Low Concentrations Of Acetylcholine Research Articles

Simultaneous Arrival of Multiple Agonists: How the Endothelium Senses and Responds

The endothelium is the innermost layer of all blood vessels. It controls numerous cardiovascular functions including vascular contractility, haemostasis, inflammation and the exchange of nutrients and waste products between circulating blood and tissue. To control each cardiovascular function, the endothelium processes and responds to endless streams of information that originate from multiple sources (i.e., blood cells, hormones, neighbouring endothelial cells or underlying smooth muscle cells). Although each endothelial cell has only a limited number of interconnected neighbors (on average six) it is able to decipher and respond to multiple streams of information. To process information, specific cells are primed to detect specific activators and these spatially-distinct populations communicate across the endothelial network to coordinate vascular responses. However, how the endothelium integrates and deciphers information when multiple activators are present simultaneously is not understood. Here, by examining the Ca2+ response in thousands of endothelial cells in intact arteries, we show that the interactions of individual endothelial cells give rise to new behaviours that are absent when cells are examined in isolation. In our study, we examined the Ca²⁺ activity in thousands of endothelial cells in intact rat mesenteric arteries exposed to low concentrations (EC25) of acetylcholine, ATP, or both simultaneously. Our findings reveal that cells which respond to acetylcholine but not ATP (‘acetylcholine-sensitive’), exhibit an enhanced Ca²⁺ response when both activators are present together. The same is true for ‘ATP-sensitive’ cells (i.e., cells that do not respond to acetylcholine, EC25) - the Ca2+ response is augmented when both agonists are present. In addition, the number of neighbours that ‘acetylcholine-sensitive’ or ‘ATP-sensitive’ cells have that respond to either agonist, significantly impacts how the Ca2+ response is altered. Interestingly, the augmented response does not conform to a simple linear summation or average of the acetylcholine/ ATP responses. Instead, a new distinct signal is generated. This suggests that cells perform signaling computations and can combine information from multiple sources. New features that appear in a system and which differ from the expectations of the sum of the components are called ‘emergent properties’. Emergent systems arise from non-linear interactions and create new collective behaviors that make the whole (the endothelium) much greater than the sum of the parts (individual endothelial cells). Our data reveals how communication among neighboring endothelial cells results in emergent behavior across the endothelium, and how this novel behavior plays a significant role in influencing cardiovascular function. British Heart Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Label free impedance based acetylcholinesterase enzymatic biosensors for the detection of acetylcholine

Realtime monitoring of neurotransmitters is of great interest for understanding their fundamental role in a wide range of biological processes in the central and peripheral nervous system, as well as their role, in several degenerative brain diseases. The measurement of acetylcholine in the brain is particularly challenging due to the complex environment of the brain and the low concentration and short lifetime of acetylcholine. In this paper, we demonstrated a novel, label-free biosensor for the detection of Ach using a single enzyme, acetylcholinesterase (ACHE), and electrochemical impedance spectroscopy (EIS). Acetylcholinesterase was covalently immobilized onto the surface of gold microelectrodes through an amine-reactive crosslinker dithiobis(succinimidyl propionate) (DSP). Passivation of the gold electrode with SuperBlock eliminated or reduced any non-specific response to other major interfering neurotransmitter molecules such as dopamine (DA), norepinephrine (NE) and epinephrine (EH). The sensors were able to detect acetylcholine over a wide concentration range (5.5–550 μM) in sample volumes as small as 300 μL by applying a 10 mV AC voltage at a frequency of 500 Hz. The sensors showed a linear relationship between Ach concentration and ΔZmod(R2 = 0.99) in PBS. The sensor responded to acetylcholine not only when evaluated in a simple buffer (PBS buffer) but in several more complex environments such as rat brain slurry and rat whole blood. The sensor remained responsive to acetylcholine after being implanted ex vivo in rat brain tissue. These results bode well for the future application of these novel sensors for real time in vivo monitoring of acetylcholine.

Cell-Type Specific Neuromodulation of Excitatory and Inhibitory Neurons via Muscarinic Acetylcholine Receptors in Layer 4 of Rat Barrel Cortex.

