Development and Optimization of Methscopolamine Bromide Gastroretentive Floating Tablets Using 32 Factorial Design.

PURPOSE: The aim of this study was to formulate methscopolamine floating drug delivery system to increase its gastro retention for further enhancement of absorption and overall bioavailability. METHOD: Direct compression method was used to formulate floating drug delivery system of methscopolamine bromide.: Different amount of HPMC, PVP K25, and MCC were used for preparation of tablets. RESULT: The prepared tablets were evaluated for thickness, hardness, weight variation, floating lag time, swelling index and in-vitro drug release. All the formulations showed less than 10% of weight variation. The hardness and thickness of all the formulations were within the range of 3.7-4.2 kg/cm2 and 3.63-3.83 mm respectively. Floating lag time for all the formulations was reported in seconds. The degree of swelling was reported in range of 82.10-85.83%. In vitro release was carried out for 24 h. The maximum release was shown by F1 (93.947%) while the minimum release was observed for F4 (90.420%). The best formulation was optimized on the basis of percentage cumulative drug release, floating lag time and swelling index. F1 found to be the best formulation. Further on analyzing the drug release mechanism, F1 found to exhibit korsmeyer peppas model of drug release. CONCLUSION: Floating gastroretentive tablet of methscopolamine bromide was successfully developed using direct compression method with potential to enhance the drug absorption and effective treatment of peptic ulcer.

Widespread Changes in Positive Allosteric Modulation of the Muscarinic M1 Receptor in Some Participants With Schizophrenia.

BACKGROUND: Preclinical and some human data suggest allosteric modulation of the muscarinic M1 receptor (CHRM1) is a promising approach for the treatment of schizophrenia. However, it is suggested there is a subgroup of participants with schizophrenia who have profound loss of cortical CHRM1 (MRDS). This raises the possibility that some participants with schizophrenia may not respond optimally to CHRM1 allosteric modulation. Here we describe a novel methodology to measure positive allosteric modulation of CHRM1 in human CNS and the measurement of that response in the cortex, hippocampus, and striatum from participants with MRDS, non-MRDS and controls. METHODS: The cortex (Brodmann's area 6), hippocampus, and striatum from 40 participants with schizophrenia (20 MRDS and 20 non-MRDS) and 20 controls were used to measure benzyl quinolone carboxylic acid-mediated shift in acetylcholine displacement of [3H]N-methylscopolamine using a novel in situ radioligand binding with autoradiography methodology. RESULTS: Compared with controls, participants with schizophrenia had lower levels of specific [3H]N-methylscopolamine binding in all CNS regions, whilst benzyl quinolone carboxylic acid-modulated binding was less in the striatum, Brodmann's area 6, dentate gyrus, and subiculum. When divided by subgroup, only in MRDS was there lower specific [3H]N-methylscopolamine binding and less benzyl quinolone carboxylic acid-modulated binding in all cortical and subcortical regions studied. CONCLUSIONS: In a subgroup of participants with schizophrenia, there is a widespread decreased responsiveness to a positive allosteric modulator at the CHRM1. This finding may have ramifications it positive allosteric modulators of the CHRM1 are used in clinical trials to treat schizophrenia as some participants may not have an optimal response.

Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats.

Vagus nerve stimulation (VNS) enhances extinction of conditioned fear in rats. Previous findings support the hypothesis that VNS effects on extinction are due to enhanced consolidation of extinction memories through promotion of plasticity in extinction-related brain pathways however, alternative explanations are plausible. According to one hypothesis, VNS may produce a hedonic effect and enhance extinction through counter-conditioning. According to another hypothesis, VNS reduces anxiety during exposure and this weakens the association of conditioned stimuli with aversive conditioned responses. The present set of experiments (1) used conditioned place preference (CPP) to identify potential rewarding effects associated with VNS and (2) examined the peripheral effects of VNS on anxiety and extinction enhancement. Male Sprague-Dawley rats were surgically implanted with cuff electrodes around the vagus nerve and subjected to a CPP task in which VNS and sham stimulation were each paired with one of two distinct contexts over the course of 5 d. Following this procedure, rats did not show a place preference, suggesting that VNS is not rewarding or aversive. The role of the peripheral parasympathetic system in the anxiolytic effect of VNS on the elevated plus maze was examined by blocking peripheral muscarinic receptors with intraperitoneal administration of methyl scopolamine prior to VNS. Methyl scopolamine blocked the VNS-induced reduction in anxiety but did not interfere with VNS enhancement of extinction of conditioned fear, indicating that the anxiety-reducing effect of VNS is not necessary for the extinction enhancement.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor.

