In a rat model of bypass DuraGraft ameliorates endothelial dysfunction of arterial grafts.

Coronary artery bypass surgery can result in endothelial dysfunction due to ischemia/reperfusion (IR) injury. Previous studies have demonstrated that DuraGraft helps maintain endothelial integrity of saphenous vein grafts during ischemic conditions. In this study, we investigated the potential of DuraGraft to mitigate endothelial dysfunction in arterial grafts after IR injury using an aortic transplantation model. Lewis rats (n = 7-9/group) were divided in three groups. Aortic arches from the control group were prepared and rings were immediately placed in organ baths, while the aortic arches of IR and IR + DuraGraft rats were preserved in saline or DuraGraft, respectively, for 1 h before being transplanted heterotopically. After 1 h after reperfusion, the grafts were explanted, rings were prepared, and mounted in organ baths. Our results demonstrated that the maximum endothelium-dependent vasorelaxation to acetylcholine was significantly impaired in the IR group compared to the control group, but DuraGraft improved it (control: 89 ± 2%; IR: 24 ± 1%; IR + DuraGraft: 48 ± 1%, p < 0.05). Immunohistochemical analysis revealed decreased intercellular adhesion molecule-1, 4-hydroxy-2-nonenal, caspase-3 and caspase-8 expression, while endothelial cell adhesion molecule-1 immunoreactivity was increased in the IR + DuraGraft grafts compared to the IR-group. DuraGraft mitigates endothelial dysfunction following IR injury in a rat bypass model. Its protective effect may be attributed, at least in part, to its ability to reduce the inflammatory response, oxidative stress, and apoptosis.

Osteoprotegerin Is Essential for the Development of Endothelial Dysfunction Induced by Angiotensin II in Mice.

Opinions on the effects of osteoprotegerin (OPG) have evolved over the years from a protein protecting the vasculature from calcification to a cardiovascular risk factor contributing to inflammation within the vascular wall. Nowadays, the link between OPG and angiotensin II (Ang II) appears to be particularly important. In this study, the endothelial function was investigated in OPG-knockout mice (B6.129.S4-OPG, OPG-) and wild-type (C57BL/6J, OPG+) mice under basic conditions and after Ang II exposure by assessing the endothelium-dependent diastolic response of aortic rings to acetylcholine in vitro. A further aim of the study was to compare the effect of Ang II on the expression of cytokines in the aortic wall of both groups of mice. Our study shows that rings from OPG- mice had their normal endothelial function preserved after incubation with Ang II, whereas those from OPG+ mice showed significant endothelial dysfunction. We conclude that the absence of OPG, although associated with a pro-inflammatory cytokine profile in the vascular wall, simultaneously protects against Ang II-induced increases in pro-inflammatory cytokines in the murine vascular wall. Our study also demonstrates that the absence of OPG can result in a decrease in the concentration of pro-inflammatory cytokines in the vascular wall after Ang II exposure. The presence of OPG is therefore crucial for the development of Ang II-induced inflammation in the vascular wall and for the development of Ang II-induced endothelial dysfunction.

Allosteric modulators of M1 muscarinic receptors enhance acetylcholine efficacy and decrease locomotor activity and turning behaviors in zebrafish.

Allosteric modulation of muscarinic acetylcholine receptors (mAChR) has been identified as a potential strategy for regulating cholinergic signaling in the treatment of various neurological disorders. Most positive allosteric modulators (PAMs) of mAChR enhance agonist affinity and potency, while very few PAMs (e.g., amiodarone) selectively enhance G protein coupling efficacy. The key structural features of amiodarone responsible for enhancement of mAChR efficacy were examined in CHO cells expressing M1 receptors. Subsequent incorporation of these structural features into previously identified allosteric modulators of potency (i.e., n-benzyl isatins) generated ligands that demonstrated similar or better enhancement of mAChR efficacy, lower in vivo toxicity, and higher allosteric binding affinity relative to amiodarone. Notable ligands include 8a, c which respectively demonstrated the strongest binding affinity and the most robust enhancement of mAChR efficacy as calculated from an allosteric operational model. Amiodarone derivatives and hybrid ligands were additionally screened in wildtype zebrafish (Danio rerio) to provide preliminary in vivo toxicity data as well as to observe effects on locomotor and turning behaviors relative to other mAChR PAMs. Several compounds, including 8a, c, reduced locomotor activity and increased measures of turning behaviors in zebrafish, suggesting that allosteric modulation of muscarinic receptor efficacy might be useful in the treatment of repetitive behaviors associated with autism spectrum disorder (ASD) and other neuropsychiatric disorders.

