Pfliigers Arch (1992) 421:394--396 Journal of Physiology 9 Springer-Verlag 1992 Short communication Shear stress induced membrane currents and calcium transients in human vascular endothelial cells Gero Sehwarz 1, Guy Droogmans 2, and Bernd Nilius 1 * Max Planck Group "Molecular and Cellular Physiology", 0-6900 Jena, Federal Republic of Germany 2 Catholic University Leuven, Department of Physiology, B-3000 Leuven, Belgium Received May 18, 1992/Accepted June 11, 1992 Abstract. We have measured membrane currents induced by shear stress together with intracellular calcium signals in endo- thelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca2+]o), shear stress induced an in- ward current at a holding potential of 0 mV which is accom- panied by a rise in intracellular Ca 2+ ([Ca2+]i). In the absence of extracellular calcium shear stress was unable to evoke a cal- cium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and duri.~g application of shear stress. Its reversa! potential Erev shifted from -2.3 * 0.8 mV (n=4) in a nominally Ca 2+ free solution to +1.5 -+ 1.6 mV at 1.5 mM [Ca2+]o (n=4) and to +21.9 _+4.4 mV (n=7) at 10 mM [Ca2+]o. From our data we conclude that shear stress opens an ion channel that is 12.5 -+ 2.9 (n=7) times more permeable for calcium than for sodium or cesium. Key words: endothelium - shear stress - Ca2+-permeable ion channels - intracellular calcium Introduction. Shear stress mediates several biological effects in human endo- thelial cells, such as EDRF release, prostacydin synthesis, ex- pression of tissue plasminogen activator (for a review see [1]. In a recent paper, we have described shear stress mediated Ca 2+ transients in vascular endothelial cells from human umbilical veins [2]. In these cells shear stress induced quenching of Fura II fluorescence by divalents, such as cobalt and nickel, indicat- ing the activation of a pathway preferentially permeable for di- valents. In this report we describe a membrane current that is activated by shear stress and carries calcium more than 10 times better than monovalent cations. This current could me- diate intracellular Ca 2+ transients stimulated by shear stress and could therefore couple mechanical events to biological effects in endothelial cells. Offprint requests to B. Nilius, Laboratorium voor Fysiologie, Campus Gasthuisberg, B-3000 Leuven (Belgium). Methods. Endothelial cells were prepared from human umbilical cord veins by a collagenase digestion procedure as described previ- ously [3]. Under our culture conditions, cells were not conflu- ent between day 1 and 4 and single endothelial cells could be used for our experiments. We only used cells from primary culture up to the second passage. For [Ca2+]i measurements, cells were incubated with 2 laM of the acetoxymethylester Fura-2/AM (Molecular Probes, Eugene, OR, USA ). The bath perfusion solution (Krebs' solution) used in all experiments had the following composition '(in retool/l): 140 NaC1, 1.5 or 10 CaCI2, 5.9 KC1, 1.2 MgC12, 11.5 Hepes-NaOH, 10 glu- cose, titrated to pH 7.3 with NaOH. The Ca 2+ measurement was coupled with simultaneous current measurements by the patch clamp technique in the whole cell mode. The technique used was described in detail elsewhere [2,4,5]. The following pipette solution was used (in mmol/l): 100 Cs aspartate, 40 mM CsCI, 5 NaCI, 5.5 MgC12, 10 Hepes, 5 Na2ATP, 0.1 EGTA buffered at pH 7.2 with CsOH. Under resting condi- tions the cells were superfused by a slow bath perfusion. Shear stress was induced by directing a stream of Krebs' solution along the surface of a single endothelial cell through a multi- barrelled pipette with a common opening of about 150 lam. The flow system and the generation of shear stress are described in detail elsewhere [2]. Mean +_standard error of the mean were calculated from pooled data. For tests of signifi- cance, we used the unpaired t-test. Permeation ratios were cal- culated from the measured reversal potentials by using a method described in detail elsewhere [6]. Results and discussion. Figure 1A shows the first simultaneous measurement of shear stress induced membrane currents and intracellular Ca 2+ sig- nals in a single endothelial cell. At a holding potential of 0 mV, shear stress (approximately 10 dyne/cm 2 as calculated from "r =4pQ/z~r3 where p represents the fluid viscosity, Q the rate of flow, r the radius of the cylinder of fluid that streams out of the pipette) evoked an inward current together with a slowly