NO regulates PDGF-induced activation of PKB but not ERK in A7r5 cells: implications for vascular growth arrest

Abstract

In addition to the well-documented role of nitric oxide (NO) as a vasodilator, NO has also been implicated in vascular smooth muscle cell (VSMC) growth arrest. Signaling mechanisms responsible for growth factor receptor-mediated VSMC proliferation include the extracellular signal-regulated kinase (ERK) and possibly the protein kinase B (PKB) cascade. Thus the present study was designed to test the hypothesis that, in A7r5 vascular smooth muscle-derived cells, platelet-derived growth factor (PDGF)-induced activation of either ERK or PKB is regulated by NO, which then modulates cellular proliferation and/or apoptosis. PKB-α was the predominant isoform of PKB expressed in A7r5 cells and was also expressed in rabbit carotid arteries and aortae. Phosphorylation of PKB-α and ERK induced by PDGF-BB was maximal within 5–15 min in A7r5 cells. Preincubation of A7r5 cells with the NO donorS-nitroso-N-acetylpenicillamine (SNAP) resulted in a biphasic regulation of PDGF-stimulated PKB-α phosphorylation and bioactivity. Acute exposure to SNAP significantly augmented PDGF-induced activation of PKB-α, whereas prolonged incubation led to a marked diminution in PDGF-induced activation of PKB-α. In contrast, SNAP did not affect PDGF-induced activation of ERK at any time point. The cGMP-independent effects of SNAP on PDGF-induced activation of PKB-α were established with the use of an inhibitor of soluble guanylyl cyclase, ODQ, as well as a cell-permeable analog of cGMP, 8-bromo-cGMP. Prolonged treatment of A7r5 cells with SNAP led to a significant decrease in DNA synthesis without an appreciable increase in apoptosis. These data suggest that, after prolonged exposure to SNAP, NO selectively attenuates PDGF-induced increase in PKB-α activation, which in turn may contribute to diminished VSMC proliferation by mechanisms involving growth arrest but not apoptosis.