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acetylcholine chloride [keywords]
- Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalisation and Subcellular Trafficking. [JOURNAL ARTICLE]
- J Biol Chem 2014 Apr 21.
Allosteric modulators are an attractive approach to achieve receptor-subtype selective targeting of G protein-coupled receptors (GPCRs). Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favourable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalisation and endocytic trafficking. In the present study, we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a BRET approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalised, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalisation is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterisation of novel allosteric modulators of GPCRs to encapsulate the consequences of chronic exposure to this family of ligands.
- Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons. [JOURNAL ARTICLE]
- J Biol Chem 2014 Apr 21.
Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labelling techniques and confocal microscopy we demonstrate that pseudotyping with rabies virus glycoprotein (RV-G) enables the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process reveals that these vectors traffic through Rab5-positive endosomes and accumulate within a non-acidic Rab7 compartment. RV-G pseudotyped vectors are co-transported with both the tetanus neurotoxin binding fragment as well as the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultures in compartmentalised chambers, we demonstrate that axonal retrograde transport of this vectors is rapid and efficient, however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, and suggests a novel area for improvement of gene therapy vectors.
- Insights into a highly conserved network of hydrogen bonds in the agonist binding site of nicotinic acetylcholine receptors: A structural and theoretical study. [JOURNAL ARTICLE]
- Proteins 2014 Apr 21.
Structural and theoretical studies on the geometrical features of a hydrogen-bond network occurring in the binding site of nicotinic acetylcholine receptors (nAChRs) and composed of interconnected WxPD (Trp-x-Pro-Asp) and SWyz (Ser-Trp-yz) sequences from loops A and B, respectively, have been carried out. Multiple sequence alignments using as template the sequence of the apoform of Aplysia californica Acetylcholine binding protein (Ac-AChBP) show the strict conservation of Serine and Tryptophan residues of the loop B SWyz sequence. Considering a sample of 19 high resolution AChBP structures, the strong conformational preferences of the key Tryptophan residue has been pointing out, whatever the form, free or bounded, of AChBP. The geometry of the motif hydrogen-bond network has been characterised through the analyses of seven distances. The robustness of the various hydrogen-bond interactions is pointed out, the one involving the Aspartate carboxylate group and the Serine residue being the shortest of the network. The role of a cooperative effect involving a NH(His145)…OH(Ser142) hydrogen bond is highlighted. Density Functional Theory (DFT) calculations on several simplified models based on the motif hydrogen-bond network allow probing the importance of the various hydrogen-bond interactions. The removal of the Ser142 hydroxyl group induces strong structural rearrangements, in agreement with the structural observations. Molecular electrostatic potential calculations on model systems highlight the importance of a cooperative effect in the whole hydrogen-bond network. More precisely, the key role of the Ser142 hydroxyl group, involved in several hydrogen bonds, is underlined. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
- The neuroanatomical basis for cholinergic modulation of locomotor networks by sacral relay neurons with ascending lumbar projections. [JOURNAL ARTICLE]
- J Comp Neurol 2014 Apr 18.
Synaptic excitation by sacrocaudal-afferent (SCA) input of sacral relay neurons projecting rostrally through the ventral white-matter funiculi (VF-neurons) is a potent activator of the hindlimb central pattern generators (CPGs) in rodent spinal cords lacking descending supraspinal control. Using electrophysiological recordings from the sacral and lumbar spinal segments we show that the motor output of the lumbar segments produced by SCA-stimulation is enhanced by exposing the sacral segments of the neonatal rat spinal cord to the acetylcholinesterase inhibitor, edrophonium (EDR). Histochemical- and immunostaining of the sacral cord reveals expression of acetylcholinesterase activity, ability to synthesize acetylcholine, and/or innervation by cholinergic synaptic inputs in significant proportions of fluorescently back-labeled sacral VF-neurons. Moreover, the majority of the VF neurons express M2 muscarinic receptors, raising the possibility that the elevated acetylcholine levels resulting from inhibition of acetylcholinesterase act on such receptors. Indeed, sacral application of atropine or the M2 -type receptor antagonist, methoctramine, was found to reverse the effects of EDR. We suggest that variations in the sacral level of acetylcholine modulate the SCA-induced locomotor rhythm via muscarinic receptor-dependent mechanisms, and that the modified activity of sacral VF-neurons in the presence of an acetylcholinesterase inhibitor can be partially ascribed to the cholinergic components associated with them. Thus, pharmacological manipulations of the sacral cholinergic-system may be used to modulate the locomotor related motor output in the absence of descending supraspinal control. J. Comp. Neurol., 2014. © 2014 Wiley Periodicals, Inc.
- Effects of Allopurinol on Endothelial Dysfunction: A Meta-Analysis. [JOURNAL ARTICLE]
- Am J Nephrol 2014 Apr 17; 39(4):348-356.
