ACh Responses Research Articles

Identification and Functional Expression of a Family of Nicotinic Acetylcholine Receptor Subunits in the Central Nervous System of the Mollusc Lymnaea stagnalis

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.

Enhanced acetylcholine-induced dilation in afferent arterioles in simvastatin-fed rats

Objective 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to increase renal blood flow and glomerular filtration rate independent of their lipid lowering effects. The purpose of this study was to determine the effect of simvastatin on acetylcholine-induced vasodilation in the renal microcirculation. The hypothesis of the study was that simvastatin would increase acetylcholine-induced vasodilation in the renal microcirculation. Methods The hydronephrotic kidney preparation was used. On the day of the experiment the kidney was prepared for videomicroscopy and dose–response curves were done with acetylcholine and sodium nitroprusside (10 − 10 M to10 − 5 M) in simvastatin-fed rats ??( n = 8) and control rats ( n = 13). The vasodilator responses of afferent and efferent arterioles were directly quantitated using videomicroscopy. l-NAME; ( N(omega)-nitro- l-arginine methyl ester) was also given in a group of simvastatin-fed rats (10 −5 M) to determine if it would block the acetylcholine (Ach)-induced vasodilation. Results Simvastatin enhanced Ach-induced vasodilation in the afferent arteriole compared to control rat responses to Ach. At 10 − 7 M, ACH caused a 31.6 ± 7.2% increase from baseline diameter in the afferent arteriole in the simvastatin-fed rats compared to a 23 ± 8.1% vasodilation in the control rats ( p < 0.05). There were no differences in the response to ACH in the efferent arteriole between the two groups. l-NAME completely abolished the ACH response in the simvastatin-fed rats. Conclusions: The increase in renal blood flow and glomerular filtration rate observed with simvastatin may be due to its preglomerular vasodilator effects. This may be due to an increase in nitric oxide production.

Contribution of capacitative and non-capacitative Ca 2+-entry to M 3-receptor-mediated contraction of porcine coronary smooth muscle

We studied the contribution of store-operated or capacitative Ca 2+-entry (SOCE or CCE, respectively) through store-operated Ca 2+ channels (SOCCs) and the contribution of Ca 2+-entry through receptor-operated, non-selective cation channels (ROCCs or NSCCs, respectively), on the M 3-receptor-mediated (270 nM Ach) contractile response of porcine coronary smooth muscle strips by means of the respective inhibitors. In the presence of L-VOCC blockade (1 μM verapamil), LOE 908 (inhibition of NSCCs) decreased the contractile response to 75 ± 5% ( p < 0.01, n = 6), 2-APB (inhibition of SOCCs) and SK&F 96365 (inhibition of SOCCs and of NSCCs) decreased the response to 45 ± 4% ( p < 0.001, n = 10) and to 23 ± 2% ( p < 0.001, n = 5), respectively (control: Ach response in the presence of verapamil alone). In the absence of L-VOCC blockade, LOE 908 reduced the Ach-response to 49 ± 7% ( p < 0.001, n = 8) and SK&F 96365 to 3 ± 2% ( p < 0.001, n = 4) of control, whereas 2-APB transiently increased the response (peak effect: 130 ± 11%; p < 0.05, n = 8). We conclude: (1) the main source of activator Ca 2+ during the M 3-receptor-mediated contractile response is the Ca 2+ influx through L-VOCCs; (2) however, in the presence of L-VOCC blockade, the contractile response is mainly due to Ca 2+-entry through SOCCs; (3) NSCCs may be considerably involved in M 3-receptor-mediated contraction as they may serve to depolarize the membrane potential and, thus, to open L-VOCCs; (4) in primary tissue of vascular smooth muscle, both, SOCE and Ca 2+-entry through NSCCs are activated during M 3-receptor stimulation.