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4ß2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

ON and OFF starburst amacrine cells are controlled by distinct cholinergic pathways.

Cholinergic signaling in the retina is mediated by acetylcholine (ACh) released from starburst amacrine cells (SACs), which are key neurons for motion detection. SACs comprise ON and OFF subtypes, which morphologically show mirror symmetry to each other. Although many physiological studies on SACs have targeted ON cells only, the synaptic computation of ON and OFF SACs is assumed to be similar. Recent studies demonstrated that gene expression patterns and receptor types differed between ON and OFF SACs, suggesting differences in their functions. Here, we compared cholinergic signaling pathways between ON and OFF SACs in the mouse retina using the patch clamp technique. The application of ACh increased GABAergic feedback, observed as postsynaptic currents to SACs, in both ON and OFF SACs; however, the mode of GABAergic feedback differed. Nicotinic receptors mediated GABAergic feedback in both ON and OFF SACs, while muscarinic receptors mediated GABAergic feedback in ON SACs only in adults. Neither tetrodotoxin, which blocked action potentials, nor LY354740, which blocked neurotransmitter release from SACs, eliminated ACh-induced GABAergic feedback in SACs. These results suggest that ACh-induced GABAergic feedback in ON and OFF SACs is regulated by different feedback mechanisms in adults and mediated by non-spiking amacrine cells other than SACs.

PI3 kinase inhibitor PI828 uncouples aminergic GPCRs and Ca2+ mobilization irrespectively of its primary target.

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied Ca2+ signaling initiated by aminergic agonists in a variety of different cells and analyzed effects of the PI3K inhibitor PI828 on cell responsiveness. It turned out that PI828 inhibited Ca2+ transients elicited by acetylcholine (ACh), histamine, and serotonin, but did not affect Ca2+ responses to norepinephrine and ATP. Another PI3K inhibitor wortmannin negligibly affected Ca2+ signaling initiated by any one of the tested agonists. Using the genetically encoded PIP3 sensor PH(Akt)-Venus, we confirmed that both PI828 and wortmannin effectively inhibited PI3K and ascertained that this kinase negligibly contributed to ACh transduction. These findings suggested that PI828 inhibited Ca2+ responses to aminergic agonists tested, involving an unknown cellular mechanism unrelated to the PI3K inhibition. Complementary physiological experiments provided evidence that PI828 could inhibit Ca2+ signals induced by certain agonists, by acting extracellularly, presumably, through their surface receptors. For the muscarinic M3 receptor, this possibility was verified with molecular docking and molecular dynamics. As demonstrated with these tools, wortmannin could be bound in the extracellular vestibule at the muscarinic M3 receptor but this did not preclude binding of ACh to the M3 receptor followed by its activation. In contrast, PI828 could sterically block the passage of ACh into the allosteric site, preventing activation of the muscarinic M3 receptor.

Noninvasive assessment of human microvascular function in health and disease using incident dark-field microscopy.