The neuromodulator acetylcholine (ACh) plays an important role in arousal, attention, vigilance, learning and memory. ACh is released during different behavioural states and affects the brain microcircuit by regulating neuronal and synaptic properties. Here, we investigated how a low concentration of ACh (30 μM) affects the intrinsic properties of electrophysiologically and morphologically identified excitatory and inhibitory neurons in layer 4 (L4) of rat barrel cortex. ACh altered the membrane potential of L4 neurons in a heterogeneous manner. Nearly all L4 regular spiking (RS) excitatory neurons responded to bath-application of ACh with a M4 muscarinic ACh receptor-mediated hyperpolarisation. In contrast, in the majority of L4 fast spiking (FS) and non-fast spiking (nFS) interneurons 30 μM ACh induced a depolarisation while the remainder showed a hyperpolarisation or no response. The ACh-induced depolarisation of L4 FS interneurons was much weaker than that in L4 nFS interneurons. There was no clear difference in the response to ACh for three morphological subtypes of L4 FS interneurons. However, in four morpho-electrophysiological subtypes of L4 nFS interneurons, VIP+-like interneurons showed the strongest ACh-induced depolarisation; occasionally, even action potential firing was elicited. The ACh-induced depolarisation in L4 FS interneurons was exclusively mediated by M1 muscarinic ACh receptors; in L4 nFS interneurons it was mainly mediated by M1 and/or M3/5 muscarinic ACh receptors. In a subset of L4 nFS interneurons, a co-operative activation of muscarinic and nicotinic ACh receptors was also observed. The present study demonstrates that low-concentrations of ACh affect different L4 neuron types in a cell-type specific way. These effects result from a specific expression of different muscarinic and/or nicotinic ACh receptors on the somatodendritic compartments of L4 neurons. This suggests that even at low concentrations ACh may tune the excitability of L4 excitatory and inhibitory neurons and their synaptic microcircuits differentially depending on the behavioural state during which ACh is released.

Abstract MP54: Pro-resolving Lipid Mediators Ameliorate Endothelium Dysfunction In Arteries From Hypertensive Rats

Resolution of inflammation is an active phenomenon to switch off the inflammatory processes andfacilitate the return to homeostasis. Increasing the levels of pro-resolving lipid mediators topromote the resolution of inflammation is emerging as a novel therapeutic approach. Arachidonicacid (AA) and docosahexaenoic acid (DHA) are substrates to produce the pro-resolving lipidmediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), respectively. However, it is unknown if thesemediators can ameliorate dysfunction in arteries from hypertensive animals. Therefore, wehypothesized that pro-resolving lipid mediators decrease acetylcholine-induced contractions inarteries from spontaneously hypertensive rats (SHR). Mesenteric resistance arteries from maleSHR and Wistar Kyoto (WKY) (14-weeks old, n=6-8) were used for vascular function via wiremyograph. MRA were incubated with either RvD1 or LXA4 (10 nM, 1 hour) or vehicle prior toconcentration response curves to acetylcholine or phenylephrine (1 nM - 3 μM). We alsoperformed lipidomic analysis on plasma from WKY and SHR. As expected, low concentrations (≤100 nM) of acetylcholine induced relaxation in arteries from both groups, however highconcentrations (≥ 1 μM) of acetylcholine induced contraction in arteries from SHR, but not in WKY[Relaxation to acetylcholine [Area Under the curve (AUC)]: WKY: 396.6 ± 17.7 vs. SHR-control:296.0 ± 26.7*, t-test *vs. WKY, p=0.03). Treatment with the mediators did not change relaxationin arteries from WKY (AUC: Vehicle: 396.6 ±17.7 vs. RvD1: 402.8 ± 18.0; LXA4: 399.0 ± 18.4,p>0.05). However, incubation with RvD1 or LXA4 reduced acetylcholine induced-contraction inarteries from SHR (AUC: Vehicle: 296.0 ± 26.7 vs. RvD1: 372.5 ± 18.5*; LXA4: 376.8 ± 23.6*, t-test *vs. vehicle, p<0.05). Lipidomic analysis showed that precursors for pro-resolving lipidmediators are decreased in SHR [DHA (pmol/mL): WKY: 5637.5 ± 275.3 vs. SHR: 4509.2 ±453.9*, AA: WKY:16018.3 ± 351.3 vs. SHR: 13928.5 ± 959.5 *vs. WKY, p<0.05]. Overall, theseresults suggest that resolution of inflammation and the pro-resolving lipid mediators, RvD1 orLXA4, may be used as a new therapeutic tool to improve vascular function in hypertension.

Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

In the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

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.

Cholinergic modulation of Up-Down states in the mouse medial entorhinal cortex in vitro.

Cholinergic tone is high during wake and rapid eye movement sleep and lower during slow wave sleep (SWS). Nevertheless, the low tone of acetylcholine during SWS modulates sharp wave ripple incidence in the hippocampus and slow wave activity in the neocortex. Linking the hippocampus and neocortex, the medial entorhinal cortex (mEC) regulates the coupling between these structures during SWS, alternating between silent Down states and active Up states, which outlast neocortical ones. Here, we investigated how low physiological concentrations of acetylcholine (ACh; 100-500nM) modulate Up and Down states in a mEC slice preparation. We find that ACh has a dual effect on mEC activity: it prolongs apparent Up state duration as recorded in individual cells and decreases the total synaptic charge transfer, without affecting the duration of detectable synaptic activity. The overall outcome of ACh application is excitatory and we show that ACh increases Up state incidence via muscarinic receptor activation. The mean firing rate of principal neurons increased in around half of the cells while the other half showed a decrease in firing rate. Using two-photon calcium imaging of population activity, we found that population-wide network events are more frequent and rhythmic during ACh and confirmed that ACh modulates cell participation in these network events, consistent with a role for cholinergic modulation in regulating information flow between the hippocampus and neocortex during SWS.

Sulodexide promotes arterial relaxation via endothelium-dependent nitric oxide-mediated pathway

Sulodexide (SDX) is a highly purified glycosaminoglycan with antithrombotic and profibrinolytic properties and reported benefits in thrombotic and atherosclerotic vascular disorders. However, the effects of SDX on vascular function are unclear. We tested whether SDX affects vascular relaxation and examined the potential underlying mechanisms. Isolated segments of male rat abdominal aorta and mesenteric artery were suspended in a tissue bath, and the changes in arterial contraction/relaxation were measured. The α-adrenergic receptor agonist phenylephrine (Phe) (10−9–10−5 M) caused concentration-dependent aortic and mesenteric artery contraction that was reduced in tissues pretreated with SDX (1 mg/ml). In aortic and mesenteric arterial segments precontracted with submaximal concentration of Phe (3 × 10−7–6 × 10−7 M), SDX (0.001–1 mg/ml) caused concentration-dependent relaxation. To test the role of endothelium, SDX-induced relaxation was compared with that of acetylcholine (ACh), a known activator of endothelium-dependent relaxation. In Phe precontracted aorta, ACh relaxation was abolished and SDX relaxation was significantly inhibited by endothelium removal or the nitric oxide synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (L-NAME), suggesting a role of NO. In mesenteric artery, ACh relaxation was abolished by endothelium removal, partially blocked by L-NAME, and completely blocked by a mixture of indomethacin, a cyclooxygenase inhibitor and blocker of the PGI2-cAMP pathway, and tetraethylammonium, a blocker of K+ channels and EDHF-dependent hyperpolarization pathway. In comparison, SDX relaxation of mesenteric artery was almost completely inhibited by endothelium removal or NOS inhibitor L-NAME. SDX enhanced vascular relaxation and increased nitrate/nitrite production in response to all ACh concentrations in the aorta, but only to low ACh concentrations (<10−7 M) in mesenteric artery. SDX did not affect aortic or mesenteric artery endothelium-independent relaxation to the NO donor sodium nitroprusside. Thus, SDX promotes arterial relaxation via a mechanism involving endothelium-dependent NO production; an effect that could enhance vasodilation and decrease vasoconstriction in vascular disorders.

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.