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

Utility of an "Allosteric Site-Impaired" M2 Muscarinic Acetylcholine Receptor as a Novel Construct for Validating Mechanisms of Action of Synthetic and Putative Endogenous Allosteric Modulators.

Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structure-based approach, we previously designed a mutant M2 mAChR (N410K+T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-l-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5'-O-[γ-thio]triphosphate [35S] binding and extracellular signal-regulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.

Characterization of methanthelinium binding and function at human M1-M5 muscarinic acetylcholine receptors.

Firstly, it was determined whether methanthelinium bromide (MB) binds to human M1-M5 (hM1-hM5) muscarinic acetylcholine receptors in comparison to the classical muscarinic antagonist N-methylscopolamine (NMS). [3H]NMS dissociation binding experiments revealed an allosteric retardation of dissociation at 100 µM of MB ranging from none in hM3 to 4.6-fold in hM2 receptors. Accordingly, global non-linear regression analysis of equilibrium inhibition binding curves between [3H]NMS (0.2 and 2.0 nM) and MB was applied and compared using either an allosteric or a competitive model. The allosteric cooperativity of MB binding within MB/NMS/hM receptor complexes was strongly negative and undistinguishable from a competitive interaction throughout all subtypes. Applying the competitive model to the equilibrium binding data of MB and NMS, suggested competition at all hM subtypes: logKI (± S.E.) hM3 = 8.71 ± 0.15, hM1 = 8.68 ± 0.14, hM5 = 8.58 ± 0.07, hM2 = 8.27 ± 0.07 to hM4 = 8.25 ± 0.11. Secondly, the effects of MB on acetylcholine (ACh) induced hM receptor function showed very strong negative allosteric cooperativity at all subtypes pointing against an allosteric antagonism of MB with ACh. Competition with ACh was characterized by logKB: hM1 = 9.53 ± 0.05, hM4 = 9.33 ± 0.05, hM5 = 8.80 ± 0.05, hM2 = 8,79 ± 0.06, to hM3 = 8.43 ± 0.04. In conclusion, MB, below 1 µM, binds competitively and non-selectively (except for the difference between hM3 vs. hM4) to all five hM receptor subtypes with nanomolar affinity and is able to functionally inhibit ACh responses in a competitive fashion, with a slight subtype preference for hM1 and hM4.

Synthesis of novel and functionally selective non-competitive muscarinic antagonists as chemical probes.

Muscarinic receptors are known to play important biological roles and are drug targets for several human diseases. In a pilot study, novel muscarinic antagonists were synthesized and used as chemical probes to obtain additional information of the muscarinic pharmacophore. The design of these ligands made use of current orthosteric and allosteric models of drug-receptor interactions together with chemical motifs known to achieve muscarinic receptor selectivity. This approach has led to the discovery of several non-competitive muscarinic ligands that strongly bind at a secondary receptor site. These compounds were found to be non-competitive antagonists that completely abolished carbachol activation in functional assays. Several of these compounds antagonized functional response to carbachol with great potency at M1 and M4 than at the rest of receptor subtypes.

Structure-Based Design and Discovery of New M2 Receptor Agonists.

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

Mice with conditional NeuroD1 knockout display reduced aberrant hippocampal neurogenesis but no change in epileptic seizures.

