Highest Dose Of Acetylcholine Research Articles

Effect of chronic administration of magnesium supplement (magnesium glycinate) on male albino wistar rats’ intestinal (Ileum) motility, body weight changes, food and water intake

Recent researches suggests magnesium as an adjuvant medication for COVID 19 patients. Magnesium relaxes skeletal muscles, an effect when prolonged in intestinal smooth muscles can cause severe discomfort such as bloating, vomiting, constipation and nausea. The objectives of this study was to ascertain if magnesium will cause relaxation of the intestinal (ileum) smooth muscles as it does in skeletal muscles. Also, this research seeks to find out the receptor pathway through which magnesium will alter motility in the gut using acetylcholine, atropine and propranolol. Ten male albino wistar rats (100–150 g) were randomly assigned into two groups (control and magnesium treated) (n = 5). Animals were acclimatized for two weeks before treatment which lasted for 6 weeks. Magnesium treated animals received oral magnesium glycinate (1600 mg/70 kg) daily while control group receive normal saline of equal volume. All animals had free access to food and water ad libitum. Results were analyzed at statistical level of P < 0.05. Body weight changes, food and water intake were not statistically significant. Basal contractions of ileum in magnesium treated group were significantly lower compared to control group. Propranolol significantly increased the percentage relaxation in magnesium treated group compared to the control. Atropine significantly decrease the percentage relaxation in magnesium treated group compared to the control. Higher doses of acetylcholine (10−5 and 10−4) increased the contractions in magnesium treated group. Conclusively, magnesium decreases motility of the intestine through beta adrenergic receptor pathway. Intake of magnesium for long period should be closely monitored to avoid the discomforting symptoms earlier stated.

α7 nicotinic receptors play a role in regulation of cardiac hemodynamics.

The α7 nicotinic receptors (NR) have been confirmed in the heart but their role in cardiac functions has been contradictory. To address these contradictory findings, we analyzed cardiac functions in α7 NR knockout mice (α7-/-) in vivo and ex vivo in isolated hearts. A standard limb leads electrocardiogram was used, and the pressure curves were recorded in vivo, in Arteria carotis and in the left ventricle, or ex vivo, in the left ventricle of the spontaneously beating isolated hearts perfused following Langedorff's method. Experiments were performed under basic conditions, hypercholinergic conditions, and adrenergic stress. The relative expression levels of α and β NR subunits, muscarinic receptors, β1 adrenergic receptors, and acetylcholine life cycle markers were determined using RT-qPCR. Our results revealed a prolonged QT interval in α7-/- mice. All in vivo hemodynamic parameters were preserved under all studied conditions. The only difference in ex vivo heart rate between genotypes was the loss of bradycardia in prolonged incubation of isoproterenol-pretreated hearts with high doses of acetylcholine. In contrast, left ventricular systolic pressure was lower under basal conditions and showed a significantly higher increase during adrenergic stimulation. No changes in mRNA expression were observed. In conclusion, α7 NR has no major effect on heart rate, except when stressed hearts are exposed to a prolonged hypercholinergic state, suggesting a role in acetylcholine spillover control. In the absence of extracardiac regulatory mechanisms, left ventricular systolic impairment is revealed.

Biphasic Cholinergic Modulation of Reverberatory Activity in Neuronal Networks

Acetylcholine (ACh) is an important neuromodulator in various cognitive functions. However, it is unclear how ACh influences neural circuit dynamics by altering cellular properties. Here, we investigated how ACh influences reverberatory activity in cultured neuronal networks. We found that ACh suppressed the occurrence of evoked reverberation at low to moderate doses, but to a much lesser extent at high doses. Moreover, high doses of ACh caused a longer duration of evoked reverberation, and a higher occurrence of spontaneous activity. With whole-cell recording from single neurons, we found that ACh inhibited excitatory postsynaptic currents (EPSCs) while elevating neuronal firing in a dose-dependent manner. Furthermore, all ACh-induced cellular and network changes were blocked by muscarinic, but not nicotinic receptor antagonists. With computational modeling, we found that simulated changes in EPSCs and the excitability of single cells mimicking the effects of ACh indeed modulated the evoked network reverberation similar to experimental observations. Thus, ACh modulates network dynamics in a biphasic fashion, probably by inhibiting excitatory synaptic transmission and facilitating neuronal excitability through muscarinic signaling pathways.

Haemodynamic characterisation of different endotypes in coronary artery vasospasm in reaction to acetylcholine.

