1 P2-Adrenoceptor agonists may exacerbate asthma by reducing the release of the anti-proliferative and anti-inflammatory molecule, heparin from mast cells in the airway. In this study, the direct effects of the clinically used bronchodilator, salbutamol, on the proliferation of airway smooth muscle cells grown in culture and stimulated with a range of mitogens have been examined.2 In mitogen-stimulated cells, salbutamol (0.1-I00 nM) inhibited [3H]-thymidine incorporation in a concentration-dependent manner. Salbutamol (100 nM) pretreatment reduced the mitogenic responses to thrombin (0.3 u ml-1), epidermal growth factor (EGF) (300 pM) and U46619 (100 nM) by 61.7 ± 6.1%, 46.9 ± 13.9% and 57.6 ± 12.7%, respectively. However, salbutamol pretreatment did not appear to reduce the small mitogenic response to endothelin-1. 3 Increases in [3H]-leucine incorporation in thrombin (0.3 u ml-')-stimulated cells were reduced by salbutamol (100 nM) by 27.7 ± 2.8%. Similarly, thrombin (0.3 u ml`)-stimulated increases in cell number were also inhibited by salbutamol (100 nM) pretreatment. Thus, the effect of salbutamol in decreasing thrombin-induced [3H]-leucine incorporation may, at least in part, be explained by inhibition of cell proliferation. 4 The inhibition of cell proliferation by salbutamol was prevented by pretreatment with either the non-selective P-adrenoceptor antagonist, propranolol (0.3 JAM) or the selective P2-adrenoceptor antagonist, ICI 118551 (50 nM).5 These results indicate that salbutamol, through activation of a P2-adrenoceptor, has a direct inhibitory effect on proliferation elicited by the mitogens thrombin, EGF, and U46619. Thus, it seems likely that this direct inhibitory action of P2-adrenoceptor agonists would override any indirect action to accelerate airway smooth muscle proliferation. These observations lead us to suggest that R2-adrenoceptor agonists exacerbate asthma by mechanisms unrelated to airway smooth muscle proliferation.

1 Airway smooth muscle proliferation is a significant component of the airway wall remodelling that occurs in asthma. In this study, the effects of glucocorticoids on mitogenic responses of human airway smooth muscle have been examined.

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1 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 b 2 -adrenoceptor agonist, salbutamol inhibits mitogen-induced proliferation of airway smooth muscle cells. In the present study, the e ects of variable durations and repeated periods of exposure to b 2 -adrenoceptor agonists on DNA synthesis in human cultured airway smooth muscle have been investigated to model some of the possible pharmacokinetic pro®les of these agents following inhalation. DNA synthesis was measured by [ 3 H]-thymidine incorporation. 2 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 71 ) stimulation had no e ect on the subsequent increase in [ 3 H]-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 b 2 -adrenoceptor agonist or when epidermal growth factor (300 pM) was used in place of thrombin as the mitogen. Salbutamol had no e ect on thrombinstimulated [ 3 H]-leucine incorporation after 8.5 h of exposure, but a statistically signi®cant e ect was observed after 48 h of exposure. 3 Experiments in which DNA synthesis was measured up to 52 h after the addition of thrombin indicated that exposure to salbutamol during the ®rst 8 h of mitogen stimulation delayed rather than inhibited the DNA synthesis. 4 Addition of salbutamol (100 nM) at di erent times either before or up to 24 h after the addition of thrombin indicated that [ 3 H]-thymidine incorporation (measured between 24 and 28 h after thrombin) could be signi®cantly attenuated when salbutamol was added as late as 18 h after the addition of thrombin.5 The e ects 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 signi®cant di erences in the level of inhibition of thrombin-stimulated [ 3 H]-thymidine incorporation between continuous and intermittent salbutamol over the 3 day period and the inhibition was also not di erent to that achieved with a single continuous exposure to salbutamol over 28 h. 6 These results indicate that although exposure to b 2 -adrenoceptor agonists during the ®rst 8 h of mitogen stimulation does not have a sustained inhibitory e ect on DNA synthesis, repeated intermittent or prolonged continuous exposures to salbutamol do inhibit DNA synthesis, without evidence of marked desensitization.

Airway wall remodeling, including hyperplasia of airway smooth muscle, is regarded as an important contributor to airway hyperresponsiveness in asthmatic patients. The effects of the proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha) on the mitogenic responses of human cultured airway smooth muscle have been investigated. Lower concentrations of TNF alpha (0.3 to 30 pM) had a small, delayed (48-h incubation), stimulatory effect on DNA synthesis that was blocked by dexamethasone (1 microM), aspirin (100 microM), or primaquine (30 microM) pretreatment, indicating that this effect was secondary to the release of cyclooxygenase products. TNF alpha (300 pM; 24- to 48-h incubation) alone had no effect on cell number or DNA or protein synthesis, but markedly reduced the stimulatory effects of thrombin (0.3 U/ml). TNF alpha (300 pM) also inhibited mitogenic responses to fetal calf serum (10%), epidermal growth factor (300 pM), and the thromboxane A2 mimetic U46619 (100 nM), indicating a nonselective effect. The inhibitory effects of TNF alpha (300 pM) were not blocked by pretreating the cells with the cyclooxygenase inhibitor aspirin (100 microM), the 5-lipoxygenase inhibitor CGS 8515 (3 microM), or the nitric oxide synthase inhibitor nitro-iminoethyl-L-ornithine (100 microM), suggesting that neither arachidonic acid metabolites nor nitric oxide were mediators of the inhibitory effect. The phospholipase A2 inhibitor primaquine (30 microM) had no effect on the inhibitory responses to TNF alpha, whereas the anti-inflammatory steroid dexamethasone (1 microM) prevented TNF alpha inhibition of mitogenic responses. Thus, concentrations of TNF alpha, within the range detected in bronchoalveolar lavage fluid from asthmatics, suppress mitogenic responses by a mechanism that is sensitive to inhibition by anti-inflammatory steroids, but does not appear to involve established targets for modulation by steroids, including arachidonic acid metabolism or induction of nitric oxide synthase.

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The ability of platelet-activating factor (PAF) receptor antagonists to protect rats from the cardiovascular collapse induced by large doses of endothelin 1 led us to examine the capacity of rat cultured vascular smooth muscle cells to produce PAF and also to evaluate its potential functional roles in this cell type. Adenosine triphosphate and the vasoactive peptides, endothelin 1, angiotensin II, and arginine vasopressin, each elicited an increase in the PAF level in extracts of rat cultured vascular smooth muscle cells as determined by bioassay. PAF was not detectable (above 20 fmol/mg protein) in the supernatants of these cells. The identity of the bioactivity as PAF was confirmed by GC/MS which indicated that more than 80% of the PAF was 1-O-hexadecyl-2-acetyl-3-sn-glyceryl-phosphorylcholine. Exogenous PAF (100 nM) elicited increases in intracellular calcium that were inhibited by WEB 2086 (10 µM). Endothelin 1, at a concentration which stimulated PAF synthesis, (1 nM), elicited increases in intracellular calcium levels that were not inhibited by WEB 2086 (10 µM). Thus, endogenous PAF is unlikely to be involved in the endothelin-1-induced calcium increases. Although WEB 2086 (3–100 µM) inhibited concentration dependently fetal calf serum (10% v/v) induced [³H]-thymidine incorporation, reaching a maximum effect at 30 µM of 40-50% reduction, in parallel experiments WEB 2086 had no effect on serum-induced increases in cell numbers. We conclude that PAF is produced and retained by cultured rat vascular smooth muscle and that it is unlikely to contribute to the signaling of increases in intracellular calcium or proliferation.