Acetylcholine Sensitivity Research Articles

Mild hyperbaric oxygen exposure protects heart during ischemia/reperfusion and affects vascular relaxation.

Mild hyperbaric oxygen therapy (mHBOT) is an adjuvant therapy used in conditions where tissue oxygenation is reduced and is implemented using pressures less than 1.5 ATA and 100% O2 (instead of the classical HBOT at 1.9-3 ATA) which results in cheaper, easier to implement, and equally effective. mHBOT is offered for wellness and beauty and as an anti-aging strategy, in spite of the absence of studies on the cardiovascular system. Consequently, we investigated the impact of mHBOT on the cardiovascular system. Mechanical and energetic parameters of isolated heart submitted to ischemia/reperfusion injury and arterial contractile response from mHBOT-exposed rats were evaluated. In the heart, mHBOT increased pre-ischemic velocity of contraction and ischemic end-diastolic pressure and developed pressure and contractile economy during reperfusion. mHBOT decreased infarct size and increased the plasma nitrite levels. In the artery, mHBOT increased acetylcholine sensitivity. mHBOT protects the heart during ischemia/reperfusion and affects vascular relaxation.

Deletion of Ascl1 in pancreatic β-cells improves insulin secretion, promotes parasympathetic innervation, and attenuates dedifferentiation during metabolic stress

ObjectiveASCL1, a pioneer transcription factor, is essential for neural cell differentiation and function. Previous studies have shown that Ascl1 expression is increased in pancreatic β-cells lacking functional KATP channels or after feeding of a high fat diet (HFD) suggesting that it may contribute to the metabolic stress response of β-cells. MethodsWe generated β-cell-specific Ascl1 knockout mice (Ascl1βKO) and assessed their glucose homeostasis, islet morphology and gene expression after feeding either a normal diet or HFD for 12 weeks, or in combination with a genetic disruption of Abcc8, an essential KATP channel component. ResultsAscl1 expression is increased in response to both a HFD and membrane depolarization and requires CREB-dependent Ca2+ signaling. No differences in glucose homeostasis or islet morphology were observed in Ascl1βKO mice fed a normal diet or in the absence of KATP channels. However, male Ascl1βKO mice fed a HFD exhibited decreased blood glucose levels, improved glucose tolerance, and increased β-cell proliferation. Bulk RNA-seq analysis of islets from Ascl1βKO mice from three studied conditions showed alterations in genes associated with the secretory function. HFD-fed Ascl1βKO mice showed the most extensive changes with increased expression of genes necessary for glucose sensing, insulin secretion and β-cell proliferation, and a decrease in genes associated with β-cell dysfunction, inflammation and dedifferentiation. HFD-fed Ascl1βKO mice also displayed increased expression of parasympathetic neural markers and cholinergic receptors that was accompanied by increased insulin secretion in response to acetylcholine and an increase in islet innervation. ConclusionsAscl1 expression is induced by stimuli that cause Ca2+-signaling to the nucleus and contributes in a multifactorial manner to the loss of β-cell function by promoting the expression of genes associated with cellular dedifferentiation, attenuating β-cells proliferation, suppressing acetylcholine sensitivity, and repressing parasympathetic innervation of islets. Thus, the removal of Ascl1 from β-cells improves their function in response to metabolic stress.

Abstract P313: Chronic Circadian Disruption Impairs Blood Pressure Rhythm And Leads To Vascular Disease In Mice