Adult neurogenesis is significantly increased in the hippocampus of rodent models of temporal lobe epilepsy (TLE). These adult-generated neurons have recently been shown to play a contributing role in the development of spontaneous recurrent seizures (SRS). In order to eventually target pro-epileptic adult neurogenesis in the clinical setting, it will be important to identify molecular players involved in the control of aberrant neurogenesis after seizures. Here, we focused on NeuroD1 (ND1), a member of the bHLH family of transcription factors previously shown to play an essential role in the differentiation and maturation of adult-generated neurons in the hippocampus. Wild-type mice treated with pilocarpine to induce status epilepticus (SE) showed a significant up-regulation of NeuroD1+ immature neuroblasts located in both the granule cell layer (GCL), and ectopically localized to the hilar region of the hippocampus. As expected, conditional knockout (cKO) of NeuroD1 in Nestin-expressing stem/progenitors and their progeny led to a reduction in the number of NeuroD1+ adult-generated neurons after pilocarpine treatment compared to WT littermates. Surprisingly, there was no change in SRS in NeuroD1 cKO mice, suggesting that NeuroD1 cKO fails to reduce aberrant neurogenesis below the threshold needed to impact SRS. Consistent with this conclusion, the total number of adult-generated neurons in the pilocarpine model, especially the total number of Prox1+ hilar ectopic granule cells were unchanged after NeuroD1 cKO, suggesting strategies to reduce SRS will need to achieve a greater removal of aberrant adult-generated neurons.

Therapeutic effects of anti-HMGB1 monoclonal antibody on pilocarpine-induced status epilepticus in mice.

Inflammatory processes in brain tissue have been described in human epilepsy of various etiologies and in experimental models of seizures. High mobility group box-1 (HMGB1) is now recognized as representative of damage-associated molecular patterns (DAMPs). In the present study, we focused on whether anti-HMGB1 antibody treatment could relieve status epilepticus- triggered BBB breakdown and inflammation response in addition to the seizure behavior itself. Pilocarpine and methyl-scopolamine were used to establish the acute seizure model. Anti-HMGB1 mAb showed inhibitory effects on leakage of the BBB, and on the HMGB1 translocation induced by pilocarpine. The expression of inflammation-related factors, such as MCP-1, CXCL-1, TLR-4, and IL-6 in hippocampus and cerebral cortex were down-regulated by anti-HMGB1 mAb associated with the number of activated astrocytes, microglial cells as well as the expression of IL-1ß. Both hematoxylin & eosin and TUNEL staining showed that the apoptotic cells could be reduced after anti-HMGB1 mAb treatment. The onset and latency of Racine stage five were significantly prolonged in the anti-HMGB1 mAb group. These results suggested that anti-HMGB1 mAb prevented the BBB permeability, reduced HMGB1 translocation while inhibiting the expression of inflammation-related factors, protected against neural cell apoptosis and prolonged Racine stage 5 seizure onset and latency.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors.

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

Reduced NPY Y1 receptor hippocampal expression and signs of decreased vagal modulation of heart rate in mice.

Central neuropeptide Y (NPY) signaling participates in the regulation of cardiac autonomic outflow, particularly via activation of NPY-Y1 receptors (Y1Rs). However, the specific brain areas and neural pathways involved have not been completely identified yet. Here, we evaluate the role of hippocampal Y1Rs in the modulation of the autonomic control of cardiac function using a conditional knockout mouse model. Radiotelemetric transmitters were implanted in 4-month-old male mice exhibiting reduced forebrain expression (rfb) of the Y1R (Npy1rrfb, n=10) and their corresponding controls (Npy1r2lox, n=8). ECG signals were recorded (i) during resting conditions, (ii) under selective pharmacological manipulation of cardiac vagal activity, and (iii) during acute and chronic psychosocial stress challenges, and analyzed via time- and frequency-domain analysis of heart rate variability. Npy1rrfb mice showed a lower Npy1r mRNA density in the dentate gyrus and in the CA1 region of the hippocampus. Under resting undisturbed conditions, Npy1rrfb mice exhibited (i) a higher heart rate, (ii) a reduced overall heart rate variability, and (iii) lower values of the indices of vagal modulation compared to Npy1r2lox counterparts. Following pharmacological vagal inhibition, heart rate was higher in control but not in Npy1rrfb mice compared to their respective baseline values, suggesting that tonic vagal influences on heart rate were reduced in Npy1rrfb mice. The magnitude of the heart rate response to acute stressors was smaller in Npy1rrfb mice compared to Npy1r2lox counterparts, likely due to a concurrent lower vagal withdrawal. These findings suggest that reduced Y1R expression leads to a decrease in resting vagal modulation and heart rate variability, which, in turn, may determine a reduced cardiac autonomic responsiveness to acute stress challenges.

