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.

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.

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.

Brain acetylcholine and choline concentrations and dynamics in a murine model of the Fragile X syndrome: age, sex and region-specific changes.

Fragile X syndrome is a learning disability caused by excess of CGG repeats in the 5' untranslated region of the Fragile X gene (FMR1) silencing its transcription and translation. We used a murine model of this condition, Fmr1 knock-out mice (KO) to study acetylcholine (ACh) metabolism and compared it to that of wild-type control mice (WT). Brain endogenous ACh (D0ACh), free choline (D0Ch), their deuterated variants D4ACh and D4Ch and mole ratios (AChMR and ChMR) were measured by gas chromatography-mass spectrometry in the cerebral hemisphere, cerebral cortex, hippocampus and cerebellum, following D4Ch administration. Regression analysis indicated a significant decrease with age (negative slope) of D4ACh, AChMR, D4Ch and ChMR in WT mice. Age dependence was only present for D4ACh and AChMR in KO mice. Analysis of variance with age as covariate indicated a significant greater D4Ch in the cerebral cortex of KO females when compared to WT females. Contrasts between sexes within genotypes indicated lower D0Ch in cortex and cerebellum of female KO mice but not in WT and lower D4Ch in hippocampus of female KO and WT mice. In conclusion, after adjusting for age, D0ACh concentrations and synthesis from deuterium-labeled Ch were similar in KO and control WT mice in all brain regions. In contrast, significant changes in Ch dynamics were found in hippocampus and cerebral cortex of KO mice that might contribute to the pathogenesis of FXS.

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.

The concept behind sugammadex / El concepto detrás de sugammadex

Sugammadex is the first selective relaxant binding agent. It allows rapid reversal of any degree of neuromuscular blockade induced by steroidal neuromuscular blocking agents. Sugammadex acts by encapsulation of the neuromuscular blocking agent. This prevents the drug from acting on prejunctional and postjunctional nicotinic receptors, allowing acetylcholine to activate these receptors, and resulting in reversal of the neuromuscular blockade. Objective monitoring of the degree of neuromuscular blockade is strongly recommended to determine the optimal dose of sugammadex. A good understanding of the concept behind sugammadex is essential in order to use this reversal agent in clinical practice (AU)

Sugammadex es el primer agente farmacológico de unión selectiva a los bloqueantes neuromusculares. Permite la reversión rápida de cualquier grado de bloqueo neuromuscular producido por agentes bloqueantes neuromusculares esteroideos. Sugammadex actúa mediante la encapsulación del bloqueante neuromuscular. Esto evita la acción de dichos fármacos en los receptores nicotínicos prejuncionales y posjuncionales, permitiendo que la acetilcolina active estos receptores, lo cual resulta en la reversión del bloqueo neuromuscular. Para determinar la dosis óptima de sugammadex se recomienda encarecidamente la monitorización objetiva del grado de bloqueo neuromuscular. Un adecuado conocimiento del concepto en que se basa sugammadex es esencial para el empleo de este reversor en la práctica clínica (AU)

Rehabilitación cognitiva de las personas con síndrome de Down: Perspectivas disciplinares de normalización / No disponible

Los autores reivindican la necesidad de una intervención específica, rehabilitadora y multidisciplinar de las personas con síndrome de Down, que vaya fundamentada en los recientes avances consegudios en las áreas de la neuropsicología, la educación, la genética, la bioquímica, la farmacología y la logopedia), que vaya orientada a la normalización parcial de las personas con síndrome de Down, y tenga en cuenta la totalidad del desarrollo, pero sin descartar el criterio de precocidad de forma absoluta. Animan a las asociaciones que defienden y apoyan a las personas con síndrome de Down a que exijan a las instituciones públicas la financiación necesaria para que: 1) se formen profesionales en estas técnicas de rehabilitación, y 2) se garantice el acceso a todas las personas que lo necesitan a estos nuevos programas de normalización. Es algo de interés general, incluso visto desde una perspectiva puramente económica, ya que cuanto más evolucionada y capaz sea una persona con síndrome de Down desde múltiples puntos de vista, más autónoma será y, por tanto, menos costosa para la sociedad. Asimismo las asociaciones deberían orientar y guiar a los padres en su particular aproximación a las nuevas perspectivas terapéuticas (AU)

No disponible

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

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.

Site-directed decapsulation of bolaamphiphilic vesicles with enzymatic cleavable surface groups.

