Quinuclidinyl Benzilate Research Articles

Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro.

Intraperitoneal injection of choline (30-90 mg.kg-1) produced a dose-dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg.kg-1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg.kg-1), pirenzepine (2 mg.kg-1) and 4-diphenylacetoxy-N-methylpiperidine (2 mg.kg-1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg.kg-1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g.kg-1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10-130 microm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1-10 mm). Hemicholium-3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1-32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.

Upregulation of muscarinic receptors by long-term nitric oxide inhibition in the rat ileum.

1. The aim of the present study was to examine the effects of long-term nitric oxide (NO) blockade on contractions of the rat ileum induced by muscarinic agonists. 2. Male Wistar rats received the NO synthesis inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 20 mg/rat per day) in drinking water for 7, 15, 30 and 60 days. Concentration-responses curves to methacholine and carbachol were obtained and pEC50 values were calculated. Saturation binding assays were performed in membranes prepared from rat ileum after 60 days of l-NAME treatment and the dissociation constant (KD) and maximal number of binding sites (Bmax) were determined by Scatchard analysis. 3. The NO synthase activity of the ileum was markedly reduced in all l-NAME-treated groups. At 60 days after l-NAME treatment, a significant increase in the potency of methacholine (fourfold) and carbachol (threefold) was observed. In binding studies, we found a significant increase in Bmax for [3H]-quinuclidinyl benzilate of approximately 57% in the l-NAME treated group without any significant change in KD values. The contractile response to methacholine was not modified by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (3 micro mol/L). No morphological alterations in the rat ileum were observed in l-NAME-treated rats. 4. Our findings suggest that treatment with l-NAME for 60 days induces a marked increase in the potency of methacholine and carbachol, as well as an increase in receptor number in the rat ileum.

Dual effects of nicotine on oxidative stress and neuroprotection in PC12 cells

In order to identify the properties of nicotine in relation to oxidative stress or neuroprotection, differentiated PC12 cells were treated with nicotine, beta-amyloid peptide (Aβ 25–35), free radical inducer and antioxidant by a separate addition or a combination way. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lipid peroxidation, [ 3 H ]epibatidine binding sites for nicotinic receptor and [ 3 H ]quinuclidinyl benzilate (QNB) for muscarinic receptor have been detected. The significant decrease of MTT reduction and increase of lipid peroxidation in PC12 cells were only observed at treatments with high concentrations of nicotine (1 and 10 mM), while Vitamin E (VitE), an antioxidant, can prevent the neurotoxic effects. In addition, nicotine in low dosage (10 μM) rescued the decreased rates of cell viability and inhibited the production of lipid peroxidation resulted from H 2O 2 and Aβ in the cultured cells. Significant increases in [ 3 H ]epibatidine binding sites were observed in PC12 cells exposed to nicotine, while no change was detected in [ 3 H ]QNB. The decreased number of nicotinic receptor binding sites due to the toxicity of Aβ was prevented by the addition of nicotine with low concentration. It is plausible that nicotine treatment may play dual effects on oxidative stress and neuroprotection, in which the effects are dependent on the differences in dosage of the drug used and their mechanisms of action. Generally, high dose of nicotine may induce neurotoxicity and stimulate oxidative stress, while reasonably low concentration may act as an antioxidant and play an important role for neuroprotective effect.

Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

The inhibitory effects of tramadol on muscarinic receptor-induced responses in Xenopus oocytes expressing cloned M(3) receptors.

