[³H]Bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters

Vascular endothelial cells previously have been shown to possess a prominent Na-K-Cl cotransport system which mediates a K⁺ influx of approximately 20 μmol/g of protein/min. Endothelial cell cotransport has also been shown to be regulated by a variety of vasoactive agents and their second messengers, suggesting that the transport system may have an important role in endothelial cell function. In the present study we investigated the possibility that the high level of cotransport in these cells is due to a large number of Na-K-Cl cotransporters in the plasma membrane. This was done by evaluating specific saturable binding of [³H]bumetanide to cultured bovine aortic endothelial cells. We found a maximal [³H]bumetanide binding of 0.83 pmol/mg protein with a dissociation constant of 0.13 μM. From these data, the number of [³H]bumetanide binding sites/endothelial cell was determined to be approximately 230,000, and the turnover number for cotransport activity was calculated to be 300 K⁺ ions/site/s. These findings indicate that endothelial cells do indeed exhibit a large number of Na-K-Cl cotransporters/cell relative to other cell types. We also investigated the effects on [³H]bumetanide binding of agents known to modulate Na-K-Cl cotransport activity. Saturable binding of [³H]bumetanide was found to be reduced significantly by treatment of the cells with 8-bromo-cyclic AMP, 8-bromo-cyclic GMP, phorbol esters, norepinephrine, or rat atriopeptin III, all of which have been shown to inhibit Na-K-Cl cotransport-mediated K⁺ influx.