ACh Responses Research Articles

Allosteric Modulator Desformylflustrabromine Relieves the Inhibition of α2β2 and α4β2 Nicotinic Acetylcholine Receptors by β-Amyloid1–42 Peptide

Nicotinic acetylcholine receptors (nAChRs) are pentameric transmembrane proteins that belong to the cys-loop ligand-gated ion channel family. These receptors are widely expressed in the brain and implicated in the pathophysiology of many neurological conditions, including Alzheimer's disease (AD), where typical symptoms include the loss of cognitive function and dementia. The presence of extracellular neuritic plaques composed of β amyloid (Aβ(1-42)) peptide is a characteristic feature of AD. Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) for α4β2 nAChRs since it increases peak ACh responses without inducing a response on its own. Previously, the effect of dFBr on the α2β2 nAChR subtype was not known. The action of dFBr was tested on α2β2 receptors expressed in Xenopus oocytes. It was found that dFBr is also a PAM for the α2β2 receptor. Next we tested whether dFBr had any effect on the previously known block of both the α4β2 and α2β2 receptors by Aβ(1-42). We found that the functional blockade of ACh-induced currents in oocytes expressing α4β2 and α2β2 receptors by Aβ(1-42) was prevented by dFBr. We conclude that dFBr is a positive allosteric modulator for both α4β2 and α2β2 subtypes of nAChRs and that it also relieves the blockade of these receptors by Aβ(1-42). This study demonstrates that PAMs for the non-α7 nAChRs have the potential to develop into clinically applicable drugs for AD and other disorders.

Homocysteine-dependent endothelial dysfunction induced by renal ischemia/reperfusion injury

Elevation of serum homocysteine is considered to contribute to endothelial dysfunction, which is considered to be the initial event in vascular disease following renal transplantation. We sought to investigate whether an association existed between serum homocysteine levels and endothelial dysfunction after renal ischemia/reperfusion (I/R) injury. Acetylcholine (Ach)-induced endothelium-dependent and sodium nitroprusside (SNP)-induced endothelial-independent relaxation responses were determined in thoracic aortas from different I/R groups. A correlation analysis was performed between Ach responses and homocysteine levels. Long-term I/R injury decreased the responses to acetylcholine and the pD2 values of the concentration response curves compared with controls. While vascular responses to SNP were unchanged among all groups. Homocysteine levels correlated with the pD2 values of acetylcholine among control and I/R groups, indicating that the increase in homocysteine was associated with decreased sensitivity to acetylcholine. In short-term I/R rats, no association was observed between these parameters. These data suggest a possible link between serum homocysteine and decreased vascular reactivity to endothelium-dependent relaxation in I/R aorta.

Changes in the Muscarinic Receptors on the Colonic Smooth Muscles of Rats with Spinal Cord Injury

ObjectiveTo investigate changes in (1) the colonic response to acetylcholine (Ach), (2) the muscarinic (M) receptors in the colon, and (3) the levels of colonic contraction-related proteins after a spinal cord injury (SCI).MethodWe divided 16 Sprague-Dawley rats into 2 groups: the control group and the SCI group. A spinal cord transection was performed surgically at the T10 vertebral level. After 1 week, the entire colon was divided into 2 segments, the proximal and distal colon. Each segment was mounted in a longitudinal or circular muscle direction in a 10-ml organ bath. We determined the intergroup differences as percentage changes in contractility after Ach treatment alone, Ach treatment with M2 receptor antagonist (AQ-RA741) pretreatment, and Ach treatment with M3 receptor antagonist (4-DAMP) pretreatment. Western blot analyses were performed to determine the expression level of RhoA, and heat shock protein 27 (HSP27).ResultsCompared to the control rats, the SCI rats showed an increased response to Ach along both the directions in the proximal colon (p<0.05). Compared to the control group, in the SCI group, the Ach response was significantly different in the proximal segment under AQ-RA741 pretreatment (p<0.05) and in the distal segment under 4-DAMP pretreatment (p<0.05). Findings of the western blot analyses showed a significant decrease in the level of protein gene product 9.5 in the proximal and distal colon and a significant increase in the level of RhoA and HSP27 in the proximal colon of the SCI rats.ConclusionOur results suggest that changes in colonic contractility after SCI are partly attributable to changes in the M receptor subtypes.

