ANNEXIN I IN FIBROTIC RAT LUNG AND CULTURED LUNG FIBROBLASTS FOLLOWING IRRADIATION

Radiation-induced lung fibrosis is a result of collagen accumulation i n the interstitium, partly due to increased collagen synthesis by fibr oblasts. One feature of active collagen synthesis is increased membran e trafficking in the fibroblasts. A group of proteins called annexins is believed to play a regulatory role in membrane fusion and exocytosi s. Therefore, increased annexin activity might be expected in the fibr otic lung. We tested this hypothesis by measuring annexin I levels, hy droxyproline content and ultrastructural changes in radiation-induced pulmonary fibrosis in rat. Three months after a single exposure to 30 Gy of X-rays to the right hemithorax, the right lung of the rat was at rophied and fibrotic with a concomitant increase in size of the shield ed left lung. Electron micrographs revealed that the irradiated lung w as ladened with interstitial collagen fibrils, with increased number o f fibroblasts amongst them. Hydroxyproline concentration in the irradi ated lung was nearly twice that in the sham-irradiated lung. Annexin I in the irradiated lung, on the other hand, was markedly reduced, and barely detectable on immunoblots. Since increased annexin I might prec ede enhanced collagen production, we also measured annexin I levels in rat lungs 3 days after 30 Gy irradiation and correlated that with hyd roxyproline concentration. We found no appreciable difference in annex in I levels and hydroxyproline content between sham-irradiated and irr adiated lungs at 3 days. To determine whether annexin I levels in cult ured fibroblasts were altered by irradiation, we assayed annexin I in cultured rat lung fibroblasts 3 days after 0-10 Gy exposure, with conc omitant measurement of C-14-proline incorporation. The annexin I level in fibroblasts irradiated with 10 Gy X-rays was 55% higher than in sh am-irradiated fibroblasts. However, incorporation of C-14-proline into collagenase-sensitive macromolecules in the culture medium and extrac ellular matrix was not different between these two groups of cells. Th ese data demonstrate a radiation-induced increase in immunoreactive an nexin I in cultured lung fibroblasts, but fail to support the hypothes is of a positive correlation between annexin I concentration and fibro sis in irradiated rat lung.