Tramadol is a widely used analgesic, but its mechanism of action is not completely understood. Muscarinic receptors are involved in neuronal function in the brain and autonomic nervous system, and much attention has been paid to these receptors as targets of analgesic drugs in the central nervous system. In this study, we investigated the effects of tramadol on type-3 muscarinic (M(3)) receptors using the Xenopus oocyte expression system. Tramadol (10 nM-100 micro M) inhibited acetylcholine-induced currents in oocytes expressing M(3) receptor. Although GF109203X, a protein kinase C inhibitor, increased the basal current, it had little effect on the inhibition of acetylcholine-induced currents by tramadol. Moreover, tramadol inhibited the specific binding sites of [(3)H]quinuclidinyl benzilate. These findings suggest that tramadol at clinically relevant concentrations inhibits M(3) function via quinuclidinyl benzilate-binding sites. This may explain the modulation of neuronal function and the anticholinergic effects of tramadol. Muscarinic receptors are involved in neuronal function and are targets of analgesic drugs. We here report that tramadol inhibits type-3 muscarinic receptors function via quinuclidinyl benzilate-binding sites at clinically relevant concentrations. These findings may explain the modulation of neuronal function and the anticholinergic effects of tramadol.

Muscarinic toxin-like proteins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene organization.

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37-Ala38 in BM8; Lys37-Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchR-binding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with alpha-neurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the protein-coding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.

Effects of N-Ethylmaleimide on Conformational Equilibria in Purified Cardiac Muscarinic Receptors

Muscarinic receptors purified from porcine atria and devoid of G protein underwent a 9-27-fold decrease in their apparent affinity for the antagonists quinuclidinyl benzilate, N-methylscopolamine, and scopolamine when treated with the thiol-selective reagent N-ethylmaleimide. Their apparent affinity for the agonists carbachol and oxotremorine-M was unchanged. Conversely, the rate of alkylation by N-ethylmaleimide, as monitored by the binding of [(3)H]quinuclidinyl benzilate, was decreased by antagonists while agonists were without effect. The receptor also underwent a time-dependent inactivation that was hastened by N-ethylmaleimide but slowed by quinuclidinyl benzilate and N-methylscopolamine. The destabilizing effect of N-ethylmaleimide was counteracted fully or nearly so at saturating concentrations of each antagonist and the agonist carbachol. Similar effects occurred with human M(2) receptors differentially tagged with the c-Myc and FLAG epitopes, coexpressed in Sf9 cells, and extracted in digitonin/cholate. The degree of coimmunoprecipitation was unchanged by N-ethylmaleimide, which therefore was without discernible effect on oligomeric size. The data are quantitatively consistent with a model in which the purified receptor from porcine atria interconverts spontaneously between two states (i.e. R R*). Antagonists favor the R state; agonists and N-ethylmaleimide favor the comparatively unstable R* state, which predominates after purification. Occupancy by a ligand stabilizes both states, and antagonists impede alkylation by favoring R over R*. Similarities with constitutively active receptors suggest that R and R* are akin to the inactive and active states, respectively. Purified M(2) receptors therefore appear to exist predominantly in their active state.

Comparative distribution of binding of the muscarinic receptor ligands pirenzepine, AF-DX 384, (R,R)-I-QNB and (R,S)-I-QNB to human brain

Quinuclidinyl benzilate (QNB) and its derivatives are being developed to investigate muscarinic receptor changes in vivo in Alzheimer's disease and dementia with Lewy bodies. This is the first study of [ 125I]-(R,R)-I-QNB and [ 125I]-(R,S)-I-QNB binding in vitro in human brain. We have compared the in vitro binding of the muscarinic ligands [ 3H]pirenzepine and [ 3H]AF-DX 384, which have selectivity for the M1 and M2/M4 receptor subtypes, respectively, to the binding of [ 125I]-(R,R)-I-QNB and [ 125I]-(R,S)-I-QNB. This will provide a guide to the interpretation of in vivo SPET images generated with [ 123I]-(R,R)-I-QNB and [ 123I]-(R,S)-I-QNB. Binding was investigated in striatum, globus pallidus, thalamus and cerebellum, and cingulate, insula, temporal and occipital cortical areas, which show different proportions of muscarinic receptor subtypes, in post-mortem brain from normal individuals. M1 receptors are of high density in cortex and striatum and are relatively low in the thalamus and cerebellum, while M4 receptors are mainly expressed in the striatum, and M2 receptors are most evident in the cerebellum and thalamus. [ 125I]-(R,R)-I-QNB and [ 125I]-(R,S)-I-QNB density distribution patterns were consistent with binding to both M1 and M4 receptors, with [ 125I]-(R,R)-I-QNB additionally binding to a non-cholinergic site not displaceable by atropine. This distribution can be exploited by in vivo imaging, developing ligands for both SPET and PET, to reveal muscarinic receptor changes in Alzheimer's disease and dementia with Lewy bodies during the disease process and following cholinergic therapy.

