Acetylcholine (ACh) shortens action potential duration (APD) in human atria. APD shortening facilitates atrial fibrillation (AF) by reducing the wavelength for reentry. However, the influence of ACh on electrical conduction in human atria and its contribution to AF are unclear, particularly when combined with impaired conduction from interstitial fibrosis.

Understanding insect nicotinic acetylcholine receptor (nAChR) subtypes is of major interest because they are the main target of several insecticides. In this study, we have cloned a cockroach Pameα7 subunit that encodes a 518 amino acid protein with futures typical of nAChR subunit, and sequence homology to α7 subunit. Pameα7 is differently expressed in the cockroach nervous system, in particular in the antennal lobes, optical lobes and the mushroom bodies where specific expression was found in the non-compact Kenyon cells. In addition, we found that cockroach Pameα7 subunits expressed in Xenopus laevis oocytes can assemble to form homomeric receptors. Electrophysiological recordings using the two-electrode voltage clamp method demonstrated that nicotine induced an I max current of -92 ± 27 nA at 1 mM. Despite that currents are low with the endogenous ligand, ACh, this study provides information on the first expression of cockroach α7 homomeric receptor.

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve-muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction.

Acetylcholine (ACh), released from axonal terminals of motor neurons in neuromuscular junctions regulates the efficacy of neurotransmission through activation of presynaptic nicotinic and muscarinic autoreceptors. Receptor-mediated presynaptic regulation could reflect either direct action on exocytotic machinery or modulation of Ca2+ entry and resulting intra-terminal Ca2+ dynamics. We have measured free intra-terminal cytosolic Ca2+ ([Ca2+]i) using Oregon-Green 488 microfluorimetry, in parallel with voltage-clamp recordings of spontaneous (mEPC) and evoked (EPC) postsynaptic currents in post-junctional skeletal muscle fiber. Activation of presynaptic muscarinic and nicotinic receptors with exogenous acetylcholine and its non-hydrolized analog carbachol reduced amplitude of the intra-terminal [Ca2+]i transients and decreased quantal content (calculated by dividing the area under EPC curve by the area under mEPC curve). Pharmacological analysis revealed the role of muscarinic receptors of M2 subtype as well as d-tubocurarine-sensitive nicotinic receptor in presynaptic modulation of [Ca2+]i transients. Modulation of synaptic transmission efficacy by ACh receptors was completely eliminated by pharmacological inhibition of N-type Ca2+ channels. We conclude that ACh receptor-mediated reduction of Ca2+ entry into the nerve terminal through N-type Ca2+ channels represents one of possible mechanism of presynaptic modulation in frog neuromuscular junction.

The balance of sympathetic and parasympathetic tone provides exquisite control of heart rate and contractility and has also been shown to modulate coronary flow and inflammation. Understanding how autonomic balance is altered by cardiac disease is an active area of research, and developing new ways to control this balance provides insights into disease therapies. However, achieving acute neuron-specific stimulation of autonomic neurons can be difficult in experiments that measure the acute effects of nerve stimulation on the heart. Conventional electrical and pharmacological approaches can be spatially and temporally non-selective. Cell-specific expression of light-activated channels (channelrhodopsin, ChR2) is a powerful approach that enables control of the timing and distribution of cellular stimulation using light. We present such an optogenetic approach where parasympathetic cardiac neurons are selectively photoactivated at high temporal precision to initiate cholinergic-mediated slowing of heart rate. Mice were crossbred to express ChR2 in peripheral cholinergic neurons using Cre-Lox recombination driven by a choline acetyltransferase (ChAT) promoter. Hearts from adult mice were excised, perfused, and the epicardium was illuminated (peak 460-465 nm) to photoactivate ChR2. In one set of studies, hearts were illuminated using a large-field LED light source. In other studies, a micro LED was placed on the right atrium to selectively illuminate the junction of the superior vena cava (SVC) and right atrium. The ECG was acquired before, during, and after tissue illumination to measure changes in heart rate. Upon illumination, hearts exhibited sudden and dramatic reductions in heart rate with restoration of normal heart rate after cessation of illumination. Delays in atrioventricular conduction were also observed. Heart rate reductions at the highest irradiance levels were similar to heart rate reductions caused by application of bethanechol (10 μM) or acetylcholine (800 μM). Atropine (50 nM) completely blocked the effect of ChR2 photoactivation, confirming cholinergic mediation. Optogenetic activation of intrinsic parasympathetic neurons reduced heart rate in an immediate, dose-dependent fashion, resembling the slowing of sinus rate in response to acetylcholine. Our results demonstrate a new approach for controlling parasympathetic modulation of cardiac function by selectively activating the endogenous release of acetylcholine from intrinsic cardiac cholinergic neurons. Key Message: Optogenetic photoactivation of intrinsic cardiac neurons provides immediate, tissue-specific stimulation with minimal cross-reactivity. Our results demonstrate that selective expression of channelrhodopsin within cardiac cholinergic neurons enables photoactivated release of acetylcholine, thereby instantaneously slowing sinus rate and altering atrioventricular conduction. This provides for in-depth examination of the endogenous interplay between cardiac autonomic neurons and the functional outcomes of downstream post-synaptic receptor activation.

Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca2+ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca2+ safely remain unclear. Our previous work contradicted the dogma that Ca2+ is ferried through the cytosol of Ca2+-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca2+ stores and their concomitant refilling by store-operated Ca2+ entry (SOCE) mediated by Ca2+ release activated Ca2+ (CRAC) channels. Given that Ca2+ handling is maximal during the enamel-mineralization stage (maturation), we anticipated that SOCE would also be elevated then. Confirmation was obtained here using single-cell recordings of cytosolic Ca2+ concentration ([Ca2+]cyt) in rat ameloblasts. A candidate SOCE agonist, cholecystokinin (CCK), was found to be upregulated during maturation, with Cck transcript abundance reaching 30% of that in brain. CCK-receptor transcripts were also detected and Ca2+ imaging showed that CCK stimulation increased [Ca2+]cyt in a dose-responsive manner that was sensitive to CRAC-channel inhibitors. Similar effects were observed with two other SOCE activators, acetylcholine and ATP, whose receptors were also found in enamel cells. These results provide the first evidence of a potential regulatory system for SOCE in enamel cells and so strengthen the Ca2+ transcytosis paradigm for ER-based transport of bulk Ca2+. Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca2+ transport.

Preeclampsia (PE) and gestational diabetes (GD) are complications in advanced pregnancy while miscarriage for early pregnancy. However, the etiological factors are not well understood. Smoking has been associated with these complications as well as the sudden intrauterine deaths, sudden infant death, miscarriages, and still births. However, the immunolocalization of alpha 7 nicotine acetylcholine receptor (α7-nAChR) is not studied. Materials and Methods: α7-nAChR subunit expression was evaluated in 10 paraffin-embedded placental tissues after delivery and 10 tissue samples of products of conception during first trimester by immunohistochemistry. Among the placental tissues, two samples were normal placental tissue, four from PE mother, and four from GD mother. The expression of α7-nAChR was compared between the two groups in general and within the subgroups of placenta as well. Protein expression was evaluated using the nuclear labeling index (%) of villi with positive cells stained, positive cells in the decidua, and intensity of staining in the outer villous trophoblast layer. Results: The expression of α7-nAChR protein was high in all the cases of placenta and products of conception (POCs). α7-nAChR expression showed no notable differences among different cases of miscarriages irrespective of the mother's age and gestational age at which the event occurred. However, there were some changes among the normal, PE, and GD placental groups in the linings of the blood vessels. Changes were restricted to the villi (as opposed to the decidua) lining cells, both cytotrophoblast and syncytiotrophoblast, and were specific to the α7 subunit. PE blood vessel lining was thicker and showed more expression of this receptor in endothelial cells and myofibroblasts in PE and GD groups. In POCs, the strong expression was observed in the decidua myocytes of maternal blood vessels and in syncytiotrophoblast and cytotrophoblast of chronic villi. Conclusion: Nicotine acetyl choline receptors are found to be expressed highly in the placental tissues and in products of conception. They may be associated with the sudden perinatal deaths and miscarriages or complications of pregnancy.

