Structural Insights into M1 Muscarinic Acetylcholine Receptor Signaling Bias between Gαq and ß-Arrestin through BRET Assays and Molecular Docking.

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and ß-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and ß-arrestin2. Pilocarpine preferentially promoted the recruitment of ß-arrestin2 (∆∆RAi = -0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to ß-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and ß-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias.

McN-A-343, a muscarinic agonist, reduces inflammation and oxidative stress in an experimental model of ulcerative colitis.

AIM: The aim of the present study was to investigate the anti-inflammatory response mediated of the M1 muscarinic acetylcholine receptor (mAChR) during experimental colitis. MATERIAL AND METHODS: After the induction of 6% acetic acid colitis, mice were treated with McN-A-343 0.5, 1.0, and 1.5 mg/kg or dexamethasone (DEXA, 2.0 mg/kg) or pirenzepine (PIR, 10 mg/kg; M1 mAChR antagonist). Colonic inflammation was assessed by macroscopic and microscopic lesion scores, colonic wet weight, myeloperoxidase (MPO) activity, interleukin-1 beta (IL1-ß) levels and tumor necrosis factor alpha (TNF-α), glutathione (GSH), malondialdehyde (MDA) and nitrate and nitrite (NO3/NO2), mRNA expression of IKKα, nuclear factor kappa beta (NF-kB) and cyclooxygenase-2 (COX-2), as well protein expression of NF-kB and COX-2. RESULTS: Treatment with McN-A-343 at a concentration of 1.5 mg/kg showed a significant reduction in intestinal damage as well as a decrease in wet weight, MPO activity, pro-inflammatory cytokine concentration, markers of oxidative stress and expression of inflammatory mediators. The action of the M1 agonist by the administration of pirenzepine, which promoted the blocking of the mAChR M1-mediated anti-inflammatory response, has also been proven. CONCLUSION: The results suggest that peripheral colonic M1 mAChR is involved in reversing the pro-inflammatory effect of experimentally induced colitis, which may represent a promising therapeutic alternative for patients with ulcerative colitis.

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.

Stimulating muscarinic M1 receptors in the anterior cingulate cortex reduces mechanical hypersensitivity via GABAergic transmission in nerve injury rats.

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A2A and Muscarinic M1 Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters.

BACKGROUND/AIMS: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. METHODS: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500-750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. RESULTS: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 µmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 µmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 µmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 µmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 µmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 µmol/L) was also prevented by HC-3 (4 µmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 µmol/L). CONCLUSION: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

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.

Muscarinic M1 Receptor Overstimulation Disrupts Working Memory Activity for Rules in Primate Prefrontal Cortex.

Acetylcholine release in the prefrontal cortex (PFC), acting through muscarinic receptors, has an essential role in regulating flexible behavior and working memory (WM). General muscarinic receptor blockade disrupts PFC WM representations, while selective stimulation of muscarinic receptor subtypes is of great interest for the treatment of cognitive dysfunction in Alzheimer's disease. Here, we tested selective stimulation and blockade of muscarinic M1 receptors (M1Rs) in macaque PFC, during performance of a cognitive control task in which rules maintained in WM specified saccadic responses. We hypothesized that M1R blockade and stimulation would disrupt and enhance rule representation in WM, respectively. Unexpectedly, M1R blockade did not consistently affect PFC neuronal rule selectivity. Moreover, M1R stimulation suppressed PFC activity, and at higher doses, degraded rule representations. Our results suggest that, in primates, the deleterious effects of general muscarinic blockade on PFC WM activity are not mediated by M1Rs, while their overstimulation deteriorates PFC rule maintenance.

Activations of muscarinic M1 receptors in the anterior cingulate cortex contribute to the antinociceptive effect via GABAergic transmission.

Background Cholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods. Results We found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex. Conclusions These results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior cingulate cortex.

Activation of M1/4 receptors phase advances the hamster circadian clock during the day.