Midazolam-ketamine dual therapy stops cholinergic status epilepticus and reduces Morris water maze deficits.

OBJECTIVE: Pharmacoresistance remains an unsolved therapeutic challenge in status epilepticus (SE) and in cholinergic SE induced by nerve agent intoxication. SE triggers a rapid internalization of synaptic γ-aminobutyric acid A (GABAA ) receptors and externalization of N-methyl-d-aspartate (NMDA) receptors that may explain the loss of potency of standard antiepileptic drugs (AEDs). We hypothesized that a drug combination aimed at correcting the consequences of receptor trafficking would reduce SE severity and its long-term consequences. METHODS: A severe model of SE was induced in adult Sprague-Dawley rats with a high dose of lithium and pilocarpine. The GABAA receptor agonist midazolam, the NMDA receptor antagonist ketamine, and/or the AED valproate were injected 40 min after SE onset in combination or as monotherapy. Measures of SE severity were the primary outcome. Secondary outcomes were acute neuronal injury, spontaneous recurrent seizures (SRS), and Morris water maze (MWM) deficits. RESULTS: Midazolam-ketamine dual therapy was more efficient than double-dose midazolam or ketamine monotherapy or than valproate-midazolam or valproate-ketamine dual therapy in reducing several parameters of SE severity, suggesting a synergistic mechanism. In addition, midazolam-ketamine dual therapy reduced SE-induced acute neuronal injury, epileptogenesis, and MWM deficits. SIGNIFICANCE: This study showed that a treatment aimed at correcting maladaptive GABAA receptor and NMDA receptor trafficking can stop SE and reduce its long-term consequences. Early midazolam-ketamine dual therapy may be superior to monotherapy in the treatment of benzodiazepine-refractory SE.

Characterization of a Novel M1 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator Radioligand, [3H]PT-1284.

Selective activation of the M1 muscarinic acetylcholine receptor (mAChR) via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. Herein, we describe the characterization of an M1 PAM radioligand, 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one ([(3)H]PT-1284), as a tool for characterizing the M1 allosteric binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 ( 1: )) was shown to potentiate acetylcholine (ACh) in an M1 fluorometric imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and carbachol in a hippocampal slice electrophysiology assay (EC50, 165 nM). PT-1284 ( 1: ) also reduced the concentration of ACh required to inhibit [(3)H]N-methylscopolamine ([(3)H]NMS) binding to M1, left-shifting the ACh Ki approximately 19-fold at 10 µM. Saturation analysis of a human M1 mAChR stable cell line showed that [(3)H]PT-1284 bound to M1 mAChR in the presence of 1 mM ACh with Kd, 4.23 nM, and saturable binding capacity (Bmax), 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit [(3)H]PT-1284 binding in a concentration-responsive manner, whereas M1 allosteric and orthosteric agonists showed weak affinity (>30 µM). A strong positive correlation (R(2) = 0.86) was found to exist between affinity values generated for nineteen M1 PAMs in the [(3)H]PT-1284 binding assay and the EC50 values of these ligands in a FLIPR functional potentiation assay. These data indicate that there is a strong positive correlation between M1 PAM binding affinity and functional activity, and that [(3)H]PT-1284 can serve as a tool for pharmacological investigation of M1 mAChR PAMs.

Pharmacological evidence is consistent with a prominent role of spatial memory in complex navigation.

