M1 M5 Research Articles

A novel clinical syndrome revealing a deficiency of the muscarinic M3 receptor

No clinical disorders have been caused by dysfunction of any of the 5 subtypes (M1-M5) of muscarinic receptors. We present a patient with a novel clinical syndrome that we suggest results from a deficiency of the muscarinic M3 receptor. We conducted a comprehensive workup of autonomic function. The patient's disorder was compared to the phenotypic features of male M3 knockout mice. M3 protein quantity was assessed by Western blot and radioligand binding in peripheral blood lymphocytes. Tests for autoantibodies and genetic abnormalities were performed. The disease pattern was characterized by disturbances in micturition, pupil constriction, body weight, and sudomotor function, with normal accommodation, gastrointestinal motility, salivation, and lacrimation, similar to features of male M3 knockout mice. M3 protein quantity was reduced. Genetic tests were unrevealing, but unspecific antinuclear antibodies were present. The presented clinical syndrome suggests a deficiency of the muscarinic M3 receptor. These results and future evaluation of patients with autonomic deficits may provide insights into the site and functional role of the muscarinic M3 receptor in humans.

Antipsychotic-induced catalepsy is attenuated in mice lacking the M4 muscarinic acetylcholine receptor

A delicate balance exists between the central dopaminergic and cholinergic neurotransmitter systems with respect to motor function. An imbalance can result in motor dysfunction as observed in Parkinson's disease patients and in patients treated with antipsychotic compounds. Cholinergic receptor antagonists can alleviate extrapyramidal symptoms in Parkinson's disease and motor side effects induced by antipsychotics. The effects of anticholinergics are mediated by muscarinic receptors of which five subtypes (M1–M5) exist. Muscarinic M4 receptors are found at high concentrations in motor parts of the striatum, suggesting a role for muscarinic M4 receptors in the motor side effects of antipsychotics, and in the alleviation of these side effects by anticholinergics. Here we investigated the potential role of the muscarinic M4 receptor in catalepsy induced by antipsychotics (haloperidol and risperidone) as well as the anti-cataleptic effects of the non-selective anticholinergic drug scopolamine in fully backcrossed muscarinic M4 receptor knockout mice. The drug-induced catalepsy was strongly attenuated, but not abolished, in M4 knockout mice as compared to wild-type controls. Scopolamine further attenuated the cataleptic response in M4 knockout mice, suggesting that non-M4 muscarinic receptors also participate in the anti-cataleptic effects. In conclusion, these data indicate an important role for M4 receptors in antipsychotic-induced motor side effects and suggest that M4 receptors could be a target for future pharmacological treatment of antipsychotic-induced as well as idiopathic parkinsonism.

Photorefractive effect in triphenylamine-based monolithic molecular glasses with low Tg

A novel series of triphenylamine-based photorefractive molecules M1–M5 with the different push-pull structures were designed and prepared. These molecules can form stable glasses with low glass transition temperature and their optical absorptions as well as photoconductivities at 633 nm increase systematically with increasing acceptor strength and conjugation length. As an unambiguous evidence, the two-beam-coupling experiment was performed to prove the photorefractive effect in each compound with or without an additional sensitizer. Obvious two-beam-coupling effects were obtained for the molecules with good film-forming ability and long-term stability, and among them single-component M1 which has a highly asymmetric structure using both a nitrobenzene and a cyano group as the acceptor showed the best PR performance with a gain coefficient of 52 cm −1 at 0 V μm −1 and 165 cm −1 at 40 V μm −1. Fast response time of 0.025–0.23 s were also obtained at low electric field for these glasses. The correlation of the molecular structure with the PR property was discussed, and a possible explanation for the unique energy transfer observed in M1 and M2 under zero field was proposed.

