Abstract 3637: Expression of the NOS3 Activator S100A1 is Negatively Regulated by a NO-dependent Posttranslational Mechanism in Cardiac Endothelial Cells
Background: The calcium (Ca) sensor protein S100A1 has recently been found to be expressed in endothelial cells (ECs) and loss of EC S100A1 diminishes NO availability and results in impaired vascular relaxation and hypertension in S100A1 knockout mice. Vice versa, S100A1 gene transfer to ECs results in enhanced NOS3 activity and NO production. Given the importance of NO homeostasis for regulation and integrity of vascular function, we hypothesized that EC NO levels might directly regulate expression of the NOS3 regulator S100A1.
Methods and Results: Rat cardiac ECs were enzymatically isolated and S100A1 mRNA and protein abundance were determined by semiquantitative RT-PCR analysis and Western blotting normalized to unchanged 18s and GAPDH signals. Exposure of cultured ECs to the NO-donor GSNO (10 μM) and SNP (10 μM) resulted in a significant 55±2%* and −49±7%* decrease of S100A1/18s mRNA after 8 and 24 hours (*P<0.05, n=5) in accord with accelerated NO release from GSNO. In further support of our hypothesis, the NOS inhibitor L-NAME (100 μM) significantly enhanced EC S100A1 mRNA levels by 2-fold after 24 hours (P<0.05 vs. control, n=3). Moreover, the inhibitory effect of GSNO on EC S100A1 transcription was blocked by coincubation with the guanylyl cyclase inhibitor ODQ (10 μM) and ODQ alone enhanced S100A1 mRNA levels by 2-fold (*P<0.05, n=3) indicating that NO might negatively regulate EC S100A1 transcription through the GC-cGMP-PKG dependent pathway. Application of the transcriptional inhibitor actinomycin D (10 μM), did not alter the inhibitory effect of GSNO suggesting that the NO-dependent decrease in EC S100A1 mRNA abundance is mediated through a posttranslational mechanism. In contrast, GSNO neither altered abundance of SERCA nor VEGF or S100A4 and S100A6 mRNA levels in ECs supporting the specificity of our finding. As expected, the NO-donor SNP decreased EC S100A1 protein levels after 72 hours in culture by 2-fold compared to control.
Conclusion: Given the importance of S100A1 in regulating EC NO-homeostasis and vascular function in vivo, we have identified a novel NO-dependent negative feedback mechanism in EC suppressing abundance of the NOS3 activator S100A1 through a posttranslational mechanism, potentially relying on GC-cGMP-PKG signaling.