High Concentrations Of Acetylcholine Research Articles

Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, andEDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 μM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 μM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

Interactions with amyloid beta peptide and acetylcholinesterase increase alkaline phosphatase activity.

Multiple studies have shown that the activity of alkaline phosphatase (AP) increases during Alzheimer's disease (AD). In this paper, using UV-Visible spectroscopy, we show that this increase in activity is due to its interaction with key components of AD such as amyloid β peptide and acetylcholinesterase. Activity increase also occurs due to high concentrations of acetylcholine and choline. These conditions are present in AD or could occur due to drugs used for treating AD.

Abstract 15832: Optogenetic Stimulation of Intrinsic Cardiac Autonomic Neurons Reveals the Interaction Between Catecholaminergic and Cholinergic Pathways

Autonomic tone, through the interaction of catecholaminergic and cholinergic pathways, is a powerful modulator of cardiac function. Until recently, selective activation of cardiac autonomic pathways has required administration of exogeneous neurotransmitters or careful placement of electrodes to stimulate specific neural populations. Optogenetics enables broader exploration of autonomic interactions through the selective expression of channelrhodopsin (ChR2) in neural populations. Our objective was to simultaneously activate catecholaminergic and cholinergic pathways, by exogenous perfusion of neurotransmitter and photostimulation of opposing autonomic neurons, to test the hypothesis that simultaneous activation will reveal the fast kinetics of cholinergic modulation of catecholaminergic increases in heart rate (HR). Norepinephrine (NE) and acetylcholine (ACh) were administered to perfused transgenic mouse hearts that expressed ChR2 within sympathetic tyrosine hydroxylase neurons (TH+ChR2) or parasympathetic choline acetyltransferase (ChAT+ChR2) neurons. Intrinsic neurons were photostimulated by illuminating the SA node with a 465nm high power microLED. The ECG was recorded to measure beat-to-beat changes in HR. Moderate doses of exogeneous NE increased HR 26.77±5.61% (n=3) and high doses increased HR 59.74±8.77% (n=3). At all NE doses, photostimulation of ChAT+ChR2 neurons immediately reduced HR to values equivalent to, or lower than, pre-NE HR. Arrhythmias were not observed. In companion studies, moderate doses of exogeneous ACh reduced HR 19.98±4.68% (n=7) and high doses reduced HR 51.31±4.07% (n=7). HR increases caused by TH+ChR2 neuron photostimulation were diminished by exogenous ACh in a dose-dependent manner. Increased dosing of exogeneous ACh, with simultaneous TH+ChR2 neuron photostimulation, dramatically shortened the onset of 2 nd degree AV block and increased its severity. At high concentrations of exogeneous ACh, non-sustained arrhythmias were observed upon the cessation of TH+ChR2 neuron photostimulation. These results provide new insight into the kinetics of intrinsic cardiac autonomic interactions and reveal powerful modulation of catecholaminergic activity by cholinergic activation.

Autophagy, Acute Pancreatitis and the Metamorphoses of a Trypsinogen-Activating Organelle.

Recent studies have highlighted the importance of autophagy and particularly non-canonical autophagy in the development and progression of acute pancreatitis (a frequent disease with considerable morbidity and significant mortality). An important early event in the development of acute pancreatitis is the intrapancreatic activation of trypsinogen, (i.e., formation of trypsin) leading to the autodigestion of the organ. Another prominent phenomenon associated with the initiation of this disease is vacuolisation and specifically the formation of giant endocytic vacuoles in pancreatic acinar cells. These organelles develop in acinar cells exposed to several inducers of acute pancreatitis (including taurolithocholic acid and high concentrations of secretagogues cholecystokinin and acetylcholine). Notably, early trypsinogen activation occurs in the endocytic vacuoles. These trypsinogen-activating organelles undergo activation, long-distance trafficking, and non-canonical autophagy. In this review, we will discuss the role of autophagy in acute pancreatitis and particularly focus on the recently discovered LAP-like non-canonical autophagy (LNCA) of endocytic vacuoles.