Enhanced cyclooxygenase 2-mediated vasorelaxation in coronary arteries from insulin-resistant obese Zucker rats

Obesity and metabolic syndrome increase the risk of coronary heart disease and lead to a proinflammatory state of the vascular wall. Endothelial dysfunction is associated with up-regulation of cyclooxygenase-2 (COX-2) and enhanced synthesis of constrictor prostaglandins in systemic arteries in diabetes. The present study assessed whether changes in the arachidonic acid (AA) metabolism via COX-1 and COX-2 may affect endothelial function of coronary arteries in obesity. Intramyocardial arteries from obese Zucker rats (OZR) and from lean Zucker rats (LZR) were mounted in microvascular myographs to assess vascular function and COX expression was determined by both immunohistochemistry and Western blot. AA elicited relaxations of similar magnitude in arteries from LZR and OZR which were abolished by endothelial cell removal. Selective inhibition of COX-1 enhanced the AA relaxant responses and inhibited the 5-hydroxytryptamine (5-HT)-induced vasoconstriction in arteries from both LZR and OZR. Antagonism of the TXA 2/PGH 2 (TP) receptor mimicked the effects of COX-1 blockade in arteries from LZR but not OZR. Selective inhibition of COX-2 markedly reduced the vasodilatation induced by AA in OZR, but not in LZR, without altering 5-HT or ACh responses. COX-1 was widely distributed throughout the endothelial layer of coronary arteries from both LZR and OZR, while COX-2 protein, which was predominantly expressed in the endothelium, was significantly increased in arteries from OZR. Whereas AA is mainly metabolised to vasoconstrictor prostanoids via COX-1 in coronary arteries from healthy animals, endothelial COX-2 is up-regulated to produce vasodilator prostaglandins thus protecting coronary arteries in insulin resistant obese rats.

Interleukin-10 inhibits the in vivo and in vitro adverse effects of TNF-α on the endothelium of murine aorta

TNF-α is a proinflammatory cytokine and is an important mediator of maternal endothelial dysfunction leading to preeclampsia. In this study, we tested whether IL-10 protects against TNF-α-induced endothelial dysfunction in murine aorta. In in vitro experiments, aortic rings of C57BL/6 female mice were incubated in Dulbecco's modified Eagle's medium in the presence of either vehicle (distilled H(2)O), TNF-α (4 nmol/l), or recombinant mouse IL-10 (300 ng/ml) or in the presence of both TNF-α and IL-10 for 22 h at 37°C. In in vivo experiments C57BL6/IL-10 knockout female mice were treated with saline or TNF-α (220 ng·kg(-1)·day(-1)) for 14 days. Aortic rings were isolated from in vitro and in vivo experiments and mounted in a wire myograph (Danish Myotech) and stretched to a tension of 5 mN. Endothelium-dependent relaxation was assessed by constructing cumulative concentration-response curves to acetylcholine (ACh, 0.001-10 μmol/l) during phenylephrine (10 μmol/l)-induced contraction. As a result, overnight exposure of aortic rings to TNF-α resulted in significant blunted maximal relaxing responses (E(max)) to ACh compared with untreated rings (22 ± 4 vs. 82 ± 3%, respectively). IL-10 knockout mice treated with TNF-α showed significant impairment in ACh responses (E(max)) compared with C57BL/6 mice treated with TNF-α (51 ± 3 vs. 72 ± 3%, respectively). Western blot analysis showed that endothelial nitric oxide synthase (eNOS) expression was reduced by TNF-α in in vitro and in vivo experiments, whereas IL-10 restored the eNOS expression. In conclusion, the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation caused by TNF-α by protecting eNOS expression.

166 Successful Implantation of Intrinsically Innervated Physiologically Functional Bioengineered Human Internal Anal Sphincter (IAS)

Background: We have bioengineered intrinsically innervated IAS tissue by co-culturing human IAS circular smooth muscle and immorto mouse fetal enteric neurons (IM-FENs) Objective: We provide proof of concept that intrinsically innervated bioengineered human IAS rings implanted in RAG1-/mice preserve physiological functionality of constituent myogenic and neuronal components. Methods: Intrinsically innervated human IAS rings were bioengineered by embedding human IAS smooth muscle cells and de-differentiated IM-FENs on a collagen/laminin matrix. IM-FENs were allowed to differentiate at 39C for 12 days. Bioengineered rings were implanted for 25 days under the skin of RAG1-/mice. Results: Histology of harvested post-implant IAS revealed neovascularization. Real-time force acquisition from these constructs revealed that physiological responses of post-implant innervated IAS were similar to control non-implanted innervated IAS rings: (1)Spontaneous basal tone generated was tetrodotoxin (TTX)-insensitive and hence purely myogenic; (2)Vasoactive Intestinal Peptide(VIP) caused a relaxation of basal tone upto 300μN. VIPinduced relaxation in the presence of TTX was only 20% of the original, indicating dual involvement of neuronal and myogenic component in VIP-induced relaxation;(3)Electrical Field Stimulation(10Hz,0.3ms) caused relaxation of basal tone upto 153μN. EFS response was abolished in presence of TTX indicating selective stimulation of neuron mediated relaxation pathway; (4)Acetylcholine(Ach) response was dose-dependent and elicited a rapidrising, sustained contraction of 477±77μN. Contraction was attenuated by 90% in the presence of TTX, implying synergistic involvement of both myogenic and neuronal components in cholinergic contraction; (5)30mM KCl elicited a rapid rising spike contraction, indicating preservation of physiologically relevant ion channels. Summary: Implanted intrinsically innervated IAS maintained: 1-Spontaneous generation of myogenic basal tone, a constitutive property of IAS. 2Neurotransmitter-mediated contraction and relaxation, and their TTX sensitivity displaying distinct contributions of viable intrinsic innervation (IM-FEN) and constituent myogenic component (human IAS smooth muscle).Conclusion: Intrinsically innervated bioengineered human IAS tissue preserved the integrity and physiological functionality of both myogenic and neuronal components, upon implantation into RAG1-/mice. This is the first report of implantation of intrinsically innervated human IAS bioengineered construct, with vast potential to create functional autologous graft tissue to repair degenerated IAS. Supported by NIHRO1DK071614 and 1RC1DK087151

