Endothelial cell shrinkage increases permeability through a Ca²⁺-dependent pathway in single frog mesenteric microvessels

1. We tested whether calcium (Ca²⁺)-dependent mechanisms were essential for our previous observation that a change in the endothelial cell (EC)-extracellular matrix (ECM) attachment caused an increase in microvessel hydraulic permeability (L(p)) after exposure to hypertonic solutions in single perfused mesenteric microvessels in pithed frogs (Rana pipiens). 2. In microvessels where integrin-dependent EC-ECM attachments were disrupted by pretreatment with the peptide Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP; 0.3 mmol l^-1), we measured microvessel L(p) after exposure to hypertonic solutions under experimental conditions that reduced Ca²⁺ influx into endothelial cells. 3. High K⁺ solutions (59.7 and 100 mmol l^-1 K⁺) were used to depolarize the endothelial membrane and therefore to reduce the electrochemical driving force for Ca²⁺ influx through conductive Ca²⁺ channels. These solutions abolished the increase in L(p) caused by hypertonic solutions in the microvessels pretreated with GRGDTP. 4. We previously suggested that the removal of albumin from the perfusate may reduce EC-ECM attachment because hypertonic solutions increased the L(p) of microvessels above that due to removal of albumin alone. This additional increase in L(p) was attenuated by the 59.7 mmol l^-1 K⁺ solution and was completely abolished by the 100 mmol l^-1 K⁺ solution. 5. Bumetanide, an inhibitor of the Na⁺-K⁺-2Cl^- co-transporter and one of the mechanisms of regulatory volume increase after exposure to hypertonic solutions in endothelial cells, did not change the response of microvessels to high K⁺ solutions. 6. Our findings indicate that Ca²⁺ entry into endothelial cells via passive conductance channels is necessary to increase microvessel L(p) after exposure to hypertonic solutions in microvessels where EC-ECM attachments are disrupted.