piperidines and hexahydrosiladifenidol

Topics: Animals; Atropine; Carbachol; Cell Line; Humans; Oxotremorine; Piperidines; Pirenzepine; Receptors, Muscarinic; Recombinant Proteins; Second Messenger Systems; Transfection

A series of hexahydro-difenidol (HHD) and hexahydro-sila-difenidol (HHSiD) analogues modified in the amino group, the phenyl ring and in the alkylene chain were investigated for their binding and functional properties at muscarinic M1, M2 and M3 receptors. Novel muscarinic receptor antagonists were obtained which exhibited different receptor selectivity profiles from the parent compounds HHD and HHSiD (M1 congruent to M3 greater than M2), e.g. HHD and HHSiD methiodides, M1 greater than M2 congruent to M3; p-fluoro-HHSiD, M3 greater than M1 greater than M2; trans-hexbutenol, M1 greater than M3 greater than M2; and (s)-p-fluoro-hexbutinol, M3 greater than M2 congruent to M1. Stereoselectivity ratios [(R)/(S)] for the enantiomers of HHD, hexbutinol and p-fluoro-hexbutinol were highest at M1, intermediate at M3 and lowest at M2 receptors.

Topics: Animals; Humans; Parasympatholytics; Piperidines

In an attempt to examine whether the muscarinic receptor-activated intestinal function is altered by aging, we studied the changes in (1) contractile responses to acetylcholine (Ach), (2) muscarinic cholinoceptors and (3) cholinesterase (ChE) activities, in jejunum and colon of the young (2-3 months) and aged (24-28 months) Fischer 344 rats. In the physiological contraction experiments of jejunum and colon, Ach concentration-dependently increased the force of contraction, and the contractile responses to Ach were not affected by aging. In addition, the true- and pseudo-ChE activities were not significantly changed by aging. The Ach-induced contraction was competitively inhibited by muscarinic M3-selective antagonist hexahydro-sila-difenidolhydrochloride p-fluoroanalog (p-F-HHSiD), suggesting that the contractile responses in the rat jejunum and colon were mediated through M3-cholinoceptor. Age-related changes in muscarinic cholinoceptors of jejunum and colon were determined with the use of specific muscarinic radioligand [3H]-quinuclidinylbenzilate (QNB). The [3H]QNB saturation binding experiments revealed that the maximal binding (B(max)) was increased only in aged jejunum without changes in K(D) values. These results suggest that aging may not attenuate the Ach-induced intestinal contraction via muscarinic M3 receptor, although the expression of muscarinic cholinoceptor is differentially modulated in jejunum and colon.

Topics: Acetylcholine; Aging; Animals; Atropine; Cholinesterases; Colon; Diamines; Dose-Response Relationship, Drug; Gastrointestinal Motility; In Vitro Techniques; Intestine, Large; Jejunum; Male; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Pirenzepine; Rats; Rats, Inbred F344

1. Functional experiments have been conducted to assess the effects of acetylcholine and carbachol, and the receptors on which they act to facilitate neurotransmission to the stromal smooth muscle of the prostate gland of the guinea-pig. 2. Acetylcholine and carbachol (0.1 microM - 0.1 mM) enhanced contractions evoked by trains of electrical field stimulation (20 pulses of 0.5 ms at 10 Hz every 50 s with a dial setting of 60 V) of nerve terminals within the guinea-pig isolated prostate. In these concentrations they had negligible effects on prostatic smooth muscle tone. 3. The facilitatory effects of acetylcholine, but not those of carbachol, were further enhanced in the presence of physostigmine (10 microM). 3. The facilitatory effects of carbachol were unaffected by the neuropeptide Y Y(1) receptor antagonist BIBP 3226 ((R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-arginina mide) (0.3 microM, n=3) or suramin (100 microM, n=5). Prazosin (0.1 microM, n=5) and guanethidine (10 microM, n=5) alone and in combination (n=4), reduced responses to field stimulation and produced rightward shifts of the log concentration-response curves to carbachol. 4. The rank orders of potency of subtype-preferring muscarinic receptor antagonists in inhibiting the facilitatory actions of acetylcholine and carbachol were: pirenzepine > HHSiD (hexahydrosiladifenidol) > pF-HHSiD (para-fluoro-hexahydrosiladifenidol)>/= 5 himbacine, and pirenzepine > HHSiD > himbacine>/= 5 pF-HHSiD, respectively. These profiles suggest that muscarinic receptors of the M(1)-subtype mediate the facilitatory effects of acetylcholine and carbachol on neurotransmission to the smooth muscle of the guinea-pig prostate.

Topics: Acetylcholine; Animals; Carbachol; Cholinergic Agonists; Electric Stimulation; Guinea Pigs; Male; Muscle Contraction; Muscle, Smooth; Piperidines; Prostate; Receptor, Muscarinic M1; Receptors, Muscarinic; Synaptic Transmission

SNI-2011 induces the long-lasting increase in the amount of aquaporin-5 (AQP5) in apical plasma membranes (APMs) of rat parotid acini in a concentration-dependent manner. This induction was inhibited by p-F-HHSiD, U73122, TMB-8, or dantrolene but not by bisindolmaleimide or H-7, indicating that SNI-2011 acting at M(3) muscarinic receptors induced translocation of AQP5 via [Ca(2+)](i) elevation but not via the activation of protein kinase C. In contrast, acetylcholine induced a transient translocation of AQP5 to APMs. SNI-2011 induces long-lasting oscillations of [Ca(2+)](i) in the presence of extracellular Ca(2+). Thus, SNI-2011 induces a long-lasting translocation of AQP5 to APMs coupled with persistent [Ca(2+)](i) oscillations.

Topics: Animals; Aquaporin 5; Aquaporins; Atropine; Calcium; Cell Membrane; Cells, Cultured; Inositol 1,4,5-Trisphosphate; Male; Membrane Proteins; Muscarinic Agonists; Parotid Gland; Piperidines; Quinuclidines; Rats; Rats, Wistar; Thiophenes

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Atropine; Binding, Competitive; Calcium; Cells, Cultured; DNA Primers; Epitopes; Flow Cytometry; Gene Expression; Green Fluorescent Proteins; Histidine; Humans; Indicators and Reagents; Kidney; Luminescent Proteins; Microscopy, Confocal; Muscarinic Antagonists; Mutagenesis, Site-Directed; Piperidines; Pirenzepine; Radioligand Assay; Receptor, Muscarinic M1; Receptors, Muscarinic; Recombinant Fusion Proteins; Tritium

1. Blind patch clamp recordings were made from substantia gelatinosa (SG) neurones in the adult rat spinal cord slice to study the mechanisms of cholinergic modulation of GABAergic inhibition. 2. In the majority of SG neurones tested, carbachol (10 microM) increased the frequency (677 % of control) of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs). A portion of these events appeared to result from the generation of spikes by GABAergic interneurones, since large amplitude IPSCs were eliminated by tetrodotoxin (1 microM). 3. The effect of carbachol on spontaneous IPSCs was mimicked by neostigmine, suggesting that GABAergic interneurones are under tonic regulation by cholinergic systems. 4. The frequency of GABAergic miniature IPSCs in the presence of tetrodotoxin (1 microM) was also increased by carbachol without affecting amplitude distribution, indicating that acetylcholine facilitates quantal release of GABA through presynaptic mechanisms. 5. Neither the M1 receptor agonist McN-A-343 (10-300 microM) nor the M2 receptor agonist, arecaidine (10-100 microM), mimicked the effects of carbachol. All effects of carbachol and neostigmine were antagonized by atropine (1 muM), while pirenzepine (100 nM), methoctramine (1 microM) and hexahydrosiladifenidol hydrochloride, p-fluoro-analog (100 nM) had no effect. 6. Focal stimulation of deep dorsal horn, but not dorsolateral funiculus, evoked a similar increase in IPSC frequency to that evoked by carbachol and neostigmine. The stimulation-induced facilitation of GABAergic transmission lasted for 2-3 min post stimulation, and the effect was antagonized by atropine (100 nM). 7. Our observations suggest that GABAergic interneurones possess muscarinic receptors on both axon terminals and somatodendritic sites, that the activation of these receptors increases the excitability of inhibitory interneurones and enhances GABA release in SG and that the GABAergic inhibitory system is further controlled by cholinergic neurones located in the deep dorsal horn. Those effects may be responsible for the antinociceptive action produced by the intrathecal administration of muscarinic agonists and acetylcholinesterase inhibitors.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Atropine; Carbachol; Diamines; Evoked Potentials; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Muscarinic Agonists; Neostigmine; Neurons; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptor, Muscarinic M1; Receptors, Muscarinic; Substantia Gelatinosa; Synaptic Transmission; Tetrodotoxin

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.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Analysis of Variance; Animals; Atropine; Bethanechol; Carbachol; Chlorides; Diamines; Duodenum; Ileum; Intestinal Mucosa; Ion Transport; Jejunum; Male; Muscarinic Agonists; Muscarinic Antagonists; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptor, Muscarinic M3; Receptors, Muscarinic; Structure-Activity Relationship; Tetrodotoxin

The primary goal of this study was to determine the extent that selective muscarinic receptor antagonists could discriminate between the chronotropic and coronary vasoconstrictor responses to acetylcholine in isolated rat hearts perfused at constant flow rate. Bolus injections of acetylcholine caused dose-dependent decreases in heart rate and increases in perfusion pressure. The ED50 (95% confidence) of acetylcholine for decreasing rate was 0.463 (0.336-0.640) nmol and the dose that increased perfusion pressure by 30 mm Hg (ED30 mmHg) was 3.19 (2.00-5.08) nmol. The M2 selective antagonist methoctramine (3.16 microM) produced a 307-fold increase in the ED50 for bradycardia but had no significant effect on the pressor response to acetylcholine. In marked contrast, the M3 antagonist hexahydrosiladifenidol displayed a distinct preference for inhibiting coronary vasoconstrictor responses to acetylcholine. When present at 316 nM, this drug produced a 66-fold increase in the ED30 mmHg but only a 6-fold increase in the ED50 for bradycardia. The M1 selective antagonist pirenzepine (316 nM) produced a 5- to 7-fold increase in both parameters. Pretreatment with pertussis toxin (25 microg/kg, i.p.) essentially eliminated acetylcholine-evoked bradycardia although pressor responses persisted with some reduction. These observations demonstrate that cardiac and coronary vascular effects of acetylcholine can be clearly discriminated with specific muscarinic antagonists. Furthermore, they provide evidence that the M3 receptor subtype mediates the vasoconstrictor effect of acetylcholine on resistance vessels in rat heart.

