Administration Of Methacholine Research Articles

Standard technical specifications for methacholine chloride (Methacholine) bronchial challenge test (2023)

Standard technical specifications for methacholine chloride (Methacholine) bronchial challenge test (2023)

Muc5b Contributes to Mucus Abnormality in Rat Models of Cystic Fibrosis.

Cystic fibrosis (CF) airway disease is characterized by excessive and accumulative mucus in the airways. Mucociliary clearance becomes defective as mucus secretions become hyperconcentrated and viscosity increases. The CFTR-knockout (KO) rat has been previously shown to progressively develop delayed mucociliary transport, secondary to increased viscoelasticity of airway secretions. The humanized-G551D CFTR rat model has demonstrated that abnormal mucociliary clearance and hyperviscosity is reversed by ivacaftor treatment. In this study, we sought to identify the components of mucus that changes as the rat ages to contribute to these abnormalities. We found that Muc5b concentrations, and to a lesser extent Muc5ac, in the airway were increased in the KO rat compared to WT, and that Muc5b concentration was directly related to the viscosity of the mucus. Additionally, we found that methacholine administration to the airway exacerbates these characteristics of disease in the KO, but not WT rat trachea. Lastly we determined that at 6 months of age, CF rats had mucus that was adherent to the airway epithelium, a process that is reversed by ivacaftor therapy in the hG551D rat. Overall, these data indicate that accumulation of Muc5b initiates the muco-obstructive process in the CF lung prior to infection.

In vivo paracrine effects of ATP-induced urothelial acetylcholine in the rat urinary bladder.

Mechanical stretch of the urothelium induces the release of ATP that activates bladder afferent nerves. In the rat urinary bladder, ATP is also a contractile co-transmitter in the parasympathetic innervation. In isolated preparations, ATP evokes a urothelial release of acetylcholine that substantially contributes to ATP-evoked contractile responses. Currently we aimed to further examine the interactions of ATP and acetylcholine in the rat urinary bladder in two in vivo models. In the whole bladder preparation, atropine reduced ATP-evoked responses by about 50% in intact but denervated bladders, while atropine had no effect after denudation of the urothelium. In a split bladder preparation, reflex-evoked responses of the contralateral half were studied by applying stimuli (agonists or stretch) to the ipsilateral half. Topical administration of ATP and methacholine as well as of stretch induced contralateral reflex-evoked contractions. While topical administration of atropine ipsilaterally reduced the ATP- and stretch-induced contralateral contractions by 27 and 39%, respectively, the P2X purinoceptor antagonist PPADS reduced them by 74 and 84%. In contrary, the muscarinic M2-(M4)-selective receptor antagonist methoctramine increased the responses by 38% (ATP) and 75% (stretch). Pirenzepine (M1-selective antagonist) had no effect on the reflex. In vitro, in the absence of the reflex, methoctramine did not affect the ATP-induced responses. It is concluded that urothelial ATP potently induces the micturition reflex and stimulates urothelial release of acetylcholine. Acetylcholine subsequently acts on afferents and on the detrusor muscle. While muscarinic M2 and/or M4 receptors in the sensory innervation exert inhibitory modulation, muscarinic M3 receptors cause excitation.

A Novel Approach for Investigating Upper Airway Hyperresponsiveness Using Micro-CT in Eosinophilic Upper Airway Inflammation such as Allergic Rhinitis Model.

Airway hyperresponsiveness (AHR) has been proposed as a feature of pathogenesis of eosinophilic upper airway inflammation such as allergic rhinitis (AR). The measurement system for upper AHR (UAHR) in rodents is poorly developed, although measurements of nasal resistance have been reported. Here we assessed UAHR by direct measurement of swelling of the nasal mucosa induced by intranasal methacholine (MCh) using micro-computed tomography (micro-CT). Micro-CT analysis was performed in both naïve and ovalbumin-induced AR mice following intranasal administration of MCh. The nasal cavity was segmented into two-dimensional horizontal and axial planes, and the data for nasal mucosa were acquired for the region of interest threshold. Then, a ratio between the nasal mucosa area and nasal cavity area was calculated as nasal mucosa index. Using our novel method, nasal cavity structure was clearly identified on micro-CT, and dose-dependent increased swelling of the nasal mucosa was observed upon MCh treatment. Moreover, the nasal mucosa index was significantly increased in AR mice compared to controls following MCh treatment, while ovalbumin administration did not affect swelling of the nasal mucosa in either group. This UAHR following MCh treatment was completely reversed by pretreatment with glucocorticoids. This novel approach using micro-CT for investigating UAHR reflects a precise assessment system for swelling of the nasal mucosa following MCh treatment; it not only sheds light on the mechanism of AR but also contributes to the development of new therapeutic drugs in AR patients.