Effect of sildenafil on the isolated rat aortic rings

Sildenafil, a highly selective inhibitor of PDE 5, is effective in the treatment of erectile dysfunction. Penile erection involves relaxation of smooth muscle of corpus cavernosum and its associated arterioles. The objective of this study was to investigate the effect of sildenafil on nitric oxide/cyclic guanosine monophosphate (NO/cGMP)-dependent relaxation of rat aortic rings. The contribution of sildenafil to the vasorelaxation of rat aortic rings was also investigated. Sildenafil produced significant potentiation of acetylcholine (ACh, 2 x 10(-6) m)-induced relaxation at concentration > or =1 x 10(-8) m. Addition of sildenafil (1 x 10(-7) m) to aortic rings failed to alter the effect of N(G)-nitro-L-arginine (l-NNA, 3 x 10(-5) m) or methylene blue (MB, 3 x 10(-5) m) on ACh response. Similarly, sildenafil (1 x 10(-7) m) augmented significantly the vasorelaxation induced by sodium nitroprusside over the range of 1 x 10(-9)-1 x 10(-8) m. When added to phenylephrine (3 x 10(-6) m)-precontracted rat aortic rings, sildenafil (1 x 10(-9)-1 x 10(-4) m) induced concentration-dependent relaxation reaching a maximum of 96.48 +/- 1.44%. These relaxations were not significantly attenuated by previous incubation with L-NNA (3 x 10(-5) m) or MB (3 x 10(-5) m). Denudation did not significantly affect the vasorelaxant effect of sildenafil. Sildenafil may act in the rat aortic rings through the amplification of NO/cGMP pathway. It may augment both basal endothelial NO function and exogenous NO-dependent vasodilatation. However, sildenafil may act by a mechanism independent of NO/cGMP pathway and this mechanism contributes to its smooth muscle relaxant effect.

Modulation of nicotinic acetylcholine and N-methyl- d-aspartate receptors by some Hymenopteran venoms

The effect of 19 venoms from solitary wasps, solitary bees, social wasps and ants were investigated for their effects on nicotinic acetylcholine receptors (nAChR) and ionotropic glutamate receptors (IGRs) of both the N-methyl- d-aspartate (NMDAR) and non-NMDAR type. Whole-cell patch clamp of human muscle TE671 cells was used to study nAChR, and of rat cortical and cerebellar granule cells for IGRs. Solitary wasp venoms caused significant voltage-dependent antagonism of nAChR responses to 10 μM ACh and NMDAR responses to 100 μM NMDA (+10 μM glycine) when co-applied at 1 μg/ml with the agonists. At positive holding potentials ( V H) potentiation of these receptors was observed with some venoms. Solitary bee venoms only affected nAChR by causing either voltage-independent antagonism or potentiation of their responses to 10 μM ACh. Of four social wasp venoms, one acted on nAChR by potentiating responses to 10 ACh, while another generated an ACh-like response when applied alone. They had no effect on IGRs. Of the two ant venoms, one caused voltage-independent inhibition of nAChR. Neither affected IGRs. The data indicate the presence of nAChR agonists and antagonists and NMDAR antagonists in Hymenopteran venoms and warrant further investigation to separate and identify these venom components.

Chronic hypoxia exposure depresses aortic endothelium-dependent vasorelaxation in both sedentary and trained rats: involvement of l-arginine

This study was designed to test the hypothesis that the previously demonstrated training-induced improvement of the endothelium vasodilator function would be blunted under conditions of chronic hypoxia exposure as a result of deleterious effects of hypoxia per se on the nitric oxide pathway. Sea-level-native rats were randomly assigned to N (living in normoxia), NT (living and training 5 days/wk for 5 wk in normoxia), CH (living in hypoxia, 2,800 m), and CHT (living and training 5 days/wk for 5 wk in hypoxia, 2,800 m) groups. Concentration-response curves to acetylcholine (ACh; 10(-9) to 10(-4) M) with or without L-arginine (10(-3) to 10(-5) M) and/or nitro-L-arginine methyl ester (10(-5) M) were assessed on aortic isolated rings. The main finding was that chronic hypoxia severely depressed maximal ACh-responses of aortic rings in both sedentary and trained groups. However, chronic hypoxia did not interfere with training-induced increases in maximal ACh responses, considering that maximal ACh vasorelaxation was improved in CHT rats to the same extent as in NT rats when both groups were directly compared with their sedentary counterparts. It should be pointed out that the vasodilator response to ACh was restored in CH and CHT rats to the level obtained in N and NT rats, respectively, by an in vitro L-arginine addition. A hypoxia-induced decrease in L-arginine bioavailability resulting from acclimatization at altitude may be involved in this limitation of the NO pathway in CH and CHT rats. These results are of importance for aerobic performance as the specific vascular adaptations to training at altitude could contribute to limit peripheral vasodilatation and subsequently blood flow during exercise.