Topics: Acetylcholine; Animals; Atropine; Coronary Vessels; Depression, Chemical; Diamines; Heart Rate; In Vitro Techniques; Male; Pertussis Toxin; Piperidines; Pirenzepine; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Vasoconstriction; Virulence Factors, Bordetella

In cross-chopped slices from rat thalamus and in the presence of 10 mM LiC1, the cholinergic agonist carbachol stimulated the accumulation of total [3H]inositol phosphates ([3H]IP2 = [3H]IP1 + [3H]IP2 + [3H]IP3). Best-fit values for the concentration-response curve for carbachol after 60 min incubation yielded an EC50 of 44 +/- 6 microM, maximum effect of 199 +/- 6% of basal accumulation and Hill coefficient (nH) of 1.1 +/- 0.1. Carbachol-induced [3H]IPs accumulation was inhibited by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; pKi 9.1) and the p-fluoro analogue of hexahydro-sila-difenidol (pF-HHSiD; pKi 8.1). Concentration-response curves for carbachol were shifted to the right in a parallel fashion by pirenzepine (100, 300 and 100 nM). A Schild plot of the data was linear (slope 0.95 +/- 0.04) and yielded a log KD for pirenzepine of -6.8 +/- 0.1. Taken together, these results suggest that carbachol-induced inositol phosphate accumulation in rat thalamus is mediated by muscarinic M3-receptors.

Topics: Animals; Binding, Competitive; Carbachol; Dose-Response Relationship, Drug; Inositol Phosphates; Male; Muscarinic Agonists; Muscarinic Antagonists; Organ Culture Techniques; Piperidines; Pirenzepine; Rats; Rats, Wistar; Receptors, Muscarinic; Thalamus; Tritium

Carbachol increased ventricular automaticity in a concentration-dependent fashion from a control rate of 72 +/- 5 (mean +/- SEM) to 86 +/- 4 beats per minute at 10(-4) M carbachol. Pirenzepine, an M1-selective antagonist, and AFDX 116, an M2-selective antagonist, both at 10(-7) M, did not block the carbachol-induced positive chronotropic response. In contrast, 10(-7) M HHSiD, an M3-selective antagonist, completely blocked the positive chronotropic effect of carbachol. Carbachol stimulated the accumulation of IP1 in a concentration-dependent manner at concentrations > or = 3 x 10(-6) M. AFDX 116 had no effect on carbachol-induced IP1 accumulation. HHSiD significantly inhibited IP1 accumulation at concentrations > or = 3 x 10(-8) M, while pirenzepine inhibited IP1 accumulation only at concentrations > or = 10(-5) M. McN A343 and methacholine, two muscarinic receptor agonists with minimal M2 activities, and carbachol did not alter basal cAMP concentration, but all three agonists significantly attenuated the increase in cAMP accumulation in response to isoproterenol. Carbachol inhibited isoproterenol-mediated cAMP accumulation at concentrations > or = 10(-7) M. AFDX 116, HHSiD, and pirenzepine blocked the carbachol-induced inhibition of isoproterenol-stimulated cAMP accumulation. At equimolar concentrations, the inhibitory effects of HHSiD and AFDX-116 were similar, while that of pirenzepine was much less. Pretreatment with pertussis toxin for 24 h did not prevent the carbachol-mediated positive chronotropic response or accumulation of IP1 but completely abolished the inhibition of isoproterenol-stimulated cAMP accumulation. These results indicate that (a) neonatal ventricular myocytes in culture have a heterogeneous population of muscarinic (M2 and M3) receptors, (b) the M3 receptor is coupled to pertussis toxin-sensitive and pertussis toxin-insensitive G proteins, (c) M3 receptor stimulation activates phosphoinositide hydrolysis and increases automaticity via a pertussis toxin-insensitive G protein-dependent pathway, and (d) both M2 and M3 receptors couple to pertussis toxin-sensitive G protein(s) to mediate the inhibition of intracellular cAMP accumulation in response to isoproterenol stimulation.

Topics: Animals; Animals, Newborn; Carbachol; Cells, Cultured; Cyclic AMP; GTP-Binding Proteins; Heart Rate; Inositol Phosphates; Muscarinic Agonists; Muscarinic Antagonists; Myocardium; Pertussis Toxin; Piperidines; Rats; Receptor, Muscarinic M2; Receptors, Muscarinic; Signal Transduction; Stimulation, Chemical; Virulence Factors, Bordetella

1. The functional antagonism between methacholine- or histamine-induced contraction and beta-adrenoceptor-mediated relaxation was evaluated in bovine tracheal smooth muscle in vitro. In addition, the putative contribution of muscarinic M2 receptors mediating inhibition of beta-adrenoceptor-induced biochemical responses to this functional antagonism was investigated with the selective muscarinic antagonists, pirenzepine (M1 over M2), AF-DX 116 and gallamine (M2 over M3), and hexahydrosiladiphenidol (M3 over M2). 2. By use of isotonic tension measurement, contractions were induced with various concentrations of methacholine or histamine, and isoprenaline concentration-relaxation curves were obtained in the absence or presence of the muscarinic antagonists. Antagonist concentrations were chosen so as to produce selective blockade of M2 receptors (AF-DX 116 0.1 microM, gallamine 30 microM), or half-maximal blockade of M3 receptors (pirenzepine 0.1 microM, AF-DX 116 0.5 microM, hexahydrosiladiphenidol 0.03 microM). Since these latter antagonist concentrations mimicked KB values towards bovine tracheal smooth muscle M3 receptors, antagonist-induced decreases in contractile tone were compensated for by doubling the agonist concentration. 3. It was found that isoprenaline-induced relaxation of bovine tracheal smooth muscle preparations was dependent on the nature and the concentration of the contractile agonist used. Thus, isoprenaline pD2 (-log EC50) values were decreased 3.7 log units as a result of increasing cholinergic tone from 22 to 106%, and 2.4 log units by increasing histamine tone over a similar range. Furthermore, maximal relaxability of cholinergic tone decreased gradually from 100% at low to only 1.3% at supramaximal contraction levels, whereas with histamine almost complete relaxation was maintained at all concentrations applied. As a result, isoprenaline relaxation was clearly hampered with methacholine compared to histamine at equal levels of contractile tone.4. In the presence of gallamine, isoprenaline relaxation was facilitated for most concentrations of methacholine, and for all concentrations of histamine. These changes could be explained by the decreased contraction levels for both contractile agonists in the presence of gallamine.5. Isoprenaline-induced relaxation of cholinergic contraction was also facilitated by AF-DX 116 as well as by pirenzepine and hexahydrosiladiphenidol, and these (small) changes were again related to the(small) dec

Topics: Animals; Bronchoconstrictor Agents; Cattle; Dose-Response Relationship, Drug; Gallamine Triethiodide; Histamine; In Vitro Techniques; Isoproterenol; Methacholine Chloride; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Nicotinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Adrenergic, beta; Receptors, Muscarinic; Trachea

The effects of subtype-selective muscarinic receptor antagonists on electrically evoked release of acetylcholine and muscle contraction were compared in circular muscle preparations of the guinea-pig ileum. Incubation of the preparation with [3H]choline resulted in the formation of [3H]acetylcholine. Electrical stimulation caused the release of [3H]acetylcholine which was abolished by tetrodotoxin and omission of calcium from the medium. 5-Hydroxytryptamine (10 microM) and the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (300 microM) did not change acetylcholine release. The muscarinic antagonists pirenzepine (M1 selective), AF-DX 116 (M2 selective) and hexahydrosiladifenidol (M3 selective) caused concentration-dependent increases in the evoked release of acetylcholine, and inhibitions of the circular muscle contraction. The postjunctional affinity constants (pA2 values) obtained for hexahydrosiladifenidol (8.06), pirenzepine (6.95) and AF-DX 116 (6.60) identified the muscular receptor as an M3 subtype. Pirenzepine was more potent in facilitating the evoked release than hexahydrosiladifenidol and AF-DX 116. These findings suggest that the release of acetylcholine in the circular muscle is inhibited by M1 muscarinic autoreceptors whereas muscle contraction is mediated by M3 receptors.

Topics: Acetylcholine; Animals; Choline; Female; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neuromuscular Junction; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic

1. Methoctramine, AF-DX 116 and hexahydrosiladifenidol (HHSiD) are the muscarinic antagonists most widely used to study muscarinic receptor subtypes. 2. The present study was undertaken to examine the mode of antagonism of these compounds in guinea-pig left atrium and ileum by comparison of the Schild and resultant analysis. With this method the effect of various concentrations of the test antagonist on the antagonism produced by specific concentrations of a reference antagonist was measured and the equilibrium dissociation constant of the test antagonist-receptor complex estimated. Atropine was used for comparative purposes and scopolamine as the reference antagonist. 3. At the cardiac level the affinity values obtained by Schild and resultant analysis for methoctramine and AF-DX 116, as for atropine, are very similar: these results indicate that the two cardio-selective antagonists and the non-selective antagonist, atropine, bind at a common site with the reference antagonist scopolamine. The resultant plot for the ileo-selective HHSiD has a slope considerably less than unity: this finding might indicate that this antagonist binds to a site different from that of scopolamine and it should be considered like an allosteric antagonist. 4. At the ileal level the affinity values obtained by Schild and resultant analysis are identical for the ileo-selective antagonist HHSiD as for atropine but not for methoctramine and AF-DX 116. This indicates a mutual binding site with scopolamine for HHSiD and atropine but not for the two cardio-selective antagonists. However, it is worth emphasizing that the difference between affinity values obtained by Schild and resultant analysis is seen when relatively high concentrations are required: a dual mode of interaction (both competitive and allosteric) could be involved.