On the relationship between inhibition and receptor occupancy by nondepolarizing neuromuscular blocking drugs

BackgroundNondepolarizing neuromuscular blocking drugs (NDNBs) are clinically used to produce muscle relaxation during general anesthesia. To better understand clinical properties of NDNBs, comparative in vitro pharmacologic studies have been performed. In these studies, a receptor binding model, which relies on the assumption that the inhibition, i.e., the effect of an NDNB, is proportional to the receptor occupancy by the drug, has been effectively used to describe obtained experimental data. However, it has not been studied in literature under which conditions the above assumption can be justified nor the assumption still holds in vivo. The purpose of this study is to explore the in vivo relationship between the inhibition and the receptor occupancy by an NDNB and to draw implications on how in vitro experimental results can be used to discuss the in vivo properties of NDNBs.MethodsAn ordinary differential equation model is employed to simulate physiologic processes of the activation of receptors by acetylcholine (ACh) as well as inhibition by an NDNB. With this model, the degree of inhibition is quantified by the fractional amount of receptors that are not activated by ACh due to the presence of an NDNB. The results are visualized by plotting the fractional amounts of the activated receptors as a function of the receptor occupancy.ResultsNumerical investigations reflecting in vivo conditions show that the degree of inhibition is not proportional to the receptor occupancy, i.e., there is a nonlinear relationship between the inhibition and the receptor occupancy. However, under a setting of high concentration of ACh reflecting a typical situation of in vitro experiments, the relationship between the inhibition and the receptor occupancy becomes linear, suggesting the validity of the receptor binding model. Also, it is found that the extent of nonlinearity depends on the selectivity of NDNBs for the two binding sites of the receptors.ConclusionsWhile the receptor binding model may be effective for estimating affinity of an NDNB through in vitro experiments, these models do not directly describe in vivo properties of NDNBs, because the nonlinearity between the inhibition and the receptor occupancy causes the modulation of the resultant concentration-effect relationships of NDNBs.

Pro‐Resolving Lipid Mediators Reduce Acetylcholine‐Induced Contractions in Resistance Arteries from Hypertensive Rats

Aims It is well known that low-grade chronic inflammation induces vascular dysfunction and contributes to hypertension. On the other hand, resolution of inflammation is an active phenomenon to switch off the inflammatory processes once the harmful stimuli is removed and facilitate the return to homeostasis. Increasing the levels of pro-resolving mediators to promote the resolution of inflammation is emerging as a novel therapeutic approach. Arachidonic acid, docosahexaenoic acid, and eicosapentaenoic acid produce pro-resolving lipid mediators lipoxin A4 (LXA4), resolvin D1 (RvD1), and resolvin E1 (RvE1), respectively, through the 5-lipoxygenase enzymatic pathway. However, it is unknown if pro-resolving lipid mediators can ameliorate dysfunction in arteries from hypertensive animals. Therefore, we hypothesized that pro-resolving lipid mediators decrease acetylcholine-induced contractions in arteries from spontaneously hypertensive rats (SHR). Methods Mesenteric resistance arteries (MRA) (lumen diameter ~ 250 µm) from male SHR and Wistar Kyoto (WKY) (14-weeks old, n=3) were collected to assess vascular function via wire myograph. After testing vascular smooth muscle cell integrity, MRA were incubated with either RvD1, RvE1, or LXA4 (10 nM, 1 hour) or vehicle prior to concentration response curves to acetylcholine (1 nM - 3 µM). Phenylephrine (10 µM) was used to contract the arteries prior relaxation curves. Results As expected, low concentrations (≤ 100 nM) of acetylcholine induced relaxation in arteries from both groups, however high concentrations (≥ 1 µM) of acetylcholine induced contraction in arteries from SHR, but not in WKY [Maximum response (Emax): WKY: 94.5 ± 3.3 vs. SHR-control: 44.9 ± 12.5* %, t-test *vs. WKY, p=0.01). Treatment with the pro-resolving lipid mediators did not change acetylcholine-induced relaxation in arteries from WKY (Vehicle: 94.5 ±3.3 vs. RvD1: 95.8 ± 0.8; RvE1: 90.1 ± 7.1; LXA4: 93.5 ± 3.9 %, p>0.05). However, incubation with RvE1 abolished acetylcholine induced-contraction in arteries from SHR and promoted relaxation (Vehicle: 44.9 ± 12.5 vs. RvE1: 94.1 ± 3.5%,* t-test *vs. vehicle, p<0.05). While acetylcholine-induced contraction abolishment from incubation with RvD1 and LXA4 did not reach significance due to small sample size in arteries from SHR, we did observe a tendency towards improvement. Conclusion Overall, these results suggest that pro-resolving lipid mediators biosynthesized from the 5-lipoxygenase enzymatic pathway improved endothelium-dependent relaxation in arteries from hypertensive animals. As docosahexaenoic acid and eicosapentaenoic acid are already recommended to protect against many cardiovascular diseases, our work suggests that RvD1, RvE1, or LXA4 may also be used as a new therapeutic tool to specifically improve vascular function in hypertension.

