Nitric oxide production is increased in septic shock. Arginine availability is rate limiting to the production of nitric oxide. We recently reported an increase in arginine flux through y⁺ cationic amino acid transport in peripheral blood mononuclear cells (PBMCs) obtained from patients with septic shock (Reade et al. 2001). We now report the results of our study of the molecular basis of this increased y⁺ activity.

PBMCs were isolated from seven septic patients and from eight healthy volunteers. RNA was extracted, treated with DNase and reverse-transcribed. Real-time quantitative PCR was carried out using cDNA specific primers and probes to CAT1, CAT2 (A and B), and CAT4, the known genes encoding proteins with y⁺ activity, along with CD98 heavy chain, the ubiquitous component of the protein complex encoding proteins with y⁺L activity. 18S cDNA was used as the internal control. The quantity of each mRNA in the PBMCs was compared with the amount present in human placenta, a tissue known to express all y⁺ and y⁺L mRNAs at high levels.

All four primers strongly amplified cDNA from the placental standard, over a 1-1/1000 range of dilutions. As shown in Table 1, CAT1 mRNA was expressed at a high level in both normal and septic cells: approximately 90 % of the amount seen in placenta. There was no difference between septic and control cells. Five of the seven septic patients expressed CAT2 mRNA, which was not seen in any of the controls.

mRNA for CAT2 is increased in PBMCs from septic patients. While the amount of CAT2 mRNA appears quantitatively small, it is not possible to conclude from this experiment whether this is functionally significant. We suggest the data presented here, combined with our previous functional data demonstrating increased y⁺ activity in human PBMCs, show that CAT2 is an attractive target to selectively reduce the upregulated component of nitric oxide production in patients with septic shock.

All work using human tissue was approved by the Central Oxfordshire Research Ethics Committee, which gave exemption from the need for informed consent from patients not competent to give it. M.C.R. is funded by a Brasenose College Oxford Graduate Scholarship and a University of Sydney Travelling Fellowship.

Reade, M.C., Young, J.D. & Boyd, C.A.R. (2001). J. Physiol. 531.P, 116P.