Shift workers have an elevated risk of developing hypertension and higher incidence of cardiovascular disease. Circadian rhythm disruption increases cardiometabolic disease risk in both humans and animals. We hypothesized that chronic circadian disruption (CCD) impairs cardiovascular rhythms and leads to vascular disease. Male 8-week old C57BL/6J mice were subjected to a standard light/dark cycle (12-h light, 12-h dark, control) or a CCD protocol (10-h light, 10-h dark, T20) with ad libitum food and water for 10 wks. T20 mice had an increased rate of weight gain (n=6, p=0.04; time: p<0.001, light cycle: p<0.001). Mean arterial pressure (MAP), heart rate (HR), and activity were measured by telemetry after 10 weeks of CCD. Control mice (12:12 LD) had significant diurnal variation in MAP, while T20 mice (10:10 LD) lacked diurnal variation (control 119±3 vs.101±2 mm Hg, dark vs. light, p<0.001; T20 114±13 vs.113±12 mm Hg, dark vs. light, p=0.86; n=3-4). Control mice had a diurnal variation in HR which was absent in T20 mice (Control 584±11 vs. 511±4 bpm, dark vs. light, p=0.001; T20- 537±14 vs. 526±5 bpm, dark vs. light, p=0.48; n=3-4). Dark phase HR was significantly lower in T20 mice (p=0.01, control vs. T20). Control mice had a light-dark difference in activity, while T20 mice lacked diurnal variation in activity with significantly higher light phase activity (p=0.04, control vs. T20). Aortic pulse wave velocity (PWV), a measure of vascular stiffness, was significantly higher after 6 weeks in T20 mice compared to control mice (Control: 1.60±0.1 m/s; T20: 2.25±0.1 m/s; n=6, p=0.0037), although aortic wall thickness was similar between groups. Wire myography in isolated aortic rings was used to assess vascular reactivity with cumulative concentration responses to phenylephrine (PE) and acetylcholine (ACh). CCD reduced sensitivity (EC50) to PE-induced vasoconstriction (Control: -6.92±0.06 M; T20: -6.74±0.04 M, n=6, p=0.03), while there were no significant changes seen in the ACh sensitivity or maximal response. These data show that CCD impairs cardiovascular and behavioral rhythms linked to the development of aortic stiffness.

A potential cost of evolving epibatidine resistance in poison frogs

BackgroundSome dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor β2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4β2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration–response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the β2 S108C into the α4β2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs.ResultsThe introduction of S108C into the α4β2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4β2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity.ConclusionsThe most parsimonious explanation of our current and previous work is that the S108C substitution renders the β2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while β2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4β2 receptor function.

Effects of amiloride on acetylcholine-dependent arterial vasodilation evolve over time in mice on a high salt diet.

The maintenance of endothelial health is required for normal vascular function and blood pressure regulation. The epithelial Na+ channel (ENaC) in endothelial cells has emerged as a new molecular player in the regulation of endothelial nitric oxide production and vascular stiffness. While ENaC expression in the kidney is negatively regulated by high [Na+], ENaC expression in isolated endothelial cells has been shown to increase in response to a high extracellular [Na+]. In culture, this increased expression leads to cellular stiffening and decreased nitric oxide release. In vivo, the effects of high salt diet on endothelial ENaC expression and activity have varied depending on the animal model utilized. Our aim in the present study was to examine the role of endothelial ENaC in mediating vasorelaxation in the C57Bl/6 mouse strain. We utilized pressure myography to test the responsiveness of thoracodorsal arteries to acetylcholine in mice with increased sodium consumption both in the presence and absence of increased aldosterone. ENaC’s contribution was assessed with the use of the specific inhibitor amiloride. We found that while aldosterone had very little effect on ENaC's contribution to acetylcholine sensitivity, a high salt diet led to an amiloride‐dependent shift in the acetylcholine response of vessels. However, the direction of this shift was dependent on the length of high salt diet administration. Overall, our studies reveal that ENaC's role in the endothelium may be more complicated than previously thought. The channel does not simply inhibit nitric oxide generation, but instead helps preserve a homeostatic response.

Long-Chain Acylcholines Link Butyrylcholinesterase to Regulation of Non-neuronal Cholinergic Signaling.

Acylcholines are comprised of an acyl chain esterified to a choline moiety; acetylcholine is the best-characterized member of this class, functioning as a neurotransmitter in the central and peripheral nervous systems as well as an inhibitor of cytokine production by macrophages and other innate immune cells. Acylcholines are metabolized by a class of cholinesterases, including acetylcholinesterase (a specific regulator of acetylcholine levels) and butyrylcholinesterase (BChE, an enigmatic enzyme whose function has not been resolved by genetic knockout models). BChE provides reserve capacity to hydrolyze acetylcholine, but its importance is arguable given acetylcholinesterase is the most catalytically efficient enzyme characterized to date. While known to be substrates of BChE in vitro, endogenous production of long-chain acylcholines is a recent discovery enabled by untargeted metabolomics. Compared to acetylcholine, long-chain acylcholines show greater stability in circulation with homeostatic levels-dictated by synthesis and clearance-suggested to impact cholinergic receptor sensitivity of acetylcholine with varying levels of antagonism. Acylcholines then provide a link between BChE and non-neuronal acetylcholine signaling, filling a gap in understanding around how imbalances between acylcholines and BChE could modulate inflammatory disease, such as the "cytokine storm" identified in severe COVID-19. Areas for further research, development, and clinical testing are outlined.

