Induction of cyclo-oxygenase-2 in human endothelial cells by SIN-1 in the absence of prostaglandin production

1 Nitric oxide (NO) regulates cyclo-oxygenase (COX) activity in various cel l systems and reports conflict in regard to its stimulatory versus inhibito ry role. Incubation of human umbilical vein endothelial cells (HUVEC) with SIN-1 (3-morpholinosydnonimine), a donor of NO, resulted in a rapid and dos e-dependent increase in the expression of COX-2 as analysed by Western and Northern blotting. 2 Incubation of HUVEC with SIN-1 and interleukine (IL)-1 alpha resulted in increased induction of COX-2 compared with IL-1 alpha alone and corresponde d to an additive effect. The COX-2 induction was dependent on a de novo syn thesis since cycloheximide, an inhibitor of protein synthesis, blocked the enzyme expression. The increase in COX-2 expression was not accompanied by a corresponding change in prostaglandin (PG) production. However, the COX a ctivity was partially recovered when immunoprecipitated COX-2 was incubated with arachidonic acid and haematin. 3 Peroxynitrite, a highly reactive nitrogen molecule derived from the inter action of NO and superoxide anion, significantly increased COX-2 expression . Under these conditions and within the limit of detection of the antibody, selective antibody for nitrotyrosine failed to detect nitrated COX-2 in im munoprecipitated COX-2 when cells where incubated with SIN-1 or SIN-1 + IL- 1 alpha. 4 Ro 31-8220, a specific inhibitor of protein kinase (PK) C, blocked the in duction of COX-2. Also, SB203580, the selective inhibitor of p38 MAP kinase , strongly blocked the induction of COX-2 by SIN-I in the presence or absen ce of IL-1 alpha, whereas the MEK-1 inhibitor, PD 98059, affected it to a l esser extent. These data demonstrate that SIN-1 induces COX-2 in HUVEC in t he absence of PG formation and suggest a complex regulation of COX-2 expres sion and PG formation by NO in endothelial cells.