The ability to learn about the spatial environment plays an important role in navigation, migration, dispersal, and foraging. However, our understanding of both the role of cognition in the development of navigation strategies and the mechanisms underlying these strategies is limited. We tested the hypothesis that complex navigation is facilitated by spatial memory in a population of Chrysemys picta that navigate with extreme precision (±3.5 m) using specific routes that must be learned prior to age three. We used scopolamine, a muscarinic acetylcholine receptor antagonist, to manipulate the cognitive spatial abilities of free-living turtles during naturally occurring overland movements. Experienced adults treated with scopolamine diverted markedly from their precise navigation routes. Naive juveniles lacking experience (and memory) were not affected by scopolamine, and thereby served as controls for perceptual or non-spatial cognitive processes associated with navigation. Further, neither adult nor juvenile movement was affected by methylscopolamine, a form of scopolamine that does not cross the blood-brain barrier, a control for the peripheral effects of scopolamine. Together, these results are consistent with a role of spatial cognition in complex navigation and highlight a cellular mechanism that might underlie spatial cognition. Overall, our findings expand our understanding of the development of complex cognitive abilities of vertebrates and the neurological mechanisms of navigation.

Apolipoprotein E4 reduces evoked hippocampal acetylcholine release in adult mice.

Apolipoprotein E4 (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease. We utilized apoE4-targeted replacement mice (approved by the Tel Aviv University Animal Care Committee) to investigate whether cholinergic dysfunction, which increases during aging and is a hallmark of Alzheimer's disease, is accentuated by apoE4. This revealed that levels of the pre-synaptic cholinergic marker, vesicular acetylcholine transporter in the hippocampus and the corresponding electrically evoked release of acetylcholine, are similar in 4-month-old apoE4 and apolipoprotein E3 (apoE3) mice. Both parameters decrease with age. This decrease is, however, significantly more pronounced in the apoE4 mice. The levels of cholinacetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) were similar in the hippocampus of young apoE4 and apoE3 mice and decreased during aging. For ChAT, this decrease was similar in the apoE4 and apoE3 mice, whereas it was more pronounced in the apoE4 mice, regarding their corresponding AChE and BuChE levels. The level of muscarinic receptors was higher in the apoE4 than in the apoE3 mice at 4 months and increased to similar levels with age. However, the relative representation of the M1 receptor subtype decreased during aging in apoE4 mice. These results demonstrate impairment of the evoked release of acetylcholine in hippocampus by apoE4 in 12-month-old mice but not in 4-month-old mice. The levels of ChAT and the extent of the M2 receptor-mediated autoregulation of ACh release were similar in the adult mice, suggesting that the apoE4-related inhibition of hippocampal ACh release in these mice is not driven by these parameters. Evoked ACh release from hippocampal and cortical slices is similar in 4-month-old apoE4 and apoE3 mice but is specifically and significantly reduced in hippocampus, but not cortex, of 12-month-old apoE4 mice. This effect is accompanied by decreased VAChT levels. These findings show that the hipocampal cholinergic nerve terminals are specifically affected by apoE4 and that this effect is age dependent.

Changes in BQCA Allosteric Modulation of [(3)H]NMS Binding to Human Cortex within Schizophrenia and by Divalent Cations.

