ACh Responses Research Articles

Microvascular function in younger adults with obesity and metabolic syndrome: role of oxidative stress

Older adults with cardiovascular disease exhibit microvascular dysfunction and increased levels of reactive oxygen species (ROS). We hypothesized that microvascular impairments begin early in the disease process and can be improved by scavenging ROS. Forearm blood flow (Doppler ultrasound) was measured in 45 young (32 ± 2 yr old) adults (n = 15/group) classified as lean, obese, and metabolic syndrome (MetSyn). Vasodilation in response to endothelial (ACh) and vascular smooth muscle [nitroprusside (NTP) and epoprostenol (Epo)] agonists was tested before and after intra-arterial infusion of ascorbic acid to scavenge ROS. Vasodilation was assessed as a rise in relative vascular conductance (ml·min(-1)·dl(-1)·100 mmHg(-1)). ACh and NTP responses were preserved (P = 0.825 and P = 0.924, respectively), whereas Epo responses were lower in obese and MetSyn adults (P < 0.05) than in lean controls. Scavenging of ROS via infusion of ascorbic acid resulted in an increase in ACh-mediated (P < 0.001) and NTP-mediated (P < 0.001) relative vascular conductance across all groups, suggesting that oxidative stress influences vascular responsiveness in adults with and without overt cardiovascular disease risk. Ascorbic acid had no effect on Epo-mediated vasodilation (P = 0.267). These results suggest that obese and MetSyn adults exhibit preserved endothelium-dependent vasodilation with reduced dependence on prostacyclin and are consistent with an upregulation of compensatory vascular control mechanisms.

Nicotinic acetylcholine receptors containing the α6 subunit contribute to ethanol activation of ventral tegmental area dopaminergic neurons

Nicotine and alcohol are often co-abused suggesting a common mechanism of action may underlie their reinforcing properties. Both drugs acutely increase activity of ventral tegmental area (VTA) dopaminergic (DAergic) neurons, a phenomenon associated with reward behavior. Recent evidence indicates that nicotinic acetylcholine receptors (nAChRs), ligand-gated cation channels activated by ACh and nicotine, may contribute to ethanol-mediated activation of VTA DAergic neurons although the nAChR subtype(s) involved has not been fully elucidated. Here we show that expression and activation of nAChRs containing the α6 subunit contribute to ethanol-induced activation of VTA DAergic neurons. In wild-type (WT) mouse midbrain sections that contain the VTA, ethanol (50 or 100mM) significantly increased firing frequency of DAergic neurons. In contrast, ethanol did not significantly increase activity of VTA DAergic neurons in mice that do not express CHRNA6, the gene encoding the α6 nAChR subunit (α6 knock-out (KO) mice). Ethanol-induced activity in WT slices was also reduced by pre-application of the α6 subtype-selective nAChR antagonist, α-conotoxin MII[E11A]. When co-applied, ethanol potentiated the response to ACh in WT DAergic neurons; whereas co-application of ACh and ethanol failed to significantly increase activity of DAergic neurons in α6 KO slices. Finally, pre-application of α-conotoxin MII[E11A] in WT slices reduced ethanol potentiation of ACh responses. Together our data indicate that α6-subunit containing nAChRs may contribute to ethanol activation of VTA DAergic neurons. These receptors are predominantly expressed in DAergic neurons and known to be critical for nicotine reinforcement, providing a potential common therapeutic molecular target to reduce nicotine and alcohol co-abuse

Statins attenuate the development of atherosclerosis and endothelial dysfunction induced by exposure to urban particulate matter (PM10)

