Jaime Chávez

Androgens are bronchoactive drugs that act by relaxing airway smooth muscle and preventing bronchospasm

Changes in the androgen levels in asthmatic men may be associated with the severity of asthma. Androgens induce a nongenomic relaxation in airway smooth muscle, but the underlying mechanisms remain unclear. The aim of this study was to investigate the potential bronchorelaxing action of testosterone (TES) and its metabolites (5α- and 5β-dihydrotestosterone (DHT). A preventive effect on ovalbumin (OVA)-induced bronchospasm was observed in sensitized guinea pigs for each androgen. Androgens were studied in response to bronchoconstrictors: carbachol (CCh) and KCl in isolated trachea rings with and without epithelium from non-sensitized and sensitized animals as well as on OVA-induced contraction. Androgens concentration-dependently abolished the contraction in response to CCh, KCl, and OVA. There were significant differences in the sensitivity to the relaxation induced by each androgen. 5β-DHT was more potent for relaxing KCl-induced contraction, while TES and 5α-DHT were more potent for CCh- and OVA-induced contraction. No differences were found in preparations with and without epithelium or in the presence of a nitric oxide (NO) synthase inhibitor or an inhibitor of K(+) channels. These data indicate the absence of involvement of the epithelium-, NO- and K(+) channels-dependent pathway in androgen-induced relaxation. However, in dissociated tracheal myocytes loaded with the calcium-binding fluorescent dye Fura -2, physiological concentrations of androgens decreased the KCl-induced [Ca(2+)]i increment. 5β-DHT was the most potent at decreasing KCl-induced [Ca(2+)]i increment and preventing bronchospasm. We suggest that androgen-induced brochorelaxation was mediated via decreased Ca(2+) influx through L-type Ca(2+)channels but additional Ca(2+) entry blockade may be involved. Molecular changes in androgen structure may determine its preferential site of action.

Non‐quantal release of acetylcholine in guinea‐pig airways: role of choline transporter

In the resting state, motor neurons continuously release ACh through quantal and non‐quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non‐quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea‐pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage‐dependent Na+ channel blocker) and ω‐conotoxin GVIA (N‐type Ca2+ channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon‐induced contraction. We confirmed that tetrodotoxin, and to some extent ω‐conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium‐3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration‐dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium‐3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon‐induced contraction was not due to depletion of neuronal vesicles. Non‐neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium‐free medium abolished the effect of paraoxon, and NiCl2, 2‐aminoethyl diphenyl‐borate and SKF 96365 partly inhibited it, suggesting that non‐specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca2+‐dependent NQR of ACh is present in cholinergic nerves from guinea‐pig airways, and that ChT is involved in this phenomenon.

Guinea pig lung resistance shows circadian rhythmicity not influenced by ozone.

Increased circadian variability of airway caliber is a key feature of asthmatic patients, but it has not been addressed in animal models of asthma. Furthermore, animal studies on circadian rhythmicity of airway resistance are very scanty. We used a plethysmographic method for unrestrained guinea pigs to monitor a lung resistance index (iRL) during 24 h. We found circadian variability of iRL values, which were fitted by a sinusoidal curve. Acrophase and bathyphase, characterizing the timing of narrowest and widest airway caliber, respectively, were found at 02:03, and 15:34 h. iRL values at these time-points were statistically different (P < 10(-5)). Moreover, average resistance during the dark period was significantly higher (P < 0.0001) than during the light period. Immediately after an acute ozone exposure (3 ppm for 1 h) an increase in iRL was demonstrated (P < 0.01), which lasted for 2 h, and tended to remain high for the next hour. After guinea pigs recovered from this obstruction, the circadian rhythm and variability of airway caliber were unaffected. Our results show that a circadian rhythm of iRL takes place in guinea pigs, greatly resembling what occurs in humans, and that ozone exposure causes a transient airway obstruction, but fails to reproduce the increased variability of airway caliber observed in asthmatic patients.

