Effects of AIT-082, a purine derivative, on tremor induced by arecoline or oxotremorine in mice.

The effects of AIT-082, a hypoxanthine derivative, on tremor in mice were investigated. The mice received intragastric administration of AIT-082 for consecutive 60 days at doses of 150, 300 and 600 mg.kg(-1). The results showed that AIT-082 not only effectively inhibited the tremor induced by arecoline or oxotremorine, but also alleviated the tremor intensity and significantly shortened the tremor durations. The inhibition of tremor was perhaps associated with the central cholinergic nerve depressant effects as well as the stimulation of proliferation and differentiation of nerve cells.

Comparative study on pharmacological effects of DM-phencynonate hydrochloride and its optical isomers.

AIM: The 3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (DM-phencynonate hydrochloride, DMCPG) is a demethylated metabolite of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (phencynonate hydrochloride, CPG). (+/-)DMCPG had one chiral center and two enantiomers [R(-) and S(+)DMCPG]. Here we carried out a comparative study of the pharmacological profiles of these optical isomers. METHODS: Affinity and relative efficacy were tested using a radioligand-binding assay with muscarinic acetylcholine receptors from the rat cerebral cortex. Pharmacological activity was assessed in three individual experiments: (1) potentiating the effect of a sub-threshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol. RESULTS: In the competitive binding assay, R(-)DMCPG (K(i)=763.75 nmol/L) was 4- and 2-fold more potent than (+/-)DMCPG (K(i)=3186 nmol/L) and S(+)DMCPG (K(i)=1699 nmol/L) in inhibiting the binding of [(3)H]QNB. The R(-) and S (+) configurations showed positive cooperation (n(H)>1) with the muscarinic receptor, whereas (+/-)DMCPG had a negative cooperation (n(H)<1) relationship with the muscarinic receptor in a radio-binding assay. Both the R(-) and S(+) configurations could potentiate the effect of sub-threshold hypnotic dose of sodium pentobarbital in a dose-dependent manner (the ED(50) values were 2.53 and 18.65 mg/kg, respectively), but (+/-)DMCPG did not display significant central depressant effects at doses from 10 to 29.15 mg/kg (P>0.05). (+/-)DMCPG and its optical isomers suppressed the guinea pig ileum contractile response to carbachol. The IC(50) values were 7.78 x 10(-9), 1.88 x 10(-7), and 1.038 x 10(-7) nmol/L, respectively. In the anti-salivation study, (+/-)DMCPG and its enantiomers depressed oxotremorine- induced salivation in a dose-dependent manner, and the order of potency was R(-)DMCPG (ED(50)=0.44 mg/kg) > (+/-)DMCPG (ED(50)=2.88 mg/kg) >S(+)DMCPG (ED(50)=5.05 mg/kg). CONCLUSION: (+/-)DMCPG and its optical isomers have differences in their pharmacological potencies as anticholinergic agents, and the R(-) configuration is more active than the S(+) configuration.

In vivo antimuscarinic actions of the third generation antihistaminergic agent, desloratadine.

