Muscarinic Modulation Research Articles

Abstract P148: Muscarinic Acetylcholine Receptors are Localized to Primary Endothelial Cilia and Involved in Nitric Oxide Signaling

Primary cilia are considered sensory hubs housing a variety of mechanosensory proteins, chemosensory receptors, and ion channels to translate extracellular stimuli into an intracellular biochemical signals such as the synthesis and release of nitric oxide (NO). Malformations in cilia structure or defects in cilia function lead to ciliopathies such as Polycystic Kidney Disease (PKD) and hypertension. Muscarinic receptors 1 and 3 play an essential role in regulating cardiovascular function by mediating both dilation and constriction in the vasculature. However, nothing is known about the relationship between primary cilia and muscarinic receptors. Supported by our exciting discovery of the mAChRs in the cilia, we hypothesize that primary cilia in the vascular system play important roles in regulating blood pressure through NO biosynthesis. Our objective is to unravel the mechanism by which cilia dysfunction contribute to hypertension and to introduce cilia as a novel therapeutic target for PKD. To explore the relationship between mAChRs and primary cilia, the effects of muscarinic modulators on cilia length and function in wildtype, and cilia mutant endothelial cells, Pkd1 – / – (dysfunctional cilia) and Tg737 orpk/orpk (cilia-less) were examined. We show that both AChM1R and AChM3R localize to primary endothelial cilia. AChM1R and AChM3R activation lead to a significant increase in cilia length in endothelial cells treated with cdd0102a, an AChM1R and AChM3R agonist (2.84±0.02 vs. 3.47± 0.04 for wildtype, 2.31± 0.03 vs. 2.53± 0.03 for Pkd1 – / – , and 0.024 ± 0.005 vs. 0.3 ± 0.004 for Tg737 orpk/orpk ) compared to control cells. Treatment with pirenzepine, an AChM1R antagonist, led to a significant decrease of cilia length (2.66± 0.02 vs. 2.48± 0.03 in wildtype, and 2.12± 0.02 vs. 1.93± 0.02 in Pkd1 – / – ) compared to control cells. Treatment with cdd0102a also significantly upregulated the expression of AChM1R, AChM3R and phosphorylated eNOS in wildtype and Pkd1 - / - cells. More importantly, cdd0102a treatment rescued NO response in Pkd1 - / - cells in response to fluid shear stress. Our data clearly suggest that modulating cilia sensory function could have a positive influence on nitric oxide generation and blood pressure regulation.

Muscarinic modulation of the Xenopus laevis tadpole spinal mechanosensory pathway

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in spinal functions like locomotion. In Xenopus laevis tadpoles at two days old, a model vertebrate for motor control research, we investigated the role of mAChRs in the skin mechanosensory pathway. We found that mAChR activation by carbachol did not affect the sensory Rohon-Beard neuron properties. However, carbachol could hyperpolarise sensory interneurons and decrease their voltage responses to outward currents. Carbachol could increase the threshold for the mechanosensory pathway to start swimming, preventing the initiation of swimming at higher concentrations altogether. Recording from the sensory interneurons in carbachol showed that their spiking after skin stimulation was depressed. However, the general muscarinic antagonist atropine did not have a clear effect on the swimming threshold or the modulation of sensory interneuron membrane conductance. Our results suggest the skin mechanosensory pathway may be subject to muscarinic modulation in this simple vertebrate system.

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.

Abstract P184: Muscarinic Receptor Modulators’ Effect on Cilia Structure and Nitric Oxide Release

Primary endothelial cilia are microtubule-based organelles that act as mechano-sensors to help detect, and respond to changes in the extracellular environment. One of these responses includes its ability to detect fluid shear stress and translating that signal into a biochemical process to synthesize nitric oxide, an endogenous vasodilator. Abnormal cilia structure and function has been known to cause a myriad of human disorders, including polycystic kidney disease and hypertension. It is also well documented that muscarinic acetylcholine receptors subtype M1 also plays a key role in producing a vasodilation response in the vasculature. However, nothing is known about the relationship between primary endothelial cilia and the M1 receptor. To further explore the relationship between AChM1R and the mechanosensory function of primary cilia, the effects of muscarinic modulators on cilia length and function in wild-type, and mechano-insensitive cilia mutant endothelial cells, Pkd1 – / – (dysfunctional cilia) and Tg737 orpk/orpk (cilia-less) were examined. We show that AChM1R localizes to primary endothelial cilia. AChM1R activation lead to the significant increase cilia length in endothelial cells treated with CDD102A, an AChM1R agonist (1.21 ± 0.07 vs. 2.33 ± 0.04 for wild-type, 1.02 ± 0.01 vs. 1.44 ± 0.01 for Pkd1 – / – , and 0.024 ± 0.005 vs. 0.3 ± 0.004 for Tg737 orpk/orpk ) compared to non-treated cells. Treating endothelial cells with pirenzepine, an AChM1R antagonist, led to a significant decrease of cilia length (1.34 ± 0.02 vs. 1.15 ± 0.01 in wild-type, and 1.16 ± 0.01 vs. 0.71 ± 0.01 in Pkd1 – / – ) compared to non-treated cells. Treatment with CDD102A also significantly upregulated the expression of AChM1R, and phosphorylated eNOS. Our data clearly suggested that modulating cilia sensory function could have a positive influence on nitric oxide generation and blood pressure regulation.

