Figure 3.

Leukotriene synthesis by mouse retina. (A) Western blot analysis (left) and RT-PCR (right) analysis of mouse retina (lane 1) did not detect 5-lipoxygenase expression. As anticipated, 5-lipoxygenase was detected in mouse bone marrow cells (lane 2). Data are representative of the results in retinas isolated from six different mice. (B) Mass spectrometric analysis of methanol extracts of mouse retina did not detect leukotriene metabolites. Upper tracing: internal standard deuterated LTB₄ ([d₄]-LTB₄). After the addition of LTA₄ to the mouse retina, LTB₄ was produced (lower tracing). Nonenzymatic generation of Δ⁶-trans-LTB₄ was also detected. (C) Increasing the dose of exogenous LTA₄ (0–300 nM) resulted in an increase in endogenous LTB₄ production. The dose-response curve was generated using six retinas, with each retina receiving a different dose of LTA₄. (C, inset) After the addition of 100 nM LTA₄, LTB₄ synthesis was increased in retinas from diabetic mice (D, black bar) compared with retinas from nondiabetic mice (N, white bar; n = 3 retinas per group; *P < 0.02). (D) By Western blot analysis, the expression of LTA₄ hydrolase in retinas of diabetic mice (D, n = 6) was increased compared with that of nondiabetic mice (N, n = 6; *P < 0.001). The level of protein expression is depicted relative to the reference protein β-tubulin.