BACKGROUND: Muscarinic receptor mediated adverse effects, such as sedation and xerostomia, significantly hinder the therapeutic usefulness of first generation antihistamines. Therefore, second and third generation antihistamines which effectively antagonize the H1 receptor without significant affinity for muscarinic receptors have been developed. However, both in vitro and in vivo experimentation indicates that the third generation antihistamine, desloratadine, antagonizes muscarinic receptors. To fully examine the in vivo antimuscarinic efficacy of desloratadine, two murine and two rat models were utilized. The murine models sought to determine the efficacy of desloratadine to antagonize muscarinic agonist induced salivation, lacrimation, and tremor. Desloratadine's effect on the cardiovascular system was explored in both rodent models. RESULTS: In the pithed rat, both desloratadine (1.0 mg/kg, i.v.) and the muscarinic M2 selective antagonist, methoctramine (0.5 mg/kg, i.v.), inhibited negative inotropic (left ventricular dP/dt) effects caused by oxotremorine, a nonselective muscarinic agonist (p < 0.05). Negative chronotropic effects caused by oxotremorine were inhibited by desloratadine, methoctramine, and the muscarinic M3 selective antagonist, 4-DAMP (1.0 mg/kg, i.v.). A late positive inotropic event observed after the initial decrease was inhibited by all three test compounds with desloratadine and 4-DAMP being the most efficacious. In the conscious animal, inhibition of baroreflex-mediated bradycardia was evaluated. Unlike atropine (0.5 mg/kg, i.v.), desloratadine did not alter this bradycardia. The antimuscarinic action of desloratadine on salivation, lacrimation, and tremor was also explored. In urethane-anesthetized (1.5 g/kg, i.p.) male ICR mice (25-35 g) desloratadine (1.0, 5.0 mg/kg) did not inhibit oxotremorine-induced (0.5 mg/kg, s.c.) salivation, unlike atropine (0.5 mg/kg) and 4-DAMP (1.0 mg/kg). In conscious mice, desloratadine failed to inhibit oxotremorine-induced (0.5 mg/kg, s.c.) salivation, lacrimation, and tremor. However, desloratadine did inhibit oxotremorine-induced tremor in phenylephrine pretreated animals. CONCLUSION: The presented data demonstrate that the third generation antihistamine, desloratadine, does not significantly antagonize peripheral muscarinic receptors mediating salivation and lacrimation, therefore, xerostomia and dry eyes should not be observed with therapeutic use of desloratadine. Our data also indicate when administered to a patient with a compromised blood-brain barrier, desloratadine may cause sedation. Patients with compromised cardiovascular systems should be closely monitored when administered desloratadine based on our results that desloratadine has the ability to interfere with normal cardiovascular function mediated by muscarinic receptors.

Pharmacological profiles of an anticholinergic agent, phencynonate hydrochloride, and its optical isomers.

AIM: To comparatively study the pharmacological profiles of 3-methyl-3-azabicyclo(3,3,1)nonanyl-9-alpha-yl-alpha-cyclopentyl-alpha-phenyl-alpha-glycolate (phencynonate hydrochloride, CPG), an anticholinergic agent, and its enantiomers [R(-)-and S(+)-CPG]. METHODS: The affinity and relative efficacy were tested using radioligand-binding assay with muscarinic acetylcholine receptors from rat cerebral cortex. The pharmacological activities were assessed in three individual experiments: (1) potentiating the effect of subthreshold hypnotic dose of sodium pentobarbital; (2) inhibiting oxotremorine-induced salivation; and (3) inhibiting the contractile response to carbachol. RESULTS: The order of potency of phencynonate hydrochloride and its optical isomers to inhibit the binding of [3H]quinuclidinyl benzilate ([3H]QNB) was R(-)-CPG (K(i)=46.49+/-1.27 nmol/L)>CPG(K(i)=271.37+/-72.30nmol/L)>S(+)-CPG(K(i)=1263.12+/-131.64 nmol/L). The results showed that R(-)-CPG had the highest affinity to central muscarinic receptors among the three compounds, but did not show any central depressant effects at dose from 10.00 to 29.15 mg/kg. CPG increased the effects of subthreshold hypnotic dose of sodium pentobarbital induced-sleeping [the ED50+/-95% LC value was 21.06+/-3.04 mg/kg]. CPG and R(-)-CPG displayed nearly equipotent effect in depressing oxotremorine-induced salivation [the ED50 +/-95% LC for R(-) and CPG were 1.10+/-0.28 and 1.07+/-0.15 mg/kg, respectively], and the contractile response to carbachol (pA(2) values for R (-) and CPG were 6.84 and 6.80, respectively). S(+)-CPG presented the lowest anticholinergic profiles, but could potentate effects of its enantiomers in some manner. CONCLUSIONS: These data suggested that R(-)-CPG acted as an eutomer in racemate and a competitive antagonist to acetylcholine muscarinic receptors, but S(+)-CPG was less active in comparison to R(-)-CPG and its racemate. The central depressant effects of R(-)-CPG and S(+)-CPG were lower in comparison to its racemate.

