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 Cl− concentrations was always close to the Cl−-equilibrium potential. These currents are therefore mainly carried by Cl−. DIDS only weakly blocked the current (Kl =120 µM), while another Cl− channel blocker, DCDPC (20 µM) 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 Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 µmol/liter GTPγS. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 µmol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 µmol/liter pBPB, a PLA2 inhibitor. NDGA (4 µmol/liter) and indomethacin (5 µmol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 µmol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl− current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current. It is therefore unlikely that HTS is signaled via a PKA-dependent pathway. Verapamil (100 µmol/liter) and DDFSK (60 µmol/liter), both inhibitors of MDRl-gene encoded ATP-dependent ABC-transporters (P-glycoprotein), completely blocked the HTS-induced currents. HTS could still activate a current in the absence of ATP in the patch pipette. However, the rate of onset of the current became slower and its amplitude gradually declined during repeated exposure to HTS. We propose that swelling of endothelial cells activates a chloride current, that may be related to P-glycoprotein. It is modulated via a G-protein-activated enzyme, but the nature of the second messenger is unknown. The PKC and PKA pathways are not involved in the regulation of the current.
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