N-methylscopolamine Research Articles

Effects of CAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

The effects of increases in intracellular adenosine 3′,5′-cyclic monophosphate (CAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca 2+ ([Ca 2+] i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The CAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent CAMP formation with half-maximal effects (-IogEC 50) at concentrations of 7.6 ± 1.3 g/ml and 4.8 ± 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca 2+] i with half-maximal inhibition (-IogEC 50 at 5.2 ± 0.7 M. Pretreatment of TSMCs with either CTX (10 μ/ml, 4 h), forskolin (10–100 μM, 30 min), or dibutyryl CAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca 2+ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC 50 values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca 2+ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 μM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 μM], an inhibitor of CAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of CAMP/PKA was involved in these inhibitory effects of forskolin. The K D and B max values of the muscarinic receptor (mAChR) for [ 3H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF 4-stimulated IPs accumulation in canine TSMCs. The AIF 4-induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF4 and uncoupled to phospholipase C by forskolin treatment. We conclude that CAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca 2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca 2+]; are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of CAMP on tracheal smooth muscle function.

Chronic (-)-isoprenaline infusion down-regulates beta 1- and beta 2-adrenoceptors but does not transregulate muscarinic cholinoceptors in rat heart.

Regulation of beta-adrenoceptor (beta-ar) subtypes and transregulation of muscarinic cholinoceptors (mAchr) was examined in regions of rat heart after chronic infusion of (-)-isoprenaline (450 micrograms/kg per hour) for 14 days. Following (-)-isoprenaline infusion systolic blood pressure was reduced for 10 days but then gradually returned to control levels, whereas heart rate was increased for 7 days before declining to a level significantly above control. Heart weight to body weight ratio was increased in (-)-isoprenaline treated rats. beta-ar subtype densities were measured by quantitative autoradiography with [125I]-cyanopindolol (CYP) in sinoatrial node (SA), atrioventricular node (AV), bundle of His (BH), left (LB) and right (RB) bundle branches, interventricular (IVS) and interatrial (IAS) septa, right atria (RA), apex (AX) and mitral valve (MV). beta 1-ars were reduced by 59.1-74.2% in the AV conducting regions, 53.4% in the SA node and 43.3-53.4% in myocardial areas, beta 2-ars were markedly reduced in myocardial regions (93.2-98.5%) and in pacemaker and conducting regions (87.7-97.8%). No changes in mAchr densities measured using [3H]-N-methyl scopolamine (NMS) occurred in the AV node, BH, LB, RB, IVS and IAS following (-)-isoprenaline infusion. Densities of beta 1- and beta 2-ars and mAchrs were also measured in ventricular homogenates from control and (-)-isoprenaline treated animals. beta-ar levels were significantly reduced (P < 0.05) in treated animals and the ratio of beta 1- to beta 2-ars increased after treatment. mAchr density in ventricular homogenates measured using either [3H]-NMS or [3H]-quinuclidinyl [phenyl-4-3H]benzilate (QNB) was unchanged. Homogenates of left and right ventricle also showed no change using [3H]-NMS. Organ bath studies were used to investigate the effect of (-)-isoprenaline infusion on negative inotropic and chronotropic effects of the non-selective muscarinic receptor agonist bethanechol in left and right atria, respectively. Lower concentrations of bethanechol (3 x 10(-10) to 10(-6) M) produced a negative inotropic response in isolated electrically driven left atria from (-)-isoprenaline treated rats, but not from control rats, with the slope of the curves being significantly different between groups (ANCOVA, P = 0.037). At concentrations of bethanechol from 10(-6) to 3 x 10(-4) M the negative inotropic response was not changed between (-)-isoprenaline treated and control animals. Bethanechol also produced a negative chronotropic response at lower concentrations (10(-10) to 10(-6) M) in (-)-isoprenaline treated rats, but not in controls. A second, steeper phase of the negative chronotropic response occurred at concentrations of bethanechol greater than 10(-6) M and was also seen in control rats. Expression of M2 (cardiac) mAchrs (m2Achr) in left and right ventricular tissues measured using a quantitative non-competitive polymerase chain reaction (PCR) assay showed a significant (P = 0.001) 28.5% increase in expression in left ventricle and a significant (P = 0.003) 21.5% decrease in expression in right ventricle after (-)-isoprenaline treatment, compared to controls. There was no significant difference in total ventricular m2Achr expression between the two groups of rats. The results suggest that chronic beta-ar stimulation down-regulates both beta 1- and beta 2-ars, and appears to differentially transregulate m2Achr expression, but not mAchr protein. Following (-)-isoprenaline infusion, muscarinic receptor mediated responses were sensitised, with no change in receptor densities, suggesting changes occur in the cell signalling system beyond the level of the receptor.