Objective: Several studies have assessed the effect of allopurinol on endothelial function, but these studies were relatively small in size and used different methods of evaluating endothelial function. We conducted a meta-analysis to investigate the effect of allopurinol on both endothelial-dependent and -independent vasodilatation. Methods: Electronic databases, Medline, PubMed, EMBASE, SCOPUS, EBSCO and the Cochrane Library Central Register of Clinical Trials were searched from January 1985 to July 2013 on clinical trials (randomized and non-randomized) which assessed the effect of allopurinol on endothelial function. We conducted a sensitivity analysis to assess the contribution of each study to the pooled treatment effect by excluding each study one at a time and recalculating the pooled treatment effect for the remaining studies. Treatment effect was significant if p < 0.05. We assessed for heterogeneity in treatment estimates using the Cochran Q test and the χ(2) statistic (with substantial heterogeneity defined as values >50%). Results: The final analysis consisted of 11 studies (2 observational and 9 randomized). For the endothelial-dependent vasodilatation there were 6 studies, including 257 patients, that evaluated flow-mediated dilatation and 5 studies with 87 patients that reported data on forearm blood flow response to acetylcholine or flow-dependent vasodilatation. Overall, there was a significant increase in the endothelium-dependent vasodilatation with allopurinol treatment (MD 2.69%, 95% CI 2.49, 2.89%, p < 0.001; heterogeneity χ(2) = 319.1, I(2) = 96%, p < 0.001). There was only 1 study (100 patients) assessing nitrate-mediated dilatation and 4 studies (73 patients) evaluating forearm blood flow response to sodium nitroprusside as measures of endothelial-independent vasodilatation. The overall analysis (MD -0.08, 95% CI -0.50, 0.34, p = 0.70; heterogeneity χ(2) = 9.0, I(2) = 44%, p = 0.11) showed no effect of allopurinol treatment on endothelium-independent vasodilatation. Conclusions: We found that treatment of hyperuricemia with allopurinol is associated with an improvement in the endothelial-dependent, but not with the endothelial-independent vasodilatation. © 2014 S. Karger AG, Basel.
- Anti-M3 muscarinic acetylcholine receptor antibodies in patients with primary biliary cirrhosis. [JOURNAL ARTICLE]
- Hepatol Res 2014 Apr 21.
M3 muscarinic acetylcholine receptor (M3R) is expressed in biliary tracts as well as in exocrine glands. It is reported some patients with primary biliary cirrhosis (PBC) carry auto-antibodies against M3R. The aim of this study is to clarify the presence, potential use as diagnostic marker, and clinical roles of anti-M3R antibodies in PBC.We synthesized peptides encoding the extracellular domains of human-M3R, including the N-terminal region, the first, second, and third extracellular loops. Antibodies against these regions were examined by peptide-based ELISA in sera of 90 patients with PBC and 40 with chronic hepatitis C (CHC), 21 with nonalcoholic steatohepatitis (NASH), 10 with primary sclerosing cholangitis (PSC), 14 with obstructive jaundice, 10 with drug induced liver injury, and 42 healthy controls.Antibodies to the N-terminal, first, second and third loop were detected in 90.0% (81/90), 73.3% (66/90), 76.7% (69/90), and 66.7% (60/90) of PBC, in 67.5% (27/40), 10.0% (4/40), 67.5% (27/40), and 27.5% (11/40) of CHC, in 85.7% (18/21), 9.5% (2/21), 4.8% (1/21), and 57.1% (12/21) of NASH, in 60.0% (6/10), 20.0% (2/10), 60.0% (6/10), and 60.0% (6/10) of PSC, in 100.0% (14/14), 0% (0/14), 64.3% (9/14), and 78.6% (11/14) of obstructive jaundice, in 100.0% (10/10), 0% (0/10), 30.0% (3/10), and 10.0% (1/10) of drug induced liver injury, and in 4.8% (2/42), 7.1% (3/42), 2.4% (1/42), and 2.4% (1/42) of the controls, respectively.A high frequency of PBC carried anti-M3R antibodies. Anti-M3R antibodies against the first loop of M3R are potentially useful diagnostic maker for PBC.
- Role of α4- and α6-containing nicotinic receptors in the acquisition and maintenance of nicotine self-administration. [JOURNAL ARTICLE]
- Addict Biol 2014 Apr 22.
Tobacco smoking is a major cause of death and disease and as such there is a critical need for the development of new therapeutic approaches to treat nicotine addiction. Here, we utilize genetic and pharmacological tools to further investigate the nicotinic acetylcholine receptor (nAChR) subtypes that support intravenous self-administration of nicotine. α4-S248F mice contain a point mutation within the α4 nAChR subunit which confers increased sensitivity to nicotine and resistance to mecamylamine. Here, we show that acute administration of mecamylamine (2 mg/kg, i.p.) reduces established nicotine self-administration (0.05 mg/kg/infusion) in wild-type (WT), but not in α4-S248F heterozygous mice, demonstrating a role for α4* nAChRs in the modulation of ongoing nicotine self-administration. Administration of N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), a selective α6β2* nAChR antagonist, dose dependently (5 and 10 mg/kg, i.p.) impairs the acquisition of nicotine self-administration and reduces established nicotine self-administration in WT mice when administered acutely (10 mg/kg, i.p.). This was not due to a general reduction in locomotor activity and the same dose of bPiDI did not affect operant responding for sucrose. bPiDI treatment (10 mg/kg, i.p.) also impaired both the acquisition and maintenance of nicotine self-administration in α4-S248F heterozygous mice. This provides further evidence for the involvement of α6β2* nAChRs in the reinforcing effects of nicotine that underlies its ability to support ongoing self-administration. Taken together, selective targeting of α6β2* or α4α6β2* nAChRs may prove to be an effective strategy for the development of smoking cessation therapies.