Abstract 5222: Genetic Deficiency of Arginase II Reduces Endothelial Dysfunction and Atherosclerosis in ApoE-Knockout Mice

Introduction: Arginase II is known to be one of the key mediator in vascular pathophysiology of cholesterol-medicated endothelial dysfunction by reciprocal regulation of NOS and NO production through substrate competition. We aimed to investigate whether the transgenic Arginase II inhibition could reduce the endothelial dysfunction and atherosclerosis in apolipoprotein E-knockout (ApoE-KO) mice. Methods and Results: ApoE-KO mice were bred on an arginase II KO (ArgII-KO) background. Mice that were homozygous deleted at both alleles were used in the study. Male 8 – 12 wk old Arg-KO mice, ApoE-KO mice, Arg/ApoE-KO (Dbl-KO) mice fed high cholesterol diet for 16 weeks, and age-matched wild type (WT) mice fed normal diet were enrolled. Arginase activity was significantly higher in the aorta of ApoE-KO mice than compared to WT mice (1612.2±445.9 pmol Urea/mg protein/min vs. 836.3±12.2 pmol Urea/mg protein/min, n=5, p &lt; 0.01), but not detected in Arg-KO and Dbl-KO mice. We measured NO an ROS in vascular strips pinned down endothelial side up using the fluorescent probes DAF-FM diacetate and DHE. Dbl-KO mice showed significantly higher NO production (0.0737±0.0056 intensity/sec vs. 0.0256±0.0029 intensity/sec, n=6, p &lt; 0.05) and lower ROS production than ApoE-KO mice (0.0676±0.0065 intensity/sec vs. 0.1147±0.01619 intensity/sec, n=6, p &lt; 0.05). We tested endothelial dependent vasorelaxation in aortic rings in organ chambers. The vasorelaxation response to acetylcholine (Ach) in Dbl-KO mice was greater than in ApoE-KO mice (log EC 50 , −5.68 vs. −5.13, p &lt;0.05), although it was less than in Arg-KO and WT mice. N G -nitro-L-arginine methyl ester (L-NAME) incubation didn’t only impair Ach response, but also abolished the inter-group difference. The aortic atheromatous plaque burden as measured by Sudan IV staining was significantly reduced in Dbl-KO group compared to ApoE-KO group (38.4±3.8% vs. 52.0±2.8%, n=6, p &lt;0.01), No significant plaque was detectable in Arg-KO and WT mice. Conclusion: The genetic inhibition of Arginase II enhanced NO production, reduced ROS produnction restored endothelial function and lowered atheromatous plaque burden. Therefore, Arginase II represents a crtical novel target for atherosclerosis therapy.

Abstract 4886: Hydrogen Peroxide Prevents Impaired Endothelium-dependent Dilation Following Acute Exertion in Chronic Exercising but Not in Sedentary Subjects

