Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells

It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-1 expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-1 deficient mice are indistinguishable from those isolated from normal mice in morphology and growth characteristics. In this study, human AoSMCs were grown from passage 9 (p9) to passage 15 (p15) and rat AoSMCs were isolated and cultured from p1 through p15. Western blotting and immunofluorescence microscopy showed little difference in PKG-1 expression among different passages. Next, rat AoSMCs of p4 were grown and harvested at different cell densities. Western blotting again showed little difference among cells seeded or harvested at different densities. To test the effect of cell passage on PKG-1 activation, rat AoSMCs of p4 and p] I were treated with cGMP and analyzed by Western blotting for phosphorylated vasodilator-stimulated phosphoprotein (P-VASP). The results showed that p4 had higher level of PKG-1 activation than p11. (C) 2004 Wiley-Liss, Inc.