Medical prevention of space motion sickness--animal model of therapeutic effect of a new medicine on motion sickness.

Space motion sickness (MS) is one of the most important problems in the field of space medicine. In order to prevent space MS, a new medicine, PMPA, has been prepared by means of synthesizing in our laboratory. The purposes of this study were to set up animal models of PMPA against MS, and to observe its effects on anti-MS, and to prove its function of antagonism to choline. Eight cats, forty rabbits and two hundred and ten rats were selected as animal subjects. The parallel swing stimulus, a method causing the reversal syndromes and tests of anti-choline function were used in our experiments. The results are as follows: (1) The score of MS symptoms in cats with PMPA or scopolamine (SCOP) is significantly lower than that in cats with placebo (p<0.01), while the incidences of efficiency and prevention of PMPA (87.5%, 75%) are higher than those of SCOP (75.0%, 50%) in cats. (2) PMPA of 1.6 mg/kg or 0.8 mg/kg could antagonize the reversal syndromes and repress reversal rotation significantly in rabbits like SCOP in comparison with placebo (p<0.01). (3) PMPA could inhibit tremor evoked by oxotremorine or by nicotine-procaine in rats like SCOP, and play an important role in the antagonism to central M-choline and N-choline receptors. The animal experiments demonstrate that PMPA is an effective medicine against MS with antagonism function to choline.

Inhibition of oxotremorine-induced desensitization of guinea-pig ileal longitudinal muscle in Ca2+-free conditions.

The aim of this study was to investigate the differences between oxotremorine-induced and acetylcholine (ACh)-induced desensitization, particularly under Ca2+-free conditions, in guinea-pig ileal longitudinal muscle, and to elucidate the different mechanisms of desensitization that might exist between these two muscarinic agonists. Pretreatment of the tissue with 10(-7)-10(-5) M oxotremorine (desensitizing treatment) in normal Tyrode solution caused desensitization of the responses to ACh, as did the desensitizing treatment with ACh. However, Ca2+-free conditions significantly reduced oxotremorine-induced desensitization, contrary to the previous findings that Ca2+-free conditions enhanced ACh-induced desensitization. The desensitizing treatment with oxotremorine caused suppression of the responses to high K+ (tonic phase), as did the ACh treatment. Ca2+-free conditions removed this suppression, whereasthis condition enhanced ACh-induced suppression of the K+ response. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10(-4) M) had no effect on oxotremorine-induced desensitization of the ACh response. The results suggest that a voltage-gated Ca2+ channel was involved in oxotremorine-induced desensitization, as in ACh-induced desensitization, but that the process of inactivation of Ca2+ channels was different between oxotremorine and ACh, and that oxotremorine-induced desensitization was due not only to Ca2+ channel, but also to other unknown factors. Protein kinase C did not participate in oxotremorine-induced desensitization.

Magnesium activation of GTP hydrolysis or incubation in S-adenosyl-l-methionine reverses iron-56-particle-induced decrements in oxotremorine enhancement of K+-evoked striatal release of dopamine.

