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Atrial natriuretic peptide antifibrotic properties are mainly described in cardiac myocytes or in induced cardiac myofibroblasts (Angiotensin II or TGF-beta induced differentiation). In the present work, we investigate the effects of ANP/NPRA/cGMP system in modulating rat cardiac fibroblasts function. Cardiac fibroblasts were isolated from adult Wistar male rats and cultured in the presence of serum in order to induce fibroblasts differentiation. Cultures were then treated with ANP (1 microM), 8-Br-cGMP (100 microM) or IBMX (100 microM), a non-specific phosphodiesterases inhibitor. ANP significantly decreased proliferation rate and collagen secretion. Its effect was mimicked by the cGMP analog, while combining ANP with 8-Br-cGMP did not lead to additional effects. Moreover intracellular cGMP levels were elevated when cells were incubated with ANP confirming that ANP intracellular pathway is mediated by cGMP. Additionally, immunoblotting and immunofluorescence were used to confirm the presence of guanylyl cyclase specific natriuretic peptide receptors A and B. Finally we scanned specific cGMP dependent PDEs via RT-qPCR, and noticed that inhibiting all PDEs led to an important decrease in proliferation rate. Effect of ANP became more prominent after 10 culture days, confirming the importance of ANP in fibroblasts to myofibroblasts differentiation. Uncovering cellular aspects of ANP/NPRA/cGMP signaling system provided more elements to help understand cardiac fibrotic process.