Activation of a Cl- current by hypotonic volume increase in human endothelial cells.

Abstract

We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after ~50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular C1-concentrations was always close to the Cl--eqnilibrium potential. These currents are therefore mainly carried by CI-. DIDS only weakly blocked the current (KI = 120 I~M), while another C1--channel blocker, DCDPC (20 IxM) was ineffective.We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca 2+ independent.A similar current was also activated with iso-osmotic pipette solutions containing 300 ~mol/liter GTP~/S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 ixmol/liter) was also ineffective in modulation of this current.The HTS-induced current was completely blocked by 10 Ixmol/liter pBPB, a PLA2 inhibitor. NDGA (4 tzmol/liter) and indomethacin (5 txmol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 I~mol/liter) were variable. In 12 out of 40 cells it either directly activated a CI-current or potentiated the current activated by HTS. The membrane current was decreased at all potentials

Volume regulation in a toad epithelial cell line: role of coactivation of K+ and Cl‐ channels.

“…On the other hand, small conductance Cl-channels activated by HTS have been described in many cell types (< 4 pS; Kunzelmann, Kubitz, Grolik, Warth & Greger, 1992;Lewis, Ross & Cahalan, 1993;Nilius et al 1994a, b). Marunaka & Eaton (1990) (Christensen & Hoffmann, 1992), in MDCK cells (Banderali & Roy, 1992a) and also in proximal tubule cells from Necturus kidney (Fillipovic & Sackin, 1992 will shift the membrane potential to a value close to the equilibrium potential of the other ion.…”

“…Electrophysiological methods have been described in detail elsewhere (Nilius, Oike, Zahradnik & Droogmans, 1994a). Wholecell membrane currents were measured using ruptured patches or patches perforated with nystatin to avoid disturbance of the intracellular Ca2+-buffering mechanisms.…”

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

“…2＋ measurements have previously been described in detail (Nilius et al, 1994). Membrane potential was monitored in current clamp mode or controlled in voltage clamp mode with an EPC-9 (HEKA Elektronik, Lambrecht, Germany), using a nystatin-perforated patch (100 mg/ml).…”

Caffeine‐evoked, calcium‐sensitive membrane currents in rabbit aortic endothelial cells

“…Additionally, we found that prolonged exposure of endothelial cells to caffeine induces cell shrinkage. Although the mechanism underlying the caffeine-induced shrinkage is unknown, it has been shown that hypotonic volume increases activate a Cl-current in human endothelial cells (Nilius et al, 1994) suggesting that caffeine-sensitive, Ca2+-activated Cl-currents may regulate cell volume.…”