- The alpha3beta4* nicotinic acetylcholine receptor subtype mediates physical dependence to morphine: mouse and human studies. [JOURNAL ARTICLE]
- Br J Pharmacol 2014 Apr 21.
Recent data have indicated that α3β4* neuronal nicotinic acetylcholine receptors (nAChRs) may play a role in morphine dependence. Here we investigated if nAChRs modulate morphine physical withdrawal.To assess the role of α3β4* neuronal nicotinic acetylcholine receptors in morphine withdrawal, we used a genetic correlation approach using publically available data sets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Lastly, male and female European-American (n=2,772) and African-American (n=1,309) subjects from the SAGE dataset were used to assess the possible association of polymorphisms in the 15q25 gene cluster and opioid dependence.BXD recombinant mouse lines demonstrated an increase in expression of α3, β4, and α5 nAChR mRNA in the forebrain and midbrain, which significantly correlates with an increase in defecation in mice undergoing morphine withdrawal. Indeed, mice with overexpression of the gene cluster CHRNA5/A3/B4 exhibited an increase in somatic signs of withdrawal. Furthermore, α5 and β4 nAChR knockout mice expressed decreased somatic withdrawal signs compared to their wildtype counterparts. Moreover, selective α3β4* nAChR antagonists, α-conotoxin AuIB (AuIB) and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nAChR subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nAChR subtype is not involved in morphine somatic withdrawal signs.Overall, our findings suggest an important role for the α3β4* nAChR subtype in morphine physical dependence.
- Evaluation of HemogloBind™ treatment for preparation of samples for cholinesterase analysis. [JOURNAL ARTICLE]
- Adv Biosci Biotechnol 2013 Dec 1; 4(12):1020-1023.
Acetylcholine is an essential neurotransmitter found throughout the nervous system. Its action on postsynaptic receptors is regulated through hydrolysis by various carboxylesterases, especially cholinesterases (ChEs). The acute toxicity of organophosphate (OP) compounds is directly linked to their action as inhibitors of ChE. One widely used assay for evaluating ChE activity is a spectrophotometric method developed by Ellman et al. When the enzyme source is from tissues or, in particular, blood, hemoglobin displays a spectrophotometric peak at the same wavelength used to analyze cholinergic activity. This creates a substantial background that interferes with the Ellman's assay and must be overcome in order to accurately monitor cholinesterase activity. Herein, we directly compare blood processing methods: classical method (1.67 ± 0.30 U/mL) and HemogloBind™ treatment (1.51 ± 0.17 U/mL), and clearly demonstrate that pretreatment of blood samples with Hemoglobind™ is both a sufficient and rapid sample preparation method for the assessment of ChE activity using the Ellman's method.
- Brain natriuretic peptide is a potent vasodilator in aged human microcirculation and shows a blunted response in heart failure patients. [Journal Article]
- J Geriatr Cardiol 2014 Mar; 11(1):50-6.
Brain natriuretic peptide (BNP) is normally present in low levels in the circulation, but it is elevated in parallel with the degree of congestion in heart failure subjects (CHF). BNP has natriuretic effects and is a potent vasodilator. It is suggested that BNP could be a therapeutic alternative in CHF. However, we postulated that the high levels of circulating BNP in CHF may downregulate the response of microvascular natriuretic receptors. This was tested by comparing 15 CHF patients (BNP > 3000 ng/L) with 10 matched, healthy controls.Cutaneous microvascular blood flow in the forearm was measured by laser Doppler Flowmetry. Local heating (+44°C, 10 min) was used to evoke a maximum local dilator response.Non-invasive iontophoretic administration of either BNP or acetylcholine (ACh), a known endothelium-dependent dilator, elicited an increase in local flow. The nitric oxide synthase inhibitor, l-N-Arginine- methyl-ester (L-NAME), blocked the BNP response (in controls). Interestingly, responses to BNP in CHF patients were reduced to about one third of those seen in healthy controls (increase in flow: 251% in CHF vs. 908% in controls; P < 0.001). In contrast, the vasodilator responses to ACh and to local heating were only somewhat attenuated in CHF patients. Thus, dilator capacity and nitric oxide signalling were not affected to the same extent as BNP-mediated dilation, indicating a specific downregulation of the latter response.The findings show for the first time that microvascular responses to BNP are markedly reduced in CHF patients. This is consistent with the hypothesis of BNP receptor function is downregulated in CHF.