Exposure to ambient air particulate matter (particles less than 10μm or PM10) has been shown to be an independent risk factor for the development and progression of atherosclerosis. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have well-established anti-inflammatory properties. The aim of this study was to determine the impact of statins on the adverse functional and morphological changes in blood vessels induced by PM10. New Zealand White rabbits fed with a high fat diet were subjected to balloon injury to their abdominal aorta followed by PM10/saline exposure for 4weeks±lovastatin (5mg/kg/day) treatment. PM10 exposure accelerated balloon catheter induced plaque formation and increased intimal macrophages and lipid accumulation while lovastatin attenuated these changes and promoted smooth muscle cell recruitment into plaques. PM10 impaired vascular acetylcholine (Ach) responses and increased vasoconstriction induced by phenylephrine as assessed by wire myograph. Supplementation of nitric oxide improved the impaired Ach responses. PM10 increased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in blood vessels and increased the plasma levels of endothelin-1 (ET-1). Incubation with specific inhibitors for iNOS, COX-2 or ET-1 in the myograph chambers significantly improved the impaired vascular function. Lovastatin decreased the expression of these mediators in atherosclerotic lesions and improved endothelial dysfunction. However, lovastatin was unable to reduce blood lipid levels to the baseline level in rabbits exposed to PM10. Taken together, statins protect against PM10-induced cardiovascular disease by reducing atherosclerosis and improving endothelial function via their anti-inflammatory properties.

Salt Loading in Canola Oil Fed SHRSP Rats Induces Endothelial Dysfunction

This study aimed to determine if 50 days of canola oil intake in the absence or presence of salt loading affects: (1) antioxidant and oxidative stress markers, (2) aortic mRNA of NADPH oxidase (NOX) subunits and superoxide dismutase (SOD) isoforms and (3) endothelial function in SHRSP rats. SHRSP rats were fed a diet containing 10 wt/wt% soybean oil or 10 wt/wt% canola oil, and given tap water or water containing 1% NaCl for 50 days. Without salt, canola oil significantly increased RBC SOD, plasma cholesterol and triglycerides, aortic p22phox, NOX2 and CuZn-SOD mRNA, and decreased RBC glutathione peroxidase activity. With salt, canola oil reduced RBC SOD and catalase activity, LDL-C, and p22phox mRNA compared with canola oil alone, whereas plasma malondialdehyde (MDA) was reduced and RBC MDA and LDL-C were higher. With salt, the canola oil group had significantly reduced endothelium-dependent vasodilating responses to ACh and contractile responses to norepinephrine compared with the canola oil group without salt and to the WKY rats. These results indicate that ingestion of canola oil increases O2 − generation, and that canola oil ingestion in combination with salt leads to endothelial dysfunction in the SHRSP model.

Layer-Specific Interference with Cholinergic Signaling in the Prefrontal Cortex by Smoking Concentrations of Nicotine

Adolescence is a period in which the developing prefrontal cortex (PFC) is sensitive to maladaptive changes when exposed to nicotine. Nicotine affects PFC function and repeated exposure to nicotine during adolescence impairs attention performance and impulse control during adulthood. Nicotine concentrations experienced by smokers are known to desensitize nicotinic acetylcholine receptors (nAChRs), but the impact thereof on PFC circuits is poorly understood. Here, we investigated how smoking concentrations of nicotine (100-300 nm) interfere with cholinergic signaling in the mouse PFC. nAChR desensitization depends on subunit composition. Since nAChR subunits are differentially expressed across layers of the PFC neuronal network, we hypothesized that cholinergic signaling through nAChRs across layers would suffer differentially from exposure to nicotine. Throughout the PFC, nicotine strongly desensitized responses to ACh in neurons expressing β2* nAChRs, whereas ACh responses mediated by α7 nAChRs were not hampered. The amount of desensitization of β2* nAChR currents depended on neuron type and cortical layer. β2*-mediated responses of interneurons in LII-III and LVI completely desensitized, while cholinergic responses in LV interneurons and LVI pyramidal cells showed less desensitization. This discrepancy depended on α5 subunit expression. Two-photon imaging of neuronal population activity showed that prolonged exposure to nicotine limited cholinergic signaling through β2* nAChRs to deep PFC layers where α5 subunits were expressed. Together, our results demonstrate a layer-dependent decrease in cholinergic activation of the PFC through nAChRs by nicotine. These mechanisms may be one of the first steps leading up to the pathophysiological changes associated with nicotine exposure during adolescence.