Reduced peripheral microvascular reactivity is associated with an increased risk for major adverse cardiac events (MACEs). Tools for noninvasive assessment of peripheral microvascular function are limited, and existing technology is poorly validated in both healthy populations and patients with cardiovascular disease (CVD). Here, we used a handheld incident dark-field imaging tool (CytoCam) to test the hypothesis that, compared with healthy individuals (no risk factors for CVD), subjects formally diagnosed with coronary artery disease (CAD) or those with ≥2 risk factors for CAD (at risk) would exhibit impaired peripheral microvascular reactivity. A total of 17 participants (11 healthy, 6 at risk) were included in this pilot study. CytoCam was used to measure sublingual microvascular total vessel density (TVD), perfused vessel density (PVD), and microvascular flow index (MFI) in response to the topical application of acetylcholine (ACh) and sublingual administration of nitroglycerin (NTG). Baseline MFI and PVD were significantly reduced in the at-risk cohort compared with healthy individuals. Surprisingly, following the application of acetylcholine and nitroglycerin, both groups showed a significant improvement in all three microvascular perfusion parameters. These results suggest that, despite baseline reductions in both microvascular density and perfusion, human in vivo peripheral microvascular reactivity to both endothelial-dependent and -independent vasoactive agents remains intact in individuals with CAD or multiple risk factors for disease.NEW & NOTEWORTHY To our knowledge, this is the first study to comprehensively characterize in vivo sublingual microvascular structure and function (endothelium-dependent and -independent) in healthy patients and those with CVD. Importantly, we used an easy-to-use handheld device that can be easily translated to clinical settings. Our results indicate that baseline microvascular impairments in structure and function can be detected using the CytoCam technology, although reactivity to acetylcholine may be maintained even during disease in the peripheral microcirculation.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

WNK kinase, ion channels and arachidonic acid metabolites choreographically execute endothelium-dependent vasodilation.

The smooth muscle-walled blood vessels control blood pressure. The vessel lumen is lined by an endothelial cell (ECs) layer, interconnected to the surrounding smooth muscle cells (SMCs) by myoendothelial gap junctions. Gap junctions also maintain homo-cellular ECs-ECs and SMCs-SMCs connections. This gap junction network nearly equalises both cells' membrane potential and cytosolic ionic composition, whether in resting or stimulated conditions. When acetylcholine (ACh) activates ECs M3 receptors, a complex signalling cascade involving second messengers and ion channels is triggered to induce vasodilation.

Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner.

Several studies have shown an inverse correlation between the likelihood of developing a neurodegenerative disorder and cancer. We previously reported that the levels of amyloid beta (Aß), at the center of Alzheimer's disease pathophysiology, are regulated by acetylcholinesterase (AChE) in non-small cell lung cancer (NSCLC). Here, we examined the effect of Aß or its fragments on the levels of ACh in A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell media. ACh levels were reduced by cell treatment with Aß 1-42, Aß 1-40, Aß 1-28, and Aß 25-35. AChE and p53 activities increased upon A549 cell treatment with Aß, while knockdown of p53 in A549 cells increased ACh levels, decreased AChE activity, and diminished the Aß effects. Aß increased the ratio of phospho/total p38 MAPK and decreased the activity of PKC. Inhibiting p38 MAPK reduced the activity of p53 in A549 cells and increased ACh levels in the media of both cell lines, while opposite effects were found upon inhibiting PKC. ACh decreased the activity of p53 in A549 cells, decreased p38 MAPK activity, increased PKC activity, and diminished the effect of Aß on those activities. Moreover, the negative effect of Aß on cell viability was diminished by cell co-treatment with ACh.

Multiple cholinergic receptor subtypes coordinate dual modulation of acetylcholine on anterior and posterior paraventricular thalamic neurons.