Vasoreactivity testing with high-dose acetylcholine is considered vasospasm provocation and low-dose as endothelial function testing. To assess the changes in reaction to low- and high-dose acetylcholine in the endotypes of CAS as defined by the Coronary Vasomotor Disorders International Study Group (COVADIS) working group. Changes in coronary epicardial diameter, coronary blood flow (CBF) and vascular resistance were determined at low-dose acetylcholine. A total of 88 ANOCA patients were included in this analysis. In the negative group (n=14) incremental infusion of acetylcholine produced a progressive increase in CBF (p=0.008). In reaction to low-dose acetylcholine, the epicardial vasospasm group (n=30) is characterised by epicardial vasoconstriction that is significantly more severe compared to the microvascular vasospasm group (p=0.004)(n=23). The equivocal group (n=21) is characterised by an increase in CBF and reduction in vascular resistance that are both significantly different compared to the epicardial vasospasm group (p=0.036 and p=0.007, respectively). High-dose acetylcholine decreased epicardial diameter and CBF significantly in the epicardial vasospasm, microvascular vasospasm and in the equivocal group (all p<0.05. Vascular resistance increased significantly in the epicardial vasospasm group (p<0.001) and equivocal group (p=0.009). In reaction to low-dose acetylcholine the negative and equivocal endotype has haemodynamic changes that suggest intact endothelium. In reaction to high-dose acetylcholine the epicardial vasospasm, microvascular vasospasm and equivocal endotype have hemodynamic changes that suggest VSMC-hyperreactivity. These results suggest that the equivocal endotype is a positive test comparable to microvascular vasospasm in the presence of normal endothelial function.

Presence of Coronary Endothelial Dysfunction, Coronary Vasospasm, and Adenosine-Mediated Vasodilatory Disorders in Patients With Ischemia and Nonobstructive Coronary Arteries.

Coronary function testing in patients with ischemia and nonobstructive coronary arteries (INOCA) commonly includes assessment of adenosine-mediated vasodilation and acetylcholine spasm provocation. The purpose of this study was to evaluate the diagnostic value of additional endothelial function testing for the diagnosis of vasomotor dysfunction in patients with INOCA. In this retrospective cohort study, we included patients with INOCA who underwent clinically indicated comprehensive coronary function testing. Endothelial dysfunction was defined as a <50% increase in coronary blood flow, determined by Doppler flow, and/or epicardial vasoconstriction compared to baseline, in response to low-dose acetylcholine. Coronary artery spasm (CAS) was defined as vasospastic angina or microvascular angina in response to coronary high-dose acetylcholine. An impaired adenosine-mediated vasodilation was defined as a coronary flow reserve <2.5 and/or hyperemic microvascular resistance ≥2.5. Among all 110 patients, 79% had endothelial dysfunction, 62% had CAS, and 29% had an impaired adenosine-mediated vasodilation. Endothelial dysfunction was present in 80% of patients who tested positively for CAS and/or an impaired adenosine-mediated vasodilation. Endothelial function testing increases the diagnostic yield of coronary function testing that only incorporates adenosine testing and spasm provocation by 17% to 92%. Of patients with normal adenosine-mediated vasodilation and no inducible CAS, 68% had endothelial dysfunction. Concomitant endothelial dysfunction was prevalent in the vast majority of patients with INOCA with inducible CAS and/or an impaired adenosine-mediated vasodilation. In patients with INOCA without inducible CAS and normal adenosine-mediated vasodilation, two-thirds had endothelial dysfunction. These results indicate the relevance to perform endothelial function testing in patients with INOCA in view of its therapeutic implication.

Sex-differences in cholinergic, nicotinic, and β-adrenergic cutaneous vasodilation: Roles of nitric oxide synthase, cyclooxygenase, and K+ channels

Previous studies indicate that sex-related differences exist in the regulation of cutaneous vasodilation, however, the mechanisms remain unresolved. We assessed if sex-differences in young adults exist for cholinergic, nicotinic, and β-adrenergic cutaneous vasodilation with a focus on nitric oxide synthase (NOS), cyclooxygenase (COX), and K+ channel mechanisms. In twelve young men and thirteen young women, four intradermal forearm skin sites were perfused with the following: 1) lactated Ringer's solution (control), 2) 10 mM Nω-nitro-l-arginine, a non-selective NOS inhibitor, 3) 10 mM ketorolac, a non-selective COX inhibitor, or 4) 50 mM BaCl2, a nonspecific K+ channel blocker. At all four sites, cutaneous vasodilation was induced by 1) 10 mM nicotine, a nicotinic receptor agonist, 2) 100 μM isoproterenol, a nonselective β-adrenergic receptor agonist, and 3) 2 mM and 2000 mM acetylcholine, an acetylcholine receptor agonist. Nicotine and isoproterenol were administered for 3 min, whereas each acetylcholine dose was administered for 25 min. Regardless of treatment site, cutaneous vasodilation in response to nicotine and a high dose of acetylcholine (2000 mM) were lower in women than men. By contrast, isoproterenol induced cutaneous vasodilation was greater in women vs. men. Irrespective of sex, NOS inhibition or K+ channel blockade attenuated isoproterenol-mediated cutaneous vasodilation, whereas K+ channel blockade decreased nicotine-induced cutaneous vasodilation. Taken together, our findings indicate that while the mechanisms underlying cutaneous vasodilation are comparable between young men and women, sex-related differences in the magnitude of cutaneous vasodilation do exist and this response differs as a function of the receptor agonist.