Chapter 12 Allosteric regulation of muscarinic receptors

This chapter describes some of the work on the location of the allosteric site on muscarinic receptors and presents recent data on the binding of strychnine to this site. Much of the work on allosteric interactions at muscarinic receptors, including the initial studies, has utilized the neuromuscular blocker gallamine. This is a highly charged ligand, bearing three positive charges, and exhibits negatively cooperative interactions with a range of muscarinic agonists and antagonists. Paradoxically, gallamine dramatically slows down the association and dissociation kinetics of quaternized muscarinic antagonists, such as N-methyl scopolamine (NMS), when it binds to muscarinic receptors—in most instances decreases in affinity are associated with faster, and not slower, kinetics. The chapter concludes that strychnine, in addition to its actions on glycine receptors, acts as an allosteric ligand at ml-m4 muscarinic receptors and is able to quantitate its binding properties. Even smaller effect of strychnine on the binding properties of NMS and acetylcholine (ACh) at ml receptors is illustrated in the chapter.

Constitutive activity of the M 1–M 4 subtypes of muscarinic receptors in transfected CHO cells and of muscarinic receptors in the heart cells revealed by negative antagonists

We investigated whether muscarinic receptors of the M 1–M 4 receptor subtypes are constitutively active. We have found that the synthesis of cyclic AMP was enhanced by the muscarinic antagonists atropine and N-methylscopolamine (NMS) in Chinese hamster ovary (CHO) cells stably transfected with human m2 and m4 muscarinic receptor genes and in rat cardiomyocytes expressing the M 2 receptor subtype, and that the production of inositol phosphates was inhibited by atropine and NMS in CHO cells stably transfected with human m1 and m3 and with rat m1 muscarinic receptor genes. The muscarinic antagonists quinuclidinyl benzilate and AF-DX 116 had no effect in some cases and acted like atropine and NMS in others. We conclude that the M 1–M 4 subtypes of muscarinic receptors are constitutively active in the CHO cell lines expressing them and in cardiomyocytes and that atropine and NMS act as negative antagonists on these receptor subtypes by stabilizing them in the inactive conformation.

The role of the cholinergic system in the development of the human cerebellum

High affinity (−)nicotine ([ 3H]nicotine), α-bungarotoxin ([ 1251]α-bungarotoxin) and muscarinic binding ([ 3H] N-methyl scopolamine) in the human cerebellum were compared between the foetal period (23–39 weeks gestation) and young adulthood (14–34 years) in an autoradiographic study. To estimate proportions of muscarinic receptor subtypes variable wash times and displacement with pirenzepine were employed. [ 3H]Nicotine binding and total muscarinic binding in foetuses exceeded that in young adults by a factor of 6 and 2 respectively in the dentate nucleus, and by a factor of 3 in white matter. [ 3H]Nicotine and muscarinic binding was also higher in the foetal external granule cell layer than in the internal granule cell layer of adult. [ 125I]α-Bungarotoxin binding was raised in the dentate nucleus of the foetus compared with the adult. The M 2 subtype appeared to be the predominant muscarinic receptor in the cerebellum, however it tended to represent a lower proportion of the muscarinic binding in the foetus than the adult. All 3 receptor types were highest in the foetal brainstem where the M 3 + M 4 muscarinic subtypes appeared to predominate. The p75 nerve growth factor receptor, measured by immunocytochemistry, in common with cholinergic receptors, paralleled choline acetyltransferase activity which has previously been reported to be high in the cerebellum during late foetal development and to fall in adulthood.