Previous reports suggest that diabetes may differentially affect the vascular beds of females and males. The objectives of this study were to examine whether there were (1) sex differences in aortic function and (2) alterations in the relative contribution of endothelium-derived relaxing factors in modulating aortic reactivity in UC Davis Type 2 Diabetes Mellitus (UCD-T2DM) rats. Endothelium-dependent vasorelaxation (EDV) in response to acetylcholine (ACh) was measured in aortic rings before and after exposure to pharmacological inhibitors. Relaxation responses to sodium nitroprusside were assessed in endothelium-denuded rings. Moreover, contractile responses to phenylephrine (PE) were measured before and after incubation of aortic rings with a nitric oxide synthase (NOS) inhibitor in the presence of indomethacin. Metabolic parameters and expression of molecules associated with vascular and insulin signaling as well as reactive oxygen species generation were determined. Diabetes slightly but significantly impaired EDV in response to ACh in aortas from females but potentiated the relaxation response in males. The potentiation of EDV in diabetic male aortas was accompanied by a traces of nitric oxide (NO)- and prostanoid-independent relaxation and elevated aortic expression of small- and intermediate conductance Ca2+-activated K+ channels in this group. The smooth muscle sensitivity to NO was not altered, whereas the responsiveness to PE was significantly enhanced in aortas of diabetic groups in both sexes. Endothelium-derived NO during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was reduced in aortas of diabetic rats regardless of sex. Accordingly, decreases in pAkt and peNOS were observed in aortas from diabetic rats in both sexes compared with controls. Our data suggest that a decrease in insulin sensitivity via pAkt-peNOS-dependent signaling and an increase in oxidative stress may contribute to the elevated contractile responses observed in diabetic aortas in both sexes. This study demonstrates that aortic function in UCD-T2DM rats is altered in both sexes. Here, we provide the first evidence of sexual dimorphism in aortic relaxation in UCD-T2DM rats.

Muscarinic acetylcholine receptor (mAChR) regulates many neurophysiological functions in insects. In this report, a full-length cDNA encoding an A-type mAChR was cloned from the oriental armyworm, Mythimna separata. Pharmacological properties studies revealed that nanomolar to micromolar concentrations of carbachol or muscarine induced an increase of intracellular Ca2+ concentration ([Ca2+] i ), with the EC50 values of 124.6 and 388.1 nM, respectively. The increases of [Ca2+] i can be greatly blocked by the antagonist atropine, with an IC50 value of 0.09 nM. The receptor mRNA is expressed in all developmental stages, with great differential expression between male and female adults. The tissue expression analysis identified novel target tissues for this receptor, including ovaries and Malpighian tubules. The distribution of Ms A-type mAChR protein in the male brain may suggest the neurophysiological roles that are mediated by this receptor. However, the receptor protein was found to be distributed on the membranes of oocytes that are not innervated by neurons at all. These results indicate that Ms A-type mAChR selectively mediates intracellular Ca2+ mobilization. And the high level of receptor protein in the membrane of oocytes may indicate a possible non-neuronal role of A-type mAChR in the reproductive system of M. separata.

Insulin resistance plays a key role in the pathogenesis of type 2 diabetes and is also related to other health problems like obesity, hypertension, and metabolic syndrome. Imbalance between insulin vascular actions via the phosphatidylinositol 3-Kinase (PI3K) and the mitogen activated protein kinase (MAPK) signaling pathways during insulin resistant states results in impaired endothelial PI3K/eNOS- and augmented MAPK/endothelin 1 pathways leading to endothelial dysfunction and abnormal vasoconstriction. The role of PI3K, MAPK, and protein kinase C (PKC) in Ca2+ handling of resistance arteries involved in blood pressure regulation is poorly understood. Therefore, we assessed here whether PI3K, MAPK, and PKC play a role in the Ca2+ signaling pathways linked to adrenergic vasoconstriction in resistance arteries. Simultaneous measurements of intracellular calcium concentration ([Ca2+]i) in vascular smooth muscle (VSM) and tension were performed in endothelium-denuded branches of mesenteric arteries from Wistar rats mounted in a microvascular myographs. Responses to CaCl2 were assessed in arteries activated with phenylephrine (PE) and kept in Ca2+-free solution, in the absence and presence of the selective antagonist of L-type Ca2+ channels nifedipine, cyclopiazonic acid (CPA) to block sarcoplasmic reticulum (SR) intracellular Ca2+ release or specific inhibitors of PI3K, ERK-MAPK, or PKC. Activation of α1-adrenoceptors with PE stimulated both intracellular Ca2+ mobilization and Ca2+ entry along with contraction in resistance arteries. Both [Ca2+]i and contractile responses were inhibited by nifedipine while CPA abolished intracellular Ca2+ mobilization and modestly reduced Ca2+ entry suggesting that α1-adrenergic vasoconstriction is largely dependent Ca2+ influx through L-type Ca2+ channel and to a lesser extent through store-operated Ca2+ channels. Inhibition of ERK-MAPK did not alter intracellular Ca2+ mobilization but largely reduced L-type Ca2+ entry elicited by PE without altering vasoconstriction. The PI3K blocker LY-294002 moderately reduced intracellular Ca2+ release, Ca2+ entry and contraction induced by the α1-adrenoceptor agonist, while PKC inhibition decreased PE-elicited Ca2+ entry and to a lesser extent contraction without affecting intracellular Ca2+ mobilization. Under conditions of ryanodine receptor (RyR) blockade to inhibit Ca2+-induced Ca2+-release (CICR), inhibitors of PI3K, ERK-MAPK, or PKC significantly reduced [Ca2+]i increases but not contraction elicited by high K+ depolarization suggesting an activation of L-type Ca2+ entry in VSM independent of RyR. In summary, our results demonstrate that PI3K, ERK-MAPK, and PKC regulate Ca2+ handling coupled to the α1-adrenoceptor in VSM of resistance arteries and related to both contractile and non-contractile functions. These kinases represent potential pharmacological targets in pathologies associated to vascular dysfunction and abnormal Ca2+ handling such as obesity, hypertension and diabetes mellitus, in which these signaling pathways are profoundly impaired.

