Tracheal relaxation of five medicinal plants used in Mexico for the treatment of several diseases.

OBJECTIVE: To assess the relaxant effect of several organic extracts obtained from Agastache mexicana (A. mexicana), Cochlospermum vitifolium (C. vitifolium), Cordia morelosana (C. morelosana), Lepechinia caulescens (L. caulescens) and Talauma mexicana (T. mexicana) used in Mexican traditional medicine for the treatment of several diseases. METHODS: Extracts were obtained by maceration at room temperature using hexane, dichloromethane and methanol for each plant material. The organic extracts were evaluated ex vivo to determine their relaxant activity on the contractions induced by carbachol (cholinergic receptor agonist, 1 µ mol/L) in isolated rat tracheal rings. RESULTS: A total of 15 extracts were evaluated (three for each species). All test samples showed significant relaxant effect, in a concentration-dependent manner, on the contractions induced by 1 µ mol/L carbachol, with exception of extracts from C. morelosana. Active extracts were less potent than theophylline [phosphodiesterase inhibitor, EC50: (28.79±0.82) µg/mL] that was used as positive control. Concentration-response curves revealed that the extracts with more significant effects were dichloromethanic extracts of T. mexicana [Emax: (103.03±3.32)% and EC50: (159.39±3.72) µg/mL) and C. vitifolium [Emax: (106.58±2.42)% and EC50: (219.54±7.61) µg/mL]. Finally, hexanic and dichloromethanic extracts from A. mexicana were fully effective but less potent than T. mexicana and C. vitifolium. CONCLUSIONS: Less polar extracts obtained from A. mexicana, T. mexicana and C. vitifolium exhibited greater relaxant effect on tracheal rat rings, which allows us to suggest them as sources for the isolation of bioactive molecules with potential therapeutic value in the treatment of asthma.

The calcium channel blocking and phosphodiesterase inhibitory activities of the extract of Andropogon muricatus explains its medicinal use in airways disorders.

The present work was carried out to provide a pharmacological base for the medicinal use of Andropogon muricatus in airways disorders, such as asthma. In isolated guinea-pig tracheal strips, the crude extract of Andropogon muricatus exhibited a non-specific relaxant effect against carbachol (1 µM) and high K⁺ precontractions, with EC50 values of 0.10 (0.07-0.11) and 0.15 mg/mL (0.11-0.18), respectively, similar to papaverine, while verapamil was more potent against high K⁺. This suggests the involvement of a non-specific relaxant effect, mediated possibly through Ca⁺⁺ channel blockade and phosphodiesterase inhibition. The functional nature of the relaxant effect was further confirmed through indirect evidence when pretreatment of the tissues with the plant extract caused potentiation of the isoprenaline inhibitory response curves, similar to papaverine, while the effect of verapamil remained unchanged. These data indicate that the crude extract of Andropogon muricatus contains constituent(s) that mediate the tracheal relaxant effect, possibly through dual inhibition of Ca⁺⁺ channels and phosphodiesterase and provide pharmacological evidence for its medicinal use in airways disorders, particularly asthma.

Analysis of the tracheal contents using headspace gas chromatography-mass spectrometry to screen for accelerant use.

We describe here the usefulness of analysis of tracheal contents in a case of death by fire, which revealed that the deceased had used the accelerants. The analysis of tracheal contents provides useful information for the determination of the circumstances of the scene.

Continuous pulmonary infusion of L-arginine during deep hypothermia and circulatory arrest improves pulmonary surfactant integrity in piglets.