Modulation of cardiac M 2 muscarinic receptor binding by progesterone-related steroids

We have previously shown that progesterone, but not estradiol or testosterone, can compete with [ 3H]N-methyl scopolamine (NMS) for the cardiac M 2 muscarinic binding site. Experiments have been carried out to investigate the inhibitory effects of a large variety of progesterone-like steroids on the M 2 muscarinic receptor. These studies were performed with the aid of a new binding assay which uses intact tissue in the form of ventricular micropunches. Our data show that synthetic, clinically-used progestins such as Provera, norgesterel and cyproterone are largely ineffective at the M 2 binding site whereas the naturally occurring progesterone derivatives 17a hydroxy progesterone and Reichstein's Substance S are highly active. (K 1 values 5 × 10 −6M and 1 × 10 −6M, respectively). Minor structural modifications such as acetylation of the 17α hydroxy group abolishes activity. Steroids known to exert cell membrane effects, such as alfaxalone and pregnenolone sulfate, had no influence on [ 3H] NMS binding. The progesterone antagonist RU-486 did not block the inhibitory effect of progesterone. Moreover, this putative receptor may be located in the cardiomyocyte membrane since 17α hydroxy progesterone induces rapid dissociation of [ 3H] NMS from its binding site (30% reduction in 5 min). We attempted to further localize the inhibitory locus to the M 2 receptor itself by means of the irreversible antagonist propylbenzilylcholine mustard (PrBCM). Ninety percent of the M 2 receptor could be blocked by PrBCM (10 −6 M), an effect reversible by the specific muscarinic antagonist scopolamine methyl bromide but not by progesterone. These results suggested that progeserone does not interact directly with the [ 3H] NMS labelled M 2 binding site. Atropine-induced dissociation of [ 3H] NMS binding was not affected by progesterone, suggesting the lack of an allosteric effect. We conclude that (a) some naturally-occurring, but not synthetic, progesterone-like molecules inhibit [ 3H] NMS binding to cardiac M 2 receptors (b) this inhibition is not mediated via the [ 3H] NMS binding site nor at an allosteric site (c) the modulatory effect is possibly non-genomic.

Muscarinic cholinergic receptors in brain and atrial membranes of adult brook trout ( Salvelinus fontinalis) measured by radioligand binding techniques

Muscarinic cholinergic receptors were measured by radioligand binding techniques in crude membrane particulate preparations of brain and atrial tissues from laboratory reared brook trout (Salvelinus fontinalis). The radioligand [ 3 H]N- methyl scopolamine was used to determine number and affinity of receptors in saturation experiments. The affinity of the radioligand did not differ in brain and atrial preparations (96 ± 8 and 60 ± 4 pM, respectively). However, the number of binding sites was greater in atrium compared with brain (269 ± 19 and 166 ± 7 fmol/mg protein, respectively). The rank order of potency of competing drugs in inhibition experiments was similar for antagonists with atropine ≥ scopolamine, >, pirenzepine. Pirenzepine, an M 1-selective drug had a 3-fold higher affinity in brain than atrium. The agonists oxotremorine and carbachol each bound to two sites in both tissues. In contrast, pilocarpine bound to only one site in brain and two in atrial tissue. These results are compared with those observed in other nonmammalian species and discussed with reference to conservation of proteins that serve important cellular roles.

Functional Expression of a Cloned Drosophila Muscarinic Acetylcholine Receptor in a Stable Drosophila Cell Line

A cloned Drosophila muscarinic acetylcholine receptor (mAChR) has been stably expressed in a Drosophila cell line (S2) under the control of an inducible Drosophila metallothionein promoter. A clonal cell line (S2-Dm1-1) has been isolated which, after induction of mAChR expression with CuSO4, exhibits high-affinity, saturable, specific binding of the muscarinic antagonist N-methyl scopolamine (NMS). The apparent molecular mass of the expressed protein, calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), is in good agreement with the apparent molecular mass of mAChRs purified from Drosophila brain. Functional expression of the cloned mAChR in this stable cell line has been demonstrated by quantitative fluorescence ratio-imaging of Fura-2-loaded cells. We have observed transient, agonist-induced elevations in intracellular Ca2+ levels which can be completely blocked by atropine, whereas AFDX-116, a muscarinic antagonist which binds preferentially to the vertebrate mAChR M2 subtype, has little effect at 100 mumol l-1. The suitability of this stable Drosophila expression system for the characterization of neurotransmitter receptors is discussed.

