA) release in cat femoral artery. For this purpose, the following receptor antagonists were used, atropine, pirenzepine (M1-antagonist), AF-DX 116 (M2-antagonist) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3-antagonist). The order of potency (pA2 values) of these drugs at postjunctional level was: atropine (9.7) greater than or equal to 4-DAMP (9.6) greater than pirenzepine (7.2) greater than AF-DX 116 (6.0), and at prejunctional level (pIC50 values) was: 4-DAMP (9.3) greater than atropine (8.5) greater than AF-DX 116 (7.1) greater than pirenzepine (5.9). These findings indicate that the muscarinic receptors mediating the vasodilation induced by ACh and the carbachol inhibition of NA release are of the M3-subtype.     

Presynaptic muscarinic receptor subtypes involved in the inhibition of acetylcholine and noradrenaline release in bovine cerebral arteries

Summary Experiments were performed in bovine cerebral arteries preincubated with [³H]-choline or [³H]-noradrenaline to analyze the presynaptic muscarinic receptors involved in inhibition of acetylcholine and noradrenaline release induced by electrical stimulation (4 Hz, 200 mA, 0.3 ms, 1 min). For this purpose, the actions of several muscarinic receptor antagonists on the ³H overflow and on the carbacol-induced inhibition of this overflow were assessed. The evoked [³H]-acetylcholine release and [³H]-noradrenaline release were markedly reduced by the presence of tetrodotoxin, Ca²⁺-free medium, and the inhibitor of both choline transport and choline acetyltransferase, AF64A. Chemical sympathetic denervation with 6-hydroxydopamine (6-OHDA) decreased the uptake of[³H]-noradrenaline, and AF64A reduced mainly the uptake of [³H]-choline, but also of [³H]-noradrenaline. Carbachol reduced the evoked [³H]-noradrenaline and [³H]-acetylcholine release; the IC₅₀ values were 0.37 and 0.43 mol/l, respectively.Atropine and 4-DAMP, but not AF DX 116, methoctramine or pirenzepine, increased the evoked [³H]-acetylcholine release. However, these muscarinic antagonists failed to modify the evoked [³H]-noradrenaline release. Carbachol inhibited the release of both acetylcholine and noradrenaline. The inhibition was blocked by the antagonists. The rank orders of potency (based on plC₅₀ values) were, in the case of [³H]-acetylcholine release, atropine > 4-DAMP >AF-DX 116 >- pirenzepine >- methoctramine, and, in the case of [³H]-noradrenaline release, atropine > 4-DAMP > AF-DX 116 >- methoctramine >-pirenzepine. These results suggest (1) that the prosynaptic receptors that modulate endogenous acetylcholine release are likely of the M₃ subtype, whilst those involved on the effect of the exogenous agonist Carbachol are of M₂ subtype, and (2) that those which inhibit noradrenaline release are probably a mixture of M₂ and M₃ subtypes as well. The autoinhibition of the acetylcholine release was funtionally active under our experimental conditions, while noradrenaline release does not appear to be modulated by muscarinic receptors in physiological conditions.Send offprint requests to G. Balfagón at the above address     

Characterization of porcine coronary muscarinic receptors

Summary To determine the muscarinic receptor subtype involved in the contractile response of coronary smooth muscle, we investigated the profiles of various muscarinic receptor antagonists competing for [³H]N-methyl-scopolamine ([³H]NMS) binding to membrane preparations from porcine coronary arteries. [³H]NMS binds to a single population of muscarinic binding sites with a K_D of 135 pM and a B_max of 57 fmol/mg. The affinity profiles of AF-DX 116 [11-2((–((diethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one], atropine, 4-DAMP [4-diphenylacetoxy-N-methylpiperidine methiodide], methoctramine [N,N-bis (6-((2-methoxybenzyl) amino)hexyl)-1,8-octane-diamine tetrahydrochloride], HHSiD [hexahydrosiladi-fenidol] and pirenzepine are consistent with binding to a mixed population of muscarinic binding sites, namely of the M₂ and M₃ subtype.Binding curves for AF-DX 116 and methoctramine are shallow with Hill-coefficients significantly less than unity. Comparison of data from binding studies with results obtained in functional experiments, i.e. antagonism of methacholine induced contraction of porcine coronary artery rings, it was found that only the low-affinity pK_i values of AF-DX 116 (6.26) and methoctramine (6.51) correlated well with functional pA₂ values.It is concluded that a mixed population of the M₂ and M₃ muscarinic receptor subtypes is present in porcine coronary arteries. Functional experiments do not support the contribution of the M₂ subtype to the contractile response. Cholinergic induced contractions of porcine coronary arteries appear to be evoked via stimulation of the muscarinic M₃ receptor subtype. However, since the compounds investigated here do not markedly discriminate between cloned m₃, m₄ and m₅ receptors the involvement of muscarinic receptors different from M₁, M₂ and M₃ cannot be excluded. Send offprint requests to M. Entzeroth at the above address     

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of [³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of [³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [³²P]NAD caused the [³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address     

Effect of muscarinic receptor blockade on canine gastric tone and compliance in vivo.