BACKGROUND: The integrity of pulmonary surfactant (PS) is impaired during deep hypothermia and circulatory arrest (DHCA), a preferred bypass strategy for infants undergoing complex cardiac operations, due mainly to bypass-induced systemic inflammation. The requirement of L-arginine, a precursor of nitric oxide, is elevated during acute pulmonary inflammation. We hypothesized that continuous intrapulmonary supplementation of L-arginine during DHCA can maintain the integrity of PS metabolism and thus protect the pulmonary function. METHODS: Sixteen piglets underwent 90-minute circulatory arrest at 18 degrees C before rewarming. During circulatory arrest, antegrade infusion of Ringer's lactate solution alone (n = 8) or containing L-arginine (1 mg/kg/min, n = 8) was initiated into the pulmonary circulation. Disaturated phosphatidylcholine, total phospholipids, and total proteins from tracheal aspirates were measured serially until the experiment ended (4 hours after rewarming). Various variables of pulmonary function were also monitored. RESULTS: L-arginine led to less decrement of disaturated phosphatidylcholine/total phospholipids and disaturated phosphatidylcholine/total proteins after DHCA. At 4 hours after rewarming, L-arginine had significantly mitigated the deterioration of pulmonary static compliance (3.6 +/- 0.5 vs 3.3 +/- 0.3 mL/cm H2O) and partial pressure of arterial oxygen/fraction of inspired oxygen (330 +/- 48 vs 296 +/- 32 mm Hg). Pulmonary retention of water (6.2 +/- 1.0 vs 5.5 +/- 1.2) was significantly reduced. The L-arginine-treated group showed an increase in NO metabolites (NO2-/NO3-) from the pulmonary circulation, the extent of which is correlated to PS content. CONCLUSIONS: Continuous L-arginine supplementation during DHCA attenuated PS depletion and, therefore, ameliorated postoperative pulmonary dysfunction.

Characterization and purification of glycosaminoglycans from crude biological samples.

Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using fluorophore-assisted carbohydrate electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common supplement.

Ionic composition of rat airway surface liquid determined by X-ray microanalysis.

The thin layer of liquid that lines the conducting airway epithelium, the airway surface liquid (ASL), is important for mucociliary clearance. Altered ionic composition and/ or volume of the ASL play a major role in the pathology of airway diseases such as cystic fibrosis. Since the ASL is a thin layer, it has been difficult to exactly determine its composition. The present paper describes two techniques that have been developed and used to study ASL composition: X-ray microanalysis of frozen hydrated rat trachea, and an ion-exchange (dextran) bead method, where dextran beads were placed on the airway epithelium to equilibrate with the ASL; the beads were then collected under silicone oil, dried and analyzed by X-ray microanalysis. The results from both frozen-hydrated specimens and from the dextran beads showed that ASL from rat trachea is hypotonic. Concentrations of Na, P, S, and K were higher in the frozen-hydrated ASL, in which mainly the mucus layer is analyzed, compared with the dextran bead method, in which mainly the periciliary liquid is sampled. Also the composition of rat nasal fluid was investigated by the dextran bead method. This fluid was somewhat hypertonic because of a high K concentration. The ionic composition of the nasal and tracheal fluid can be manipulated by cholinergic or alpha- or beta-adrenergic stimulation. Collecting ASL with dextran beads did not disturb the integrity of the airway epithelium. The ionic composition of the collected beads remained stable for several days during storage in silicone oil. It is concluded that X-ray microanalysis is a suitable method to determine the ionic composition of ASL.

Attenuating effect of H+K+ATPase inhibitors on airway cough hypersensitivity induced by allergic airway inflammation in guinea-pigs.

BACKGROUND: Gastrooesophageal reflux (GER) is a frequent cause of chronic cough. Several investigators have indicated that inhibitors of H(+)K(+)ATPase (proton pump inhibitors; PPIs) could relieve coughing via inhibition of acid reflux. However, we considered that PPIs might directly inhibit increased cough reflex sensitivity. OBJECTIVE: The present study was designed to examine whether PPIs directly inhibit antigen-induced increase in cough reflex sensitivity and to elucidate the mechanism. METHODS: Actively sensitized guinea-pigs were challenged with aerosol antigen (ovalbumin, OVA) and cough reflex sensitivity to inhaled capsaicin was measured 24 h later. The PPIs (omeprazole and rabeprazole) or the histamine H(2) blocker cimetidine were administered intraperitoneally 1 h before OVA challenge and before measuring cough reflex sensitivity, then bronchoalveolar lavage fluid (BALF) was immediately collected. The pH of the fluid obtained by bronchial washing was determined after examining the effect of rabeprazole on the cough response to capsaicin. RESULTS: The number of coughs elicited by capsaicin was significantly increased 24 h after challenge with OVA compared with saline, indicating antigen-induced increase in cough reflex sensitivity. Both PPIs dose dependently and significantly inhibited antigen-induced cough hypersensitivity. Omeprazole did not influence the antigen-induced increase in the total number of cells or ratio (%) of eosinophils in BALF. Cimetidine did not affect the antigen-induced cough hypersensitivity or cellular components of BALF. The pH of the bronchial washing fluid was significantly decreased in antigen-challenged animals. Rabeprazole did not affect the antigen-induced decrease in the pH of bronchial washing fluid. CONCLUSION: These findings show that PPIs, but not histamine H(2) blockers, can directly decrease antigen-induced cough reflex hypersensitivity, while the mechanism remains unclear.