The 2002 M5 Au Sable Forks, NY, earthquake sequence: Source scaling relationships and energy budget

We find invariant high stress drops and radiated energies for a sequence of intraplate earthquakes. We estimate source parameters of the Au Sable Forks, NY, earthquake (M5, 2002) and aftershocks. This intraplate earthquake was the largest to occur in Eastern North America since 1988 and the largest to be recorded by regional broadband networks. We use the empirical Green's function (EGF) method and define a set of qualitative and quantitative rules for the selection of EGF earthquake pairs and for the quality verification of the obtained EGF spectral ratio. We use a multitaper code that performs the complex spectral division with minimum frequency leakage and allows transformation back to the time domain to check the validity of the EGF event. We estimate source parameters for 22 earthquakes (M1–M5) in the sequence. The median stress drop of 104 MPa (using Madariaga source model, and 19 MPa for a Brune model) is significantly larger than estimates for interplate earthquakes. The lower crustal strain rates, and longer fault healing times in intraplate environments, may be responsible for this high average value. We find constant stress drop between M2 and M5, up to the bandwidth resolution limit (80 Hz) of the study, and no evidence of stress drop breakdown for M2 to M1 earthquakes. We find consistently high radiated seismic energy and apparent stress, and a median radiated energy to seismic moment ratio of 9 × 10−5. This is significantly larger than estimates for interplate earthquakes (∼2 × 10−5) and consistent with higher stress drops and stronger faults.

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C-terminal regions of second intracellular loops. This binding relies on Ca(2+) activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca(2+) recruits CaMKIIalpha to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity-dependent manner. Our data identify a new model of protein-protein interactions. In a Ca(2+)-sensitive manner, CaMKIIalpha regulates M4R efficacy and controls the acetylcholine-dopamine balance in the basal ganglia and also the dynamics of movement.

Prediction of skin sensitization potential using D-optimal design and GA-kNN classification methods

Modelling of skin sensitization data of 255 diverse compounds and 450 calculated descriptors was performed to develop global predictive classification models that are applicable to whole chemical space. With this aim, we employed two automated procedures, (a) D-optimal design to select optimal members of the training and test sets and (b) k-Nearest Neighbour classification (kNN) method along with Genetic Algorithms (GA-kNN Classification) to select significant and independent descriptors in order to build the models. This methodology helped us to derive multiple models, M1–M5, that are stable and robust. The best among them, model M1 (CCR train = 84.3%, CCR test = 87.2% and CCR ext = 80.4%), is based on six neighbours and nine descriptors and further suggests that: (a) it is stable and robust and performs better than the reported models in literature, and (b) the combination of D-optimal design and GA-kNN classification approach is a very promising approach. Consensus prediction based on the models M1–M5 improved the CCR of training, test and external validation datasets by 3.8%, 4.45% and 3.85%, respectively, over M1. From the analysis of the physical meaning of the selected descriptors, it is inferred that the skin sensitization potential of small organic compounds can be accurately predicted using calculated descriptors that code for the following fundamental properties: (i) lipophilicity, (ii) atomic polarizability, (iii) shape, (iii) electrostatic interactions, and (iv) chemical reactivity.

Analysis Techniques for Congruence of the Patellofemoral Joint

Quantifying joint congruence may help to understand the relationship between joint function and health. In previous studies, a congruence index (CI) has been used to define subject-specific joint congruence. However, the sensitivity of the CI algorithm to surface representation was unknown. The purpose of this study was to assess the effects of applying five modifications (M1-M5) to the CI algorithm to determine whether the magnitude and variability of the patellofemoral CI is dependent on the surface representation used. The five modifications focused on calculating the CI based on the principal curvature (M1) at the centroid of the contact region, (M2) using an root mean square value for the contact region, (M3) using a mean value for the contact region, (M4) using all digitized points of the patellar surface, and (M5) using all digitized points in contact. The CI found using the contact area (M1, M2, M3, and M5) provides a local measure for congruence, which was shown to increase (decreasing CI) with increasing joint angle. In ten healthy subjects measured with magnetic resonance (MR) images, the patellofemoral joint became significantly more congruent as the knee angle increased from 15 deg to 45 deg using method M5. The magnitude and variability of the patellofemoral CI was dependent on the surface representation used, suggesting that standardization of the surface representation is important to provide a consistent measure. Specifically, M5 provides a local measure of joint congruence, which can account for joint position and orientation. M5 balances the ability to detect differences in congruence between knee angles without introducing high variability.

