M1 Muscarinic Receptor Antagonist Research Articles

DMPP-evoked increases in postganglionic sympathetic nerve activity and blood pressure occurs by two mechanisms in the rat.

1. Intravenous administration of the ganglionic nicotinic receptor agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide) into urethane-anaesthetized rats evoked dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). 2. The ganglionic nicotinic receptor antagonists pentolinium and hexamethonium either alone or combined did not inhibit the increase in RSNA and MAP evoked by 50 to 200 micrograms kg-1 doses of DMPP. The increase in renal sympathetic nerve activity evoked by DMPP occurred as a brief burst in firing. 3. The increase in MAP, but not RSNA, evoked by DMPP in the presence of pentolinium was inhibited by the selective alpha 1-adrenergic receptor antagonist prazosin. 4. The non-selective alpha-adrenoceptor and NPY receptor antagonist benextramine also inhibited the increase in MAP without inhibiting the increase in RSNA. Surprisingly, the combination of benextramine and pentolinium, or benextramine and hexamethonium, completely blocked the DMPP-evoked increase in RSNA and thus the increase in MAP. 5. The uptake1 antagonist desipramine combined with pentolinium did not affect the DMPP-evoked increases in MAP or RSNA when compared to the responses evoked in the presence of pentolinium alone. 6. Adding the selective M1 muscarinic receptor antagonist telenzepine to pentolinium and prazosin did not inhibit the increase in RSNA evoked by a 100 micrograms kg-1 dose of DMPP. 7. While the DMPP-evoked increase in MAP in the presence of ganglionic nicotinic receptor antagonists is primarily dependent upon activation of alpha 1-adrenoceptors, the increase in RSNA occurs via activation of ganglionic nicotinic receptors and activation of a mechanism susceptible to blockade by benextramine.

Selective M1 muscarinic receptor antagonists disrupt memory consolidation of inhibitory avoidance in rats

The effect of three different M1 muscarinic antagonists, pirenzepine, biperiden, and trihexyphenidyl on memory consolidation was investigated. Rats were trained in a one-trial step-through inhibitory avoidance task and injected intraperitoneally immediately afterwards, either with pirenzepine, biperiden, or trihexyphenidyl (dose range from 0 to 16 mg/kg). The non-selective antimuscarinic compound scopolamine, was also administered for comparison. One day later, rats were tested for retention. Results show that biperiden, trihexyphenidyl and scopolamine produced a dose-dependent impairment of inhibitory avoidance consolidation, while pirenzepine had no effect. The amnestic state produced by biperiden and trihexyphenidyl was comparable to that observed after the administration of scopolamine. These results indicate that the selective blockade of the central M1 muscarinic receptors interfere with memory consolidation of inhibitory avoidance and suggest that this receptor subtype is critically involved in mnemonic functions.

Rebound stimulation of the cAMP-regulated Cl- current by acetylcholine in guinea-pig ventricular myocytes.

1. Acetylcholine (ACh)-induced rebound stimulation of the cAMP-regulated Cl- current was studied in isolated guinea-pig ventricular myocytes using dialysing and dialysis-limiting configurations of the whole-cell patch-clamp technique. 2. Exposure to and subsequent washout of ACh produced a transient rebound stimulation of the Cl- current. However, this rebound response was only observed in the presence of submaximally stimulating concentrations of the cAMP-producing agonists isoprenaline (Iso) or histamine. ACh-induced rebound stimulation was not observed in the presence of maximally stimulating concentrations of Iso, nor was it observed in the absence of Iso. 3. To prevent saturation of responses during rebound, the effects of ACh were studied in the presence of a subthreshold concentration of Iso (0.001 microM). Varying the duration of exposure to ACh before washout demonstrated that the stimulatory effect of 1 microM ACh approaches steady state with a time constant of 34 s. Exposing myocytes to varying concentrations of ACh for 90 s demonstrated that the EC50 for the stimulatory effect of ACh was 0.15 microM with a maximum response equal to 67% of that obtained by a maximally stimulating concentration of Iso alone. 4. Rebound stimulation of the Cl- current could also be elicited by washing in 2 microM atropine during exposure to ACh, instead of washing out ACh. Furthermore, ACh-induced rebound was blocked by the M2 muscarinic receptor antagonist methoctramine but not by the M1 receptor antagonist pirenzepine. Rebound was also blocked in pertussis toxin (PTX)-treated myocytes. 5. ACh-induced rebound stimulation was not blocked by: (a) L-NMMA, an inhibitor of nitric oxide synthase activity; (b) Methylene Blue, LY-83583, and ODQ, inhibitors of cGMP production; or (c) milrinone, an inhibitor of cGMP-dependent phosphodiesterase activity. 6. These results indicate that ACh can stimulate cAMP-regulated ion channel activity in cardiac ventricular myocytes by facilitating beta-adrenergic and histaminergic responses. This is opposite to the inhibitory actions more typically associated with muscarinic receptor stimulation in ventricular myocardium. This stimulatory effect of ACh is mediated through M2 muscarinic receptors and a PTX-sensitive G-protein, but it does not appear to involve the production of nitric oxide or cGMP.

