Acetylcholine ameliorated hypoxia-induced oxidative stress and apoptosis in trophoblast cells via p38 MAPK/NF-κB pathway.

Hypoxia-induced oxidative stress and apoptosis of trophoblast are involved in the pathogenesis of preeclampsia (PE). Extensive research reports that the principal vagal neurotransmitter acetylcholine (ACh) shows anti-oxidative and anti-apoptotic effects in various diseases models. However, the role of ACh in hypoxic trophoblast remains unknown. Here, we examined the apoptotic levels of human placenta and explored the role(s) of ACh on cobalt chloride (CoCl2)-treated (trophoblast-derived) HTR-8/SVneo cells for mimicking hypoxic injuries. Cell counting kit-8 (CCK-8), dihydroethidium (DHE) probe, western blotting, immunofluorescence staining, migration and invasion assay were employed in the current study. Our data showed that placentas from PE women exhibited increased level of reactive oxygen species (ROS) and apoptotic index than those in normal pregnancy. Our in vitro study showed that CoCl2 enhanced ROS generation and apoptosis in HTR-8/SVneo cells through the activation of the p38 mitogen-activated protein kinase (p38 MAPK)/nuclear factor-κB (NF-κB) pathway. ACh significantly decreased hypoxia-induced ROS generation and the resulting apoptosis, accompanied by lowered phosphorylation of p38 MAPK and NF-κB. Western blotting analysis further confirmed that ACh decreased the ratio of pp38 MAPK/p38 MAPK, p-NF-κB/NF-κB, Bax/Bcl-2 and cleaved Caspase-3/Caspase-3. Besides, ACh promoted cell invasion and migration ability under hypoxic conditions. Atropine, the muscarinic receptor antagonist, abolished ACh's effects mentioned above. Overall, our data showed that ACh exerted protective effects on hypoxia-induced oxidative stress and apoptosis in trophoblast cells via muscarinic receptors, indicating that improved vagal activity may be of therapeutic value in PE management.

Ephx2-gene deletion affects acetylcholine-induced relaxation in angiotensin-II infused mice: role of nitric oxide and CYP-epoxygenases.

Previously, we showed that adenosine A2A receptor induces relaxation independent of NO in soluble epoxide hydrolase-null mice (Nayeem et al. in Am J Physiol Regul Integr Comp Physiol 304:R23-R32, 2013). Currently, we hypothesize that Ephx2-gene deletion affects acetylcholine (Ach)-induced relaxation which is independent of A2AAR but dependent on NO and CYP-epoxygenases. Ephx2-/- aortas showed a lack of sEH (97.1%, P < 0.05) but an increase in microsomal epoxide hydrolase (mEH, 37%, P < 0.05) proteins compared to C57Bl/6 mice, and no change in CYP2C29 and CYP2J protein (P > 0.05). Ach-induced response was tested with nitro-L-arginine methyl ester (L-NAME) NO-inhibitor; 10-4 M), N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH) (CYP-epoxygenase inhibitor; 10-5 M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10-5 M), SCH-58261 (A2AAR-antagonist; 10-6 M), and angiotensin-II (Ang-II, 10-6 M). In Ephx2-/- mice, Ach-induced relaxation was not different from C57Bl/6 mice except at 10-5 M (92.75 ± 2.41 vs. 76.12 ± 3.34, P < 0.05). However, Ach-induced relaxation was inhibited with L-NAME (Ephx2-/-: 23.74 ± 3.76% and C57Bl/6: 11.61 ± 2.82%), MS-PPOH (Ephx2-/-: 48.16 ± 6.53% and C57Bl/6: 52.27 ± 7.47%), and 14,15-EEZE (Ephx2-/-: 44.29 ± 8.33% and C57Bl/6: 39.27 ± 7.47%) vs. non-treated (P < 0.05). But, it did not block with SCH-58261 (Ephx2-/-: 68.75 ± 11.41% and C57Bl/6: 66.26 ± 9.43%, P > 0.05) vs. non-treated (P > 0.05). Interestingly, Ang-II attenuates less relaxation in Ehx2-/- vs. C57Bl/6 mice (58.80 ± 7.81% vs. 45.92 ± 7.76, P < 0.05). Our data suggest that Ach-induced relaxation in Ephx2-/- mice depends on NO and CYP-epoxygenases but not on A2A AR, and Ephx2-gene deletion attenuates less Ach-induced relaxation in Ang-II-infused mice.