Topics: Animals; Diamines; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Myocardial Contraction; Parasympatholytics; Piperidines; Pirenzepine

The effects of muscarine on whole-cell Ca2+ channel currents in SH-SY5Y cells were studied using conventional and perforated-patch-clamp techniques, with 10 mM Ba2+ as charge carrier. Muscarine (10-300 microM) caused concentration-dependent inhibitions of Ca2+ channel currents which were only reversible when perforated-patch recordings were used. Inhibition of currents was associated with slowing of activation kinetics in approximately 50% of cells. In the presence of 5 microM nifedipine, muscarine was still able to inhibit currents, but after pre-exposure of cells to 1 microM omega-conotoxin GVIA the inhibitory effects of muscarine were almost completely lost. In the presence of 100 microM muscarine, Bay K 8644 (5 microM) was still able to enhance current amplitudes. Pre-treatment of cells with pertussis toxin (250 ng/ml for 16-24 hr) or inclusion of 1 mM GDP-beta-S in the patch-pipette prevented the inhibitory actions of muscarine. Hexahydrosiladifenidol (0.1-1 microM) antagonized the actions of muscarine (calculated pA2 7.1) but the presence of 10 microM pirenzipine or 0.1 microM methoctramine in the bath solution did not alter the degree of current inhibition caused by 100 microM muscarine. In summary, these results indicate that muscarine in SH-SY5Y cells causes inhibition of N-type Ca2+ channels via a M3 receptor coupled to a pertussis toxin-sensitive G-protein.

Topics: Brain Neoplasms; Calcium Channel Blockers; GTP-Binding Proteins; Humans; Muscarine; Muscarinic Agonists; Neuroblastoma; Parasympatholytics; Patch-Clamp Techniques; Pertussis Toxin; Piperidines; Receptors, Muscarinic; Tumor Cells, Cultured; Virulence Factors, Bordetella

Muscarinic autoreceptors on horse airway cholinergic nerves were studied by examining the effects of muscarinic receptor antagonists on electrical field stimulation (EFS)-induced acetylcholine (ACh) release in trachealis preparations. All the antagonists including atropine (non-selective), pirenzepine (M1-selective), AF-DX 116 (M2-selective), and hexahydrosiladifenidol (M3-selective) augmented ACh release concentration-dependently. The augmentation was not due to displacement of ACh molecules from tissue receptors into the bath liquid because incubation with atropine after EFS had no influence on the measured amount of ACh. Hexahydrosiladifenidol was more potent in inhibiting ACh-induced muscle contraction, which is known to be mediated by M3 receptors, than in augmenting ACh release. The maximal ACh release rate in response to the selective antagonists was much less than that following atropine. Furthermore, the concentrations of the selective antagonists required to augment ACh release far exceeded their KdS for M1, M2, or M3 receptors. These observations suggest that the muscarinic autoreceptors on horse airway cholinergic nerves may belong to a novel subtype.

Topics: Acetylcholine; Animals; Atropine; Cholinergic Fibers; Electric Stimulation; Horses; In Vitro Techniques; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

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. 5. The pharmacological profile of antagonist affinities at the muscarinic receptor population responding to muscarinic agonist-evoked contraction is similar to that widely accepted as characterizing the activation of an M3 muscarinic receptor subtype, although pA2 values of some antagonists are lower than that seen in other investigations.

Topics: Alkaloids; Atropine; Carbachol; Carcinoma; Colon; Colonic Neoplasms; Dose-Response Relationship, Drug; Furans; Humans; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Naphthalenes; Piperidines; Pirenzepine

The prejunctional muscarine receptor on sympathetic nerves in the rat caudal artery was characterized using several selective antagonists. The inhibitory response of carbachol on vasoconstriction elicited by sympathetic nerve stimulation was antagonized by benzhexol (trihexyphenidyl; pKB 7.1), heptane-1,7-bis(dimethyl-3'-phthalimidopropyl ammonium bromide) (C7/3-phth; pKB 6.5) and hexahydrosiladifenidol (HHSiD; apparent pKB 6.0). These pKB values suggest that the receptor most closely resembles the muscarine M2 receptor subtype rather than the muscarine M1, M3 or M4 receptor subtypes.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Arteries; Atropine; Bis-Trimethylammonium Compounds; Carbachol; Female; In Vitro Techniques; Isoindoles; Male; Muscarinic Antagonists; Muscle, Smooth, Vascular; Nerve Endings; Parasympatholytics; Piperidines; Rats; Rats, Wistar; Receptors, Muscarinic; Sympathetic Nervous System; Tail; Trihexyphenidyl

In previous studies carbachol-induced stimulation of gastrin release from antral G-cells in primary culture suggested the presence of muscarinic acetylcholine receptors on this cell type. Therefore, we attempted to pharmacologically characterize the muscarinic acetylcholine receptor subtype involved. Enzymatically isolated rabbit antral mucosal cells (0.8% G-cells) were separated by counterflow elutriation yielding a fraction (1.7% G-cells) that was placed in culture on collagen-coated well plates. After 24-36 h of culture 13.0 +/- 2.4% of total adherent cells were immunoreactive for gastrin as shown by immunocytochemical staining using the avidin-biotin complex method. In this preparation basal gastrin release ranged from 3.3 +/- 0.3 to 4.1 +/- 0.3% of total cellular content. Maximal gastrin release in response to the acetylcholine receptor agonist carbachol (10(-4) M) or the selective muscarinic receptor agonist arecaidine propargyl ester (10(-4) M) was 8.5 +/- 0.4% and 7.6 +/- 0.4% of total cellular content, respectively. The EC50 values were 3.7 +/- 0.5 x 10(-6) M carbachol and 1.8 +/- 0.4 x 10(-6) M arecaidine propargyl ester. At a concentration of 10(-6) M the non-selective muscarinic receptor antagonist atropine and the muscarinic M3 receptor preferring antagonist hexahydro-sila-difenidol (HHSiD; M3 > or = M1 > M2) completely inhibited gastrin release in response to carbachol (Ki values: 52 x 10(-9) M atropine and 29 x 10(-9) M HHSiD) and arecaidine propargyl ester (Ki values: 11 x 10(-9) M atropine and 13 x 10(-9) M HHSiD).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arecoline; Atropine; Benzodiazepinones; Carbachol; Cell Adhesion; Cell Separation; Cells, Cultured; Gastrins; Immunohistochemistry; Male; Muscarinic Antagonists; Parasympatholytics; Piperidines; Pirenzepine; Pyloric Antrum; Rabbits; Receptors, Muscarinic

The aim of this study was to characterise the muscarinic receptor present in the uterus of the virgin rat. Homogenate binding studies were undertaken using [3H]quinuclidinyl benzilate as the radioligand and atropine (10 microM) to determine non-specific binding. [3H]Quinuclidinyl benzilate binding was saturable with a Kd of 63 pM and a Bmax of 3 fmol/mg protein. The pKi values obtained using antagonists with high affinity for differing muscarinic receptor subtypes were pirenzepine, 6.2; hexahydrosiladifenidol, 6.9; AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]5,11-dihydro-6H - pyrido[2,3-b][1,4]benzodiazepine-6-one), 7.0; and himbacine, 7.8. These findings suggest that muscarinic M2 receptors are present in rat uterus.

Topics: Alkaloids; Animals; Atropine; Binding, Competitive; Computer Simulation; Female; Furans; In Vitro Techniques; Ligands; Muscarinic Antagonists; Naphthalenes; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Uterus

We have used the pilocarpine-induced seizure model in mice and i.c.v. injection of subtype-specific receptor antagonists to investigate the muscarinic receptor subtype specificity of cholinergically-activated seizures. The rank order potencies of antagonists for inhibition of pilocarpine-induced seizures are atropine = telenzepine > 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine (4-DAMP) > pirenzepine with ID50's of 8.6, 12.0, 29.9, and 83.0 nmol/mouse, respectively. The M3-specific antagonists hexahydrosila-difenidol and its p-fluoro analog showed no effect on pilocarpine-induced seizures. The M2-specific antagonists gallamine and methoctramine cause seizures in mice in the absence of a pilocarpine injection. These seizures could be inhibited by coinjection of methoctramine with the M1-specific antagonist, pirenzepine. These data suggest a role of muscarinic M1 receptors in mediating pilocarpine-induced seizures and a role of the muscarinic M2 receptors in modulating neuronal activity.

Topics: Animals; Atropine; Cerebral Ventricles; Injections, Intraventricular; Male; Mice; Parasympatholytics; Pilocarpine; Piperidines; Pirenzepine; Receptors, Muscarinic; Seizures

Muscarinic cholinergic receptors were identified and characterized by radioligand receptor binding assay using [3H]quinuclidinyl benzilate (QNB) in rat vas deferens membrane particulates of three experimental groups: 1) 8-week diabetic, 2) 8-week diabetic insulin-treated and 3) age-matched control. Diabetes was induced by the intravenous injection of 65 mg./kg. streptozotocin (STZ). The density of muscarinic receptors (Bmax values), as determined by saturation experiments with [3H]QNB, was demonstrated to be higher in the vas deferens of diabetic rats than in the vas deferens of control and diabetic insulin-treated rats. The equilibrium dissociation constants (KD values), however, were similar in all three groups. Muscarinic cholinergic antagonists competed with [3H]QNB binding sites in the vas deferens membrane particulates with the following rank order of Ki values: atropine < methoctramine < or = 4-DAMP < AF-DX 116 < HHSiD < pirenzepine = pfHHSiD. The pharmacological profile of muscarinic receptors was similar in all three groups. Additional pharmacological studies showed a similar rank order of Ki values for vas deferens, bladder dome and heart, but this rank order was significantly different in cerebral cortex and prostate. This is consistent with the predominance of the M2 muscarinic cholinergic receptor subtype in the rat vas deferens. It is concluded that STZ-induced diabetes causes an upregulation of muscarinic cholinergic receptor density in the rat vas deferens that can be prevented by the administration of insulin.