Bacterial respiratory tract inflammation in neonatal rat model is attenuated by benzofuran through inhibition of GATA3

The current study was aimed to investigate the effect of benzofuran on asthma neonatal rat model. Twenty-five neonatal rats were assigned into five groups; Normal control, untreated, 1 mg/kg, 8 mg/kg and 10 mg/kg treatment groups. Methacholine was administered orally to the rats of untreated and treatment groups. Animals in the normal control group were given PBS as a vehicle. FlexiVent system employing a computer-controlled mouse ventilator along with respiratory mechanics was used for the analysis of airway resistance in the rats. Cytokine level and IFN-γ in the rat serum samples was performed by ELISA in accordance with the instructions of manufacturer. Methacholine administration into the rats caused a marked increase in lung airway resistance. However, treatment with 8 and 10 mg/kg doses of benzofuran led to marked decrease in the airway resistance. Benzofuran treatment prevented accumulation of macrophages and inflammatory cells in the lung airways. Inhibition of inflammation in methacholine administered rats by benzofuran was also confirmed by hematoxylin & eosin-staining. Examination of the rat serum showed significantly higher level of Th2 cytokines (IL-4, -5 and -13) in the untreated rats. However, treatment of methacholine administered rats with benzofuran significantly inhibited Th2 cytokine expression. The level of IFN-γ was increased by benzofuran treatment in methacholine administered rats. In methacholine administered rats the level of IgE was markedly higher however treatment of asthma rats with benzofuran inhibited up-regulation of IgE significantly. The expression of T-bet is decreased and that of GATA-3 is increased by methacholine administration in the rat lungs. Benzofuran treatment of methacholine administered rats prevented reduction in T-bet and up-regulation of GATA-3 expression in the rat lungs. The effect of benzofuran was significant at the doses of 8 and 10 mg/kg and non-significant at 1 mg/kg. These finding suggest that benzofuran inhibits expression of dominant T-helper 2 cytokines through targeting GATA-binding protein 3 transcription factor. Thus benzofuran can be of therapeutic importance for the treatment of asthma.

The spatial pattern of methacholine bronchoconstriction recurs when supine independently of posture during provocation but does not recur between postures.

The location of lung regions with compromised ventilation (often called ventilation defects) during a bronchoconstriction event may be influenced by posture. We aimed to determine the effect of prone versus supine posture on the spatial pattern of methacholine-induced bronchoconstriction in six healthy adults (ages 21-41, 3 women) using specific ventilation imaging. Three postural conditions were chosen to assign the effect of posture to the drug administration and/or imaging phase of the experiment: supine methacholine administration followed by supine imaging, prone methacholine administration followed by supine imaging, and prone methacholine administration followed by prone imaging. The two conditions in which imaging was performed supine had similar spatial patterns of bronchoconstriction despite a change in posture during methacholine administration; the odds ratio for recurrent constriction was mean (SD) = 7.4 (3.9). Conversely, dissimilar spatial patterns of bronchoconstriction emerged when posture during imaging was changed; the odds ratio for recurrent constriction between the prone methacholine/supine imaging condition and the prone methacholine/prone imaging condition was 1.2 (0.9). Logistic regression showed that height above the dependent lung border was a significant negative predictor of constriction in the two supine imaging conditions (P < 0.001 for each) but not in the prone imaging condition (P = 0.20). These results show that the spatial pattern of methacholine bronchoconstriction is recurrent in the supine posture, regardless of whether methacholine is given prone or supine but that prone posture during imaging eliminates that recurrent pattern and reduces its dependence on gravitational height.NEW & NOTEWORTHY The spatial pattern of methacholine bronchoconstriction in the supine posture is recurrent and skewed toward the dependent lung, regardless of whether inhaled methacholine is administered while supine or while prone. However, both the recurrent pattern and the gravitational skew are eliminated if imaging is performed prone. These results suggest that gravitational influence on regional lung inflation and airway topography at the time of measurement play a role in determining regional bronchoconstriction in the healthy lung.