Nicotinic ACh receptors in area postrema neurons of immature rat brain

The electrophysiological properties of nicotinic ACh receptors (nAChR) were investigated in acutely dissociated area postrema (AP) neurons of the immature rat brain using the whole-cell patch-clamp recording method. ACh induced a transient inward current exhibiting a strong inward rectification. The ACh response was mimicked by nicotine and cytisine, and was inhibited by nAChR antagonists, but not by 10 −7 M atropine. Muscarinic AChR agonists did not induce any current. We confirmed the Ca 2+ permeability of nAChR. These results indicate the presence of nAChR on AP neurons, and suggest that the activation of nAChR play important roles in cardiovascular functions in rats.

Modification of the acetylcholine-induced current of the snail Helix pomatia L. by fast temperature changes

Using the single electrode voltage clamp method, we found that acetylcholine (aCh) induces transient inward dose-dependent current on the membrane of the identified Helix pomatia Br neuron. We analyzed the effects of fast cooling and heating as well as thermal acclimation on the aCh inward current. the experiments were conducted on active and dormant snails acclimated to either 20 or 7?C for at least four weeks. the Hill coefficient remained approximately 1 in all cases, which means that there is a single aCh binding site on the membrane. Fast temperature alternations induce binding affinity changes. in the work presented, we analyzed the effects of cooling on the aCh-induced inward current. the amplitude of aCh-induced inward current was markedly reduced after cooling, and the speed of decay of the aCh response was lower. &lt;br&gt;&lt;br&gt;&lt;font color="red"&gt;&lt;b&gt; This article has been retracted. Link to the retraction &lt;u&gt;&lt;a href="http://dx.doi.org/10.2298/ABS1501341E"&gt;10.2298/ABS1501341E&lt;/a&gt;&lt;u&gt;&lt;/b&gt;&lt;/font&gt;

Cardiovascular risk factors and endothelial dysfunction.

Endothelial dysfunction is a feature of atherosclerosis and is associated with CHD (coronary heart disease) risk factors. This study aimed to determine the relationship between the degree of endothelial dysfunction and calculated cardiovascular risk. Endothelial function, as determined by the ACh/NP (acetycholine/sodium nitroprusside response) ratio on brachial plethysmography, was compared with cardiovascular risk as calculated from the Framingham, PROCAM (Prospective Cardiovascular Munster) and MRFIT (Multiple Risk Factor Intervention Trial) algorithms in 246 (187 male) patients, including 44 (22%) with established CHD. Endothelial dysfunction correlated with the total number of risk factors (r2=0.22; P=0.002) and was related to LDL (low-density lipoprotein)-cholesterol in men and triacylglycerols (triglycerides) in women. The ACh/NP ratio correlated with the occurrence of diabetes, CHD and the LDL-cholesterol concentration (r2=0.58; P<0.001). Endothelial dysfunction was associated with presence of CHD on receiver-operating characteristic plot analysis (area=0.706+/-0.04; P=0.001). There was no correlation between ACh/NP ratio and CHD risk calculated with the Framingham algorithm in men, although both ACh and NP response correlated separately with risk in women. The endothelial ACh/NP ratio correlated with absolute risk in the PROCAM algorithm (r2=0.41; P<0.005). Intermediate results were obtained with MRFIT. Individual risk factors make different contributions to endothelial dysfunction compared with their role in risk calculators. The stronger relationship of endothelial dysfunction with PROCAM risk reflects the contribution of male sex, LDL-cholesterol and triacylglycerols to risk calculated by this algorithm.

Link between free radicals and protein kinase C in glucose-induced alteration of vascular dilation