Previous research has determined that the deficits in motor behavior seen in aged animals irradiated with (56)Fe particles involved alterations in muscarinic receptor sensitivity. In the present experiments, we determined whether increasing either membrane fluidity by exposure of striatal slices from irradiated ((56)Fe particles) animals to S-adenosyl-l-methionine (SAM) or GTP hydrolysis with Mg(2+) would reverse this (56)Fe-particle-induced loss of muscarinic receptor sensitivity, as has been observed in aged animals. Results indicated that, while increasing Mg(2+) concentrations in the incubation medium was effective in reducing the radiation effects, SAM was able to effect some reversal of the radiation effects only at the lower concentration (200 microM). These results suggest that similar mechanisms may be involved in the deficits in signal transduction seen after (56)Fe-particle irradiation to those seen in aging, and that these may include changes in the membrane structure or composition that could alter subsequent responsiveness of transduction pathways. The results further suggest that, as has been reported previously, (56)Fe-particle irradiation may accelerate brain aging, and that since these HZE particles contribute at least 1% of the dose that astronauts would receive from cosmic rays, long-term exposure on extended space flights (e.g. to Mars) may produce similar deficits that could have immediate or delayed effects on behavior.

Cholinergic/serotonergic interactions in hypothermia: implications for rat models of depression.

This article reviews published reports and presents new evidence that support a number of commonalties between lines of rats selectively bred for differences in cholinergic (muscarinic) and serotonergic (5-HT1A) sensitivity. The Flinders Sensitive Line (FSL) rat, a genetic animal model of depression derived for cholinergic supersensitivity, is more sensitive to both cholinergic and serotonergic agonists, and exhibits exaggerated immobility in the forced swim test relative to the control, Flinders Resistant Line (FRL), rat. Similar exaggerated responses are seen in a line of rats recently selected for increased sensitivity to the 5-HT1A agonist, 8-OH-DPAT (High DPAT Sensitive--HDS), relative to lines selectively bred for either low (Low DPAT Sensitive--LDS) or random (Random DPAT Sensitive--RDS) sensitivity to 8-OH-DPAT. For both the FSL and HDS rats, their exaggerated immobility in the forced swim test is reduced following chronic treatment with antidepressants. The present studies examined further the interaction between cholinergic and serotonergic systems in the above lines. Supersensitive hypothermic responses to 8-OH-DPAT were observed very early (postnatal day 18) in FSL rats, suggesting that both muscarinic and serotonergic supersensitivity are inherent characteristics of these rats. Scopolamine, a muscarinic antagonist, completely blocked the hypothermic effects of the muscarinic agonist oxotremorine in FSL and FRL rats, but had no effect on the hypothermic responses to 8-OH-DPAT, suggesting an independence of muscarinic and 5-HT1A systems. On the other hand, genetic selection of genetically heterogeneous rats for differential hypothermic responses to the muscarinic agonist oxotremorine were accompanied by differential hypothermic responses to 8-OH-DPAT, suggesting an interaction between muscarinic and 5-HT1A systems. Overall, these studies argue for an inherent interaction between muscarinic and 5-HT1A systems, which probably occurs beyond the postsynaptic receptors, possibly at the level of G proteins.

Pharmacological studies on YM992, a novel antidepressant with selective serotonin re-uptake inhibitory and 5-HT2A receptor antagonistic activity.

YM992 ((S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride) is a novel compound that has selective serotonin (5-hydroxytryptamine, 5-HT) re-uptake inhibition and 5-HT2A receptor antagonistic activity in vivo. YM992, fluoxetine and citalopram showed 5-HT uptake inhibition activity in l-5-hydroxy-tryptophan (l-5-HTP)-treated mice. YM992 and trazodone attenuated 5-HT2A/2C receptor agonist-induced head-twitches in mice, indicating that these drugs had 5-HT2A receptor antagonistic activity. YM992 and amitriptyline were highly active in the mouse tail suspension test. In contrast, fluoxetine and citalopram showed only a tendency to reduce the immobility time. Single treatment with YM992 as well as trazodone and fluoxetine ameliorated the learning deficit of olfactory-bulbectomized rats, whereas citalopram and amitriptyline showed an ameliorative effect only after chronic treatment. Although YM992 has moderate affinity for alpha1-adrenoceptors, alpha1-adrenoceptor antagonism of YM992 in vivo was 10 times weaker than that of trazodone. These results demonstrate that YM992 has 5-HT uptake inhibition and 5-HT2A receptor antagonistic activity in vivo, and suggest that YM992 may be a novel antidepressant with high efficacy in clinical use.