Autonomic Blockade Reverses Endothelial Dysfunction in Obesity-Associated Hypertension.

Impaired nitric oxide (NO) vasodilation (endothelial dysfunction) is associated with obesity and thought to be a factor in the development of hypertension. We previously found that NO synthesis inhibition had similar pressor effects in obese hypertensives compared with healthy control during autonomic blockade, suggesting that impaired NO vasodilation is secondary to sympathetic activation. We tested this hypothesis by determining the effect of autonomic blockade (trimethaphan 4 mg/min IV) on NO-mediated vasodilation (increase in forearm blood flow to intrabrachial acetylcholine) compared with endothelial-independent vasodilation (intrabrachial sodium nitroprusside) in obese hypertensive subjects (30<body mass index<40 kg/m(2)). Acetylcholine and sodium nitroprusside were given at equipotent doses (10, 30, and 50 μg/min and 1, 2, and 3 μg/min, respectively) to 14 obese subjects (49±3.6 years, 34±1 kg/m(2), 165/94±7/6 mm Hg), on separate occasions 1 month apart, randomly assigned. Autonomic blockade increased basal forearm blood flow (from 3.9±0.7 to 5.2±1.2 mL/100 mL per minute, P=0.078). As expected, NO-mediated vasodilation was blunted on the intact day compared with NO-independent vasodilation; forearm blood flow increased from 3.6±0.6 to 10.1±1.1 with the highest dose of nitroprusside, but only from 3.7±0.4 to 7.2±0.8 mL/100 mL per minute with the highest dose of acetylcholine, P<0.05. In contrast, forearm blood flow responses to acetylcholine were restored by autonomic blockade and were no longer different to nitroprusside (from 6.2±1.1 to 11.4±1.6 mL/100 mL per minute and from 5.2±0.9 to 12.5±0.9, respectively, P=0.58). Our results support the concept that sympathetic activation contributes to the impairment in NO-mediated vasodilation seen in obesity-associated hypertension and provides further rationale to explore it as a therapeutic target.

The impact of high-dose acetylcholine on bovine corneal epithelium.

Ion channels are formed when acetylcholine (ACh) combines with specific receptors on the postsynaptic membrane. We are testing whether this occurs in vitro in corneal epithelial cells when exposed to ACh, using intracellular ascorbic acid (AA) as a tracer. The corneal epithelium was separated from Bowman's membrane as an intact sheet with thermolysin (TL). These cells were incubated in a medium containing phospholine iodide (Phi) to block acetylcholinesterase (AChE) activity, and then, five different groups of specimens were examined: the basic level of ACh and AA in the cells was tested in groups I and II, respectively. In groups III-V, the culture medium was supplemented with AA, ACh and ACh+AA, respectively, and following 2 hr of incubation the cells were tested for AA. AA was determined by high-performance liquid chromatography (HPLC) with UV detection, and ACh by LC-MS/MS. The ACh concentration in the corneal epithelial cells was 23.7 ng/mg wet weight. AA values were as follows: preexposure 0.17 mg/g. After exposure to AA, ACh and ACh+AA, the values were 0.30, 0.12 and 0.21 mg/g, respectively. The AA concentration mechanism of the corneal epithelium was intact despite exposure to Phi and ACh. The main observation is that the AA content of the corneal epithelium drops in response to ACh exposure. Various explanations are discussed, in particular the possibility that ACh exposure may cause cell membrane leakage through pores (nicotinic receptors). This would be similar to the moderate membrane leakage (depolarization) in neural synapses in response to ACh release. Interesting in vivo consequences may occur when considering that AChE is extremely UV sensitive, as this substance could be a target at which actinic radiation may influence the ACh-AChE complex in this epithelium.

