Effect of extracellular hypertonicity and alkalosis on endothelial-derived EA.hy 926 cells in vitro

Endothelial and local metabolic mechanisms contribute in concert to the regulation of blood flow. In vivo extracellular alkalosis induces a vasoconstriction, hyperosmolarity a vasodilatation. The interaction between local metabolic and endothelial mechanisms is poorly understood. Therefore we investigated in endothelial-derived EA.hy926 cells the secretion of endothelial modulators of vascular tone under hypertonic stress with and without alkalosis: hyperosmolality was generated by either the addition of NaHCO subset 3 (25, 50, 100 mM, pH up to > 8) or mannitol (50, 100, 200 mM) to the cell culture media. The cells were studied using automated cell counting, measurement of the activity of the lactate dehydrogenase (LDH) and a bromo-deoxyuridine (BrdU) cell proliferation assay. Endothelin and cGMP, a surrogate marker for nitric oxide (NO), were measured with specific ELISAs. EA.hy 926 cells formed stable monolayers in vitro. The secretion of endothelin, but not of cGMP was inversely correlated with the osmolality of the incubation media: the endothelin concentration in the supernatants decreased in both mannitol- and NaHCO subset 3 -treated cells in a concentration-dependent manner (152.4 +/- 6.2 pg/ml (control) to 24.4 +/- 2.4 pg/ml (200 mM mannitol), res. to 18.2 +/- 2.7 pg/ml (100 mM NaHCO subset 3). Neither hypertonic bicarbonate nor mannitol solutions decreased the monolayer cell density or cell viability during the 6 hour incubation period. In conclusion, EA.hy926 cells are quite resistant against a 6-hour hypertonic/alkaline stress. Hypertonicity decreases the secretion of endothelin and has no effect on cGMP. At each level of hypertonicity the endothelin concentration was similar in the NaHCO subset 3 and mannitol media arguing against a direct role of endothelin in alkalosis-induced vasoconstriction in vivo. The decreased secretion of endothelin during hypertonicity could contribute to the hyperosmolal vasodilation seen in vivo.