The characterization of oxotremorine-induced hypothermic response in the rat.

Oxotremorine is a muscarinic receptor agonist that induces a variety of physiological and behavioural effects including hypothermia in mice. These effects are antagonized dose-dependently by classical anticholinergic compounds such as atropine. Although the oxotremorine-induced hypothermic response has been demonstrated in mice, few studies of the effects of this muscarinic agonist have been made in the rat. The following studies were made in male Sprague Dawley rats: 1. an investigation of the dose-response relationship between oxotremorine and hypothermia; 2. an examination of the effect of housing on the oxotremorine-induced hypothermic response, and 3, an investigation of the acute administration of various doses of atropine sulphate on the hypothermia caused by oxotremorine. The results indicate that the dose-response relationship between oxotremorine and the antagonism of hypothermia is similar in rat as it is in mice. The results also showed that this effect did not occur in group-housed animals.

Functional interactions between muscarinic M2 receptors and 5-hydroxytryptamine (5-HT)4 receptors and beta 3-adrenoceptors in isolated oesophageal muscularis mucosae of the rat.

1. Relaxations of isolated oesophageal muscularis mucosae of rat are mediated by 5-hydroxytryptamine (5-HT), acting at 5-HT4 receptors, and isoprenaline, principally acting via beta 3-adrenoceptors. The aim of this study was to investigate the hypothesis that muscarinic M2 receptors, also present in this tissue, functionally oppose 5-HT and beta-adrenoceptor-relaxant effects in this preparation. 2. Contractions of rat oesophageal muscularis mucosae were induced, in a concentration-dependent manner, by the muscarinic receptor agonist, oxotremorine M (pEC50 = 6.7 +/- 0.1). The contractile responses to oxotremorine M were surmountably antagonized by the following compounds, (pKB values in parentheses): atropine (9.1 +/- 0.2), 4-DAMP (4-diphenylacetoxy-N-methyl piperidine methiodide, 8.7 +/- 0.1), p-F-HHSiD (para-fluoro-hexa-hydro-siladifenidol, 7.5 +/- 0.1), zamifenacin (8.6 +/- 0.3), himbacine (7.2 +/- 0.2), pirenzepine (6.8 +/- 0.3) and methoctramine (6.2 +/- 0.2). These data are consistent with a role for muscarinic M3 receptors mediating contractions to oxotremorine M. The contractile response was associated with a low receptor reserve, since the responses were shifted to the right and virtually abolished by the alkylating agent, 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl) piperidine, 40 nM; 60 min equilibration). 3. In tissues precontracted with U46619 (0.7 microM; approx. EC90), isoprenaline (pEC50 = 8.0 +/- 0.1) and 5-HT (pEC50 = 7.5 +/- 0.2) induced concentration-dependent relaxations. The isoprenaline potency was slightly, but significantly, different in tissues precontracted with oxotremorine M (isoprenaline, pEC50 = 7.4 +/- 0.2). In contrast, the potency of 5-HT (pEC50 = 7.5 +/- 0.2), in tissues that were precontracted with 1 microM (EC90) oxotremorine M, was identical. When these experiments were repeated in the presence of the muscarinic M2 receptor antagonist, methoctramine (1 microM), there was no effect on the relaxant potencies to either 5-HT or isoprenaline. Collectively, these data suggest that muscarinic M2 receptors do not, under these conditions, modulate relaxant potencies to either 5-HT or isoprenaline. 4. In a second protocol, tissues were pre-contracted with U46619 (0.7 microM) and relaxed with either 5-HT (0.1 microM) or isoprenaline (0.1 microM). In these tissues (in which the muscarinic M3 receptor population was extensively depleted by alkylation), oxotremorine M caused concentration-dependent re-contractions (i.e. reversal of relaxations). In tissues relaxed with 5-HT, the potency of oxtremorine M was 5.9 +/- 0.2, while in tissues relaxed with isoprenaline, the potency (pEC50) = 5.6 +/- 0.3. These re-contractions were antagonized, in a surmountable fashion, by methoctramine (1 microM; pKB = 7.6 +/- 0.1). Similar observations were seen when relaxations were induced by isoprenaline (1 microM; pKB = 7.5 +/- 0.2). Under these conditions, therefore, the pKB values are consistent with activation of muscarinic M2 receptors, and inconsistent with activation of M3 receptors. 5. It is concluded that in isolated oesophageal muscularis mucosae of rat, muscarinic M3 receptors mediate direct contractions and are associated with a low receptor reserve. When this population is depleted, and the tissues relaxed via activation of receptors that augment adenylyl cyclase activity, a functional role for muscarinic M2 receptors is revealed.