The Safety Factor for Neuromuscular Transmission: Effects of Dimethylsulphoxide, Cannabinoids and Synaptic Homeostasis.

BackgroundIn myasthenia gravis, impaired postsynaptic sensitivity to acetylcholine results in failure of neuromuscular transmission and fatiguing muscle weakness.ObjectiveDevelop an ex vivo muscle contraction assay to test cannabinoids and other substances that might act on the myasthenic neuromuscular junction to restore control of the muscle.MethodsTubocurarine was added to an ex vivo, mouse phrenic nerve-hemidiaphragm muscle preparation to reduce acetylcholine sensitivity. This produced a myasthenia-like decrement in twitch force during a train of 10 nerve impulses (3 / sec). Endplate potential (EPP) recordings were used to confirm and extend the findings.ResultsSurprisingly, addition to the bath of dimethylsulphoxide (DMSO), at concentrations as low as 0.1%(v/v), partially reversed the decrement in nerve-evoked force. Intracellular electrophysiology, conducted in the presence of tubocurarine, showed that DMSO increased the amplitudes of both the spontaneous miniature EPP (MEPP) and the (nerve-evoked) EPP. In the absence of tubocurarine (synaptic potentials at physiological levels), an adaptive fall in quantal content negated the DMSO-induced rise in EPP amplitude. The effects of cannabinoid receptor agonists (solubilized with DMSO) in the contraction assay do not support their further exploration as useful therapeutic agents for myasthenia gravis. CP 55,940 (a dual agonist for cannabinoid receptor types 1 and 2) reversed the beneficial effects of DMSO.Conclusions:We demonstrate a powerful effect of DMSO upon quantal amplitude that might mislead pharmacological studies of synaptic function wherever DMSO is used as a drug vehicle. Our results also show that compounds targeting impaired neuromuscular transmission should be tested under myasthenic-like conditions, so as to avoid confounding effects of synaptic homeostasis.

Sex differences in the cardiac cholinergic response to adrenalin-induced myocardial necrosis and light desynchronosis

The aim was to identify sex differences in the cardiac response to cholinergic stimuli in the development of myocardial necrosis and light desynchronosis.Materials and methods. Experiments were conducted in 144 albino rats (72 males, 72 females). Myocardial necrosis was induced by adrenaline (0.5 mg/kg, intramuscularly).The intensity of bradycardia that occurred as a response to intravenous acetylcholine injection and electrical stimulation of vagus nerve was measured. The animals were divided into 4 groups: 1 – rats that were in a light balance (day/night); 2 – rats exposed to light deprivation for 10 days; 3 – rats exposed to permanent lighting for 10 days; 4 – animals exposed to permanent lighting for 10 days and injected with melatonin (5 mg/kg) intraperitoneally 1 hour before the necrosis modeling.Results. The development of myocardial necrosis when occurring in light deprivation was characterized by an increased responsiveness of the rat heart to cholinergic stimulation due to an increase in both cholinoreceptors sensitivity and release of acetylcholine from the vagus nerve terminals in females, and due to only an increased cholinoreceptors sensitivity in males. The development of myocardial necrosis in rats exposed to permanent lighting was characterized by significantly higher sensitivity of cholinergic structures of the heart than that in light balance, especially in females. Melatonin injection in the development of myocardial necrosis contributed to the heart cholinergic structures response to the stimulation, which was close to that observed in conditions of light balance, though it remained somewhat higher: due to the facilitation of acetylcholine release from vagus nerve in females, and it was combined with a higher sensitivity of cholinoreceptors in males.Conclusions. The development of myocardial necrosis in light desynchronosis (light deprivation or permanent lighting) causes an increased heart response to cholinergic stimulation in rats. Such effects are stronger, especially in females in conditions of permanent lighting. The injection of melatonin in rats with myocardial necrosis and permanent lighting results in an approximation (but not restoration) of the heart sensitivity to cholinergic stimuli parameters to those that are observed in light balance. However, the heart response to cholinergic stimulation remains higher, especially in males.

