Endogenous synthesis of tetrahydrobiopterin (BH4) is an important requirement for cytokine-stimulated nitric oxide (NO) production in mesangial cells. We have shown that inducible NO synthase is expressed in mesangial cells in response to two principal classes of activating signals, inflammatory cytokines such as interleukin 1β (IL-1β) and agents that elevate cellular levels of cAMP [Kunz, Mühl, Walker and Pfeilschifter (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5387–5391]. In the present paper we demonstrate that IL-1β and cAMP similarly increase the steady-state mRNA levels of GTP cyclohydrolase I (EC 3.5.4.16), the rate-limiting enzyme in BH4 biosynthesis, as measured by a sensitive and quantitative nuclease protection assay. Stimulation of cells with a combination of IL-1β plus cAMP revealed an additive induction profile of GTP cyclohydrolase I mRNA. Message stability studies established that GTP cyclohydrolase I mRNA induced by cAMP has a longer half-life than the IL-1β-induced message. Moreover, cAMP exposure markedly prolonged the half-life of GTP cyclohydrolase I mRNA, from 1.5 to 3.4 h. In a next step we generated a rabbit polyclonal antibody against rat GTP cyclohydrolase I expressed in Escherichia coli and demonstrated that IL-1β and cAMP elevated GTP cyclohydrolase I protein levels in mesangial cells. Furthermore, IL-1β and cAMP led to a marked increase in GTP cyclohydrolase I activity and to increased accumulation of biopterin in mesangial cells. Combinations of IL-1β and cAMP resulted in a synergistic stimulation of GTP cyclohydrolase I activity. This may suggest that, in addition to transcriptional and post-transcriptional regulation, there is a prominent post-translational modulation of enzyme activity.

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