Type 2 diabetes specifically attenuates purinergic skin vasodilatation without affecting muscarinic and nicotinic skin vasodilatation and sweating.

What is the central question of this study? It remains to be determined whether type 2 diabetes attenuates muscarinic and nicotinic cutaneous vasodilatation and sweating as well as purinergic cutaneous vasodilatation. What is the main finding and its importance? We show that type 2 diabetes specifically attenuates purinergic cutaneous vasodilatation without influencing muscarinic and nicotinic cutaneous vasodilatation and sweating. Our results provide valuable new information regarding the receptor-specific influence of type 2 diabetes on microvascular and sudomotor function. The present study evaluated whether type 2 diabetes (T2D) attenuates muscarinic and/or nicotinic cutaneous vasodilatation and sweating as well as purinergic cutaneous vasodilatation. Cutaneous vascular conductance and sweat rate were evaluated in 12 healthy non-diabetic older adults (Control, 60±8years) and 13 older adults with T2D (62±10years) at three intradermal forearm skin sites perfused with the following: (i) methacholine (muscarinic receptor agonist, five doses: 0.0125, 0.25, 5, 100 and 2000mm); (ii) nicotine (nicotinic receptor agonist, five doses: 1.2, 3.6, 11, 33 and 100mm); or (iii) ATP (purinergic receptor agonist, five doses: 0.03, 0.3, 3, 30 and 300mm). Each agonist was administered for 25min per dose. At the end of the protocol, 50mm sodium nitroprusside was administered to all skin sites to elicit maximal cutaneous vasodilatation. Cutaneous vascular conductance during methacholine and nicotine administration did not differ between groups (all P>0.05). In contrast, cutaneous vascular conductance during administration of 30mm (42±28 versus 63±26% maximum, P≤0.05) and 300mm ATP (56±24 versus 71±20% maximum, P≤0.05) was attenuated in individuals with T2D in comparison to the Control participants. Furthermore, cutaneous vascular conductance during administration of 50mm sodium nitroprusside was lower in individuals with T2D relative to Control subjects (P=0.04). Methacholine- and nicotine-induced sweating was similar between groups (all P>0.05). Thus, T2D attenuates purinergic cutaneous vasodilatation without affecting muscarinic and nicotinic cutaneous vascular and sweating responses.

Detection of smoothly distributed spatial outliers, with applications to identifying the distribution of parenchymal hyperinflation following an airway challenge in asthmatics.

Methacholine challenge tests are used to measure changes in pulmonary function that indicate symptoms of asthma. In addition to pulmonary function tests, which measure global changes in pulmonary function, computed tomography images taken at full inspiration before and after administration of methacholine provide local air volume changes (hyper-inflation post methacholine) at individual acinar units, indicating local airway hyperresponsiveness. Some of the acini may have extreme air volume changes relative to the global average, indicating hyperresponsiveness, and those extreme values may occur in clusters. We propose a Gaussian mixture model with a spatial smoothness penalty to improve prediction of hyperresponsive locations that occur in spatial clusters. A simulation study provides evidence that the spatial smoothness penalty improves prediction under different data-generating mechanisms. We apply this method to computed tomography data from Seoul National University Hospital on five healthy and ten asthmatic subjects. Copyright © 2017 John Wiley & Sons, Ltd.

Endothelin-1 modulates methacholine-induced cutaneous vasodilatation but not sweating in young human skin.