Pharmacological characterization of muscarinic acetylcholine binding sites in human and bovine cerebral microvessels.

The aim of this study was to investigate the presence of muscarinic receptors in human brain microvessels (MVs) and capillaries (CAPs) and, further, to pharmacologically characterize these receptors as well as those in bovine cerebral microvascular beds. For this purpose, the binding of [3H]N-methyl scopolamine ([3H]NMS) was assessed in isolated human and bovine cerebral MVs and CAPs and competition studies were performed against [3H]NMS binding with several well characterized muscarinic antagonists. The antagonist cerebrovascular affinity constants (pKD) were determined with the computer-fitting software LIGAND and then compared by correlation analyses to their reported affinities (pKi) at the five cloned muscarinic receptors. The specific binding of [3H]NMS to human and bovine MVs and CAPs was saturable, of high affinity and competitively inhibited by muscarinic antagonists. Heterogeneous populations of muscarinic binding sites were found in the microvascular tissues from both species. In human cortical MVs, the pharmacological binding profile obtained from various muscarinic receptor antagonists was best correlated to that of the cloned ml (r = 0.95; p < 0.001) and less so m5 (r = 0.77; p = 0.025) receptor subtypes while in bovine MVs, the presence of the m1 subtype was strongly suggested. Cerebrovascular affinities obtained for selected muscarinic antagonists in single preparations of human and bovine CAPs were suggestive of the presence of M1/m1 and M3/m3 receptor subtypes, and possibly the m5 subtype in bovine CAPs. The detection of M1/m1, M3/m3 and possibly m5 muscarinic receptor subtypes in brain microcirculation is consistent with reports where these receptors have been shown to mediate vasoconstriction, vasodilatation, and activation of nitric oxide synthase, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic infusion of (-)-isoprenaline on rat cardiac muscarinic (M2)-cholinoceptors and beta 1- and beta 2-adrenoceptors.

1. The effects of chronic (-)-isoprenaline infusion (400 micrograms kg-1 h-1; 4 h-14 days) on rat ventricular muscarinic M2-cholinoceptors and beta 1- and beta 2-adrenoceptors was determined. [3H]-N-methylscopolamine (NMS) was used to measure M2-cholinoceptor binding and (-)-[125I]-cyanopindolol (CYP) was used for beta 1- and beta 2-adrenoceptor binding. 2. Chronic infusion of (-)-isoprenaline did not affect either the affinity of [3H]-NMS for M2-cholinoceptors, or the maximal density of these receptors (Bmax) at all treatment periods investigated (4 and 20 h; 7 and 14 days). The affinity of (-)-[125I]-CYP for beta-adrenoceptors was not changed following chronic (-)-isoprenaline infusion (4, 12 and 20 hours; 7 and 14 days). 3. Competition between (-)-[125I]-CYP and the selective beta 1-adrenoceptor antagonist CGP 20712A resulted in biphasic curves at all treatment periods corresponding to the presence of beta 1- and beta 2-adrenoceptor binding sites. beta 1-adrenoceptors made up the greater proportion of beta-adrenoceptors in rat ventricle. There was no change in the ratio of beta 1- to beta 2-adrenoceptors following 4 h (-)-isoprenaline infusion [(beta 1:beta 2), vehicle: 77.5 +/- 4.0%:22.5 +/- 4.0%, n = 6; (-)-isoprenaline: 79.2 +/- 1.2%:20.8 +/- 1.2%, n = 6], however infusion for 12 h increased the ratio [(beta 1:beta 2), vehicle: 65.2 +/- 6.1%:34.8 +/- 6.1%, n = 6; (-)-isoprenaline: 85.6 +/- 1.8%:14.4 +/- 1.8%, n = 6, P < 0.05], which was maintained for longer infusion periods (20 h-14 days). 4. Both beta 1- and beta 2-adrenoceptors were down-regulated in rat ventricular tissue following (-)-isoprenaline infusion. beta 2-adrenoceptors were down-regulated by 12 h (vehicle: 11.8 +/- 2.4 fmol mg protein-1, n = 6; (-)-isoprenaline: 3.5 +/- 0.4 fmol mg protein-1, n = 6, P < 0.05) however beta 1-adrenoceptors were not affected (vehicle: 21.7 +/- 2.0 fmol mg protein-1, n = 6; (-)-isoprenaline: 20.9 +/- 0.6 fmol mg protein-1, n = 6, P > 0.05) until 20 h infusion (vehicle: 19.4 +/- 2.2 fmol mg protein-1, n = 6; (-)-isoprenaline: 12.6 +/- 2.2 fmol mg protein-1, n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