Positive modulation of the α9α10 nicotinic cholinergic receptor by ascorbic acid

The activation of α9α10 nicotinic cholinergic receptors (nAChRs) present at the synapse between efferent olivocochlear fibres and cochlear hair cells can prevent acoustic trauma. Hence, pharmacological potentiators of these receptors could be useful therapeutically. In this work, we characterize ascorbic acid as a positive modulator of recombinant α9α10 nAChRs. ACh-evoked responses were analysed under two-electrode voltage-clamp recordings in Xenopus laevis oocytes injected with α9 and α10 cRNAs. Ascorbic acid potentiated ACh responses in X. laevis oocytes expressing α9α10 (but not α4β2 or α7) nAChRs, in a concentration-dependent manner, with an effective concentration range of 1-30 mM. The compound did not affect the receptor's current-voltage profile nor its apparent affinity for ACh, but it significantly enhanced the maximal evoked currents (percentage of ACh maximal response, 240 ± 20%). This effect was specific for the L form of reduced ascorbic acid. Substitution of the extracellular cysteine residues present in loop C of the ACh binding site did not affect the potentiation. Ascorbic acid turned into a partial agonist of α9α10 nAChRs bearing a point mutation at the pore domain of the channel (TM2 V13'T mutant). A positive allosteric mechanism of action rather than an antioxidant effect of ascorbic acid is proposed. The present work describes one of the few agents that activates or potentiates α9α10 nAChRs and leads to new avenues for designing drugs with potential therapeutic use in inner ear disorders.

Attenuated vascular responsiveness to K+ channel openers in diabetes mellitus: the differential role of reactive oxygen species

The current study examined the responsiveness of blood vessels from diabetic rats to K+ channel openers and explored whether ROS might be involved in any changes. Responses were measured in aortic rings isolated from four weeks streptozotocin (65 mg/kg)-induced diabetic rats. Relaxation to levcromakalim (ATP-sensitive potassium channel KATP opener, 10(-9)-10(-5) mol/l) and (+/-)-naringenin (large conductance calcium-activated channel BKCa opener, 10(-8)-10(-3) mol/l) were recorded in phenylephrine (1 µmol/l) pre-contracted segments in the absence and presence of superoxide dismutase (SOD, 100 µmol/l) and apocynin (an antioxidant and inhibitor of NADPH oxidase, 100 µmol/l). Contractions to phenylephrine (10(-9)-10(-5) mol/l) and relaxation to acetylcholine (ACh, 10(-9)-10(-5) mol/l) were also recorded. Relaxation curves for levcromakalim, naringenin and ACh for the diabetic group were shifted to the right (p < 0.05) compared with the control. Contractions to phenylephrine were enhanced in the diabetic group (p < 0.01). SOD restored the ACh response but not those of K+ channel openers. On the other hand, apocynin restored the relaxation to naringenin but had no effect on both levcromakalim and ACh responses. The results suggest that both KATP and BKCa activities are attenuated in diabetes mellitus and that ROS appears to contribute only to the change in BKCa function.

Adenosine A2A receptor modulates vascular response in soluble epoxide hydrolase-null mice through CYP-epoxygenases and PPARγ