Repeated effects of asenapine on adrenergic and cholinergic muscarinic receptors

Adrenergic (alpha1 and alpha2) and cholinergic muscarinic (M1-M5) receptor binding in rat forebrain was quantified after 4 wk of twice-daily subcutaneous administration of asenapine or vehicle. Asenapine (0.03, 0.1, and 0.3 mg/kg) produced increases in [3H]prazosin binding to alpha1-adrenergic receptors in the medial prefrontal cortex (mPFC: 30%, 39%, 57%) and dorsolateral frontal cortex (DFC: 27%, 37%, 53%) and increased [3H]RX821002 binding to alpha2-adrenergic receptors in mPFC (36%, 43%, 50%) and DFC (41%, 44%, 52%). Despite showing no appreciable affinity for muscarinic receptors, asenapine produced regionally selective increases in binding of [3H]QNB to M1-M5 receptors in mPFC (26%, 31%, 43%), DFC (27%, 34%, 41%), and hippocampal CA1 (40%, 44%, 42%) and CA3 (25%, 52%, 48%) regions. These regionally selective effects of asenapine on adrenergic and cholinergic muscarinic receptor subtypes may contribute to its beneficial clinical effects in the treatment of schizophrenia and bipolar disorder.

Paraoxon induced cell death and alterations of M1–M5 lymphocytic cholinergic muscarinic subtypes in human leukemic T-lymphocytes

Paraoxon induced cell death and alterations of M1–M5 lymphocytic cholinergic muscarinic subtypes in human leukemic T-lymphocytes

The Role of Kalirin9 in p75/Nogo Receptor-mediated RhoA Activation in Cerebellar Granule Neurons

p75 and the Nogo receptor form a signaling unit for myelin inhibitory molecules, with p75 being responsible for RhoA activation. Because p75 lacks the GDP/GTP exchange factor domain, it has remained unclear how p75 activates RhoA. Here, we report that Kalirin9, a dual RhoGEF, binds p75 directly and regulates p75-Nogo receptor-dependent RhoA activation and neurite inhibition in response to myelin-associated glycoprotein. The region of p75 that Kalirin9 binds includes its mastoparan-like fifth helix, which was shown to recruit RhoGDI-RhoA. As predicted from the presence of a shared binding site, we found that Kalirin9 competes with RhoGDI for p75 binding in a dose-dependent manner in vitro. In line with these data, myelin-associated glycoprotein addition to cerebellar granule neurons resulted in a reduction in the association of Kalirin9 with p75, and a simultaneous increase in the binding of RhoGDI to p75. These results reveal a mechanism by which the fifth helix of p75 regulates RhoA activation.

Involvement of the Lymphocytic Muscarinic Acetylcholine Receptor in Methylmercury-Induced c-Fos Expression and Apoptosis in Human Leukemic T Cells

Methylmercury (MeHg) is an environmental toxicant that is known to induce lymphocyte apoptosis; however, little is known about the molecular mechanism involved. Data showed that MOLT-3 cells were more sensitive to MeHg-induced cytotoxic effects than Jurkat clone E6-1 cells, suggesting that the lymphocytic muscarinic cholinergic system may be involved since the expressions of five subtypes (M1–M5) of muscarinic acetylcholine receptor (mAChR) in MOLT-3 cells are higher than in Jurkat cells. The role of mAChR-linked pathways in MeHg-induced apoptosis in human leukemic T cells was examined in this study. Treatment of the MOLT-3 cells with 1 μM MeHg produced induction of c-Fos expression, apoptotic cell death, and downregulation of mAChR. MeHg-induced c-Fos expression was significantly reduced by pretreatment with atropine (a nonselective mAChR antagonist), or 4-DAMP (a selective M1/M3 mAChR antagonist), whereas pirenzipine (a selective M1 mAChR antagonist) or himbazine (a selective M2/M4 mAChR antagonist) did not reduce this induction, suggesting that MeHg-induced c-Fos expression through the activation of the mAChR, at least M3 subtype, is involved. Pretreatment with 4-DAMP or SB 203580 (a specific p38 inhibitor) resulted in decreases in the level of phosphorylated p38, c-Fos expression, and apoptotic cell death induced by MeHg. Taken together, these data suggest that the mAChR-p38-dependent pathway participates in the increase of c-Fos expression, which is involved in MeHg-induced lymphocyte apoptosis. In addition, a noncytotoxic concentration of MeHg (0.1 μM) inhibited PHA/PMA-stimulated interleukin (IL)-2 production, and this inhibition was reversed by pretreatment with atropine or 4-DAMP. Overall, this study provides initial evidence that MeHg may alter the immune system by targeting the lymphocytic mAChR.