High glucose effects on endothelium-dependent vasorelaxation in isolated thoracic aorta of spontaneously hypertensive rats

There is an increasing evidence regarding the relationship between metabolic syndrome components and cardiovascular complications leading to vascular disorders and type 2 diabetes. Cardio-metabolic risk factors have been reported to promote the development of endothelial dysfunction. Nevertheless, the direct impact of these factors on the mechanisms underlying endothelial dysfunction remain poorly understood. The aim was to identify the impact of hyperglycaemia on endothelial function in spontaneously hypertensive rat (SHR). For this we investigated the direct effect of high glucose short-term incubation on endothelium-dependent relaxation on isolated thoracic aorta. Thirty-eight SHR males, 12-weeks old (weighting on average 330 g) were used for this study. Thoracic aortae were isolated and cut into rings. Aortic rings were precontracted with 1 μM phenylephrine and cumulative concentration-response curves (CCRCs) to acetylcholine (1 nM to 0.1 mM) and sodium nitroprusside (10 nM to 10 μM) were performed in aortic rings exposed during two hours to a normal (11 mM) or to a high glucose concentration (25 mM). A non-linear mixed effects model was used to compare the CCRCs parameters (pD2 and Emax). A significant level of P = 0.05 was used for statistical comparison. In phenylephrine-precontracted rings, acetylcholine produced a concentration-dependent relaxation (Emax = 87.06 ± 3.04% and pD2 was 6.99 ± 0.06; n = 20). Incubation of aortic rings in high glucose for 2 hours led to an impairment of the acetylcholine-induced relaxation (pD2 = 6.57 ± 0.08 and Emax = 79.95 ± 4.19%; P < 0.001 vs. control; n = 20). Pretreatment of aortic rings with apocynin (30 μM), a NADPH oxidase inhibitor, or indomethacin (1 μM), a non-selective COX inhibitor, increased the maximal effect of acetylcholine-induced relaxation (Emax = 101.06 ± 5.20% and Emax = 97,98 ± 4.70% after apocynin and indomethacin respectively, P < 0.01 vs. control) in control group. The vasorelaxation was also improved in the high glucose group (Emax = 97.96 ± 4.70% and 102.34 ± 5.05% respectively, P < 0.001 vs. high glucose group after apocynin or indomethacin respectively), with an increase of acetylcholine sensitivity by indomethacin (pD2 = 7.23 ± 0.10; P < 0.001 vs. high glucose group after indomethacin). We found that short-term incubation with high glucose led to an impairment of endothelium-dependent relaxation of SHR thoracic aorta. This impairment could involve NADPH oxidase and COX products, that may be responsible for a decrease of nitric oxide bioavailability and its vasorelaxant activity.

Non-enzymatic simultaneous detection of acetylcholine and ascorbic acid using ZnO·CuO nanoleaves: Real sample analysis

A facile wet-chemical technique used to prepare the zinc oxide doped copper oxide nanoleaves (ZnO·CuO nanoleaves) in alkaline phase. ZnO·CuO nanoleaves were examined using a variety of conventional techniques for example UV–Visible, FTIR, XRD, FESEM equipped XEDS, and XPS. A non-enzymatic sensor was prepared with modification of a slightly deposited of ZnO·CuO nanoleaves onto a flat GCE, which fabricated through a conducting nafion polymer matrix in order to detect selective acetylcholine and ascorbic acid simultaneously. Analytical performances such as sensitivity, LOD, LOQ, LDR, and durability of the selective acetylcholine and ascorbic acid sensors were examined through a conventional current–voltage method. Calibration graphs of acetylcholine and ascorbic acid sensors were found linear (R2 = 0.9049 and 0.9201) over a good concentration range (100.0 pM–100.0 mM) respectively. Sensitivity (317.0 and 94.94 pAμM-1cm−2), LOD (14.7 and 12.0 pM), and LOQ (490.0 and 367.0 mM) of acetylcholine and ascorbic acid sensors were calculated correspondingly from the slope of the linear portion of calibration graph. Preparation of ZnO·CuO nanoleaves using wet-chemical technique is an excellent approach for the advancement of nanomaterial based on sensor development in support of enzyme free detection of biological molecules for the safety in healthcare and biomedical fields. This expected sensor applied for the specific determination of acetylcholine and ascorbic acid in real samples (Human, mouse, and rabbit serum, orange juice, and urine) and found satisfactory and acceptable results.

