Muscarinic M1 receptors stimulated by intracerebroventricular administration of McN-A-343 reduces the nerve injury-induced mechanical hypersensitivity via GABAB receptors rather than GABAA receptors in mice.

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.

Medial septal cholinergic mediation of hippocampal theta rhythm induced by vagal nerve stimulation.

BACKGROUND: Electrical vagal nerve stimulation (VNS) has been used for years to treat patients with drug-resistant epilepsy. This technique also remains under investigation as a specific treatment of patients with Alzheimer's disease. Recently we discovered that VNS induced hippocampal formation (HPC) type II theta rhythm, which is involved in memory consolidation. In the present study, we have extended our previous observation and addressed the neuronal substrate and pharmacological profile of HPC type II theta rhythm induced by VNS in anesthetized rats. METHODS: Male Wistar rats were implanted with a VNS cuff electrode around the left vagus nerve, a tungsten microelectrode for recording the HPC field activity, and a medial septal (MS) cannula for the injection of a local anesthetic, procaine, and muscarinic agents. A direct, brief effect of VNS on the HPC field potential was evaluated before and after medial-septal drug injection. RESULTS: Medial septal injection of local anesthetic, procaine, reversibly abolished VNS-induced HPC theta rhythm. With the use of cholinergic muscarinic agonist and antagonists, we demonstrated that medial septal M1 receptors are involved in the mediation of the VNS effect on HPC theta field potential. CONCLUSION: The MS cholinergic M1 receptor mechanism integrates not only central inputs from the brainstem synchronizing pathway, which underlies the production of HPC type II theta rhythm, but also the input from the vagal afferents in the brain stem.

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate.

At the rat motor nerve terminals, activation of muscarinic M(1) receptors negatively modulates the activity of inhibitory muscarinic M(2) receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involved endogenous adenosine tonically activating A(1) receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [(3)H]-choline (2.5 microCi/ml). Selective activation of muscarinic M(1) and adenosine A(1) receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 microM) and R-N(6)-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [(3)H]-ACh release induced by muscarinic M(2) receptor activation with oxotremorine (10 microM). Attenuation of the inhibitory effect of oxotremorine (10 microM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M(1) autoreceptors, suggesting that suppression of M(2)-inhibiton by A(1) receptor activation is independent on muscarinic M(1) receptor activity. Conversely, the negative crosstalk between M(1) and M(2) autoreceptors seems to involve endogenous adenosine tonically activating A(1) receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 microM) by McN-A-343 (3 microM) was suppressed by the A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 microM). The results suggest that the negative crosstalk between muscarinic M(1) and M(2) autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A(1) receptors at the rat motor endplate.

The spinal muscarinic M(1) receptors and GABA(A) receptors contribute to the McN-A-343-induced antinociceptive effects during thermal stimulation of mice.

The present study was undertaken to clarify how spinal muscarinic receptors are involved in the antinociceptive effects in thermal stimulation. Intrathecal (i.t.) injection of the muscarinic agonist McN-A-343 inhibited the tail-flick response to noxious thermal stimulation in a dose-dependent manner (31.5 - 63.0 nmol). This McN-A-343-induced antinociceptive effect was dose-dependently inhibited by intrathecal (i.t.) injection of a nonselective muscarinic receptor antagonist atropine, the selective muscarinic M(1) antagonist pirenzepine, or the M(4) antagonist himbacine. The inhibition of pirenzepine was greater than that of himbacine. In contrast, the selective muscarinic M(2) antagonist methoctramine did not inhibit the antinociceptive effects of McN-A-343. In addition, the McN-A-343-induced antinociceptive effect was attenuated by i.t. injection of the GABA(A) antagonist bicuculline, but not by injection of the GABA(B) antagonist CGP35348. These results suggest that McN-A-343 produces its antinociceptive effect on the response to thermal stimulation via spinal muscarinic M(1) receptors and, at least in part, through neuronal pathways involving spinal GABA(A) receptors in mice.

Cotinine inhibits catecholamine release evoked by cholinergic stimulation from the rat adrenal medulla.

The aim of the present study was to clarify whether cotinine affects the release of catecholamines (CA) from the isolated perfused rat adrenal gland, and to establish the mechanism of its action, in comparison with the response of nicotine. Cotinine (0.3-3 mM), when perfused into an adrenal vein for 60 min, inhibited CA secretory responses evoked by ACh (5.32 mM), DMPP (a selective neuronal nicotinic agonist, 100 microM for 2 min) and McN-A-343 (a selective muscarinic M1-agonist, 100 microM for 2 min) in dose- and time-dependent manners. However, cotinine did not affect CA secretion by high K+ (56 mM). Cotinine itself also failed to affect basal CA output. Furthermore, in the presence of cotinine (1 mM), CA secretory responses evoked by Bay-K-8644 (an activator of L-type Ca2+ channels, 10 microM) and cyclopiazonic acid (an inhibitor of cytoplasmic Ca2+-ATPase, 10 microM) were relative time-dependently attenuated. However, nicotine (30 microM), given into the adrenal gland for 60 min, initially rather enhanced CA secretory responses evoked by ACh and high K+, followed by the inhibition later, while it time-dependently depressed the CA release evoked by McN-A-343 and DMPP. Taken together, these results suggest that cotinine inhibits greatly CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, but does fail to affect that by the direct membrane-depolarization. It seems that this inhibitory effect of cotinine may be exerted by the cholinergic blockade, which is associated with blocking both the calcium influx into the rat adrenal medullary chromaffin cells and Ca2+ release from the cytoplasmic calcium store. It also seems that there is a big difference in the mode of action between cotinine and nicotine in the rat adrenomedullary CA secretion.