Beat to beat variability of cardiac tissue or isolated cells is frequently investigated by determining time intervals from electrode measurements in order to compute scale dependent or scale independent parameters. In this study, we utilize high-speed video camera recordings to investigate the variability of intervals as well as mechanical contraction strengths and relative contraction strengths with nonlinear analyses. Additionally, the video setup allowed us simultaneous electrode registrations of extracellular potentials. Sinoatrial node tissue under control and acetylcholine treated conditions was used to perform variability analyses by computing sample entropies and Higuchi dimensions. Beat to beat interval variabilities measured by the two recording techniques correlated very well, and therefore, validated the video analyses for this purpose. Acetylcholine treatment induced a reduction of beating rate and contraction strength, but the impact on interval variability was negligible. Nevertheless, the variability analyses of contraction strengths revealed significant differences in sample entropies and Higuchi dimensions between control and acetylcholine treated tissue. Therefore, the proposed high-speed video camera technique might represent a non-invasive tool that allows long-lasting recordings for detecting variations in beating behavior over a large range of scales.

Diabetic bladder dysfunction (DBD) is one of the most common complication of diabetes. Methylglyoxal (MGO), a highly reactive dicarbonyl compound formed as a by-product of glycolysis, is found at high levels in plasma of diabetic patients. Here, we explored the effects of chronic administration of MGO on micturition pattern (cystometry) and bladder contractility in vitro in healthy male C57/BL6 mice. Methylglyoxal was given at 0.5% in drinking water for 4 weeks. Exposure to MGO led to bladder tissue disorganization, edema of lamina propria, partial loss of urothelium and multiple leukocyte infiltrates. Filling cystometry revealed significant increases of micturition frequency and number of non-voiding contractions (NVCs) in the MGO group, clearly indicating an overactive bladder profile. Bladder contractions induced by electrical-field stimulation (EFS) and carbachol were significantly higher in the MGO group, while the muscarinic M2 and M3 mRNA expressions remained unchanged between groups. Additionally, MGO exposure induced upregulation of TRPA1 and down-regulation of TRPV1 and TRPV4 in bladder tissues. Methylglyoxal did not change the mRNA expression of the advanced glycation end products receptor (RAGE), but markedly increased its downstream NF-κB - iNOS signaling. The mRNA expression of cyclooxygenase-2 (COX-2) and reactive-oxygen species (ROS) levels remained unchanged. Altogether, our data show that 4-week MGO intake in mice produces an overactive bladder phenotype in addition to bladder inflammation and increased NF-kB/iNOS signaling. TRPA1 up-regulation and TRPV1/TRPV4 down-regulation may account for the MGO-induced bladder overactivity. Scavengers of MGO could be an option to ameliorate bladder dysfunction in diabetic conditions.