Cholinergic modulation of spatial learning, memory and navigation.

Spatial learning, including encoding and retrieval of spatial memories as well as holding spatial information in working memory generally serving navigation under a broad range of circumstances, relies on a network of structures. While central to this network are medial temporal lobe structures with a widely appreciated crucial function of the hippocampus, neocortical areas such as the posterior parietal cortex and the retrosplenial cortex also play essential roles. Since the hippocampus receives its main subcortical input from the medial septum of the basal forebrain (BF) cholinergic system, it is not surprising that the potential role of the septo-hippocampal pathway in spatial navigation has been investigated in many studies. Much less is known of the involvement in spatial cognition of the parallel projection system linking the posterior BF with neocortical areas. Here we review the current state of the art of the division of labour within this complex 'navigation system', with special focus on how subcortical cholinergic inputs may regulate various aspects of spatial learning, memory and navigation.

Modeling Perfusion Dynamics in the Skin During Iontophoresis of Vasoactive Drugs Using Single-Pulse and Multiple-Pulse Protocols.

OBJECTIVE: After iontophoresis of vasoactive drugs into the skin, a decrease in perfusion is commonly observed. We delivered vaso-active drugs by iontophoresis using different delivery protocols to study how these affect this decrease in perfusion as measured using LDF. METHODS: We measured skin perfusion during iontophoresis of (ACh), MCh, and NA using a single pulse or separate pulses at different skin sites, and during repeated delivery of ACh at the same site. RESULTS: Perfusion half-life was 6.1 (5.6-6.6) minutes for ACh and 41 (29-69) minutes for MCh (p < 0.001). The maximum response with multiple pulses of ACh iontophoresis was lower than with a single pulse, 30 (22-37) PU vs. 43 (36-50) PU, p < 0.001. Vasoconstriction to NA was more rapid with a single pulse than with multiple pulses. The perfusion half-life of ACh decreased with repeated delivery of ACh at the same site-first 16 (14-18), second 5.9 (5.1-6-9) and third 3.2 (2.9-3.5) minutes, p < 0.001. CONCLUSIONS: The drug delivery protocol affects microvascular responses to iontophoresis, possibly as a result of differences in the dynamics of local drug concentrations. Perfusion half-life may be used as a measure to quantify the rate of perfusion recovery after iontophoresis of vasoactive drugs.

Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3)-AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.

Assessment of lower limb microcirculation: exploring the reproducibility and clinical application of laser Doppler techniques.

PURPOSE OF STUDY: Non-invasive laser Doppler fluximetry (LDF) and laser Doppler imaging (LDI), combined with iontophoresis, have been used to study the microcirculation in a range of clinical conditions including lower limb venous disease. A prerequisite for an accurate measurement tool is that it is reproducible. However, there is currently no literature with respect to the reproducibility of LDF and LDI combined with iontophoresis in the lower limb (in general) and in the upright position (in specific). Furthermore, the two techniques have been used interchangeably by researchers and the association between these two different measurement methods has not been explored, nor have the factors that affect them been well described. Thus the aim of this study was to determine the reproducibility of LDF and LDI with iontophoresis in the lower limb and investigate factors that influence their clinical application. PROCEDURES: Cutaneous microvascular responses in the lower limb were measured in the supine and standing positions using LDF and LDI combined with iontophoretic administration of endothelial-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside) vasodilators in 25 patients with uncomplicated isolated superficial venous incompetence (ISVI) and 26 healthy controls. RESULTS: Maximum perfusion had the best reproducibility assessed by LDF (CV 20.5-24.3%) and LDI (15.8-17.6%). Both techniques were positively influenced by iontophoretic dose (e.g. p = 0.0001 for LDF) and the use of vasodilator agents (e.g. p = 0.0001 for LDF), but negatively influenced in the standing position and/or in the presence of ISVI (p = 0.0016 and 0.045, respectively, for LDF). There was a statistically significant positive relationship between the two techniques, for example ACh maximum perfusion versus LDF ACh maximum perfusion (r = 0.404, p = 0.016). CONCLUSIONS: Both techniques are reproducible, in line with similar studies undertaken in other areas of the human body, and provide useful information for the study of the lower-limb microcirculation. Direct comparison between techniques based on absolute numbers should be avoided and the technique choice should be based on individual study needs.