Abstract P215: Hyperglycemic Rats Have Increased Fetal Demise But Not Hypertension During Pregnancy

While obesity is a major cause for pregnancy complications including preeclampsia and fetal demise, it is not exactly clear the precise obesity-related metabolic factors that promote these adverse outcomes. Epidemiological studies have pointed toward hyperglycemia as one such factor. Therefore, we tested the hypothesis that hyperglycemic rats have hypertension and fetal demise during pregnancy. For this purpose, we utilized the type II diabetic model, the Goto-Kakizaki (GK) rat (N=16), compared to normoglycemic Wistar Hannover (WH) rats (N=9), which were maintained on Envigo 8640 standard chow. The GK rat allows for assessment of hyperglycemia on pregnancy without confounding obesity. Maternal fasting glucose levels were significantly greater (P&lt;0.05) in GK (97±8 mg/dL) vs. WH (72±9 mg/dL) rats by gestational day 19. Body weight was lower (P&lt;0.05) in GK (248±4 g) versus WH (289±4 g) pregnant rats, whereas perirenal fat (1.56±0.07 g vs. 1.38±0.07 g, P&gt;0.05) and circulating levels of the adipokine, leptin (1.6±0.2 ng/mL vs. 2.2±0.3 ng/mL, P&gt;0.05) were similar between GK and WH pregnant groups, respectively. Endothelial-dependent relaxation to acetylcholine (sensitivity as logEC50: -5.2±0.3 M vs -5.2±0.4 M) and endothelial-independent relaxation to the nitric oxide-donor sodium nitroprusside (logEC50: -7.2±0.2 M vs. -7.5±0.1 M) were similar (P&gt;0.05) in uterine arteries isolated from GK and WH rats, respectively. It was then determined if reduced uterine perfusion pressure (RUPP)-induced placental ischemia, a significant contributor to the development of preeclampsia, promoted greater maternal hypertension in GK rats. RUPP was conducted on gestational day 14 and blood pressure assessed on day 19. RUPP produced hypertension to a similar extent (P&gt;0.05) in GK (116±5 mmHg vs. Sham 102±5 mmHg) and WH (124±4 mmHg vs. Sham 100±2 mmHg) groups. Blood pressure was similar under Sham conditions. Fetal demise was already greater in Sham GK vs. Sham WH pregnant rats (% absorptions: 13±2 vs. 2±2, P&lt;0.05) but increased similarly following RUPP in GK (61±11 %) and WH (65±5 %) pregnant rats. In conclusion, these data suggest that high glucose levels promote fetal demise during pregnancy but do not exaggerate the outcomes of placental ischemia-induced hypertension.

The 4Hz–Pulsing Magnetic Field (PMF) has Common therapeutic effects on Patients Subjected to Different Pain Stress

Previously, we have suggested a hypothesis that the metabolic net water efflux from the cells is a key mechanism for metabolic controlling semipermeable properties of the cell membrane, dysfunction of which is a common consequence of cell pathology. The existence of the electrogenic Na/K-pump in the cell membrane, beating high ATP utilizing the machine in cell, which by activation of mitochondrial function stimulate endogen water formation making it as a key mechanism for the generation of metabolic water efflux from the cells and its dysfunction as a primary step of cell pathology. It is known that the intracellular concentration Ca2+([Ca]i) which is a strong inhibitor for Na/K-pump, controlling by intracellular cyclic nucleotides dependent systems that are extra sensitive to weak chemical and physical signals, unable to activate ionic channels in the membrane. In our earlier studies, we have shown that the activation of both cGMP-activated Ca-pump in the cell membrane and Na/Ca exchange in forwarding (F) mode and cAMP-activated Na/Ca exchange in reverse (R) mode by generating water efflux from the cells, having key roles in controlling low permeability membrane for inward Na+ current (INa) controlling semipermeable properties of membrane and gradient these ions on the membrane, i.e they have a protective function of cell preceding to inward water-induced decrease Na gradient on the cell membrane. It was shown that the soluble guanylate cyclase (sGC) which is sensitive even to water structure changes of cell bathing medium, serves as a sensor for activation cGMP-dependent FNa/Ca exchange while G proteins in the membrane are sensors for activation cAMP-dependent RNa/Ca exchange. The cGMP induced activation of Ca-pump and FNa/Ca exchange by the decrease of [Ca]i–induced activation of Na/K pump activate water efflux from the cell and controlling the Na gradient on the membrane. While the same function doing the cAMP-dependent RNa/Ca exchange because of its electrogenicity and [Ca]i–induced activation of mitochondrial formation water molecules. By our previous study have shown that the activation of cGMP-dependent FNa/Ca exchange has pain relief while the activation of cAMP-dependent RNa/Ca exchange pain generation effects. It was also shown also that 4Hz pulsing magnetic fields, which activation of cGMP- dependent FNa/Ca exchange have relaxation effects on heart muscles and depressed acetylcholine (Ach) sensitivity cell membrane sensitivity, pain relief and antitumor effects. The accumulation of our and literature data on pain and stress relief, antitumor and antivirus effects of 4Hz PMF allow us to hypothesizes that 4Hz–induced activation of cGMP-dependent F Na/Ca exchange can serve as a universal therapeutic tool to increase the resistance of the cells to pathological factors directed to reverse water efflux into water influx in the cells and increases inward Na current which is the primary step of cell pathology. To check this hypothesis, in this present work the 4Hz effect on voluntary patients who have been subjected by different stress on biochemical characteristics of blood we have studied which serve as a marker for pain–the stress of organism.

