Insulin stimulates nitric oxide (NO) synthesis, via the endothelial NO synthase (eNOS), and L-arginine transport, via the cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) in human umbilical vein endothelial cells (HUVECs) (Sobrevia et al. 1996; Flores et al. 2001). Biological actions of insulin involve activation of phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) pathway in mammalian cells (Bevan, 2001). We investigated the involvement of PI3K, PKC and p42/p44^mapk in the effect of insulin on activity and expression of CATs in HUVECs.

Cells were isolated (0.2 mg ml^-1 collagenase) from normal pregnancies (Ethics Committee approval and informed patient consent were obtained) and cultured in medium 199 (M199) supplemented with sera. L-[³H]Arginine transport (100 µM, 2 µCi ml^-1, 37 °C, 1 min) and mRNA for hCAT-1 and hCAT-2B were quantified by real time PCR in the presence or absence of insulin (0.1 nM, 8 h), wortmannin (50 nM, 30 min, PI3K inhibitor), PD-98059 (10 µM, 30 min, MAPK kinase inhibitor), calphostin C (100 nM, 30 min, PKC inhibitor) or N^G-nitro-L-arginine methyl ester (L-NAME, 100 µM, 30 min, eNOS inhibitor). The effect of insulin on p42/44^mapk activation was also assayed by Western blot.

Insulin increased the V_max (21 ± 1 vs. 4 ± 0.2 pmol (µg protein)^-1 min^-1, n = 5, P < 0.05, means ± S.E.M., Student’s unpaired t test) with no significant changes in the apparent K_m (control: 138 ± 21 µM, insulin: 101 ± 21 µM, P > 0.05). Insulin-increased V_max was blocked (P < 0.05) by wortmannin (2.8 ± 0.6 pmol (µg protein)^-1 min^-1), calphostin C (2.3 ± 0.4 pmol (µg protein)^-1 min^-1 and L-NAME (3.5 ± 0.5 pmol (µg protein)^-1 min^-1). Insulin also increased hCAT-1 (3-fold) and hCAT-2B (1.7-fold) mRNA number of copies, effects that were blocked (P < 0.05) by PD-98059 and calphostin C. Insulin also increased eNOS mRNA number of copies (2.3-fold) and protein (1.7-fold) level, and eNOS activity (2.1-fold), and induced phosphorylation of p42/44^mapk.

In summary, increased activity of L-arginine transport induced by insulin involves PI3K, PKC and eNOS activity in HUVECs. In addition, insulin-induced increase of L-arginine transport may be due to increased expression of hCAT-1 transporters.

This work was supported by FONDECYT (1030781, 1030607, 7030004, 7030109) and DIUC-University of Concepción (201.084.003-1.0)-Chile, and The Welcome Trust (UK).