Endothelin-1 (ET-1) is a potent endothelial-derived vasoconstrictor that may modulate cholinergic cutaneous vascular regulation. Endothelin receptors are also expressed on the human eccrine sweat gland, although it remains unclear whether ET-1 modulates cholinergic sweating. We investigated whether ET-1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)-dependent mechanism. Our findings show that ET-1 attenuates methacholine-induced cutaneous vasodilatation through a NOS-independent mechanism. We also demonstrate that ET-1 attenuates cutaneous vasodilatation in response to sodium nitroprusside, suggesting that ET-1 diminishes the dilatation capacity of vascular smooth muscle cells. We show that ET-1 does not modulate methacholine-induced sweating at any of the administered concentrations. Our findings advance our knowledge pertaining to the peripheral control underpinning the regulation of cutaneous blood flow and sweating and infer that ET-1 may attenuate the heat loss responses of cutaneous blood flow, but not sweating. The present study investigated the effect of endothelin-1 (ET-1) on cholinergic mechanisms of end-organs (i.e. skin blood vessels and sweat glands) for heat dissipation. We evaluated the hypothesis that ET-1 attenuates cholinergic cutaneous vasodilatation and sweating through a nitric oxide synthase (NOS)-dependent mechanism. Cutaneous vascular conductance (CVC) and sweat rate were assessed in three protocols: in Protocol 1 (n=8), microdialysis sites were perfused with lactated Ringer solution (Control), 40pm, 4nm or 400nm ET-1; in Protocol 2 (n=11) sites were perfused with lactated Ringer solution (Control), 400nm ET-1, 10mm N(G) -nitro-l-arginine (l-NNA; a NOS inhibitor) or a combination of 400nm ET-1 and 10mm l-NNA; in Protocol 3 (n=8), only two sites (Control and 400nm ET-1) were utilized to assess the influence of ET-1 on the dilatation capacity of vascular smooth muscle cells (sodium nitroprusside; SNP). Methacholine (MCh) was co-administered in a dose-dependent manner (0.0125, 0.25, 5, 100, 2000mm, each for 25min) at all skin sites. ET-1 at 400nm (P<0.05) compared to lower doses (40pm and 4nm) (all P>0.05) significantly attenuated increases in CVC in response to 0.25 and 5mm MCh. A high dose of ET-1 (400nm) co-infused with l-NNA further attenuated CVC during 0.25, 5 and 100mm MCh administration relative to the ET-1 site (all P<0.05). Cutaneous vasodilatation in response to SNP was significantly blunted after administration of 400nm ET-1 (P<0.05). We show that ET-1 attenuates cutaneous vasodilatation through a NOS-independent mechanism, possibly through a vascular smooth muscle cell-dependent mechanism, and methacholine-induced sweating is not altered by ET-1.

Effects of methacholine infusion on desflurane pharmacokinetics in piglets.

The data of a corresponding animal experiment demonstrates that nebulized methacholine (MCh) induced severe bronchoconstriction and significant inhomogeneous ventilation and pulmonary perfusion (V̇A/Q̇) distribution in pigs, which is similar to findings in human asthma. The inhalation of MCh induced bronchoconstriction and delayed both uptake and elimination of desflurane (Kretzschmar et al., 2015) [1].The objective of the present data is to determine V̇A/Q̇ matching by Multiple Inert Gas Elimination Technique (MIGET) in piglets before and during methacholine- (MCh-) induced bronchoconstriction, induced by MCh infusion, and to assess the blood concentration profiles for desflurane (DES) by Micropore Membrane Inlet Mass Spectrometry (MMIMS).Healthy piglets (n=4) under general anesthesia were instrumented with arterial, central venous, and pulmonary artery lines. The airway was secured via median tracheostomy with an endotracheal tube, and animals were mechanically ventilated with intermittent positive pressure ventilation (IPPV) with a FiO2 of 0.4, tidal volume (VT)=10 ml/kg and PEEP of 5cmH2O using an open system. The determination of V.A/Q. was done by MIGET: before desflurane application and at plateau in both healthy state and during MCh infusion. Arterial blood was sampled at 0, 1, 2, 5, 10, 20, and 30 min during wash-in and washout, respectively.Bronchoconstriction was established by MCH infusion aiming at doubling the peak airway pressure, after which wash-in and washout of the anesthetic gas was repeated. Anesthesia gas concentrations were measured by MMIMS. Data were analyzed by ANOVA, paired t-test, and by nonparametric Friedman׳s test and Wilcoxon׳s matched pairs test.We measured airway pressures, pulmonary resistance, and mean paO2 as well as hemodynamic variables in all pigs before desflurane application and at plateau in both healthy state and during methacholine administration by infusion. By MIGET, fractional alveolar ventilation and pulmonary perfusion in relation to the V.A/Q. compartments, data of logSDQ̇ and logSDV̇ (the second moments describing global dispersion, i.e. heterogeneity of distribution) were estimated prior to and after MCh infusion. The uptake and elimination of desflurane was determined by MMIMS.

