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Effect of cell passage and density on protein kinase G expression and activation in vascular smooth muscle cells
Guiting Lin1, Sylvia Chow, Jackie Lin
1Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, California 94143-1695, USA.
Abstract:
It has been shown that rat aortic smooth muscle cells (AoSMCs) lost PKG-I expression when propagated repetitively or grown at low densities. Conversely, AoSMCs isolated from PKG-I 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-I 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-I activation, rat AoSMCs of p4 and p11 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-I activation than p11.
Insights
Aortic smooth muscle cells (AoSMCs) show stable cyclic GMP-dependent protein kinase 1 (PKG-I) expression across passages and densities. However, PKG-I activation decreases with increasing cell passage.
Area of Science:
- Vascular Biology
- Cellular Physiology
- Molecular Cardiology
Background:
- Previous studies suggested cyclic GMP-dependent protein kinase 1 (PKG-I) expression decreases in propagated rat aortic smooth muscle cells (AoSMCs).
- However, PKG-I deficient mice AoSMCs exhibit normal morphology and growth, questioning the impact of PKG-I loss.
Purpose of the Study:
- To investigate the effect of cell passage and density on PKG-I expression and activation in human and rat AoSMCs.
- To determine if cell passage influences PKG-I mediated signaling.
Main Methods:
- Western blotting and immunofluorescence microscopy were used to assess PKG-I expression in human and rat AoSMCs across passages (p9-p15 and p1-p15, respectively).
- Rat AoSMCs were cultured at varying densities to evaluate PKG-I expression.
- PKG-I activation was assessed by measuring phosphorylated vasodilator-stimulated phosphoprotein (P-VASP) levels in response to cGMP stimulation in cells from different passages (p4 vs. p11).
Main Results:
- Little difference in PKG-I expression was observed among different passages (p9-p15 for human, p1-p15 for rat) and cell densities.
- Rat AoSMCs from passage 4 (p4) exhibited higher PKG-I activation compared to those from passage 11 (p11), as indicated by P-VASP levels.
Conclusions:
- Cell passage and density do not significantly alter PKG-I expression levels in AoSMCs.
- Higher cell passage number is associated with reduced PKG-I activation, suggesting a potential decline in signaling capacity over time.
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