Development of vascular complications in diabetes has been linked to the quality of glucose regulation and characterized by endothelial dysfunction. The exact mechanism behind vascular complications in diabetes is poorly understood. However, alteration of nitric oxide (NO) biosynthesis or bioactivity is strongly implicated and the mechanism behind such alterations is still a subject for research investigations. In the present study, we tested the hypothesis that glucose-induced attenuation of vascular relaxation involves protein kinase C (PKC)-linked generation of free radicals. Vascular relaxation to acetylcholine (ACh; 10 −9–10 −5 M), isoproterenol (10 −9–10 −5 M), or NO donor, sodium nitropruside (SNP; 10 −9–10 −6 M) was determined in phenylephrine (PE, 10 −7 M) pre-constricted aortic rings from Sprague-Dawley rats in the presence or absence of 30 mM glucose (30 min), L-nitro-arginine methyl ester (L-NAME; 10 −4 M for 15 min), a NO synthase inhibitor, or xanthine (10 −5 M), a free radical generator. ACh dose-dependently caused relaxation that was attenuated by L-NAME, glucose, or xanthine. Pre-incubation (15 min) of the rings with vitamin C (10 −4 M), an antioxidant or calphostin C (10 −6 M), a PKC inhibitor, restored the ACh responses. However, high glucose had no significant effects on SNP or isoproterenol-induced relaxation. ACh-induced NO production by aortic ring was significantly reduced by glucose or xanthine. The reduced NO production was restored by pretreatment with vitamin C or calphostin C in the presence of glucose, but not xanthine. These data demonstrate that oxidants or PKC contribute to glucose-induced attenuation of vasorelaxation which could be mediated via impaired endothelial NO production and bioavailability. Thus, pathogenesis of glucose-induced vasculopathy involves PKC-coupled generation of oxygen free radicals which inhibit NO production and selectively inhibit NO-dependent relaxation.

Heme oxygenase-mediated endothelial dysfunction in DOCA-salt, but not in spontaneously hypertensive, rat arterioles.

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide. Carbon monoxide inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. We reported HO-1-mediated endothelial dysfunction in Dahl salt-sensitive hypertension. Previous studies suggested that salt-sensitive hypertensive rats, but not spontaneously hypertensive rats (SHR), display endothelial dysfunction. This study examines the hypothesis that HO-1-mediated arteriolar endothelial dysfunction develops in deoxycorticosterone acetate (DOCA)-salt hypertensive (DOCA) rats, but not in SHR. Uninephrectomized (isoflurane anesthesia) male Sprague-Dawley rats received DOCA injections and saline drinking solution for 4 wk. Rats subjected to sham surgery received vehicle injections and tap water. Blood pressure was elevated in DOCA rats and SHR compared with sham and Wistar-Kyoto (WKY) groups. Aortic HO-1 expression and blood carboxyhemoglobin levels were elevated in the DOCA group, but not in SHR. In isolated gracilis muscle arterioles, ACh caused concentration-related vasodilation in all groups, with attenuated maximum responses in DOCA, but not in SHR, arterioles. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, restored ACh-induced responses in DOCA arterioles to sham levels. ACh responses remained the same in SHR and WKY arterioles after chromium mesoporphyrin treatment. These data show that HO-1 levels and activity are increased and arteriolar responses to ACh are decreased in DOCA rats, but not in SHR. Furthermore, in DOCA arterioles, an inhibitor of HO restores ACh-induced vasodilation to sham levels. These results suggest that elevated HO-1 levels and activity, not resulting from hypertension per se, contribute to endothelial dysfunction in DOCA rats.

The effects of resuscitation with a blood substitute on vasomotor physiology in a swine model of arterial hemorrhage

Introduction: The purpose of this study was to determine if a hemoglobin based oxygen carrier, HBOC-201, alters endothelial function and nitric oxide (NO) physiology following hemorrhagic shock. Methods: Swine (Sus scrofa) underwent catheterization of the aorta and the femoral, circumflex iliac, and pulmonary arteries. Control animals (n = 3) underwent instrumentation only. Study animals underwent hemorrhage to a mean arterial pressure (MAP) of 30 ± 5 mmHg; maintained for 45 minutes; resuscitated to baseline MAP and maintained for 4 hours. Resuscitation fluids included: Lactated Ringers (LR) (n = 8), Shed Blood (SB) (n = 8), Lactated Ringers, 40 cc/kg plus Shed Blood (LRSB) (n = 8), and HBOC (n = 8). At baseline, one, and four hours after resuscitation, acetylcholine (Ach) was infused through the circumflex iliac artery and vasorelaxation measured by M-mode ultrasound. NO levels were determined by chemiluminescent assay. Data were analyzed using repeated measures ANOVA with Tukey’s post-hoc analysis. Values listed are ± SEM, p values < 0.05 were considered significant. Results: All animals survived the experiment. HBOC, SB, and LRSB provided equivalent resuscitation. Animals receiving HBOC required the lowest resuscitation volume (HBOC – 14.6 ± 2.13 cc/kg; SB – 41.5 ± 3.49 cc/kg; LRSB – 76.4 ± 1.10 cc/kg p < 0.01). For all groups, the mean baseline diameter of the external iliac artery was 0.52 ± 0.01 cm. Maximal vessel contraction occurred at the completion of shock (mean 0.27 ± 0.01 cm). Vessel diameter (0.48 ± 0.01 cm) and response to acetylcholine (100% recovery of baseline diameter) was restored to normal in the SB and LRSB groups at the end of resuscitation. The HBOC group had persistent vasoconstriction (0.35 ± 0.02 cm) and diminished Ach response (21% recovery at end resuscitation, p < 0.01). No levels did not differ between study groups (p = 0.69). Conclusion: HBOC may be an alternative resuscitation agent in patients with hemorrhagic shock. Resuscitation with HBOC requires less volume than blood or crystalloid. These data suggest HBOC-201 has a vasoconstrictive effect without nitric oxide scavenging.

