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.     

Different muscarinic receptor subtypes mediating the phasic activity and basal tone of pig isolated intravesical ureter.

1. We have studied the effects of muscarinic cholinoceptor agonists and specific antagonists on both phasic activity and basal tone of the isolated intravesical ureter of the pig by means of isometric techniques in vitro. 2. Acetylcholine in the presence and absence of physostigmine increased both phasic activity and basal tone of ureteral strips in a concentration-dependent manner. Moreover carbachol, methacholine and oxotremorine-M increased both contractile parameters while bethanechol and McN-A-343 evoked only increases in tone without affecting the frequency of the phasic contractions. 3. The nicotinic receptor blocker, hexamethonium (10(-6)-10(-4) M), failed to modify the contractions evoked by a single dose of carbachol (10(-5) M), whilst the muscarinic antagonist, atropine inhibited both phasic and tonic responses. 4. The muscarinic M1 (pirenzepine), M2 (AF-DX 116 and methoctramine), M3 (4-DAMP, HHSiD and p-F-HHSiD), and putative M4 receptor (tropicamide) antagonists significantly reversed increases in both frequency of phasic activity and baseline tone induced by a submaximal dose of carbachol (10(-5) M). The pIC50 values for inhibition of the induced phasic activity were: atropine (10.16) > 4-DAMP (9.12) > HHSiD (8.22) = methoctramine (7.98) = p-F-HHSiD (7.88 > tropicamide (7.62) = pirenzepine (7.53) = AF-DX 116 (7.45) and for inhibition of basal tone were: atropine (10.73) > 4-DAMP (9.32) > HHSiD (8.65) = pirenzepine (8.43) = p-F-HHSiD (8.38) > methoctramine (7.79) > tropicamide (7.53) > AF-DX 116 (7.04).(ABSTRACT TRUNCATED AT 250 WORDS)     

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 the muscarinic receptor subtype that mediates the contractile response of acetylcholine in the swine myometrium

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([3H]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (-logEC50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA2 values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3 b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (-logIC50) was 4-DAMP (8.35) > tropicamide (6.72) > himbacine (6.54) > pirenzepine (6.31)> AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M2 and/or M4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (Kd = 0.92 nM; Bmax = 126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pKi value and Hill coefficient in parentheses): atropine (8.22 and 0.93) > 4-DAMP (8.18 and 0.94) > tropicamide (6.78 and 0.93) > pirenzepine (5.46 and 0.92) > AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M3 receptors present on smooth muscle cells.     

Muscarinic M3 receptors mediate contractions in rabbit endothelium-denuded aorta in vitro

1 Muscarinic receptors mediating contraction of rabbit endothelium-denuded aorta have been characterized functionally, in vitro, using a range of antagonists (atropine, pirenzepine, methoctramine, himbacine, 4-diphenyl-acetoxy-N-methyl piperidine methiodide (4-DAMP) and para-fluoro-hexahydro-siladifenidol (p-F-HHSiD). 2 The non-selective muscarinic agonist, (+)cis-dioxolane, induced concentration-dependent contractions of endothelium-denuded aortic rings. The potency (EC₅₀) of (+)cis-dioxolane was 1.0 ± 0.4 μM and the maximal increase in isometric tension was 944 ± 98 mg (mean ± SEM, n= 25). The concentration–effect curves to (+)cis-dioxolane were shifted to the right in the presence of antagonists, in a concentration-dependent manner. The following affinities (– log K_B) were calculated; atropine, 9.4; pirenzepine, 6.6; methoctramine, 5.9; himbacine, 7.1; 4-DAMP, 9.2; and p-F-HHSiD, 7.7. 3 It is concluded that muscarinic M₃ receptors mediate contractions of endothelium-denuded aorta. The low potency of (+)cis-dioxolane, when compared to its potency in other M₃ receptor assays, suggests that the efficiency of receptor coupling, associated with contraction of this tissue, is poor.     

Interaction of selective compounds with muscarinic receptors at dispersed intestinal smooth muscle cells.