Introduction: Exercise-induced hypertension increases reactive oxygen species (ROS) generation and threatens vascular endothelial health. Recent studies indicate conditioned subjects (EX) are protected from endothelial dysfunction observed in sedentary (SED) subjects after acute hypertension induced by weight lifting (WL). Hypothesis: We hypothesized that: chronic exercise protects against endothelial dysfunction induced by acute exertion and the mechanism of maintained dilation after acute hypertension involves hydrogen peroxide (H 2 O 2 ) in microvessels from exercise trained subjects. Methods: Healthy, lean SED and EX subjects (runners &gt; 15 miles/week or WL &gt; 3x/week), underwent a single progressive 15 minute leg press WL session. Brachial artery flow- mediated dilation (FMD) and nitroglycerin (NTG; 0.4 mg) dilations were measured with ultrasound. Isolated arterioles (gluteal fad pad biopsies) from the same subjects were cannulated to assess dilation to acetylcholine (ACh; 10 −9 –10 −4 M) pre and post WL. Separately, H 2 O 2 production was assessed with DCF fluorescence. Results: All subjects were free of hypertension with similar blood pressure responses to WL (max SBP: 183.7±5 mmHg). Brachial artery FMD was maintained after WL in EX (+2.0±0.3%; p&lt;0.001 vs. SED; n=48) but not SED (−3.0±0.2%; n = 28). Dilation to ACh was maintained in EX [Max dilation (MD): 87±3 %; n = 10] but not SED (50±5%; n = 10) post WL. Inhibition of NOS with LNAME eliminated baseline ACh dilation in EX but not post WL (73±6%). Indomethacin and 17-ODYA had no effect on post WL ACh responses. In EX subjects, WL increased hydrogen peroxide (DCF fluorescence ratio: 3.4±0.4; p = 0.001 vs. baseline; n = 9). The H 2 O 2 scavenger PEG-catalase (500 U/ml) reduced DCF fluorescence and ACh dilation post WL (29±7%; p&lt;0.001 vs. pre WL) but had no effect before WL. Endothelium-independent responses to papaverine were similar pre and post WL. In conclusion, these data indicate chronic exercise protects against microvascular endothelial dysfunction after exertion and although NOS is responsible for ACh dilation before acute weight lifting in EX, H 2 O 2 maintains endothelium-dependent dilation in microvessels after exposure to exercise-induced hypertension.

Obese But Not Normal-Weight Women With Polycystic Ovary Syndrome Are Characterized by Metabolic and Microvascular Insulin Resistance

A high proportion of women with the polycystic ovary syndrome (PCOS) are obese. It is unclear whether normal-weight women with PCOS are at a greater risk of diabetes and cardiovascular disease than obese women without PCOS. It is also unclear whether risks of diabetes and cardiovascular disease in obese women and those with PCOS are additive. To determine whether the risk of diabetes and cardiovascular disease in PCOS is independent of the risk associated with obesity, this prospective controlled study compared insulin sensitivity and microvascular function in normal-weight and obese women with and without PCOS. The 4 PCOS groups were comprised of 19 normal-weight and 16 obese women with PCOS, and 14 normal-weight and 13 obese women controls without PCOS matched for age and body mass index. Before and after insulin infusion to produce hyperinsulinemia, metabolic insulin sensitivity (M/I value) was measured by means of the isoglycemic hyperinsulinemic clamp, and microvascular insulin sensitivity was assessed by analysis of microvascular endothelium-dependent and independent vasodilatation using iontophoresis of acetylcholine (Ach) and sodium nitroprusside (SNP) in combination with laser Doppler flowometry in the skin. For the latter, area under the response curves to Ach and SNP were measured. Analysis of metabolic insulin sensitivity showed that both groups of obese women had a significantly lower M/I value than normal-weight women (P ≤ 0.001) and that obese PCOS women had a lower M/I value than obese controls (P ≤ 0.02). In contrast, there was no difference in the M/I value between normal-weight PCOS women and controls. Before insulin infusion, there was no significant difference in microvascular responses to Ach among the 4 groups. During insulin infusion, Ach responses were significantly reduced in obese compared with normal-weight women; a similar difference was found for normal-weight women with or without PCOS. Compared to baseline values, SNP-dependent vasodilatation was not increased during insulin infusion in the 4 groups (P = ns). This report is the first to show that normal-weight women with PCOS may be at relatively normal risk of metabolic and cardiovascular disease and that obese woman with PCOS may be at increased risk. The investigators conclude from these findings that the focus of management in women with PCOS should be on weight control.

ORIGINAL RESEARCH–BASIC SCIENCE: Cavernous Neurotomy in the Rat is Associated with the Onset of an Overt Condition of Hypogonadism

ABSTRACT Background Most men following radical retropubic prostatectomy (RRP) are afflicted by erectile dysfunction (ED). RRP-related ED occurs as a result of surgically elicited neuropraxia, leading to histological changes in the penis, including collagenization of smooth muscle and endothelial damage. Aim To verify whether hypogonadism could contribute to the pathogenesis of RRP-ED. Methods Effects of testosterone (T), alone or in association with long-term tadalafil (Tad) treatment in a rat model of bilateral cavernous neurotomy (BCN). Main Outcome Measures Penile tissues from rats were harvested for vasoreactivity studies 3 months post-BCN. Penile oxygenation was evaluated by hypoxyprobe immunostaining. Phosphodiesterase type 5 (PDE5), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS) mRNA expression were quantified by Real Time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results In BCN rats, we observed the onset of an overt condition of hypogonadism, characterized by reduced T plasma level, reduced ventral prostate weight, reduced testis function (including testis weight and number of Leydig cells), with an inadequate compensatory increase of luteinizing hormone. BCN induced massive penile hypoxia, decreased muscle/fiber ratio, nNOS, eNOS, PDE5 expression, increased sensitivity to the nitric oxide donor, sodium nitroprusside (SNP), and reduced the relaxant response to acetylcholine (Ach), as well as unresponsiveness to acute Tad dosing. In BCN rats, chronic Tad-administration normalizes penile oxygenation, smooth muscle loss, PDE5 expression, SNP sensitivity, and the responsiveness to the acute Tad administration. Chronic Tad treatment was ineffective in counteracting the reduction of nNOS and eNOS expression, along with Ach responsiveness. T supplementation, in combination with Tad, reverted some of the aforementioned alterations, restoring smooth muscle content, eNOS expression, as well as the relaxant response of penile strips to Ach, but not nNOS expression. Conclusion BCN was associated with hypogonadism, probably of central origin. T supplementation in hypogonadal BCN rats ameliorates some aspects of BCN-induced ED, including collagenization of penile smooth muscle and endothelial dysfunction, except surgically induced altered nNOS expression.

Endothelium‐dependent vasodilatation and exercise hyperaemia in ageing humans: impact of acute ascorbic acid administration

Age-related increases in oxidative stress impair endothelium-dependent vasodilatation in humans, leading to the speculation that endothelial dysfunction contributes to impaired muscle blood flow and vascular control during exercise in older adults. We directly tested this hypothesis in 14 young (22 +/- 1 years) and 14 healthy older men and women (65 +/- 2 years). We measured forearm blood flow (FBF; Doppler ultrasound) and calculated vascular conductance (FVC) responses to single muscle contractions at 10, 20 and 40% maximum voluntary contraction (MVC) before and during ascorbic acid (AA) infusion, and we also determined the effects of AA on muscle blood flow during mild (10% MVC) continuous rhythmic handgrip exercise. For single contractions, the peak rapid hyperaemic responses to all contraction intensities were impaired approximately 45% in the older adults (all P < 0.05), and AA infusion did not impact the responses in either age group. For the rhythmic exercise trial, FBF (approximately 28%) and FVC (approximately 31%) were lower (P = 0.06 and 0.05) in older versus young adults after 5 min of steady-state exercise with saline. Subsequently, AA was infused via brachial artery catheter for 10 min during continued exercise. AA administration did not significantly influence FBF or FVC in young adults (1-3%; P = 0.24-0.59), whereas FBF increased 34 +/- 7% in older adults at end-exercise, and this was due to an increase in FVC (32 +/- 7%; both P < 0.05). This increase in FBF and FVC during exercise in older adults was associated with improvements in vasodilator responses to acetylcholine (ACh; endothelium dependent) but not sodium nitroprusside (SNP; endothelium independent). AA had no effect on ACh or SNP responses in the young. We conclude that acute AA administration does not impact the observed age-related impairment in the rapid hyperaemic response to brief muscle contractions in humans; however, it does significantly increase muscle blood flow during continuous dynamic exercise in older adults, and this is probably due (in part) to an improvement in endothelium-dependent vasodilatation.

Mechanisms of Microvascular Endothelial Dysfunction in Subcutaneous Fat during Human Obesity

Microvascular endothelial dysfunction contributes to cardiovascular disease risk during obesity. Inflammatory cytokines (CRP and IL‐6) and NADPH oxidase generation of reactive oxygen species (ROS) reduces endothelial function during obesity. Therefore, we hypothesized that pro‐ inflammatory cytokines and NADPH oxidase generation of ROS contribute to microvascular dysfunction in subcutaneous (SQ) fat during obesity. Isolated resistance arteries were obtained from SQ biopsies in lean (LN; BMI&lt;27) and during laproscopic band surgery in obese (OB; BMI&gt;40) women (mean age: 36±1). Microvessels were cannulated for vascular reactivity measurements to acetylcholine (ACh; 10−9‐10−4 M) in the absence and presence of the SOD mimetic Tiron (10−4 M), NADPH oxidase inhibitor apocynin (10−6 M), and the xanthine oxidase inhibitor allopurinol (10−4 M). Superoxide production was assessed with hydroethidine fluorescence in the presence and absence of the same inhibitors. Blood measurements of CRP and IL‐6 were determined with ELISA. Maximum dilation to ACh was reduced in OB (46 ± 6%; n=9; p&lt;0.05) vs. LN (79 ± 9%; n=5) patients and was restored by Tiron. Superoxide production was increased in microvessels from OB subjects. Tiron and apocynin restored the dilator response to ACh and reduced baseline and ACh‐ stimulated superoxide fluorescence of OB. Allopurinol had no effect on ACh responses in OB microvessels. There was no association between IL‐6 or CRP and superoxide fluorescence. These data indicate that NADPH oxidase generation of ROS impairs endothelial function in SQ microvessels during obesity.