Detrimental Effects of Nearby Construction Activity on Endothelial and Vascular Smooth Muscle Function in Cerebral Arteries of Sprague‐Dawley (S‐D) Rats

The present study assessed the effect of nearby construction activity on endothelium‐dependent vasodilation to acetylcholine (ACh), a crucial diagnostic test, in middle cerebral arteries (MCA) of male Sprague‐Dawley (S‐D) rats. Construction activity began on October 2, 2017 and consisted of installing a utility tunnel for a new heating system near the MCW basic science building, followed by additional construction in front of the adjacent medical education building. The project involved extensive digging and tunneling, and the use of heavy equipment (steam shovels, bulldozers, dump trucks, front end loaders, etc.). Various construction activities continued until the end of May 2018. Most of the construction activity generated high levels of vibration and noise in the patio area just outside the MCW Biomedical Resource Center, which houses numerous rodents and other species used in research projects at the institution. Vibrations were monitored using a system (RPi) designed in our laboratory and consisting of a small computing unit attached to a rolling sensor (low sensitivity) and a Piezo Film Sensor (high sensitivity). The sensor was placed in an empty rat cage and vibrations were quantified as Hz. During the construction period, normal endothelium‐dependent vasodilation in response to ACh was abolished or dramatically reduced in isolated MCA of S‐D rats fed a normal salt diet. MCA from rats studied after completion of construction but housed in the vivarium during construction showed a partial recovery of ACh‐induced dilation, but MCA responses to ACh and vascular NO sensitivity, assessed via vasodilator responses to the NO donor sodium nitroprusside (SNP), in those animals were significantly reduced compared to rats obtained, housed, and studied after completion of the construction project. In separate experiments, activity of Ca2+‐activated maxi K+ channels was lost in isolated cerebral artery smooth muscle cells from S‐D rats housed and studied during the construction period. During the construction period, the RPi system recorded high levels of vibration that corresponded to construction activity outside the building. Following cessation of construction, baseline levels of vibration returned and endothelium‐dependent vasodilation to ACh and vessel NO sensitivity recovered to normal levels. The results of this study show that the noise and vibration associated with nearby construction can have highly deleterious effects upon crucial physiological phenotypes.Support or Funding InformationNIH #R01‐HL128242This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

170 Short-term Exposure to High Glucose Alters Acetylcholine Sensitivity and Maximal Relaxation in Male and Female Rat Pudendal Artery, Respectively, Which is Reversed With Testosterone Incubation

170 Short-term Exposure to High Glucose Alters Acetylcholine Sensitivity and Maximal Relaxation in Male and Female Rat Pudendal Artery, Respectively, Which is Reversed With Testosterone Incubation

Short‐term Exposure to High Glucose Decreases Acetylcholine Sensitivity in Male Rat Pudendal Artery