Effects of the κ-opioid dynorphin A(1–13) on learning and memory in mice

The effects of intracerebroventricular administration of dynorphin A(1–13) on scopolamine- and pirenzepine-induced amnesia were investigated in mice by observing the step-down-type passive avoidance response and spontaneous alternation performance. The pre- or post-training, or preretention administration of dynorphin A(1–13) (0.3–10μg) alone failed to affect the passive avoidance response, while scopolamine (1 mg/kg) significantly inhibited it. Dynorphin A(1–13) (1 μg) given 15 min before training and retention tests, but not immediately after training, significantly improved the scopolamine (1 mg/kg)-induced impairment of passive avoidance response, indicating the anti-amnesic effects of dynorphin A(1–13). A lower dose (1 mg/kg) of the x-opioid receptor antagonist (-)-(1R,5R,9R)-5,9-diethyl-2-(3-furyl-methyl)-2′-hydroxy-6,7-benzomorphan reversed the anti-amnesic effects of dynorphin A(1–13) (1 μg). In contrast, although dynorphin A(1–13) (1, 3 and 10 μg) did not influence spontaneous alternation performance, scopolamine (1 mg/kg) and the muscarinic M1 receptor antagonist pirenzepine (3 μg) markedly decreased spontaneous alternation performance. Dynorphin A(1–13) (3, 5.6 and/or 10μg) significantly improved the scopolamine (1 mg/kg)- and pirenzepine (3 μg) induced impairment of spontaneous alternation performance. The improving effects of dynorphin A(1–13) (3 μg) were almost completely reversed by pretreatment with nor-binaltorphimine (4 μg), (σ1) κ-selective opioid receptor antagonist. These results suggest that the stimulation of κ-opioid receptors improves memory dysfunctions resulting from the blockade of muscarinic M1 receptors.

Synthesis of novel succinamide derivatives having the 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one skeleton as potent and selective M2 muscarinic receptor antagonists. I.

A series of 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one derivatives containing the succinamide skeleton has been synthesized and evaluated for M1, M2 and M3 muscarinic receptor binding affinities (in vitro) and M2 and M3 muscarinic receptor antagonistic activities (in vivo). Some of them showed higher and more selective binding affinities for M(2) muscarinic receptors than that of AF-DX 116. Among them, 11-[3-[N-[2-(N-benzyl-N- methylamino)ethyl]-N-ethylcarbamoyl]propionyl]-5,11-dihydro-6H-pyr ido [2,3-b][1,4]benzodiazepin-6-one (68) was found to be the most potent and selective M2 muscarinic receptor antagonist in vitro. This compound also strongly inhibited the oxotremorine-induced bradycardia after intravenous administration and showed 130-fold selectivity for M2 muscarinic receptors over M3 muscarinic receptors in vivo.