Topics: Animals; Atropine; Binding, Competitive; Diabetes Mellitus, Experimental; Diamines; Insulin; Male; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Up-Regulation; Vas Deferens

Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Astrocytoma; Atropine; Carbachol; Diamines; Humans; Hydrolysis; Parasympathomimetics; Phosphatidylinositols; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine; Tumor Cells, Cultured

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of different selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60-80%) correspond to M2 muscarinic subtypes, and the rest is constituted by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.

Topics: Adrenal Medulla; Animals; Atropine; Binding Sites; Binding, Competitive; Cattle; Cell Membrane; Dicyclomine; Muscarine; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Receptors, Muscarinic

To investigate the nature of the muscarinic receptors present on parietal cell membranes, binding studies and polymerase chain reaction (PCR) amplification of parietal cell messenger (m) RNA were undertaken. Displacement of N-[3H]methylscopolamine by various muscarinic antagonists showed displacement with a single affinity. The apparent dissociation constant values were as follows: atropine (nonselective), 1.95 +/- 0.28 nmol/L; pirenzepine (M1), 169 +/- 24 nmol/L; AF-DX 116 (M2), 1542 +/- 33 nmol/L; and hexahydrosiladifenidol (M3), 29 +/- 3.4 nmol/L. These data confirmed the existence of only an M3 receptor linked to acid secretion as defined pharmacologically. PCR amplification of parietal cell mRNA with primers designed for detection of all known muscarinic receptor subtypes showed that only m3 fragments were produced from parietal cell mRNA, whereas m1 and m2 products could be detected in brain or cardiac mRNA. The m3 nature of the PCR product was confirmed by Southern blotting with 32P-labeled human m3 complementary DNA. Hence the two carbachol affinities and the separable cellular responses following muscarinic activation are caused by separate coupling pathways of the M3 receptor.

Topics: Animals; Atropine; Base Sequence; Blotting, Southern; Molecular Sequence Data; N-Methylscopolamine; Parietal Cells, Gastric; Piperidines; Pirenzepine; Polymerase Chain Reaction; Rabbits; Receptors, Muscarinic; RNA, Messenger; Scopolamine Derivatives

To enhance our understanding of cholinergic mechanisms and muscarinic receptors in bronchoconstriction, we have characterized the muscarinic receptor subtypes in rabbit tracheal smooth muscle using radioligand binding and functional assays. The Kd for [3H]quinuclidinyl benzilate ([3H](-)QNB) binding determined from saturation isotherms was 12.6 x/divided by 1.1 pM (geometric mean x/divided by SEM), and the Bmax was 269 +/- 7 fmol/mg protein (arithmetic mean +/- SEM). Competitive inhibition studies with the muscarinic antagonists pirenzepine (PZ), 11[[2-[(diethylamino)-methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116), 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP), and hexahydrosiladifenidol (HHSiD) demonstrated heterogeneity of muscarinic receptor subtypes in rabbit tracheal smooth muscle. PZ bound with low affinity to a single receptor site, indicative of an absence of M1 receptors. AF-DX116 (M2 selective) bound with high affinity to approximately 83% of muscarinic binding sites, and 4-DAMP and HHSiD (M3 antagonists) bound with high affinity to approximately 24 and 28% of muscarinic binding sites, respectively. Additionally, direct binding studies with [3H]4-DAMP demonstrated high-affinity binding with 23% of muscarinic binding sites. Thus, the majority of muscarinic receptors in rabbit tracheal smooth muscle bound with high affinity to an M2-selective antagonist, and the remaining receptor sites bound with high affinity to M3 antagonists. The inhibitory effects of atropine, PZ, AF-DX116, and 4-DAMP on methacholine-induced contraction of rabbit tracheal rings were compared. 4-DAMP was a potent inhibitor of methacholine-induced contraction, but PZ and AF-DX116 demonstrated low potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Female; Male; Methacholine Chloride; Muscle Contraction; Muscle, Smooth; Myocardium; Parasympatholytics; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rabbits; Radioligand Assay; Receptors, Muscarinic; Trachea

Classification of muscarinic receptors in the central airways has revealed the coexistence of M2 and M3 muscarinic receptors in this tissue, with the M2 subtype being predominant. Although M3 muscarinic receptors have been linked to airway smooth muscle contraction, a functional role for the M2 subtype in this tissue has been unclear. In nonairway smooth muscle, stimulation of the M2 muscarinic receptor has been shown to be associated with inhibition of adenylyl cyclase. In the present study, characterization of muscarinic receptors in canine tracheal smooth muscle confirmed that the majority of these muscarinic receptors were of the M2 subtype (89 +/- 3%), with a minor population of M3 receptors (11 +/- 3%). In functional studies, both isoproterenol and forskolin cause a dose-dependent relaxation of precontracted airway smooth muscle. In tissues precontracted with methacholine, 11-([[2-(diethylamino)methyl]-1-piperidinyl]acetyl)5,11- dihydro-6H-pyrido[2,3-6][1,4]benzodiazepine-6-one (AF-DX 116), a selective M2 antagonist, shifted dose-response curves to both isoproterenol and forskolin significantly to the left. In contrast, AF-DX 116 did not alter relaxation induced by the K+ channel opener BRL 38227. Furthermore, the ability of AF-DX 116 to enhance isoproterenol-induced relaxation appears to be limited to smooth muscle precontracted with muscarinic agonists because AF-DX 116 had no effect on isoproterenol dose-response curves in muscle strips precontracted with histamine. Hexahydrosiladifenidol (HHSiD), a selective antagonist for M3 receptors, did not shift the isoproterenol dose-response curve in muscle precontracted with methacholine. This study demonstrates that stimulation of M2 muscarinic receptors in canine airway smooth muscle plays an important role in functional antagonism by reducing the relaxation caused by agents such as isoproterenol and forskolin.

Topics: Animals; Dogs; Drug Interactions; Isoproterenol; Methacholine Chloride; Muscle Relaxation; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

We previously showed that M1 and M2 muscarinic receptors in dissociated cells of the adult rat cortex couple to phosphoinositide (Pl) and cyclic AMP (cAMP) metabolism, respectively. To further classify these receptors according to probable subtype, we have employed a group of selective muscarinic antagonists to obtain pharmacological profiles of the cortical M1 and M2 receptors, and to compare them with the muscarinic receptors in N1E-115 cells, which contain M1 receptors mediating cyclic GMP elevation and M4 receptors inhibiting cAMP levels. The M2-mediated inhibition of cAMP levels in cortex was blocked by 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) with higher potency (0.29 nM) than for reported potency in cardiac tissue (approximately 10 nM), indicating that this cortical response is probably not mediated by the m2 gene product. Similarly, the potency of hexahydrosiladiphenidol (HSD) at the cortical M2 receptor (159 nM) was somewhat greater than the reported potency in cardiac tissue (295 nM). The cardioselective drugs AF-DX 116 and methoctramine blocked the cortical M2 response less potently (135 nM and 229 nM, respectively) than would be expected for involvement of the m2 gene product. Thus, the potencies of AF-DX 116, methoctramine, 4-DAMP and HSD suggest that the cortical M2 response, like the striatal M2 receptor, is mediated by a noncardiac M2 receptor, perhaps by the m4 gene product. This postulate was supported by the significant correlations between cortical and striatal M2 receptors as compared to the M4 receptor in N1E-115 cells (r = 0.92 and 0.99, respectively, P less than .025).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cerebral Cortex; Cyclic AMP; Diamines; In Vitro Techniques; Muscarinic Antagonists; Parasympatholytics; Phenethylamines; Phosphatidylinositols; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic; Tumor Cells, Cultured

Dose-dependent depression of the electrically evoked surface-negative field potential (N-wave) produced by bath-superfusion of carbachol was measured in guinea-pig olfactory cortex slices maintained in vitro. The possible involvement of M3 (smooth muscle/glandular) type muscarinic receptors in partly mediating this response was investigated by testing the effectiveness of the muscarinic M3 receptor antagonists hexahydro-sila-difenidol (HHSiD) and p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD). Low doses of HHSiD (10-100 nM) or p-F-HHSiD (up to 1 microM), pre-applied for 30 min, produced no obvious antagonism of carbachol responses. However, a clear competitive-type inhibition of carbachol effects was observed in 250 nM-1 microM HHSiD or 10-50 microM p-F-HHSiD respectively. Schild plot analysis (regression slope constrained to unity) of pooled data yielded pA2 values of 6.6 for HHSiD (n = 6 slices) and 5.5 for p-F-HHSiD (n = 6 slices) respectively, suggesting a weak competitive antagonism by both compounds. In addition, combination experiments using either HHSiD or p-F-HHSiD with atropine, produced dose-ratio shifts close to those predicted for two antagonists competing for a common receptor site. By comparison, another suggested M3-receptor antagonist, 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) was a potent competitive blocker of carbachol responses. Schild analysis for 4-DAMP versus carbachol gave a pA2 of 7.9 (n = 6 slices). It is concluded that the muscarinic receptors involved in the suppression of the olfactory cortical N-wave possess a low affinity for HHSiD and particularly for p-F-HHSiD, but not 4-DAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Action Potentials; Animals; Carbachol; Cerebral Cortex; Evoked Potentials; Guinea Pigs; In Vitro Techniques; Olfactory Bulb; Parasympatholytics; Piperidines; Receptors, Muscarinic; Synaptic Transmission