Ascorbic acid in nasal and tracheobronchial airway lining fluids.

Ascorbic acid (AA) is thought to be an important antioxidant in the respiratory tract, whose regulation is yet to be fully characterized. We investigated whether AA in respiratory tract lining fluids (RTLFs) can be augmented by oral supplementation with AA. Plasma, nasal lavage fluids (NLFs), induced sputum (IS), and saliva were analyzed for AA immediately before and 2 h after ingestion of 2 g of AA in 13 healthy subjects. Concentrations of AA (median and range) were 52.5 (16.0-88.5), 2.4 (0.18-4.66), 2.4 (0.18-6.00), and 0.55 (0.18-18.90) micromol/l, respectively. Two hours after ingestion of AA, plasma AA increased 2-fold (p = .004), NLF AA increased 3-fold (p = .039), but IS and saliva AA did not increase. As AA concentrations in saliva and tracheobronchial secretions were low compared with other common extracellular components (such as urate), we evaluated the fate of AA in these fluids. Addition of AA to freshly obtained saliva or IS resulted in rapid depletion, which could be largely prevented or reversed by sodium azide or dithiothreitol. These findings suggest that oxidant-producing systems in saliva and airway secretions, such as heme peroxidases and other oxidizing substances, rapidly consume AA. Whereas oral supplementation resulted in detectable increases of AA in NLFs, its levels in tracheobronchial lining fluid, as measured by IS, were unaffected and remained relatively low, suggesting that AA may play a less significant antioxidant role in this compartment as compared with most other extracellular compartments.

Antioxidant supplementation and pulmonary function at rest and exercise.

Antioxidants have been implicated in the reduction and prevention of oxidative stress during exercise. We hypothesised that a dietary supplement containing a mixture of natural antioxidants together with vitamins E, C and selenium, given for 4 weeks, would increase the systemic and pulmonary antioxidant capacity leading to a reduction in markers of oxidative damage and an improvement in pulmonary function during exercise. In 6 healthy horses studied, the antioxidant supplement significantly increased plasma concentrations of ascorbic acid (from mean +/- s.d. 16 +/- 7 to 23 +/- 4 micromol/l; P = 0.007) and alpha-tocopherol (from 10 +/- 3 to 14 +/- 3 micromol/l; P = 0.02) and increased the bronchoalveolar lavage pulmonary epithelial lining fluid (ELF) concentration of ascorbic acid compared to a placebo, but not significantly (2.0 +/- 0.9 mmol/l and 1.2 +/- 0.9 mmol/l, respectively; P>0.05). Alpha-tocopherol was not detected in ELF either before or after supplementation or exercise. The mean concentration of malondialdehyde (MDA) in ELF was lower following antioxidant supplementation compared to placebo and control periods, but not significantly. An intermittent exercise test consisting of 2 min at 70, 80 and 90% of the horses' individual maximum oxygen uptake, failed to induce significant systemic or pulmonary oxidative stress (based on the glutathione redox ratio (GRR) and the ascorbic acid redox ratio (ARR)) and lipid peroxidation (based on the concentration of thiobarbituric acid reactive substances in plasma and MDA in ELF) either for placebo or antioxidant treatments. There was a strong correlation between GRR and ARR in the pulmonary epithelial lining fluid (r = 0.89; P<0.0001). In healthy horses on a diet containing adequate levels of antioxidants, additional antioxidant supplementation has no apparent beneficial or detrimental effect on pulmonary function during moderate intensity exercise. The importance of antioxidant supplementation may only become apparent if the diet is deficient in antioxidants, if exercise intensity is higher or more prolonged, or if disease or additional stresses are present.

CLA reduces antigen-induced histamine and PGE(2) release from sensitized guinea pig tracheae.

