Transmembrane signalling mechanisms regulating expression of cationic amino acid transporters and inducible nitric oxide synthase in rat vascular smooth muscle cells

Abstract

The signalling mechanisms involved in the induction of nitric oxide synthase (iNOS) and Larginine transport were investigated in bacterial lypopolysaccharide (LPS) and interferon-γ (IFN-γ)- stimulated rat cultured aortic smooth muscle cells (RASMC). The expression profile of transcripts for cationic amino acid transporters (CATs) and their regulation by LPS and IFN-γ were also examined. Control RASMC expressed mRNA for CAT-1, CAT-2A and CAT-2B. Levels of all three transcripts were significantly elevated in activated cells. Stimulated CAT mRNA expression and L-arginine transport occurred independently of protein kinase C (PKC), protein tyrosine kinase (PTK) and p44/42 mitogen activated kinases (MAPK), but were inhibited by the p38 MAPK inhibitor SB203580, which at 3 μM caused maximum inhibition of both responses. Induction of NO synthesis was independent of p44/42 MAPK activation and only marginally dependent on PKC, but was attenuated markedly by the PTK inhibitors, genistein and herbimycin A. SB203580 differentially regulated iNOS expression and NO production, potentiating both processes at low μM concentrations and inhibiting at concentrations of ≥ 1μM. In conclusion, our data suggest that RASMC constitutively express transcripts for CAT-1, CAT-2A and CAT-2B, and that expression of these transcripts is significantly enhanced by LPS and IFN-γ. Moreover, stimulation of Larginine transport and induction of NO synthesis by LPS and IFN-γ appears to be under critical regulation by the p38 MAPK, since both processes were significantly modified by SB203580 at concentrations so far shown to have no effect on other signalling pathways. Thus, in RASMCs, the p38 MAPK cascade represents an important signalling mechanism, regulating both enhanced L-arginine transport and induced NO synthesis.