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) can be reset by the cholinergic agonist carbachol. In hamsters, intraSCN carbachol produces phase advances during the day. This phenomenon has previously been attributed to the muscarinic receptors, as carbachol-induced phase shifts are blocked by pretreatment with the muscarinic antagonist atropine. The SCN contains all five muscarinic receptors, leaving open the question as to which muscarinic receptors mediate these shifts. Here we test two selective muscarinic agonists, the M1/4 agonist McN-A-343 and the M2/3 agonist bethanechol, in addition to the non-selective cholinergic agonist carbachol. Consistent with previous reports, carbachol produced significant phase advances when injected to the SCN during the mid-subjective day. At the doses used here, McN-A-343, but not bethanechol, also produced significant phase shifts when injected to the SCN during the mid-subjective day. Phase shifts to McN-A-343 were as large as those produced by carbachol, suggesting that activation of the M1/4 receptors alone can fully account for the daytime phase advances produced by cholinergic agonists. Given acetylcholine's role in arousal, and the similarity between phase advances to carbachol/McN-A-343 and to exercise and arousal manipulations, it is possible that acetylcholine may contribute to non-photic resetting of the circadian clock.

Central muscarinic cholinergic activation alters interaction between splenic dendritic cell and CD4+CD25- T cells in experimental colitis.

BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4+/CD25-T cell activation in the context of experimental colitis. METHODS: The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25-T cell co-culture were determined. RESULTS: McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25-T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy. CONCLUSIONS: Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.

Activation of central muscarinic receptor type 1 prevents development of endotoxin tolerance in rat liver.

Endotoxin tolerance is a mechanism in which cells receiving low doses of endotoxin, enter a transient phase with less inflammatory response to the next endotoxin challenges. Central nervous system is known to modulate systemic inflammation through activation of the cholinergic system; however, the role of central anti-inflammatory pathway in pathophysiology of hepatic endotoxin tolerance is unknown. Our study was designed to assess the effect central muscarinic type 1 receptor (M1) activation on development of endotoxin tolerance in rat liver. Endotoxin tolerance was induced by daily intraperitoneal injection of endotoxin (1 mg/kg) for 5 days. Animals were randomly divided into two groups which received intracerebroventricular injection of either MCNA-343 (an M1 agonist, 5 ng/kg) or saline 1h after intraperitoneal injection of saline or endotoxin. The responsiveness to endotoxin was assessed by measuring hepatic MCP-1 (monocyte chemotactic protein-1), iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis-α) mRNA expression 3h after intraperitoneal administration of endotoxin using quantitative RT-PCR. A significant reduction in hepatic expression of MCP-1, iNOS and TNF-α was observed in rats with 5 days endotoxin challenge in comparison with rats given a single dose of endotoxin. There was no significant difference in hepatic expression of MCP-1, iNOS or TNF-α between acute and chronic LPS-treated groups in rats given MCNA-343. Central MCNA-343 stimulation could prevent the induction of hepatic endotoxin tolerance in animals receiving repeated doses of endotoxin. This indicates that M1 cholinergic receptor activation in the central nervous system can modulate endotoxin tolerance in rat liver.

Regulation of persistent activity in hippocampal mossy cells by inhibitory synaptic potentials.

The hippocampal formation receives strong cholinergic input from the septal/diagonal band complex. Although the functional effects of cholinergic activation have been extensively studied in pyramidal neurons within the hippocampus and entorhinal cortex, less is known about the role of cholinergic receptors on dentate gyrus neurons. Using intracellular recordings from rat dentate hilar neurons, we find that activation of m1-type muscarinic receptors selectively increases the excitability of glutamatergic mossy cells but not of hilar interneurons. Following brief stimuli, cholinergic modulation reveals a latent afterdepolarization response in mossy cells that can extend the duration of stimulus-evoked depolarization by >100 msec. Depolarizing stimuli also could trigger persistent firing in mossy cells exposed to carbachol or an m1 receptor agonist. Evoked IPSPs attenuated the ADP response in mossy cells. The functional effect of IPSPs was amplified during ADP responses triggered in the presence of cholinergic receptor agonists but not during slowly decaying simulated ADPs, suggesting that modulation of ADP responses by IPSPs arises from destabilization of the intrinsic currents underlying the ADP. Evoked IPSPs also could halt persistent firing triggered by depolarizing stimuli. These results show that through intrinsic properties modulated by muscarinic receptors, mossy cells can prolong depolarizing responses to excitatory input and extend the time window where multiple synaptic inputs can summate. By actively regulating the intrinsic response to synaptic input, inhibitory synaptic input can dynamically control the integration window that enables detection of coincident inputs and shape the spatial pattern of hilar cell activity.