Consciousness, Anesthesia, and Acetylcholine.

Consciousness, Anesthesia, and Acetylcholine.

The Cholinergic System Contributes to the Immunopathological Progression of Experimental Pulmonary Tuberculosis.

The cholinergic system is present in both bacteria and mammals and regulates inflammation during bacterial respiratory infections through neuronal and non-neuronal production of acetylcholine (ACh) and its receptors. However, the presence of this system during the immunopathogenesis of pulmonary tuberculosis (TB) in vivo and in its causative agent Mycobacterium tuberculosis (Mtb) has not been studied. Therefore, we used an experimental model of progressive pulmonary TB in BALB/c mice to quantify pulmonary ACh using high-performance liquid chromatography during the course of the disease. In addition, we performed immunohistochemistry in lung tissue to determine the cellular expression of cholinergic system components, and then administered nicotinic receptor (nAChR) antagonists to validate their effect on lung bacterial burden, inflammation, and pro-inflammatory cytokines. Finally, we subjected Mtb cultures to colorimetric analysis to reveal the production of ACh and the effect of ACh and nAChR antagonists on Mtb growth. Our results show high concentrations of ACh and expression of its synthesizing enzyme choline acetyltransferase (ChAT) during early infection in lung epithelial cells and macrophages. During late progressive TB, lung ACh upregulation was even higher and coincided with ChAT and α7 nAChR subunit expression in immune cells. Moreover, the administration of nAChR antagonists increased pro-inflammatory cytokines, reduced bacillary loads and synergized with antibiotic therapy in multidrug resistant TB. Finally, in vitro studies revealed that the bacteria is capable of producing nanomolar concentrations of ACh in liquid culture. In addition, the administration of ACh and nicotinic antagonists to Mtb cultures induced or inhibited bacterial proliferation, respectively. These results suggest that Mtb possesses a cholinergic system and upregulates the lung non-neuronal cholinergic system, particularly during late progressive TB. The upregulation of the cholinergic system during infection could aid both bacterial growth and immunomodulation within the lung to favor disease progression. Furthermore, the therapeutic efficacy of modulating this system suggests that it could be a target for treating the disease.

Acetylcholinesterase and α-Amylase inhibitors from Mouriri elliptica Martius leaf extract

Population growth has raised food production, and new sources are needed to increase quantity and quality of agricultural products. Carbamates and organophosphates are insecticide classes used worldwide as acetylcholinesterase (AChE) inhibitors. Plants have a natural resistance to insects, which can be employed in pest control as a new alternative to reduce the use of chemicals. An alternative may be the use of α-amylase inhibitors, which are digestive enzymes that impair pest species growth and development. Another would be acetylcholinesterase inhibitors since they damage the normal functioning of the central and peripheral nervous system, by releasing high concentrations of acetylcholine in cholinergic synapses. This substance accumulation increases stimulations that lead to behavioral changes, asphyxia, hyperactivity, and death. Botanical agrochemicals are believed to have advantages over synthetic ones, as they are rapidly degraded in the environment. In this scenario, plants have played an important role in pest control as sources of interest for the synthesis of new molecules for agricultural use. The present study evaluated acetylcholinesterase and α-amylase inhibition by microplate method, from leaf extracts of Mouriri elliptica Martius with different polarities.