Conjugated linoleic acid (CLA) has been shown to enhance immune reactions such as lymphocyte blastogenesis and delayed-type hypersensitivity. We investigated the role of CLA in type I (immediate) hypersensitivity, using a guinea pig tracheal superfusion model for measuring antigen-induced airway smooth muscle contraction and inflammatory mediator release. Female Hartley guinea pigs were fed a diet supplemented with 0.25 g corn oil or linoleic acid/100 g of diet (control) or 0.25 g CLA/100 g of diet for at least 1 wk before and during active sensitization to ovalbumin antigen. Tracheae from sensitized guinea pigs were suspended in air-filled water-jacketed (37 degrees C) tissue chambers in a superfusion apparatus. Tracheae were superfused with buffer containing antigen, and tissue contraction was recorded. Superfusate was collected at 90-s intervals for evaluation of histamine and PGE(2) release. CLA did not affect antigen-induced tracheal contractions when expressed as gram contraction per gram tissue. CLA significantly reduced antigen-induced histamine and PGE(2) release. CLA appears to decrease release of some inflammatory mediators during type I hypersensitivity reactions.

Age-dependent changes of elements in human trachea.

To elucidate compositional changes of human trachea by aging, element contents in tracheae were determined by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The sulfur content of the tracheae decreased gradually with aging. In regard to calcium and phosphorus, both the contents increased to about threefold amounts in their seventies compared with those in their sixties, and decreased thereafter. The contents of calcium and phosphorus were the highest in their seventies. Therefore, it is likely that surplus calcium released from bones is deposited temporally in the trachea, and the deposits are released from the trachea at older age. Based on our results of human cartilages, there are two types in regard to calcium accumulation: The first type is that calcium accumulation occurs progressively with aging; the second one is that calcium accumulation becomes the highest in the seventies or eighties, and decreases thereafter. Therefore, the trachea belongs to the second type. Furthermore, the magnesium content remained constant through the age range.

X-ray microanalysis of native airway surface liquid collected by cryotechnique.

The airway surface liquid (ASL) that lines the surface epithelium of the tracheobronchial tree is of vital importance to the airway defence against microbial invasion and damage due to environmental factors. Little is known about the ASL collected in situ in native conditions, owing to difficulties in collecting ASL without causing damage to the airway mucosa. We have developed a method to collect and analyse the elemental composition of tracheal ASL in pathogen-free mice. A specially designed cryoprobe, adapted to the internal curvature of the mouse trachea, was used to collect the native ASL from the tracheal surface. The complete ASL elemental composition including [Na] = 5.5 +/- 0.3, [Cl] = 1.3 +/- 0.3, [K] = 1.1 +/- 0.2, [Ca] = 1.2 +/- 0.3, [P] = 1.5 +/- 0.8, [S] = 1.7 +/- 0.4 and [Mg] = 1.3 +/- 0.4 mmol L-1 was determined by X-ray micro-analysis. We demonstrate here that the technique that we used for ASL collection maintained perfectly the airway epithelial integrity and functionality.

Vitamin D toxicosis in cats: natural outbreak and experimental study.

A pathological study on 5 of 21 cats affected naturally with systemic calcinosis was performed. The animals ranged in age from 1 to 9 years. Hematology and serum chemistry analyses showed the elevated values of phosphorus, blood urea nitrogen and serum creatinine. X-ray examination disclosed the increased density of systemic bones. Histologically, marked calcification was present at the vascular walls of almost all the organs including the lungs, trachea, kidneys, heart, aorta, alimentary tracts, choroid plexus and bones. In the lungs, kidneys and stomach, the calcified lesions were associated with deposition of oxalate crystals. Serum chemistry showed more elevated values of 25-hydroxycholecalciferol (vitamin D) of the affected cats than the normal level. Retrospective examination revealed that these cats had been fed the commercial pet foods containing a large amount of vitamin D (6,370 IU/100 g diet) from their young age, and its value was about ten times as much as that of the control food (680 IU/100 g diet). Pathological changes found in the cats from the experimental vitamin D3 toxicosis were similar to those in the natural cases. In addition, tissue levels of calcium, phosphorous and zinc in the lungs and kidneys were markedly elevated in both natural and vitamin D-intoxicated cases. These findings suggest that long-term feeding of the pet food containing excessive vitamin D was responsible for the outbreak of the systemic calcinosis in the cats.

Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes?