Vasopeptidase Inhibitor Improves Coronary Dysfunction in Zucker Obese but not Zucker Diabetic Fatty (ZDF) Rats

Previous studies have shown endothelial dysfunction in coronary and mesenteric near resistance arteries in Zucker obese and ZDF rats. Our hypothesis is that vascular dysfunction is due in part to upreglulation of neutral endopeptidase and treatment with a vasopeptidase inhibitor will improve vascular function. 20‐week‐old Zucker obese and 12‐week‐old ZDF rats were treated with the vasopeptidase inhibitor AVE7688 (500mg/kg) for 12 weeks. Afterwards, coronary and mesenteric function was evaluated using an isolated vessel preparation. Vessels (75 ‐ 175µm intraluminal diameter and ~1 mm in length) were studied under hydrostatic pressure (40mm Hg) and no flow, and internal diameter was measured. Dilator responses were evaluated for acetylcholine (Ach, 10−10‐10−4 M), and calcitonin gene‐related peptide (CGRP, 10−11‐10−9 M) after constriction with U46619.ResultsACh (10μM) responses were attenuated in arteries from untreated rats compared to responses from lean rats (coronary: Zucker obese = 49.4%, ZDF = 50.7% decrease and mesenteric: Zucker obese = 27.4%, ZDF = 33.5% decrease). ACh response was augmented in coronary vessels from AVE7688 treated Zucker obese but not ZDF rats when compared to vessels from untreated rats. Our data suggest that coronary artery endothelial dysfunction is improved when Zucker obese but not ZDF rats were treated with AVE7688. Supported by VA Merits to Dr. Oltman and Dr. Yorek.

Superoxide from uncoupled nitric oxide synthase reduces endothelium‐dependent arteriolar dilation in mice fed high salt

We have previously reported that the impaired endothelium‐dependent dilation of arterioles in rodents on a high salt diet is due to oxidation of nitric oxide (NO) by superoxide anion (O2−) in the arteriolar wall. The aim of this study was to test the hypothesis that "uncoupled" nitric oxide synthase (NOS) is the predominant source of this O2−. Arterial pressure in C57BL/6J mice fed a high salt (4%, HS) diet for 4 weeks was not significantly different from that in mice fed a normal salt (0.45%, NS) diet. However, compared to NS mice, spinotrapezius muscle arterioles in HS mice had elevated arteriolar wall oxidant activity (assessed by hydroethidine oxidation) and a large (75‐80%) reduction in dilator responses to acetylcholine. In HS mice, arteriolar oxidant stress was abolished by acute application of the NOS inhibitor NG monomethyl L‐arginine and by long‐term supplementation of L‐arginine intake (1.51% in drinking water for 4 weeks), which has been shown to increase vascular tetrahydrobiopterin (BH4) levels. L‐arginine supplementation also prevented the reduction in arteriolar ACh responses in HS mice but had no effect on these responses in NS mice. These findings suggest that NOS uncoupling due to reduced BH4 availability is primarily responsible for O2− accumulation and the resultant impairment of NO‐dependent arteriolar dilation in mice fed a high salt diet. (Support AHA Grant‐in‐Aid 0755264B and NIH ES‐015022)

Vascular Endothelial Dysfunction in Cyclosporine-Treated Rat Aortas Is Not Associated With Serum Total Homocysteine Levels

Objectives Elevation of serum total homocysteine (tHcy) is considered to contribute to endothelial cell dysfunction, which is considered to be the initial event in posttransplant vascular disease. We sought to investigate whether an association existed between serum tHcy levels and vascular endothelial function during cyclosporine (CsA) treatment. Materials and Methods Endothelium-dependent and -independent relaxation responses (to acetylcholine [ACh] and sodium nitroprusside [SNP]) were determined on thoracic aortae from CsA-treated rats (5 mg kg/d, subcutaneously, for 14 days). A correlation analysis was performed between ACh responses and tHcy levels. Results CsA decreased the responses to ACh and the p D 2 values of the concentration–response curves compared with controls ( P < .05). Responses to SNP and serum tHcy levels were unchanged among the groups. tHcy negatively correlated with the ACh p D 2 values among control ( r = −0.69; P < .05) and vehicle ( r = −0.73; P < .05) groups, indicating that the increase in tHcy was associated with decreased sensitivity to ACh. In CsA-treated rats, no association was observed between these parameters. Also, no correlation was noted between CsA concentrations and tHcy levels. Conclusion These data suggested a possible link between serum tHcy and decreased vascular sensitivity to endothelium-dependent vasorelaxation in control aortae, but CsA-induced vascular endothelial dysfunction was not associated with an effect of the drug on homocysteine metabolism.