The interaction between adenosine and soluble epoxide hydrolase (sEH) in vascular response is not known. Therefore, we hypothesized that lack of sEH in mice enhances adenosine-induced relaxation through A(2A) adenosine receptors (AR) via CYP-epoxygenases and peroxisome proliferator-activated receptor γ (PPARγ). sEH(-/-) showed an increase in A(2A) AR, CYP2J, and PPARγ by 31%, 65%, and 36%, respectively, and a decrease in A(1)AR and PPARα (30% and 27%, respectively) vs. sEH(+/+). 5'-N-ethylcarboxamidoadenosine (NECA, an adenosine receptor agonist), CGS 21680 (A(2A) AR-agonist), and GW 7647 (PPARα-agonist)-induced responses were tested with nitro-l-arginine methyl ester (l-NAME) (NO-inhibitor; 10(-4) M), ZM-241385, SCH-58261 (A(2A) AR-antagonists; 10(-6) M), 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an epoxyeicosatrienoic acid-antagonist; 10(-5) M), 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA; 10 μM) or trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB, sEH-inhibitors; 10(-5) M), and T0070907 (PPARγ-antagonist; 10(-7) M). In sEH(-/-) mice, ACh response was not different from sEH(+/+) (P > 0.05), and l-NAME blocked ACh-responses in both sEH(-/-) and sEH(+/+) mice (P < 0.05). NECA (10(-6) M)-induced relaxation was higher in sEH(-/-) (+12.94 ± 3.2%) vs. sEH(+/+) mice (-5.35 ± 5.2%); however, it was blocked by ZM-241385 (-22.42 ± 1.9%) and SCH-58261(-30.04 ± 4.2%). CGS-21680 (10(-6) M)-induced relaxation was higher in sEH(-/-) (+37.4 ± 5.4%) vs. sEH(+/+) (+2.14 ± 2.8%). l-NAME (sEH(-/-), +30.28 ± 4.8%, P > 0.05) did not block CGS-21680-induced response, whereas 14,15-EEZE (-7.1 ± 3.7%, P < 0.05) did. Also, AUDA and t-AUCB did not change CGS-21680-induced response in sEH(-/-) (P > 0.05), but reversed in sEH(+/+) (from +2.14 ± 2.8% to +45.33 ± 4.1%, and +63.37 ± 7.2, respectively). PPARα-agonist did not relax as CGS 21680 (-2.48 ± 1.1 vs. +37.4 ± 5.4%) in sEH(-/-), and PPARγ-antagonist blocked (from +37.4 ± 5.4% to +9.40 ± 3.1) CGS 21680-induced relaxation in sEH(-/-). Our data suggest that adenosine-induced relaxation in sEH(-/-) may depend on the upregulation of A(2A) AR, CYP2J, and PPARγ, and the downregulation of A(1) AR and PPARα.

Effect of aqueous fraction of Rosa damascena on ileum contractile response of guinea pigs.

The use of drugs with herbal origin is increasing for treatment of gastrointestinal (GI) disorders. Rosa damascena (R. damascena) is a well-known plant suggested to have beneficial effect on GI system. In this study, the effect of aqueous fraction of R. damascena on the contractions of isolated guinea pig ileum was investigated. Aqueous fraction of plant was obtained from ethanolic extract after ethyl acetate and n-butanol fractions were discarded. To evaluate effect of this fraction on ileum contraction, guinea pig ileum was removed and mounted on organ bath and its contraction was recorded. Effect of various concentrations (0.66, 0.83, and 1.3 mg/ml) of aqueous fraction on ileum contraction in comparison with Ach in presence and absence of atropine, a muscarinic antagonist of cholinergic, was evaluated. The response of ileum to 1 µg/ml of acetylcholine was considered as 100% response. Our results showed that aqueous fractions of R. damascena dose-dependently increased basal guinea pigs ileum contractions (p<0.05 to p<0.001). Maximal contraction of fraction (1.3 mg/ml) induced 23.4 % of maximal Ach response. The contraction of ileum to aqueous fraction was significant decreased in presence 0.001 µg/ml of atropine. It is concluded that aqueous fraction of R. damascena has mild excitatory effect on ileum contraction and this fraction may be beneficial as a mild laxative agent.

Altered sensitivity to nitric oxide donors, induced by intravascular infusion of quantum dots, in murine mesenteric arteries

Quantum dots (QDs) are utilised in imaging diagnostics, tissue engineering and medical therapeutics, however, their influence on vascular function is not ascertained. Here, we examined small mesenteric arterial responses after acute intravascular exposure to QDs. Incubation in mercaptoundecanoic acid (MUA)-coated QDs (at 15μg/mL) had no influence on endothelial-dependent dilator responses (Acetylcholine; Ach) but led to an attenuated relaxation to the nitric oxide donor, sodium nitroprusside (SNP). Conversely, incubation in POSS-PCU coated QDs (at 15μg/mL) led to attenuated Ach responses (10−11–10−3M; n=5, P<0.05), but had no influence on SNP-induced relaxation. At lower concentrations of POSS-PCU coated QDs (5μg/mL), Ach responses were preserved. We demonstrate that acute exposure to QDs, can attenuate vasodilation but not vasoconstriction, and is dependent on their surface coatings. Our findings have implications in QD use for imaging diagnostics in disease states, where SNP based drugs are used in therapeutic intervention. From the Clinical EditorIn this paper, the influence of quantum dots on vascular function is investigated---an important aspect to consider with the growing utility of quantum dots in imaging diagnostics, tissue engineering and medical therapeutics.