Studies were performed in the rabbit aortic rings, precontracted with norepinephrine, to determine the subtype(s) of muscarinic receptors involved in endothelium-dependent relaxation and contraction in the absence of endothelium elicited by cholinergic stimuli. Acetylcholine (ACh) and arecaidine propargyl ester (APE), a M2 and M3 agonist, produced a dose-dependent relaxation and contraction in endothelium-intact and endothelium-denuded rabbit aortic rings, respectively. Both of these responses were blocked by the muscarinic receptor antagonist atropine. M1 selective agonist McN-A-343 [4-[N-(3-chlorophenyl)carbamoyloxy]-2-butinyltrimethylammonium+ ++ chloride] did not produce any effect on the tone of precontracted aortic rings. ACh- and APE-induced relaxation in aortic rings with intact endothelium was selectively blocked by M3 receptor antagonists hexahydrosila-difenidol and p-fluoro-hexahydro-sila-difenidol (pA2 of 7.84 and 7.18) but not by M1 antagonist pirenzepine or M2 receptor antagonists AF-DX 116 [11-(2-[(diethylamino)methyl]- 1-piperidinyl]acetyl)-5, 11-dihydro-6H-pyrido-[2,3-b][1,4]-benzo-diazepin-6-one] and methoctramine. ACh- and APE-induced contraction was inhibited by M2 receptor antagonists AF-DX 116 and methoctramine (pA2 of 7.11 and 6.71) but not by pirenzepine, hexahydro-sila-difenidol or p-fluoro-hexahydro-sila-difenidol. ACh- and APE-induced relaxation or contraction were not altered by nicotinic receptor antagonist hexamethonium or cyclooxygenase inhibitor indomethacin. These data suggest that relaxation elicited by cholinergic stimulin in endothelium-intact aortic rings is mediated via release of endothelium-derived relaxing factor consequent to activation of M3 receptors located on endothelial cells, whereas the contraction in aortic rings denuded of their endothelium is mediated via stimulation of M2 receptors located on smooth muscle cells.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Antihypertensive Agents; Aorta, Abdominal; Arecoline; Atropine; Diamines; Endothelium, Vascular; Hexamethonium; Hexamethonium Compounds; Indomethacin; Male; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Norepinephrine; Parasympatholytics; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic

1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors. Careful attention should therefore be paid to the experimental conditions in binding assays used to assess the affinities and selectivities of new muscarinic antagonists in order to avoid misleading results. 9. The ability to produce enhanced or attenuated affinities and selectivities of antagonists, resulting from the induction of different conformations of the receptor by a variety of physical, chemical or molecular biological perturbations may lead to a better understanding of the structural basis of drug receptor interactions.

Topics: Animals; Centrifugation; Cerebral Cortex; Cholic Acids; Digitonin; Gallamine Triethiodide; Heart; In Vitro Techniques; Lacrimal Apparatus; Membranes; Muscarinic Antagonists; Myocardium; Parasympatholytics; Piperidines; Pirenzepine; Rats; Structure-Activity Relationship

The antimuscarinic properties of the methoctramine with high selectivity for cardiac muscarinic M2 receptors were investigated on cholinergically induced changes in prostaglandin (PG) synthesis and mechanical function in the isolated perfused rabbit heart. Acetylcholine (ACh)- and arecaidine propargyl ester (APE)-induced increases in PG synthesis were significantly attenuated by methoctramine in a concentration-dependent manner. Methoctramine at a low concentration of 0.1 microM potentiated ACh-induced PG synthesis, which was blocked by simultaneous infusion of hexahydro-sila-difenidol (HHSiD), a M3 receptor antagonist. Methoctramine produced an additive effect with HHSiD in diminishing the ACh- or APE-induced PG synthesis. Methoctramine displayed a potent antagonistic activity at M2 receptors that mediate the decrease in heart rate and increase in coronary perfusion pressure in isolated perfused rabbit heart. Methoctramine also minimized ACh- and APE-induced decrease in developed tension. In contrast, at 0.1-0.75 microM it exhibited no antagonistic activity at vascular muscarinic receptors (M3) mediating vasodilation in response to ACh or APE. These data suggest that methoctramine has a high affinity for cardiac M2 receptors mediating increases in PG output and coronary perfusion pressure as well as decrease in heart rate and developed tension and has a low affinity for M3 receptors mediating coronary vasodilator response.

Topics: 6-Ketoprostaglandin F1 alpha; Acetylcholine; Animals; Arachidonic Acid; Arachidonic Acids; Arecoline; Coronary Circulation; Diamines; Heart; Heart Rate; In Vitro Techniques; Isoproterenol; Male; Myocardium; Parasympatholytics; Piperidines; Prostaglandins; Rabbits; Receptors, Muscarinic

The distribution of cholinergic nerve fibres, as well as the characterization of the muscarinic receptors responsible for the contraction, were determined in the detrusor smooth muscle of the sheep. The results obtained demonstrated a rich presence of acetylcholinesterase (AChE)-positive fibres distributed throughout the bladder body forming dense neuromuscular, subepithelial and perivascular plexuses. Furthermore, intramural ganglia containing AChE-positive cell bodies were identified. However, acetylcholine and carbachol induced a dose-dependent contraction of detrusor smooth muscle. The effect observed with carbachol was competitively antagonized by atropine (pA2: 8.94), pirenzepine (pA2: 7.38), AF-DX 116 (pA2: 7.35), 4-DAMP (pA2: 9.26) and hexahydroxiladifenidol (HHSiD) (pA2: 8.49). The pA2 value for pirenzepine is intermediate between M1- and M2-receptors which suggests that this antagonist does not act on M1- or M2-receptors, but that it does on M3-receptors. The pA2 value for AF-DX 116 is consistent with the presence of M2-receptors in this tissue. Moreover, the pA2 values obtained for both 4-DAMP and HHSiD are in agreement with the presence of M3-receptors, due to the lack of effect of pirenzepine on M1-muscarinic receptors. These results indicate the existence of a rich parasympathetic innervation in the sheep detrusor muscle and suggest that its contraction could be mediated by the stimulation of muscarinic receptors belonging to both M3- and M2-subtypes.

Topics: Acetylcholine; Acetylcholinesterase; Animals; Atropine; Carbachol; Cholinesterase Inhibitors; Ganglia, Parasympathetic; Male; Muscle Contraction; Muscle, Smooth; Nerve Tissue Proteins; Piperidines; Pirenzepine; Receptors, Muscarinic; Sheep; Tetraisopropylpyrophosphamide; Urinary Bladder

1. The tritiated muscarinic antagonist N-methyl scopolamine, [3H]-NMS, was used to characterize the muscarinic receptors associated with the intact dog tracheal smooth muscle cells. Based on receptor binding assays, the intact tracheal smooth muscle cells had specific, saturable, high-affinity binding sites for [3H]-NMS. 2. Specific binding was cell concentration- and time-dependent. The specific binding of [3H]-NMS was increased linearly with increasing cell concentrations. The equilibrium for association of [3H]-NMS with the muscarinic receptors was attained within 30 min at 37 degrees C. 3. Binding was saturable with respect to [3H]-NMS concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 320 +/- 20 pM and a maximum receptor density (Bmax) of 13.7 +/- 1.4 fmole per 5 x 10(4) cells. The Hill coefficient for [3H]-NMS binding was 1.00 +/- 0.01. The association (K1) and dissociation (K-1) rate constants were determined to be (1.19 +/- 0.23) x 10(8) M-1 min-1 and 0.034 +/- 0.09 min-1, respectively. KD, calculated from the ratio of K-1 and K1, was 286 +/- 65 pM; this value is close to the value of KD calculated from Scatchard plots of binding isotherms. 4. The non-selective muscarinic antagonist atropine and M1 selective antagonist pirenzepine did not reveal any selectivity of these muscarinic receptors. Pirenzepine competed with [3H]-NMS for a single binding site with a Ki value of (6.02 +/- 0.69) x 10(-7) M which is close to the value of M2 or M3 receptors, indicating that the M1 receptor subtype did not exist in the intact tracheal smooth muscle cells. 5. Competition with cardioselective antagonist (M2), methoctramine; smooth muscle selective antagonists (M3), hexahydrodifenidol and hexahydrosiladifenidol; as well as carbachol, were best fit by a two-binding site model. The results suggest that both M2 and M3 receptor subtypes exist at the cell surface of tracheal smooth muscle cells.

Topics: Animals; Atropine; Carbachol; Diamines; Dogs; In Vitro Techniques; Kinetics; Muscle, Smooth; N-Methylscopolamine; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine Derivatives; Trachea

1. The muscarine receptor mediating electrically-stimulated acid secretion in the mouse isolated stomach was characterized using a variety of muscarine receptor antagonists confirming the M1 nature of the antagonist effect of telenzepine. 2. Field stimulation (7 V, 10 Hz, 0.5 ms) resulted in a plateau acid secretion over at least 90 min which was completely blocked by either 1 mumol/l TTX or H2 receptor antagonists (100 mumol/l cimetidine or 10 mumol/l lupitidine). Ranitidine, which is known to potently inhibit mucosal acetylcholine esterase, was ineffective. Compound 48/80 at 100 mumol/l, which depletes mucosal histamine stores, initially mimicked electrical stimulation but subsequently prevented it from inducing acid secretion. 3. 10 muscarine receptor antagonists with differing relative affinities for M1, M2 and M3 receptors were introduced at 1 mumol/l to inhibit electrically-stimulated acid secretion. The percentages inhibition were plotted against binding affinities of the antagonists at either M1, M2 or M3 binding sites. A statistically significant correlation between functional and binding data was detected only when based on M1 affinities. 4. It is concluded that field stimulation, which probably mimicks vagal drive, results in muscarinic M1 receptor activation on paracrine cells to release histamine. Histamine then stimulates parietal cells to secrete acid. Hence, according to the present and our previous data, telenzepine inhibits acid secretion under these conditions by blocking M1 receptors at least partially located on histamine-releasing paracrine cells.

Topics: Acetylcholine; Alkaloids; Animals; Anti-Ulcer Agents; Electric Stimulation; Furans; Gastric Acid; Gastric Mucosa; Histamine; Mice; Muscarinic Antagonists; Naphthalenes; p-Methoxy-N-methylphenethylamine; Parasympatholytics; Piperidines; Pirenzepine; Stomach; Thiazepines; Vagus Nerve

A method was developed to determine the affinities of antimuscarinic drugs at M1 receptors. [3H](+/-)-Telenzepine served as radioligand in crude preparations of calf superior cervical ganglia and showed high affinity for a single receptor population, consisting of M1 receptors (KD = 1.12 nM). Kinetic experiments showed monophasic association (k1 = 0.017 min-1 nM-1) and dissociation (k-1 = 0.017 min-1) kinetics, the half-life of dissociation being 41 min at 37 degrees C. The kinetic KD value amounted to 1.00 nM. M1 affinities for pirenzepine, methoctramine, hexahydro-sila-difenidol and p-fluoro-hexahydro-sila-difenidol determined in competition experiments were similar to those found in functional studies with M1 receptors in rabbit isolated vas deferens. The binding assay was used to determine the affinities of the (R) and (S) enantiomers of tertiary (trihexyphenidyl, hexahydro-difenidol, hexbutinol, p-fluoro-hexbutinol) and quaternary muscarinic antagonists (trihexyphenidyl methiodide, hexbutinol methiodide). Comparison of results obtained with the rabbit vas deferens suggested that the ionic environment may influence the affinities.