Stable nano-sized vesicles with a monolayer encapsulating membrane were prepared from novel bolaamphiphiles with choline ester head groups. The head groups were covalently bound to the alkyl chain of the bolaamphiphiles either via the nitrogen atom of the choline moiety, or via the choline ester's methyl group. Both types of bolaamphiphiles competed with acetylthiocholine for binding to acetylcholine esterase (AChE), yet, only the choline ester head groups bound to the alkyl chain via the nitrogen atom of the choline moiety were hydrolyzed by the enzyme. Likewise, only vesicles composed of bolaamphiphiles with head groups that were hydrolyzed by AChE released their encapsulated material upon exposure to the enzyme. Injection of carboxyfluorescein (CF)-loaded vesicles with cleavable choline ester head groups into mice resulted in the accumulation of CF in tissues that express high AChE activity, including the brain. By comparison, when vesicles with choline ester head groups that are not hydrolyzed by AChE were injected into mice, there was no accumulation of CF in tissues that highly express the enzyme. These results imply that bolaamphiphilic vesicles with surface groups that are substrates to enzymes which are highly expressed in target organs may potentially be used as a drug delivery system with controlled site-directed drug release.

Development and validation of a sample stabilization strategy and a UPLC-MS/MS method for the simultaneous quantitation of acetylcholine (ACh), histamine (HA), and its metabolites in rat cerebrospinal fluid (CSF).

A UPLC-MS/MS assay was developed and validated for simultaneous quantification of acetylcholine (ACh), histamine (HA), tele-methylhistamine (t-mHA), and tele-methylimidazolacetic acid (t-MIAA) in rat cerebrospinal fluid (CSF). The biological stability of ACh in rat CSF was investigated. Following fit-for-purpose validation, the method was applied to monitor the drug-induced changes in ACh, HA, t-mHA, and t-MIAA in rat CSF following administration of donepezil or prucalopride. The quantitative method utilizes hydrophilic interaction chromatography (HILIC) Core-Shell HPLC column technology and a UPLC system to achieve separation with detection by positive ESI LC-MS/MS. This UPLC-MS/MS method does not require extraction or derivatization, utilizes a stable isotopically labeled internal standard (IS) for each analyte, and allows for rapid throughput with a 4 min run time. Without an acetylcholinesterase (AChE) inhibitor present, ACh was found to have 1.9±0.4 min in vitro half life in rat CSF. Stability studies and processing modification, including the use of AChE inhibitor eserine, extended this half life to more than 60 min. The UPLC-MS/MS method, including stabilization procedure, was validated over a linear concentration range of 0.025-5 ng/mL for ACh and 0.05-10 ng/mL for HA, t-mHA, and t-MIAA. The intra-run precision and accuracy for all analytes were 1.9-12.3% CV and -10.2 to 9.4% RE, respectively, while inter-run precision and accuracy were 4.0-16.0% CV and -5.3 to 13.4% RE, respectively. By using this developed and validated method, donepezil caused increases in ACh levels at 0.5, 1, 2, and 4h post dose as compared to the corresponding vehicle group, while prucalopride produced approximately 1.6- and 3.1-fold increases in the concentrations of ACh and t-mHA at 1h post dose, respectively, compared to the vehicle control. Overall, this methodology enables investigations into the use of CSF ACh and HA as biomarkers in the study of these neurotransmitter systems and related drug discovery efforts.

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.

Caracterización farmacológica y análisis funcional de los receptores nicotínicos α 9 α 10 en células romafines aisladas de la médula adrenal de rata / Pharmacological characterization and functional analysis of α 9 α 10 acetylcholine nicotinic receptors in isolated chromaffin cells from the rat adrenal medulla

El descubrimiento de receptores nicotínicos (nAChRs) formadospor las subunidades α9 y α10 en las células de los epitelios sensitivosdel sistema vestibular y auditivo ha motivado su búsqueda enestructuras del sistema nervioso autónomo, como las células cromafinesde la médula adrenal, en las que podrían inducir la hiperpolarizaciónde la membrana celular mediante la activación de canales deK+ dependientes de Ca2+ de baja conductancia iónica (canales SK). Elobjetivo fundamental de nuestro trabajo ha sido determinar la presenciay, en su caso, caracterizar funcionalmente el nAChR α9α10 enlas células cromafines de la médula adrenal de la rata. Con ese propósito,hemos empleado cultivos primarios de células cromafines obtenidasde la glándula adrenal de rata y recurrido a la técnica electrofisiológicade patch-clamp para registrar las corrientes iónicasgeneradas por la activación de los nAChRs del conjunto de la membranacelular. Asimismo, hemos empleado la α-conotoxina RgIA, unpéptido capaz de bloquear de forma selectiva los nAChRs formadospor las subunidades α9 y α10. Los resultados obtenidos aportan evidenciasfarmacológicas que permiten concluir que las células cromafinesde la rata expresan nAChRs α9α10 funcionales que, además, desempeñarían un papel modulador comportamiento eléctrico de dichascélulas(AU)

