β2 -Adrenoceptor agonist-mediated inhibition of human airway smooth muscle cell proliferation: importance of the duration of β 2 -adrenoceptor stimulation

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Abstract
Airway hyperresponsiveness in asthma has been ascribed to airway wall thickening as a result of smooth muscle proliferation and hypertrophy. We have previously shown that continuous exposure to the β2‐adrenoceptor agonist, salbutamol inhibits mitogen‐induced proliferation of airway smooth muscle cells. In the present study, the effects of variable durations and repeated periods of exposure to β2‐adrenoceptor agonists on DNA synthesis in human cultured airway smooth muscle have been investigated to model some of the possible pharmacokinetic profiles of these agents following inhalation. DNA synthesis was measured by [3H]‐thymidine incorporation. Shorter periods of exposure (up to 2.5 h) of airway smooth muscle cells to salbutamol (100 nM) commencing 30 min before thrombin (0.3 u ml−1) stimulation had no effect on the subsequent increase in [3H]‐thymidine incorporation. However, inhibition by salbutamol was evident with a 4.5 h exposure and was maximal after an 8.5 h exposure. Similar patterns of results were observed when fenoterol (100 nM) was used in place of salbutamol as the β2‐adrenoceptor agonist or when epidermal growth factor (300 pM) was used in place of thrombin as the mitogen. Salbutamol had no effect on thrombin‐stimulated [3H]‐leucine incorporation after 8.5 h of exposure, but a statistically significant effect was observed after 48 h of exposure. Experiments in which DNA synthesis was measured up to 52 h after the addition of thrombin indicated that exposure to salbutamol during the first 8 h of mitogen stimulation delayed rather than inhibited the DNA synthesis. Addition of salbutamol (100 nM) at different times either before or up to 24 h after the addition of thrombin indicated that [3H]‐thymidine incorporation (measured between 24 and 28 h after thrombin) could be significantly attenuated when salbutamol was added as late as 18 h after the addition of thrombin. The effects of more prolonged exposure to salbutamol were investigated by the addition of salbutamol for either 15 or 24 h per day for a total of 3 days. There were no significant differences in the level of inhibition of thrombin‐stimulated [3H]‐thymidine incorporation between continuous and intermittent salbutamol over the 3 day period and the inhibition was also not different to that achieved with a single continuous exposure to salbutamol over 28 h. These results indicate that although exposure to β2‐adrenoceptor agonists during the first 8 h of mitogen stimulation does not have a sustained inhibitory effect on DNA synthesis, repeated intermittent or prolonged continuous exposures to salbutamol do inhibit DNA synthesis, without evidence of marked desensitization.