Synthesis of Novel Succinamide Derivatives Having a 5,11-Dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one Skeleton as Potent and Selective M2 Muscarinic Receptor Antagonists. II

A series of succinamide derivatives containing the 5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one skeleton (6a-z) was prepared and evaluated for binding affinity to muscarinic receptors in vitro and for antagonism of bradycardia and salivation in vivo in comparison with AF-DX 116 (1a). Structure-activity relationships (SAR) studies in vitro indicated that the 4-(4-alkyl-1-piperazinyl)benzylamino moiety plays a crucial role in enhancing the affinity for M2 muscarinic receptors. Compound 6y, containing a 4-(4-isopropyl-1-piperazinyl)benzylmethylamino moiety, exhibited the highest affinity for M2 muscarinic receptors (pKi = 9.2), being 200 times as potent as 1a, and compound 6u, containing a 4-(4-ethyl-1-piperazinyl)benzylethylamino moiety, showed the highest selectivity for M2 over M3 muscarinic receptors (M3/M2 ratio = 320). Both 6y and 6u antagonized the oxotremorine-induced bradycardia in rats after intravenous or oral administration. Oral evaluation in conscious dogs showed that the efficacy for increasing the heart rate was at least 3-fold greater than that of 1a.

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.

M1 acetylcholine receptor stimulation increases the extracellular concentrations of glutamate and GABA in the medial prefrontal cortex of the rat.

The present study was undertaken to examine the effects of different muscarinic receptor agonists on glutamate and GABA concentrations in the medial prefrontal cortex of the rat. In vivo perfusions were made in the conscious rat using a concentric push-pull cannulae system. Amino acid concentrations in samples were determined by HPLC with fluorometric detection. The intracortical perfusion of arecoline, a M1-M2 muscarinic receptor agonist, produced a significant increase in extracellular [GLU] and [GABA]. McN-A-343, a M1 muscarinic receptor agonist, but not the M2 muscarinic receptor agonist, oxotremorine, produced a significant increase in extracellular [GLU] and [GABA]. The effects of McN-A-343 on extracellular [GLU] and [GABA] were blocked by pirenzepine, a M1 muscarinic receptor antagonist. These results suggest that M1 muscarinic receptor stimulation increases the extracellular concentrations of GLU and GABA in the medial prefrontal cortex of the rat.

Cholinergic modulation of synaptic transmission in horizontal connections of rat motor cortex.

The influence of compounds interacting with cholinergic systems on field potentials evoked in layer II/III horizontal connections was investigated in rat motor cortex in vitro. The cholinesterase inhibitor eserine (10 microM) decreased field responses by 20 +/- 2%. This effect could be prevented by preincubation with atropine (10 microM). Application of 5 microM carbachol resulted in reduction of the responses by 30 +/- 1%. These reductions were reversible, repeatable and independent of stimulus intensity; they could be blocked by the M1 muscarinic receptor antagonist pirenzepine (3 microM) but not by the M2 muscarinic receptor antagonist gallamine (10 microM). During carbachol application, paired-pulse facilitation (40 ms interpulse interval) was increased. The results indicate that endogenous acetylcholine may modulate excitatory synaptic transmission in horizontal connections of rat motor cortex, most likely by acting upon M1 receptors located presynaptically on glutamatergic terminals, and may contribute both to information processing and synaptic plasticity within the motor cortex.

Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors.

1. Whole cell recordings of glutamatergic excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation in the reticular formation were made from visualized hypoglossal motoneurons (HMs) in rat brain stem slices. 2. Carbachol, muscarine, or physostigmine reduced EPSC amplitude to 50 +/- 3%, 37 +/- 3%, and 54 +/- 7% (mean +/- SE) of control, respectively; effects of carbachol and physostigmine were antagonized by atropine (1-2 microM). EPSC depression was most effectively antagonized by methoctramine, an M2 muscarinic acetylcholine receptor (mAChR) antagonist with a high affinity constant (pKB) of 8.07 for the receptor mediating this response, whereas pirenzepine, an M1 mAChR antagonist, had a pKB of < 7.0, showing that EPSC depression was mediated by the M2 mAChR. 3. Postsynaptic properties of HMs (holding current and input resistance), EPSCs (reversal potential, rise time, half-width, and decay time constant), and postsynaptic glutamate-gated currents (amplitude and waveform) were not altered by carbachol or muscarine. 4. Muscarine did not decrease presynaptic neuron excitability, because the frequency of spontaneous EPSCs in HMs in the absence of tetrodotoxin (TTX) was either unchanged or increased. Leak and action currents of reticular formation neurons were not significantly altered by muscarine. In contrast, with TTX present, the frequency of spontaneous miniature glutamatergic EPSCs in HMs was decreased by both carbachol (mean change = 203 +/- 46%) and muscarine (mean change = 185 +/- 26%), with no change in miniature EPSC amplitude distribution. 5. Muscarinic depression of excitatory transmission to HMs thus occurs at the presynaptic terminal, most probably affecting release mechanisms downstream from calcium entry, and is likely to be significant during rapid eye movement sleep, possibly underlying the loss of tongue tone and inspiratory activity during this state.