Muscarinic pathways are involved in maintaining gastric tone during fasting and atropine is known to decrease gastric tone via blockade of a tonic vagal cholinergic input. Our aim was to assess the role of different muscarinic receptors in modulating canine gastric tone and compliance in vivo by using "selective" muscarinic receptor antagonists (telenzepine, AF-DX 116 and 4-DAMP for M1, M2, and M3 receptors, respectively) and the non-selective muscarinic receptor antagonist atropine. In four fasting, conscious dogs, we characterized the pressure-volume relationship in the proximal stomach by using a barostat. Drug effects were investigated by studying pressure-volume relationships before and 15 min after intravenous administration telenzepine, AF-DX 116, 4-DAMP or atropine. Pressure-volume curves were fitted by non-linear regression analysis. Before drug administration, the curve that best fitted the pressure-volume relationship was exponential. Atropine (100 microg kg-1) immediately decreased baseline gastric tone, i.e. relaxed the stomach (Deltavolume at 2 mmHg=236+/-15 ml; P<0.05), and significantly (P<0.01) shifted the pressure-volume curve to the left. Telenzepine, at the lowest dose (3 microg kg-1), shifted the pressure-volume curve to the right (P<0.01). AF-DX 116 at the lower dose (422 microg kg-1) had no effect on baseline gastric tone or the gastric pressure-volume curve, whereas the higher dose (2532 microg kg-1) significantly shifted the pressure-volume curve to the left (P<0.01), but did not increase baseline gastric volume. Finally, 4-DAMP (13.5, 45, 135 microg kg-1) immediately decreased baseline gastric tone (Deltavolume at 2 mmHg=97+/-29 ml, 110+/-35 ml and 155+/-21 ml, respectively) and significantly shifted the pressure-volume curve to the left (P<0.01). We conclude that muscarinic pathways are involved in modulating canine gastric tone and compliance during fasting: M3 receptors seem to play a key role in excitatory pathways, whereas the shift of pressure-volume curve to the right observed with the lowest dose of telenzepine is consistent with the existence of M1 receptors on inhibitory pathways.     

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart

Summary The effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [³H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (±)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenidol > AF-DX 116. The comparison with known pA₂ values for M₁-, M₂- and M₃-receptors revealed that the presynaptic autoreceptor meets the criteria of an M₁-receptor. Basal, not electrically evoked overflow of unlabelled ACh into the perfusate was caused by leakage release (non-exocytotic), as it was independent of extracellular Ca²⁺ . Muscarinic receptor antagonists failed to enhance basel overflow. In contrast, when ChE activity was inhibited by 10^–6M tacrine or pretreatment with 10^–4M DFP, the ACh overflow was partially Ca²⁺-dependent and was reduced by tetrodotoxine. Moreover, block of the inhibitory muscarinic autoreceptors by (±)-telenzepine or pirenzepine caused a several-fold enhancement of the ACh release. The potencies of these antagonists were identical to those found for the electrically evoked [³H]-ACh release. The rate of ACh release enhanced by ChE inhibition plus telenzepine corresponds to about 12% of the total ACh pool per min, which is about the maximum amount of ACh that is available for any kind of stimuli. The release was dependent on the presence of exogenous choline. Hence elevation of ACh release led to a correspondingly enhanced ACh synthesis. The dramatic enhancement of ACh release by the ChE inhibition in combination with a block of presynaptic muscarinic autoinhibition was not inhibited by (+)-tubocurarine but by atropine (10^–9 to 10^–7 M) or 10^–6 M telenzepine. It is concluded that basal release of ACh in the heart was due to non-exocytotic leakage release. Inhibition of ChE led to a marked stimulation of excitatory muscarinic receptors of the intrinsic parasympathetic neuron with a consecutive postganglionic release of ACh. The strong postganglionic excitation was obvious when the inhibitory muscarinic autoreceptors were selectively blocked. Of the two described muscarinic receptors found in the parasympathetic postganglionic neuron of the chicken heart only the inhibitory was classified as being M₁, whereas the subtype of the excitatory one is unlike M₁ and remains to be identified.Preliminary results have been presented at the Spring meeting of the German Pharmacological Society in 1992 (Brehm and Lindmar 1992) Correspondence to R. Lindmar at the above address     

Muscarinic M3 receptors mediate total inositol phosphates accumulation in murine HSDM1C1 fibrosarcoma cells

