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

'The final version of record is available at: http://www.biochemj.org/bj/tocprev/toc1999.htm ' --Copyright Biochemical SocietyThe 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.Peer reviewe

Biochemical characterization of a virus-induced osteosarcoma-like osseous lesion <em>in vitro</em>.

Chondroprogenitor cells present in the apical and lateral parts of the mandibular condyle from neonatal mice differentiate towards the osteoblastic lineage and from bone within 7 days in culture. Infection of condylar explants with the FBR osteosarcoma virus (FBR MSV) results in the transformation of cell in the progenitor zone, previously identified as the target for the virus [1], and the formation of a transplantable osteosarcoma-like lesion. Morphological and biochemical changes in this system were investigated in the course of tumor development. Virus infection was followed by a significant increase in cell density and 3H-thymidine incorporation within the progenitor zone at the early stage of culture. In later stages, cell density and 3H-thymidine incorporation were lower than in control tissue. The 3H-thymidine labeling index gave similar results in infected and control tissues until day 7. Then, a significantly higher labeling index was found in the progenitor zone of infected condyles. At this stage, the proliferative effect of the virus even affected the cartilagenous core of the tissue. Quantitative alkaline phosphatase activity increased between day 3 and day 7 and was particularly high in the zone of infected cells. In addition, infected tissues consistently revealed a higher uptake of 45Ca, and deposition of the radioisotope along irregularly formed bone trabecules in the transformed tissue. The results suggest that there is an enhancement of tissue maturation following infection with the FBR osteosarcoma virus. Although biochemical investigations of whole condyles showed few differences in the total values of alkaline phosphatase activity, 3H-thymidine incorporation, DNA content, and 45Ca uptake, the histochemical assays revealed clear differences in the distributional pattern of these parameters within infected and control condyles

Characterization of <em>fos</em>-induced osteogenic tumours and tumour-derived murine cell lines.

Osteogenic tumours from c-fos (MT-c-fos-LTR)-transgenic mice and from mice infected with the v-fos-bearing FBR murine osteosarcoma virus (FBR MSV) showed close morphological and neoplastic similarities. Fos mRNA expression was elevated in both types of tumours, and expression of several genes characteristic of differentiated bone cells was either lower, enhanced, or not detectable in comparison to that in normal bone. Tumour-derived cell lines showed variable levels of exogenous fos expression; bone-cell-specific genes were similarly expressed in both primary tumours and tumour-derived cell lines. Upon transplantation the tumour cells formed fibrosarcomas, some of which contained areas of focal osseochondrous differentiation. Non-tumorigenic cell lines established from bone tissue of normal and MT-c-fos-LTR transgenic mice showed osteoblastic characteristics, whereas no parathyroid hormone (PTH) response was observed in transgenic tumour cell lines in spite of high alkaline phosphatase activity. These data indicate that deregulated fos expression interferes with terminal osteogenic differentiation in v-fos- and c-fos-induced bone tumours

Differential regulation of L-arginine transport and nitric oxide synthase induction in cultured smooth muscle cells

"The Version of Record (VoR) is available at www.biochemj.org" [Full text of this article is not available in the UHRA]Peer reviewe

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y(+)), while no information exists on the role of the heterodimeric transporters of system y(+)L (4F2hc/y(+)LAT1 and 4F2hc/y(+)LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y(+)L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y(+)), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y(+)LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y(+)LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y(+) (CAT1 transporter) and system y(+)L (4F2hc/y(+)LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid