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 metabolism as a result of its inhibitory effect on phospholipase A₂. Synthesis of prostaglandin is increased in irradiated tissue, but the mechanism underlying this increase has not been delineated. It is conceivable that a decrease in the level of annexin I resulting in increased phospholipase activity may be responsible for the enhanced synthesis of prostaglandin. Accordingly, we measured the level of a lung 36 kDa PLBP, which possesses characteristics of annexin I, as well as the activity of phospholipase and the synthesis of thromboxane${\rm A}_{2}\ ({\rm TXA}_{2})$ 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 TXA₂ by minced lung tissue was determined by radioimmunoassay of its stable metabolite TXB₂. Phospholipase activity was assayed by hydrolysis of [¹⁴ C]dioleoylphosphatidylcholine. Our results showed that PLBP activity in the lungs irradiated with 30 Gy was lower than that in the lungs irradiated 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 irradiated with 0 Gy (0.23 ± 0.01 vs 0.32 ± 0.01 nmol phosphatidylcholine liberated/mg protein/min, P < 0.001). Reduced phospholipase activity was observed not only in the cytosolic or soluble fraction of lung homogenate, but also in precipitates obtained after 21,000g and 100,000g centrifugation. Despite this decline in phospholipase activity, there was a 2.8-fold increase in the synthesis of thromboxane (367 ± 65 compared to 1076 ± 143 pg${\rm TXB}_{2}/{\rm mg}$ tissue/10 min for lungs irradiated with 0 and 30 Gy, respectively). These results are not consistent with the hypothesis that increased synthesis of thromboxane A₂ in irradiated rat lung is a direct result of elevated phospholipase activity. In fact, phospholipase activity is decreased in the irradiated lung, despite a decline in the concentration of annexin I, its putative inhibitor.