Topics: Animals; Binding, Competitive; Cattle; Ganglia; Half-Life; In Vitro Techniques; Male; Neck; Piperidines; Pirenzepine; Rabbits; Radioligand Assay; Receptors, Muscarinic; Tritium; Vas Deferens

The present study investigated the effect of selective muscarinic antagonists on natriuresis, kaliuresis and antidiuresis induced by intracerebroventricular (i.c.v.) injection of carbachol in the rat. The muscarinic antagonists were given by i.c.v. injection 1 min before carbachol (1 microgram/rat). 4-Diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a rather selective M1 and M3 receptor antagonist, was the most potent inhibitor of carbachol-induced natriuresis, kaliuresis and antidiuresis, its ID50 being respectively 0.12, 0.04 and 0.56 nmol/rat. Pirenzepine, a selective M1 antagonist, potently inhibited the above mentioned carbachol effects, its ID50 being 1.85, 3.25 and 1.49 nmol/rat, respectively. On the other hand, the M2-selective antagonist methoctramine and the M3-selective antagonist p-fluoro-hexahydro-sila-difenidol were very weak inhibitors. Methoctramine at doses up to 60 nmol/rat produced non statistically significant inhibition of carbachol-induced natriuresis, kaliuresis and antidiuresis. Para-fluoro-hexahydro-sila-diphenidol showed an ID50 of 64.4 nmol/rat on carbachol-induced natriuresis, while at the maximum dose employed, 100 nmol/rat, the inhibition of carbachol-induced kaliuresis and antidiuresis was lower than 50%. The rank order of potency of the antagonists tested proved to be related to their pA2 values for muscarinic M1 receptors, suggesting that this receptor subtype mediates the central effects of cholinergic mechanisms on water and electrolyte excretion.

Topics: Animals; Carbachol; Diamines; Diuresis; Dose-Response Relationship, Drug; Injections, Intraventricular; Male; Muscarinic Antagonists; Natriuresis; Piperidines; Potassium; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Hexahydro-sila-difenidol and eight analogues behaved as simple competitive inhibitors of [3H]N-methyl-scopolamine binding to homogenates from human neuroblastoma NB-OK 1 cells (M1 sites), rat heart (M2 sites), rat pancreas (M3 sites), and rat striatum 'B' sites (M4 sites). Pyrrolidino- and hexamethyleneimino analogues showed the same selectivity profile as the parent compound. Hexahydro-sila-difenidol methiodide and the methiodide of p-fluoro-hexahydro-sila-difenidol had a higher affinity but a lower selectivity than the tertiary amines. Compounds containing a p-methoxy, p-chloro or p-fluoro substituent in the phenyl ring of hexahydro-sila-difenidol showed a qualitatively similar selectivity profile as the parent compound (i.e., M1 = M3 = M4 greater than M2), but up to 16-fold lower affinities. o-Methoxy-hexahydro-sila-difenidol has a lower affinity than hexahydro-sila-difenidol at the four binding sites. Its selectivity profile (M4 greater than M1, M3 greater than M2) was different from hexahydro-sila-difenidol. Replacement of the central silicon atom of hexahydro-sila-difenidol, p-fluoro-hexahydro-sila-difenidol and their quaternary (N-methylated) analogues by a carbon atom did not change their binding affinities significantly. The four muscarinic receptors showed a higher affinity for the (R)- than for the (S)-enantiomers of hexahydro-difenidol, p-fluorohexahydro-difenidol and their methiodides. The stereoselectivity varied depending on the receptor subtype and drug considered.

Topics: Animals; Binding, Competitive; Brain; Humans; In Vitro Techniques; Kinetics; Myocardium; Pancreas; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

We have attempted to characterize a muscarinic receptor subtype involved in Cl- secretion in isolated epithelium of hen trachea, taking advantage of drugs developed in the last 15 yr. Hen trachea can be stimulated to secrete Cl- equal to approximately 80-90 microA/cm2 by application of acetylcholine (ACh) to the serosal side. The process has an apparent dissociation constant (Kd) for ACh of 740 nM. The Cl- secretion is completely inhibited by 20 microM bumetanide at the serosal side. Of five selective antagonists for muscarinic receptors, pirenzepine, hexahydrosiladifenidol, dicyclomine, 11-([2-[(diethylamino)-methyl]-1-piperidinyl]acetyl)-5,11- dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, and 4 diphenyl acetoxy-N-methylpiperidine methobromide, only the latter had a high affinity for the functional receptor with an apparent Kd of 3 nM. The receptor may be classified as an M4-muscarinic receptor subtype and probably belongs to a group of muscarinic receptors on exocrine glands and mucosal cells involved in ion transport. All the functional responses caused by muscarinic agonists and antagonists tested exhibited exponents (apparent Hill coefficients) in the range from 1.3 to 2.4, indicating a gain in the stimulus secretion coupling mechanism, an aspect of muscarinic receptor function that is not revealed in radioligand binding studies.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Acetylcholine; Animals; Atropine; Bethanechol; Bethanechol Compounds; Bumetanide; Calcium Channels; Chickens; Chlorides; Dicyclomine; Female; In Vitro Techniques; Ion Channels; Kinetics; Mucous Membrane; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

Cholinergic receptors on canine bronchial smooth muscle (mediating excitation) or on the cholinergic nerve endings (modulating acetylcholine release) have not yet been characterized. In this study, the effects of agents selective for the various muscarinic receptors on mechanical and electrical responses in isolated segments of canine bronchial smooth muscle (3rd-6th order) were investigated. Carbachol (Cch), bethanechol (Bch), oxotremorine (Oxo), and McNeil A343 (McN) produced membrane depolarization and contractions with a ranked potency order of Oxo approximately Cch greater than Bch greater than McN. Oxo-induced contractions were antagonized by pirenzepine (Pir), AF-DX 116 (AF-DX), or hexahydrosiladifenidol (Hexa); Schild analysis yielded pA2 values of 6.61, 6.81, and 7.40, respectively. Contractions induced by field stimulation (FS) were antagonized by Pir, AF-DX, or Hexa with a potency order of Hexa greater than AF-DX approximately Pir. FS responses were potentiated by lower (i.e., M1-selective) concentrations of Pir, and antagonized by McN. The non-M1 agonists and antagonists had no such effects. These findings indicate that the postjunctional receptors are of the M3 subtype, and that the inhibitory receptors on the cholinergic nerve endings are of the M1 subtype.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Animals; Bethanechol Compounds; Bronchi; Carbachol; Dogs; Electric Stimulation; In Vitro Techniques; Membrane Potentials; Microelectrodes; Muscle Contraction; Muscle, Smooth; Oxotremorine; Piperidines; Pirenzepine; Receptors, Muscarinic

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.

Topics: Bronchi; Humans; In Vitro Techniques; Ipratropium; Muscle Contraction; Muscle, Smooth; Parasympatholytics; Piperidines; Pirenzepine; Receptors, Muscarinic; Trachea

The pharmacokinetics of tritiated hexahydro-sila-difenidol [( 3H]-HHSiD) were examined in rats. Furthermore, the distribution of radioactivity was studied by means of whole body autoradiography. After i.v. administration of 2.9 mg/kg HHSiD plus [3H]-HHSiD to anaesthetized rats bearing a catheter implanted in the ductus choledochus and receiving a mannitol infusion, HHSiD was rapidly distributed and metabolized. Only 5% of the radioactivity was recovered in blood after 23 s and 0.4% after 2.5 h. 64% of the plasma radioactivity could be extracted with hexane from the samples taken 23 s after administration. 52% of the radioactivity was eliminated within 2.5 h, 13% by urinary and 39% by biliary excretion. Following oral administration of 8.6 mg/kg HHSiD plus [3H]-HHSiD there was an absorption of approximately one fourth of the administered radioactivity within 4 h. By means of whole body autoradiography (i.v. injection) as well as by tissue distribution measurement the highest levels of radioactivity were found in bile, urine, lung, kidney, adrenals, liver and pancreas. Thus, after i.v. administration to rats HHSiD is rather quickly distributed, metabolized and excreted. This explains its low antimuscarinic potency in vivo.

Topics: Administration, Oral; Anesthesia; Animals; Autoradiography; Bile; Injections, Intravenous; Male; Parasympatholytics; Piperidines; Rats; Rats, Inbred Strains; Tissue Distribution

Five subtypes of muscarinic receptors have been distinguished by pharmacological and molecular biological methods. This report characterizes the muscarinic subtype present in human gastric mucosa by radioligand binding studies. The receptor density was 27 +/- 6 fmol/mg protein and the tritiated ligand N-methylscopolamine had an affinity of (KD) 0.39 +/- 0.08 nM (n = 11). The M1 receptor selective antagonist pirenzepine and the M2 receptor selective ligand AF-DX 116 had low affinities of 148 +/- 32 nM (n = 13) and 4043 +/- 1011 nM (n = 3) KD, respectively. The glandular M3 antagonists hexahydrosiladifenidol and silahexocyclium had high affinities of KD 78 +/- 23 nM (n = 5) and 5.6 +/- 1.8 nM (n = 3). The agonist carbachol interacted with a single low-affinity site and binding was insensitive to modulation by guanine nucleotides. Antagonist and agonist binding studies thus showed an affinity profile typical of M3 receptors of the glandular type.