Cerebrolysin prevents deficits in social behavior, repetitive conduct, and synaptic inhibition in a rat model of autism.

Autism spectrum disorder (ASD) is a syndrome of diverse neuropsychiatric diseases of growing incidence characterized by repetitive conduct and impaired social behavior and communication for which effective pharmacological treatment is still unavailable. While the mechanisms and etiology of ASD are still unknown, a consensus is emerging about the synaptic nature of the syndrome, suggesting a possible avenue for pharmacological treatment with synaptogenic compounds. The peptidic mixture cerebrolysin (CBL) has been successfully used during the last three decades in the treatment of stroke and neurodegenerative disease. Animal experiments indicate that at least one possible mechanism of action of CBL is through neuroprotection and/or synaptogenesis. In the present study, we tested the effect of CBL treatment (daily injection of 2.5 mL/Kg i.p. during 15 days) on a rat model of ASD. This was based on the offspring (43 male and 51 female pups) of a pregnant female rat injected with valproic acid (VPA, 600 mg/Kg) at the embryonic day 12.5, which previous work has shown to display extensive behavioral, as well as synaptic impairment. Comparison between saline vs. CBL-injected VPA animals shows that CBL treatment improves behavioral as well as synaptic impairments, measured by behavioral performance (social interaction, Y-maze, plus-maze), maximal response of inhibitory γ-amino butyric acid type A receptor (GABAA R)-mediated synaptic currents, as well as their kinetic properties and adrenergic and muscarinic modulation. We speculate that CBL might be a viable and effective candidate for pharmacological treatment or co-treatment of ASD patients. © 2017 Wiley Periodicals, Inc.

Mice Lacking M1 and M3 Muscarinic Acetylcholine Receptors Have Impaired Odor Discrimination and Learning.

The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1−/− and the M1/M3−/− knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing.

Pi(4,5)P2 Modulates Hysteresis and Pharmacology of KV7 Channels

KV7 channels are critical components of the plasma membrane in many excitable cells. The main function of KV7 channels is to contribute to the maintenance of the resting potential, thus regulating cellular electrical excitability. In Central Nervous System, the activity of the heteromeric KV7.2/KV7.3 channel gives rise to M-currents. These K+-currents are subject to muscarinic modulation as the phosphoinositide PI(4,5)P2, a signaling lipid dephosphorylated during muscarinic activity, is required by KV7.2/KV7.3 channels to be functional. On the other hand, Corbin-Leftwich and colleagues (JGP, 2016) have recently reported that the deactivation rate of KV7.2/KV7.3 channels depends on the duration of activation. This observation revealed that this heteromeric channel displays a remarkable hysteretic behavior. In addition, it was shown that these channels have, at least, two open modes and that these modes differ in stability, as deactivation from one mode is slower than the other, and in pharmacology, as the anticonvulsant Retigabine preferentially acts on the slower-deactivating mode. Since, (1) the hysteretic behavior of KV7.2/KV7.3 depends of activity and, in turn, (2) activity depends on PI(4,5)P2, an clear next step was to determine whether PI(4,5)P2 can modulate the effect of the anticonvulsant Retigabine. In addressing this question, here, it is presented that each of the open modes of KV7.2/KV7.3 channels displays distinct apparent affinities for PI(4,5)P2. Also, it was found that decreasing the PI(4,5)P2 concentration either pharmacologically or enzymatically, reduces the effect of Retigabine on channel activity when applied in low doses. Furthermore, here it is shown that the hysteretic behavior of the KV7.2/KV7.3 channels does not emerge from being heteromeric, as the individual components display hysteretic behavior as well. The work presented here provides evidence the leads to propose that muscarinic modulation of M-current is likely dependent on the hysteretic behavior of KV7.2/KV7.3 channels.

Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release

Muscarinic receptors represent a promising therapeutic target for schizophrenia, but the mechanisms underlying the antipsychotic efficacy of muscarinic modulators are not well understood. Here, we report that activation of M4 receptors on striatal spiny projection neurons results in a novel form of dopaminergic regulation resulting in a sustained depression of striatal dopamine release that is observed more than 30min after removal of the muscarinic receptor agonist. Furthermore, both the M4-mediated sustained inhibition of dopamine release and the antipsychotic-like efficacy of M4 activators were found to require intact signaling through CB2 cannabinoid receptors. These findings highlight a novel mechanism by which striatal cholinergic and cannabinoid signaling leads to sustained reductions in dopaminergic transmission and concurrent behavioral effects predictive of antipsychotic efficacy.

Data regarding M1 muscarinic receptor-mediated modulation of hepatic catalase activity in response to oxidative stress.

We recently demonstrated the role of M1 muscarinic receptors (M1R) in modulating oxidative stress in liver and hepatocytes (Urrunaga et al., 2015) [1]. Here we provide data regarding the effect of a novel M1R agonist, VU0357017 (Lebois et al., 2010) [2], on H2O2-mediated hepatocyte injury, the effect of an M1R antagonist VU0255035 (Sheffler et al., 2009) [3] on catalase and super oxide dismutase (SOD) activities in H2O2–treated hepatocytes in vitro, and finally, the effect of M1R ablation on hepatic catalase activity in acetaminophen (APAP)-treated mice.

Differential Muscarinic Modulation in the Olfactory Bulb.

State-dependent cholinergic modulation of brain circuits is critical for several high-level cognitive functions, including attention and memory. Here, we provide new evidence that cholinergic modulation differentially regulates two parallel circuits that process chemosensory information, the accessory and main olfactory bulb (AOB and MOB, respectively). These circuits consist of remarkably similar synaptic arrangement and neuronal types, yet cholinergic regulation produced strikingly opposing effects in output and intrinsic neurons. Despite these differences, the chemogenetic reduction of cholinergic activity in freely behaving animals disrupted odor discrimination of simple odors, and the investigation of social odors associated with behaviors signaled by the Vomeronasal system.

Distribution and effects of the muscarinic receptor subtypes in the primary visual cortex

Muscarinic cholinergic receptors modulate the activity and plasticity of the visual cortex. Muscarinic receptors are divided into five subtypes that are not homogeneously distributed throughout the cortical layers and cells types. This distribution results in complex action of the muscarinic receptors in the integration of visual stimuli. Selective activation of the different subtypes can either strengthen or weaken cortical connectivity (e.g., thalamocortical vs. corticocortical), i.e., it can influence the processing of certain stimuli over others. Moreover, muscarinic receptors differentially modulate some functional properties of neurons during experience-dependent activity and cognitive processes and they contribute to the fine-tuning of visual processing. These functions are involved in the mechanisms of attention, maturation and learning in the visual cortex. This minireview describes the anatomo-functional aspects of muscarinic modulation of the primary visual cortex’s (V1) microcircuitry.

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons.

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M-currents and voltage-dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M-current inhibition. Here, we report the presence of a riluzole-activated current (IRIL ) that flows through the TREK-2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine-M (Oxo-M) inhibited the IRIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single-channel TREK-2 currents. IRIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2-aminoethoxydiphenylborane. Nevertheless, the IRIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET-18-OCH3. Finally, the phosphatidylinositol-3-kinase/phosphatidylinositol-4-kinase inhibitor wortmannin strongly attenuated the IRIL , whereas blocking phosphatidylinositol 4,5-bisphosphate (PIP2 ) depletion consistently prevented IRIL inhibition by Oxo-M. These results demonstrate that TREK-2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP2 levels via a PLC-dependent pathway. The similarities between the signaling pathways regulating the IRIL and the M-current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability.

Enhancement of long-term potentiation via muscarinic modulation in the hippocampus of HCNP precursor transgenic mice

