Natriuretic Peptide Receptors in vascular Smooth Muscle Cells - The Influence of Cell Culture.

Relaxation of vascular smooth muscle cells (vSMCs) by cGMP is either mediated by NO and sGC or by ANP/BNP and GC-A as well as by CNP and GC-B.

We aimed to investigate culturing effects on the expression and activity of the GCs and compared intact aortic media with corresponding primary vSMCs isolated from the same tissue. To rule out sex-specific differences we included a male and female cohort.

Gene expression of cGMP pathway components (GC-A, GC-B, sGC) and the natriuretic peptide (NP) clearance receptor NPRC was quantified by RT-qPCR in intact media and derived primary vSMCs. cGMP production was measured by ELISA.

We found that U2 different to commonly used housekeeping genes for RT-qPCR enabled a direct comparison of gene expression in intact aortic media vs. corresponding primary cells. Interestingly, GC-A was stably expressed in both intact media and cultured vSMCs while all other genes investigated were upregulated in culture. In intact media, GC-A exceeded GC-B, sGC and NPRC. Data were mirrored by cGMP production. However, cultured vSMCs of the same aortae displayed higher GC-B, sGC and NPRC expression compared to the stable GC-A. No significant difference between both sexes was found but expression levels seemed to show a larger spread in females.

Culturing of vSMCs significantly alters their gene expression regarding cGMP signaling components and affects cGMP production in response to NP. Hence, total tissue or in vivo setups reflect NP related signalling more realistically. Hints towards sex-specific differences merit further investigation.