Topics: Carbachol; Gastric Mucosa; Humans; In Vitro Techniques; N-Methylscopolamine; Parasympatholytics; Piperazines; Piperidines; Pirenzepine; Receptors, Muscarinic; Scopolamine Derivatives

Five subtypes of muscarinic receptors have been identified by pharmacological and molecular biological methods. The muscarinic receptor subtype mediating acid secretion at the level of the parietal cell was unknown. Therefore, this study was performed to characterize muscarinic receptors on rat gastric parietal cells using the 3 subtype-selective antagonists hexahydrosiladifenidol and silahexocyclium, which have high affinity for glandular M3 subtypes, and AF-DX 116, which has high affinity to cardiac M2 receptors. The affinity of these antagonists was determined by radioligand binding experiments. In addition, their inhibitory potency on carbachol-stimulated inositol phosphate production was investigated. Inhibition of carbachol-stimulated aminopyrine uptake was used as an indirect measure of proton production. Both M3 antagonists, hexahydrosiladifenidol and silahexocyclium, had nanomolar affinities for parietal cell muscarinic receptors and potently antagonized inositol phosphate production with nanomolar Ki values. Silahexocyclium similarly antagonized aminopyrine accumulation while hexahydrosiladifenidol behaved as a noncompetitive antagonist. AF-DX 116 was a low-affinity ligand and a weak competitive antagonist at parietal-cell muscarinic receptors. It was concluded that muscarinic M3 receptors mediate acid secretion probably by activation of the phosphoinositide second messenger system in rat gastric parietal cells.

Topics: Animals; Gastric Acid; Parasympatholytics; Parietal Cells, Gastric; Piperazines; Piperidines; Radioligand Assay; Rats; Receptors, Muscarinic

The potency and selectivity of (-)cis-2,3-dihydro-3-(4-methylpiperazinylmethyl)-2-phenyl-1,5 benzothiazepin-4-(5H)one HCl (BTM-1086) for muscarinic receptor subtypes was compared in functional assay systems, in guinea pig peripheral tissues, to known reference drugs: atropine (nonselective), pirenzepine (M1), AF-DX 116 (M2) and HHSiD (M3). Like atropine, BTM-1086 was a potent, nonselective, competitive muscarinic antagonist with no detectable antispasmodic activity in urinary bladder or ileal muscle. In vivo, in the guinea pig cystometrogram, BTM-1086 depressed intravesical bladder pressure (PvesP) with the same efficacy and potency as oxybutynin, a drug used clinically for the treatment of urinary incontinence. The pharmacological profile of BTM-1086, however, suggests that it may not be suitable for development for bladder dysfunction disorders.

Topics: Animals; Atropine; Calcium; Female; Guinea Pigs; Heart Atria; Ileum; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neurons; Parasympatholytics; Piperidines; Pirenzepine; Structure-Activity Relationship; Thiazepines; Tumor Cells, Cultured; Urinary Bladder; Vas Deferens

Pharmacological profiles of the striatal and brainstem M2 receptors were developed with a group of selective muscarinic antagonists. The striatal M2 muscarinic receptor was identified by its inhibition of [3H]cyclic AMP levels, whereas the brainstem M2 receptor was characterized using competition with [3H]quinuclidinyl benzilate binding. The potency of pirenzepine does not differentiate clearly between the striatal M2 receptor (Ki approximately 300 nM) and the brainstem M2 receptor (Ki = 219 nM) or peripheral M2 receptors. In the present study, we used 4-diphenylacetoxy-N-methylpiperidine methbromide, hexahydrosiladifenidol, AF-DX 116 and methoctramine to characterize the striatal and brainstem M2 receptors in more pharmacological detail. For comparison, the potencies of these antagonists were also measured at cortical M2 receptors (using competition with [3H]pirenzepine binding). The potencies of 4-diphenylacetoxy-N-methylpiperidine methbromide (KB = 0.19 nM) and hexahydrosiladifenidol (KB = 14 nM) in blocking the striatal M2 receptor suggested similarity to those M2 receptors localized in certain smooth muscles or in glands. However, AF-DX 116 (KB = 155 nM) and methoctramine (KB = 47 nM) were considerably more potent in blocking the striatal M2 receptor than as reported in functional studies in smooth muscle or glands. Thus, the profile of the striatal M2 receptor obtained with these antagonists did not match in all respects with either glandular (probable M4 gene product) or cardiac (probable M2 gene product) muscarinic receptors. In contrast, our data with the brainstem M2 receptor was highly correlated (r = 0.93) with literature data regarding the cardiac muscarinic system.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain Stem; Corpus Striatum; Cyclic AMP; Diamines; Parasympatholytics; Piperidines; Pirenzepine; Rats; Receptors, Muscarinic

The cardioselective muscarinic antagonist, AF-DX 116 [11[2-[(diethyl-amino)-methyl]-O-1-piperidinyl]-5,11-dihydro-6H-pyrido- [2,3-b][1,4]-benzodiazepine-6-one), was weak at blocking the M2 muscarinic receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) formation in mouse neuroblastoma cells (clone N1E-115). In contrast, the glandular-selective antagonists, hexahydro-sila-difenidol (HHSiD) and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), were quite potent at inhibiting this response, being 14- and 318-fold more potent than AF-DX 116 in this regard, respectively. According to the rank order of potency of these two classes of antagonists, these data provide the first pharmacological evidence that inhibition of cAMP formation in a neuronal tissue is mediated by a non-cardiac M2 muscarinic receptor subtype.

Topics: Animals; Binding, Competitive; Cyclic AMP; Mice; Muscarinic Antagonists; N-Methylscopolamine; Neuroblastoma; Piperidines; Receptors, Muscarinic; Scopolamine Derivatives; Tumor Cells, Cultured

In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a series of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M1-(rabbit vas deferens), M2- (guinea-pig atria) and M3- (guinea-pig ileum) receptors. All compounds were competitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenyl ring with a methoxy group or a chlorine atom as well as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 approximately M3 greater than M2. A different selectivity pattern was observed for p-fluoro-hexahydro-sila-difenidol: M3 greater than M1 greater than M2. This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA2 = 6.68) and lowest affinity for the M2-receptors in guinea-pig atria (pA2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2) and ileum (M3) of the rat. Furthermore, dose ratios obtained with either pirenzepine (M1) or hexahydrosila-difenidol (M2 and M3) and the p-fluoro analogue used in combination suggested that the antagonism was additive, implying mutual competition with a single population of muscarinic receptor subtypes. These results indicate that p-fluoro-hexahydro-sila-difenidol represents a valuable tool for characterization of muscarinic receptor subtypes.

Topics: Animals; Diamines; Female; Ganglia, Sympathetic; Guinea Pigs; Heart; Ileum; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Piperidines; Pirenzepine; Rabbits; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Vas Deferens

To investigate the muscarine receptor type mediating inhibition of [3H]-noradrenaline release from the isolated rat and guinea-pig iris we have determined the potency of antimuscarinic drugs to antagonize the methacholine-induced inhibition of [3H]-noradrenaline overflow evoked by field stimulation (3 Hz, 2 min). The prejunctional apparent affinities were compared with those obtained for postjunctional muscarine receptors mediating the methacholine-induced contraction of the isolated rabbit iris sphincter muscle. Prejunctional apparent affinity constants of pirenzepine (6.67), himbacine (8.51), methoctramine (7.92), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 8.00), hexahydro-difenidol enantiomers (6.92, (R); 5.77, (S)) in the rat iris and methoctramine (7.58) in the guinea-pig iris indicate the presence of M2 receptors. Although the postjunctional affinity constants in the rabbit iris sphincter of methoctramine (5.93), gallamine (3.92), and 4-DAMP (9.07) confirm our previous suggestions of the presence of M3-like receptors, the results obtained with the hexahydro-difenidol enantiomers do not agree with that concept. The postjunctional affinity constants of the hexahydro-difenidol enantiomers were not different from the prejunctional values (6.86, (R); 5.55, (S)), indicating a similar and low degree of stereoselectivity for these stereoisomers at both receptor sites (14 and 17, (R)/(S)-ratios, respectively). Hence, the postjunctional muscarine receptor in the rabbit iris sphincter fails to exhibit the high degree of stereoselectivity observed for hexahydro-difenidol enantiomers at M3 receptors on other smooth muscles.

Topics: Animals; Electric Stimulation; In Vitro Techniques; Iris; Kinetics; Methacholine Compounds; Muscle Contraction; Muscle, Smooth; Norepinephrine; Piperidines; Rabbits; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Muscarinic receptor subtypes in human and guinea pig lung membranes were characterised using selective muscarinic antagonists. Competition experiments were carried out against [3H](-)-quinuclidinyl benzilate binding at 25 degrees C in Tris-HCl buffer; non-specific binding was determined in the presence of 1 microM atropine. Of all the antagonists examined, only the M1-selective antagonist pirenzepine exhibited a heterogeneous binding profile (nH less than 1.0), best described by two-binding sites of high and low affinity. Binding of [3H]pirenzepine confirmed the presence of a high proportion of high affinity (M1) receptors (60% of total) in human peripheral lung. The high potency of M3-selective antagonists 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) and hexahydrosiladifenidol suggested the presence of M3 receptors, but the low potency of AF-DX 116 and methoctramine indicated that there was no significant population of M2 receptors present. The existence of muscarinic receptor subtypes in lung may have important clinical implication but their cellular localisation remains to be determined.

Topics: Adamantane; Adult; Aged; Animals; Atropine; Guinea Pigs; Humans; In Vitro Techniques; Lung; Middle Aged; Parasympatholytics; Piperidines; Pirenzepine; Protease Inhibitors; Quinuclidinyl Benzilate; Receptors, Muscarinic; Species Specificity

1. In an attempt to assess the structural requirements for the muscarinic receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a series of structurally related C/Si pairs were investigated, along with atropine, pirenzepine and methoctramine, for their binding affinities in NB-OK 1 cells as well as in rat heart and pancreas. 2. The action of these antagonists at muscarinic receptors mediating negative inotropic responses in guinea-pig atria and ileal contractions has also been assessed. 3. Antagonist binding data indicated that NB-OK 1 cells (M1 type) as well as rat heart (cardiac type) and pancreas (glandular/smooth muscle type) possess different muscarinic receptor subtypes. 4. A highly significant correlation was found between the binding affinities of the antagonists to muscarinic receptors in rat heart and pancreas, respectively, and the affinities to muscarinic receptors in guinea-pig atria and ileum. This implies that the muscarinic binding sites in rat heart and the receptors in guinea-pig atria are essentially similar, but different from those in pancreas and ileum. 5. The antimuscarinic potency of hexahydro-diphenidol and hexahydro-sila-diphenidol at the three subtypes was influenced differently by structural modifications (e.g. quaternization). Different selectivity profiles for the antagonists were obtained, which makes these compounds useful tools to investigate further muscarinic receptor heterogeneity. Indeed, the tertiary analogues hexahydro-diphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) had an M1 = glandular/smooth muscle greater than cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M1 preferring (M1 greater than glandular/smooth muscle, cardiac).