Nitric oxide synthase, arginase and cyclooxygenase are involved in muscarinic receptor activation in different murine mammary adenocarcinoma cell lines

Investigations on the influence of the parasympathetic nervous system via muscarinic signaling in tumor progression have produced contradictory evidence. We investigated the expression of muscarinic acetylcholine receptors (mAchR) and their intracellular transduction pathways, in two murine mammary adenocarcinoma cell lines, LM3 and LM2 in comparison with the normal murine mammary epithelial cell line: NMuMG. Saturation binding assays with the tritiated muscarinic antagonist quinuclidinyl benzilate ([3H]-QNB) indicate that LM3 cells express higher amounts of mAchR than LM2 cells. Muscarinic receptor activation with carbachol (CARB) enhanced basal production of citrulline to a greater extent in LM3 cells than in LM2 cells. The nitric oxide synthase (NOS) inhibitor, NGmono-methyl-L-arginine (L-NMMA), blunted this effect only in LM3 cells while in LM2 cells the action of CARB was blocked by Nomega hydroxy-L-arginine (L-OH-Arg), which is known to inhibit the arginase pathway. Atropine blocks the action of CARB in both cell lines. Additionally, mAchR activation stimulates prostaglandin E2 (PGE2) synthesis only in LM2 cells. NMuMG cells show detectable basal amounts of nitric oxide and PGE2, but they did not respond to CARB. Binding experiments confirm the absence of mAchR in these cells. The findings indicate that mAchR expression in tumor cells, and its control on arginine metabolism, via NOS/arginase, and on PGE2 synthesis by COX activation, could be a switch on mechanism that might lead mammary cells from normal to malignant phenotype. Moreover, mAchR coupling to distinct effectors might be associated with differences in aggressiveness of tumor cells.

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin.

gamma-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with beta-sheet structure as is that of snake venom alpha-neurotoxins and cardiotoxins. gamma-Bungarotoxin exhibits activity on inhibiting the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated alpha-bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC50 of approximately 200 nM. The genomic DNA encoding the gamma-bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for alpha-neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that gamma-bungarotoxin, alpha-neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.

Evaluation of the cholinomimetic actions of trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana (Gastropoda, Opisthobranchia).

Trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana, negatively modulates vagal response, indicating a probable ability to inhibit cholinergic responses. In the present study, the pharmacological profile of trimethylsulfonium was characterized on muscarinic and nicotinic acetylcholine receptors. In rat jejunum the contractile response induced by trimethylsulfonium (pD2 = 2.46 +/- 0.12 and maximal response = 2.14 +/- 0.32 g) was not antagonized competitively by atropine. The maximal response (Emax) to trimethylsulfonium was diminished in the presence of increasing doses of atropine (P<0.05), suggesting that trimethylsulfonium-induced contraction was not related to muscarinic stimulation, but might be caused by acetylcholine release due to presynaptic stimulation. Trimethylsulfonium displaced [3H]-quinuclidinyl benzilate from rat cortex membranes with a low affinity (Ki = 0.5 mM). Furthermore, it caused contraction of frog rectus abdominis muscles (pD2 = 2.70 +/- 0.06 and Emax = 4.16 +/- 0.9 g), which was competitively antagonized by d-tubocurarine (1, 3 or 10 microM) with a pA2 of 5.79, suggesting a positive interaction with nicotinic receptors. In fact, trimethylsulfonium displaced [3H]-nicotine from rat diaphragm muscle membranes with a Ki of 27.1 microM. These results suggest that trimethylsulfonium acts as an agonist on nicotinic receptors, and thus contracts frog skeletal rectus abdominis muscle and rat jejunum smooth muscle via stimulation of postjunctional and neuronal prejunctional nicotinic cholinoreceptors, respectively.