Sexual dysfunction is one of the most prevalent complications of diabetes. The combination of diabetes and sexual dysfunction affects family planning and overall quality of life, and sexual dysfunction is now considered an early marker for cardiovascular diseases. In diabetic males, erectile dysfunction is associated with impaired endothelium‐dependent vasodilation. Diabetes also increases the risk of female sexual dysfunction however, whether there is impaired endothelium function in females is less clear. The objective of this research is to determine if these distinct phenotypes of diabetes are modulated by testosterone. In the male, low testosterone is associated with hyperglycemia and erectile dysfunction and impaired endothelial function. In the female, high levels of testosterone are associated with hyperglycemia but no data exist characterizing mechanistic explanations for reduced clitoral blood flow responses. To test this hypothesis we used pudendal arteries (important source of blood flow to sexual tissues) from 12 week old Wistar male and female rats. Functional studies were performed in pudendal arterial rings mounted onto a myograph and incubated in high glucose (22 mM) for one hour in the presence or absence of testosterone (10−7 M). Concentration‐response curves were then performed in these tissues to phenylephrine (10−9 M‐10−4 M) and acetylcholine (10−9 M‐10−4 M). Male pudendal arteries incubated in high glucose exhibited a decreased sensitivity to acetylcholine. This suggests that short term exposure to high glucose induces responses which inhibit the vasodilater function in males.

Attenuation of Compulsive-Like Behavior Through Positive Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors in Non-Induced Compulsive-Like Mice.

Nicotinic α4β2 receptors are the most abundant subtypes of nicotinic acetylcholine receptors (nAChRs) expressed in brain regions implicated in obsessive compulsive disorder (OCD). These receptors are known to modify normal and addictive behaviors by modulating neuronal excitability. Desformylflustrabromine (dFBr) is a novel, positive allosteric modulator (PAM) of high acetylcholine sensitivity (HS) and low acetylcholine sensitivity (LS) α4β2 nAChRs. The present study tested the hypothesis that positive allosteric modulation of α4β2 receptors by dFBr will attenuate compulsive-like behavior in a non-induced compulsive-like mouse model. Male mice (Mus musculus) selected for compulsive-like nesting behavior (NB; 48 animals; 12 per group) received acute (once) and chronic (every day for 32 days) subcutaneous injection of dFBr at 2, 4 and 6 mg/kg doses. Saline was used as a control (0 mg/kg). Compulsive-like NB was assessed after 1, 2, 3, 4, 5 and 24 h, while compulsive-like marble burying (MB) and anxiety-like open field (OF) behaviors were performed 2 h after dFBr administration. In the acute administration protocol, dFBr dose dependently attenuated NB and MB. Rapid effects (1–2 h after drug administration) of dFBr on MB and NB were observed for the chronic administration which was in congruence with the acute study. Chronic administration also revealed sustained suppression of NB by dFBr following 5 weeks of treatment. In both the acute and chronic regimen dFBr did not modulate OF behaviors. This research demonstrates the novel role of positive allosteric modulation of α4β2 nicotinic receptors by dFBr as a translational potential for OCD.

Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies

Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

Adverse vascular remodelling is more sensitive than endothelial dysfunction to hyperglycaemia in diabetic rat mesenteric arteries

Increased vascular stiffness and reduced endothelial nitric oxide (NO) bioavailability are characteristic of diabetes. Whether these are evident at a more moderate levels of hyperglycaemia has not been investigated. The objectives of this study were to examine the association between the level of glycaemia and resistance vasculature phenotype, incorporating both arterial stiffness and endothelial function. Diabetes was induced in male Sprague Dawley rats with streptozotocin (STZ; 55mg/kg i.v.) and followed for 8 weeks. One week post STZ, diabetic rats were allocated to either moderate (⿼20mM blood glucose, 6⿿7U/insulins.c. daily) or severe hyperglycaemia (⿼30mM blood glucose, 1⿿2U/insulins.c. daily as required). At study end, rats were anesthetized, and the mesenteric arcade was collected. Passive mechanical wall properties were assessed by pressure myography. Responses to the endothelium-dependent vasodilator acetylcholine (ACh) were assessed using wire myography. Our results demonstrated for the first time that mesenteric arteries from both moderate and severely hyperglycaemic diabetic rats exhibited outward hypertrophic remodelling and increased axial stiffness compared to arteries from non-diabetic rats. Secondly, mesenteric arteries from severely (⿼30mM blood glucose), but not moderately hyperglycaemic (⿼20mM blood glucose) rats exhibit a significant reduction to ACh sensitivity compared to their non-diabetic counterparts. This endothelial dysfunction was associated with significant reduction in endothelium-derived hyperpolarisation and endothelium-dependent NO-mediated relaxation. Interestingly, endothelium-derived nitroxyl (HNO)-mediated relaxation was intact. Therefore, moderate hyperglycaemia is sufficient to induce adverse structural changes in the mesenteric vasculature, but more severe hyperglycaemia is essential to cause endothelial dysfunction.