Distinctive single-channel properties of α4β2-nicotinic acetylcholine receptor isoforms.

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings.

Investigation of the Roles of Non-neuronal Acetylcholine in Chronic Myeloid Leukemic Cells and their Erythroid or Megakaryocytic Differentiated Lines.

Many studies suggested that Acetylcholine (ACh) might serve as an autocrine/ paracrine growth factor in several types of tumors or tumor cell lines. High levels of Acetylcholinesterase (AChE) activity have been reported in primary brain tumors, ovarian, colon and lung tumors. The role of cholinergic signaling needs to be clarified in in leukemia. K562 cells were derived from a chronic myelogenous leukemia patient during blast crisis serving as pluripotent hematopoietic stem cells. K562 cells were incubated with various cholinergic agonists or antagonists to investigate the role of ACh in different differentiated cell lines. Our experiments showed that AChE activity was increased in response to ACh in undifferentiated K562 cells, but in the erythroid differentiated K562 cells a high concentration of ACh (1 mM) decreased the AChE activity. ACh failed to elevate the AChE activity in the megakaryocytic differentiated K562 cells. An AChE inhibitor, eserine, also suppressed the AChE activity in a concentration-dependent manner. Choline uptake inhibition by hemicholinium did increase the AChE activity but not in the erythroid differentiated K562 cell line. Likewise, megakaryocytic differentiated K562 cells also displayed a similar pattern. Vesamicole, a vesicular choline uptake inhibitor, produced similar results. Curare, a nicotinic antagonist, elevated the cell counts of the megakaryocytic differentiated cells. Our findings may suggest excess extracellular ACh will decrease the cell growth in undifferentiated and megakaryocytic differentiated K562 cell lines through nicotinic type cholinoceptors.

Data-Driven Modeling of Cholinergic Modulation of Neural Microcircuits: Bridging Neurons, Synapses and Network Activity.

Neuromodulators, such as acetylcholine (ACh), control information processing in neural microcircuits by regulating neuronal and synaptic physiology. Computational models and simulations enable predictions on the potential role of ACh in reconfiguring network activity. As a prelude into investigating how the cellular and synaptic effects of ACh collectively influence emergent network dynamics, we developed a data-driven framework incorporating phenomenological models of the physiology of cholinergic modulation of neocortical cells and synapses. The first-draft models were integrated into a biologically detailed tissue model of neocortical microcircuitry to investigate the effects of levels of ACh on diverse neuron types and synapses, and consequently on emergent network activity. Preliminary simulations from the framework, which was not tuned to reproduce any specific ACh-induced network effects, not only corroborate the long-standing notion that ACh desynchronizes spontaneous network activity, but also predict that a dose-dependent activation of ACh gives rise to a spectrum of neocortical network activity. We show that low levels of ACh, such as during non-rapid eye movement (nREM) sleep, drive microcircuit activity into slow oscillations and network synchrony, whereas high ACh concentrations, such as during wakefulness and REM sleep, govern fast oscillations and network asynchrony. In addition, spontaneous network activity modulated by ACh levels shape spike-time cross-correlations across distinct neuronal populations in strikingly different ways. These effects are likely due to the regulation of neurons and synapses caused by increasing levels of ACh, which enhances cellular excitability and decreases the efficacy of local synaptic transmission. We conclude by discussing future directions to refine the biological accuracy of the framework, which will extend its utility and foster the development of hypotheses to investigate the role of neuromodulators in neural information processing.