Indirect muscarinic receptor activation by pentamidine on airway smooth muscle.

1. Pentamidine is routinely used to reduce the incidence of Pneumocystis carinii pneumonia in patients infected with human immunodeficiency virus, but it has been described as inducing pulmonary adverse effects, such as cough and bronchospasm. 2. In this paper we have investigated the effects of pentamidine on guinea-pig isolated main bronchi and human isolated bronchi. Pentamidine induced a concentration-dependent contraction in both preparations with pD2 values of 9.64 +/- 0.07 (n = 8) and 9.73 +/- 0.06 (n = 8) and a maximal effect (Emax) of 40 +/- 4% and 34 +/- 5% of the response to acetylcholine (1 mM) in guinea-pig and human bronchi respectively. Atropine (0.01 to 0.1 microM) and the muscarinic M3 receptor antagonist, hexahydro-siladiphenidol (0.1 and 1 microM) inhibited pentamidine-induced concentration-responses in both preparations in a non-competitive manner, whereas only high concentrations of the M1 receptor antagonist pirenzipine (1 microM) inhibited pentamidine concentration-response curves. 3. The cholinesterase inhibitor, tacrine (1 microM), potentiated the effect of pentamidine; in contrast, morphine inhibited pentamidine-induced responses. 4. The bronchoconstrictor effect of pentamidine on guinea-pig and human isolated bronchi was not modified by the H1 histamine receptor antagonist, mepyramine, by indomethacin or by the neurokinin NK1 and NK2 receptor antagonists, CP-96,345 and SR 48969 respectively, suggesting that neither histamine receptor stimulation, arachidonic acid derivative formation, nor tachykinin release are involved in pentamidine-induced contraction of human and guinea-pig airways. 5. Our overall results suggest that pentamidine induces contraction of guinea-pig and human isolated bronchi through prejunctional cholinergic nerve stimulation.

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.

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.

Influence of methoctramine on the acetylcholine-isoprenaline functional antagonism in the guinea pig isolated trachea.

It has been suggested that activation of muscarinic M2 receptors is one of the components of the functional antagonism between muscarinic and beta-adrenoceptor agonists in canine and guinea pig tracheal smooth muscle. The aim of the present study was to determine in the guinea pig trachea the importance of this component according to the magnitude of the acetylcholine-induced contraction. Cumulative concentration-response curves for isoprenaline were obtained in the absence or presence of the muscarinic M2 receptor antagonist methoctramine (3 x 10(-7) M) in tracheal rings under basal tension or precontracted by acetylcholine 2 x 10(-7), 3 x 10(-6) and 10(-4) M, giving contractions of 25, 50 Or 75%, respectively, of the maximal tension induced by acetylcholine 3 x 10(-3) M. In the absence of methoctramine, acetylcholine induced a concentration-dependent shift of the concentration-response curves of isoprenaline (-log EC50 of isoprenaline are 8.09 +/- 0.07, 7.85 +/- 0.08, 7.38 +/- 0.12 and 6.49 +/- 0.12, n = 6 for basal tension and for acetylcholine concentrations of 2 x 10(-7), 3 x 10(-6) and 10(-4) M, respectively). In the presence of methoctramine, the basal -log EC50 of isoprenaline was unmodified, whereas the acetylcholine induced shifts of concentration-response curves of isoprenaline were abolished for low levels of contraction (25%) and significantly reduced to 50 and 75% levels of contraction. Under similar conditions, acetylcholine-induced shifts of concentration-response curves of isoprenaline were unmodified by the muscarinic M1 receptor antagonist pirenzepine (10(-7) M). These results suggest that the inhibitory effect of M2 receptors on beta-adrenoceptor agonists effects is important for low contraction levels induced by acetylcholine, and that this effect becomes less important for higher concentrations of acetylcholine.