Endothelial-derived hyperpolarization contributes to acetylcholine-mediated vasodilation in human skin in a dose-dependent manner

Cutaneous acetylcholine (ACh)-mediated dilation is commonly used to assess microvascular function, but the mechanisms of dilation are poorly understood. Depending on dose and method of administration, nitric oxide (NO) and prostanoids are involved to varying extents and the roles of endothelial-derived hyperpolarizing factors (EDHFs) are unclear. In the present study, five incremental doses of ACh (0.01-100 mM) were delivered either as a 1-min bolus (protocol 1, n = 12) or as a ≥20-min continuous infusion (protocol 2, n = 10) via microdialysis fibers infused with 1) lactated Ringer, 2) tetraethylammonium (TEA) [a calcium-activated potassium channel (KCa) and EDHF inhibitor], 3) L-NNA+ketorolac [NO synthase (NOS) and cyclooxygenase (COX) inhibitors], and 4) TEA+L-NNA+Ketorolac. The hyperemic response was characterized as peak and area under the curve (AUC) cutaneous vascular conductance (CVC) for bolus infusions or plateau CVC for continuous infusions, and reported as %maximal CVC. In protocol 1, TEA, alone and combined with NOS+COX inhibition, attenuated peak CVC (100 mM Ringer 59 ± 6% vs. TEA 43 ± 5%, P < 0.05; L-NNA+ketorolac 35 ± 4% vs. TEA+L-NNA+ketorolac 25 ± 4%, P < 0.05) and AUC (Ringer 25,414 ± 3,528 vs. TEA 21,403 ± 3,416%·s, P < 0.05; L-NNA+ketorolac 25,628 ± 3,828%(.)s vs. TEA+L-NNA+ketorolac 20,772 ± 3,711%·s, P < 0.05), although these effects were only significant at the highest dose of ACh. At lower doses, TEA lengthened the total time of the hyperemic response (10 mM Ringer 609 ± 78 s vs. TEA 860 ± 67 s, P < 0.05). In protocol 2, TEA alone did not affect plateau CVC, but attenuated plateau in combination with NOS+COX inhibition (100 mM 50.4 ± 6.6% vs. 30.9 ± 6.3%, P < 0.05). Therefore, EDHFs contribute to cutaneous ACh-mediated dilation, but their relative contribution is altered by the dose and infusion procedure.

Suspected Microvascular Coronary Spasm Diagnosed by Spasm Provocation Test with High Dose of Acetylcholine

A 71-year-old Japanese man was admitted to our hospital complaining of chest oppression on exercise, such as cycling uphill, wh ich had continued for 9 years. We diagnosed him as having microvascular spasm according to the results of coronary angiography with an acetylcholine provocation test with a high dose (150 μg) of acetylcholine administrate d at a time during 20 seconds.Electrocardiographic changes during the spasm provocation test resembled that during exercise tests performed upon admission and 9 years previously. He was treated with a calcium channel blocker and partial symptom relief was obtained.

Comparison of low and high dose intracoronary adenosine and acetylcholine in women undergoing coronary reactivity testing: Results from the NHLBI-sponsored Women's Ischemia Syndrome Evaluation (WISE)