Identification of human cytochrome P450 enzymes involved in the metabolism of IN-1130, a novel activin receptor-like kinase-5 (ALK5) inhibitor

1. The in vitro metabolism of 3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130), a selective activin receptor-like kinase-5 (ALK5) inhibitor and a candidate drug for fibrotic disease, was studied.2. The cytochrome P450s (CYPs) responsible for metabolism of IN-1130 in liver microsomes of rat, mouse, dog, monkey and human, and in human CYP supersomes™, were identified using specific CYP inhibitors. The order of disappearance of IN-1130 in various liver microsomal systems studied was as follows: monkey, mouse, rat, human, and dog.3. Five distinct metabolites (M1–M5) were identified in all the above microsomes and their production was substantially inhibited by CYP inhibitors such as SKF-525A and ketoconazole. Among nine human CYP supersomes™ examined, CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19 were involved in the metabolism of IN-1130, and the production of metabolites were significantly inhibited by specific CYP inhibitors. IN-1130 disappeared fastest in CYP2C8 supersomes. CYP3A4 produced four metabolites of IN-1130 (M1–M4), whereas supersomes expressing human FMO cDNAs, such as FMO1, FMO3, and FMO5, produced no metabolites.4. Hence, it is concluded that metabolism of IN-1130 is mediated by CYP3A4, CYP2C8, CYP2D6*1, and CYP2C19.

Blockade of M1 muscarinic acetylcholine receptors modulates the methamphetamine-induced psychomotor stimulant effect

Muscarinic acetylcholine receptors (M1–M5) regulate many key functions of the CNS and peripheral nervous system. In the present study, the role of M1 muscarinic receptors (M1R) in the psychomotor stimulant and sensitizing properties of methamphetamine (METH) is investigated using molecular, neurochemical, and behavioral approaches. Acute and repeated treatment with METH increased M1R mRNA expression in the frontal cortex and the CA2 region of the hippocampus. Repeated treatment with METH also increased M1R mRNA expression in the dentate gyrus. Dicyclomine, an M1R antagonist, did not affect the psychomotor effect of METH, but it attenuated METH-induced increases in the dopamine (DA) efflux in the nucleus accumbens (NAc). Dicyclomine enhanced the psychomotor effect of METH after repeated treatment with METH and 8.0 mg/kg of dicyclomine, and also augmented the increase in the NAc DA overflow evoked by repeated METH treatment. These results suggest that M1R plays a role in the METH-induced psychomotor stimulant effect by changing the release of DA in the NAc of mice.

Neurochemistry of the afferents to the rat cochlear root nucleus: Possible synaptic modulation of the acoustic startle

Afferents to the primary startle circuit are essential for the elicitation and modulation of the acoustic startle reflex (ASR). In the rat, cochlear root neurons (CRNs) comprise the first component of the acoustic startle circuit and play a crucial role in mediating the ASR. Nevertheless, the neurochemical pattern of their afferents remains unclear. To determine the distribution of excitatory and inhibitory inputs, we used confocal microscopy to analyze the immunostaining for vesicular glutamate and GABA transporter proteins (VGLUT1 and VGAT) on retrogradely labeled CRNs. We also used reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry to detect and localize specific neurotransmitter receptor subunits in the cochlear root. Our results show differential distributions of VGLUT1- and VGAT-immunoreactive endings around cell bodies and dendrites. The RT-PCR data showed a positive band for several ionotropic glutamate receptor subunits, M1–M5 muscarinic receptor subtypes, the glycine receptor α1 subunit (GlyRα1), GABAA, GABAB, and subunits of α2 and β-noradrenergic receptors. By immunohistochemistry, we confirmed that CRN cell bodies exhibit positive immunoreaction for the glutamate receptor (GluR) 3 and NR1 GluR subunits. Cell bodies and dendrites were also positive for M2 and M4, and GlyRα1. Other subunits, such as GluR1 and GluR4 of the AMPA GluRs, were observed in glial cells neighboring unlabeled CRN cell bodies. We further confirmed the existence of noradrenergic afferents onto CRNs from the locus coeruleus by combining tyrosine hydroxylase immunohistochemistry and tract-tracing experiments. Our results provide valuable information toward understanding how CRNs might integrate excitatory and inhibitory inputs, and hence how they could elicit and modulate the ASR.