Endothelial Dysfunction as a result of Hypercaloric Intake: Underlying Mechanism in Absence of Hyperglycemia

Endothelial dysfunction is a common occurrence in diabetes, secondary to hyperglycemia. However, little is known about the mechanism underlying the early endothelial damage occurring in the course of diabetes development prior to impaired glycemic control. We used a high‐calorie (HC) diet rat model with delayed development of hyperglycemia to address this question. Aortic rings from HC rats exhibited impaired endothelium‐dependent relaxation of phenylephrine (PE)‐induced tone to acetylcholine (ACh), where higher concentrations of ACh caused an increase in contractile tone. On the other hand, similar relaxation patterns to the exogenous nitric oxide (NO) donor, sodium nitroprusside, were observed indicating that the defect is endothelial in origin. Hence, the possible role of an endothelium‐derived contractile agent, such as endothelin‐1 and 20‐HETE, or an increased production of reactive oxygen species due to uncoupling of endothelial NO synthase, as the basis of increased tone caused by higher ACh concentrations was ruled out. In aortic rings from control rats, endothelium‐dependent relaxation to ACh was comprised of a major NO component with a lesser contribution from inward rectifier potassium channel (Kir)‐dependent mechanisms, particularly at higher ACh concentrations. While both components of the response were affected, HC‐feeding appears to abolish the Kir‐dependent pathway completely. Protein levels of Kir 2.1 subunit were down‐regulated in aortic tissues from HC‐fed rats. Using U‐46619, a thromboxane analogue, to evoke contraction involving calcium sensitization, rather than depolarization, aortic rings from control rats demonstrated a NO‐dependent, hyperpolarization‐independent pattern of relaxation in response to ACh. Despite a slightly more blunted response to ACh, HC rings pre‐contracted with U‐46619 did not show the terminal contraction observed in tissues contracted with PE. These results potentially implicate impaired myoendothelial coupling as the underlying mechanism for endothelial dysfunction in diabetes. Further examination of the role and expression of gap junctions in these tissues in under way.Support or Funding InformationSupported by AUB MPP fund #320148This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High‐calorie Diet Induces Vascular and Hemodynamic Abnormalities in Absence of Change in Blood Glucose or Insulin Levels: Modulation by Oral Anti‐hyperglycemic Drugs

While the effect of hyperglycemia associated with diabetes on vascular function is well‐established by experimental and clinical evidence, few studies examined the early onset vascular changes occurring within the context of diabetes development before the establishment of frank hyperglycemia. Moreover, recent clinical evidence suggests a vasculoprotective effect for anti‐hyperglycemic drugs that is not a direct consequence of their effect on blood glucose level. Thus, an early intervention with diabetic vascular complications, be it through anti‐hyperglycemic drugs or otherwise, requires an adequate understanding of the underlying pathophysiological mechanism. In this study, we used a high calorie‐fed rat model, known to develop stable fasting hyperglycemia on long‐term feeding, to study changes in vascular function prior to the development of diabetes. Rats were fed a high fat (15% saturated fat), high fructose (20%) diet for 12 weeks, at which point there was no increase in fasting serum glucose, serum insulin, nor mean arterial pressure (MAP). Aortic rings from these rats showed an increased contractile response to phenylephrine (PE) compared to rings from age‐matched control animals, with an increased sensitivity (reduced EC50). The PE‐induced contractions were resistant to Ca‐free physiological solution and appeared to be more sensitive to Rho kinase inhibition. In parallel, the hypercontractile phenotype was manifested as an increase in the pressor effect of PE upon administration in vivo with a poor reflex bradycardiac response, despite the absence of a change in basal MAP. On the other hand, aortic rings from the high‐calorie fed rats showed an impaired endothelium‐dependent relaxation in response to acetylcholine (ACh), with increased constriction with higher ACh concentrations. Cyclo‐oxygenase‐mediated relaxation appeared to be most affected in these vessels, whereas nitric oxide donor‐mediated relaxations were not affected. Both ACh‐mediated relaxation and constriction were abolished in denuded rings from high‐calorie fed rats or upon treatment with a nitric oxide synthase inhibitor. Again, the altered dilatory effect was reflected in a reduced in vivo depressor response. Histopathology and immunohistochemistry showed an increased intimal thickness of aorta from these rats together with increased TGF‐β1 and Smad 3 expression. A two‐week treatment with either pioglitazone or metformin reversed the increased contractility to PE, increased intimal thickness, and increased TGF‐β1 expression with no change in fasting serum glucose. Our results suggest that early vascular dysfunction associated with high calorie intake involve inflammatory changes that could be linked to adipose inflammation. Anti‐hyperglycemic drugs showed a possible vascular anti‐inflammatory effect that is distinct from their effect on glycemic control.Support or Funding InformationFaculty of Medicine Bridge Fund, American University of Beirut