A potential strategy for decreasing milk production in the ewe at weaning using a growth hormone release blocker2

Following the loss or removal of ewes' lambs, ewes frequently continue to lactate for a short time, which potentially predisposes them to mastitis. Therapies that stop or reduce milk production in such situations would be beneficial. Milk production in ruminants is positively correlated with serum concentrations of growth hormone (GH). We sought to determine whether methscopolamine bromide (MB), an anticholinergic agent reported to block GH secretion, would selectively and reversibly reduce milk yield in lactating ewes. White-face ewes (n = 24) that were nursing lambs were assigned on d 59 +/- 2 postpartum (d 0) to receive s.c. injections of 96 mg of MB on d 2, 3, and 4 (1630). On d 0, 2, 5, and 7 (0900), ewes were sequentially separated from their lambs, treated with 40 IU oxytocin, and 30 s later were milked. Ewes remained separated from their lambs for 6 h, after which (1500) ewes were again milked and quantity of milk was determined. Milk yields at d 0, 2, 5, and 7 were 267 +/- 16.9, 266 +/- 15.5, 225 +/- 11.9, and 244 +/- 13.4 g, respectively. It was concluded that treatment of ewes with MB reduced milk yield (P < .01), and the effect was reversible. A second experiment was performed to determine the acute effects of MB on serum concentrations of GH. Hampshire ewes (n = 14) that were nursing lambs were assigned on d 55 +/- 1 postpartum to receive a.s.c. injection of saline (n = 7) or 96 mg of MB (n = 7) at min 0.(ABSTRACT TRUNCATED AT 250 WORDS)

A snake toxin against muscarinic acetylcholine receptors: Amino acid sequence, subtype specificity and effect on guinea-pig ileum

The sequence of muscarinic toxin 1 (MT1) from Dendroaspis angusticeps (green mamba) was determined (66 amino acids, M r 7509). The central part, peptide 25–40, is rich in hydrophobic amino acids, which is a characteristic of muscarinic toxins. MT1 started to inhibit [ 3H]-NMS ( N-methylscopolamine) binding to synaptosomal membranes of porcine brain (contains all five receptor subtypes) at about 1 nM and to membranes from pig heart muscle (only subtype m2) at about 1 μM. Binding of [ 3H]-AF-DX 384 to heart was inhibited with an ic 50 of 14 μM and to brain in two steps. In the first step (IC 50 = 32 nM) binding decreased by 37%, indicating that the toxin acted on m1 or m4 receptors, each accounting for about 40% of total receptor content. The second step was similar to the effect on heart. Pirenzepine inhibited binding of [ 125I]-MT1 to brain receptors with an ic 50 of 6.5 nM, corresponding to a K i, of about 6 nM. Literature values of K i for pirenzepine are 16–18 nM for m1 and ⩾ 120 mM for other subtypes. This indicates binding to m1 receptors. [ 125I]-MT1 bound to brain with a K d of 20 nM and a Hill coefficient of 1.0 i.e. one toxin molecule per receptor. In guinea-pig ileum, MT1 (670 nM) produced a rapid contraction, reversible by atropine. The toxin may be an agonist, but might also cause contraction by inducing acetylcholine release by a different mechanism.