Binding of σ receptor ligands and their effects on muscarine-induced Ca 2+ changes in SH-SY5Y cells

In human neuroblastoma SH-SY5Y cell preparations, σ 1 receptor agonists (+)-pentazocine and 1 S,2 R-(−)- cis- N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737) competed for [ 3H]haloperidol binding with K i values of 67±10 and 14±10 nM, respectively. (+)-Pentazocine or BD737 up to 10 μM did not affect basal levels of intracellular Ca 2+ concentration ([Ca 2+] i) in these cells, but they significantly reduced muscarine-induced [Ca 2+] i changes in a dose-related manner. However, the reduction by (+)-pentazocine was not reversed by the σ 1 receptor antagonist haloperidol. Further studies showed (+)-pentazocine, BD737 and haloperidol could compete for [ 3H]quinuclidinyl benzilate binding in SH-SY5Y cells with K i values of 0.51±0.06, 0.32±0.07 and 4.4±2.3 μM, respectively. Thus, the inhibitory effects on muscarine-induced [Ca 2+] i changes by (+)-pentazocine and BD737 in SH-SY5Y cells were likely due to blockade of muscarinic receptors, not due to σ 1 receptor activation by these ligands.

Age-related effects of chlorpyrifos on muscarinic receptor-mediated signaling in rat cortex.

Chlorpyrifos (CPF) is a widely used organophosphorus pesticide. Earlier work from our laboratory and others has demonstrated that the sensitivity to CPF exposure changes markedly during maturation. A number of studies suggest that in addition to inhibiting acetylcholinesterase (AChE), CPF oxon may also interact directly with m2 and/or m4 subtypes of muscarinic acetylcholine receptors (mAChRs). In the present study, we investigated the in vivo effects of CPF exposure on phosphoinositide (PI) hydrolysis and cAMP formation, second-messenger systems coupled to m1, m3 and m5 (PI hydrolysis) or m2 and m4 (cAMP formation) mAChRs. Neonatal (7-day), juvenile (21-day) and adult (90-day) rats were treated with either peanut oil s.c. or CPF s.c. at 0.3x or 1x the maximum tolerated dosage (MTD: 45, 127 and 279 mg/kg for 7-day, 21-day and 90-day rats, respectively). Neurochemical end-points including AChE activity, muscarinic receptor ([3H]quinuclidinyl benzilate, and [3H]oxotremorine) binding, PI hydrolysis, and cAMP formation in cortex were evaluated at 4 h, 24 h, or 96 h after treatment. Under these conditions, relatively similar maximal degrees of cholinesterase (ChE) inhibition were noted, but times to peak inhibition varied among these age groups (24 h in neonates and juveniles, 96 h in adults). Total muscarinic receptor (QNB) binding was reduced in all three age groups with 1x MTD exposure, at both 24 h and 96 h in neonates and juveniles, but only at 96 h in adults. Oxotremorine binding was also reduced at 96 h after MTD exposure in all three age groups. Neither basal nor carbachol-stimulated IP accumulation was affected in any age group or at any time point following CPF exposure. In contrast, basal cAMP formation was significantly increased by MTD exposure in all three age groups 4 h after exposure, and at 4 h, 24 h, and 96 h after exposure in juveniles. Forskolin/Mn2+-stimulated cAMP formation was increased in neonates and juveniles at 96 h, and in juveniles also at 24 h, but was significantly decreased in adults at 96 h after MTD exposure. Oxotremorine-mediated inhibition of cAMP formation was significantly greater at 96 h after MTD exposure in all three age groups. These results provide further evidence that the cortical cAMP signaling pathway may be particularly sensitive to CPF exposure in neonatal, juvenile, and adult rats, possibly due to a direct interaction between CPF (or its oxon) and mAChRs or other components of the adenylyl cyclase cascade.