Mutation in neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes blocks ethanol action.

Amino acids (AAs) in the extracellular portion of the transmembrane domain of several inhibitory ligand-gated ion channels participate in an alcohol binding site. To extend these studies to neuronal nicotinic acetylcholine receptors (nAChRs), we focused on an AA (L262) located in the same region of the second transmembrane domain of the alpha2 subunit of neuronal nAChRs. Single-point mutation of alpha2L262 was carried out, the resulting alpha2 subunits co-expressed with wild-type beta4 subunits in Xenopus laevis oocytes, and studied using two-electrode voltage clamp. Ethanol enhancement of ACh responses was diminished [alpha2(L262F)beta4] or abolished [alpha2(L262G)beta4, alpha2(L2625)beta4 and alpha2(L262A)beta4]. Mutation of the homologous AA in beta4 [beta4(L258A)] did not modify the ethanol modulation and the mutation in alpha2 was dominant, because ethanol did not enhance ACh responses in alpha2(L262A)beta4(L258A) nAChRs. n-Alcohols (ethanol through octanol) were applied to alpha2(L262A)beta4 nAChRs. As described previously for other nAChRs, short-chain alcohols enhanced, intermediate-chain alcohols had no effect and long-chain alcohols inhibited ACh responses in the wild-type receptor. For alpha2(L262A)beta4 nAChRs the alcohol enhancing effect was absent, and the alcohol inhibitory action was increased. Although this suggests removal of an alcohol enhancing site through mutagenesis, we cannot rule out the enhancement of action at an alcohol inhibitory site.

Five ADNFLE mutations reduce the Ca2+ dependence of the mammalian alpha4beta2 acetylcholine response.

Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild-type (WT) subunit (alpha4 or beta2), and studied the Ca2+ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca2+-induced increases in the 30 microM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca2+-activated Cl- currents. The S256L mutation also abolished 2 mM Ba2+ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration-response relationship but all three mutations shifted the concentration-response relationship to the left at [ACh] >= 30 microM. Co-expression of the V262M mutation with a mutation (E180Q) that abolished Ca2+ potentiation resulted in 2 mM Ca2+ block, rather than potentiation, of the 30 microM ACh response, suggesting that the ADNFLE mutations reduce Ca2+ potentiation by enhancing Ca2+ block of the alpha4beta2 nAChR. Ca2+ modulation may prevent presynaptic alpha4beta2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca2+ dependence of the ACh response could trigger seizures by increasing alpha4beta2-mediated glutamate release during such bouts.

Effects of posture and venous insufficiency on endothelial-dependent and -independent cutaneous vasodilation in the perimalleolar region