Binding of muscarinic toxins MT×1 and MT×2 from the venom of the green mamba Dendroaspis angusticeps to cloned human muscarinic cholinoceptors

Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Previous competition binding studies indicate that the MTxs may be selective for the M, subtype of muscarinic acetylcholine receptors. The present work was undertaken in order to clarify the muscarinic subtype specificity and functional effects of MTx1 and MTx2. Binding interactions were determined using 3 H- N-methyl scopolamine (NMS) and cloned human muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary functional studies were performed on rabbit vas deferens preparations, which contain M 1 cholinoceptors. MTx1 and MTx2 inhibited 3H-NMS binding to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for ml receptors: K for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and K i for MTx2 were 364 nM at m1 and 1.2μM at m3 receptors. At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit vas deferens preparations, MTx1 and MTx2 at concentrations above 50 nM behaved in a similar way to the relatively selective M 1-agonists M cN-A-343 and CPCP (4-[ N-(4-chlorophenyl)carba-moyloxy]-4-pent-2-ynyl-trimethylammonium iodide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 receptors, but they also reveal a slightly weaker effect at m3 receptors. The interaction at m1 receptors apears to be essentially irreversible, implying that the toxins could be useful tools in studies of the functional role of m1 muscarinic receptors.

Antimuscarinic action of liriodenine, isolated from Fissistigma glaucescens, in canine tracheal smooth muscle.

1. The antimuscarinic properties of liriodenine, isolated from Fissistigma glaucescens, were compared with methoctramine (cardioselective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, smooth muscle selective M3 antagonist) by radioligand binding tests, functional tests and measurements of second messenger generation in canine cultured tracheal smooth muscle cells. 2. Liriodenine, pirenzepine, methoctramine and 4-DAMP displaced [3H]-N-methyl scopolamine ([3H]-NMS) binding in a concentration-dependent manner with Ki values of 2.2 +/- 0.4 x 10(-6), 3.3 +/- 0.7 x 10(-7), 8.9 +/- 2.3 x 10(-8) and 2.3 +/- 0.6 x 10(-9) M, respectively. The curves for competitive inhibition of [3H]-NMS with liriodenine, methoctramine and 4-DAMP were best fitted according to a two site model of binding, but pirenzepine was best fitted according to a model with one site. 3. Liriodenine and 4-DAMP displayed a high affinity for blocking tracheal contraction (pKB = 5.9 and 9.1, respectively) and inositol phosphate formation (pKB = 6.0 and 8.9, respectively), but a low affinity for antagonism of cyclic AMP inhibition (pKB = 4.7 and 7.8, respectively). 4. Methoctramine blocked cyclic AMP inhibition with a high affinity (pKB = 7.4), but it antagonized tracheal contraction and inositol phosphate formation with a low affinity (pKB = 6.1 and 6.0, respectively). 5. In conclusion, both M2 and M3 muscarinic receptor subtypes coexist in canine tracheal smooth muscle and are coupled to the inhibition of cyclic AMP formation and phosphoinositide breakdown, respectively. The antimuscarinic characteristics of liriodenine are similar to those of 4-DAMP. It may act as a selective M3 receptor antagonist in canine tracheal smooth muscle.

Effects of disrupting the cholinergic system on short-term spatial memory in rats

The effects of disrupting the muscarinic or nicotinic systems on short-term spatial memory were investigated using a delayed matching to position (DMTP) procedure. Rats were trained on the DMTP until stability and then divided into two groups: one group was implanted with an indwelling cannula aimed at the lateral ventricle. The cannulated group received injections of selective muscarinic antagonists (pirenzepine, M1; AFDX 116, M2; UH-AH 37, M1/M3) or hemicholinium-3 (a choline uptake inhibitor). The remaining animals were treated with conventional muscarinic antagonists (scopolamine, methyl scopolamine) or nicotinic channel blockers (mecamylamine, hexamethonium). Scopolamine, methyl scopolamine and UH-AH 37 disrupted all performance parameters in a non-specific but dose related manner. Pirenzepine disrupted accuracy in a delay, but not dose dependent manner, and exerted no other negative effects on performance. Hemicholinium-3-induced performance deficits showed some elements of effects seen following pirenzepine and scopolamine (delay dependent effects on accuracy, some negative effects on other motoric aspects of performance). AFDX 116 and hexamethonium had no significant effects on performance with respect to control. Mecamylamine reduced accuracy and increased response latencies at the highest dose tested. These data indicate that muscarinic antagonists are more effective at disrupting mnemonic performance than nicotinic blockers, and moreover, that distinct muscarinic receptors may have differential effects on cognitive performance.

