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 approximately 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 (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) 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 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/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.(ABSTRACT TRUNCATED AT 400 WORDS)
Single-channel measurements and whole-cell experiments with the two suction electrode, voltage clamp technique were used to investigate the effects of external and internal proton concentrations on T-type Ca channels in heart muscle cells of the guinea pig. As in the L-type Ca channel, an increase in the external proton concentration decreases T-type currents, while external alkalinization enlarges the currents. In contrast to the L-type Ca channel, however, a change in the internal proton concentration does not modulate T-type Ca currents. The T-type Ca channel is much more sensitive to variations in pHo than the L-type Ca channel. By the combination of single-channel and whole-cell experiments we can conclude that the observed changes in macroscopic currents are due to (a) changes in the single-channel conductance and in the probability of the T-type Ca channel being open, and (b) the titration of the negative surface charges in the neighborhood of the T-type Ca channel with shifts of both the activation and inactivation processes of the channel. The pHo-induced changes in the maximal conductance (gmax) of the T-type Ca channel show an apparent pKa in the range of 7.1-7.5, while the titration of the negative surface charges near the channel shows an apparent pKa of 7.1 with a concomitant surface potential of -24.6 mV at 5.4 mM [Ca]o. These pKa values, less acid than the pKa values found for the pHo-induced, L-type Ca channel modulation, might imply a physiological importance of this novel type of channel modulation.