Mutation Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Reduces Low-Sensitivity α4β2, and Increases α5α4β2, Nicotinic Receptor Surface Expression.

A number of mutations in α4β2-containing (α4β2*) nicotinic acetylcholine (ACh) receptors (nAChRs) are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), including one in the β2 subunit called β2V287L. Two α4β2* subtypes with different subunit stoichiometries and ACh sensitivities co-exist in the brain, a high-sensitivity subtype with (α4)2(β2)3 subunit stoichiometry and a low-sensitivity subtype with (α4)3(β2)2 stoichiometry. The α5 nicotinic subunit also co-assembles with α4β2 to form a high-sensitivity α5α4β2 nAChR. Previous studies suggest that the β2V287L mutation suppresses low-sensitivity α4β2* nAChR expression in a knock-in mouse model and also that α5 co-expression improves the surface expression of ADNFLE mutant nAChRs in a cell line. To test these hypotheses further, we expressed mutant and wild-type (WT) nAChRs in oocytes and mammalian cell lines, and measured the effects of the β2V287L mutation on surface receptor expression and the ACh response using electrophysiology, a voltage-sensitive fluorescent dye, and superecliptic pHluorin (SEP). The β2V287L mutation reduced the EC50 values of high- and low-sensitivity α4β2 nAChRs expressed in Xenopus oocytes for ACh by a similar factor and suppressed low-sensitivity α4β2 expression. In contrast, it did not affect the EC50 of α5α4β2 nAChRs for ACh. Measurements of the ACh responses of WT and mutant nAChRs expressed in mammalian cell lines using a voltage-sensitive fluorescent dye and whole-cell patch-clamping confirm the oocyte data. They also show that, despite reducing the maximum response, β2V287L increased the α4β2 response to a sub-saturating ACh concentration (1 μM). Finally, imaging SEP-tagged α5, α4, β2, and β2V287L subunits showed that β2V287L reduced total α4β2 nAChR surface expression, increased the number of β2 subunits per α4β2 receptor, and increased surface α5α4β2 nAChR expression. Thus, the β2V287L mutation alters the subunit composition and sensitivity of α4β2 nAChRs, and increases α5α4β2 surface expression.

Desformylflustrabromine Modulates α4β2 Neuronal Nicotinic Acetylcholine Receptor High- and Low-Sensitivity Isoforms at Allosteric Clefts Containing the β2 Subunit.

Alterations in expression patterns of α4β2 nicotinic acetylcholine receptors have been demonstrated to alter cholinergic neurotransmission and are implicated in neurologic disorders, including autism, nicotine addiction, Alzheimer's disease, and Parkinson's disease. Positive allosteric modulators (PAMs) represent promising new leads in the development of therapeutic agents for the treatment of these disorders. This study investigates the involvement of the β2-containing subunit interfaces of α4β2 receptors in the modulation of acetylcholine (ACh)-induced responses by the PAM desformylflustrabromine (dFBr). Eight amino acids on the principal face of the β2 subunit were mutated to alanine to explore the involvement of this region in the potentiation of ACh-induced currents by dFBr. ACh-induced responses obtained from wild-type and mutant α4β2 receptors expressed in Xenopus laevis oocytes were recorded in the presence and absence of dFBr using two-electrode voltage clamp electrophysiology. Wild-type and mutant receptors were expressed in both high and low ACh sensitivity isoforms by using biased injection ratios of 1:5 or 5:1 α4 to β2 complementary RNA. Mutations were made in the B, C, and A loops of the principal face of the β2 subunit, which are regions not involved in the binding of ACh. Mutant β2(Y120A) significantly eliminated dFBr potency in both isoform preparations. Several other mutations altered dFBr potentiation levels in both preparations. Our findings support the involvement of the principal face of the β2 subunit in dFBr modulation of ACh-induced responses. Findings from this study will aid in the improved design of dFBr-like PAMs for potential therapeutic use.