ROCK1 translocates from non-caveolar to caveolar regions upon KCl stimulation in airway smooth muscle.

Airway smooth muscle (ASM) membrane depolarization through KCl opens L-type voltage dependent Ca2+ channels (Ca(v)1.2); its opening was considered the cause of KCl contraction. This substance is used to bypass intracellular second messenger pathways. It is now clear that KCl also activates RhoA/Rho kinase (ROCK) pathway. ROCK isoforms are characterized as ROCK1 and ROCK2. Because ROCK1 seems the most abundant isotype in lung, we studied its participation in KCl stimulated bovine ASM. With methyl-beta-cyclodextrin (MbetaCD) we disrupted caveolae, a membrane compartment considered as the RhoA/ROCK assembly site, and found that KCl contraction was reduced to the same extent (~26%) as Y-27632 (ROCK inhibitor) treated tissues. We confirmed that KCl induces ROCK activation and this effect was annulled by Y-27632 or MbetaCD. In isolated plasmalemma, ROCK1 was localized in non-caveolar membrane fractions in Western blots from control tissues, but it transferred to caveolae in samples from tissues stimulated with KCl. Ca(v)1.2 was found at the non-caveolar membrane fractions in control and MbetaCD treated tissues. In MbetaCD treated tissues stimulated with KCl, contraction was abolished by nifedipine; only the response to Ca(v)1.2 opening remained as the ROCK component disappeared. Our results show that, in ASM, the KCl contraction involves the translocation of ROCK1 from non-caveolar to caveolar regions and that the proper physiological response depends on this translocation.

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.

Chronic exposure to ozone causes tolerance to airway hyperresponsiveness in guinea pigs: lack of SOD role.

Tolerance to respiratory effects of O3 has been demonstrated for anatomic and functional changes, but information about tolerance to O3-induced airway hyperresponsiveness (AHR) is scarce. In guinea pigs exposed to air or O3 (0.3 parts/million, 4 h/day, for 1, 3, 6, 12, 24, or 48 days, studied 16-18 h later), pulmonary insufflation pressure changes induced by intravenous substance P (SP, 0.032-3.2 micro ug/kg) were measured, then the animals were subjected to bronchoalveolar lavage (BAL). Bronchial rings with or without phosphoramidon were also evaluated 3 h after air or a single O3 exposure. O3 caused in vivo AHR (increased sensitivity) to SP after 1, 3, 6, 12, and 24 days of exposure compared with control. However, after 48 days of exposure, O3 no longer caused AHR. Total cell, macrophage, neutrophil, and eosinophil counts in BAL were increased in most O3-exposed groups. When data from all animals were pooled, we found a highly significant correlation between degree of airway responsiveness and total cells (r = 0.55), macrophages (r = 0.54), neutrophils (r = 0.47), and eosinophils (r = 0.53), suggesting that airway inflammation is involved in development of AHR to SP. Superoxide dismutase (SOD) levels in BAL fluids were increased (P < 0.05) after 1, 3, 6, and 12 days of O3 exposure and returned to basal levels after 24 and 48 days of exposure. O3 failed to induce hyperresponsiveness to SP in bronchial rings, and phosphoramidon increased responses to SP in air- and O3-exposed groups, suggesting that neutral endopeptidase inactivation was not involved in O3-induced AHR to SP in vivo. We conclude that chronic exposure to 0. 3 ppm O3, a concentration found in highly polluted cities, resulted in tolerance to AHR to SP in guinea pigs by an SOD-independent mechanism.