Effects of oxotremorine and pilocarpine on striatal acetylcholine release as studied by brain dialysis in anesthetized rats.

1. The effects of oxotremorine and pilocarpine on striatal acetylcholine (ACh) release were investigated using brain microdialysis techniques in urethan-anesthetized rats. 2. Oxotremorine (0.1 and 0.5 mg/kg, IV), a preferential M2 agonist, dose-dependently decreased ACh release in the striatum. On the other hand, pilocarpine, at 5 mg/kg (IV), showed a tendency to decrease ACh release in the striatum but, at 7.5 and 10 mg/kg (IV), significantly enhanced release in a dose-dependent manner. 3. The effect of oxotremorine was blocked by scopolamine (0.1 mg/kg, IV) but not by pirenzepine (10 mg/kg, IV), a selective M1 antagonist. 4. Pilocarpine (10 mg/kg, IV) enhancement of striatal ACh release was not affected by 10 mg/kg pirenzepine, but 5 mg/kg pilocarpine significantly increased ACh release in scopolamine (0.1 mg/kg)-pretreated rats without affecting the release by itself. 5. These results suggest that oxotremorine-induced decrease in striatal ACh release is due to stimulation of presynaptic M2 autoreceptor, and that the increase of striatal ACh release by pilocarpine is mediated by mechanism(s) other than effects on muscarinic ACh receptors.

Synthesis of new serotonergic 2-substituted 4,6-diaryl pyridazin-3-ones.

A series of 4,6-diaryl pyridazin-3-ones substituted in the 2-position by [4-(4-aryl piperazin-1-yl]-but-2-ynyl moieties was synthesized and evaluated for antidepressant activity. The structures of these new pyridazine derivatives were confirmed by IR, 1H-NMR spectra and by elementary analysis. At 150 mg/kg i.p., they induced little or no reduction of the duration of immobility of mice in the forced swimming test. Head twitches produced by L-5-hydroxytryptophan in mice pretreated with pargyline were significantly potentiated by most of the tested compounds. In addition, pyridazine derivatives did not antagonize reserpine-induced palpebral ptosis or enhance the toxic effects of yohimbine and were almost devoid of anticholinergic properties in mice.

Differential in vivo induction of immediate early genes by oxotremorine in the central nervous system of long- and short-sleep mice.