Bronchoconstriction induced by inhaled methacholine delays desflurane uptake and elimination in a piglet model

Bronchoconstriction is a hallmark of asthma and impairs gas exchange. We hypothesized that pharmacokinetics of volatile anesthetics would be affected by bronchoconstriction. Ventilation/perfusion (VA/Q) ratios and pharmacokinetics of desflurane in both healthy state and during inhalational administration of methacholine (MCh) to double peak airway pressure were studied in a piglet model.In piglets, MCh administration by inhalation (100μg/ml, n=6) increased respiratory resistance, impaired VA/Q distribution, increased shunt, and decreased paO2 in all animals. The uptake and elimination of desflurane in arterial blood was delayed by nebulization of MCh, as determined by Micropore Membrane Inlet Mass Spectrometry (wash-in time to P50, healthy vs. inhalation: 0.5min vs. 1.1min, to P90: 4.0min vs. 14.8min). Volatile elimination was accordingly delayed.Inhaled methacholine induced severe bronchoconstriction and marked inhomogeneous VA/Q distribution in pigs, which is similar to findings in human asthma exacerbation. Furthermore, MCh-induced bronchoconstriction delayed both uptake and elimination of desflurane. These findings might be considered when administering inhalational anesthesia to asthmatic patients.

Reprint of “Non-invasive measure of respiratory mechanics and conventional respiratory parameters in conscious large animals by high frequency Airwave Oscillometry”

IntroductionA number of drugs in clinical trials are discontinued due to potentially life-threatening airway obstruction. As some drugs may not cause changes in core battery parameters such as tidal volume (Vt), respiratory rate (RR) or minute ventilation (MV), including measurements of respiratory mechanics in safety pharmacology studies represents an opportunity for design refinement. The present study aimed to test a novel non-invasive methodology to concomitantly measure respiratory system resistance (Rrs) and conventional respiratory parameters (Vt, RR, MV) in conscious Beagle dogs and cynomolgus monkeys. MethodsAn Airwave Oscillometry system (tremoFlo; THORASYS Inc., Montreal, Canada) was used to concomitantly assess Rrs and conventional respiratory parameters before and after intravenous treatment with a bronchoactive agent. Respiratory mechanics measurements were performed by applying a short (i.e. 16s) single high frequency (19Hz) waveform at the subject's airway opening via a face mask. During measurements, pressure and flow signals were recorded. After collection of baseline measurements, methacholine was administered intravenously to Beagle dogs (n=6) and cynomolgus monkeys (n=4) at 8 and 68μg/kg, respectively. ResultsIn dogs, methacholine induced significant increases in Vt, RR and MV while in monkeys, it only augmented RR. A significant increase in Rrs was observed after methacholine administration in both species with mean percentage peak increases from baseline of 88 (53)% for dogs and 28 (16)% for cynomolgus monkeys. ConclusionAirwave Oscillometry appears to be a promising non-invasive methodology to enable respiratory mechanics measurements in conscious large animals, a valuable refinement in respiratory safety pharmacology.

Age-related differences in postsynaptic increases in sweating and skin blood flow postexercise.