Pharmacokinetics of Tramadol and its Metabolites M1, M2 and M5 in Horses Following Intravenous, Immediate Release (Fasted/Fed) and Sustained Release Single Dose Administration

Tramadol (T) is a centrally acting analgesic structurally related to codeine and morphine. This drug displays a weak affinity for the μ and δ-opioid receptors, and weaker affinity for the κ-subtype; it also interferes with the neuronal release and reuptake of serotonin and noradrenaline in the descending inhibitory pathways. The metabolism of this drug has been investigated in different animals (rats, mice, Syrian hamsters, guinea pigs, rabbits, and dogs) and humans; similar metabolites are produced but in different amounts. The major metabolic pathways involved in phase I metabolite production (M1–M5) are O-demethylation, N-demethylation, and N,N-demethylation. The aim of the current study is to evaluate the pharmacokinetic profile of T in the horse, and its M1, M2, and M5 metabolites after single-dose administration (5 mg/kg body weight [BW]) by intravenous, sustained-release tablets and immediate-release capsules. We also will investigate the potential effects of fasting and feeding on bioavailability of immediate-release capsules. The study design was divided into four randomized phases. Twenty-four gelding Italian trotter race horses were divided into four groups (6 animals each) and administered T intravenously, with T immediate-release capsules in a fasting status, T immediate-release capsules in a feeding status, and T sustained-release in fasting status. Blood samples were collected at different times and analyzed by high-pressure liquid chromatography (HPLC) with fluorimetric detection. The limit of quantification was 5 ng/ml for T, M1, and M2, and 10 ng/ml for M5. A one-compartment model best fit the plasma concentrations of T and M2 after all treatments. Unfortunately, for M1 and M5, it was not always possible to fit plasma curves because of very low and variable concentrations. M2 was the main metabolite produced in the four different treatments and its concentration was higher than the concentration of T after sustained-release administration. Conversely, M1, the main metabolite in humans, and M5 seemed to be only marginally produced in the horse. When T was administered in both fasted and fed states, variations in some pharmacokinetic parameters were not considered clinically significant. We concluded that T could be administered in either a fasted or a fed condition.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity of α2–α7, β2, β4 nAChR and M1–M5 mAChR subtypes during neuronal differentiation. Nicotinic α3 and β2 mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression of α2, α4–α7, β4 nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+ flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+]i) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+]i response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation.

Antiaflatoxigenic property of food grade antioxidants under different conditions of water activity in peanut grains

Analytical grade (AG) and industrial grade (IG) of three-food grade antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl paraben (PP) were analyzed to prove their fungitoxic effect on Aspergillus section Flavi strains. The effect of interactions among 10 antioxidant treatments at water activity levels (0.982, 0.955, 0.937 a W) for 11 and 35 days of incubation and at 25 °C in peanut grains on mycelial growth (CFU g −1) and aflatoxin B 1 (AFB 1) accumulation were evaluated. Both antioxidant grade treatments had a significant effect ( P < 0.001) on fungal count. All antioxidant treatments showed the highest effectiveness on control of growth of peanut aflatoxigenic strains at 0.937 a W and at 11 days of incubation. Overall, AG and IG binary mixtures M3 (20 + 10 mM), M4 (20 + 20 mM) and ternary mixtures M5 (10 + 10 +10 mM), M6 (10 + 20 + 10 mM), M7 (20 + 10 + 10 mM) and M8 (20 + 20 + 10 mM) were the treatments most effective at inhibiting growth of Aspergillus section Flavi strains. Industrial grade BHA 10 and 20 mM, binary mixtures M1 (10 + 10 mM), M2 (10 + 20 mM), M3 (20 + 10 mM), M4 (20 + 20 mM) and ternary mixtures M5 (10 + 10 + 10 mM), M6 (10 + 20 + 10 mM), M7 (20 + 10 + 10 mM) and M8 (20 + 20 + 10 mM) completely inhibited AFB 1 production. The studied results suggest that IG antioxidant mixtures have potential for controlling growth of these mycotoxigenic species and prevent aflatoxin accumulation at the peanut storage system.

