Celia Sanz

Non-specific hyperreactivity to pharmacological stimuli in tracheal and lung parenchymal strips of actively sensitized guinea-pigs

The responsiveness of tracheal and lung parenchymal strips isolated from actively sensitized guinea‐pigs to CaCl2 (in K+‐depolarized tissue), KCl, acetylcholine and histamine was compared with that of strips from unsensitized animals. The concentration‐response curves to the mentioned agonists exhibited, in the sensitized group, a left upward displacement (greater maximal effect, lesser effective concentration 50% and a steeper slope) compared with those obtained in the unsensitized group. These results indicate the existence of a non‐specific increase in responsiveness in the airway smooth muscle from sensitized animals.

Cooling-induced contraction of trachea isolated from normal and sensitized guinea-pigs

SummaryFast (−7°C/min) cooling of guinea-pig isolated trachea produced a rapidly developing, transient contraction followed by relaxation. Cooling-induced contraction was dependent on temperature (30, 20 or 10°C) and responses in trachea obtained from actively sensitized guinea pigs were significantly greater (20 and 10°-C) than those observed in normal trachea. Cooling to 20°C was selected for subsequent experiments. Pre-treatment with sufficient concentrations of atropine, clemastine, cromoglycate, indomethacin, or nordihydroguaiaretic acid did not depress contraction to cooling in either normal or sensitized trachea. This indicates a direct effect of cooling. The contraction. produced by cooling was resistant to verapamil (1 μmol/l) or dantrolene (0.3 mmol/l). Calmodulin antagonists (trifluoperazine, W-7 and calmidazolium; all of them at 10–100 μmol/l) inhibited contraction in sensitized and normal trachea. Activators of protein kinase C (phorbol 12,13-diacetate, 1 μmol/l) enhanced while inhibitors (H-7, 20 μmol/l; staurosporine, 10 μmol/l) depressed cooling-induced contraction in both normal and sensitized tissues. Incubation (20 min) in a Ca2+ -free solution inhibited cooling-induced contraction in normal but not in sensitized trachea. Exposure to a low Na+ (25 mmol/l) or a K+-free medium abolished contraction to cooling in normal and sensitized trachea. Ouabain (0.1–10 μmol/l) and vanadate (0.01–5 mmol/l) inhibited cooling-induced-contraction to a greater extent in normal than in sensitized trachea. Polymyxin B (0.5 mmol/l) selectively depressed responses to cooling in sensitized trachea. In a separate series of experiments, it was shown that sensitized trachea was hyperresponsive to ouabain and vanadate. Previous cooling to 20°C abolished responses to ouabain but only attenuated those to vanadate. These results are compatible with an enhancement of Na+,K+-ATPase and Ca2+-ATPase activities in sensitized trachea and further support the notion that intracellularly stored Ca2+ plays a decisive role in the activation of sensitized tracheal muscle.

EFFECTS OF DANTROLENE ON THE RESPONSES TO METHYLXANTHINES IN THE ISOLATED GUINEA-PIG TRACHEA

The effect of dantrolene on the responses to methylxanthines (caffeine and theophylline) and a beta-adrenoceptor agonist (salbutamol) was studied in isolated guinea-pig trachea. Caffeine and theophylline (1 microM-10 mM) and salbutamol (1 nM-10 microM) produced concentration-dependent relaxation of spontaneous and stimulated (acetylcholine 1 mM) tone. Responses to high concentrations (10 mM) of caffeine and theophylline added to tracheal strips either unstimulated (spontaneous tone) or indomethacin (2.8 microM)-treated were reversed to contractions in the presence of dantrolene (3 microM-0.3 mM). This effect was not observed for salbutamol or when relaxant responses to the agonists were generated in pre-contracted (acetylcholine 1 mM) strips. Dantrolene inhibited concentration dependently the contraction evoked by caffeine (1 mM) in indomethacin (2.8 microM)-treated strips at 20 degrees C. This effect of dantrolene was attenuated in a low Ca2+ (0.8 mM) medium or when the bath temperature was lowered to 10 degrees C. Although an intracellular site of action cannot be excluded these results suggest that dantrolene mainly interferes with transmembrane Ca2+ movements in the guinea-pig trachea.

Modification by indomethacin of airway contractile responses in normal and sensitized guinea-pigs

Active sensitization of guinea-pigs resulted in an increase in responsiveness and sensitivity of tracheal and lung parenchymal strips to CaCl2 (in K+-depolarised tissue), KCl, acetylcholine and histamine. Indomethacin (5 microM) preferentially enhanced the response of tracheal strips from normal animals to histamine and to a lesser extent acetylcholine but not to CaCl2 or KCl. A similar trend was observed in sensitized tissues. Indomethacin pretreatment did not cause changes in responsiveness or sensitivity of lung parenchymal strips from normal or sensitized guinea-pigs to the agonists tested. It is concluded that immunological sensitization produced a non-specific hyperresponsiveness in trachea and lung parenchymal strips. Conversely, cyclooxygenase inhibition by indomethacin elicited a selective increase in the responsiveness to certain agonists in central but not in the peripheral airways.