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells

In cultured canine tracheal smooth muscle cells (TSMCs), muscarinic receptor stimulation led to phosphoinositide (PI) hydrolysis, formation of inositol phosphates (IPs), and mobilization of intracellular Ca2+. Desensitization of IPs accumulation and Ca2+ mobilization evoked by carbachol was investigated using [3H]inositol labelling and Ca(2+)-sensitive dye fura-2. Treatment of TSMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min blocked the carbachol-stimulated formation of IPs and mobilization of Ca2+. The concentrations of PMA that gave half-maximal and maximal inhibition of carbachol-induced IPs accumulation were 70 nM and 1 microM, respectively. The inhibitory effect of PMA on carbachol-induced responses was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. Treatment of TSMCs with PMA for 24 h, the cells remained the ability to response to carbachol-induced IPs accumulation and Ca2+ mobilization with the same extent as that observed in the control group. Inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit the responses. The KD and Bmax of the muscarinic receptor for [3H]N-methyl scopolamine binding were not significantly changed by PMA treatment for either 30 min or 24 h. The locus of this inhibition was further investigated by examining the effect of PMA on AlF4(-)-stimulated IPs accumulation in canine TSMCs. AlF4(-)-induced response was inhibited by PMA treatment, supporting that G protein(s) can be directly activated by AlF4- which was uncoupled to phospholipase C (PLC) by PMA treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of ventricular M2 muscarinic receptors in cardiomyopathic hamster (CHF 147) at the necrotic stage of the myopathy

We have previously demonstrated that isolated ventricular myocytes from cardiomyopathic hamsters (CHF 147) during the necrotic stage (70-100 days) exhibit an attenuated contractile response to muscarinic stimulation. In the present study we have investigated whether this dysfunction may be related to a change in the density (or affinity) of cardiac muscarinic receptors. Thus, we have characterized and quantified the binding of the muscarinic antagonist [3H]-N-methyl scopolamine (NMS) to M2 muscarinic receptors in cardiac micropunches and in suspensions of isolated intact cardiomyocytes obtained from cardiomyopathic (CHF 147) and Golden Syrian hamsters. The hamsters were either 70-100 days old, when the cardiomyopathy had reached the cytolytic and necrotic stage or 30 days old, i.e. before the onset of the cardiomyopathy. In both preparations (micropunches and dissociated cardiomyocytes) the specific binding of [3H]-NMS was stereospecific, reversible, saturable, of high affinity and linearly dependent upon increasing amounts of tissue and cells. The binding site also possessed the drug specificity typical of an M2 muscarinic receptor. Saturation binding analysis revealed that the hearts of the older CHF 147 hamsters contain significantly fewer M2 muscarinic receptors than the control Golden Syrian hamsters while the affinity (Kd) was not altered. This reduction of M2 receptor number was not observed in CHF 147 hamsters at 30 days. Further, we found no differences in beta-adrenergic or in alpha 1-adrenergic binding in the two strains of hamster at either age. Thus, our results indicate that the parasympathetic regulation of cardiac function in CHF 147 hamsters may be compromised by a decreased number of muscarinic receptors at the necrotic stage of the cardiomyopathy.

The involvement of muscarinic receptor subtypes in the mediation of hypothermia, tremor, and salivation in male mice.