Acetylcholine may contribute to the increase in regional cerebral blood flow (rCBF) during cerebral activation since glycopyrrolate, a potent inhibitor of acetylcholine, abolishes the exercise-induced increase in middle cerebral artery mean flow velocity. We tested the hypothesis that cholinergic vasodilatation is important for the increase in rCBF during cerebral activation. The subjects were 11 young healthy males at an age of 24 ± 3 years (mean ± SD). We used arterial spin labeling and blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to evaluate rCBF with and without intravenous glycopyrrolate during a handgrip motor task and visual stimulation. Glycopyrrolate increased heart rate from 56 ± 9 to 114 ± 14 beats/min (mean ± SD; p < 0.001), mean arterial pressure from 86 ± 8 to 92 ± 12 mmHg, and cardiac output from 5.6 ± 1.4 to 8.0 ± 1.7 l/min. Glycopyrrolate had, however, no effect on the arterial spin labeling or BOLD responses to the handgrip motor task or to visual stimulation. This study indicates that during a handgrip motor task and visual stimulation, the increase in rCBF is unaffected by blockade of acetylcholine receptors by glycopyrrolate. Further studies on the effect of glycopyrrolate on middle cerebral artery diameter are needed to evaluate the influence of glycopyrrolate on mean flow velocity during intense exercise.

Background: We previously reported that bilateral sympathetic stellate ganglionectomy attenuated cardiac remodeling and fibrosis in rats with chronic volume overload. Transforming growth factor beta 1 (TGF-β1) is a polypeptide member of the transforming growth factor beta superfamily of cytokines and actively involved in many pathological processes of cardiovascular diseases. The present study explored the impact of bilateral sympathetic stellate ganglionectomy on the TGF-β1 pathway in this model. Methods and Results: Male Sprague-Dawley rats were randomly divided into sham (S) group, abdominal aorta-cava fistula (AV) group, and bilateral sympathetic stellate ganglionectomy after abdominal aorta-cava fistula (AD) group. Twelve weeks after the abdominal aorta-cava fistula surgery, the myocardial expressions of norepinephrine (NE) and hydroxyproline were significantly higher, while acetylcholine was downregulated in the AV group compared to the S group; the above changes were partly reversed in the AD group. The myocardial expression of TGF-β1 and activity of Smad2/3 phosphorylation were also upregulated in the AV group compared to the S group, which could be reversed by bilateral sympathetic stellate ganglionectomy. In vitro, the TGF-β1 expression in cultured myocardial fibroblasts and the proliferation of myocardial fibroblasts were significantly increased post-stimulation with NE in a dose-dependent manner, and these effects could be blunted by co-treatment with a TGF-β1 inhibitor. Conclusion: Our study results indicate that stellate ganglionectomy decreases cardiac norepinephrine release, which leads to decreased TGF-β1 release and reduced fibrosis in rats with chronic volume overload.

Background: Endothelial dysfunction plays a pivotal role in the initiation of atherosclerosis. Vascular insulin resistance might contribute to a reduction in endothelial nitric oxide (NO) production, leading to impaired endothelium-dependent relaxation in cardiometabolic diseases. Because perivascular adipose tissue (PVAT) controls endothelial function and NO bioavailability, we hypothesized a role for this fat deposit in the vascular complications associated with the initial stages of atherosclerosis. Therefore, we investigated the potential involvement of PVAT in the early endothelial dysfunction in hypercholesterolemic LDL receptor knockout mice (LDLr-KO). Methods: Thoracic aortas with and without PVAT were isolated from 4-month-old C57BL/6J (WT) and LDLr-KO mice. The contribution of PVAT to relaxation responses to acetylcholine, insulin, and sodium nitroprusside was investigated. Western blotting was used to examine endothelial NO synthase (eNOS) and adiponectin expression, as well the insulin signaling pathway in aortic PVAT. Results: PVAT-free aortas of LDLr-KO mice exhibited impaired acetylcholine- and insulin-induced relaxation compared with those of WT mice. Both vasodilatory responses were restored by the presence of PVAT in LDLr-KO mice, associated with enhanced acetylcholine-induced NO levels. PVAT did not change vasodilatory responses to acetylcholine and insulin in WT mice, while vascular relaxation evoked by the NO donor sodium nitroprusside was not modified by either genotype or PVAT. The expression of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), AKT, ERK1/2, phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204), and adiponectin was similar in the PVAT of WT and LDLr-KO mice, suggesting no changes in PVAT insulin signaling. However, eNOS expression was enhanced in the PVAT of LDLr-KO mice, while eNOS expression was less abundant in PVAT-free aortas. Conclusion: These results suggest that elevated eNOS-derived NO production in aortic PVAT might be a compensatory mechanism for the endothelial dysfunction and impaired vasodilator action of insulin in hypercholesterolemic LDLr-deficient mice. This protective effect may limit the progression of atherosclerosis in genetic hypercholesterolemia in the absence of an atherogenic diet.