Muscarinic receptors in murine fibrosarcoma HSDM1C1 cells were characterized using both radioligand binding and total inositol phosphates accumulation (IPs). Muscarinic agonists elicited a concentration-dependent enhancement of IPs accumulation with a maximum of 14-fold stimulation above basal level. The following potencies ( - log EC₅₀) were observed for the full agonists: ( + )-cis-dioxolane 5.4, oxotremorine-M 5.3, ( + )-muscarine 5.2 and carbachol 5.0. Bethanechol (4.1) and arecoline (5.0) were partial agonists, evoling 43 and 55%, respectively of the maximum level of stimulation to ( + )-cis-dioxolane, whereas pilocarpine and McN-A-343 were inactive as agonists (1 μmol/1-1 mmol/1) The apparent affinities for muscarinic antagonists ( - log K_B) estimated by Schild regression were: 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) 9.2, dicyclomine 7.0, pirenzepine 6.9, (±)-p-F-HHSiD (para-fluoro-hexahydro-siladifenidol) 7.0, AF-DX 116 6.2, methoctramine 5.7. In saturation binding studies using [³H]N-methylscopolamine a homogeneous population of sites was identified, with a density of 145 pmol/mg protein. In competition radioligand binding studies, the following apparent affinities ( - log K_i) were observed: 4-DAMP 9.7, dicyclomine 8.3, (±)-p-F-HHSiD 7.6, AF-DX 116 6.8, methoctramine 6.6 and gallamine 6.8. In binding studies all antagonists studied recognized a single population of sites, as judged by the Hill coefficients from the displacement isotherms. These data are consistent with HSDM1C1 cells expressing an apparent homogeneous muscarinic M₃ population that mediates a large level of total IPs accumulation. This clonal line may provide a useful model to further elucidate relationship between endogenous muscarinic M₃ receptor stimulation and IPs accumulation. Additionally, these studies illustrate the importance of characterizing muscarinic receptor subtypes by null methods when using biochemical responses in isolated cells.     

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     

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     

Novel pharmacological profile of muscarinic receptors mediating contraction of the guinea-pig uterus

Summary The present study was designed to further characterize the muscarinic receptors mediating contraction of the guinea-pig uterus. The affinities of various selective muscarinic antagonists were determined and compared with those obtained at M₁ (rabbit vas deferens), M₂ (guinea-pig atria) and M₃ receptors (guinea-pig ileum).The contractile responses of uterine smooth muscle from immature guinea-pigs to carbachol (pD₂ = 5.73) were competitively antagonized by pirenzepine (pA₂ = 7.04), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]- 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzo. diazepin-6-one) (pA₂ = 6.96), himbacine (pA₂ = 7.92), methoctramine (pA₂ = 7.52), 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) (pA₂ = 8.87) and sila-hexocyclium (pA₂ = 8.81). A comparison of affinity values indicates that the muscarinic receptors present in guinea-pig uterus display a novel pharmacological profile which is not consistent with the presence of either an M₁, M₂ or M₃ receptor. The affinities determined for the different antagonists rather showed a close similarity to those obtained at muscarinic receptors present in rat striatum and NG108-15 cells which are considered pharmacological equivalents (M₄ receptors) of the m4 gene product. We thus hypothesize that the guinea-pig isolated uterus preparation may serve as a simple functional assay system to study the pharmacology of M₄ receptors.This work has been presented in part at the Spring Meeting of the German Society for Pharmacology and Toxicology in Mainz, March 1990 (Dörje et al. 1990) Send offprint requests to F. Dörje at the present address     

Binding of muscarine receptor antagonists to pig coronary smooth muscle

Summary In order to characterize the muscarinic binding site on coronary smooth muscle, we investigated the binding properties of (³H)quinuclidinyl benzilate (QNB) in membrane preparations of pig coronary arteries and atria. Scatchard analysis and Hill plot showed that (³H)QNB binds to a single population of sites in both tissues. The binding profiles of the muscarine receptor antagonists atropine, 11-((2-((dimethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b) (1,4)benzodiazepine-6-one (AF-DX 116), pirenzepine, and 4-diphenylacetoxy-Nmethylpiperidine methiobromide (4-DAMP) in both tissues were compared with binding data from other tissues, representative for different muscarinic binding site subtypes. It is concluded that the pig coronary smooth muscle muscarinic binding site is different from M₁ and M₂ binding sites investigated so far. Send offprint requests to I. Rinner     

Neuromuscular facilitation induced by muscarinic antagonists in the rat isolated diaphragm

• 1.1. Atropine (EC₅₀ = 87 μM), pirenzepine (447 μM), and AF-DX 116 (95.5 μM), but not 4-DAMP (at concentrations of up to 110 μM), produced neuromuscular facilitation and antagonized the oxotremorine-induced neuromuscular blockade in the rat isolated diaphragm.
• 2.2. Atropine, pirenzepine, and AF-DX 116 did not change the responses of curarized diaphragms to direct stimulation, or the twitch tension produced by retrograde injection of acetylcholine.
• 3.3. These results indicate that neuromuscular facilitation induced by muscarinic antagonists may depend on drug interaction with the M2 subtype of muscarinic autoreceptors to increase acetylcholine output in the neuromuscular junction.