Design, Synthesis and Evaluation of 4-Aminopyridine Analogues as Cholinesterase Inhibitors for Management of Alzheimer’s Diseases

Abstract: Introduction: Alzheimer’s disease (AD) is a slowly progressive devasting neurodegenerative disorder of central nervous system manifested by deterioration of memory, cognitive functions, behaviour change and impairment in performing activities of daily life. Neurochemical studies of patients suffering from AD demonstrate selective loss of cholinergic neurons, low concentration of acetylcholine (Ach) in the selective areas of brain such as cortex and hippocampus. Objective: A series of new semicarbazones of 4-aminopyridine has been designed, synthesized and evaluated for cognition enhancing activities through the inhibition of acetylcholinesterase (AChE) and by passive avoidance model. Material and Methods: In the present study, ten new 4-Aminopyridine analogues were synthesized and characterized by analytic methods such as UV, IR, NMR, elemental analysis and for their inhibitory role on Acetylcholinesterase activity by applying the molecular docking studies and performed enzyme kinetics study by Ellman’s spectrophotometric method. The synthesized analogues were then evaluated for antiamnesic and cognition enhancing activities by passive avoidance test. Results: The results illustrated a significant cognition enhancing effect on passive avoidance test with a significant reversal of scopolamine-induced amnesia, which is comparable with standard drug rivastigmine. The in-vitro study of synthesized analogues showed maximum activity of compound-3 and compound-9 compared to standard drug rivastigmine, whereas its enzyme kinetic study revealed a non-competitive inhibition of acetylcholinesterase (AChE) is held responsible to a possible interaction of analogue with the peripheral anionic site (PAS) of AChE and was also confirmed by molecular docking studies. Conclusion: On the basis of present study, we are concluding that hydroxyl substituted Compound 3 and 9 identified as most potent drug which can leads to the discovery and development of new Cognition enhancers in near future. Key words: 4-aminopyridine, Acetylcholinesterase, Passive avoidance test, Rivastigmine.

Fenugreek (Trigonella Foenum-Graecum) Seed Flour and Diosgenin Preserve Endothelium-Dependent Arterial Relaxation in a Rat Model of Early-Stage Metabolic Syndrome.

Fenugreek is a common herb possessing several bioactive components including diosgenin. Here, dietary fenugreek seed flour and diosgenin were evaluated on a model of endothelium-dependent vasorelaxation by abdominal aortas isolated from rats receiving high-fat, high-sugar diet (HFHSD). 60 male Wistar rats were randomized into six groups: (i) negative control getting conventional rat feed regimen; (ii) positive control receiving HFHSD; (iii) a test group fed 2 g/kg bw/day fenugreek seed flour (containing 10 mg/kg bw/day diosgenin) + HFHSD; (iv) three test groups fed 1, 10 and 50 mg/kg bw/day diosgenin + HFHSD. Alimentary treatments were carried out for six weeks. The abdominal aortas were isolated, and 2 mm wide rings were sectioned off and mounted at a resting tension of 10 mN in organ baths containing Krebs solution (36 °C) exposed to 95% O2 and 5% CO2. After 60-min incubation, a norepinephrine concentration-response (E/c) curve was generated to determine their half-maximal effective concentration (EC50) value. After 60-min wash-out, a pre-contraction with norepinephrine EC50 was made, followed by an acetylcholine E/c curve. Plasma glutathione levels, glutathione-handling enzyme activities and blood antioxidant capacities were also determined. HFHSD significantly decreased the dilatory response to acetylcholine and increased plasma glutathione levels and these effects were significantly reversed by fenugreek seed flour, 10 and 50 mg/kg bw/day diosgenin. Both fenugreek and diosgenin treatments prevent HFHSD-induced endothelial dysfunction and redox changes. As fenugreek treatment was more effective at lower acetylcholine concentrations than diosgenin treatments, components of fenugreek other than diosgenin may contribute to the beneficial effects of dietary fenugreek seed flour.

R-(+) and S-(-) isomers of cotinine augment cholinergic responses in vitro and in vivo.