Women with signs and symptoms of myocardial ischemia who are referred for invasive coronary angiography often have no evidence of obstructive coronary artery disease (CAD) [1]. Forty-five to sixty percent of these patients have abnormal coronary vasomotion due to endothelial or non-endothelial dependent macro- or microvascular coronary dysfunction [1–3]. Abnormal coronary vasomotion is linked with adverse cardiovascular outcomes, suggesting that additional coronary reactivity testing (CRT), beyond standard angiography, is needed in clinical practice to appropriately risk stratify these patients [2–6]. CRT with intra-coronary adenosine and acetylcholine (ACh) is used to identify vascular dysfunction, yet ascending dosing of these agents is time-consuming. We aimed to determine if 1) coronary flow reserve (CFR) was different after low or high dose adenosine, and 2) change in coronary artery cross sectional area (CSA) and coronary blood flow (CBF) was different after low or high dose ACh. Eighty -six women with no obstructive CAD, who were enrolled in the WISE Coronary Vascular Dysfunction Study (WISE-CVD, www.clinicaltrials.gov Identifier {type:clinical-trial,attrs:{text:NCT00832702,term_id:NCT00832702}}NCT00832702), underwent CRT using a Doppler guide wire (Volcano ®FloWire 300cm) in a proximal left coronary. CFR was recorded in response to low dose (18μg) and high dose (36μg) intra-coronary adenosine. Change in CSA and CBF were determined after intra-coronary low dose ACh (10−6 mol/L infusion for total of 0.364μg) and high dose ACh (10−4 mol/L infusion for total of 36.4μg). The protocol was approved by the local institutional review board, and written informed consent was obtained from each participant. On average, the women included in this analysis were 54 (±12.2) years old; 36.7% had hypertension; 13.9% had dyslipidemia; 45.6% had a history of tobacco use; 70.9% were Caucasian; and 10% had diabetes. The average CFR after low dose adenosine was 2.6 ± 0.6, and the average CFR after high dose adenosine was 2.5 ± 0.6. The CFR obtained after low dose adenosine was not significantly different from the CFR obtained after high dose adenosine in the same subject (mean of paired difference 0.06 ± 0.3, p-value = 0.11). The changes in CBF and CSA after low and high dose ACh are shown in the Table. On average CBF increased 15% ± 32% with low dose ACh, but increased 92% ± 102% with high dose ACh. In the same subject the increase in CBF was 77% higher after high dose ACh compared with after low dose ACh (p-value < 0.0001). The CSA of the proximal left anterior descending artery (LAD) and the LAD 5 mm distal to the flow wire remained essentially the same after low dose ACh, but decreased slightly after high dose ACh. On average, in the same subject the change in CSA of the proximal LAD was 4% smaller after high dose ACh compared with after low dose ACh (p-value = 0.05). Table Comparison of cross-sectional area and coronary blood flow change with use of low vs. high dose acetylcholine. Our data indicate that escalating dosing of adenosine to detect non-endothelial dependent microvascular coronary dysfunction may not be needed. These findings are consistent with previous work that suggests that intracoronary doses of adenosine as low as 16 μg achieve maximal hyperemia [7]. For the purpose of our testing, we have used relatively lower doses of adenosine (18 μg and 36 μg) that test its non-endothelial dependent effects on the coronary circulation. At higher doses of adenosine, it becomes an epicardial coronary vasodilator, and would test hyperemic CBF due to both endothelial and non-endothelial dependent effects [8]. Our ACh dosing was selected based on the pioneering work by Ludmer et al. that demonstrated that patients with atherosclerosis and suspected endothelial dysfunction have paradoxical vasoconstriction in response to intra-coronary infusions of ACh at doses of 10−6 mol/L or less [9]. Other studies have demonstrated a very high incidence of abnormal coronary spasm in response to intracoronary ACh in patients with suspected endothelial dysfunction when using even higher doses of intracoronary ACh (100–200 μg) [1,10]. The results of our analysis also suggest that higher dose ACh (36.4μg [10−4 mol/L]) has greater diagnostic utility than lower dose ACh (0.364μg [10−6 mol/L]), as higher dose ACh resulted in a significantly greater increase in CBF compared with low dose ACh, and on average only high dose ACh demonstrated an inappropriate decrease in CSA in this group of women with suspected endothelial dysfunction. Among women with evidence of ischemia but no obstructive CAD, low and high dose adenosine produced similar augmentation in coronary flow velocity, but low and high dose ACh produced different results. For clinically indicated CRT, low dose adenosine but high dose ACh appear to be indicated.

Eleutheroside B or E enhances learning and memory in experimentally aged rats.

Eleutheroside B or E, the main component of Acanthopanax, can relieve fatigue, enhance memory, and improve human cognition. Numerous studies have confirmed that high doses of acetylcholine significantly attenuate clinical symptoms and delay the progression of Alzheimer's disease. The present study replicated a rat model of aging induced by injecting quinolinic acid into the hippocampal CA1 region. These rats were intraperitoneally injected with low, medium and high doses of eleutheroside B or E (50, 100, 200 mg/kg), and rats injected with Huperzine A or PBS were used as controls. At 4 weeks after administration, behavioral tests showed that the escape latencies and errors in searching for the platform in a Morris water maze were dose-dependently reduced in rats treated with medium and high-dose eleutheroside B or E. Hematoxylin-eosin staining showed that the number of surviving hippocampal neurons was greater and pathological injury was milder in three eleutheroside B or E groups compared with model group. Hippocampal homogenates showed enhanced cholinesterase activity, and dose-dependent increases in acetylcholine content and decreases in choline content following eleutheroside B or E treatment, similar to those seen in the Huperzine A group. These findings indicate that eleutheroside B or E improves learning and memory in aged rats. These effects of eleutheroside B or E may be mediated by activation of cholinesterase or enhanced reuse of choline to accelerate the synthesis of acetylcholine in hippocampal neurons.

