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* From the Institute for Environmental Medicine (Drs. Abraham, DeBolt, and Koval), Departments of Pediatrics (Dr. Savani) and Physiology (Dr. Koval), University of Pennsylvania School of Medicine, Philadelphia, PA.
Correspondence to: Michael Koval, PhD, University of Pennsylvania Medical Center, 1 John Morgan Bldg/6068, 3620 Hamilton Walk, Philadelphia, PA 19104
Gap junction proteins (connexins) form channels that allow flow of aqueous cytoplasmic small molecules between neighboring cells. To examine phenotypic differences in gap junction expression, primary rat type II alveolar epithelial cells were cultured either in minimum essential medium (MEM) + 10% fetal bovine serum or on Madin-Darby canine kidney-derived extracellular matrix in MEM + 2% fetal bovine serum + 5 ng/mL keratinocyte growth factor (KGF). Type II cells grown in KGF retained surfactant protein C (SP-C) expression, while cells cultured in MEM showed little, if any, SP-C production. By reverse transcriptase-polymerase chain reaction analysis, type II cells cultured in MEM showed increasing expression of Cx43 and Cx46 messenger RNA and decreasing expression of Cx26, Cx32, and Cx37. In contrast, cells cultured in KGF retained expression of Cx26 and Cx32. By immunofluorescence microscopy, type II cells cultured for 4 days in MEM had Cx43 and Cx46 present at the cell surface. Cx32 and Cx43 were present in the plasma membrane of type II cells cultured in KGF; however, Cx46 was retained in an intracellular compartment, most likely corresponding to the trans Golgi network.
These differences in gap junction composition had functional consequences, since type II cells grown in MEM showed higher levels of Lucifer yellow dye transfer (12 to 13 cells per microinjection) than cells grown in KGF (3 to 4 cells per microinjection).
Bleomycin treatment of Sprague-Dawley rats was used as a whole animal model system for studying changes in connexin expression that occur during lung injury. Rats were instilled intratracheally with 2 U of bleomycin in 250 µL saline solution vehicle, or with vehicle alone. Lungs were then examined 2 to 14 days following treatment for connexin expression using diaminobenzidine immunohistochemical staining. Cx43 was found to be expressed throughout the alveolar airspace, while Cx46 had a distribution consistent with expression by type II cells. In saline solution-treated animals, Cx46 appeared to translocate to the type II cell surface, prior to cell proliferation. Bleomycin-treated animals showed no Cx46 translocation and had lost most Cx46 and SP-C-positive cells by 7 days following treatment.
These data taken together indicate that the regulated control of gap junctions is important for recovery from lung injury. This suggests that disruption of normal gap junctional control by bleomycin plays a role in pulmonary fibrosis.
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