Comparison of Inhibitory Effects between Enalapril and Losartan on Adrenal Catecholamine Secretion

BACKGROUND: The present study was attempted to compare enalapril, an angiotensin-converting enzyme inhibitor with losartan an angiotensin II (Ang II) receptor blocker in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. METHODS: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. RESULTS: Both enalapril and losartan during perfusion into an adrenal vein for 90 minutes inhibited the CA release evoked by acetylcholine (ACh), 1.1-dimethyl-4-phenyl piperazinium (DMPP, a selective Nn agonist), high K+ (a direct membrane-depolarizer), 3-(m-chloro-phenyl-carbamoyl-oxy-2-butynyl-trimethyl ammonium (McN-A-343, a selective M1 agonist), and Ang II in a time-dependent manner. Also, in the presence of enalapril or losartan, the CA release evoked by veratridine (an activator of voltage-dependent Na+ channels), 6-dimethyl-3-nitro-4-(2-trifluoromethyl-phenyl)-pyridine-5-carboxylate (BAY-K-8644, an L-type Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly reduced. Based on the same concentration of enalapril and losartan, for the CA release evoked by ACh, high K+, DMPP, McN-A-343, Ang II, veratridine, BAY-K-8644, and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: losartan > enalapril. In the simultaneous presence of enalapril and losartan, ACh-evoked CA secretion was more strongly inhibited compared with that of enalapril- or losartan-treated alone. CONCLUSIONS: Collectively, these results demonstrate that both enalapril and losartan inhibit the CA secretion evoked by activation of both cholinergic and Ang II type-1 receptors stimulation in the perfused rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, the inhibitory effect of losartan on the CA secretion seems to be more potent than that of enalapril.

Influence of Fimasartan (a Novel AT1 Receptor Blocker) on Catecholamine Release in the Adrenal Medulla of Spontaneously Hypertensive Rats

The aim of this study was to determine whether fimasartan, a newly developed AT1 receptor blocker, can affect the CA release in the isolated perfused model of the adrenal medulla of spontaneously hypertensive rats (SHRs). Fimasartan (5~50 microM) perfused into an adrenal vein for 90 min produced dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high K+ (56 mM, a direct membrane depolarizer), DMPP (100 microM) and McN-A-343 (100 microM). Fimasartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with fimasartan (15 microM), the CA secretory responses evoked by Bay-K-8644 (10 microM, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 microM, an inhibitor of cytoplasmic Ca(2+)-ATPase), and veratridine (100 microM, an activator of Na+ channels) as well as by angiotensin II (Ang II, 100 nM), were markedly inhibited. In simultaneous presence of fimasartan (15 microM) and L-NAME (30 microM, an inhibitor of NO synthase), the CA secretory responses evoked by ACh, high K+, DMPP, Ang II, Bay-K-8644, and veratridine was not affected in comparison of data obtained from treatment with fimasartan (15 microM) alone. Also there was no difference in NO release between before and after treatment with fimasartan (15 microM). Collectively, these experimental results suggest that fimasartan inhibits the CA secretion evoked by Ang II, and cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by membrane depolarization from the rat adrenal medulla. It seems that this inhibitory effect of fimasartan may be mediated by blocking the influx of both Na+ and Ca2+ through their ion channels into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from the cytoplasmic calcium store, which is relevant to AT1 receptor blockade without NO release.

Influence of PD 123319 (AT2-Receptor Antagonist) on Catecholamine Secretion in the Perfused Rat Adrenal Medulla