Reproducibility of laser Doppler fluximetry and the process of iontophoresis in assessing microvascular endothelial function using low current strength.

Iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP) combined with laser Doppler fluximetry (LDF) is a tool used to determine microvascular endothelial function. Our aim was to study the reproducibility of different parameters of this technique using iontophoresis with low current strength on the forearm skin of healthy subjects. Baseline skin perfusion was done before application of five current pulses with 1 min of current-free interval. Current strength of 0.007 mA, current density of 0.01 mA/cm(2) and charge density of 6 mC/cm(2) were used, along with 1% ACh and 1% SNP. The absolute maximum change in perfusion (max), percent change in perfusion (% change), peak change in perfusion (peak) and area under the curve during iontophoresis (AUC) at the anodal and cathodal leads were recorded. Measurements were performed in three sessions for 2 days. The coefficient of variation (CV) was calculated for each parameter. Among the parameters studied, maximum change in perfusion and peak flux were the most reproducible parameters.

A time-response model for analysis of drug transport and blood flow response during iontophoresis of acetylcholine and sodium nitroprusside.

BACKGROUND/AIMS: The analysis of blood flow responses to iontophoresis of vasoactive drugs is often limited to evaluation of maximum responses. In this study, a time-response model is proposed for the blood flow responses to vasoactive drugs applied by iontophoresis. METHODS: The microvascular bed is represented as a single compartment with a zero-order influx of the drugs from the electrode and a first-order clearance due to diffusion and blood flow. The blood flow response to the local drug dose is described using the E(max) model. RESULTS: The model accurately describes the blood flow responses to acetylcholine and sodium nitroprusside during a single iontophoretic current pulse. There is a significant clearance out of the microvascular bed during iontophoresis which depends on the type of drug administered. CONCLUSION: The model enables an accurate estimation of response parameters such as ED50 and maximum response, even if the true maximum blood flow is not obtained. The results suggest that due to clearance from the microvascular bed, the local drug dose during a single pulse of current is not linearly proportional to current strength multiplied by pulse duration.

Quantification of eccrine sweat glands with acetylcholine sweat-spot test and anatomical redistribution of sweating after T2-T3 thoracoscopic sympathicolysis.

BACKGROUND: In this study, patients treated by thoracoscopic sympathicolysis for palmar hyperhidrosis were evaluated to determine the number and response of sweat glands to intradermal acetylcholine stimulus. METHODS: A total of 30 patients were included in the study. Group A consisted of 10 patients with palmar hyperhidrosis who underwent thoracoscopic sympathicolysis in October 2005, and group B consisted of 20 patients who underwent surgery during the years 1999, 2000, and 2001. The study procedure involved applying iodine alcohol to the palm and then intradermally injecting 0.1 ml 1% acetylcholine. This activated the sweat glands, which were then photographed and counted. The study procedure was performed prospectively over different periods in group A and retrospectively in group B. RESULTS: In group A, the mean number of glands activated 1, 3, 6, and 12 months after surgery were 41, 174.20, 522.8, and 747.2, respectively; this gradual increase was statistically significant over the first 6 months (p = 0.004) but not between months 6 and 12 (p = 0.255). The trend towards an increasing number of active glands occurred in both groups, with a mean of 1369.8 active glands in group B compared to 747.2 (p = 0.095) in group A after 12 months. CONCLUSION: It is well-known that Cannon's law of denervation (1939) is not applicable to the sweat glands, that is, there is no hyperactivation following intradermal acetylcholine stimulation. However, some response, which increased over the first 6 months following surgery, was observed in our study. Nevertheless, this activation is subsequently self-limiting, resulting in no gland atrophy, and reinnervation occurs without patient awareness.

The relationship between in vivo nasal drug clearance and in vitro nasal mucociliary clearance: Application to the prediction of nasal drug absorption.