Endothelial dysfunction, which leads to ischemic events under atherosclerotic conditions, can be attenuated by antagonizing the thromboxane-prostanoid receptor (TP) that mediates the vasoconstrictor effect of prostanoids including prostacyclin (PGI2). This study aimed to determine whether antagonizing the E prostanoid receptor-3 (EP3; which can also be activated by PGI2) adds to the above effect of TP deficiency (TP-/-) under atherosclerotic conditions and if so, the underlying mechanism(s). Atherosclerosis was induced in ApoE-/- mice and those with ApoE-/- and TP-/-. Here, we show that in phenylephrine pre-contracted abdominal aortic rings with atherosclerotic lesions of ApoE-/-/TP-/- mice, although an increase of force (which was larger than that of non-atherosclerotic controls) evoked by the endothelial muscarinic agonist acetylcholine to blunt the concurrently activated relaxation in ApoE-/- counterparts was largely removed, the relaxation evoked by the agonist was still smaller than that of non-atherosclerotic TP-/- mice. EP3 antagonism not only increased the above relaxation, but also reversed the contractile response evoked by acetylcholine in NO synthase-inhibited atherosclerotic ApoE-/-/TP-/- rings into a relaxation sensitive to I prostanoid receptor antagonism. In ApoE-/- atherosclerotic vessels the expression of endothelial NO synthase was decreased, yet the production of PGI2 (which evokes contraction via both TP and EP3) evoked by acetylcholine was unaltered compared to non-atherosclerotic conditions. These results demonstrate that EP3 blockade adds to the effect of TP-/- in uncovering the dilator action of natively produced PGI2 to alleviate endothelial dysfunction in atherosclerotic conditions.

Acetaldehyde (AA) is a small, ubiquitous compound present in foods, beverages, as a gas phase combustion product, and also endogenously generated from metabolism as from ethanol (EtOH). Acetate is a short chain fatty acid derived from AA oxidation, and acetate levels were significantly higher in urine collected overnight with food provided ad libitum compared with urine collected after 9 h fasting. Feeding increases gastrointestinal blood flow, and thus, we explored the direct effects of AA (and acetate) in isolated murine superior mesenteric artery (SMA). Over the concentration range of 1-100 mM, AA strongly, and reversibly relaxed agonist-induced contractions of SMA including phenylephrine (PE), thromboxane A2 analog (U46,619) and high potassium (High K+) without toxicity. The sensitivity (EC50) but not the efficacy (>90% relaxation of PE-precontraction) of AA-induced relaxations was dependent on blood vessel (SMA was 3× more sensitive than aorta) and contractile agonist (PE EC50 = 3.3 ± 0.4 mM; U46,619 EC50 = 14.9 ± 1.5 mM; and High K+ EC50 = 17.7 ± 0.5 mM) yet independent of circadian cycle and sex. The most sensitive component of the AA-induced relaxation was inhibited significantly by: (1) a mechanically impaired endothelium; (2) nitric oxide synthase (NOS) inhibitor (L-NAME); and (3) a guanylyl cyclase (GC) inhibitor (ODQ). Both acetate and EtOH stimulated much weaker relaxations in SMA than did AA, yet these relaxations were significantly inhibited by L-NAME as well. Neither EtOH nor acetate relaxed pre-contracted aorta. Although neither cyanamide, a non-specific aldehyde dehydrogenase (ALDH) enzyme inhibitor, nor Alda-1, a specific activator of ALDH2 activity, had any effect on either sensitivity or efficacy of AA-induced relaxation in SMA, cyanamide significantly blocked both EtOH- and acetate-induced relaxations in SMA implicating a role of ALDH activity in vasorelaxation. These data show that AA relaxes SMA via an endothelium- and NO-dependent mechanism indicating that AA may be one component of the complex post-prandial hyperemia reflex via vasodilatation of mesenteric vasculature.