&lt;em&gt;In Vitro&lt;/em&gt; Anticholinergic and Antihistaminic Activities of &lt;em&gt;Acorus Calamus&lt;/em&gt; Linn. Leaves Extracts

The present investigation was aimed at determining the effects of hexane, acetone, methanol and aqueous extracts of Acorus calamus leaves (ACHE, ACAE, ACME and ACAQE) on cholinergic and histaminic system using isolated frog rectus abdominis muscle and guinea pig ileum. A dose dependent potentiation of Ach response (anticholinesterase like effect) was found with ACAE and ACME at 0.25, 0.5, 0.75 and 1 mg/ml, but at higher dose of ACAE, ACME, ACAQE and ACHE (5, 20 mg/ml) inhibit the Ach response (antinicotinic effect). These results revealed biphasic effect of Acorus calamus leaves extracts on acetylcholine induced contractile response in isolated frog rectus abdominis muscle preparation (i.e. potentiation effect at lower dose and inhibitory effect at higher dose). Studies on isolated guinea pig ileum demonstrated antihistaminic effect in a dose dependent manner (100-1000 µg/ml) with ACAE, ACME and ACAQE. In addition, the dose dependent inhibition of Ach response (antimuscarinic effect) was observed with ACAE and ACME. In conclusion, Acorus calamus leaves extracts exerts antinicotinic, anticholinesterase like activities in isolated frog rectus abdominis muscle and antihistaminic, antimuscarinic effect in guinea pig ileum. It has been suggested that these observed activities can be further studied for therapeutic potential of Acorus calamus leaves in the treatment of cognitive disorders and asthma.

Increasing pressure in newborn carotid arteries promotes endothelial cell maturation

The endothelium of newborn arteries has an unusual phenotype, which includes immature cell:cell junctions, prominent stress fibers and reduced NO‐mediated endothelial dilation to acetylcholine (Ach). To investigate mechanisms regulating postnatal endothelial maturation, carotid arteries were isolated from newborn (postnatal day 1, P1), P7 and P21 mice, and analyzed in a myograph at a transmural pressure (P™) of 20 mmHg (mean blood pressure BP at P1). Ach caused dilation in maturing (P7, P21), but not newborn arteries. This was associated with increased Akt activity in maturing arteries. Indeed, inhibition of PI3K/Akt (wortmannin, LY294002) markedly reduced dilation to Ach at P7. A brief, transient exposure of P1 arteries to increased P™ (50 mmHg, equivalent to systolic BP at P7, 60 min) uncovered powerful dilation to Ach. The effect of increased P™ to rescue Ach responses in P1 arteries was prevented by inhibition of PI3K/Akt or by a VE‐cadherin function‐blocking antibody, which disrupts endothelial adherens junctions. The antibody also inhibited Ach responses in P7 arteries. Our results suggest that maturation of newborn arterial endothelium, including increased NO‐mediated endothelial dilation, may be induced by increases in BP in the early postnatal period. Furthermore, the effect of increasing pressure may be mediated by increased VE‐Cadherin signaling at adherens junctions. Funded by NIH HL102715

Cerebrovascular oxidative stress and endothelial dysfunction in response to aldosterone is Nox2‐mediated

Aldosterone is thought to be an important contributor to hypertension. There is an association between high aldosterone levels and stroke risk that is blood pressure‐independent. High aldosterone levels may increase stroke risk through effects on the cerebral vasculature involving oxidative stress (via Nox2‐oxidase) and endothelial dysfunction. We tested whether aldosterone produces cerebrovascular oxidative stress and endothelial dysfunction, and Nox2‐oxidase involvement. Aldosterone treatment (0.28 mg/kg/day, 2 wks) had no effect on blood pressure. Aldosterone impaired dilation of isolated, pressurised basilar arteries to acetylcholine (ACh; endothelium‐dependent agonist) compared to vehicle treatment in control mice, indicating aldosterone causes endothelial dysfunction. In contrast, aldosterone had no effect on ACh responses in Nox2‐deficient mice, indicating this effect is Nox2‐dependent. Basal and Nox2‐stimulated superoxide levels were greater in cerebral arteries of aldosterone vs vehicle‐treated mice, effects that were abolished in Nox2‐deficient mice. These data provide the first evidence suggesting aldosterone causes oxidative stress and endothelial dysfunction in brain via Nox2‐oxidase.