Endothelial dysfunction of internal thoracic artery graft in patients with chronic kidney disease

ObjectivesThe present study was designed to evaluate the association between chronic kidney disease and the endothelial function of internal thoracic artery (ITA) grafts in patients undergoing coronary bypass surgery. An isometric tension study was performed in ITA strips obtained during surgery. Concentration-response curves for acetylcholine (ACh) and sodium nitroprusside were constructed in ITA strips partially precontracted with phenylephrine under the inhibition of cyclooxygenase. The integrity of the endothelium was verified histologically by en-face staining of the luminal surface with the use of silver nitrate solution. ResultsIn endothelium-intact ITA strips, ACh produced a concentration-dependent relaxation in patients with glomerular filtration rate (GFR, mL/min/1.73 m2) > 60. A concentration-dependent relaxation response also was observed in patients with GFR 30 to 60, but it was reduced significantly compared with those with GFR > 60. In both groups, removal of endothelium or treatment with nitric oxide (NO) synthase inhibitors almost abolished the ACh-induced relaxation. On the other hand, in patients with GFR < 30, mild contraction rather than relaxation was induced at a high concentration of ACh, which was modified neither by treatment with NO synthase inhibitors nor by removal of the endothelium. Vasodilator responses to sodium nitroprusside were comparable among the 3 groups. The relaxation of endothelium-intact strips to a peak ACh concentration correlated positively with GFR. This relationship held true in a multiple linear regression model, and interaction terms between GFR and other risk factors were not statistically significant. ConclusionsEndothelial function of ITA grafts to release NO is impaired at the time of surgery in patients with chronic kidney disease.

Honeybees Produce Millimolar Concentrations of Non-Neuronal Acetylcholine for Breeding: Possible Adverse Effects of Neonicotinoids.

The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4–8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75–90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food.

Inducibility of human atrial fibrillation in an in silico model reflecting local acetylcholine distribution and concentration.

Vagal nerve activity has been known to play a crucial role in the induction and maintenance of atrial fibrillation (AF). However, it is unclear how the distribution and concentration of local acetylcholine (ACh) promotes AF. In this study, we investigated the effect of the spatial distribution and concentration of ACh on fibrillation patterns in an in silico human atrial model. A human atrial action potential model with an ACh-dependent K+ current (IKAch) was used to examine the effect of vagal activation. A simulation of cardiac wave dynamics was performed in a realistic 3D model of the atrium. A model of the ganglionated plexus (GP) and nerve was developed based on the "octopus hypothesis". The pattern of cardiac wave dynamics was examined by applying vagal activation to the GP areas or randomly. AF inducibility in the octopus hypothesis-based GP and nerve model was tested. The effect of the ACh concentration level was also examined. In the single cell simulation, an increase in the ACh concentration shortened APD90 and increased the maximal slope of the restitution curve. In the 3D simulation, a random distribution of vagal activation promoted wavebreaks while ACh secretion limited to the GP areas did not induce a noticeable change in wave dynamics. The octopus hypothesis-based model of the GP and nerve exhibited AF inducibility at higher ACh concentrations. In conclusion, a 3D in silico model of the GP and parasympathetic nerve based on the octopus model exhibited higher AF inducibility with higher ACh concentrations.

Anesthetic actions of thiopental remain largely unaffected during cholinergic overstimulation in cultured cortical networks.

