Beta 2-adrenoceptor agonist-mediated inhibition of human airway smooth muscle cell proliferation: importance of the duration of beta 2-adrenoceptor stimulation.

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 beta 2-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 beta 2-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. 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-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 beta 2-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. 3. 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. 4. 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. 5. 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. 6. These results indicate that although exposure to beta 2-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.

The effect of glucocorticoids on proliferation of human cultured airway smooth muscle.

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. 2. Pretreatment of smooth muscle cells with dexamethasone (100 nM, 60 min) inhibited thrombin-induced increases in [3H]-thymidine incorporation (DNA synthesis) and cell number. 3. Inhibition of thrombin-induced [3H]-thymidine incorporation was also observed with hydrocortisone (0.01-1 microM) and methylprednisolone (0.001-0.1 microM) pretreatment. In contrast, pretreatment with either testosterone (0.001-1 microM) progesterone (0.001-1 microM), 17 beta-oestradiol (0.001-1 microM), or aldosterone (0.001-1 microM) had no effect on the response to thrombin. 4. Responses to a range of mitogens including thrombin (0.01-. 10 u ml-1), epidermal growth factor (EGF, 3-3000 pM), basic fibroblast growth factor (bFGF, 0.3-300 pM) and foetal calf serum (FCS, 0.1-10% v/v) were inhibited by dexamethasone (100 nM) pretreatment. However, the magnitude of the inhibitory effect was dependent on the mitogen, with EGF being the least, and thrombin being the most sensitive to the inhibitory effect. 5. The potency of hydrocortisone as an inhibitor of [3H]-thymidine incorporation was reduced when FCS (10% v/v, which caused a 40 fold increase in [3H]-thymidine incorporation) was used as the mitogen in place of thrombin (0.3 u ml-1, which caused a 10 fold increase in [3H]-thymidine incorporation). 6. The effect of post-treatment with dexamethasone (100 nM) indicated that addition of the glucocorticoid up to 17-19 h after thrombin (0.3 u ml-1) produced similar degrees of inhibition to those obtained when it was added as a pretreatment. Dexamethasone no longer produced an inhibitory effect if added 21 h or more after the addition of thrombin. 7. These results suggest that glucocorticoids regulate airway smooth muscle proliferation initiated by a range of stimuli. This effect may be of importance in the therapeutic actions of these compounds in asthma, particularly when they are used for prolonged periods of time.

Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels.

The link between increased usage of beta-adrenoceptor agonists and worsening of asthma symptoms has raised interest in the effects of agents such as salbutamol on airway wall remodelling, and particularly airway smooth muscle proliferation. In the present study we have investigated the role of increases in intracellular cAMP in the inhibitory effect of salbutamol on airway smooth muscle proliferation. The inhibitory effects of a combination of submaximally effective concentrations of salbutamol (10 nM) and the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 100 microM) on thrombin (0.3 U/mL)-induced mitogenesis in human cultured airway smooth muscle cells was greater than that for either agent alone. In addition, agents known to increase cAMP-dependent protein kinase activity including forskolin (10 microM), 8-bromoadenosine-3',5'-cyclic monophosphate (100 microM), and prostaglandin E2 (1 microM) have an inhibitory effect on thrombin (0.3 U/mL)-induced induced proliferation. Furthermore, the cAMP antagonist, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (300 microM) significantly reduced the inhibitory effect of salbutamol (10 nM) on thrombin (0.3 U/mL)-induced DNA synthesis. In IBMX (100 microM)-pretreated cells, salbutamol (100 nM) increased intracellular cAMP levels via stimulation of a beta 2-adrenoceptor. Salbutamol (10 microM), at concentrations supramaximally effective for inhibition of mitogenesis, had no effect on thrombin (0.3 U/mL)-induced increases in intracellular calcium levels. Therefore, our results suggest that the previously reported inhibition of mitogen-induced proliferation in human cultured airway smooth muscle cells by the beta 2-adrenoceptor agonist, salbutamol (100 nM), is at least partly due to elevation of intracellular cAMP, while there is no effect of salbutamol on initial mitogen-induced increases in intracellular calcium.

Tumor necrosis factor alpha modulates mitogenic responses of human cultured airway smooth muscle.

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.

Platelet-activating factor biosynthesis in rat vascular smooth muscle cells.

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 microM). 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 microM). Thus, endogenous PAF is unlikely to be involved in the endothelin-1-induced calcium increases. Although WEB 2086 (3-100 microM) inhibited concentration dependently fetal calf serum (10% v/v) induced [3H]-thymidine incorporation, reaching a maximum effect at 30 microM 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.

Inhibition by salbutamol of the proliferation of human airway smooth muscle cells grown in culture.

1 beta 2-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 (0.1-100 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-1)-stimulated cells were reduced by salbutamol (100 nM) by 27.7 +/- 2.8%. Similarly, thrombin (0.3 u ml-1)-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 beta-adrenoceptor antagonist, propranolol (0.3 microM) or the selective beta 2-adrenoceptor antagonist, ICI 118551 (50 nM). 5. These results indicate that salbutamol, through activation of a beta 2-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 Beta2-adrenoceptor agonists would override any indirect action to accelerate airway smooth muscle proliferation. These observations lead us to suggest that Beta2-adrenoceptor agonists exacerbate asthma by mechanisms unrelated to airway smooth muscle proliferation.

Airway wall remodelling in asthma: a novel target for the development of anti-asthma drugs.

The regulation of vascular smooth muscle proliferation has been examined extensively in recent years since the recognition that hyperplasia and hypertrophy of vascular smooth muscle are significant pathological features of hypertension and atherosclerosis. In this article, Alastair Stewart and colleagues discuss recent developments in the investigation of the pathology of asthma that indicate some parallels with the structural changes that occur in blood vessels in hypertension and atherosclerosis.