Stimulation of the cortical muscarinic M1 receptor (CHRM1) is proposed as a treatment for schizophrenia, a hypothesis testable using CHRM1 allosteric modulators. Allosteric modulators have been shown to change the activity of CHRMs using cloned human CHRMs and CHRM knockout mice but not human CNS, a prerequisite for them working in humans. Here we show in vitro that BQCA, a positive allosteric CHRM1 modulator, brings about the expected change in affinity of the CHRM1 orthosteric site for acetylcholine in human cortex. Moreover, this effect of BQCA is reduced in the cortex of a subset of subjects with schizophrenia, separated into a discrete population because of a profound loss of cortical [(3)H]pirenzepine binding. Surprisingly, there was no change in [(3)H]NMS binding to the cortex from this subset or those with schizophrenia but without a marked loss of cortical CHRM1. Hence, we explored the nature of [(3)H]pirenzepine and [(3)H]NMS binding to human cortex and showed total [(3)H]pirenzepine and [(3)H]NMS binding was reduced by Zn(2+), acetylcholine displacement of [(3)H]NMS binding was enhanced by Mg(2+) and Zn(2+), acetylcholine displacement of [(3)H]pirenzepine was reduced by Mg(2+) and enhanced by Zn(2+), whereas BQCA effects on [(3)H]NMS, but not [(3)H]pirenzepine, binding was enhanced by Mg(2+) and Zn(2+). These data suggest the orthosteric and allosteric sites on CHRMs respond differently to divalent cations and the effects of allosteric modulation of the cortical CHRM1 is reduced in a subset of people with schizophrenia, a finding that may have ramifications for the use of CHRM1 allosteric modulators in the treatment of schizophrenia.

Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber).

The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1-5 of the naked mole-rat were compared to that of the house mouse (Mus musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies with [(3)H]-N-methylscopolamine. The BLAST test revealed 95 % protein sequence homology showing the naked mole-rat to have the genetic potential to express all five muscarinic acetylcholine receptor subtypes. A significant reduction in pain behavior was demonstrated after administration of 8.4 mg/kg in the formalin test. Administration of 50 mg/kg VU0152100 resulted in a non-significant tendency towards antinociception. The antinociceptive effects were reversed by the muscarinic acetylcholine receptor antagonist atropine. Binding studies indicated presence of muscarinic acetylcholine receptors with a radioligand affinity comparable to that reported in mice. In conclusion, muscarinic acetylcholine receptor subtypes are present in the naked mole-rat and contribute to antinociception in the naked mole-rat.

Functional and biochemical characteristics of urinary bladder muscarinic receptors in long-term alloxan diabetic rats.

OBJECTIVE: To re-examine the function of the urinary bladder in vivoas well as to determine the functional and biochemical characteristics of bladder muscarinic receptors in long-term alloxan-induced diabetes rats. METHODS: Two-month-old male Wistar rats were injected with alloxan and the animals showing blood glucose levels >300mg/dL together with age-paired untreated animals were kept for 11 months. Body weight, bladder weight, blood glucose, and urinary volume over a period of 24 hours were determined in both groups of animals. A voiding cystometry in conscious control and diabetic rats was performed to determine maximal micturition pressure, micturition contraction interval and duration as well as voided and post-voiding residual volume. In addition, concentration-response curves for bethanechol in isolated bladder strips, as well as [3H]-N methyl-scopolamine binding site characteristics in bladder homogenates were determined. RESULTS: Mean bladder weight was 162.5±21.2mg versus 290±37.9mg in control and treated animals, respectively (p<0.05). Micturition contraction amplitude (34.6±4.7mmHg versus 49.6±2.5mmHg), duration (14.5±1.7 seconds versus 23.33±4.6 seconds) and interval (87.5±17.02 seconds versus 281.11±20.24 seconds) were significantly greater in alloxan diabetic rats. Voided urine volume per micturition contraction was also significantly higher in diabetic animals. However the post-voiding residual volume was not statistically different. Bethanechol potency (EC50 3µM versus 5µM) and maximal effect (31.2±5.9g/g versus 36.1±6.8g/g) in isolated bladder strips as well as number (169±4fmol/mg versus 176±3fmol/mg protein) and affinity (0.69±0.1nM versus 0.57±0.1nM) of bladder muscarinic receptors were also not statistically different. CONCLUSION: Bladder function in vivo is altered in chronic alloxan-induced diabetes rats without changes in functional and biochemical characteristics of bladder muscarinic receptors.