In case of military or terrorist use of organophosphorus (OP) compounds victims are likely to suffer from not only intoxication but physical trauma as well. Appropriate emergency care may therefore include general anesthesia to allow life-saving surgical intervention. Since there is evidence that drug potency and efficacy of several anesthetics are attenuated by high concentrations of acetylcholine in the CNS, this study was designed to evaluate the anesthetic actions of thiopental during cholinergic overstimulation. Making use of organotypic slice cultures derived from the mouse neocortex, drug effects were assessed by extracellular voltage recordings of network activity at basal cholinergic tone and during simulated cholinergic crisis (high cholinergic tone). The latter was achieved by inhibition of acetylcholinesterases via soman and an ambient acetylcholine concentration of 10μM. The induction of cholinergic crisis in vitro increased the network activity of cortical neurons significantly. Surprisingly, differences in network activity between basal and high cholinergic tone became less pronounced with rising concentrations of thiopental and drug potency and efficacy were almost equivalent. These results clearly distinguish thiopental from previously tested general anesthetics and make it a promising candidate for in vivo studies to identify suitable anesthetics for victims of OP intoxication.

The nicotinic acetylcholine receptor alpha 4 subunit contains a functionally relevant SNP Haplotype

BackgroundNon-coding single nucleotide polymorphisms within the nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4) are robustly associated with various neurological and behavioral phenotypes including schizophrenia, cognition and smoking. The most commonly associated polymorphisms are located in exon 5 and segregate as part of a haplotype. So far it is unknown if this haplotype is indeed functional, or if the observed associations are an indirect effect caused by linkage disequilibrium with not yet identified adjacent functional variants. We therefore analyzed the functional relevance of the exon 5 haplotype alleles.ResultsUsing voltage clamp experiments we were able to show that the CHRNA4 haplotype alleles differ with respect to their functional effects on receptor sensitivity including reversal of receptor sensitivity between low and high acetylcholine concentrations. The results indicate that underlying mechanisms might include differences in codon usage bias and changes in mRNA stability.ConclusionsOur data demonstrate that the complementary alleles of the CHRNA4 exon 5 haplotype are functionally relevant, and might therefore be causative for the above mentioned associations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0204-1) contains supplementary material, which is available to authorized users.

CrossTalk opposing view: Rotors have not been demonstrated to be the drivers of atrial fibrillation.

Despite extensive research, it is still not clear how atrial fibrillation (AF) perpetuates itself. Experimental studies have shown that various mechanisms may sustain this ‘eternal’ arrhythmia (Lewis et al. 1921; Moe & Abildskov, 1959; Moe, 1962; Allessie et al. 1977, 1985; Konings et al. 1994; Mandapati et al. 2000; Jalife et al. 2002; Waldo, 2003). Roughly, they can be divided into focal (repetitive ectopic discharges) and reentrant mechanisms (mother-wave, rotor, multiple wavelets). Recently, we introduced a third mechanism, independent of focal or reentrant activity (the double layer hypothesis; Allessie et al. 2010; de Groot et al. 2010; Eckstein et al. 2011). Using high-resolution optical mapping in isolated sheep hearts subjected to high concentrations of acetylcholine, Jalife and co-workers clearly demonstrated that pacing-induced AF was due to the presence of a very rapid rotor (15–20 Hz) in the left atrium (Mandapati et al. 2000; Jalife et al. 2002). However, although it is unquestionable that under experimental conditions a single rapid rotor can serve as a ‘driver’ of AF, the question still remains whether this is also the operative mechanism in patients with longlasting persistent AF. Based on a series of clinical studies, in which the electrical activity of the atria was mapped by two basket catheters, Narayan et al. (2012a,2012b,2012c, 2013) postulated that human AF is due to the presence of a stable rotor that serves as a ‘driver’ to sustain AF. Ablation of the centre of these rotors abruptly terminated or consistently slowed AF in the vast majority of cases, and substantially improved long-term freedom from AF compared to conventional ablation alone (Narayan et al. 2012b).