Objectives: to assess the effects of posture, endothelial function and venous insufficiency on cutaneous microvascular vasodilator function in the gaiter area, in particular defining factors which may affect microangiopathy and ulcer formation. Methods: endothelial-dependent and -independent vasodilator responses to incremental-doses of acetylcholine (Ach) and sodium nitroprusside (SNP) were evaluated in the perimalleolar region in the supine and standing positions in middle-aged patients with isolated superficial venous insufficiency (ISVI) (n = 25) and healthy controls (n = 28) using laser Doppler fluximetry (LDF) and iontophoresis of vasodilators. Results: the venoarteriolar reflex (vasoconstriction on standing) was equally present in both groups, and reduced the vasodilator responses to SNP in the upright position (e.g., for patients with ISVI, peak SNP response was 82 ± 11 PU [standing] vs 123 ± 15 PU [supine]). The presence of ISVI had no effect on endothelial vasodilator function in the supine position, but on standing cutaneous reactivity to Ach was significantly reduced (e.g., peak Ach response 69 ± 8 PU [ISVI] vs 109 ± 11 PU [controls], p < 0.003). Conclusions: upright posture impairs cutaneous endothelial-dependent vasodilation in the gaiter area of patients with ISVI. This may be of clinical and prognostic utility in identifying which patients with uncomplicated ISVI are at highest risk of tissue breakdown and ulcer formation in the gaiter area.Eur J Vasc Endovasc Surg 26, 100-104 (2003)

Increased plasma endothelin-1 and impaired vasorelaxation in mice featuring endothelial cell specific knockout of the endothelin B receptor

The role of the murine endothelial cell (EC) endothelin B receptor (ETBR) in vascular function and BP regulation was investigated. Floxed ETBR mice with loxP sites flanking exons 2 and 3 were generated through homologous recombination in embryonic stem cells. Flox/Wild (F/W) mice were crossed with Tie2-Cre transgenic mice to produce EC-specific ETBR knockout mice (KO) (F/F Tie2). Wild type (W/W -/-) and single transgenic (W/W Tie2 and F/F -/-) littermates acted as controls. In vitro vascular function was assessed by wire myography and in vivo BP is being measured in conscious animals using i.a. catheters. 125I ET-1 binding was measured in pulmonary EC. Plasma ET-1 was measured by RIA. We have successfully achieved EC-specific knockout of the ETBR in the mouse. This model has an impaired vasodilator response to Sx6c and ACh but maintains normal tracheal smooth muscle ETBR-mediated contraction. The mechanism underlying the impaired ACh response awaits further investigation. The EC ETBR is likely to represent the clearance receptor for circulating ET-1. The floxed ETBR KO mouse will be a useful tool for the study of the role of EC ETBR in cardiovascular function.

Postsynaptic muscarinic M1 receptors activate prefrontal cortical EEG of C57BL/6J mouse.

Recent pharmacological studies exploring the functional roles of muscarinic cholinergic receptor (mAChR) subtypes in prefrontal cortex of C57BL/6J (B6) mouse have provided evidence for a presynaptic M2 autoreceptor. The B6 mouse was chosen for these studies because it is a genetically well-characterized model that also provides the genomic background for many genetically modified mice. In addition to increasing ACh release, one functional consequence of pharmacologically blocking the cortical M2 autoreceptor is activation of the contralateral prefrontal cortical EEG. To date, the mechanisms through which M2 autoreceptor antagonism causes cortical EEG activation have not been investigated. The present study tested the hypothesis that, in the B6 mouse, prefrontal cortical ACh activates the contralateral prefrontal EEG via postsynaptic M1 receptors. This hypothesis was tested in 15 mice using in vivo microdialysis delivery of muscarinic antagonists with simultaneous quantification of ACh release, number of 7- to 14-Hz EEG spindles, and fast Fourier transformation analysis of prefrontal EEG. Dialysis delivery of the nonsubtype selective muscarinic antagonist scopolamine (10 nM) significantly (P = 0.01) increased ACh release. Quantitative EEG analysis showed that scopolamine did not alter contralateral prefrontal cortical EEG. To differentiate mAChR subtypes mediating pre- versus postsynaptic responses, additional experiments used muscarinic antagonists with different affinities for the five mAChR subtypes. Microdialysis delivery of 3 nM AF-DX 116, a muscarinic antagonist with relatively high affinity for the M2 and M4 subtypes, significantly (P < 0.01) increased prefrontal cortical ACh release and activated EEG in the contralateral prefrontal cortex. EEG activation was characterized by a significant decrease in number of 7- to 14-Hz EEG spindles (P < 0.0001) and power (Vrms) of EEG slow waves (P < 0.05). Microdialysis delivery of 3 nM AF-DX 116 plus 3 nM pirenzepine, a relatively selective M1 and M4 muscarinic antagonist, also significantly (P < 0.01) increased ACh release but did not decrease the number of EEG spindles and did not change EEG slow waves. The differential EEG and ACh responses to dialysis delivery of the muscarinic antagonists support the conclusion that, in B6 mouse, postsynaptic muscarinic receptors of the M1 subtype are a primary site by which ACh activates the EEG.