The influence of peripheral factors on the control of heat loss responses (i.e., sweating and skin blood flow) in the postexercise period remains unknown in young and older adults. Therefore, in eight young (22 ± 3 years) and eight older (65 ± 3 years) males, we examined dose‐dependent responses to the administration of acetylcholine (ACh) and methacholine (MCh) for sweating (ventilated capsule), as well as to ACh and sodium nitroprusside (SNP) for cutaneous vascular conductance (CVC, laser‐Doppler flowmetry, % of max). In order to assess if peripheral factors are involved in the modulation of thermoeffector activity postexercise, pharmacological agonists were perfused via intradermal microdialysis on two separate days: (1) at rest (DOSE) and (2) following a 30‐min bout of exercise (Ex+DOSE). No differences in sweat rate between the DOSE and Ex+DOSE conditions at either ACh or MCh were observed for the young (ACh: P =0.992 and MCh: P =0.710) or older (ACh: P =0.775 and MCh: P =0.738) adults. Similarly, CVC was not different between the DOSE and Ex+DOSE conditions for the young (ACh: P =0.123 and SNP: P =0.893) or older (ACh: P =0.113 and SNP: P =0.068) adults. Older adults had a lower sweating response for both the DOSE (ACh: P =0.049 and MCh: P =0.006) and Ex+DOSE (ACh: P =0.050 and MCh: P =0.029) conditions compared to their younger counterparts. These findings suggest that peripheral factors do not modulate postexercise sweating and skin blood flow in both young and older adults. Additionally, sweat gland function is impaired in older adults, albeit the impairments were not exacerbated during postexercise recovery.

Non-invasive measure of respiratory mechanics and conventional respiratory parameters in conscious large animals by high frequency Airwave Oscillometry

IntroductionA number of drugs in clinical trials are discontinued due to potentially life-threatening airway obstruction. As some drugs may not cause changes in core battery parameters such as tidal volume (Vt), respiratory rate (RR) or minute ventilation (MV), including measurements of respiratory mechanics in safety pharmacology studies represents an opportunity for design refinement. The present study aimed to test a novel non-invasive methodology to concomitantly measure respiratory system resistance (Rrs) and conventional respiratory parameters (Vt, RR, MV) in conscious Beagle dogs and cynomolgus monkeys. MethodsAn Airwave Oscillometry system (tremoFlo; THORASYS Inc., Montreal, Canada) was used to concomitantly assess Rrs and conventional respiratory parameters before and after intravenous treatment with a bronchoactive agent. Respiratory mechanics measurements were performed by applying a short (i.e. 16s) single high frequency (19Hz) waveform at the subject's airway opening via a face mask. During measurements, pressure and flow signals were recorded. After collection of baseline measurements, methacholine was administered intravenously to Beagle dogs (n=6) and cynomolgus monkeys (n=4) at 8 and 68μg/kg, respectively. ResultsIn dogs, methacholine induced significant increases in Vt, RR and MV while in monkeys, it only augmented RR. A significant increase in Rrs was observed after methacholine administration in both species with mean percentage peak increases from baseline of 88 (53)% for dogs and 28 (16)% for cynomolgus monkeys. ConclusionAirwave Oscillometry appears to be a promising non-invasive methodology to enable respiratory mechanics measurements in conscious large animals, a valuable refinement in respiratory safety pharmacology.

Cutaneous microvascular endothelium‐dependent vasodilation is impaired in young obese subjects (678.4)

Cutaneous microvascular endothelium dependent vasodilation is impaired in young obese subjects Jordan C. Patik, Kevin M. Christmas, and R. Matthew Brothers Environmental and Autonomic Physiology Laboratory, Department of Kinesiology and Health Education, University of Texas at Austin Microvascular dysfunction is a precursor to a variety of cardiovascular diseases and contributes to insulin resistance and type II diabetes. Obese individuals are at a greater risk for the development of these aforementioned conditions relative to their lean counterparts. The purpose of this study was to asses endothelium dependent microvascular function in young, otherwise healthy, obese individuals. Specifically, we tested the hypothesis that the cutaneous microvascular response to intradermal administration of methacholine (MCh) would be blunted in obese relative to lean individuals. 6 young obese and 5 young lean individuals (Age: 24±2.2 vs. 25±1.18 years P&gt;0.05; BMI: 34.96±1.89 vs. 22.24±0.84 kg/m2, P&lt;0.001) participated in this study. Microvascular function was assessed using laser Doppler flowmetry to calculate cutaneous vascular conductance over a range of MCh concentrations infused through microdialysis membranes. Dose response curves were constructed to determine the effective concentration that elicits 50% of maximal conductance (EC50). Young obese subjects had significantly blunted EC50 responses to MCh infusion relative to the lean individuals (Obese: ‐3.173±0.526 vs. Lean: ‐4.875 ±0.199 log molar concentration of MCh, P = 0.02). Our preliminary results support the hypothesis that impaired endothelium dependent vasodilation is present in relatively young obese individuals prior to the development of obesity related disease.