Drug absorption after nasal application is dependent on drug clearance from the nasal cavity, which is determined by nasal mucociliary clearance (MC). We previously developed an in vitro method to evaluate MC via the translocation velocity of fluorescent microspheres (VFMS) applied to excised rat nasal mucosa. In the present study, the relationship between in vivo nasal MC and in vitro VFMS was examined to optimize our PK model for the prediction of nasal drug absorption. Appropriate inhibitors (propranolol and atropine) and enhancers (terbutaline and acetylcholine chloride) of MC were utilized to modify MC. In vivo clearance of drug from the nasal cavity was determined from the disappearance of fluorescent microspheres (FMS) from the nasal cavity following nasal application to rats. The first order elimination rate constant, kmc, was determined from the disappearance profiles of FMS. kmc was decreased to 35.8% by propranolol and 52.6% by atropine, but increased to 117% by terbutaline and 168% by acetylcholine chloride. A significant linear correlation was observed between kmc and VFMS (r2 = 0.9745, p < 0.001). These results indicate that in vivo kmc can be estimated from the in vitro parameter, VFMS. By introducing linear correlation into our PK model, nasal drug absorption may be precisely estimated, even with changes in MC.

Glycine stimulates the release of labeled acetylcholine but not dopamine nor glutamate from superfused rat striatal tissue.

Glycine is known as an inhibitory neurotransmitter in the spinal cord and forebrain but its precise role in the forebrain is largely overlooked. This investigation evaluated whether glycine alters acetylcholine, glutamate or dopamine release from striatal tissue using an in vitro approach. We observed that while glycine induced a robust (3)H-acetylcholine release ((3)H-ACh) from superfused striatal tissue, it failed at releasing (3)H-glutamate or (3)H-dopamine. Glycine stimulated (3)H-ACh release in a dose- and calcium-dependent manner (EC(50)=69 microM). Tetrodotoxin (1 microM) inhibited about 75% of the release demonstrating a predominant dendritic and cell body location of glycine receptors. The prototypical glycine receptor antagonist strychnine at 10 microM completely abolished (3)H-ACh release. To further characterize the role of striatal glycine receptors in (3)H-ACh release we examined glycine effects after in vivo treatment with Haloperidol-decanoate (HD). Treatment for 30 days or more with HD decreased maximal glycine-stimulated release of (3)H-ACh suggesting a non-competitive inhibition. After 30 days of washout release parameters did not return to vehicle-treated levels. The glutamate agonist NMDA also stimulated acetylcholine release but showed slightly different behavior in HD-treated striatal tissue. These effects could be attributed to changes in chloride transporters expressed in the giant striatal cholinergic cell as well as glycine receptor subunit composition and finally, GABA/glycine co-release in this tissue.

Continuum simulations of acetylcholine diffusion with reaction-determined boundaries in neuromuscular junction models.

The reaction-diffusion system of the neuromuscular junction has been modeled in 3D using the finite element package FEtk. The numerical solution of the dynamics of acetylcholine with the detailed reaction processes of acetylcholinesterases and nicotinic acetylcholine receptors has been discussed with the reaction-determined boundary conditions. The simulation results describe the detailed acetylcholine hydrolysis process, and reveal the time-dependent interconversion of the closed and open states of the acetylcholine receptors as well as the percentages of unliganded/monoliganded/diliganded states during the neuro-transmission. The finite element method has demonstrated its flexibility and robustness in modeling large biological systems.

A simple and rapid radiochemical choline acetyltransferase (ChAT) assay screening test.

A simple radiochemical choline acetyltransferase (ChAT) assay screening test was developed by measuring for [(3)H]acetylcholine ([(3)H]ACh) formed from 0.2 mM [(3)H]acetyl-coenzyme A ([(3)H]acetyl-CoA) and 1 mM choline by 0.2 mg of rat brain homogenates containing ChAT into 96-well microplates. A simple and rapid procedure for isolating [(3)H]ACh from the incubation mixture into 96-well microplates was achieved by using a sodium tetraphenylboron (Kalibor) solution (in ethyl acetate, 0.75%, w/v) and a hydrophobic liquid scintillator mixture (1:5, v/v, 0.2 mL) as an extraction solvent. The benefits of this radiochemical method using 96-well microplates are as follows: (1) this method is reliable and reproducible; (2) many samples can be examined at the same time by this method; (3) this method is economical and effective in reducing radioactive waste. The development of a new simple radiochemical ChAT assay screening test is the first stage of development of radiolabeled ChAT mapping agent.