Functional and biochemical characteristics of urinary bladder muscarinic receptors in long-term alloxan diabetic rats / Características funcionais e bioquímicas dos receptores muscarínicos da bexiga de ratos com diabetes crônico induzido por aloxana

Objective To re-examine the function of the urinary bladder in vivoas well as to determine the functional and biochemical characteristics of bladder muscarinic receptors in long-term alloxan-induced diabetes rats.Methods Two-month-old male Wistar rats were injected with alloxan and the animals showing blood glucose levels >300mg/dL together with age-paired untreated animals were kept for 11 months. Body weight, bladder weight, blood glucose, and urinary volume over a period of 24 hours were determined in both groups of animals. A voiding cystometry in conscious control and diabetic rats was performed to determine maximal micturition pressure, micturition contraction interval and duration as well as voided and post-voiding residual volume. In addition, concentration-response curves for bethanechol in isolated bladder strips, as well as [3H]-N methyl-scopolamine binding site characteristics in bladder homogenates were determined.Results Mean bladder weight was 162.5±21.2mg versus 290±37.9mg in control and treated animals, respectively (p<0.05). Micturition contraction amplitude (34.6±4.7mmHg versus 49.6±2.5mmHg), duration (14.5±1.7 seconds versus 23.33±4.6 seconds) and interval (87.5±17.02 seconds versus 281.11±20.24 seconds) were significantly greater in alloxan diabetic rats. Voided urine volume per micturition contraction was also significantly higher in diabetic animals. However the post-voiding residual volume was not statistically different. Bethanechol potency (EC50 3µM versus 5µM) and maximal effect (31.2±5.9g/g versus 36.1±6.8g/g) in isolated bladder strips as well as number (169±4fmol/mg versus 176±3fmol/mg protein) and affinity (0.69±0.1nM versus 0.57±0.1nM) of bladder muscarinic receptors were also not statistically different.Conclusion Bladder function in vivo is altered in chronic alloxan-induced diabetes rats without changes in functional and biochemical characteristics of bladder muscarinic receptors.

Objetivo Reestudar o funcionamento da bexiga in vivo e determinar as características funcionais e bioquímicas dos receptores muscarínicos vesicais de ratos com diabetes crônico induzido por aloxana.Métodos Ratos Wistar de dois meses de idade receberam injeção de aloxana, e os animais que apresentaram glicemia >300mg/dL foram mantidos por 11 meses junto de outros não tratados e pareados por idade. Nos dois grupos de animais, peso corpóreo, peso da bexiga, glicemia e volume urinário de 24 horas foram medidos. Em ambos os grupos, realizou-se a cistometria miccional em animais não anestesiados. Foram determinados os seguintes parâmetros: pressão máxima de micção, intervalo e contração de micção, bem como o volume de esvaziamento e o volume residual pós-miccional. Além disso, foram determinadas as curvas de concentração-resposta a betanecol em preparações isoladas de bexiga e também as características dos sítios de ligação da [3H]-N-metil-escopolamina em homogenatos de bexiga.Resultados O peso médio da bexiga foi de 162,5±21,2mg versus290±37,9mg nos animais controles e tratados, respectivamente (p<0,05). A amplitude de contração (34,6±4,7mmHg versus 49,6±2,5mmHg), a duração (14,5±1,7 segundos versus 23,33±4,6 segundos) e o intervalo (87,5±17,02 segundos versus 281,11±20,24 segundos) de micção foram significantemente maiores nos ratos tratados com aloxana. O volume de urina eliminada durante a contração miccional também foi maior nos animais diabéticos. Contudo, o volume residual pós-miccional não foi estatisticamente diferente. Não foram observadas diferenças na resposta ao betanecol (EC50 3µM versus 5µM) e no seu efeito máximo (31,2±5,9g/g versus 36,1±6,8g/g) em preparações isoladas de bexiga, bem como no número total (169±43fmol/mgversus 176±3fmol/mg) e na afinidade (0,69±0,1nMversus 0,57±0,1nM) dos receptores muscarínicos da bexiga.Conclusão O funcionamento da bexiga in vivo está alterado no diabetes crônico induzido por aloxana, porém sem alterações funcionais e bioquímicas nos receptores muscarínicos da bexiga.