In vitro effects of chlorpyrifos, parathion, methyl parathion and their oxons on cardiac muscarinic receptor binding in neonatal and adult rats

Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase. Young animals are generally more sensitive than adults to these toxicants. A number of studies reported that some organophosphorus agents also bind directly to muscarinic receptors, in particular the m 2 subtype, in tissues from adult rats. As both the density and agonist affinity states of cardiac muscarinic receptors (primarily m 2) have been reported to change in an age-related manner, we evaluated the relative in vitro sensitivity of cardiac muscarinic receptors in tissues from neonatal (7–11 days of age) and adult (90 days of age) rats to selected organophosphorus compounds (chlorpyrifos, parathion, methyl parathion and their oxygen analogs or oxons). The effects of the cholinergic agonist carbachol (100 pM–5 μM) or an organophosphorus toxicant (50 pM–10 μM) on muscarinic receptor binding were determined using the nonselective muscarinic ligand [ 3H]quinuclidinyl benzilate or the m 2-preferential ligand [ 3H]oxotremorine-M acetate. Carbachol displaced [ 3H]oxotremorine labeling in adult and neonatal membranes in a relatively similar manner (IC 50=7–20 nM). The oxons all displaced [ 3H]oxotremorine binding in a concentration-dependent manner, with chlorpyrifos oxon being the most potent (IC 50: neonates, 15 nM; adults, 7 nM) and efficacious (maximum displacement: neonates, 42%; adults, 56%). Interestingly, methyl parathion was an extremely potent displacer of [ 3H]oxotremorine binding in adult tissues (IC 50=0.5 nM, maximum displacement=37%) but had no effect in neonatal tissues. The displacement of [ 3H]oxotremorine binding by chlorpyrifos oxon (10 μM) was still apparent after washing the tissues, suggesting the oxon irreversibly blocked agonist binding to the receptor while interaction with MePS appeared reversible. As effective concentrations of the oxons were relatively similar to their anticholinesterase potencies, these findings suggest that direct interaction with cardiac muscarinic receptors by some organophosphorus agents may occur at relevant exposure levels and contribute to cardiac toxicity.

Actions of the new antimuscarinic compound Lu 25-109 on isolated human and pig detrusor.

The aim of this study was to evaluate the effects of a novel antimuscarinic agent, Lu 25-109, a partial M1 receptor agonist, and M2/M3 receptor antagonist in human and pig detrusor to establish its affinity for muscarinic receptors in human detrusor and parotid gland and to compare the results with those obtained with oxybutynin. Effects on the detrusor were determined as regards the ability to inhibit carbachol-induced contractions and contractions induced by electrical field stimulation (EFS). Radioligand binding studies were performed to assess the ability to displace quinuclidinyl benzilate (3H-QNB) from muscarinic receptors in the detrusor and parotid gland. Lu 25-109 produced a concentration-dependent rightward shift of the concentration-response curves for carbachol in both human and pig detrusor, the pK(b) values being 6.2+/-0.1 (n=6) and 5.8+/-0.3 (n=6). Corresponding values for oxybutynin were 7.9+/-0.1 (n=7) and 7.8+/-0.1 (n=6). Contractions induced by EFS in human detrusor were almost completely inhibited by 100 micromol/L Lu 25-109 (84+/-4%; n=4). In contrast, EFS-induced contractions in pig detrusor were less sensitive to Lu 25-109, resulting in a final inhibition of 32+/-6% (n=9) with the highest concentration used (100 micromol/L). This difference in effect between human and pig detrusor was not observed with oxybutynin. Radioligand binding experiments demonstrated a small difference in affinity for Lu 25-109 in the parotid gland compared with the bladder, the pKi values being 6.2+/-0.1 versus 6.5+/-0.1 (n=4). Corresponding values for oxybutynin were 8.5+/-0.1 versus 8.2+/-0.1 (n=4). The results show that Lu 25-109 competitively and effectively antagonizes carbachol-induced contractions and contractions induced by EFS in human detrusor muscle. Even if Lu 25-109 were less potent than oxybutynin, it has an effect profile that makes it of interest to test its ability to counteract bladder overactivity in humans.