EDHFs contribute to ACh‐mediated vasodilation in human skin in a dose‐dependent manner

The human cutaneous microcirculation has been shown to be reflective of globalized microvascular function, but the role of EDHFs in acetylcholine (ACh) mediated vasodilation has yet to be explored. In humans and animals, EDHFs appear to play only a modest role in ACh vasodilation, with a greater contribution at higher doses of ACh and under disease or NO‐deficient conditions. Five bolus doses of ACh (0.01, 0.1, 1, 10, and 100mM) were delivered via microdialysis fibers continuously infused with 1) Lactated Ringer's, 2) tetraethylammonium (TEA; calcium‐sensitive potassium channel &amp; EDHF inhibitor), 3) L‐NNA + Ketorolac (to inhibit NO + COX), and 4) TEA + L‐NNA + Keto. The hyperemic response was characterized as area under the curve (AUC) and reported as % max cutaneous vascular conductance. In our preliminary findings (n=5), TEA had no effect on AUC compared to control at any of the lower doses of ACh, but did attenuate AUC to 100mM ACh (Control: 31,621±5,947% vs. TEA: 20,809±6,686%). We found similar results with combined NO + COX inhibition. TEA + L‐NNA + Keto did not attenuate AUC compared to L‐NNA + Keto until the 100mM dose of ACh (L‐NNA + Keto: 16,308±2831% vs. TEA + L‐NNA + Keto: 8,491±1,853%). Therefore, EDHFs appear to contribute to ACh vasodilation in the skin in a dose‐dependent manner, similarly to other vascular beds, confirming the skin to be a good model of overall microvascular function.Supported by NIH Grant HL081671

A comparison of in vitro relaxant responses to ipratropium bromide, β-adrenoceptor agonists and theophylline in feline bronchial smooth muscle

This study compares the potency and efficacy of different relaxant drugs including anticholinergic, β-adrenergic and methylxanthine agents on acetylcholine-contracted feline bronchi, and investigates the influence of the initial muscarinic-induced tone on bronchodilator response. Feline bronchi were removed from euthanased client-owned cats and were contracted with acetylcholine to cause either 40% or 80% of the acetylcholine maximal contraction. The efficacy and potency of bronchodilating drugs were obtained from cumulative dose-response curves with efficacy (E(max)) as the maximal relaxant response and potency (-logEC(50)) as the logarithm of the concentration of drug inducing 50% of maximal relaxation. Under low contractile tone (40%), all bronchodilators relaxed feline bronchi in a concentration-dependent manner with the following rank order of potency: formoterol>ipratropium bromide>fenoterol>isoprenaline>salbutamol≥salmeterol>theophylline. E(max) values ranged from 80% to 100% depending on the tested drug. Constriction of feline bronchi with high-dose acetylcholine (80%) caused a rightward and downward shift of the β(2)-mimetic dose-response curves. Significant decreases in -logEC(50) and E(max) values were reported for salbutamol, formoterol and salmeterol. This study provides evidence that existing classes of bronchodilators produce effective relaxation of acetylcholine-contracted feline bronchi and that airway responsiveness to β(2)-stimulants is dependent on the magnitude of the initial muscarinic-induced tone. The clinical relevance of these in vitro findings has yet to be explored in clinical trials.

Endothelium-dependent coronary vasomotor response and neointimal coverage of zotarolimus-eluting stents 3 months after implantation

BackgroundZotarolimus-eluting stents (ZES) have a higher rate of neointimal coverage than the first-generation drug-eluting stents on optical coherence tomography (OCT).ObjectiveTo determine whether neointimal coverage of stent struts detected by OCT...