Evidence for a differential cholecystokinin-B and -A receptor regulation of gaba release in the rat nucleus accumbens mediated via dopaminergic and cholinergic mechanisms

In the present study we characterized the cholecystokinin receptor regulation of (i) the dopamine D2 agonist binding sites in striatal sections including the nucleus accumbens and (ii) GABA and dopamine release in the central part of the rat nucleus accumbens, by combining the in vitro filter wipe-off and the in vivo microdialysis techniques. In the binding study we demonstrate that sulphated cholecystokinin octapeptide (1 nM) increased (219 +/- 30%) the KD value of the D2 agonist [3H]N-propylnorapomorphine binding sites in sections from the striatum including the accumbens. This effect was counteracted by the cholecystokinin-B antagonist PD134308 (50 nM). In a parallel study using microdialysis in the central nucleus accumbens, we found that local perfusion with sulphated cholecystokinin octapeptide (1 microM) induced an increase in GABA (135 +/- 7%) and dopamine (146 +/- 8%) release which was unaffected by the cholecystokinin-A antagonist L-364,718 (10 nM). In contrast, when the cholecystokinin-B antagonist PD134308 (10 nM) was co-perfused with the peptide it prevented the increase in dopamine and decreased GABA release (-24 +/- 2%). This reduction was counteracted by the addition to the perfusate medium of the cholecystokinin-A antagonist or the cholinergic muscarinic M2 receptor antagonist AF-DX 116 (0.1 microM). Taken together, these data demonstrate that the facilitation by sulphated cholecystokinin octapeptide of GABA and dopamine release in the central accumbens probably reflects an inhibitory effect of the peptide on both pre- and postsynaptic D2 receptors, mediated via cholecystokinin-B receptor activation. In addition, for the first time we provide evidence for a differential cholecystokinin-A and -B receptor-mediated regulation of GABA transmission in the central accumbens, where the cholecystokinin-B receptor exerts a dominant excitatory influence while the cholecystokinin-A receptor mediates an inhibition of GABA release via a local muscarinic M2 receptor.

Carbachol induces inward current in neostriatal neurons through M1-like muscarinic receptors

The effects of carbachol on rat neostriatal neurons were examined in the slice and the freshly dissociated neuron preparations using intracellular and whole-cell voltage-clamp recording methods. Superfusion of carbachol (30 microM) produced a depolarization concomitant with an increase in the rate of spontaneous action potentials. This depolarization was associated with an increase in the input resistance. The carbachol-induced membrane depolarization was blocked by pirenzepine (1 microM), a selective M1 muscarinic receptor antagonist. In other experiments, we observed that carbachol induced a transient inward current on the freshly dissociated neostriatal neuron at a holding potential of -60 mV in a concentration-dependent manner underlying the whole-cell voltage-clamp mode. The inward current caused by carbachol was not reduced by tetrodotoxin (1 microM), calcium-free recording solution or Cd2+ (100 microM). However, it was blocked by Ba2+ (100 microM). In addition, the carbachol-induced inward current reversed polarity at about the potassium equilibrium potential. The whole-cell membrane inward current in response to voltage-clamp step from -90 to -140 mV was reduced by 30 microM carbachol. With stronger hyperpolarization beyond the potassium equilibrium potential, carbachol produced a progressively greater reduction in membrane current. This inhibitory effect was also abolished by Ba2+ (100 microM). A concentration of 30 microM carbachol-induced inward current could be reversibly antagonized by the M1 muscarinic receptor antagonist pirenzepine (0.1-1 microM), with an estimated IC50 of 0.3 microM. However, other muscarinic receptor subtype (M2 or M3) antagonists could also block the carbachol-induced inward current. The rank order of antagonist potency was: pirenzepine (M1 antagonist) > 4-diphenylacetoxy-N,N-methyl-piperidine methiodide (M3/M1 antagonist) > gallamine (M2 antagonist). Based on these pharmacological data, we concluded that carbachol can act at M1-like muscarinic receptors to reduce the membrane K+ conductances and excite the neostriatal neurons.