Influence Of Chronic Nicotine On Acetylcholine‐Evoked Renal Vasodilations In Female Rats: Dose Dependency And Ovarian Hormonal Modulation

In this study we report on the interaction of chronic nicotine with cholinergically‐mediated renal vasodilations in female rats and its modulation by ovarian hormones. Dose‐vasodilatory response curves to cumulative bolus doses of acetylcholine (ACh, 0.01–2.43 nmol) were established in phenylephrine‐preconstricted isolated perfused kidneys obtained from sham‐operated (SO) rats treated with or without nicotine (0.5–4.0 mg/kg/day for 2 weeks). The interaction was also evaluated in OVX rats in the absence or presence of progesterone (P), estrogen (E2), or their combination. The vasodilatory effects of ACh were potentiated in a dose‐dependent fashion by the low‐dose range of nicotine (0.5 and 1 mg/kg/day). By contrast, higher nicotine doses (2 and 4 mg/kg/day) failed to alter Ach responses. The co‐existence of both gonadal hormones, P and E2, seems imperative for the display of the potentiating effect of nicotine on ACh vasodilations because: (i) the increase in ACh responses by nicotine was lost in OVX kidneys, and (ii) combined supplementation with P and E2, but not individual treatments, restored the nicotine‐evoked potentiation of ACh vasodilation. Together, the data suggests that nicotine elicits dose‐ and gonadal hormonal‐dependent facilitation of ACh renal vasodilations in female rats.This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No. 502.

M2 muscarinic ACh receptors sensitive BK channels mediate cholinergic inhibition of type II vestibular hair cells

There are two types of hair cells in the sensory epithelium of vestibular end organ. Type II vestibular hair cell (VHC II) is innervated by the efferent nerve endings, which employ a cholinergic inhibition mediated by SK channels through the activation of α9-containing nAChR. Our previous studies demonstrated that a BK-type cholinergic inhibition was present in guinea pig VHCs II, which may be mediated by an unknown mAChR. In this study, BK channel activities triggered by ACh were studied to determine the mAChR subtype and function. We found the BK channel was insensitive to α9-containing nAChR antagonists and m1, m3, m4 muscarinic antagonists, but potently inhibited by the m2 muscarinic antagonist. Muscarinic agonists could mimic the effect of ACh and be blocked by m2 antagonist. cAMP analog activated the BK current and adenyl cyclase (AC) inhibitor inhibited the ACh response. Inhibitor of Giα subunit failed to affect the BK current, but inhibitor of Giα and Giβγ subunits showed a potent inhibition to these currents. Our findings provide the physiological evidence that mAChRs may locate in guinea pig VHCs II, and m2 mAChRs may play a dominant role in BK-type cholinergic inhibition. The activation of m2 mAChRs may stimulate Giβγ-mediated excitation of AC/cAMP activities and lead to the phosphorylation of Ca2+ channels, resulting in the influx of Ca2+ and opening of the BK channel.

Pharmacological characterization of native α7 nicotinic ACh receptors and their contribution to depolarization‐elicited exocytosis in human chromaffin cells

Expression of α7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells. To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors. The nicotinic current provoked by 300µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4ms rise time) and inactivated component (8.5 ± 0.4ms time constant) (termed α7), which was blocked by α-Bgtx or MLA, followed by a slower component (non-α7). α7 nAChR currents were dissected by blocking the non-α7 nAChR current component of the ACh and choline response with the α6* nAChR blocker α-conotoxin (α-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an α7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. α7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1mM) and PNU-120596 (10µM), potentiated responses that were blocked by α-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited α7 nAChR currents, using choline in the presence of α-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists. Our electrophysiological recordings of pure α7 nAChR currents elicited by rapid application of agonists demonstrated that functional α7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.

