We investigated the non-linear dynamics of the pressure-vs.-hydraulic conductivity (L_p) relationship in lung microvascular endothelial cells and demonstrate that heparan sulfates, an important component of the endothelial glycocalyx, participate in pressure-sensitive mechano-transduction that results in barrier dysfunction. The pressure-vs.-L_p relationship was complex, possessing both time- and pressure-dependent components. Pretreatment of lung capillary endothelial cells with heparanase III completely abolished the pressure-induced increase in L_p. This extends our previous observation regarding heparan sulfates as mechano-transducers for shear stress (7). Inhibition of nitric oxide synthase with L-NAME and intracellular scavenging of reactive oxygen species (ROS) by TBAP significantly attenuated the pressure-induced L_p response. Intracellular NO/ROS were visualized using the fluorescent dye, DCFA, and cells demonstrated a pressure-induced increase in intracellular fluorescence. Heparanase-pretreatment significantly reduced the pressure-induced increase in intracellular fluorescence, suggesting that cell-surface heparan sulfates directly participate in mechano-transduction that results in NO/ROS production and increased permeability. This is the first report to demonstrate a role for heparan sulfates in pressure-mediated mechano-transduction and barrier regulation. These observations may have important clinical implications during conditions where pulmonary microvascular pressure is elevated.