Neuronal Ca2+ sensor VILIP-1 leads to the upregulation of functional α4β2 nicotinic acetylcholine receptors in hippocampal neurons

The neuronal Ca2+-sensor protein VILIP-1, known to affect clathrin-dependent receptor trafficking, has been shown to interact with the cytoplasmic loop of the α4-subunit of the α4β2 nicotinic acetylcholine receptor (nAChR), which is the most abundant nAChR subtype with high-affinity for nicotine in the brain. The α4β2 nAChR is crucial for nicotine addiction and the beneficial effects of nicotine on cognition. Its dysfunction has been implicated in frontal lobe epilepsy, Alzheimer's disease and schizophrenia. Here we report that overexpression of VILIP-1 enhances ACh responsiveness, whereas siRNA against VILIP-1 reduces α4β2 nAChR currents of hippocampal neurons. The underlying molecular mechanism likely involves enhanced constitutive exocytosis of α4β2 nAChRs mediated by VILIP-1. The two interaction partners co-localize in a Ca2+-dependent manner with syntaxin-6, a Golgi-SNARE protein involved in trans-Golgi membrane trafficking. Thus, we speculate that regulation of VILIP-1-expression might modulate surface expression of ligand-gated ion channels, such as the α4β2 nAChRs, possibly comprising a novel form of physiological up-regulation of ligand-gated ion channels.

Color responses of the human lateral geniculate nucleus: selective amplification of S‐cone signals between the lateral geniculate nucleno and primary visual cortex measured with high‐field fMRI

The lateral geniculate nucleus (LGN) is the primary thalamic nucleus that relays visual information from the retina to the primary visual cortex (V1) and has been extensively studied in non-human primates. A key feature of the LGN is the segregation of retinal inputs into different cellular layers characterized by their differential responses to red-green (RG) color (L/M opponent), blue-yellow (BY) color (S-cone opponent) and achromatic (Ach) contrast. In this study we use high-field functional magnetic resonance imaging (4 tesla, 3.6 × 3.6 × 3 mm3) to record simultaneously the responses of the human LGN and V1 to chromatic and Ach contrast to investigate the LGN responses to color, and how these are modified as information transfers between LGN and cortex. We find that the LGN has a robust response to RG color contrast, equal to or greater than the Ach response, but a significantly poorer sensitivity to BY contrast. In V1 at low temporal rates (2 Hz), however, the sensitivity of the BY color pathway is selectively enhanced, rising in relation to the RG and Ach responses. We find that this effect generalizes across different stimulus contrasts and spatial stimuli (1-d and 2-d patterns), but is selective for temporal frequency, as it is not found for stimuli at 8 Hz. While the mechanism of this cortical enhancement of BY color vision and its dynamic component is unknown, its role may be to compensate for a weak BY signal originating from the sparse distribution of neurons in the retina and LGN.

Exercise prevents age‐related decline in nitric‐oxide‐mediated vasodilator function in cutaneous microvessels

Ageing is associated with impaired endothelium-derived nitric oxide (NO) function in human microvessels. We investigated the impact of cardiorespiratory fitness and exercise training on physiological and pharmacological NO-mediated microvascular responses in older subjects. NO-mediated vasodilatation was examined in young, older sedentary and older fit subjects who had two microdialysis fibres embedded into the skin on the ventral aspect of the forearm and laser Doppler probes placed over these sites. Both sites were then heated to 42 degrees C, with Ringer solution infused in one probe and N-nitro-L-arginine methyl ester (L-NAME) through the second. In another study, three doses of ACh were infused in the presence or absence of L-NAME in similar subjects. The older sedentary subjects then undertook exercise training, with repeat studies at 12 and 24 weeks. The NO component of the heat-induced rise in cutaneous vascular conductance (CVC) was diminished in the older sedentary subjects after 30 min of prolonged heating at 42 degrees C (26.9 +/- 3.9%CVC(max)), compared to older fit (46.2 +/- 7.0%CVC(max), P < 0.05) and young subjects (41.2 +/- 5.2%CVC(max), P < 0.05), whereas exercise training in the older sedentary group enhanced NO-vasodilator function in response to incremental heating (P < 0.05). Similarly, the NO contribution to ACh responses was impaired in the older sedentary versus older fit subjects (low dose 3.2 +/- 1.3 versus 6.6 +/- 1.3%CVC(max); mid dose 11.4 +/- 2.4 versus 21.6 +/- 4.5%CVC(max); high dose 35.2 +/- 6.0 versus 52.6 +/- 7.9%CVC(max), P < 0.05) and training reversed this (12 weeks: 13.7 +/- 3.6, 28.9 +/- 5.3, 56.1 +/- 3.9%CVC(max), P < 0.05). These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents age-related decline in indices of physiological and pharmacological microvascular NO-mediated vasodilator function. Since higher levels of NO confer anti-atherogenic benefit, this study has potential implications for the prevention of microvascular dysfunction in humans.