Long-sleep (LS) and short-sleep (SS) mice show differential sensitivity to both acute and chronic ethanol administration. Previous data also showed differential behavioral responses to muscarinic acetylcholine receptor agonist or antagonist treatment. We now report significantly greater inductions of c-fos, c-jun, jun-B, and Egr-1, but not jun-D, mRNA in the central nervous system (CNS) of LS versus SS mice after the intraperitoneal administration of oxotremorine. These genomic responses were dose dependent and completely inhibited (in both strains) by scopolamine, a specific muscarinic receptor antagonist. In situ hybridization studies verified the greater immediate early gene (IEG) inductions in LS mice, as initially observed by Northern analysis, and specifically showed that c-fos mRNA induction occurred predominantly in the thalamus, olfactory bulb, cerebellum, and cerebral cortex. Oxotremorine-induced c-jun mRNA was increased in cerebellum, CA1 hippocampal field, and cerebral cortex of both strains. Induced jun-B and Egr-1 transcripts were determined to have very similar CNS distribution patterns. Both mRNA species were induced in the cerebral cortex, caudate nucleus and putamen, hippocampal structures, and olfactory bulb. To further determine whether these differential IEG inductions reflect regional differences in receptor numbers, we determined the distributions and levels of each of the five muscarinic receptor subtypes in both strains by in situ hybridization. These data show that differences in receptor numbers alone may not account for the differential IEG inductions observed between the strains. Differential coupling constraints among CNS muscarinic receptors in LS versus SS mouse CNS may also play a significant role in producing differential IEG inductions.

Urinary bladder-selective action of the new antimuscarinic compound vamicamide.

1. The inhibitory action of vamicamide (FK176, (+/-)-(2R*,4R*)-4-dimethylamino-2-phenyl-2-(2-pyridyl)valeramide, CAS 132373-81-0) on the responses of various tissues to the cholinergic agonists, carbachol and McN-A-343 (4-[m-chlorophenylcarbamoyloxy]-2-butynyl-trimethylammonium chloride, CAS 55-45-8), was investigated in isolated tissue preparations. Vamicamide showed competitive antagonistic actions against all the preparations tested and its pA2 value for the urinary bladder was 6.82, which was higher than that for the atria (5.94) and almost the same as that for the vas deferens (6.90) and for the stomach (6.81). The pA2 values of oxybutynin hydrochloride (oxybutynin) and atropine sulfate monohydrate (atropine) were nearly the same in all the tissues tested. 2. Oral administration of vamicamide 0.1-1.0 mg/kg inhibited dose-dependently spontaneous bladder contractions caused by raising the intravesical volume in conscious rats. Inhibitory actions were also obtained with 0.32-3.2 mg/kg of oxybutynin or 0.0032-0.032 mg/kg of atropine, but the duration of action of oxybutynin was shorter than that of vamicamide or atropine. Vamicamide further inhibited bladder contractions in rats following intravesical administration of 0.05-0.5 mg/ml solution. 3. Vamicamide had no effect or only slightly inhibited spontaneous motility of the stomach and distal colon in conscious rats, as well as heart rate and salivary secretion in conscious dogs, after oral dosing with 3.2 mg/kg of the compound. Similar results were obtained with oxybutynin, excepting the occurrence of tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Characteristics and regulation of a muscarinically activated K current in HSG-PA cells.

Whole cell currents were measured in HSG-PA cells (a proposed model for salivary gland duct cells) after muscarinic receptor activation or exposure to known signaling agents. Exposure to carbachol or oxotremorine M produced large and often oscillatory increases in outward current whose reversal potentials indicated a K current. The current was sensitive to extracellular atropine, charybdotoxin, and quinine, but not apamin, and to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette. The response was prolonged or increased by guanosine 5'-O-(3-thiotriphosphate) and mimicked by D-myo-inositol 1,4,5-trisphosphate (IP3) or heparin in the pipette and by extracellular Ca ionophores. Tetraethylammonium indirectly inhibited the response via the muscarinic receptor. Fura 2 in cell suspensions showed that muscarinic agonists increased cytosolic Ca ion concentration ([Ca2+]i) five- to sevenfold, and measurements with indo 1 in individual cells showed that the oscillatory changes in outward current were tightly correlated with parallel changes in [Ca2+]i. The results indicate that muscarinic receptor stimulation of HSG-PA cells activates Ca(2+)-activated K channels through a signaling pathway involving a G protein, IP3 production, and increased [Ca2+]i levels. These findings are similar to those in salivary gland acinar cells.