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

Total autonomic blockade eliminates the attenuated pressor response to angiotensin II in pregnant rats

Pressor responses to angiotensin II (ANG II) are markedly attenuated in reflex-intact pregnant animals, a phenomenon widely attributed to intrinsic changes in vascular reactivity. To test the hypothesis that gestational augmentation of neural reflex activity contributes importantly to this phenomenon, changes in mean arterial pressure (MAP), cardiac output (CO), and total peripheral resistance (TPR) were compared during constant infusion (25-400 ng.kg-1.min-1) of ANG II in conscious virgin and pregnant rats, using a model of total autonomic blockade (chlorisondamine chloride and methscopolamine bromide), with restoration of baseline hemodynamics by infusion of norepinephrine. Basal CO was higher and TPR lower in pregnant (CO 121.8 +/- 3.8 ml/min; TPR 0.78 +/- 0.04 mmHg.ml-1.min) compared with virgin (CO 95.9 +/- 3.9 ml/min; TPR 1.05 +/- 0.08 mmHg.ml-1.min) rats (P < 0.005). Pressor responses to ANG II were similar in both groups of reflex-blocked animals due to comparable changes in TPR and CO (not significant by repeated-measures analysis of variance). Other experiments demonstrated that changes in MAP after bolus administration of ANG II did not differ in areflexic virgin and gravid rats. Thus in the absence of autonomic control ANG II has similar effects on systemic resistance in pregnant and nonpregnant rats, suggesting that reflex neural mechanisms contribute significantly to gestational changes in pressor responsiveness. These data further suggest that pregnancy is not accompanied by a generalized decrease in vascular reactivity to all pressor agents.

Agonist-regulated alteration of the affinity of pancreatic muscarinic cholinergic receptors.

Heterologous desensitization is a term that indicates that exposure of a cell to an agonist attenuates the response of that cell to other agonists. We examined heterologous desensitization of muscarinic cholinergic receptors of pancreatic acini and characterized mechanisms that might be responsible for desensitization. Muscarinic cholinergic receptor binding was measured by using N-[3H]methscopolamine bromide ([3H]NMS). N-Methscopolamine bromide (NMS), a receptor antagonist, bound to a single class of receptors with an affinity of 0.22 +/- 0.04 nM and a capacity of 61.5 +/- 5.1 fmol/mg of protein. These parameters of NMS binding sites were not altered by an addition of cholecystokinin (CCK) octapeptide, CCK-JMV-180, vasoactive intestinal peptide, 8-bromo-cAMP, 4-bromo-A23187, thapsigargin, or 12-O-tetradecanoylphorbol-13-acetate (TPA). Analysis of competitive inhibition curve of [3H] NMS binding by carbachol showed apparently two classes of carbachol binding sites with high affinity (38.6%) and low affinity (61.4%). Simultaneous incubation of carbachol with CCK or TPA increased the relative affinity of [3H]NMS binding, and the competitive inhibition curves showed a single class of carbachol binding site. L-364,718 blocked the effect of CCK, and staurosporine blocked the effects of TPA and partially blocked the effect of CCK. CCK-JMV-180, vasoactive intestinal peptide, 8-bromo-cAMP, 4-bromo-A23187, and thapsigargin had no effects on the competitive binding. Second, the carbachol-induced sequestration of the receptors was examined. Incubation of acini with carbachol resulted in a decrease of [3H] NMS binding sites, and the addition of CCK or TPA caused an inhibition of the carbachol-induced disappearance of [3H]NMS binding sites. Finally, studies that examined the biological response of the acinar cells showed that biphasic amylase release in response to carbachol was completely suppressed by 10 nM CCK for entire range of carbachol. Taken together, these results suggest that the effect of CCK on carbachol-induced sequestration is important for the alteration of the apparent affinity of carbachol binding sites and the biological response of acinar cells to carbachol. Further, the results suggest that another factor that induces uncoupling of receptor from effector might be involved in agonist-regulated desensitization. The results, that CCK-JMV-180 or other agonists that activate the adenylate cyclase pathway did not exert these effects of CCK, suggest that protein kinase C may be one of the key factors involved in heterologous desensitization by CCK on the carbachol binding sites and the suppression of carbachol-induced amylase release.