Abnormal growth of airway smooth muscle may play an important role in the pathogenesis of human airway diseases. Little is known about the proliferative responses of cultured airway smooth muscle cells, nor of the precise pathways responsible for mitogenesis in these cells. We assessed DNA synthesis, cell proliferation, and mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes after exposure to four potential mitogens: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and 5-hydroxytryptamine (5-HT). Stimulation with either PDGF or IGF-1 induced substantial increases in DNA synthesis and cell number, as reflected by [3H]thymidine incorporation, flow cytometry, and methylene blue staining. Treatment with EGF or 5-HT, on the other hand, induced only modest DNA synthesis and no increase in cell number. Immunoblots and kinase renaturation assays of cell extracts demonstrated activation of both the 42- and 44-kDa MAP kinases within minutes of either PDGF, IGF-1, EGF, or 5-HT exposure. However, relative to EGF and 5-HT stimulation, late-phase MAP kinase activation was significantly greater after treatment with the mitogens PDGF and IGF-1. We conclude that in cultured bovine tracheal myocytes 1) PDGF and IGF-1 are potent mitogens; 2) MAP kinase may be activated subsequent to stimulation of either receptor tyrosine kinases (PDGF, EGF, IGF-1) or G protein-linked receptors lacking in known tyrosine kinase activity (5-HT); and 3) unsustained MAP kinase activation is insufficient for mitogenesis. Finally, the finding that mitogenicity correlates with the late phase of MAP kinase activation is consistent with the notion that sustained MAP kinase activation is important for bovine tracheal myocyte proliferation.

We examined the effects of the bronchoconstrictor agonists serotonin (5-hydroxytryptamine; 5-HT) and histamine on mitogen-activated protein (MAP) kinase activation in cultured bovine tracheal myocytes. Kinase renaturation assays demonstrated activation of the 42- and 44-kDa MAP kinases within 2 min of 5-HT exposure. MAP kinase activation was mimicked by alpha-methyl-5-HT and reduced by pretreatment with either phorbol 12,13-dibutyrate or forskolin, suggesting activation of the 5-HT2 receptor, protein kinase C, and Raf-1, respectively. Raf-1 activation was confirmed by measurement of Raf-1 activity, and the requirement of Raf-1 for 5-HT-induced MAP kinase activation was demonstrated by transient transfection of cells with a dominant-negative allele of Raf-1. Histamine pretreatment significantly inhibited 5-HT and insulin-derived growth factor-1-induced MAP kinase activation. Attenuation of MAP kinase activation was reversed by cimetidine, mimicked by forskolin, and accompanied by cAMP accumulation and inhibition of Raf-1, suggesting activation of the H2 receptor and cAMP-dependent protein kinase A. However, histamine treatment inhibited Raf-1 but not MAP kinase activation following treatment with either platelet-derived growth factor or epidermal growth factor, implying a Raf-1-independent MAP kinase activation pathway. In summary, our data suggest a model whereby 5-HT activates MAP kinase via a protein kinase C/Raf-1 pathway, and histamine attenuates MAP kinase activation by serotonin via activation of cAMP-dependent protein kinase A and inhibition of Raf-1.

Exposure of 21-day-old Sprague-Dawley rats to hyperoxia (> 95% O2 for 8 days) causes thickening of the airway epithelial and smooth muscle layers. To test the hypothesis that hyperoxic exposure increases airway layer DNA synthesis, we labeled the nuclei of cells undergoing S-phase by administering the thymidine analog bromodeoxyuridine (BrdU). BrdU was administered on days 3 and 4, 5 and 6, or 7 and 8 of air or O2 exposure, and the lungs were harvested immediately thereafter. Histologic sections were stained with an avidin-biotin-immunoperoxidase stain that revealed BrdU incorporation into nuclei, and a hematoxylin counterstain. After 4 days of air or O2 exposure, there was no difference in BrdU fractional labeling between control and hyperoxic animals. Thereafter, fractional BrdU labeling of the small airway (circumference < 1,000 microns) epithelium and smooth muscle layer was significantly increased in O2-exposed animals (P < 0.01, unpaired t test). The fractional labeling of larger, central airway smooth muscle layer cells was also increased after 8 days of O2 exposure (P < 0.01). In another cohort of O2-exposed animals, measurements of airway layer dimensions demonstrated increases in small airway epithelial and smooth muscle layer thickness that paralleled the time course seen for BrdU incorporation. We conclude that O2 exposure of immature rats increases airway epithelial and smooth muscle layer cellular DNA synthesis. These data suggest that hyperplasia of airway epithelial and smooth muscle layer cells may contribute to hyperoxia-induced airway remodeling.

We recently found that exposure of 21-day-old rats to hyperoxia (> 95% O2 for 8 days) significantly increased in vivo airway cholinergic responsiveness and that O2 exposure also increased airway epithelial and smooth muscle layer thicknesses in a separate cohort of animals. There was substantial variation in the magnitude of both the functional and structural responses to hyperoxia. The present study was designed to test whether the magnitude of O2-induced airway remodeling could account for individual differences in airway responsiveness after O2 exposure, as well as for the difference in responsiveness between air- and O2-exposed animals. We assessed in vivo airway responsiveness to aerosolized acetylcholine (ACh) and airway architecture in 14 O2- and 5 air-exposed, immature rats. Total respiratory system resistance was determined using a plethysmographic method. The mean thicknesses and fractional areas of the airway epithelial and smooth muscle layers were determined by contour tracing using a digitizing pad and microcomputer. Both the small (circumference < 1,000 microns) and central (circumference 1,000 to 4,000 microns) airways were studied. For O2-exposed rats, individual values of EC200 ACh correlated negatively with small airway smooth muscle layer thickness (r = -0.59, p < 0.05; ANOVA), small airway smooth muscle layer fractional area (r = -0.75, p < 0.01), small airway epithelial thickness (r = -0.54, p < 0.05), small airway epithelial fractional area (r = -0.69, p < 0.01), and central airway smooth muscle layer thickness (r = -0.53, p < 0.05). When both air- and O2-exposed animals were considered, EC200 ACh correlated negatively with all eight parameters of airway layer thickness and fractional area.(ABSTRACT TRUNCATED AT 250 WORDS)