Obesity, an important risk factor for cardiovascular disease, promotes vascular oxidative stress. Considering that free testosterone levels remain within the reference range, especially in obese young men and that testosterone stimulates reactive oxygen species (ROS) generation, we sought to investigate whether testosterone interferes with obesity-associated oxidative stress and vascular dysfunction in male mice. We hypothesized that testosterone favors ROS accumulation and vascular dysfunction in high fat diet (HFD)-fed obese mice. We also questioned whether testosterone downregulates the nuclear factor E2-related factor 2 (Nrf2), one of the major cellular defense mechanisms against oxidative stimuli. Male C57Bl/6J mice were submitted to orchiectomy or sham-operation. Mice received either a control diet (CD) or HFD for 18 weeks. Vascular function was assessed in thoracic aortic rings and molecular mechanisms by which testosterone contributes to vascular dysfunction were determined. HFD reduced acetylcholine-induced vasodilation and increased vascular ROS generation in sham mice. Castration prevented these effects. Treatment of castrated mice fed either the CD or HFD with testosterone propionate decreased acetylcholine vasodilation. HFD decreased Nrf2 nuclear accumulation, events linked to decreased mRNA expression and activity of Nrf2-regulated enzymes (catalase, heme oxygenase-1, peroxiredoxin, and thioredoxin). These events were prevented in HFD-fed castrated mice. Bardoxolone, a Nrf2 activator, increased nuclear accumulation of Nrf2, decreased ROS generation and improved acetylcholine vasodilation in HFD-fed sham mice. In vitro, testosterone increased ROS generation and decreased Nrf2 nuclear accumulation. These effects were prevented in the presence of an androgen receptor antagonist, an inhibitor of gene transcription and an inhibitor of the pro-oxidant enzyme NOX-1. These results indicate that testosterone downregulates Nrf2, leading to oxidative stress and vascular dysfunction in HFD-fed obese young mice.

Neonicotinoids are the most widely used insecticides in the world and are implicated in the widespread population declines of insects including pollinators. Neonicotinoids target nicotinic acetylcholine receptors which are expressed throughout the insect central nervous system, causing a wide range of sub-lethal effects on non-target insects. Here, we review the potential of the fruit fly Drosophila melanogaster to model the sub-lethal effects of neonicotinoids on pollinators, by utilizing its well-established assays that allow rapid identification and mechanistic characterization of these effects. We compare studies on the effects of neonicotinoids on lethality, reproduction, locomotion, immunity, learning, circadian rhythms and sleep in D. melanogaster and a range of pollinators. We also highlight how the genetic tools available in D. melanogaster, such as GAL4/UAS targeted transgene expression system combined with RNAi lines to any gene in the genome including the different nicotinic acetylcholine receptor subunit genes, are set to elucidate the mechanisms that underlie the sub-lethal effects of these common pesticides. We argue that studying pollinators and D. melanogaster in tandem allows rapid elucidation of mechanisms of action, which translate well from D. melanogaster to pollinators. We focus on the recent identification of novel and important sublethal effects of neonicotinoids on circadian rhythms and sleep. The comparison of effects between D. melanogaster and pollinators and the use of genetic tools to identify mechanisms make a powerful partnership for the future discovery and testing of more specific insecticides.

Vasodilation of lower leg arterioles is impaired in animal models of chronic peripheral ischemia. In addition to arterioles, feed arteries are a critical component of the vascular resistance network, accounting for as much as 50% of the pressure drop across the arterial circulation. Despite the critical importance of feed arteries in blood flow control, the impact of ischemia on feed artery vascular reactivity is unknown. At 14 days following unilateral resection of the femoral-saphenous artery-vein pair, functional vasodilation of the profunda femoris artery was severely impaired, 11 ± 9 versus 152 ± 22%. Although endothelial and smooth muscle-dependent vasodilation were both impaired in ischemic arteries compared to control arteries (Ach: 40 ± 14 versus 81 ± 11%, SNP: 43 ± 12 versus and 85 ± 11%), the responses to acetylcholine and sodium nitroprusside were similar, implicating impaired smooth muscle-dependent vasodilation. Conversely, vasoconstriction responses to norepinephrine were not different between ischemic and control arteries, -68 ± 3 versus -66 ± 3%, indicating that smooth muscle cells were functional following the ischemic insult. Finally, maximal dilation responses to acetylcholine, ex vivo, were significantly impaired in the ischemic artery compared to control, 71 ± 9 versus 97 ± 2%, despite a similar generation of myogenic tone to the same intravascular pressure (80 mmHg). These data indicate that ischemia impairs feed artery vasodilation by impairing the responsiveness of the vascular wall to vasodilating stimuli. Future studies to examine the mechanistic basis for the impact of ischemia on vascular reactivity or treatment strategies to improve vascular reactivity following ischemia could provide the foundation for an alternative therapeutic paradigm for peripheral arterial occlusive disease.