Characterization of muscarinic acetylcholine receptor in rat Sertoli cells.

This study was designed to characterize muscarinic acetylcholine receptors (mAChRs) in primary cultured Sertoli cells from 30-d-old rats. RT-PCR was performed, and five PCR products corresponding to m1-m5 mAChR mRNA subtypes were detected in these cells. Ribonuclease protection assay further confirmed the presence of protected products for m1, m2, m3, and m4 mAChR transcripts. Radioligand binding studies and the analysis of changes in intracellular signaling pathways after cell exposure to carbachol were performed to study mAChRs at the protein level. Scatchard analysis revealed one single class of [(3)H]quinuclidinyl benzilate binding sites. Carbachol produced a reduction on forskolin-induced intracellular cAMP accumulation in Sertoli cells. This effect was reversed by atropine, methoctramine, and tropicamide but not by p-fluoro-hexahydro-sila-difenidol or pirenzepine. Carbachol also induced an increase on total [(3)H]-inositol phosphates content, an effect antagonized by atropine, p-fluoro-hexahydro-sila-difenidol, or pirenzepine but not by methoctramine. Thus, mAChR activation in Sertoli cell is linked to both adenylyl cyclase inhibition and to phosphoinositide hydrolysis. Furthermore, gel shift assays indicated that carbachol also induced a time-dependent stimulation of the activator protein-1 DNA-binding activity, suggesting that activation of mAChRs may play a role in the modulation of gene expression in Sertoli cells. Taken together, these results indicate that mAChRs are present at mRNA and protein level in rat Sertoli cells.

Characterization of muscarinic acetylcholine receptors in the rat epididymis.

The aim of the present study was to characterize the muscarinic acetylcholine receptor subtypes present in the caput and cauda of rat epididymis. The specific binding of [3H]quinuclidinyl benzilate ([3H]QNB) to epididymal membranes was time dependent, temperature dependent, and saturable. The cauda epididymis showed higher affinity to [3H]QNB and higher muscarinic receptor density when compared to the caput region. The [3H]QNB binding was tested in competition studies with different muscarinic receptor antagonists. Each antagonist tested displaced [3H]QNB bound to caput and cauda epididymal membrane with similar affinity. Correlation among the negative logarithm of inhibition constant values (pK(i)) for these antagonists obtained in the epididymis with their correspondent published pK(i) values obtained in tissues that expressed each receptor subtype (M1, M2, M3, and M4) indicated that the muscarinic receptors present in caput and cauda epididymis belong to the muscarinic M2 receptor subtype. When reverse transcription-polymerase chain reaction was used to identify muscarinic receptor mRNA subtypes in the epididymis, only m2 transcripts were detected in the caput region, while both m2 and m3 mRNA subtypes were observed in the cauda region. In conclusion, these results demonstrate that muscarinic receptors are present in the rat epididymis, with expression levels dependent on the region of the epididymis analyzed. Thus, the cholinergic neurotransmitter in the epididymis may be a factor controlling contractility and/or the luminal fluid microenvironment.

Effects of sub-chronic in vivo chlorpyrifos exposure on muscarinic receptors and adenylate cyclase of rat striatum.