M1 muscarinic receptor stimulation decreases aspartate release in the rat neostriatum.

This study investigates the effects of different muscarinic receptor agonists on extracellular glutamate and aspartate concentrations in the rat neostriatum. In vivo intracerebral perfusions were undertaken in the conscious rat using a concentric push-pull cannulae system. Amino acid concentrations in samples were determined by HPLC with fluorometric detection. The intrastriatal perfusion of arecoline, a M1-M2 muscarinic receptor agonist, produced a significant decrease in extracellular [ASP] (45% of decrease) but not in extracellular [GLU]. These effects were blocked by scopolamine, a M1-M2 muscarinic receptor antagonist. McN-A-343, a M1 muscarinic receptor agonist, but not the M2 muscarinic receptor agonist, oxotremorine, produced a significant decrease in extracellular [ASP] (40% of decrease) but not in extracellular [GLU]. The effects of McN-A-343 on extracellular [ASP] were blocked by pirenzepine, a M1 muscarinic receptor antagonist. These results suggest that the decrease in extracellular [ASP] could be mediated, at least in part, by M1 muscarinic receptor activation in the rat neostriatum.

McN-A-343 increases renal sympathetic nerve activity and blood pressure by a muscarinic and a non-muscarinic mechanism in the rat.

1. Intravenous administration of the putative M1 muscarinic agonist McN-A-343 to conscious rats evokes an increase in mean arterial pressure (MAP) which can be blocked by muscarinic receptor antagonists. The present study was undertaken to evaluate the increase in MAP and renal sympathetic nerve activity (RSNA) evoked by intravenous administration of McN-A-343 to urethane-anaesthetized rats. 2. McN-A-343 (0.1-0.3 mg kg-1) evoked a concurrent increase in MAP and RSNA which could be inhibited by the nonselective muscarinic receptor antagonist methylatropine or the selective M1 muscarinic receptor antagonist telenzepine. Administration of higher doses of McN-A-343 (0.3-1.2 mg kg-1) in the presence of muscarinic receptor blockade evoked brief bursts in RSNA accompanied by increases in MAP. 3. The increases in MAP, but not the increases in RSNA, evoked by all doses of McN-A-343 could be attenuated by the selective alpha 1-adrenoceptor antagonist prazosin. Adding the selective alpha 2-adrenoceptor antagonist yohimbine to prazosin did not further inhibit the pressor response to the low doses of McN-A-343. 4. The irreversible alpha-adrenoceptor and NPY receptor antagonist benextramine also attenuated the pressor response evoked by the low doses of McN-A-343 but not the increases in RSNA. However, when combined with muscarinic receptor blockade, benextramine completely inhibited the brief bursts in RSNA, and thus also the increases in MAP, evoked by the high doses of McN-A-343. 5. The pressor response remaining after the administration of high doses of McN-A-343 to rats pretreated with prazosin and methylatropine was inhibited by treatment with alpha,beta-methylene ATP. 6. These results show that McN-A-343 evokes increases in RSNA by muscarinic and non-muscarinic mechanisms. Furthermore, the subsequent increase in MAP is primarily dependent upon activation of vascular alpha 1-adrenoceptors, but may also involve activation of P2 alpha receptors.

Selective blockade of muscarinic M2 receptors in vivo by the new antagonist tripitramine.

The antimuscarinic effects of tripitramine (1, 1, 24--tris[[5, 11-dihydro-6-oxo-6H-pyrido [2,3-b][1,4]- benzodiazepin-11-yl)carbonyl]methyl]-8, 17-dimethyl-1, 8, 17, 24-tetraazatetracosane tetraoxalate), a member of a series of polymethylene tetraamines with in vitro cardioselectivity, were assessed in two in vivo preparations: anaesthetized and pithed rats. The well-known M2 selective antagonist methoctramine was used in a comparative study. Tripitramine (0.0202 mumol/kg i.v.) proved to be a potent antagonist at cardiac M2 receptors that mediate the decrease in heart rate in the pithed rat; the same dose of this antagonist in the anaesthetized rat did not significantly affect the depressor action of methacholine mediated by vascular M3 receptors. In the pithed rat, this dose did not affect the ganglionic M1 receptor-mediated tachycardia and pressor response to muscarine or McN-A-343. These in vivo data are consistent with the in vitro findings and confirm that tripitramine is a more potent and selective muscarinic M2 receptor antagonist than methoctramine.

