ATP is released from many cell types in response to conditions such as hypotonic challenge, mechanical stretch, or shear stress. Some of the proposed mechanisms for ATP release pathways include: flux through connexin (CX) hemichannels, exocytosis, and/or involvement of cystic fibrosis transmembrane conductance regulator (CFTR) proteins. Once released, the extracellular ATP can act as an autocrine or paracrine agent to activate nucleotide (P2) receptors on cell membrane, resulting in activation of calcium signaling pathways. Cardiac fibroblasts (CFs) comprise approximately 90% of non-myocyte cells in the heart and are important for the regulation of cardiac function. Given the vast number of CFs in the heart, we hypothesized that CFs can be a source of ATP in the extracellular environment and that CX hemichannels may be involved in the release mechanism for ATP. Ventricular CFs were isolated from adult Sprague Dawley rats via retrograde enzymatic (collagenase) digestion. Cultured cells (12-16 days old) were plated on 60mm petri dishes or glass slides for static and fluid flow experiments, respectively. Samples exposed to selected experimental conditions were collected, and ATP levels were measured via a luminometric luciferin-luciferase ATP assay. Initial studies revealed that ATP release was increased by approximately 4-fold following 50% hypotonic challenge. ATP release was further increased when ecto-ATPase inhibitors, ARL 67156 and/or adenosine 5’-(α-β-methylene) diphosphate (AMP-CP), were present. These preliminary results suggest that the inhibition of ATP metabolism can cause further ATP release, possibly triggered by positive feedback mechanisms. Furthermore, decreasing the extracellular calcium concentration from 1 mM to 0 mM, a maneuver known to open CX hemichannels, resulted in an increase in ATP release, while 18-α-glycyrrhetinic acid and carbenoxolone, CX hemichannel blockers, markedly decreased ATP release. These studies suggest that hypotonic induced ATP release from CFs may take place through CX hemichannels. In ongoing studies, the presence of CX hemichannels and P2 receptors in cardiac fibroblasts is being evaluated with PCR analysis, western blots, and fluorescence imaging, and down regulation of CX43 expression, using small interfering RNA (siRNA), is being attempted.