The nicotine metabolite cotinine (1-methyl-5-[3-pyridynl]-2-pyrrolidinone), like its precursor, has been found to exhibit procognitive and neuroprotective effects in some model systems; however, the mechanism of these effects is unknown. In this study, both the R-(+) and S-(-) isomers of cotinine were initially evaluated in an extensive profiling screen and found to be relatively inactive across a wide range of potential pharmacologic targets. Electrophysiological studies on human α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes confirmed the absence of agonistic activity of cotinine at α4β2 or α7 nAChRs. However, a significant increase in the current evoked by a low concentration of acetylcholine was observed at α7 nAChRs exposed to 1.0 μM R-(+)- or S-(-)-cotinine. Based on these results, we used a spontaneous novel object recognition (NOR) procedure for rodents to test the hypothesis that R-(+)- or S-(-)-cotinine might improve recognition memory when administered alone or in combination with the Alzheimer's disease (AD) therapeutic agent donepezil. Although both isomers enhanced NOR performance when they were coadministered with donepezil, neither isomer was active alone. Moreover, the procognitive effects of the drug combinations were blocked by methyllycaconitine and dihydro-β-erythroidine, indicating that both α7 and α4β2 nAChRs contribute to the response. These results indicate that cotinine may sensitize α7 nAChRs to low levels of acetylcholine (a previously uncharacterized mechanism), and that cotinine could be used as an adjunctive agent to improve the effective dose range of cholinergic compounds (e.g., donepezil) in the treatment of AD and other memory disorders.

Different effects of lobeline on neuronal and muscle nicotinic receptors

Lobeline is a plant alkaloid known to interact with cholinergic system. The effect of lobeline on neuronal α3β4 receptors expressed in COS cells and muscle embryonic αβγδ receptors naturally expressed in TE671 cells was studied using a patch-clamp technique. Our results show that lobeline inhibited responses to acetylcholine in human embryonic muscle nicotinic receptor in a pseudo-noncompetitive manner. The responses of rat neuronal α3β4 receptors to a low concentration of acetylcholine were potentiated by a mixed occupation mechanism that corresponds to “competitive potentiation”. This potentiation turned into voltage-dependent inhibition for α3β4 receptors was activated by a high concentration of acetylcholine.

In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat

Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime.

Nervous System and Gastrointestinal Effects of the Insecticide Esfenvalerate on the Rat: An &amp;lt;i&amp;gt;Ex Vivo&amp;lt;/i&amp;gt; Study

Esfenvalerate belongs to the pyrethroid group of insecticides which display significant selective toxicity against insects compared to mammalian species, nevertheless, they may pose health risks, especially in case of accidental exposure. The aim of the present study was to model the effect of acute, relatively high-dose exposure of the esfenvalerate-containing formulation Sumi-Alpha?. Eventual functional alterations in the central nervous system and in the gastrointestinal tract were studied on in vitro tissue preparations at different delays after intragastric administration to rats. Neuronal effects were characterized by field potential recording in cortical and hippocampal brain slices, while gastrointestinal effects were examined by analyzing the motility and excitability of isolated ileum segments. On the brain slices originating from esfenvalerate-treated animals, changes in excitability of both inhibitory and excitatory type could be observed. Voltage thresholds necessary to evoke responses in neocortex slices were elevated, and population spike amplitudes were lower in hippocampal slices. However, epileptiform potentials with pronounced late components were also observed. A decreased long-term potentiation (LTP) could be seen in both brain areas after esfenvalerate treatment. Seizure susceptibility of the slices was not significantly altered, but tended to be somewhat higher in slices originating from treated rats. In ileum segments, both spontaneous and acetyl-choline (ACh)-elicited contractions were modified by treatment. Esfenvalerate raised the amplitude of contractions in the low ACh concentration range. However, the solvent xylene also considerably contributed to the detected changes. We can conclude that a relatively high, single oral dose of Sumi-Alpha? exerted mild and temporary effects on the elementary brain functions and intestine functions of the rat.

Synthesis, docking and acetylcholinesterase inhibitory assessment of 2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione derivatives with potential anti-Alzheimer effects

BackgroundAlzheimer’s disease (AD) as neurodegenerative disorder, is the most common form of dementia accounting for about 50-60% of the overall cases of dementia among persons over 65 years of age. Low acetylcholine (ACh) concentration in hippocampus and cortex areas of the brain is one of the main reasons for this disease. In recent years, acetylcholinesterase (AChE) inhibitors like donepezil with prevention of acetylcholine hydrolysis can enhance the duration of action of acetylcholine in synaptic cleft and improve the dementia associated with Alzheimer’s disease.ResultsDesign, synthesis and assessment of anticholinesterase activity of 2-(2-(4-Benzylpiperazin-1-yl)ethyl)isoindoline-1,3-dione derivatives showed prepared compounds can function as potential acetylcholinesterase inhibitor. Among 12 synthesized derivatives, compound 4a with ortho chlorine moiety as electron withdrawing group exhibited the highest potency in these series (IC50 = 0.91 ± 0.045 μM) compared to donepezil (IC50 = 0.14 ± 0.03 μM). The results of the enzyme inhibition test (Ellman test) showed that electron withdrawing groups like Cl, F and NO2 can render the best effect at position ortho and para of the phenyl ring. But compound 4g with methoxy group at position 3(meta) afforded a favorable potency (IC50 = 5.5 ± 0.7 μM). Furthermore, docking study confirmed a same binding mode like donepezil for compound 4a.ConclusionsSynthesized compounds 4a-4l could be proposed as potential anticholinesterase agents.

