ANNEXIN-I CONCENTRATION, PHOSPHOLIPASE-ACTIVITY AND THROMBOXANE SYNTHESIS IN IRRADIATED RAT LUNG

Annexin I, a member of a family of Ca2+-dependent phospholipid-binding proteins (PLBP), has been suggested as a regulator of prostaglandin m etabolism as a result of its inhibitory effect on phospholipase A(2). Synthesis of prostaglandin is increased in irradiated tissue, but the mechanism underlying this increase has not been delineated. It is conc eivable that a decrease in the level of annexin I resulting in increas ed phospholipase activity may be responsible for the enhanced synthesi s of prostaglandin. Accordingly, we measured the level of a lung 36 kD a PLBP, which possesses characteristics of annexin I, as well as the a ctivity of phospholipase and the synthesis of thromboxane A(2) (TXA(2) ) in irradiated rat lung. The right lung of rats was irradiated with 0 , 15 or 30 Gy of X rays and the animals were sacrificed after 3 months . Phospholipid binding protein was assayed by its ability to transfer unilamellar liposomes to multilamellar liposomes and by immunoblotting against anti-36 kDa rabbit PLBP antisera. Production of TXA(2) by min ced lung tissue was determined by radioimmunoassay of its stable metab olite TXB(2). Phospholipase activity was assayed by hydrolysis of [C-1 4]dioleoylphosphatidylcholine. Our results showed that PLBP activity i n the lungs irradiated with 30 Gy was lower than that in the lungs irr adiated with 0 and 15 Gy (8.82 +/- 0.47 compared to 9.73 +/- 0.49 and 9.95 +/- 0.78 nmol phospholipid transferred/mg protein, respectively). Western blotting demonstrated a near total depletion of annexin I in the lungs irradiated with 30 Gy. Phospholipase activity was also lower in the lungs irradiated with 30 Gy compared to that in the lungs irra diated with 0 Gy (0.23 +/- 0.01 vs 0.32 +/- 0.01 nmol phosphatidylchol ine liberated/mg protein/min, P < 0.001). Reduced phospholipase activi ty was observed not only in the cytosolic or soluble fraction of lung homogenate, but also in precipitates obtained after 21,000g and 100,00 0g centrifugation. Despite this decline in phospholipase activity, the re was a 2.8-fold increase in the synthesis of thromboxane (367 +/- 65 compared to 1076 +/- 143 pg TXB(2)/mg tissue/10 min for lungs irradia ted with 0 and 30 Gy, respectively). These results are not consistent with the hypothesis that increased synthesis of thromboxane A(2) in ir radiated rat lung is a direct result of elevated phospholipase activit y. In fact, phospholipase activity is decreased in the irradiated lung , despite a decline in the concentration of annexin I, its putative in hibitor. (C) 1995 by Radiation Research Society