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We thank van Eijk et al. for their interest in our work and appreciate their congratulation for the first investigation of vascular reactivity in septic patients [1]. Despite some evidence in favour of endothelial dysfunction during experimental sepsis, we showed that in septic shock, acetylcholine-induced vasodilation in the forearm vasculature is well preserved, albeit in a small number of patients [1]. Van Eijk et al. remark that prior studies demonstrate that in septic animals or during experimental endotoxaemia, endothelial function is impaired as indicated by decreased vasodilation in response to acetylcholine in several organ systems [2-4] and feel that this contrasts with our observations in septic patients [1]. We would like to suggest that observations made in animal models of ‘sepsis’ or during experimental endotoxaemia in human volunteers, may not reflect the complex changes seen in patients with sepsis. In fact, this was the very reason why we decided to study vascular reactivity in septic patients so as to overcome the deficiencies of several sepsis models. By studying patients with similar cardiovascular instability we thought to be able to minimize inter-individual variability in vascular function as much as possible. In fact, in patients with sepsis, acetylcholine induced endothelium-dependent relaxation was found to be intact in the skin compared to ICU patients without sepsis as well as to healthy volunteers [5]. Accordingly, preserved endothelium-dependent vasodilation in the forearm vasculature of our patients with septic shock parallels previous findings in skin microcirculation during sepsis. Of note, however, we did not study endothelial dysfunction during an early stage of sepsis. Van Eijk also suggests that lower vascular resistance in sepsis may be the reason for the decreased response to acetylcholine observed. If this were the case, one would expect impaired vasodilation to acetylcholine due to a decreased vasodilator reserve. In contrast, at least in the forearm vasculature, we observed increased blood flow in patients with septic shock but at the same time a similar response to the exogenous nitric oxide-donator nitroprusside [1]. Thus, vasodilator reserve appears to be maintained in forearm vasculature during human septic shock. Finally, van Eijk comments that a full dose-response relationship of acetylcholine cannot be characterized by the two doses administered (a point we have already addressed in our publication) and that the dose of L-N monomethylarginine given is rather low. We fully agree, but can report that blood flow measurements were indeed performed in the contralateral arm concurrently. These indicated that higher doses of acetylcholine or L-N monomethylarginine resulted in systemic effects in both patients and volunteers. Since we did not want to confuse measurements of regional effects in the forearm by systemic baroreflex mediated effects we limited interpretation and data to the lower dosages reported. Van Eijk speculates that endothelial dysfunction may have been demonstrated following administration of other endothelium-dependent vasodilators, like substance P or bradykinin. Since we studied endothelium-dependent and endothelium-independent vasodilation as well as vasoconstriction using three different vasoconstrictors in the same subjects/patients we had to limit the duration of the study to maintain stable baseline conditions and dispensed the evaluation of additional vasoactive drugs. Obviously, what effect other agents might have had is an area for speculation. In summary, we challenge prior work in animal models of sepsis and in human volunteers following lipopolysaccharide administration by demonstrating in ‘real sepsis’ that endothelial function is preserved in the forearm vasculature of septic patients. While endothelial dysfunction may still occur during earlier stages of sepsis and further studies are needed to address the issue there is nothing that beats data in real, septic humans.

No NOS Needed for This NO

Nitric oxide is a potent signaling molecule in various physiological systems. Normally, its abundance is thought to be controlled through control of NO synthase (NOS) enzymes that produce NO. NO appears to serve several functions in the pancreas, but the literature contains conflicting reports on whether NOS is present in pancreatic acinar cells and whether NO generation is sensitive to changes in intracellular concentrations of free Ca 2+ . Chvanov et al. used fluorescent NO-sensitive dyes administered by patch clamping in cultured mouse pancreatic cells to explore NO signaling. They propose that the aforementioned confusion may arise in part because, to their surprise, NOS isn't the key source of NO signals in these cells. The authors could detect increased Ca 2+ -dependent production of NO in response to acetylcholine. Although pharmacological inhibitors of NOS did affect the later phase of this response (after about 600 seconds), the initial response (about 200 seconds after stimulation) was not affected. Chvanov et al. propose that this rapidly formed NO may come from previously bound cellular NO in the form of S -nitrosothiols. In keeping with this idea, treatment of cells with an NO donor (to increase intracellular S -nitrosothiol content) increased the generation of NO in response to high doses of acetylcholine. The authors estimate that release from S -nitrosothiols in these cells may provide about a 100-nM increase in NO concentration. Further studies with pharmacological inhibitors showed that the Ca 2+ dependence of the NO response was not mediated by calmodulin or protein kinase C, but rather was sensitive to inhibitors of the Ca 2+ -sensitive protease calpain. The authors therefore speculate that this novel form of NO generation might result from partial proteolysis of S -nitrosylated proteins that reduces the stability of the S-NO bond and allows denitrosylation of thiol groups. Similar results in other mouse and bovine cell types indicated that the phenomenon is not restricted to pancreatic cells. M. Chvanov, O. V. Gerasimenko, O. H. Petersen, A. V. Tepikin, Calcium-dependent release of NO from intracellular S -nitrosothiols. EMBO J. 25 , 3024-3032 (2006). [PubMed]

The postprandial state does not impair endothelial function in women with Type 2 diabetes irrespective of glycaemic control