Hippocampal cholinergic neurostimulating peptide (HCNP) regulates acetylcholine synthesis in the septal hippocampus through the quantitative increase of choline acetyltransferase levels in the septal nucleus both in vitro and in vivo. Additionally, HCNP-precursor protein transgenic (HCNP-pp Tg) mice display depressive behavior. To examine the physiological function of HCNP and/or HCNP-pp on hippocampal neural activity, we investigated whether overexpression of HCNP-pp strengthened the efficiency of neural activity in the hippocampus. Long-term potentiation (LTP) of excitatory synaptic transmission was induced by a tetanic stimulation of the Schaffer collateral-commissural fibers (SCs) in mouse hippocampal slices. LTP in HCNP-pp Tg mice was significantly enhanced when compared with wild-type littermate (WT) mice. This facilitation of LTP in HCNP-pp Tg mice was blocked by atropine or pirenzepine, but not by mecamylamine. In contrast, LTP in WT mice was not affected by atropine, but enhanced by carbachol. However, neither difference in the input–output relationship of field excitatory postsynaptic potentials nor in the facilitation ratio in paired-pulse stimulation of the SCs was observed between HCNP-pp Tg and WT mice, indicating that presynaptic glutamate release in HCNP-pp Tg mice is similar to that of WT mice. These results suggest that muscarinic (M1) modulation of glutamatergic postsynaptic function may be involved in strengthening LTP in HCNP-pp Tg mice.

Developmental Nicotine Exposure Alters The Respiratory Frequency Response to Muscarine

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters development of the brainstem circuits that control breathing. Previous work has shown that DNE desensitizes nicotinic acetylcholine receptors (nAChRs) and alters the respiratory motor response to exogenous nicotine in neonatal rats. In the brainstem‐spinal cord (BSSC) split‐bath preparation, nicotine applied to the medullary compartment increases the frequency of respiratory related bursting recorded from the fourth cervical ventral root (C4VR), but this effect is markedly blunted by DNE. Because ACh is the endogenous ligand for both nAChRs and muscarinic acetylcholine receptors (mAChRs), and mAChRs are expressed on PreBotzinger neurons, we hypothesized that DNE also alters muscarinic modulation of C4VR frequency in this system. In neonatal rats, postnatal day 0‐6, 1 mM muscarine, a mAChR agonist, applied to the medullary compartment in the BSSC split‐bath preparation increased burst frequency recorded from C4VR in 4/4 control animals and, in some cases, the pattern of bursting became irregular. Muscarine applied to the medulla decreased burst frequency in 4/4 DNE animals. These preliminary data show that chronic stimulation of nAChRs also alters the development of muscarinic synaptic transmission by yet to be determined mechanisms.

KV7 Channels Regulate Firing during Synaptic Integration in GABAergic Striatal Neurons.

Striatal projection neurons (SPNs) process motor and cognitive information. Their activity is affected by Parkinson's disease, in which dopamine concentration is decreased and acetylcholine concentration is increased. Acetylcholine activates muscarinic receptors in SPNs. Its main source is the cholinergic interneuron that responds with a briefer latency than SPNs during a cortical command. Therefore, an important question is whether muscarinic G-protein coupled receptors and their signaling cascades are fast enough to intervene during synaptic responses to regulate synaptic integration and firing. One of the most known voltage dependent channels regulated by muscarinic receptors is the KV7/KCNQ channel. It is not known whether these channels regulate the integration of suprathreshold corticostriatal responses. Here, we study the impact of cholinergic muscarinic modulation on the synaptic response of SPNs by regulating KV7 channels. We found that KV7 channels regulate corticostriatal synaptic integration and that this modulation occurs in the dendritic/spines compartment. In contrast, it is negligible in the somatic compartment. This modulation occurs on sub- and suprathreshold responses and lasts during the whole duration of the responses, hundreds of milliseconds, greatly altering SPNs firing properties. This modulation affected the behavior of the striatal microcircuit.

Muscarinic presynaptic modulation in GABAergic pallidal synapses of the rat.

The external globus pallidus (GPe) is central for basal ganglia processing. It expresses muscarinic cholinergic receptors and receives cholinergic afferents from the pedunculopontine nuclei (PPN) and other regions. The role of these receptors and afferents is unknown. Muscarinic M1-type receptors are expressed by synapses from striatal projection neurons (SPNs). Because axons from SPNs project to the GPe, one hypothesis is that striatopallidal GABAergic terminals may be modulated by M1 receptors. Alternatively, some M1 receptors may be postsynaptic in some pallidal neurons. Evidence of muscarinic modulation in any of these elements would suggest that cholinergic afferents from the PPN, or other sources, could modulate the function of the GPe. In this study, we show this evidence using striatopallidal slice preparations: after field stimulation in the striatum, the cholinergic muscarinic receptor agonist muscarine significantly reduced the amplitude of inhibitory postsynaptic currents (IPSCs) from synapses that exhibited short-term synaptic facilitation. This inhibition was associated with significant increases in paired-pulse facilitation, and quantal content was proportional to IPSC amplitude. These actions were blocked by atropine, pirenzepine, and mamba toxin-7, suggesting that receptors involved were M1. In addition, we found that some pallidal neurons have functional postsynaptic M1 receptors. Moreover, some evoked IPSCs exhibited short-term depression and a different kind of modulation: they were indirectly modulated by muscarine via the activation of presynaptic cannabinoid CB1 receptors. Thus pallidal synapses presenting distinct forms of short-term plasticity were modulated differently.