BACKGROUND: The aim of this study was to examine whether PD 123319 (an angiotensin II type 2 [AT2] receptor antagonist) can influence the release of catecholamines (CA) from the perfused model of the rat adrenal medulla. METHODS: The adrenal gland was isolated by the modification of Wakade method, and perfused with normal Krebs-bicarbonate solution. The content of CA was measured using the fluorospectrophotometer. RESULTS: During perfusion of PD 123319 (range, 5 to 50 nM) into an adrenal vein for 90 minutes the CA secretory responses evoked by acetylcholine (ACh), high K+, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and McN-A-343 was dose- and time-dependently inhibited. Furthermore, loading with PD 123319 for 90 minutes also markedly inhibited the CA secretory responses evoked by 4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-methyl-phenyl)-pyridine-5-carboxylate (Bay-K-8644), cyclopiazonic acid, veratridine, and angiotensin II (Ang II). PD 123319 did not affect basal CA output. Simultaneous perfusion of PD 123319 and CGP 42112 perfused into an adrenal vein for 90 minutes rather more potently inhibited the CA seretory responses evoked by Ach, high K+, DMPP, Bay-K-8644, veratridine, and Ang II compared to the inhibitory effect by PD123319-treated alone. CONCLUSIONS: Taken together, these results show that PD 123319 inhibits the CA secretion evoked by both cholinergic and Ang II receptor stimulation from the perfused rat adrenal medulla. This inhibitory effect of PD 123319 seems to be exerted by blocking the influx of both Na+ and Ca2+ through their voltage-dependent channels into the rat adrenomedullary chromaffin cells as well as by reducing the Ca2+ release from its cytoplasmic calcium store, which may be relevant to AT2 receptor blockade. Based on these present data, it is thought that PD 123319 has different activity from previously known AT2 antagonist activity in the perfused adrenal medulla, and that AT2 receptors may be involved in the rat adrenomedullary CA secretion.

M-type potassium channels modulate Schaffer collateral-CA1 glutamatergic synaptic transmission.

Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents ­ Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.

The pharmacology of McN-A-343.

The unusual pharmacology of McN-A-343 was first described by Roszowski in 1961. The agonist appeared to be a selective stimulant of muscarinic receptors in sympathetic ganglia, now known to be the muscarinic M1 receptor subtype. However, subsequent research demonstrated that McN-A-343 is a partial agonist with similar affinity at all five muscarinic acetylcholine receptor subtypes and its relative selectivity depends on a higher efficacy at the M1 (and M4) subtypes. Being a partial agonist its action is also dependent on factors, such as receptor density and coupling efficacy between receptor activation and tissue response. Nevertheless, the relatively high efficacy at M1 receptors led to its widespread use as an aid to distinguish responses mediated through M1 receptors from those utilizing M2 or M3 muscarinic receptor subtypes, especially in the CNS. There is also evidence that it has an allosteric action at some receptor subtypes. Recently, it was demonstrated that McN-A-343 can bind to an allosteric site on the M2 receptor as well as to the orthosteric site and has thus been termed a "bitopic agonist". This allosteric site differs from that occupied by allosteric modulators, such as gallamine. Comparison of comparable mutagenic changes in M2 and M4 receptors also suggests that McN-A-343 utilizes different regions of the two receptors for ERK1/2 activation. McN-A-343 has a number of non-muscarinic actions. These include activation of some types of nicotinic acetylcholine receptors, antagonism of serotonin 5-HT3 and 5-HT4 receptor subtypes, inhibition of the uptake mechanism and a local anesthetic action.

[Acetylcholine induces human detrusor muscle cell proliferation: molecular and pharmacological characterization]. / L'acetilcolina induce la proliferazione delle cellule da muscolo detrusore umano: caratterizzazione molecolare e farmacologica.

INTRODUCTION: The purpose of the study is to understand whether the cholinergic stimulation is important, not only in inducing contraction of the detrusor muscle, but also in modulating the proliferation of smooth muscle cells. These results could help to better understand the role of antimuscarinic drugs, which are currently used for the treatment of many urological diseases. PATIENTS AND METHODS: Primary cultures were prepared from biopsies of human detrusor muscle of subjects >65 years. From the cell culture set-up for each patient, mRNA was extracted and both the gene expression and the influence of increasing passages on the expression of muscarinic receptor subtypes were evaluated by semi-quantitative and quantitative PCR (RT-PCR and Q-RT-PCR). The rate of cell proliferation induced by cholinergic drugs was assessed by the evaluation of the [3H]-thymidine incorporation. RESULTS: The gene expression analysis demonstrated that the range of expression of muscarinic subtypes in human detrusor smooth muscle cells (HDSMCs) is M2 > M3 > M1 > M4 >> M5. The exposure to the cholinergic agonist carbachol induced a concentration-dependent increase in cell proliferation rate. The pharmacological characterization indicated that this effect was mainly mediated by the receptor subtypes M3 and M2. DISCUSSION: The cholinergic stimulation led to an increase in HDSMC proliferation, suggesting that this phenomenon might be involved in the pathogenic mechanism through which the cervico-urethral obstruction causes a detrusor hypertrophy, followed by a loss of function. These results could then provide an indication of the use of antimuscarinic drugs in the treatment of lower urinary tract disorders.