Oxyntomodulin increases intrinsic heart rate through the glucagon receptor

Two hormones from the gastrointestinal tract, glucagon and oxyntomodulin (OXM), vigorously elevate the intrinsic heart rate (IHR) of mice. We have previously shown that OXM influences murine heart rate (HR) independent of the glucagon-like peptide 1 (GLP-1) receptor. Here, we demonstrate using radiotelemetry in mice deficient in the glucagon receptor (Gcgr −/−) that both OXM and glucagon require the glucagon receptor for their chronotropic effects on the heart. Furthermore, we found that other hormones associated with hunger and satiety (ghrelin, leptin, and PYY3-36) had no effect on IHR, while cholecystokinin moderately elevated the IHR. Finally, the resting HR of Gcgr −/− mice was higher than in control mice (Gcgr +/+ and Gcgr +/−) at thermal neutral temperature (30°C). Using atropine, we demonstrated that Gcgr −/− mice have diminished parasympathetic (PNS) influence of the heart at this temperature. Gcgr −/− mice displayed a normal bradycardia as compared to controls in response to administration of either methacholine (to activate the muscarinic acetylcholine receptor) or methoxamine (to activate the baroreflex through agonism of the α1 adrenergic receptor agonist) suggesting that vagal pathways are intact in the Gcgr −/− mice. As OXM is an agonist of the GLP-1 receptor and Gcgr with antidiabetic activity, we suggest OXM may be an alternative to glucagon in the treatment of overdose of beta-blockers to elevate HR in clinical conditions.

Glycyrrhetinic acid attenuates vascular smooth muscle vasodilatory function in healthy humans

Abnormal glucocorticoid metabolism contributes to vascular dysfunction and cardiovascular disease. Cortisol activation of vascular mineralocorticoid and glucocorticoid receptors is regulated by two types of 11beta-HSD (11-beta hydroxysteroid dehydrogenase), namely 11beta-HSD2 and 11beta-HSD1 (type 2 and type 1 11beta-HSD respectively). We hypothesized that inhibition of 11beta-HSD would attenuate vascular function in healthy humans. A total of 15 healthy subjects were treated with the selective 11beta-HSD inhibitor GA (glycyrrhetinic acid) or matching placebo in a randomized double-blinded cross-over trial. 11beta-HSD activity was assessed by the urinary cortisol/cortisone ratio, and vascular function was measured using strain-gauge plethysmography. Endothelial function was measured through incremental brachial artery administration of methacholine (0.3-10 microg/min) and vascular smooth muscle function with incremental verapamil (10-300 microg/min). GA increased the 24-h urinary cortisol/cortisone ratio compared with placebo (P=0.008). GA tended to reduce the FBF (forearm blood flow) response to methacholine (P=0.09) and significantly reduced the FBF response to verapamil compared with placebo (P=0.04). MAP (mean arterial pressure) did not differ between the study conditions. 11beta-HSD inhibition attenuated vascular smooth muscle vasodilatory function in healthy humans. Disturbances in cortisol activity resulting from 11beta-HSD inactivation is therefore a second plausible mechanism for mineralocorticoid-mediated hypertension in humans.