Fine-tuning modulation of myenteric motoneurons by endogenous adenosine: on the role of secreted adenosine deaminase.

Besides the well-characterized inhibitory effect of adenosine in the gastrointestinal tract mediated by A1 receptors, we recently demonstrated that endogenously generated adenosine facilitates [3H]acetylcholine release from myenteric neurons through preferential activation of prejunctional A2A receptors. The co-existence of both receptor subtypes on cholinergic neurons prompted the question of how does adenosine discriminate between these receptors to regulate synaptic transmission in the longitudinal muscle-myenteric plexus (LM-MP) of the rat ileum. Electrical stimulation of the LM-MP increased the outflow of adenosine, inosine and hypoxanthine. Myenteric neurons seem to be the main source of endogenous adenosine, since blockade of action potentials with tetrodotoxin (1 microM) or omission of Ca2+ (plus EGTA, 1 mM) in the buffer essentially abolished nucleosides release, while adenosine outflow remained unchanged when smooth muscle contractions were prevented by nifedipine (1 microM). Inhibition of ecto-5'-nucleotidase by concanavalin A (0.1 mg ml-1) produced only a moderate decrease (approximately 25%) on adenosine accumulation in the LM-MP, indicating that the extracellular catabolism of released ATP might not be a major source of the nucleoside. Data using the acetylcholinesterase inhibitor, physiostigmine (10 microM), and several subtype-specific muscarinic receptor antagonists, 4-DAMP (100 nM), AF-DX 116 (10 microM) and muscarinic toxin-7 (1 nM), suggest that cholinergic motoneurons are endowed with muscarinic M3 autoreceptors facilitating the outflow of adenosine. Surprisingly, bath samples collected after stimulating the LM-MP exhibited a relatively high adenosine deaminase (ADA) activity (0.60+/-0.07 U ml-1), which increased in parallel with the accumulation of adenosine and its deamination products. Our findings are in keeping with the hypothesis that ADA secretion, along with a less-efficient dipyridamole-sensitive nucleoside transport system, may restrict endogenous adenosine actions to the synaptic region channelling to facilitatory A2A receptors activation. Such a local environment may also limit diffusion of exogenously added adenosine towards the active zones, as we showed that this constrain may be overcome by inhibiting ADA activity with erythro-9(2-hydroxy-3-nonyl) adenine (50 microM).

Contribution of Cyclooxygenase End Products and Oxidative Stress to Intrahepatic Endothelial Dysfunction in Early Non-Alcoholic Fatty Liver Disease.

INTRODUCTION: Metabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD. MATERIALS AND METHODS: Sprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels. RESULTS: HFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response. CONCLUSIONS: Our study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease.

Human alpha4beta2 acetylcholine receptors formed from linked subunits.

We prepared concatamers of alpha4 and beta2 subunits for human nicotinic acetylcholine receptors (AChRs), in which the C terminus of alpha4 was linked to the N terminus of beta2, or vice versa, via a tripeptide sequence repeated 6 or 12 times, and expressed them in Xenopus oocytes. Linkage did not substantially alter channel amplitude or channel open-duration. Linkage at the C terminus of alpha4 prevented AChR potentiation by 17-beta-estradiol by disruption of its binding site. Assembly of AChRs from concatamers was less efficient, but function was much more efficient than that of unlinked subunits. With both linked and free subunits, greater ACh-induced currents per surface AChR were observed with the (alpha4)3(beta2)2 stoichiometry than with the (alpha4)2(beta2)3 stoichiometry. The (alpha4)3(beta2)2 stoichiometry exhibited much lower ACh sensitivity. When concatamers were expressed alone, dipentameric AChRs were formed in which the (alpha4)2(beta2)3 pentamer was linked to the (alpha4)3(beta2)2 pentamer. Dipentamers were selectively expressed on the cell surface, whereas most monopentamers with dangling subunits were retained intracellularly. Coexpression of concatamers with monomeric beta2, beta4, or alpha4 subunits resulted in monopentamers, the stoichiometry of which was determined by the free subunit added. Linkage between the C terminus of beta2 and the N terminus of alpha4 favored formation of ACh-binding sites within the concatamer, whereas linkage between the C terminus of alpha4 and the N terminus of beta2 favored formation of ACh-binding sites between concatamers. These protein-engineering studies provide insight into the structure and function of alpha4beta2 AChRs, emphasizing the functional differences between alpha4beta2 AChRs of different stoichiometries.

Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating.

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

Cytoplasmic unsaturated free fatty acids inhibit ATP-dependent gating of the G protein-gated K(+) channel.

This study reports the identification of an endogenous inhibitor of the G protein-gated (K(ACh)) channel and its effect on the K(ACh) channel kinetics. In the presence of acetylcholine in the pipette, K(ACh) channels in inside-out atrial patches were activated by applying GTP to the cytoplasmic side of the membrane. In these patches, addition of physiological concentration of intracellular ATP (4 mM) upregulated K(ACh) channel activity approximately fivefold and induced long-lived openings. However, such ATP-dependent gating is normally not observed in cell-attached patches, indicating that an endogenous substance that inhibits the ATP effect is present in the cell. We searched for such an inhibitor in the cell. ATP-dependent gating of the K(ACh) channel was inhibited by the addition of the cytosolic fraction of rat atrial or brain tissues. The lipid component of the cytosolic fraction was found to contain the inhibitory activity. To identify the lipid inhibitor, we tested the effect of approximately 40 different lipid molecules. Among the lipids tested, only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids (0.2-2 microM) reversibly inhibited the ATP-dependent gating of native K(ACh) channels in atrial cells and hippocampal neurons, and of recombinant K(ACh) channels (GIRK1/4 and GIRK1/2) expressed in oocytes. Unsaturated free fatty acids also inhibited phosphatidylinositol-4, 5-bisphosphate (PIP(2))-induced changes in K(ACh) channel kinetics but were ineffective against ATP-activated background K(1) channels and PIP(2)-activated K(ATP) channels. These results show that during agonist-induced activation, unsaturated free fatty acids in the cytoplasm help to keep the cardiac and neuronal K(ACh) channels downregulated by antagonizing their ATP-dependent gating. The opposing effects of ATP and free fatty acids represent a novel regulatory mechanism for the G protein-gated K(+) channel.

N-[18F] fluoroethylpiperidin-4ylmethyl acetate, a novel lipophilic acetylcholine analogue for PET measurement of brain acetylcholinesterase activity.

The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using (11)C-labeled acetylcholine analogues, N-[(11)C]methylpiperidin-4-yl acetate and propionate, and positron emission tomography (PET). Our aim was to develop an (18)F-labeled acetylcholine analogue useful for brain AChE mapping with PET, since (18)F, with a longer half-life, has advantages over (11)C. In a preliminary study, a series of N-[(14)C]ethylpiperidin-3-yl or -4-ylmethanol esters (acetyl and propionyl esters) were newly designed and evaluated in vitro regarding the reactivity with and specificity to AChE using purified human enzymes, leading to a novel (18)F-labeled acetylcholine analogue, N-[(18)F]fluoroethylpiperidin-4-ylmethyl acetate. In rat experiments, the (18)F-labeled candidate showed desirable properties for PET AChE measurement: high brain uptake of the authentic ester, high AChE specificity, a moderate hydrolysis rate, and low membrane permeability (metabolic trapping) of the metabolite.

Reactivity of urinary bladder smooth muscle in guinea pigs to acetylcholine and carbachol--participation of acetylcholinesterase.

The authors examined the influence of acetylcholinesterase inhibitor (neostigmine) on the in vitro reactivity of urinary bladder smooth muscle (UBSM) in guinea pigs. The aim of the present study was to determine the participation of pharmacokinetic properties of acetylcholine and carbachol in different UBSM reactivity to these mediators. In vitro method of organ baths was used and reactivity of UBSM strips to cumulative doses of acetylcholine and carbachol was tested before and after the incubation with neostigmine (10(-4) mol.l(-1)). Neostigmine caused a significant increase of UBSM reactivity to acetylcholine. The UBSM reactivity to acetylcholine was significantly higher at concentrations of 10(-5) and 10(-4) mol.l(-1) compared to carbachol at the same concentrations. These findings indicate that in addition to different mediator affinity to muscarinic receptors and to their different intrinsic activity, the pharmacokinetic properties of acetylcholine and carbachol also participate in UBSM reactivity.