     

Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology

The nature of the muscarinic receptor subtype mediating the endothelium-dependent relaxation of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted arterial segments. Relaxation induced by several agonists (acetylcholine (ACh), acetyl-beta-methylcholine, oxotremorine, carbachol and McN-A-343) was recorded. The ability of selective (pirenzepine, dicyclomine, adiphenine, AF-DX 116, methoctramine, gallamine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD] and non-selective antagonists (atropine, scopolamine and quinuclidinyl benzilate (QNB] to block the relaxation induced by ACh was also estimated. The weak activity of the poorly selective M1 muscarinic receptor as together with the intermediate affinity of pirenzepine and adiphenine tend to exclude the M1 muscarinic receptor as the primary mediator of the cholinergic relaxation. The low affinity of AF-DX 116 and methoctramine further suggested that the cerebrovascular muscarinic receptor does not correspond to the M2 cardiac subtype. In contrast, 4-DAMP and HHSiD potently inhibited the ACh-induced relaxation with affinities similar to those reported at the M3 glandular receptor. We conclude that a similar to the pharmacological M3 muscarinic receptor subtype is responsible for the cholinergic relaxation of the cat middle cerebral artery.     

Muscarinic excitatory and inhibitory mechanisms involved in afferent fibre-evoked depolarization of motoneurones in the neonatal rat spinal cord.

1. The involvement of acetylcholine and muscarinic receptors in spinal synaptic responses evoked by electrical and noxious sensory stimuli was investigated in the neonatal rat spinal cord in vitro. 2. Potentials were recorded extracellularly from a ventral root (L3-L5) of the isolated spinal cord, spinal cord-cutaneous nerve, and spinal cord-skin preparations of 1- to 4-day-old rats. Spinal reflexes were elicited by electrical stimulation of the ipsilateral dorsal root or the cutaneous saphenous nerve, or by noxious skin stimulation. 3. Single shock stimulation of supramaximum intensity of a dorsal root induced a mono-synaptic reflex in the corresponding ventral root. Bath-application of the muscarinic agonists, muscarine (0.3-30 microM) and (+)-cis-dioxolane (0.1-100 microM), produced an inhibition of the mono-synaptic reflex and a depolarization of motoneurones. Other muscarinic agonists, arecoline (10 nM-10 microM) and oxotremorine (10 nM-1 microM), inhibited the mono-synaptic reflex with little or no depolarization of motoneurones. Repetitive stimulation of the saphenous nerve at C-fibre strength induced a slow depolarizing response lasting about 30 s of the L3 ventral root. This slow ventral root potential (VRP) was also inhibited by arecoline (10 nM-10 microM) and oxotremorine (10 nM-1 microM). 4. In the spinal cord-saphenous nerve-skin preparation, a slow VRP was evoked by application of capsaicin (0.5 microM), bradykinin (3 microM), or noxious heat (47 degrees C) to skin. This slow VRP was depressed by the muscarinic agonists, arecoline (3 microM) and oxotremorine (1 microM). 5. Of the (+)-cis-dioxolane-induced inhibition of mono-synaptic reflex and motoneurone depolarization, the M2 antagonists, AF-DX 116 (0.1-1 microM) and methoctramine (100-300 nM), preferentially blocked the former response, whereas the M3 antagonists, 4-DAMP (3-10 nM) and p-F-HHSiD (0.3-3 microM), preferentially blocked the latter response. AF-DX 116 (0.1-1 microM) and methoctramine (100-300 nM) also effectively antagonized the arecoline- and oxotremorine-induced inhibition of the slow VRP. The pA2 values of AF-DX 116 and methoctramine against the arecoline-induced inhibition of the mono-synaptic reflex were both 6.79, and that of 4-DAMP against the (+)-cis-dioxolane-induced motoneurone depolarization was 8.16. 6. In the spinal cord-cutaneous nerve preparation, the saphenous nerve-evoked slow VRP was augmented by the anticholinesterase, edrophonium (5 microM). AF-DX 116 (1 microM) and methoctramine (100 nM) also potentiated the slow VRP, whereas 4-DAMP (10 nM) depressed the response.(ABSTRACT TRUNCATED AT 400 WORDS)