Paraventricular thalamus (PVT) plays important roles in the regulation of emotion and motivation through connecting many brain structures including the midbrain and the limbic system. Although acetylcholine (ACh) neurons of the midbrain were reported to send projections to PVT, little is known about how cholinergic signaling regulates PVT neurons. Here, we used both RNAscope and slice patch-clamp recordings to characterize cholinergic receptor expression and ACh modulation of PVT neurons in mice. We found ACh excited a majority of anterior PVT (aPVT) neurons but predominantly inhibited posterior PVT (pPVT) neurons. Compared to pPVT with more inhibitory M2 receptors, aPVT expressed higher levels of all excitatory receptor subtypes including nicotinic α4, α7, and muscarinic M1 and M3. The ACh-induced excitation was mimicked by nicotine and antagonized by selective blockers for α4ß2 and α7 nicotinic ACh receptor (nAChR) subtypes as well as selective antagonists for M1 and M3 muscarinic ACh receptors (mAChR). The ACh-induced inhibition was attenuated by selective M2 and M4 mAChR receptor antagonists. Furthermore, we found ACh increased the frequency of excitatory postsynaptic currents (EPSCs) on a majority of aPVT neurons but decreased EPSC frequency on a larger number of pPVT neurons. In addition, ACh caused an acute increase followed by a lasting reduction in inhibitory postsynaptic currents (IPSCs) on PVT neurons of both subregions. Together, these data suggest that multiple AChR subtypes coordinate a differential modulation of ACh on aPVT and pPVT neurons.

Eight weeks of treatment with mineralocorticoid receptor blockade does not alter vascular function in individuals with and without type 2 diabetes.

Aldosterone has been suggested to be involved in the microvascular complications observed in type 2 diabetes. We aimed to investigate the effect of mineralocorticoid receptor (MR) blockade on endothelial function in individuals with type 2 diabetes compared to healthy controls. We included 12 participants with type 2 diabetes and 14 controls. We measured leg hemodynamics at baseline and during femoral arterial infusion of acetylcholine and sodium nitroprusside before and 8 weeks into treatment with MR blockade (eplerenone). Acetylcholine infusion was repeated with concomitant n-acetylcysteine (antioxidant) infusion. No difference in leg blood flow or vascular conductance was detected before or after the treatment with MR blockade in both groups and there was no difference between groups. Infusion of n-acetylcysteine increased baseline blood flow and vascular conductance, but did not change the vascular response to acetylcholine before or after treatment with MR blockade. Skeletal muscle eNOS content was unaltered by MR blockade and no difference between groups was detected. In conclusion, we found no effect of MR blockade endothelial function in individuals with and without type 2 diabetes. As the individuals with type 2 diabetes did not have vascular dysfunction, these results might not apply to individuals with vascular dysfunction.

Extracellular acidification attenuates bronchial contraction via an autocrine activation of EP2 receptor: Its diminishment in murine experimental asthma.

PURPOSE: Extracellular acidification is a major component of tissue inflammation, including airway inflammation in asthmatics. However, its physiological/pathophysiological significance in bronchial function is not fully understood. Currently, the functional role of extracellular acidification on bronchial contraction was explored. METHODS: Left main bronchi were isolated from male BALB/c mice. Epithelium-removed tissues were exposed to acidic pH under submaximal contraction induced by 10-5 M acetylcholine in the presence or absence of a COX inhibitor indomethacin (10-6 M). Effects of AH6809 (10-6 M, an EP2 receptor antagonist), BW A868C (10-7 M, a DP receptor antagonist) and CAY10441 (3×10-6 M, an IP receptor antagonist) on the acidification-induced change in tension were determined. The release of prostaglandin E2 (PGE2) from epithelium-denuded tissues in response to acidic pH was assessed using an ELISA. RESULTS: In the bronchi stimulated with acetylcholine, change in the extracellular pH from 7.4 to 6.8 caused a transient augmentation of contraction followed by a sustained relaxing response. The latter inhibitory response was abolished by indomethacin and AH6809 but not by BW A868C or CAY10441. Both indomethacin and AH6809 significantly increased potency and efficacy of acetylcholine at pH 6.8. Stimulation with low pH caused an increase in PGE2 release from epithelium-denuded bronchi. Interestingly, the acidic pH-induced bronchial relaxation was significantly reduced in a murine asthma model that had a bronchial hyperresponsiveness to acetylcholine. CONCLUSION: Taken together, extracellular acidification could inhibit the bronchial contraction via autocrine activation of EP2 receptors. The diminished acidic pH-mediated inhibition of bronchial tone may contribute to excessive bronchoconstriction in inflamed airways such as asthma.

Adiponectin-Receptor Agonistic Dipeptide Tyr-Pro Stimulates the Acetylcholine Nervous System in NE-4C Cells.

The dipeptide Tyr-Pro has physiological potential for intact transportability into the brain parenchyma, prevention of cognitive impairment, and an adiponectin receptor 1 (AdipoR1) agonistic effect. The present study aimed to understand the effect of Tyr-Pro on the acetylcholine (ACh) nervous system and its underlying mechanism in NE-4C nerve cells. Concentration-dependent ACh production was induced by stimulation with Tyr-Pro and AdipoRon (an AdipoR1 agonist), along with the expression of AdipoR1 and choline acetyltransferase (ChAT) in NE-4C cells. By knocking down AdipoR1 in the cells, Tyr-Pro promoted ChAT expression, along with the activations of AMPK and ERK 1/2. Tyr-Pro did not alter acetylcholinesterase or ACh receptors, indicating that the dipeptide might operate as an ACh accelerator in nerve cells. This study provides the first evidence that the AdipoR1 agonistic Tyr-Pro is a promising dipeptide responsible for the stimulation of the ACh nervous system by AdipoR1-induced ChAT activation.

Acetylcholine Engages Distinct Amygdala Microcircuits to Gate Internal Theta Rhythm.

Acetylcholine (ACh) is released from basal forebrain cholinergic neurons in response to salient stimuli and engages brain states supporting attention and memory. These high ACh states are associated with theta oscillations, which synchronize neuronal ensembles. Theta oscillations in the basolateral amygdala (BLA) in both humans and rodents have been shown to underlie emotional memory, yet their mechanism remains unclear. Here, using brain slice electrophysiology in male and female mice, we show large ACh stimuli evoke prolonged theta oscillations in BLA local field potentials that depend upon M3 muscarinic receptor activation of cholecystokinin (CCK) interneurons (INs) without the need for external glutamate signaling. Somatostatin (SOM) INs inhibit CCK INs and are themselves inhibited by ACh, providing a functional SOM→CCK IN circuit connection gating BLA theta. Parvalbumin (PV) INs, which can drive BLA oscillations in baseline states, are not involved in the generation of ACh-induced theta, highlighting that ACh induces a cellular switch in the control of BLA oscillatory activity and establishes an internally BLA-driven theta oscillation through CCK INs. Theta activity is more readily evoked in BLA over the cortex or hippocampus, suggesting preferential activation of the BLA during high ACh states. These data reveal a SOM→CCK IN circuit in the BLA that gates internal theta oscillations and suggest a mechanism by which salient stimuli acting through ACh switch the BLA into a network state enabling emotional memory.

Relation of Vasoreactivity in the Left and Right Coronary Arteries During Acetylcholine Spasm Provocation Testing.

The diagnosis of vasospastic angina (VSA) according to Japanese guidelines involves an initial intracoronary acetylcholine (ACh) provocation test in the left coronary artery (LCA) followed by testing in the right coronary artery (RCA). However, global variations in test protocols often lead to the omission of ACh provocation in the RCA, potentially resulting in the underdiagnosis of VSA. This study assessed the validity of the LCA-only ACh provocation approach for the VSA diagnosis and whether vasoreactivity in the LCA aids in determining further provocation in the RCA. A total of 273 patients who underwent sequential intracoronary ACh provocation testing in the LCA and RCA were included. Patients with a positive ACh provocation test in the LCA were excluded. Relations between vasoreactivity in the LCA and ACh test outcomes (positivity and adverse events) in the RCA were evaluated. In patients with negative ACh test results in the LCA, subsequent ACh testing was positive in the RCA in 23 of 273 (8.4%) patients. In patients with minimal LCA vasoconstriction (<25%), only 3.0% had a positive ACh test in the RCA, whereas the ACh test in the RCA was positive in 13.5% of those with LCA constriction of 25% to 90% (p = 0.002). No major adverse events occurred during ACh testing in the RCA. In conclusion, for the VSA diagnosis, the omission of ACh provocation in the RCA may be clinically acceptable, particularly when vasoconstriction induced by ACh injection was minimal in the LCA. Further studies are needed to define ACh provocation protocols worldwide.