High-resolution spatial measurements of ventilation-perfusion heterogeneity in rats

This study was designed to validate a high-resolution method to measure regional ventilation (VA) in small laboratory animals, and to compare regional Va and perfusion (Q) before and after methacholine-induced bronchoconstriction. A mixture of two different colors of 0.04-microm fluorescent microspheres (FMS) was aerosolized and administered to five anesthetized, mechanically ventilated rats. Those rats also received an intravenous injection of a mixture of two different colors of 15-microm FMS to measure regional blood flow (Q). Five additional rats were labeled with aerosol and intravenous FMS, injected with intravenous methacholine, and then relabeled with a second pair of aerosol and intravenous FMS colors. After death, the lungs were reinflated, frozen, and sequentially sliced in 16-microm intervals on an imaging cryomicrotome set to acquire signal for each of the FMS colors. The reconstructed lung images were sampled using randomly placed 3-mm radius spheres. Va within each sphere was estimated from the aerosol fluorescence signal, and Q was estimated from the number of 15-microm FMS within each sphere. Method error ranged from 6 to 8% for Q and 0.5 to 4.0% for Va. The mean coefficient of variation for Q was 17%, and for Va was 34%. The administration of methacholine altered the distribution of both VA and Q within lung regions, with a change in Va distribution nearly twice as large as that seen for Q. The methacholine-induced changes in Va were not associated with compensatory shifts in Q. Cryomicrotome images of FMS markers provide a high-resolution, anatomically specific means of measuring regional VA/Q responses in the rat.

Two ATS Recommended Protocols for Administration of Methacholine Are Not Equal

Background. The 1999 American Thoracic Society methacholine challenge guidelines stated that the 5-breath dosimeter method of methacholine administration is similar to the 2-minute tidal breath method. Recent data has disputed this assertion. We examined the differences in the diagnosis of asthma using these two methods. Methods. Data were abstracted from a prospectively generated pulmonary function database over 4 years. During the first 2 years the 5-breath dosimeter method was used, and the subsequent 2 years the 2-minute tidal breath method was used. The effect of the delivery technique was assessed by crude and adjusted odds ratios, controlling for known confounders and group differences. Results. A total of 907 subjects underwent methacholine challenge testing during the 4-year study period: 19.3% of the subjects tested with the 5-breath dosimeter method and 31.2% of those tested with the 2-minute tidal breathing method had a PC20 ≤ 8.0 mg/mL (OR 1.90, 95% CI 1.4 to 2.58, p < 0.001). The ability to reliably exclude airway hyper-responsiveness (PC20 > 16.0 mg/mL) was also altered by the differences between the testing techniques. Using the 5-breath dosimeter method, 72.4% of subjects were ruled out for airway hyper-responsiveness, whereas only 59.9% of subjects were ruled out with the 2-minute tidal breathing technique (p < 0.001). Conclusion. The two recommended protocols for the diagnosis of asthma are not equivalent and significantly alter the rate of diagnosis of asthma as well as the severity. The differences were seen across all PC20 levels, from those with strongly positive tests (PC20 ≤ 1.0 mg/mL) as well as those with negative tests for airway hyper-responsiveness (PC20 > 16.0 mg/mL).

Comparisons of effects of intravenous and inhaled methacholine on airway physiology in a murine asthma model

Airway responses to intravenous (i.v.) and inhaled (i.h.) delivery of methacholine (MCh) in BALB/c and C57BL/6 mouse strains have been compared with and without ovalbumin (OVA)-induced airway inflammation. Bronchial reactivity to MCh was assessed in anaesthetised and tracheostomised animals by using an animal ventilator (flexiVent). We partitioned the response of the lungs into airway and parenchymal components in order to compare the contributions of the airways with those of the lung parenchyma to the pulmonary mechanical responses resulting from different routes of MCh administration. Our results indicate disparate physiological responses. Intravenous MCh delivery induced a higher maximum lung resistance than i.h. MCh in OVA-treated BALB/c mice but not in C57BL/6 mice. Inhaled MCh delivery led to a significantly larger fall in lung compliance and a greater impact on peripheral airways than i.v. MCh in both strains. In conclusion, i.v. and i.h. MCh produced disparate effects in different murine strains and variant responses in inflamed airways and healthy controls. The two methods of MCh delivery have important advantages but also certain limitations with regard to measuring airway reactivity in a murine model of allergic asthma.