Osteopontin inhibits inducible nitric oxide synthase activity in rat vascular tissue

We tested the hypothesis that osteopontin (OPN) can inhibit the induction o f inducible nitric oxide synthase (iNOS) in vascular tissue. iNOS activity was induced in rat thoracic aortas by incubation of the tissue with lipopol ysaccharide (LPS) and measured by conversion of L-[H-3]arginine to L-[H-3]c itrulline. Addition of greater than or equal to 1 nM recombinant OPN protei n significantly reduced the LPS-induced increase in iNOS activity. Western blotting and the RT-PCR were used to determine the effect of LPS with and w ithout OPN on tissue levels of iNOS protein and RNA, respectively. LPS resu lted in an increase in iNOS protein and RNA, whereas OPN dose-dependently r educed tissue levels of iNOS activity, protein, and RNA. Mutated OPN protei ns, in which the integrin-binding RGD amino acid sequence was deleted or mu tated to RGE, resulted in complete and partial loss, respectively, of the a bility of OPN to inhibit LPS-induced iNOS activity, implicating integrin bi nding in the effect. These results indicate that OPN can prevent induction of iNOS in vascular tissue.