Cholinergic activation of glucose transport in bovine chromaffin cells involves calmodulin and protein kinase Czeta signaling.

The aim of the present study was to delineate possible signaling pathways involved in acetylcholine (Ach)-induced glucose transport in chromaffin cells, a widely applied model system for sympathetic neurons. Acute Ach stimulation (10 min) enhanced the rate of glucose transport through activation of both nicotinic and muscarinic receptors. The calmodulin antagonist, W13, and the protein kinase C (PKC) inhibitor, staurosporine, each partially depressed Ach-induced glucose transport, with staurosporine exhibiting the stronger inhibitory effect. Pretreating the cells with phorbol 12-myristate 13-acetate (PMA) to downregulate PKC activity did not affect the nicotine-induced glucose transport, but completely attenuated that activated by muscarine, suggesting that Ach activation of transport involved both diacylglycerol-independent (PKCzeta) and diacylglycerol-dependent PKCs (PKCalpha/PKCepsilon). The PI 3-kinase inhibitor, wortmannin, diminished the Ach response, consistent with activation of the PKCs by the upstream PI 3-kinase-dependent phosphoinositide-dependent kinase, PDK1. Cholinergic activation strongly activated the ERK1/ERK2 cascade and p38 MAP kinase, but only p38 MAP kinase appeared to play a role, however minor, in nicotine-induced glucose uptake. The results are consistent with PKCs being more important than calmodulin in coupling cholinergic activation to glucose transport in chromaffin cells, but additional, yet unidentified, signaling pathways appear to be needed to obtain full activation of glucose transport in response to Ach.

Heat stress protection against mesenteric I/R-induced alterations in intestinal mucosa in rats.

Prior induction of heat shock protein 70 (HSP70) protects against ischemia-reperfusion (I/R) mucosal injury, but the ability of HSP70 to affect I/R-induced alterations in epithelial cell function is unknown. Rats subjected to whole body hyperthermia (41.5-42 degrees C for 6 min) increased HSP70 and heat shock factor 1 mRNA expression, reaching a maximum 2 h after heat stress and declining thereafter. HSP70 production was maximally elevated at 4 h after heat stress and remained elevated until after 12 h. Heat stress alone had no effect on mucosal function except to enhance secretion in response to ACh. Heat stress provided complete morphological protection against I/R-induced mucosal injury but did not confer a similar protection against I/R-induced decreases in mucosal resistance, sodium-linked glucose absorption, or tachykinin-mediated chloride secretion. Heat stress, however, attenuated the I/R-induced suppression of ACh response, and this effect was dependent on enteric nerves. Thus induction of heat shock protein 70 is associated with the preservation of mucosal architecture and attenuation of some specific functional alterations induced by I/R.

Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed.

During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca2+, pulses of 100 microM ACh or 100 mM K+ of increasing duration (1-5 s) caused similar exocytosis of about 3-4 microC catecholamine. Depletion of endoplasmic reticulum (ER) Ca2+ by pretreatment with 1 microM thapsigargin, 10 mM caffeine and 10 microM ryanodine more than halved the responses to ACh but did not affect the responses to K+ responses. In these ER Ca2+-depleted cells the protonophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20 microM given during the 5 s preceding each pulse) augmented the responses to ACh responses fourfold for all pulse durations applied (1-5 s) whereas responses to K+ were potentiated twofold with 1 to 2 s pulses but were not affected with longer pulse durations. ACh pulses applied to fura-2-loaded cells evoked increases of bulk cytosolic [Ca2+] ([Ca2+](c)) that were substantially smaller than those elicited by K+ pulses. Confocal microscopy of fluo-3-loaded cells showed that ACh pulses elicited discrete and more localized [Ca2+](c) elevations, whereas K+ pulses produced higher [Ca2+](c) transients that spread out quickly throughout the cytosol. These results suggest that mitochondria sense the increase of local [Ca2+](c) elicited by ACh (that evokes firing of action potentials) much better than that induced by K+ (that produces sustained cell depolarisation). This implies that mitochondria are more sensitive to the local [Ca2+](c) changes resulting from the physiological triggering of action potentials by ACh.