The postprandial state has been shown to be associated with endothelial dysfunction, a predictor of cardiovascular morbidity. In type 2 diabetes, postprandial metabolic excursions are prolonged and exaggerated, but less pronounced if glycaemic control is optimised. We investigated the impact of improved glycaemic control on endothelial function in the postprandial state. We studied 19 postmenopausal women with type 2 diabetes and ten non-diabetic subjects. Participants with diabetes were re-studied 3 months after intensive glucose regulation. We measured forearm blood flow by strain gauge plethysmography during rest, during acetylcholine infusion and post ischaemia in the fasting state, and again 3 hours after a mixed meal (660 kcal, 55% fat). Endothelium-dependent vasodilation was impaired in the diabetic group (p<0.005) and improved following an HbA1c reduction of 0.96% (p<0.05 for high-dose acetylcholine infusion). Postprandial metabolic excursions were higher in the diabetic group (p<0.001, p<0.01 and p<0.05 for glucose, insulin and triglycerides respectively). Resting forearm blood flow increased in all groups after the meal (p<0.005). There was no difference in fasting and postprandial endothelium-dependent vasodilation before and after improved glucose regulation in either group. The postprandial state does not impair endothelial function in non-diabetic women and does not make pre-existing endothelial dysfunction worse in women with type 2 diabetes, irrespective of glycaemic control.

Involvement of nitric oxide in the regulation of regional hemodynamics in streptozotocin-diabetic rats

In experimental and human diabetes mellitus, evidence for an impaired function of the vascular endothelium has been found and has been suggested to contribute to the development of vascular complications in this disease. The aim of the study was to evaluate possible regional hemodynamic in vivo differences between healthy and diabetic rats which would involve nitric oxide (NO). Central hemodynamics and regional blood flow (RBF) were studied using radioactive microspheres in early streptozotocin (STZ)-diabetic rats and compared to findings in healthy control animals. This method provides a possibility to study the total blood flow and vascular resistance (VR) in several different organs simultaneously. L-NAME iv induced widespread vasoconstriction to a similar extent in both groups. In the masseter muscle of both groups, acetylcholine 2 g/kg per min, induced a RBF increase, which was abolished by pretreatment with L-NAME, suggesting NO as a mediator of vasodilation. In the heart muscle of both groups, acetylcholine alone was without effect while the combined infusion of acetylcholine and L-arginine induced an L-NAME-sensitive increase in RBF. The vasodilation induced by high-dose acetylcholine (10 g/kg per min) in the kidney was more pronounced in the STZ-diabetic rats. The results indicate no reduction in basal vasodilating NO-tone in the circulation of early diabetic rats. The sensitivity to vasodilating effects of acetylcholine at the level of small resistance arterioles vary between tissues but was not impaired in the diabetic rats. In the heart muscle the availability of L-arginine was found to limit the vasodilatory effect of acetylcholine in both healthy and diabetic rats. In conclusion, the results indicate a normal action of NO in the investigated tissues of the early STZ-diabetic rat.

Slow depletion of endoplasmic reticulum Ca(2+) stores and block of store-operated Ca(2+) channels by 2-aminoethoxydiphenyl borate in mouse pancreatic acinar cells.

The compound 2-aminoethoxydiphenyl borate (2-APB) has been used as either a specific membrane-permeable inhibitor for InsP(3) receptors or a store-operated Ca(2+) channel blocker in some cells. In this study, we have investigated actions of 2-APB on Ca(2+) signalling in mouse pancreatic acinar cells by measuring Ca(2+) concentration in the cytosol ([Ca(2+)]c) and in the endoplasmic reticulum (ER). Although 2-APB (50 microM) inhibited or modulated [Ca(2+)]c oscillations generated by a low dose of ACh, it did not block InsP(3)-mediated Ca(2+) release from the ER elicited by high doses of acetylcholine (ACh). 2-APB alone more than 70 microM tended to increase [Ca(2+)]c. When we directly measured Ca(2+) concentration in the lumen of the ER with a low affinity Ca(2+) dye, Mag-fluo-4, 2-APB itself slowly lowered ER Ca(2+) concentration and ACh could further release Ca(2+) from the ER in the presence of 2-APB, suggesting its lack of potency to block InsP(3) receptors. When store-operated Ca(2+) entry was evoked by addition of external Ca(2+) (5 mM) after depletion of Ca(2+) stores, 2-APB (50 microM) substantially blocked the Ca(2+) influx in a reversible manner. We conclude that (a) 2-APB is a good blocker for store-operated Ca(2+) channels, (b) 2-APB could not effectively block InsP(3) receptors, and (c) low doses of 2-APB lower Ca(2+) concentration in the lumen of the ER without a significant elevation of [Ca(2+)]c.