Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K⁺ and Cl⁻ channels

Abstract

Steady laminar shear stress has been shown previously to markedly increase Na-K-Cl cotransporter mRNA and protein in human umbilical vein endothelial cells and also to rapidly increase endothelial K(+) and Cl(-) channel conductances. The present study was done to evaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and to determine whether changes in cotransporter expression may be dependent on early changes in K(+) and Cl(-) channel conductances. Confluent BAEC monolayers were exposed in a parallel-plate flow chamber to either steady shear stress (19 dyn/cm(2)) or purely oscillatory shear stress (0 +/- 19 dyn/cm(2)) for 6-48 h. After shearing, BAEC monolayers were assessed for Na-K-Cl cotransporter activity or were subjected to Western blot analysis of cotransporter protein. Steady shear stress led to a 2- to 4-fold increase in BAEC cotransporter protein levels and a 1.5- to 1.8-fold increase in cotransporter activity, increases that were sustained over the longest time periods studied. Oscillatory flow, in contrast, had no effect on cotransporter protein levels. In the presence of flow-sensitive K(+) and Cl(-) channel pharmacological blockers, the steady shear stress-induced increase in cotransporter protein was virtually abolished. These results suggest that shear stress modulates the expression of the BAEC Na-K-Cl cotransporter by mechanisms that are dependent on flow-activated ion channels.

Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells

“…Beyond being responsive to fluid mechanical shear stress, vascular ECs distinguish among and respond differently to different types of shear stress (7,11,12,14,15,20,31,38). However, the mechanisms by which ECs resolve differences among different flow waveforms remain completely unknown.…”

“…1) were consistent with this notion, we performed a limited number of current-clamp measurements in the presence of either the K ϩ channel blocker Ba 2ϩ or the Cl Ϫ channel blocker NPPB. These pharmacological agents were previously shown to effectively block flow-sensitive K ϩ and Cl Ϫ currents (5,32,38). The presence of NPPB (100 M) resulted in the absence of the depolarization so that flow led only to sustained membrane hyperpolarization ( Fig.…”

“…Similar levels of hyperpolarization were induced by steady Hyperpolarizing and depolarizing flow-sensitive currents are differentially responsive to different types of flow. Previous work in our laboratory and by others has shown that BAECs have at least two flow-sensitive currents: an inwardly rectifying K ϩ current that is activated at membrane potentials negative to the K ϩ reversal potential (E K ) and that can be completely blocked by Ba 2ϩ and an outwardly rectifying Cl Ϫ current that can be blocked by NPPB (5,16,17,26,32,38). To probe whether the membrane potential responses described above are consistent with these currents, whole cell voltage-clamp recordings were performed on BAECs in the presence of either NPPB or Ba 2ϩ…”

“…In vitro, whereas both steady and nonreversing pulsatile flow induce EC elongation and mobilize intracellular Ca 2ϩ , purely oscillatory flow does not elicit either of these responses (14, 15). Steady and oscillatory flow also impact endothelial gene expression and protein synthesis differently (7,20,38). Turbulent flow has a considerably different impact on EC gene expression, morphology, and turnover rates from laminar flow with the same time-averaged shear stress (11,12).…”

Flow-activated Chloride Channels in Vascular Endothelium

“…Steady and oscillatory flow Akt studies were performed in a parallel plate flow chamber as described elsewhere (21). Flow periods ranged from 5 to 60 min.…”