Effect of dantrolene sodium in isolated guinea-pig trachea

The effect of dantrolene sodium (3 microM-0.3 mM) on the spontaneous tone and responses to various contractile agonists was studied in isolated guinea-pig trachea. Dantrolene produced a concentration-related inhibition of the spontaneous tracheal tone, reaching a value of 94.8 +/- 4.8% of the relaxation induced by caffeine 10 mM. Removal of the epithelium did not affect the dantrolene-induced relaxation. Dantrolene did not alter the concentration-response curve for KCl and produced only small displacements of the concentration-response curves for CaCl2, acetylcholine and histamine, without affecting their maximal effects. Dantrolene dose relatedly inhibited the contraction induced by caffeine (1 mM) in Krebs solution containing indomethacin (2.8 microM) at 20 degrees C. The spasm induced by caffeine in Ca2(+)-free Krebs solution (20 degrees C, indomethacin 2.8 microM) was slightly depressed by dantrolene. Dantrolene did not depress the Ca2+ (1 microM)-induced contraction in skinned trachea. These results suggest that besides a possible intracellular site of action, the mechanism of the inhibitory effect of dantrolene in guinea-pig trachea may be related to interference with Ca2+ entry through pathways not susceptible to calcium channel blockers.

Different Ability of Trifluoperazine to Inhibit Agonist‐induced Contraction of Lung Parenchyma Strips from Control and Sensitized Guinea‐pigs

Abstract— There is increasing interest in the therapeutic potential of calcium antagonists in asthma. Among them the use of calmodulin antagonists deserves consideration. In the present work the effect of trifluoperazine on contractions generated by different mechanisms (CaCl2, KCl, acetylcholine, histamine and 5‐hydroxytryptamine) in lung parenchyma strip isolated from control and actively sensitized guinea‐pigs has been studied. Trifluoperazine produced both in unsensitized and sensitized lung strips, a concentration‐dependent, right, downward displacement of the concentration‐response curves to the agonists used, although the sensitization procedure resulted in a potentiation in the ability of trifluoperazine to inhibit agonist‐induced contractions. The basis for this greater potency of trifluoperazine in sensitized tissues remains to be elucidated but raises attention to the future use of selective calmodulin antagonists in the management of asthma.

Responses to histamine and selective H2-receptor agonists in lung parenchymal strips from normal and sensitized guinea-pigs

Histamine produces concentration-dependent contractions of lung parenchyma strips obtained from normal and sensitized guinea-pigs. The responsiveness of the sensitized lung strips to histamine was significantly increased compared to normal tissues. Clemizole (0.1 μM) was equally effective as an H1-antagonist in normal (dose-ratio 9.12) and sensitized (dose-ratio 9.77) tissues.The concentration-response curves to histamine were displaced to the left by cimetidine (0.1 μM to 0.1 mM) with similar dose-ratios in normal and sensitized tissues. Cimetidine enhanced maximal responses to histamine only in normal lung strips. The effects of submaximal equieffective concentrations of histamine were augmented to the same extent by cimetidine (0.1 mM) in normal and sensitized tissues. The responses to histamine were not modified by indomethacin (5 μM).The responsiveness and sensitivity of sensitized lung strips to isoprenaline, impromidine, 4-methylhistamine and dimaprit were not different from those of normal tissues. Cimetidine yielded, as antagonist of dimaprit, similar pA2 values in normal and sensitized tissues.In conclusion, there is no experimental evidence in favour of the existence of an impairment of H2-receptor activity in sensitized airways. Hyperreactivity to histamine is probably due to differences between normal and sensitized tissues with respect to Ca2+ entry and/or intracellular Ca2+ release in response to H1-receptor activation.

Evaluation of Mucociliary Clearance by Three Dimension Micro-CT-SPECT in Guinea Pig: Role of Bitter Taste Agonists

Different image techniques have been used to analyze mucociliary clearance (MCC) in humans, but current small animal MCC analysis using in vivo imaging has not been well defined. Bitter taste receptor (T2R) agonists increase ciliary beat frequency (CBF) and cause bronchodilation but their effects in vivo are not well understood. This work analyzes in vivo nasal and bronchial MCC in guinea pig animals using three dimension (3D) micro-CT-SPECT images and evaluates the effect of T2R agonists. Intranasal macroaggreggates of albumin-Technetium 99 metastable (MAA-Tc99m) and lung nebulized Tc99m albumin nanocolloids were used to analyze the effect of T2R agonists on nasal and bronchial MCC respectively, using 3D micro-CT-SPECT in guinea pig. MAA-Tc99m showed a nasal mucociliary transport rate of 0.36 mm/min that was increased in presence of T2R agonist to 0.66 mm/min. Tc99m albumin nanocolloids were homogeneously distributed in the lung of guinea pig and cleared with time-dependence through the bronchi and trachea of guinea pig. T2R agonist increased bronchial MCC of Tc99m albumin nanocolloids. T2R agonists increased CBF in human nasal ciliated cells in vitro and induced bronchodilation in human bronchi ex vivo. In summary, T2R agonists increase MCC in vivo as assessed by 3D micro-CT-SPECT analysis.