1. The characterization of muscarinic receptors on single cells of the guinea-pig ileum longitudinal smooth muscle, devoid of neuronal elements, was functionally studied by estimating the affinities of muscarinic antagonists on acetylcholine-induced contractions. 2. Atropine (5 x 10(-11) to 5 x 10(-6) M), 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP, 5 x 10(-8) to 5 x 10(-6) M), cyclohexyl(4-fluoro-phenyl) (3-piperidinopropyl) silanol (pFHHSiD, 5 x 10(-7) to 5 x 10(-5) M) as well as pirenzepine (5 x 10(-7) to 5 x 10(-5) M) competitively antagonized the acetylcholine-dependent contractions with different affinities (atropine > 4-DAMP > pFHHSiD > pirenzepine). 3. Methoctramine (5 x 10(-7) to 5 x 10(-5) M), and AF-DX 116 (5 x 10(-6) and 5 x 10(-5) M) also showed antagonist properties but these deviated from simple competition. These compounds, which discriminate between M2 and M3 receptors, showed a potency lower than that of pirenzepine, the rank order of potencies being pirenzepine > methoctramine > AF-DX 116. When concentrations of AF-DX 116, methoctramine and pirenzepine were increased an unspecific contractile effect occurred. 4. McN-A-343, a partial agonist on intact guinea-pig longitudinal smooth muscle strips, on this preparation induced a weak contraction (about 7% in comparison to control) that was not reversed by antimuscarinic agents. 5. These data indicate that M3 rather than M2 receptor sites are present on this tissue.     

The minor population of M3‐receptors mediate contraction of human detrusor muscle in vitro

1 The objective was to determine the role of muscarinic receptor subtypes in mediating contraction of the human detrusor smooth muscle in vitro. 2 Contractile responses of human detrusor muscle strips to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (pirenzepine, methoctramine, 4‐diphenylacetoxy‐N‐methyl piperidine methiodide (4‐DAMP), tropicamide, oxybutynin and tolterodine). Affinity estimates (pK_B values) were calculated for the antagonists and correlated with values at the cloned muscarinic receptor subtypes quoted in the literature. 3 Pirenzepine, methoctramine and tropicamide drugs that have high affinities at M₁, M₂ and M₄‐receptors, respectively, all had low affinities on the human detrusor (pK_B values of 6.8, 6.9 and 6.5, respectively), whilst the M₃‐selective antagonist 4‐DAMP had a high affinity (9.5). Schild plots for all four antagonists had slopes of unity indicating an action at a single receptor. Oxybutynin and tolterodine also acted as competitive antagonists with affinity estimates of 7.6 and 8.1, respectively. 4 When the antagonist affinities obtained on the bladder were plotted against the values published for these antagonists at the cloned muscarinic receptor subtypes, the best correlations were obtained for the m3‐ and m5‐muscarinic receptor subtypes. 5 These data suggest that direct contractile responses of the human detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M₃‐muscarinic receptor subtype with no contribution from the major M₂‐receptor population.     

In vitro characterization of parasympathetic and sympathetic responses in cyclophosphamide-induced cystitis in the rat

In cyclophosphamide-induced cystitis in the rat, detrusor function is impaired and the expression and effects of muscarinic receptors altered. Whether or not the neuronal transmission may be affected by cystitis was presently investigated. Responses of urinary strip preparations from control and cyclophosphamide-pretreated rats to electrical field stimulation and to agonists were assessed in the absence and presence of muscarinic, adrenergic and purinergic receptor antagonists. Generally, atropine reduced contractions, but in contrast to controls, it also reduced responses to low electrical field stimulation intensity (1-5 Hz) in inflamed preparations. In both types, purinoceptor desensitization with alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) caused further reductions at low frequencies (<10 Hz). The muscarinic receptor antagonists atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) ('M(1)/M(3)/M(5)-selective'), methoctramine ('M(2)-selective') and pirenzepine ('M(1)-selective') antagonized the tonic component of the electrical field stimulation-evoked contractile response more potently than the phasic component. 4-DAMP inhibited the tonic contractions in controls more potently than methoctramine and pirenzepine. In inflamed preparations, the muscarinic receptor antagonism on the phasic component of the electrical field stimulation-evoked contraction was decreased and the pirenzepine and 4-DAMP antagonism on the tonic component was much less efficient than in controls. In contrast to controls, methoctramine increased -- instead of decreased -- the tonic responses at high frequencies. While contractions to carbachol and ATP were the same in inflamed and in control strips when related to a reference potassium response, isoprenaline-induced relaxations were smaller in inflamed strips. Thus, in cystitis substantial changes of the efferent functional responses occur. While postjunctional beta-adrenoceptor-mediated relaxations are reduced, effects by prejunctional inhibitory muscarinic receptors may be increased.     

Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA₂-values characteristic of M₃ (smooth muscle/glandular) muscarinic receptors. Since these pA₂-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M₃ subtype-selective muscarinic antagonists to be used as bronchodilators.     

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     

The Sources of Ca2+ for Muscarinic Receptor-induced Contraction in the Rat Ileum

The contractile responses obtained by activation of different muscarinic receptor subtypes in the longitudinal muscle of the rat ileum and especially the responses of this muscle to acetylcholine in a Ca²⁺-free medium have been investigated. In Ca²⁺-containing solution, acetylcholine elicited similar concentration-dependent contractile responses in the duodenum, jejunum and ileum strips of the rat intestine. The response to a maximal concentration of the agonist (1 μM) consisted of a rapid phasic response followed by a slower tonic one. Nifedipine completely relaxes or inhibits the sustained response and only partially diminishes the phasic one, which suggests that the phasic contraction depends on the release of internal Ca²⁺ as well as Ca²⁺ entry from the extracellular space through voltage-dependent Ca²⁺ channels, but that the tonic contraction only depends on the influx of the external ion. In Ca²⁺-free medium, acetylcholine (1 μM) induced phasic contractions that depend on the release of this ion from internal stores. Participation of different subtypes of receptors (M₁, M₂ and M₃) in these responses depends on the inhibitory action shown by methoctramine, 4?DAMP and atropine but not by pirenzepine in two different experimental models.     

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     

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.     

Cholinergic Modulation of Electrogenic Ion Transport in Different Regions of the Rat Small Intestine

Acetylcholine acting via muscarinic receptors located in the intestinal mucosa controls ion and fluid transport. This study examined the pathway(s) by which cholinergic receptors mediate secretion in rat isolated duodenum, jejunum and ileum using the short-circuit current (Isc) as an index of electrogenic Cl^? secretion. Carbachol and bethanechol induced electrogenic Cl^? transport which was insensitive to the neural blocker tetrodotoxin, indicating their direct action on the enterocytes. Functional characterization of electrogenic secretion activated via muscarinic receptors on jejunal and ileal enterocytes was achieved by use of selective muscarinic antagonists in the presence of tetrodotoxin. In both regions the rank order of potency of these compounds (atropine > 4-diphenylacetoxy-N-piperidine methiodide (4-DAMP) > hexahydro-sila-difenidol (HHSiD) > pirenzepine > methoctramine) indicated the M₃ receptor subtype. Secretion activated by the muscarinic agonist 4-[[(3-chlorophenyl)amino]carbonyl]-N,N,N-trimethyl-2-butyn-1-ammonium chloride (McN-A-343) was sensitive to tetrodotoxin and pirenzepine but not to the ganglionic blocker, hexamethonium, indicating the M₁ receptor subtype on post ganglionic neurons. Regional differences for bethanechol-activated secretion showed an increasing gradient in secretory capacity (Isc max) in a proximal-to-distal direction along the small intestine. Responses to McN-A-343 also showed regional differences but these were unlike those of bethanechol. These results show that cholinomimetic-induced electrogenic Cl^? secretion in rat isolated small intestine appears to be mediated by two dissimilar populations of muscarinic receptor: M₃ muscarinic receptors positioned on enterocytes and M₁ muscarinic receptors sited on submucosal neurons.     

Characterization of muscarinic receptors mediating contractions of circular and longitudinal muscle of human isolated colon.