Differential Muscarinic Modulation of Synaptic Transmission in Dorsal and Ventral Regions of the Rat Nucleus Accumbens Core

The nucleus accumbens (NAc) core is critical in the control of motivated behaviors. The muscarinic acetylcholine receptors (mAChRs) modulating the excitatory inputs into the NAc core have been reported to impact such behaviors. Recent studies suggest that ventral and dorsal regions of the NAc core seem to be innervated by distinct populations of glutamatergic projection neurons. To further examine mAChRs modulation of these glutamatergic inputs to the NAc core, we employed intracellular recordings in rat NAc coronal slice preparation to characterize: 1) the effects of muscarine, an mAChRs agonist, on membrane properties of the NAc core neurons; 2) depolarizing synaptic potentials (DPSP) elicited by ventral and dorsal focal electrical stimuli; and 3) paired-pulse response with paired-pulse stimulation. Here we report that the paired-pulse ratio (PPR) elicited by dorsal stimuli was greater than that elicited by ventral stimuli. Bath application of muscarine (1-30 microM) decreased both ventral and dorsal DPSP in a concentration-dependent manner, with no effect on electrophysiological properties of NAc core neurons. Muscarine at 30 microM also elicited larger depression of dorsal DPSP than ventral DPSP. Moreover, muscarine increased the PPR of both dorsal and ventral DPSP. These data indicate that the glutamatergic afferent fibers traversing the dorsal and ventral NAc are separate, and that differential decrease of distinct afferent excitatory neurotransmission onto NAc core neurons may be mediated by presynaptic mechanisms.

Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions

The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca2+ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M2-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M2-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca2+ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies.

Muscarinic acetylcholine receptor modulators derived from natural toxins and diverse interaction modes

Muscarinic acetylcholine receptors (mAChRs) play crucial roles in various physiological functions and pathophysiological processes. Acetylcholine (ACh), as a classical ligand and one of the pivotal neurotransmitters, serves as a prototype for the elucidation of molecular interaction and the development of mimicked and antagonized agents. With the advances in medicinal chemistry and structural biology, more and more mAChRs modulators derived from natural toxins have been identified. Based on the chemical structures and the receptor-ligand interaction modes, these mAChRs modulators can be divided into orthosteric modulators, allosteric modulators and other modulators. Moreover, allosteric modulators can be further divided into three groups: alcuronium-like modulators, staurosporine-like modulators, and phlegmarine-like modulators. In this review, we focus on various mAChRs modulators derived from natural toxins on the basis of the receptor-ligand interaction modes. The understanding of the affinity, the intrinsic efficacy, and the selectivity of mAChRs modulators may lead to the discovery of new drug leads for the treatment of diseases related to mAChRs.

Somatodendritic targeting of M5 muscarinic receptor in the rat ventral tegmental area: implications for mesolimbic dopamine transmission.

Muscarinic modulation of mesolimbic dopaminergic neurons in the ventral tegmental area (VTA) plays an important role in reward, potentially mediated through the M5 muscarinic acetylcholine receptor (M5R). However, the key sites for M5R-mediated control of dopamine neurons within this region are still unknown. To address this question we examined the electron microscopic immunocytochemical localization of antipeptide antisera against M5R and the plasmalemmal dopamine transporter (DAT) in single sections through the rat VTA. M5R was located mainly to VTA somatodendritic profiles (71%; n = 627), at least one-third (33.2%; n = 208) of which also contained DAT. The M5R immunoreactivity was distributed along cytoplasmic tubulovesicular endomembrane systems in somata and large dendrites, but was more often located at plasmalemmal sites in small dendrites, the majority of which did not express DAT. The M5R-immunoreactive dendrites received a balanced input from unlabeled terminals forming either asymmetric or symmetric synapses. Compared with dendrites, M5R was less often seen in axon terminals, comprising only 10.8% (n = 102) of the total M5R-labeled profiles. These terminals were usually presynaptic to unlabeled dendrites, suggesting that M5R activation can indirectly modulate non-DAT-containing dendrites through presynaptic mechanisms. Our results provide the first ultrastructural evidence that in the VTA, M5R has a subcellular location conducive to major involvement in postsynaptic signaling in many dendrites, only some of which express DAT. These findings suggest that cognitive and rewarding effects ascribed to muscarinic activation in the VTA can primarily be credited to M5R activation at postsynaptic plasma membranes distinct from dopamine transport.