Evidence that the M1 muscarinic receptor subtype mediates the effects of oxotremorine on masculine sexual behavior.

The cholinergic system participates in the regulation of masculine sexual behavior, mainly through the muscarinic system. Recently, muscarinic receptors have been subdivided into at least two subtypes, M1 and M2, according to their differential affinity for pirenzepine. In this study, we analyzed the possible participation of the M1 muscarinic receptor subtype on masculine sexual behavior regulation. In the first experiment, trihexyphenidyl, a specific M1 antagonist, was administered to experienced adult male rats in a wide range of doses (from 0.1 to 6.4 mg/kg). No modification was observed in any of the male sexual behavior parameters recorded, with the exception of the highest dose at which an increase of the intromission frequency and a decrease of the ejaculation frequency were observed. In the second experiment, trihexyphenidyl was administered in several doses (from 0.2 to 1.6 mg/kg), before the administration of oxotremorine, a muscarinic agonist, at a dose that readily facilitates masculine sexual behavior. Trihexyphenidyl completely prevented the facilitatory effects of oxotremorine even at the smallest dose used. These results strongly suggest that the M1 muscarinic receptor subtype participates in the cholinergic facilitation of masculine sexual behavior.

Attenuation of cholinergic analgesia by nifedipine.

Nociception was tested in mice receiving oxotremorine or physostigmine either after the dihydropyridine calcium channel blocker nifedipine or the non-calcium antagonist vasodilator hydralazine. Nifedipine did not change the reaction time to thermal stimulation (tail-flick test), but attenuated the prolonging action on tail-flick latencies exerted by the two cholinomimetic agents. Hydralazine had no effect alone nor modified the action of cholinomimetics. The results suggest that attenuation of cholinergic analgesia by nifedipine might be related to not yet defined neuronal changes produced by calcium channel blockade, but changes in the pharmacokinetics of oxotremorine and physostigmine cannot be ruled out.

Effects on general behaviour and neurotransmitter functions of a new 5-hydroxytryptamine3 receptor antagonist with potential therapeutic relevance in central nervous system disturbances.

1. The general pharmacology of DAU 6215 (N-(endo-8-methyl-8-azabicyclo(3.2.1) oct-3-yl)-2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxamide hydrochloride, CAS 127618-28-4), a new 5-hydroxytryptamine3 (5-HT3) receptor antagonist, was investigated for a preliminary evaluation of its toxicity and possible side-effects related to interferences with neurotransmitter functions. 2. In vitro studies--receptor binding: DAU 6215 showed a high affinity for 5-HT3 receptors (Ki, nmol/l: 3.8) without any significant affinity for other serotonergic, adrenergic or dopaminergic receptors. A certain affinity was found for muscarinic (M) receptors (Ki, nmol/l: M1 = 95; M2 = 6,917; M3 = 181). 3. In vivo studies--general behaviour: In mice, DAU 6215 was well tolerated up to 100 mg/kg p.o. and 30 mg/kg i.v. The only noticeable symptom was mydriasis. No other major symptoms appeared up to the maximum non lethal doses (100 mg/kg p.o. and 30 mg/kg i.v.). At lethal doses, deaths occurred with signs of central nervous system excitation, like tremors, twitches and convulsions. Survived mice exhibited a complete recovery from symptoms within 1 h from administration. DAU 6215 did not exhibit any effect either on motor and exploratory activity or in the traction test in mice up to 30 mg/kg i.p. In the open field test in rats, DAU 6215 (0.01 and 10 mg/kg p.o.) failed to modify the behavioural parameters considered, with the exception of a decrease in defecations at 10 mg/kg. 4. In vivo studies--neurotransmitter functions: a) Monoaminergic transmission: reserpine-induced hypothermia in mice was not antagonized by DAU 6215 (0.01-1 mg/kg i.p.).(ABSTRACT TRUNCATED AT 250 WORDS)