Muscarinic receptor subtypes involved in regulation of colonic motility in mice: functional studies using muscarinic receptor-deficient mice.

Although muscarinic M(2) and M(3) receptors are known to be important for regulation of gastric and small intestinal motility, muscarinic receptor subtypes regulating colonic function remain to be investigated. The aim of this study was to characterize muscarinic receptors involved in regulation of colonic contractility. M(2) and/or M(3) receptor knockout (KO) and wild-type mice were used in in vivo (defecation, colonic propulsion) and in vitro (contraction) experiments. Amount of feces was significantly decreased in M(3)R-KO and M(2)/M(3)R-KO mice but not in M(2)R-KO mice. Ranking of colonic propulsion was wild-type=M(2)R-KO>M(3)R-KO>M(2)/M(3)R-KO. In vitro, the amplitude of migrating motor complexes in M(2)R-KO, M(3)R-KO and M(2)/M(3)R-KO mice was significantly lower than that in wild-type mice. Carbachol caused concentration-dependent contraction of the proximal colon and distal colon from wild-type mice. In M(2)R-KO mice, the concentration-contraction curves shifted to the right and downward. In contrast, carbachol caused non-sustained contraction and relaxation in M(3)R-KO mice depending on its concentration. Carbachol did not cause contraction but instead caused relaxation of colonic strips from M(2)/M(3)R-KO mice. 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (McN-A-343) caused a non-sustained contraction of colonic strips from wild-type mice, and this contraction was changed to a sustained contraction by tetrodotoxin, pirenzepine and L-nitroarginine methylester (L-NAME). In the colon of M(2)/M(3)R-KO mice, McN-A-343 caused only relaxation, which was decreased by tetrodotoxin, pirenzepine and L-NAME. In conclusion, M(1), M(2) and M(3) receptors regulate colonic motility of the mouse. M(2) and M(3) receptors mediate cholinergic contraction, but M(1) receptors on inhibitory nitrergic nerves counteract muscarinic contraction.

Investigation of gastrin-releasing peptide as a mediator for 5'-guanidinonaltrindole-induced compulsive scratching in mice.

Gastrin-releasing peptide (GRP) has been implicated in the itch-scratch cycle. We investigated if this gut-brain-skin peptide plays a role in the compulsive, hindleg scratching of the neck of mice by 5'-guanidinonaltrindole (GNTI), the kappa opioid receptor antagonist, and in the antipruritic activity of nalfurafine, the kappa opioid agonist. Previously, we showed that GNTI (0.03-1mg/kg, s.c.) elicits dose-related scratching and that nalfurafine (0.001-0.02mg/kg, s.c.) inhibits this behavior in mice. Utilizing immunohistochemistry, GRP positive nerve fibers were detected in mouse skin and superficial layer of the dorsal horn of the spinal cord as well as GRP positive cells in the dorsal root ganglion. Pretreating mice with either a pseudopeptide GRP receptor antagonist, RC-3095 (10-30mg/kg, s.c. at -15min), or a peptide GRP receptor antagonist, [d-Phe(6)]bombesin(6-13) methyl ester (2-100nmol, i.t. at -10min), did not suppress GNTI-induced scratching. However, pretreating mice with either antagonist inhibited scratching precipitated by the GRP receptor agonist, GRP(18-27) (2nmol, i.t.). Pretreating mice with a muscarinic M(1) receptor agonist, McN-A-343 (1.5-15µg/5µl, i.t. at -10min) antagonized GNTI-induced scratching. Norbinaltorphimine (20mg/kg, i.p. at -18 to -20h), a kappa opioid antagonist, countered the antiscratch activity of nalfurafine. We conclude that (a) the GRP receptor system does not mediate GNTI-induced scratching and (b) the kappa opioid system is involved, at least in part, in the scratch suppressing activity of nalfurafine.