To date five human mucin cDNAs (MUC2, 5A, 5B, 5C and 6) mapped to 11p15.3-15.5, so it appears that this chromosome region might contain several distinct gene loci for mucins. Three of these cDNAs, MUC5A, B and C, were cloned in our laboratory and previously published. A common number, 5, was recommended by the Human Gene Mapping Nomenclature Committee to designate them because of their common provenance from human tracheobronchial mucosa. In order to define whether they are products of the same gene locus or distinct loci, we describe in this paper physical mapping of these cDNAs using the strategy of analysis of CpG islands by pulse-field gel electrophoresis. The data suggest that MUC5A and MUC5C are part of the same gene (called MUC5AC) which is distinct from MUC5B. In the second part of this work, complete sequences of the inserts corresponding to previously described (JER47, JER58) and novel (JER62, JUL32, MAR2, MAR10 and MAR11) cDNAs of the so-called MUC5AC gene are presented and analysed. The data show that in this mucin gene, the tandem repeat domain is interrupted several times with a subdomain encoding a 130 amino acid cysteine-rich peptide in which the TR3A and TR3B peptides previously isolated by Rose et al. [Rose, Kaufman and Martin (1989) J. Biol. Chem., 264, 8193-8199] from airway mucins are found. A consensus peptide sequence for these subdomains involving invariant positions of most of the cysteines is proposed. The consensus nucleotide sequence of this subdomain is also found in the MUC2 gene and in the MUC5B gene, two other mucin genes mapped to 11p15. The functional significance for secreted mucins of these cysteine-rich subdomains and the modular organization of mucin peptides are discussed.

Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections.

Methods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X-ray microanalysis studies are presented. The cells are grown on Pioloform-covered gold grids supported on Thermanox coverslips. This provides a growth-compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze drying. The effects of different washing media (0.3 M sucrose, 0.15 M ammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled-water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe asthma.

Static respiratory compliance in the newborn. II: Its potential for improving the selection of infants for early surfactant treatment.

Static respiratory system compliance (Crs) and lecithin/sphingomyelin (L/S) ratios in tracheal aspirates were estimated in two independent groups of mechanically ventilated infants. Crs was measured rapidly at the cotside using a passive expiratory flow technique and L/S ratios were estimated in the laboratory by high performance liquid chromatography. In the reference group of 22 infants, Crs < 1.8 ml/cm H2O/m predicted surfactant deficiency with a positive predictive value of 100% and a negative predictive value of 92%. In the validation group of 23 infants, Crs < 1.8 ml/cm H2O/m predicted surfactant deficiency with a positive predictive value of 94% and a negative predictive value of 83%. Measurement of static Crs is a rapid, non-invasive technique which may usefully supplement current methods of selecting infants at high risk of respiratory distress syndrome.

Prostanoids inhibit release of endogenous norepinephrine from rat isolated trachea.

Prostanoids, of epithelial origin, are known as modulators of several processes in the airways. The present study examined whether prostanoids are involved in the local control of sympathetic neurotransmission. The release of endogenous norepinephrine from rat isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses) in the presence of yohimbine, desipramine, and tyrosine. In different series of experiments, indomethacin (3 mumol/L) increased the evoked release of endogenous norepinephrine by 70 to 80%. In the presence of indomethacin, prostaglandin E2 (PGE2) and several prostanoid receptor agonists inhibited the evoked release of norepinephrine in a concentration-dependent manner, maximally by 60 to 70%. According to the concentration producing 35% inhibition of norepinephrine release (half-maximal effect), the following rank order of potencies was observed (EC35): nocloprost (8 nmol/L), sulprostone (30 nmol/L), PGE2 (308 nmol/L), iloprost (2 mumol/L), and U46619 (> 10 mumol/L). The EP1 receptor antagonist AH 6809 (3 mumol/L) had no effect on the evoked norepinephrine release and did not affect the inhibitory effect of 1 mumol/L of sulprostone. In the absence of indomethacin, the inhibitory effect of PGE2 was similar to that observed in the presence of indomethacin. After removal of the epithelium, the evoked norepinephrine release was markedly reduced. However, no significant effect of indomethacin was observed in epithelium-denuded tracheae. In conclusion, norepinephrine release in the rat trachea is inhibited via prostaglandin receptors that have the pharmacologic characteristics of the EP3 subtype. Endogenous eicosanoids, most likely of epithelial origin, are involved in the local control of the release of norepinephrine.