PPADS, a P2X receptor antagonist, as a novel inhibitor of the reverse mode of the Na+/Ca2+ exchanger in guinea pig airway smooth muscle

The Na(+)/Ca(2+)exchanger (NCX) principal function is taking 1 Ca(2+) out of the cytoplasm and introducing 3 Na(+). The increase of cytoplasmic Na(+) concentration induces the NCX reverse mode (NCX(REV)), favoring Ca(2+) influx. NCX(REV) can be inhibited by: KB-R7943 a non-specific compound that blocks voltage-dependent and store-operated Ca(2+) channels; SEA0400 that appears to be selective for NCX(REV), but difficult to obtain and SN-6, which efficacy has been shown only in cardiomyocytes. We found that PPADS, a P2X receptor antagonist, acts as a NCX(REV) inhibitor in guinea pig tracheal myocytes. In these cells, we characterized the NCX(REV) by substituting NaCl and NaHCO(3) with LiCl, resulting in the increase of the intracellular Ca(2+) concentration ([Ca(2+)]i) using fura 2-AM. We analyzed 5 consecutive responses of the NCX(REV) every 10 min, finding no differences among them. To evaluate the effect of different NCX(REV) blockers, concentration response curves to KB-R7943 (1, 3.2 and 10 μM), and SN-6 (3.2, 10 and 30 μM) were constructed, whereas PPADS effect was characterized as time- and concentration-dependent (1, 3.2, 10 and 30 μM). PPADS had similar potency and efficacy as KB-R7943, whereas SN-6 was the least effective. Furthermore, KCl-induced contraction, sensitive to D600 and nifedipine, was blocked by KB-R7943, but not by PPADS. KCl-induced [Ca(2+)]i increment in myocytes was also significantly decreased by KBR-7943 (10 μM). Our results demonstrate that PPADS can be used as a reliable pharmacological tool to inhibit NCX(REV), with the advantage that it is more specific than KB-R7943 because it does not affect L-type Ca(2+) channels.

Spontaneous changes in guinea-pig respiratory pattern during barometric plethysmography: role of catecholamines and nitric oxide

Barometric plethysmography for unrestrained animals is a non‐invasive method that allows repetitive measurements of pulmonary function, but habituation of the conscious animal to this technique has not been explored. Respiratory frequency (fR) and ‘enhanced pause’ (Penh) were measured by barometric plethysmography for a period of 8 h in guinea‐pigs. Compared with basal values, during the first hour of recording a progressive increase in Penh (up to 25–50%) and a corresponding decrease in fR were recorded, followed by a relative plateau in each for up to 8 h. These changes were avoided by a 30‐min pretreatment with propranolol and l‐NAME (nitric oxide synthase inhibitor), with Penh values as high as this plateau phase since the beginning of recording. Atropine, salbutamol or budesonide did not modify the progressive increment in Penh. We concluded that catecholamines and nitric oxide are released when guinea‐pigs are introduced into the plethysmographic chamber, leading to initial low Penh values. These mediators probably diminish owing to habituation of the animal to the new environment, with an apparent progressive increment in Penh. These spontaneous changes in Penh and fR must be taken into account during barometric plethysmography in order to avoid misinterpretation of the results.

Possible role of substance P in the ischemia-reperfusion injury in the isolated rabbit lung

The origin of the endothelial damage leading to the ischemia-reperfusion injury after lung transplantation has not been elucidated. We postulated that neurotransmitters released during the preservation of the donor lung might explain this vascular derangement. Thus, in isolated rabbit lungs preserved over 24 hours, we evaluated the release of acetylcholine (ACh) and substance P (SP), the activity of their major degrading enzymes, acetylcholinesterase (AChE) and neutral endopeptidase (NEP), and changes in the capillary permeability. Both neurotransmitters showed the highest release rate in the first 15 minutes, followed by a sharp exponential decrement at 1, 6, 12 and 24 hours. AChE and NEP activities showed no variation at these time intervals. Basal capillary permeability significantly increased (P<0.01) after 24 hours preservation with saline. This increased permeability was avoided (P<0.01) by the SP fragment 4-11 (an SP receptors antagonist), but not by atropine. These results suggest for the first time a pathogenic role of SP in the ischemia-reperfusion injury, and thus the potential usefulness of SP antagonists as additives in the lung preservation solutions should be explored.