Topics: Animals; Cells, Cultured; Guinea Pigs; Humans; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Myocardium; Pancreas; Parasympatholytics; Piperazines; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Stereoisomerism; Structure-Activity Relationship

This study was performed to determine the subtype of M2 muscarinic receptor that is involved in the action of cholinergic agents on prostaglandin (PG) synthesis as well as on the mechanical function of the isolated rabbit heart perfused at a constant flow rate with Krebs-Henseleit buffer. The increase in PG output elicited by acetylcholine (ACh) or arecaidine propargyl ester (APE), a selective M2 agonist was attenuated by both 11-[2-[(diethylamino)methyl]-1-piperidinyl]acetyl-5,11-dihydro-6H- pyrido-[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116), an M2 alpha antagonist, and hexahydro-sila-difenidol (HHSiD), an M2 beta antagonist. The coronary vasodilating effect of ACh and APE was inhibited by HHSiD, but not by AF-DX 116, whereas the vasoconstrictor effect was blocked by AF-DX 116, but not by HHSiD. The decrease in heart rate produced by ACh or APE was blocked by AF-DX 116, but not by HHSiD; however, the decrease in developed tension produced by the cholinergic stimuli was abolished by all these muscarinic receptor antagonists. The increase in PG output or changes in the mechanical parameters of the heart produced by ACh or APE were not altered by adrenergic receptor antagonists, phentolamine and propranolol, or by the nicotinic receptor antagonist, hexamethonium. The effect of isoproterenol or exogenous arachidonic acid to enhance PG output was not altered by these M2 receptor antagonists; however, the cyclooxygenase inhibitor indomethacin abolished the output of PG elicited by these agents or by ACh or APE. These data indicate that the effect of cholinergic stimuli to promote cardiac PG synthesis and decrease developed tension is mediated through the activation of both M2 alpha and M2 beta subtypes of muscarinic receptors. The cholinergically induced vasodilating component of the coronary response is mediated through the activation of M2 beta, whereas the coronary vasoconstriction and the decrease in heart rate is mediated through the activation of M2 alpha muscarinic receptors.

Topics: Acetylcholine; Animals; Arecoline; Dose-Response Relationship, Drug; Heart; In Vitro Techniques; Male; Parasympatholytics; Parasympathomimetics; Piperidines; Pirenzepine; Prostaglandins; Rabbits; Receptors, Muscarinic

Heterogeneity in the muscarinic receptor population of guinea pig ileum longitudinal smooth muscle was found in competition binding experiments against N-methyl[3H]scopolamine using either a cardioselective (AF-DX 116) or a smooth muscle-selective (hexahydrosiladifenidol) antimuscarinic compound. AF-DX 116 recognized 65% of the total receptors with high affinity and 35% with low affinity. Hexahydrosiladifenidol distinguished 24% of the total receptors with high affinity and 76% with low affinity. The two affinity binding constants displayed in smooth muscle by the compounds were similar to those of heart and glands, suggesting that the muscarinic receptor population in the smooth muscle is formed of about 30% glandular type and 70% cardiac type of the M2 receptors. In dissociation experiments, the rate of breakdown of the N-methyl[3H]scopolamine receptor complex in the smooth muscle was rapid and similar to the dissociation of N-methyl[3H]scopolamine from muscarinic receptors in cardiac membranes, supporting the evidence for the presence of a large fraction of the cardiac receptor type in smooth muscle. To further characterize the population of the smooth muscle receptors recognized as glandular type, we performed protection experiments with hexahydrosiladifenidol, which binds to glandular M3 receptors with high affinity. Smooth muscle membranes were initially incubated with this compound and then phenoxybenzamine was added to irreversibly alkylate the remaining unprotected receptors. Data from competition and dissociation binding experiments showed that, under these conditions, this protected fraction of the total receptor population in ileum smooth muscle had all the characteristics of the glandular type, i.e., slow N-methyl[3H]scopolamine dissociation and affinity constants for a series of selective and nonselective muscarinic antagonists in the same order of magnitude as those found in the glandular tissue. These findings, together with the known observation that hexahydrosiladifenidol is more potent in inhibiting the functional activation of muscarinic receptors in smooth muscle relative to heart, lead to the hypothesis that smooth muscle contractility is mediated by a muscarinic receptor subtype similar to that found in glandular tissue.

Topics: Animals; Binding, Competitive; Guinea Pigs; Ileum; In Vitro Techniques; Kinetics; Male; Muscle, Smooth; Myocardium; N-Methylscopolamine; Parasympatholytics; Phenoxybenzamine; Piperidines; Pirenzepine; Receptors, Muscarinic; Salivary Glands; Scopolamine Derivatives

The complete amino acid sequence of porcine muscarinic acetylcholine receptor III has been deduced by cloning and sequencing the genomic DNA. The antagonist binding properties of muscarinic acetylcholine receptor III expressed from the cloned DNA in Xenopus oocytes correspond most closely to those of the pharmacologically defined M2 glandular (III) subtype.

Topics: Amino Acid Sequence; Animals; Base Sequence; DNA, Recombinant; Glycosylation; Molecular Sequence Data; Muscarine; Oocytes; Piperidines; Pirenzepine; Protein Conformation; Quinuclidinyl Benzilate; Rats; Receptors, Muscarinic; RNA, Messenger; Sequence Homology, Nucleic Acid; Swine; Xenopus laevis

The binding of hexahydrosiladifenidol, procyclidine, 4-DAMP (4-diphenylacetoxy-N-methylpiperidine) and AF-DX 116 to muscarinic receptors in the heart, ileum, urinary bladder, parotid gland and cerebral cortex from guinea pig was studied in competition experiments with (-)-[3H]QNB. The affinity of AF-DX 116 was higher in the heart than in the cortex and it was extremely low in the parotid gland. The affinities of hexahydrosiladefinidol, procyclidine and 4-DAMP were higher in the cortex and parotid gland than in the heart, bladder and ileum. Hexahydrosiladifenidol and 4-DAMP recognized two classes of muscarinic binding sites in the cortex. However, in contrast to functional data, binding results showed that 4-DAMP hexahydrosiladifenidol and procyclidine did not distinguish between the sites in the smooth muscles and those in the heart. Nevertheless, the present data support the view that the putative M2-receptors are heterogeneous, since the four drugs examined were found to distinguish between the muscarinic binding sites in the parotid gland and those in smooth muscles and heart.

Topics: Animals; Cerebral Cortex; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Myocardial Contraction; Parasympatholytics; Parotid Gland; Piperidines; Pirenzepine; Procyclidine; Quinuclidinyl Benzilate; Receptors, Muscarinic; Urinary Bladder

The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (3H)N-methylscopolamine [3)NMS) (KD values of 140 and 280 nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-([(2-[diethylamino)-methyl)-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on) (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptors also showed different (3H)NMS association and dissociation rates. These results support the concept of M2 receptor heterogeneity and confirm that M2 receptor subtypes have different binding kinetic properties.

Topics: Animals; Binding, Competitive; GTP-Binding Proteins; Kinetics; Male; Myocardium; N-Methylscopolamine; Pancreas; Parasympatholytics; Piperidines; Pirenzepine; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Type C Phospholipases

The characteristics of the muscarinic receptor in isolated gastric fundic cells from rabbit were determined by radioligand binding techniques and functional tests. The dissociation constants (KDS) of selective (hexahydrosiladifenidol and pirenzepine) and non-selective (N-methylscopolamine and atropine) muscarinic receptor antagonists obtained in competition experiments vs [3H]-N-methylscopolamine were compared with the pA2 values of the drugs as inhibitors of carbachol-stimulated [14C]-aminopyrine accumulation (an index of acid secretion) in the gastric fundic cells. Good correlations were found between the ability of the drugs to inhibit acid secretion and their affinity for muscarinic receptors in the gastric fundic cells. The rank order of potency in both tests was N-methylscopolamine greater than atropine greater than hexahydrosiladifenidol greater than pirenzepine. The character of the muscarinic receptor subtype present on gastric fundic cells was established by comparing the affinity values of the compounds for this receptor with those for the receptors in other rabbit tissues. It was found that only pirenzepine and hexahydrosiladifenidol displayed tissue selectivity in their binding profiles. The KDS for pirenzepine were 13nM for the M1 receptor of the cerebral cortex and about 500 nM for the M2 receptors of the submandibular and gastric glands and heart. Differently from pirenzepine, hexahydrosiladifenidol showed about 10-fold discrimination between the M2 subtype of the gland (KD = 31 nM) and the M2 subtype of the heart (KD = 330 nM).

Topics: Aminopyrine; Animals; Atropine; Carbachol; Gastric Fundus; Kinetics; Male; N-Methylscopolamine; Piperidines; Pirenzepine; Rabbits; Receptors, Muscarinic; Scopolamine Derivatives

Pre- and postjunctional pA2 values of the muscarinic antagonist hexahydrosiladifenidol were determined with guinea-pig ileum and rat heart. Hexahydrosiladifenidol did not discriminate between pre- and postjunctional receptors within the same organ but was more potent on the ileum (20-80 times) than on the heart. It is concluded that pre- and postjunctional muscarinic receptors in the heart may differ from those in the ileum.

Topics: Acetylcholine; Animals; Guinea Pigs; Heart; Ileum; In Vitro Techniques; Male; Muscle Contraction; Norepinephrine; Organ Specificity; Parasympatholytics; Piperidines; Rats; Rats, Inbred Strains; Receptors, Muscarinic