The role of T-type calcium channels in elderly human vascular function: A pilot randomized controlled trial.

Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8 weeks, they were given 40 mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43 ± 0.45 l/min, P = 0.006) and leg vascular conductance (Δ 5.38 ± 5.67 ml/min/mmHg, P = 0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8 weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.

Microbiota-derived acetylcholine can promote gut motility in Drosophila melanogaster.

The gut microbiota is crucial for intestinal health, including gastrointestinal (GI) motility. How commensal bacterial species influence GI motility has not been fully elucidated. A major factor of GI motility is the gut contraction promoting the propulsive movement of orally ingested materials. Here, we developed a method to monitor and quantify gut contractions in living Drosophila melanogaster larvae. We found that the culture medium of an isolated strain Lactiplantibacillus plantarum Lsi promoted gut contraction in vivo, which was not observed in Leuconostoc sp. Leui nor Acetobacter persici Ai culture medium. To identify bacteria-derived metabolites, we performed metabolome analysis of the culture media by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the 66 metabolites detected, we found that some metabolites changed in a species-specific manner. Among them, acetylcholine was specifically produced by L. plantarum. Feeding exogenous acetylcholine increased the frequency of gut contractions, which was blocked by D-tubocurarine, an inhibitor of nicotinic acetylcholine receptors. In this study, we propose a mechanism by which the gut microbiota influences Drosophila gut motility. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

The difference in endothelium-dependent relaxation components in proximal and distal thoracic aorta regions of male rats.

Aorta, the largest vessel in the body, is generally considered anatomically homogeneous, yet spatial functional differences exist. In our study, we conducted a comprehensive analysis by reexamining public RNA-SEQ data, comparing expression patterns between thoracic and abdominal aorta. Additionally, we measured acetylcholine-induced relaxations of the different regions of thoracic aorta in Wistar Rats. Our results revealed a distinct percentage difference in acetylcholine-induced relaxation in the proximal and distal segments of the thoracic aorta (p = 1.14e-4). To explain this variation, we performed differential expression analysis of previously published RNA-sequencing data between thoracic and abdominal aorta, which showed 497 differentially expressed genes between these locations. From results of RNA-Seq analysis, we draw a hypothesis that differential expressions of the potassium inward rectifying channels (KIR) and voltage gated calcium channels (VGCC) presumably located on SMC, with higher expression in the distal thoracic segments in comparison with the proximal thoracic segments of aorta, can explain differences in acetylcholine-induced relaxation. Notably, specific blockade of KIR eliminated differences between the proximal and distal regions of thoracic aorta, underscoring their significance in understanding the spatial nuances in aortic behavior, also blockade of VGCC, shows a higher effect on basal tone, in distal region of thoracic aorta in comparison with proximal.

Acetylcholine synergizes with netrin-1 to drive persistent firing in the entorhinal cortex.

The ability of the mammalian brain to maintain spatial representations of external or internal information for short periods of time has been associated with sustained neuronal spiking and reverberatory neural network activity in the medial entorhinal cortex. Here, we show that conditional genetic deletion of netrin-1 or the netrin receptor deleted-in-colorectal cancer (DCC) from forebrain excitatory neurons leads to deficits in short-term spatial memory. We then demonstrate that conditional deletion of either netrin-1 or DCC inhibits cholinergic persistent firing and show that cholinergic activation of muscarinic receptors expressed by entorhinal cortical neurons promotes persistent firing by recruiting DCC to the plasma membrane. Together, these findings indicate that normal short-term spatial memory function requires the synergistic actions of acetylcholine and netrin-1.