The identification of acetylcholine nicotinic receptors (nAChRs)formed by α9 and α10 subunits in the sensory cells of the vestibularand auditory systems, prompted us to investigate their presence inadrenomedullary chromaffin cells, in which they could mediate membranehyperpolarization through the activation of small-conductanceCa2+-dependent K+ channels (SK channels). The aim of the currentstudy has been to pharmacologically identify α9α10 nAChRs and initiatetheir functional characterization in isolated chromaffin cellsfrom the rat adrenal medulla. We have employed the patch clamptechnique to record either the ionic currents generated by the activationof nAChRs or the associated changes in membrane potential. Wetook advantage of the specificity of α-conotoxin RgIA for the nAChRsformed by α9 and α10 subunits. Our pharmacological results suggestthat the rat chromaffin cells express functional α9α10 nAChRs thatwould influence the electrical behaviour of these cells(AU)

Diffusion-convection effects on drug distribution at the cell membrane level in a patch-clamp setup.

We present a model-based method for estimating the effective concentration of the active drug applied by a pressure pulse to an individual cell in a patch-clamp setup, which could be of practical use in the analysis of ligand-induced whole-cell currents recorded in patch-clamp experiments. Our modelling results outline several important factors which may be involved in the high variability of the electric response of the cells, and indicate that with a pressure pulse duration of 1s and diameter of the perfusion tip of 600 µm, elevated amounts of drug can accumulate locally between the pipette tip and the cell. Hence, the effective agonist concentration at the cell membrane level can be consistently higher than the initial concentration inside the perfusion tubes. We performed finite-difference and finite-element simulations to investigate the diffusion/convection effects on the agonist distribution on the cell membrane. Our model can explain the delay between the commencement of acetylcholine application and the onset of the whole-cell current that we recorded on human rhabdomyosarcoma TE671 cells, and reproduce quantitatively the decrease of signal latency with the concentration of agonist in the pipette. Results also show that not only the geometry of the bath chamber and pipette tip, but also the transport parameters of the diffusive and convective phenomena in the bath solution are determinant for the amplitude and kinetics of the recorded currents and have to be accounted for when analyzing patch-clamp data.

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.

Efecto del clorhidrato de mianserina sobre preparaciones de órgano aislado de cobaya y de rata "in vitro" / Effects of mianserin hydrochloride on isolated organ preparations of guinea-pig and rat "in vitro"

OBJETIVO. Estudiar las modificaciones inducidas por Mianserina en las respuestas del ileon aislado de cobaya a acetilcolina e histamina, conducto deferente de rata a noradrenalina y útero aislado de rata a histamina. MATERIAL Y MÉTODOS. Se utilizó ileon aislado de cobaya incubado en solución de Tyrode, conducto deferente de rata incubado en solución de Krebs- Henseleit y útero aislado de rata incubado en solución de Jalón. Se realizaron curvas dosis-efecto a acetilcolina, histamina y noradrenalina en ausencia y en presencia de mianserina y se calculó el pA2. RESULTADOS. La mianserina se comporta como antagonista de los neurotransmisores estudiados. Conclusión: La mianserina se comporta como fármaco escasamente estabilizador inespecífico de membrana (AU)

OBJETIVE. Study the modifications produced by mianserin in the responses of isolated guinea-pig to acetylcholine and histamine, rat vas deferens to noradrenalinee and rat uterus to histamine. MATERIAL AND METHODS. Guinea-Pig ileum incubated in Tyrode solution were used. Dose-effect curves to acetylcholine and histamine were made in absence and in the presence of mianserin. Rat vas deferens incubated in Krebs-Henseleit solution were used. Dose-effect curves to noradrenaline were made in absence and in the presence of mianserin. Uterus of rat incubated in Jalon solution were used. Dose-effect curves to histamine were made in absence and in the presence of mianserin, pA2 was calculated. RESULTS. Mianserin behave as antagonist of acethylcholine, histamine and noradrenaline. Conclusions: Mianserin acts as few unspecific membrane stabilizer (AU)