1. The effects of seven muscarinic receptor antagonists were used to characterize the receptors which mediate carbachol-evoked contractions of intertaenial circular and taenial longitudinal muscle in human isolated colon. The effects of these antagonists were studied upon colon contractions induced by cumulatively added carbachol which had mean EC50 values of 11.7 +/- 2.3 microM (n = 8) and 12.6 +/- 2.3 microM (n = 8) respectively upon circular and longitudinal smooth muscle. 2. All antagonists displaced concentration-response curves to carbachol to the right in a parallel manner. The maximum concentration of each antagonist added (30 nM-10 microM) did not significantly suppress the maximum response. 3. In circular muscle, the M3 muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladiphenidol (HHSiD) and para-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) inhibited responses with pA2 values of 9.41 +/- 0.23, 7.17 +/- 0.07, 6.94 +/- 0.18 respectively. The M2 muscarinic receptor antagonist, AF-DX 116, the M2/M4 muscarinic receptor antagonist, himbacine, and the M1 muscarinic receptor antagonist, pirenzepine, yielded pA2 values of 7.36 +/- 0.43, 7.47 +/- 0.14 and 7.23 +/- 0.48 respectively. The non-selective antagonist, atropine, had a pA2 of 8.72 +/- 0.28. 4. In longitudinal muscle 4-DAMP, HHSiD, p-F-HHSiD, AF-DX 116, himbacine and pirenzepine gave pA2 values of 9.09 +/- 0.16, 7.45 +/- 0.43, 7.44 +/- 0.21, 6.44 +/- 0.1, 7.54 +/- 0.40, 6.87 +/- 0.38 respectively. Atropine yielded a pA2 value of 8.60 +/- 0.08.(ABSTRACT TRUNCATED AT 250 WORDS)     

M2 muscarinic receptors on the iris sphincter muscle differ from those on iris noradrenergic nerves

The pre- and postjunctional affinity constants of a series of muscarinic antagonists were determined in guinea pig and rabbit irises. Field stimulation-evoked [3H]noradrenaline release from superfused isolated irises was concentration dependently inhibited by (+/-)-methacholine, confirming the presence on the iris noradrenergic nerves of prejunctional inhibitory muscarinic receptors. The affinity constants of the antagonists at the pre- and postjunctional receptors are compatible with the coexistence in the iris of two different M2 receptors: the cardiac (M2 alpha) subtype on the noradrenergic nerves and the smooth muscle (M2 beta) subtype on the iris sphincter muscle. The rank order of potency of the antagonists studied at the prejunctional site was: atropine greater than himbacine greater than AF-DX 116 greater than pirenzepine greater than hexahydrosiladifenidol. The order of potency at the postjunctional receptors mediating the methacholine-induced isotonic contraction of the isolated rabbit iris sphincter was: atropine greater than hexahydrosiladifenidol greater than pirenzepine greater than himbacine greater than AF-DX 116.     

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     

Functional muscarinic cholinoceptors in the isolated canine ureter

The purpose of present study was to characterize the functional muscarinic cholinoceptor (mAChR) subtypes in the isolated canine ureter. Carbachol (CCh), a non-selective mAChR agonist, concentration-dependently increased the frequency of the rhythmic contractions in isolated spiral ureteral preparations, the pD(2) value being 5.78+/-0.12. We then evaluated the effects of subtype-selective mAChR antagonists on the CCh-induced rhythmic contractions. The rank order of antagonistic potencies (apparent pA(2)) was 4-diphenylacetoxy- N-methylpiperidinemethiodide (4-DAMP; M3-subtype selective; 9.31+/-0.06) >atropine (non-selective; 9.16+/-0.10) >himbacine (M4-subtype selective; 7.32+/-0.18) >pirenzepine (M1-subtype selective; 6.78+/-0.16) >methoctramine (M2-subtype selective; 5.51+/-0.43). In sharp contrast, CCh concentration-dependently reduced the 80 mM KCl-induced contraction in longitudinal ureteral preparations, the pD(2) value being 4.83+/-0.10. On this CCh-induced ureteral relaxation, the rank order of antagonistic potencies (apparent pA(2)) was atropine (8.56+/-0.09) >4-DAMP (7.63+/-0.21) >himbacine (7.46+/-0.09) >methoctramine (6.54+/-0.18) >pirenzepine (6.33+/-0.22). The nitric-oxide-synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG; 1 x 10(-4) M) had no effect on the CCh-induced ureteral relaxation. These data suggest that the CCh-induced rhythmic contraction in the spiral preparation was mediated via the M3-receptor, while the CCh-induced relaxation in the longitudinal preparation was probably mediated mainly via the M4-receptor.