Citrulline Recycling Supports Nitric Oxide Biosynthesis in Mouse PAM212 Keratinocytes

Nitric oxide synthases generate nitric oxide and citrulline from arginine using NADPH and molecular oxygen as co‐substrates. In most mammalian cells, arginine is an essential amino acid and limiting extracellular supplies of this amino acid can reduce nitric oxide biosynthesis. This can occur during inflammation and injury where a variety of factors can reduce available arginine including high levels of tissue arginase. In cells and tissue containing urea cycle enzymes, arginine can be synthesized from citrulline. Thus, in a rate limiting step, argininosuccinate synthetase catalyzes the conversion of citrulline and aspartate to argininosuccinate which is then metabolized to arginine via argininosuccinate lyase. In earlier studies we reported that, in response to γ‐interferon, PAM212 keratinocytes readily produce nitric oxide in an arginine‐dependent manner via an inducible form of nitric oxide synthase. Citrulline is formed in the cells during this process while arginine is metabolized. In the present studies we determined if citrulline was taken up by PAM212 keratinocytes and utilized for nitric oxide biosynthesis. We found that citrulline effectively supported nitric oxide production; approximately 10‐fold higher concentrations of citrulline were required to produce nitric oxide when compared to arginine. Both citrulline and arginine were taken up by PAM212 keratinocytes; at equimolar concentrations (50 µM), more arginine than citrulline was taken up by the cells. However, at higher concentrations, uptake of these amino acids was saturable; the apparent Km and Vmax for arginine uptake was 170 µM and 1.2 nmol/10 6 cells/min, respectively, and citrulline uptake was 850 µM and 0.7 nmol/10 6 cells/min, respectively. Nitric oxide production supported by citrulline was inhibited by the nitric oxide synthase inhibitors, N‐monomethylarginine, N‐nitroarginine and N‐nitroarginine benzyl ester. Each of these compounds also inhibited citrulline uptake into keratinocytes. Citrulline is known to be taken up by the L amino acid transporter, and nitric oxide synthase inhibitors compete with this transporter for uptake into keratinocytes. When pulse‐labeled with 14C‐citrulline, approximately 17% to 25% of the label accumulated as 14C‐arginine in cytoplasmic pools of PAM212 cells demonstrating that these cells contain arginosuccinate synthetase/lyase activity. Nitric oxide synthase inhibitors had no effect on citrulline recycling in the cells. These data indicate citrulline can be used for nitric oxide biosynthesis in PAM212 keratinocytes. Citrulline is less efficient than arginine in supporting nitric oxide production due to limited uptake and slow conversion to arginine. In PAM212 keratinocytes, nitric oxide synthase inhibitors have multiple modes of action: inhibition of uptake of citrulline and nitric oxide synthase activity.