Mechanism of cytokine-induced modulation of beta-adrenoceptor responsiveness in airway smooth muscle.

To elucidate the role of specific proinflammatory cytokines in regulating airway responsiveness, we examined the effects and mechanisms of action of IL-1beta, TNF-alpha, and IL-2 on the beta-adrenoceptor- and postreceptor-coupled transmembrane signaling mechanisms regulating relaxation in isolated rabbit tracheal smooth muscle (TSM) segments. During half-maximal isometric contraction of the tissues with acetylcholine, relaxation responses to isoproterenol, PGE2, and forskolin were separately compared in control (untreated) TSM and tissues incubated for 18 h with IL-1beta (10 ng/ml), TNF-(alpha (100 ng/ml), or IL-2 (200 ng/ml). Relative to controls, IL-1beta- and TNF-alpha-treated TSM, but not IL-2-treated tissues, depicted significant attenuation of their maximal relaxation and sensitivity (i.e., -log dose producing 50% maximal relaxation) to isoproterenol (P < 0.001) and PGE2 (P < 0.05); whereas the relaxation responses to direct stimulation of adenylate cyclase with forskolin were similar in the control and cytokine-treated tissues. Further, the attenuated relaxation to isoproterenol and PGE2 was ablated in the IL-1beta-treated TSM that were pretreated with either the muscarinic M2-receptor antagonist, methoctramine (10(-6) M), or pertussis toxin (100 ng/ml). Moreover, Western immunoblot analysis demonstrated that: (a) Gi protein expression was significantly enhanced in membrane fractions isolated from IL-1beta-treated TSM; and (b) the latter was largely attributed to induced enhanced expression of the Gi alpha2 and Gi alpha3 subunits. Collectively, these observations provide new evidence demonstrating that IL-lbeta and TNF-alpha induce impaired receptor-coupled airway relaxation in naive TSM, and that the latter effect is associated with increased muscarinic M2-receptor/Gi protein-coupled expression and function.

Muscarinic inhibition of glutamatergic transmissions onto rat magnocellular basal forebrain neurons in a thin-slice preparation.

We have examined excitatory and inhibitory transmission in visually identified rat magnocellular basal forebrain neurons using whole-cell patch-clamp recordings in a thin-slice preparation of the rat brain. In most cells, spontaneous excitatory and inhibitory synaptic activities could be recorded from their resting membrane potential. Following focal stimulation within the basal forebrain nucleus or directly onto visualized neighbouring neurons, postsynaptic currents were elicited in magnocellular basal forebrain cells held at -70 mb (a value close to their resting membrane potential). The synaptic responses were complex, consisting either mainly of excitatory postsynaptic currents (EPSCs), or inhibitory postsynaptic currents (IPSCs), or an EPSC-IPSC sequence. The EPSC component was consistent with the activation of AMPA/KA receptors, as it could be selectively blocked by CNQX. The IPSC component resulted in the activation of GAGAA receptors, and could be blocked by bicuculline. Since GABA-mediated trasmissions were not frequently recorded, we focused on the glutamate-mediated transmission. Studies using specific calcium channel blockers suggested that both omega-conotoxin GVIA-sensitive and omega-agatoxin VIA-sensitive calcium channels contribute to the glutamatergic transmission onto magnocellular basal forebrain neurons. Carbachol (0.3-30 microM) had no observable effect on holding current, but produced a dose-dependent inhibition of the amplitude of evoked EPSCs. This cholinergic modulation was mediated by muscarinic receptors, as it could be antagonized by atropine. The inhibitory effect of carbachol on the amplitude of EPSCs could be significantly antagonized by 100 nM methoctramine, an M2-receptor antagonist. In contrast, only a small degree of antagonism could be obtained with pirenzepine, and M1-muscarinic receptor antagonist, when present at relatively high concentration of 1 microM. Moreover, the action of carbachol was presynaptic, since the frequency of miniature postsynaptic currents was reduced without affecting their amplitude. In conclusion, the present findings indicate that glutamate-mediated transmission onto magnocellular basal forebrain neurons appeared to involve both N- and P/Q-type calcium channels, and that muscarinic modulation of glutamatergic transmission to MBF neurons is mediated by a presynaptic M2-muscarinic receptor subtypes.

Muscarinic receptors mediating depression and long-term potentiation in rat hippocampus.

1. Two concentration-dependent effects of the muscarinic agonist carbachol (CCh) were characterized in submerged slices of rat hippocampus using extracellular recordings of excitatory postsynaptic potentials (EPSPs): muscarinic long-term potentiation (LTP(m)) and depression. 2. LTP(m) of the EPSP slope was seen following long exposure (20 min) of the slice to low concentrations of CCh (0.2-0.5 microM). This LTP(m) was not accompanied by a change in the size of the afferent fibre volley or by a change in paired-pulse potentiation, consistent with a postsynaptic locus of CCh action. 3. Intracellular recordings from voltage-clamped neurons of inward current evoked by iontophoretically applied alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) revealed that, while cellular responses to NMDA rose transiently upon superfusion with 0.5 microM CCh, responses to AMPA increased gradually and remained potentiated after washout of CCh. 4. LTP(m) is mediated by an M2 muscarinic receptor. Two M2 muscarinic receptor antagonists, methoctramine and AFDX-116, blocked LTP(m). The M2 agonist oxotremorine induced LTP(m) at low agonist concentrations. None of the M1 and M3 receptor agonists and antagonists tested affected LTP(m). 5. Muscarinic fast onset depression of the EPSP was seen in response to higher concentrations of CCh (2-5 mu M). This depression was accompanied by an increase in paired-pulse potentiation, indicating a possible presynaptic locus of action. The M3 muscarinic receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) blocked the muscarinic depression of the EPSP slope. M1, M2 and M4 muscarinic antagonists did not block this response. 6. Blockade of the muscarinic depression by 4-DAMP did not uncover a suppressed LTP(m). However, addition of picrotoxin facilitated the expression of LTP(m) induced by high concentrations of CCh, indicating an involvement of interneurons in regulation of LTP(m). 7. Cholinergic denervation produced by fimbria-fornix transection resulted in supersensitivity of both M2- and M3-mediated effects, indicating that the receptors mediating these effects are not located on presynaptic cholinergic fibres. In the presence of 4-DAMP and picrotoxin the dose-response curve for CCh-induced effects in slices from lesioned animals was shifted to the left relative to that of normal animals, indicating a supersensitivity of both receptor types.

Neurohormonal modulation of rat enterochromaffin-like cell histamine secretion

BACKGROUND & AIMS: Gastric mucosal cells and nerve terminals contain at least acetylcholine, adrenergic agents, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, serotonin, gamma-amino-butyric acid, neurokinins A and B, neurotensin, neuropeptide Y, peptide YY, gastrin-releasing peptide, somatostatin, and [Met5]enkephalin. Although some of these agents have been implicated as regulators of gastric acid secretion, their site and mechanism of action is not well understood. The aim of this study was to investigate whether local gastric neurotransmitters modulate acid secretion by regulating basal and gastrin-driven enterochromaffin-like (ECL) cell histamine release. METHODS: The effects of the above agents were investigated in a short-term 90%-95% pure ECL cell culture system. Cells were incubated with either the neuromodulator alone or in combination with gastrin for 10-40 minutes, and histamine secretion was measured by enzyme immunoassay. RESULTS: Acetylcholine, isoproterenol, and vasoactive intestinal polypeptide significantly stimulated basal and gastrin-driven histamine secretion, whereas calcitonin gene-related peptide and somatostatin inhibited basal and gastrin-driven histamine secretion. The M1 muscarinic receptor antagonist pirenzepine dose dependently inhibited the action of acetylcholine, whereas the M3 receptor antagonist 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride had no effect. the rest of the evaluated agents had no effect on ECL cell histamine secretion. CONCLUSIONS: These data are consistent with the hypothesis that substantial neurohormonal modulation of ECL cell function exists. (Gastroenterology 1996 Apr;110(4):1084-92)