UP-REGULATION OF NITRIC-OXIDE SYNTHASE IN CULTURED HUMAN KERATINOCYTES AFTER ULTRAVIOLET-B AND BRADYKININ

Ultraviolet B (UVB) irradiation of the skin has been reported to upreg ulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combin ed effect of UVB and bradykinin on NOS was therefore examined in a cul tured human keratinocyte (KC) line. Activity was determined in KC homo genates by the recovery of [H-3]L-citrulline using labeled L-arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterpar ts were used to determine the isoform(s) in control, UVB, bradykin tre ated and WE and bradykinin treated KC. Human KC express NOS activity w hich is lowest at confluence and highest during proliferation. UVB inc reased NOS activity when a set dose of irradiation was administered fr om 32.2-48.3 mJ/cm(2) but was inhibitory after 64.4 and 80.5 mJ/cm(3). Thirty min after 10(-6) M bradykinin, NOS activity nearly doubled fol lowed by return of activity to control levels at 60 min. Activity afte r UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M(r) 155 kDa was det ected in control cells with the antibody for the constitutive brain en zyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent, When both stimulant s were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the exp ression of NOS activity under unstimulated conditions in human KC in c ulture is due to the constitutive NOS found in neuronal tissue, bNOS, The recovery of bNOS increased after UVB and after bradykinin while th e combination of both resulted in the synergistic increase in bNOS pro tein with only a marginal further increase in NOS activity.