In this study dosing regimens were designed such that cholinesterase inhibition following exposure to chlorpyrifos was produced in one treatment group, but was absent in the other. The higher dosing regimen inhibited plasma and brain cholinesterase activities by 51 and 70%, respectively, and resulted in decreased [3H]cis-methyldioxolane ([3H]CD) binding, which was attributable to a decrease in Bmax. No concomitant loss of [3H]quinuclidinyl benzilate ([3H]QNB) binding sites was observed, indicating that the M2 muscarinic receptor subtype to which [3H]CD binds is particularly susceptible to alterations induced by chlorpyrifos treatment. As the M2 receptor subtype is surmised to be the muscarinic autoreceptor, decreases in this receptor may exacerbate poisoning by organophosphorus agents as a result of decreased ability to terminate synaptic acetylcholine release. The ability of carbachol to inhibit striatal adenylate cyclase, which is an effector molecule associated with the M2 receptor, was unaltered in chlorpyrifos-treated rats. Decreases in M2 receptors occurred with the higher dosing regimen, in the absence of any clinical manifestations. Thus, in the absence of overt clinical signs, perturbations of the muscarinic receptor system did occur as a result of sub-chronic chlorpyrifos exposure. Such alterations may contribute to neurological impairments that develop following chronic organophosphorus exposure.

Interaction of anandamide with the M 1 and M 4 muscarinic acetylcholine receptors

The M 1 and M 4 muscarinic acetylcholine receptors are the most abundant muscarinic receptor subtypes in the brain, and are involved in learning and memory. Because cannabinoid receptors are also abundantly expressed in similar brain regions and mediate opposite effects to acetylcholine on cognition, the present study investigated whether the endocannabinoid agonist, anandamide, and its metabolically stable derivative, methanandamide, directly modified the binding properties of the human M 1 and M 4 receptors individually expressed in CHO cell membranes. Experiments utilized the antagonists, [ 3H] N-methylscopolamine and [ 3H]quinuclidinyl benzilate. When acetylcholine was used as the inhibiting ligand, shallow, biphasic isotherms were observed at both receptors, characterised by similar apparent dissociation constants for high and low affinity binding at each receptor but with a greater proportion of high affinity sites at the M 4 (40–45%) than at the M 1 receptor (17–20%). In contrast, anandamide and methanandamide inhibited the binding of both radioligands over a narrow (low micromolar) concentration range, with monophasic isotherms characterized by Hill coefficients significantly greater than 1 at both receptors. These effects were not due to the vehicle used. Further saturation binding analyses found anandamide able to significantly reduce the apparent affinity and maximal density of binding sites labeled by [ 3H]quinuclidinyl benzilate. Interestingly, no significant inhibition of radioligand binding was noted using the synthetic cannabinoid agonist, WIN55212-2, or the cannabinoid CB 1 receptor antagonist, SR141716A. These data thus provide evidence for a direct role of anandamides in modulating muscarinic receptor binding properties through a non-competitive mechanism that is unrelated to their actions on cannabinoid receptors.

Lowered Susceptibility of Muscarinic Receptor Involved in Salivary Secretion of Streptozotocin-Induced Diabetic Rats

We investigated the responses of salivary secretion and the susceptibility of the muscarinic receptors in the salivary glands of the streptozotocin (STZ)-induced diabetic rats (STZ rats). Giving water ad libitum, the amount of whole saliva with no stimulation was similar in the STZ and the control rats. Pilocarpine increased salivary secretion in both groups, although the effect in the STZ rats was two to three fold less than in the control rats. If the animals were restricted from taking water for 6 h, salivary secretion was not slightly changed in the STZ rats in spite of a remarkable increase in the control. An obvious decrease in salivary secretion of the STZ rats was negatively correlated with an increase in urination. Furthermore, salivary secretion from the parotid gland was increased in a dose-dependent manner with pilocarpine in the control rats, but not in the STZ rats. In the [3H]quinuclidinyl benzilate (QNB) binding studies for muscarinic receptor of the STZ rats, Bmax was decreased in the parotid gland and Kd was increased in the submandibular gland. Competitive inhibition of [3H]QNB binding to both glands showed an increase in IC50 of pilocarpine and carbachol. These results suggest that a decrease in salivary secretion of STZ rats is not only induced by a water loss, but also closely associated with the lowered susceptibility of the muscarinic receptors.