Obese But Not Normal-Weight Women with Polycystic Ovary Syndrome Are Characterized by Metabolic and Microvascular Insulin Resistance

Polycystic ovary syndrome (PCOS) and obesity are associated with diabetes and cardiovascular disease, but it is unclear to what extent PCOS contributes independently of obesity. The objective of the study was to investigate whether insulin sensitivity and insulin's effects on the microcirculation are impaired in normal-weight and obese women with PCOS. Thirty-five women with PCOS (19 normal weight and 16 obese) and 27 age- and body mass index-matched controls (14 normal weight and 13 obese) were included. Metabolic Insulin sensitivity (isoglycemic-hyperinsulinemic clamp) and microvascular insulin sensitivity [endothelium dependent (acetylcholine [ACh])] and endothelium-independent [sodium nitroprusside (SNP)] vasodilation with laser Doppler flowmetry was assessed at baseline and during hyperinsulinemia. Metabolic insulin sensitivity (M/I value) and the area under the response curves to ACh and SNP curves were measured to assess microcirculatory function at baseline and during insulin infusion (microvascular insulin sensitivity). Obese women were more insulin resistant than normal-weight women (P < 0.001), and obese PCOS women were more resistant than obese controls (P = 0.02). In contrast, normal-weight women with PCOS had similar insulin sensitivity, compared with normal-weight women without PCOS. Baseline responses to ACh showed no difference in the four groups. ACh responses during insulin infusion were significantly greater in normal-weight PCOS and controls than in obese PCOS and controls. PCOS per se had no significant influence on ACh responses during insulin infusion. During hyperinsulinemia, SNP-dependent vasodilatation did not significantly increase, compared with baseline in the four groups. PCOS per se was not associated with impaired metabolic insulin sensitivity in normal-weight women but aggravates impairment of metabolic insulin sensitivity in obese women. In obese but not normal-weight women, microvascular and metabolic insulin sensitivity are decreased, independent of PCOS. Therefore, obese PCOS women in particular may be at increased risk of metabolic and cardiovascular diseases.

Amino Acid Residues That Confer High Selectivity of the α6 Nicotinic Acetylcholine Receptor Subunit to α-Conotoxin MII[S4A,E11A,L15A

Nicotinic acetylcholine receptors (nAChRs) containing alpha3 and beta2 subunits are found in autonomic ganglia and mediate ganglionic transmission. The closely related alpha6 nAChR subtype is found in the central nervous system where changes in its level of expression are observed in Parkinson's disease. To obtain a ligand that discriminates between these two receptors, we designed and synthesized a novel analog ofalpha-conotoxin MII, MII[S4A,E11A,L15A], and tested it on nAChRs expressed in Xenopus oocytes. The peptide blocked chimeric alpha6/alpha3beta2beta3 nAChRs with an IC(50) of 1.2 nm; in contrast, its IC(50) on the closely related alpha3beta2 as well as non-alpha6 nAChRs was three orders of magnitude higher. We identified the residues in the receptors that are responsible for their differential sensitivity to the peptide. We constructed chimeras with increasingly longer fragments of the N-terminal ligand binding domain of the alpha3 subunit inserted into the homologous positions of the alpha6 subunit, and these were used to determine that the region downstream of the first 140 amino acids was involved. Further mutagenesis of this region revealed that the alpha6 subunit residues Glu-152, Asp-184, and Thr-195 were critical, and replacement of these three residues with their homologs from the alpha3 subunit increased the IC(50) of the peptide by >1000-fold. Conversely, when these key residues inalpha3 were replaced with those fromalpha6, the IC(50) decreased by almost 150-fold. Similar effects were seen with other alpha6-selective conotoxins, suggesting the general importance of thesealpha6 residues in conferring selective binding.