Acetylcholine Promotes Ca2+and NO-Oscillations in Adipocytes Implicating Ca2+→NO→cGMP→cADP-ribose→Ca2+ Positive Feedback Loop - Modulatory Effects of Norepinephrine and Atrial Natriuretic Peptide

PurposeThis study investigated possible mechanisms of autoregulation of Ca2+ signalling pathways in adipocytes responsible for Ca2+ and NO oscillations and switching phenomena promoted by acetylcholine (ACh), norepinephrine (NE) and atrial natriuretic peptide (ANP).MethodsFluorescent microscopy was used to detect changes in Ca2+ and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca2+-channels in Ca2+ signalling pathways.ResultsACh activating M3-muscarinic receptors and Gβγ protein dependent phosphatidylinositol 3 kinase induces Ca2+ and NO oscillations in adipocytes. At low concentrations of ACh which are insufficient to induce oscillations, NE or α1, α2-adrenergic agonists act by amplifying the effect of ACh to promote Ca2+ oscillations or switching phenomena. SNAP, 8-Br-cAMP, NAD and ANP may also produce similar set of dynamic regimes. These regimes arise from activation of the ryanodine receptor (RyR) with the implication of a long positive feedback loop (PFL): Ca2+→ NO→cGMP→cADPR→Ca2+, which determines periodic or steady operation of a short PFL based on Ca2+-induced Ca2+ release via RyR by generating cADPR, a coagonist of Ca2+ at the RyR. Interplay between these two loops may be responsible for the observed effects. Several other PFLs, based on activation of endothelial nitric oxide synthase or of protein kinase B by Ca2+-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca2+-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G.ConclusionsThis study presents a kinetic model of Ca2+-signalling system operating in adipocytes and integrating signals from various agonists, which describes it as multivariable multi feedback network with a family of nested positive feedback.

Increased susceptibility to acetylcholine in the entorhinal cortex of pilocarpine-treated rats involves alterations in KCNQ channels

In models of temporal lobe epilepsy, in-vitro exposure of the entorhinal cortex (EC) to low concentrations of acetylcholine (ACh) induces muscarinic-dependent seizure-like events. Potassium channels from the KCNQ/Kv7 family, which close upon activation of muscarinic receptors, are mutated in several epileptic syndromes such as benign familial neonatal convulsions (KCNQ2/KCNQ3) and sudden unexplained death in epilepsy (KCNQ1). Therefore, we tested the hypothesis whether the ictogenic effect of ACh involves alterations of KCNQ channels. In horizontal temporo-hippocampal slices from pilocarpine-treated chronically epileptic rats, field potential recordings of epileptiform activity were performed in response to the application of ACh, the KCNQ blocker linopirdine, and KCNQ agonists. In the EC of control rats, ACh (20 and 50μM) induced nested fast activity in the range of 15–20Hz riding on <1Hz slow oscillations. By contrast, in slices from pilocarpine-treated rats, 5μM ACh was sufficient to induce interictal discharges that frequently transformed to epileptiform events at 20μM ACh. While the non-specific KCNQ/Kv7 channel blocker linopirdine (20 and 50μM) had no effect in control animals, in slices from epileptic rats it induced interictal discharges or seizure-like events. These could be blocked by the unspecific KCNQ/Kv7 agonist retigabine and attenuated by the Kv7.1 agonist L364–373. Immunohistochemistry revealed reduced expression of KCNQ2 and KCNQ3 in the EC and of KCNQ3-positive dendrites in the subiculum of epileptic rats. These results indicate that channels of the KCNQ family are key regulators of seizure susceptibility and their decreased availability in the epileptic tissue may reduce seizure threshold and contribute to ictogenesis.