B. Nilius et al., ACTIVATION OF A CL- CURRENT BY HYPOTONIC VOLUME INCREASE IN HUMAN ENDOTHELIAL-CELLS, The Journal of general physiology, 103(5), 1994, pp. 787-805
We have used whole-cell and perforated patches to study ionic currents
induced by hypotonic extracellular solutions (HTS, 185 mOsm instead o
f 290 mOsm) in endothelial cells from human umbilical veins. These cur
rents activated within 30-50 s after application of HTS, reached a max
imum value after similar to 50-150 s and recovered completely after re
-exposing the cells to normal osmolarity. They slowly inactivated at p
otentials positive to +50 mV. The same current was also activated by b
reaking into endothelial cells with a hypertonic pipette solution (377
mOsm instead of 290 mOsm). The reversal potential of these volume-ind
uced currents using different extracellular and intracellular Cl- conc
entrations was always close to the Cl--equilibrium potential. These cu
rrents are therefore mainly carried by Cl-. DIDS only weakly blocked t
he current (K-I = 120 mu M), while another Cl--channel blocker, DCDPC
(20 mu M) was ineffective.We were unable to record single channel acti
vity in cell-attached patches but we always observed an increased curr
ent variance during I-ITS. From the mean current-variance relation of
the whole-cell current records, we determined a single channel conduct
ance of 1.1 pS. The size and kinetics of the current were not correlat
ed with the concomitant changes in intracellular calcium. Furthermore,
the currents could still be activated in the presence of 10 mmol/lite
r intracellular EGTA and are thus Ca2+ independent. A similar current
was also activated with iso-osmotic pipette solutions containing 300 m
u mol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, di
d not prevent activation of this current. TPA (4 mu mol/liter) was als
o ineffective in modulation of this current. The HTS-induced current w
as completely blocked by 10 mu mol/liter pBPB, a PLA(2) inhibitor. NDG
A (4 mu mol/liter) and indomethacin (5 mu mol/liter), blockers of lipo
xygenase and cyclo-oxygenase respectively, did however not affect the
current induced by hypotonic solutions. The effects of arachidonic aci
d (10 mu mol/liter) were variable. In 12 out of 40 cells it either dir
ectly activated a Cl- current or potentiated the current activated by
FITS. The membrane current was decreased at all potentials in 18 cells
, and was not affected in 10 cells. The HTS-induced currents may there
fore be modulated by cleavage products of PLA(2), but not by messenger
s 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 I-ITS-induced current. It is therefore unlikely that
HTS is signaled via a PKA-dependent pathway. Verapamil (100 mu mol/li
ter) and DDFSK (60 mu mol/liter), both inhibitors of MDR1-gene encoded
ATP-dependent ABC-transporters (P-glycoprotein), completely blocked t
he HTS-induced currents. HTS could still activate a current in the abs
ence of ATP in the patch pipette. However, the rate of onset of the cu
rrent became slower and its amplitude gradually declined during repeat
ed exposure to HTS. We propose that swelling of endothelial cells acti
vates a chloride current, that may be related to P-glycoprotein. It is
modulated via a G-protein-activated enzyme, but the nature of the sec
ond messenger is unknown. The PKC and PKA pathways are not involved in
the regulation of the current.