Endothelium-dependent nitric oxide and hyperpolarization-mediated venous relaxation pathways in rat inferior vena cava

The vascular endothelium plays a major role in the control of arterial tone; however, its role in venous tissues is less clear. The purpose of this study was to determine the role of endothelium in the control of venous function and the relaxation pathways involved. Circular segments of inferior vena cava (IVC) from male Sprague-Dawley rats were suspended between two wires and isometric contraction to phenylephrine (Phe; 10(-5)M) and 96 mM KCl was measured. Acetylcholine (Ach; 10(-10) to 10(-5)M) was added and the percentage of venous relaxation was measured. To determine the role of nitric oxide (NO) and prostacyclin (PGI(2)), vein relaxation was measured in the presence of the nitric oxide synthase inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME; 3 × 10(-4) M) and the cyclooxygenase inhibitor indomethacin (10(-5) M). To measure the role of hyperpolarization, vein relaxation was measured in the presence of K(+) channel activator cromakalim (10(-11) to 10(-6) M), and the nonselective K(+) channel blocker tetraethylammonium (TEA; 10(-3) M). To test for the contribution of a specific K(+) channel, the effects of K(+) channel blockers: glibenclamide (adenosine triphosphate [ATP]-sensitive K(ATP), 10(-5) M), 4-aminopyridine (4-AP; voltage-dependent K(v), 10(-3) M), apamin (small conductance Ca(2+)-dependent SK(Ca), 10(-7) M), and iberiotoxin (large conductance Ca(2+)-dependent BK(Ca), 10(-8) M) on Ach-induced relaxation were tested. Ach caused concentration-dependent relaxation of Phe contraction (maximum 49.9 ± 4.9%). Removal of endothelium abolished Ach-induced relaxation. IVC treatment with L-NAME partially reduced Ach relaxation (32.8 ± 4.9%). In IVC treated with L-NAME plus indomethacin, significant Ach-induced relaxation (33.6 ± 3.2%) could still be observed, suggesting a role of endothelium-derived hyperpolarizing factor (EDHF). In IVC treated with L-NAME, indomethacin and TEA, Ach relaxation was abolished, supporting a role of EDHF. In veins stimulated with high KCl, Ach caused relaxation (maximum 59.5 ± 3.5%) that was abolished in the presence of L-NAME and indomethacin suggesting that any Ach-induced EDHF is blocked in the presence of high KCl depolarizing solution, which does not favor outward movement of K(+) ion and membrane hyperpolarization. Cromakalim, an activator of K(ATP), caused significant IVC relaxation when applied alone or on top of maximal Ach-induced relaxation, suggesting that the Ach response may not involve K(ATP). Ach-induced relaxation was not inhibited by glibenclamide, 4-AP, or apamin, suggesting little role of K(ATP), K(v) or SK(Ca), respectively. In contrast, iberiotoxin significantly inhibited Ach-induced relaxation, suggesting a role of BK(Ca). Thus, endothelium-dependent venous relaxation plays a major role in the control of venous function. In addition to NO, an EDHF pathway involving BK(Ca) may play a role in endothelium-dependent venous relaxation.

Abstract 9743: Profibrillatory Atrial Autonomic and Cellular Electrophysiological Remodelling with Endurance Training