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

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

M2 muscarinic receptor agonists produce hypotension and bradycardia when injected into the nucleus tractus solitarii.

Bilateral microinjections (0.2-2 nmol/site) of a potent M2 muscarinic receptor agonist, cis-methyldioxolane (CD), but not those of a relatively selective M1 receptor agonist (McN-A343; 3 nmol/site), into the intermediate portion of nucleus tractus solitarii (NTS) of pentobarbital-anesthetized rats elicited a decrease in blood pressure (23-52 mm Hg) and heart rate (16-50 bpm). Previous microinjections of a selective competitive M2 receptor antagonist (AFDX-116; 0.8 nmol/site), but not those of a potent selective M1 receptor antagonist (pirenzepine; 2 nmol/site), into the NTS blocked the effects of CD. These results indicate that the muscarinic receptors in the intermediate portion of NTS are of M2 type.

Emesis induced by 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343): evidence for a predominant central muscarinic M1 mediation.

The emetic action of 4-(m-chlorophenylcarbamoyloxy)-2- butynyltrimethylammonium chloride (McN-A-343) was investigated in the unanaesthetized cat, after it was injected into the cerebral ventricles, through chronically-implanted cannulae. Intracerebroventricular injection of McN-A-343 produced dose-dependent and shortlasting emesis, which was not completely abolished after ablation of the area postrema. The predominantly selective muscarinic M1 antagonist, pirenzepine as well as the mixed muscarinic M1 and M2 antagonist, atropine, injected into the cerebral ventricles, attenuated or abolished the emesis evoked by intracerebroventricular McN-A-343. Both atropine and pirenzepine produced dose-dependent inhibition of the emesis evoked by McN-A-343. However, the ID50 value for atropine was approximately five times greater than that for pirenzepine. Abolition of McN-A-343-induced emesis only occurred with the largest dose of atropine (1 mg). On the other hand, selected ganglionic blocking agents, an alpha- and beta-adrenoceptor blocking agent, a dopamine antagonist, a 5-hydroxytryptamine antagonist and an antihistamine, all injected into the cerebral ventricles, had no significant effect on emesis evoked by McN-A-343, similarly injected. The emetic response to intracerebroventricular injection of McN-A-343 was attenuated or abolished in cats pretreated with hemicholinium-3, triethylcholine, reserpine and 6-hydroxydopamine, intracerebroventricularly. On the contrary, the emetic response to intracerebroventricular injection of McN-A-343 was not altered in cats pretreated with intracerebroventricular injections of bretylium, alpha-methyl-p-tyrosine and 5,6-dihydroxytryptamine. It is postulated that the emesis produced by McN-A-343, injected into the cerebral ventricles, is mediated through muscarinic M1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential role of M-1 and M-2 receptors in sympathetic ganglia of the pithed normotensive rat in alpha adrenoceptor-mediated vasoconstriction.

In the pithed normotensive rat the adrenoceptors involved in the hypertensive and tachycardic effects of the muscarinic ganglionic stimulants 1,1-dimethyl-4-carboxypiperidine methylester (DMCPM) and (4-m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium (McN-A-343) were analyzed. The selective alpha-1 adrenoceptor antagonist prazosin, the selective alpha-2 adrenoceptor antagonist rauwolscine, the selective beta-1 adrenoceptor antagonist atenolol and the selective beta-2 adrenoceptor antagonist ICI 118,551 were used as tools for the identification of the adrenoceptors. DMCPM elicited a release of catecholamines which activated vascular alpha-1, alpha-2 and cardiac beta-1 adrenoceptors. McN-A-343, however, was induced by a release of neurotransmitter stimulation of predominantly vascular alpha-1 and cardiac beta-1 adrenoceptors. Both DMCPM and McN-A-343 were characterized with respect to their ability to stimulate muscarinic-1 (M-1) and/or muscarinic-2 (M-2) receptors. To demonstrate the M-1 component the selective M-1 receptor antagonist pirenzepine was used. M-2 receptor activation was identified by means of the muscarinic receptor-induced bradycardia after pretreatment with a high dose of atenolol. DMCPM proved to be a mixed M-1/M-2 agonist, in contrast to McN-A-343 which behaved as a rather selective M-1 agonist. The data led us to formulate the hypothesis that the activation of ganglionic M-1 receptors elicits the stimulation of predominantly alpha-1 adrenoceptors in the vascular wall. Activation of ganglionic M-2 receptors may induce an additional stimulation of vascular alpha-2 adrenoceptors. The increase in heart rate seems to be mediated by beta-1 adrenoceptors only.