Metabolic studies of mesterolone in horses

Mesterolone (1α-methyl-5α-androstan-17β-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid–liquid extraction and analysed by gas chromatography–mass spectrometry (GC–MS) after acylation or silylation. Five metabolites (M1–M5) were detected. They were 1α-methyl-5α-androstan-3α-ol-17-one (M1), 1α-methyl-5α-androstan-3β-ol-17-one (M2), 1α-methyl-5α-androstane-3α,17β-diol (M3), 1α-methyl-5α-androstane-3β,17β-diol (M4), and 1α-methyl-5α-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1. For the in vivo metabolic studies, Proviron ® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC–MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1α-methyl-5α-androstane-3,17α-diol (M6 and M7) and 1α-methyl-5α-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1α-methyl-5α-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses.

Muscarinic acetylcholine receptor subtype expression in avian vestibular hair cells, nerve terminals and ganglion cells

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the CNS and peripheral nervous system and play an important role in modulating the cell activity and function. We have shown that the cholinergic agonist carbachol reduces the pigeon’s inwardly rectifying potassium channel (pKir2.1) ionic currents in native vestibular hair cells. We have cloned and sequenced pigeon mAChR subtypes M2–M5 and we have studied the expression of all five mAChR subtypes (M1–M5) in the pigeon vestibular end organs (semicircular canal ampullary cristae and utricular maculae), vestibular nerve fibers and the vestibular (Scarpa’s) ganglion using tissue immunohistochemistry (IH), dissociated single cell immunocytochemistry (IC) and Western blotting (WB). We found that vestibular hair cells, nerve fibers and ganglion cells each expressed all five (M1–M5) mAChR subtypes. Two of the three odd-numbered mAChRs (M1, M5) were present on the hair cell cilia, supporting cells and nerve terminals. And all three odd numbered mAChRs (M1, M3 and M5) were expressed on cuticular plates, myelin sheaths and Schwann cells. Even-numbered mAChRs were seen on the nerve terminals. M2 was also shown on the cuticular plates and supporting cells. Vestibular efferent fibers and terminals were not identified in our studies. Results from WB of the dissociated vestibular epithelia, nerve fibers and vestibular ganglia were consistent with the results from IH and IC. Our findings suggest that there is considerable co-expression of the subtypes on the neural elements of the labyrinth. Further electrophysiological and pharmacological studies should delineate the mechanisms of action of muscarinic acetylcholine receptors on structures in the labyrinth.

Expression and function of genes encoding cholinergic components in murine immune cells

It is now evident that acetylcholine (ACh) synthesized by choline acetyltransferase (ChAT) and released from T cells during antigen presentation binds to muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively) on T and B cells or dendritic cells, leading to modulation of their function. In the present study, we used reverse transcription-polymerase chain reaction (RT-PCR) to investigate whether mononuclear leukocytes (MNLs), bone marrow-derived dendritic cells (DCs) and macrophages from C57BL/6J mice express components of the cholinergic system. Expression of ChAT mRNA was detected in MNLs activated with ConA and DCs stimulated with LPS, but not in resting MNLs and DCs or in resting and stimulated macrophages. MNLs, DCs and macrophages all expressed mRNAs encoding the five mAChR subtypes (M1–M5) and the nAChR α2, α5, α6, α7, α10 and β2 subunits. Expression of VIP mRNA was detected in MNLs and macrophages, but not in DCs. MNLs, DCs and macrophages all expressed VIP receptor-1 (VPAC1) and -2 (VPAC2) mRNAs, as well as mRNAs encoding secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein (SLURP)-1 and SLURP-2, two endogenous nAChR ligands. These results suggest that the lymphocytic cholinergic system is activated by ACh via mAChR- and nAChR-mediated pathways during antigen presentation between T cells and DCs or macrophages, leading to modulation of immune cell function. Moreover, VIP released from both postganglionic cholinergic neurons and immune cells may play a role in the cholinergic anti-inflammatory reflex, acting via VPAC1 and VPAC2 on immune cells.