Background: Endurance exercise (Ex) increases AF risk, but underlying mechanisms are unknown. Here, we studied Ex induced autonomic and cellular electrophysiological changes controlling AF risk. Methods: Ex rats (daily treadmill training X 16 wks) were compared to matched sedentary (Sed) controls. Parasympathetic and sympathetic tone were assessed from heart rate (HR) changes caused by atropine and propranolol respectively. Baroreflex sensitivity was studied by HR responses to phenylephrine-induced BP changes. Ion currents (I KACh , I to , I CaL ) were measured in atrial myocytes by patch clamp. Atrial refractory period (ARP) was measured in vivo with transvenous electrode catheters. mRNA expression was quantified by qPCR. Results: Atrial myocytes were hypertrophied in Ex (129±8.7 pF vs 73±5 pF Sed, p&lt;0.001). Parasympathetic tone was significantly enhanced in Ex rats by 83%, with no changes in sympathetic tone. Reflex vagal sensitivity was enhanced (60% increase in HR-reduction vs BP-increase slope with phenylephrine boluses). Post-synaptic responses were also increased, as shown by significant increases in I KACh induced by 100 μ M acetylcholine, ACh (Fig. A). mRNA expression of beta-adrenergic (β1, β2) and muscarinic (M2, M3) receptors and associated G proteins did not differ between groups. Regulatory G signaling proteins (RGS, Fig. B), which decrease ACh responses, were downregulated in Ex. Balanced downregulation of voltage gated K + and Ca 2+ current subunits (Fig. C) and currents (Fig. D) left ARP unchanged in the absence of ACh. AF inducibility was enhanced in Ex rats, an effect that was abolished by atropine. Conclusions: Ex promotes AF by enhancing vagal tone through pre (baroreflex) and post (RGS, I KACh ) synaptic mechanisms. Ion channel expression changes in a balanced way, leaving repolarization unaffected in the absence of ACh.

Myenteric neural network activated by motilin in the stomach of Suncus murinus (house musk shrew)

It has been shown in human and canine studies that motilin, a gastroprokinetic hormone, induces gastric phase III contractions via the enteric nervous; however, the center of motilin action in the stomach has not been clearly revealed. In the present study, we investigated the neural pathway of motilin-induced gastric contraction by using Suncus murinus, a new animal model for motilin study. An isolated suncus stomach was used in vitro to determine the mechanism of motilin action through the myenteric plexus. Synthetic suncus motilin (10(-11) -10(-7) molL(-1) ) was added to an organ bath, and the spontaneous contraction response was expressed as a percent of ACh (10(-5) molL(-1) ) responses. Motilin-induced contractions were also studied by a pharmacological method using several receptor antagonists and enzyme inhibitor. Suncus motilin induced a concentration-dependent gastric contraction at concentrations from 10(-9) to 10(-7) molL(-1) . The responses to suncus motilin in the stomach were completely abolished by atropine and tetrodotoxin treatment and significantly suppressed by administration of hexamethonium, verapamil, phentolamine, yohimbine, ondansetron, and naloxone, whereas ritanserin, prazosin, timolol, and FK888 did not affect the action of motilin. Additionally, N-nitro l-arginine methylester slightly potentiated the contractions induced by motilin. The results indicate that motilin directly stimulates and modulates suncus gastric contraction through cholinergic, adrenergic, serotonergic, opioidergic, and NO neurons in the myenteric plexus.

Onset of Cholinergic Efferent Synaptic Function in Sensory Hair Cells of the Rat Cochlea

In the developing mammalian cochlea, the sensory hair cells receive efferent innervation originating in the superior olivary complex. This input is mediated by α9/α10 nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent SK2 potassium channels. We examined the acquisition of this cholinergic efferent input using whole-cell voltage-clamp recordings from inner hair cells (IHCs) in acutely excised apical turns of the rat cochlea from embryonic day 21 to postnatal day 8 (P8). Responses to 1 mm acetylcholine (ACh) were detected from P0 on in almost every IHC. The ACh-activated current amplitude increased with age and demonstrated the same pharmacology as α9-containing nAChRs. Interestingly, at P0, the ACh response was not coupled to SK2 channels, so that the initial cholinergic response was excitatory and could trigger action potentials in IHCs. Coupling to SK current was detected earliest at P1 in a subset of IHCs and by P3 in every IHC studied. Clustered nAChRs and SK2 channels were found on IHCs from P1 on using Alexa Fluor 488 conjugated α-bungarotoxin and SK2 immunohistochemistry. The number of nAChRs clusters increased with age to 16 per IHC at P8. Cholinergic efferent synaptic currents first appeared in a subset of IHCs at P1 and by P3 in every IHC studied, contemporaneously with ACh-evoked SK currents, suggesting that SK2 channels may be necessary at onset of synaptic function. An analogous pattern of development was observed for the efferent synapses that form later (P6-P8) on outer hair cells in the basal cochlea.