Cover glasses that contained glomeruli were placed into a perfusion chamber
mounted on an inverted Nikon Ti-S microscope and superfused with a physiologic saline
solution (pH 7.4). Single-channel current data were acquired as described
previously.19 (link),20 (link) After a high resistance seal was obtained, cell-attached
recording was performed immediately. The membrane resistance was monitored regularly to
ensure the quality of recording. For measurements of acute effect only one experiment was
performed per dish to avoid any possibility of examining cells whose properties might have
been altered by extended exposure to Ang II. The recordings were made in symmetric
chloride solutions. The bath solution consisted of 126 NaCl, 1 CaCl2, 10 HEPES,
2 MgCl2, 10 glucose, pH 7.4. The pipette solution contained 126 NaCl, 1.5
CaCl2, 10 HEPES, 10 glucose; pH7.4; plus added directly before the
patch-clamp experiments were 100 µM niflumic acid or DIDS (to block
Ca2+-activated Cl− channels), 10 mM TEA (to inhibit
large-conductance Ca2+-dependent K+ channel), 10 nM iberiotoxin (to
block Ca2+-activated K+ channels), 10 µM nicardipine (to
block N-type Ca2+ channels). During the patch-clamp measurements in the
single-channel mode the activity of the ion channels was first monitored in response to
the potential applied in steps of 10 or 20 mV in the range of – 90 mV to + 60 mV
in order to estimate the channel’s conductance and I-V relationship. After that,
the voltage was clamped at – 60 mV and the channels’ activity was recorded
for several minutes before the drugs were applied.
mounted on an inverted Nikon Ti-S microscope and superfused with a physiologic saline
solution (pH 7.4). Single-channel current data were acquired as described
previously.19 (link),20 (link) After a high resistance seal was obtained, cell-attached
recording was performed immediately. The membrane resistance was monitored regularly to
ensure the quality of recording. For measurements of acute effect only one experiment was
performed per dish to avoid any possibility of examining cells whose properties might have
been altered by extended exposure to Ang II. The recordings were made in symmetric
chloride solutions. The bath solution consisted of 126 NaCl, 1 CaCl2, 10 HEPES,
2 MgCl2, 10 glucose, pH 7.4. The pipette solution contained 126 NaCl, 1.5
CaCl2, 10 HEPES, 10 glucose; pH7.4; plus added directly before the
patch-clamp experiments were 100 µM niflumic acid or DIDS (to block
Ca2+-activated Cl− channels), 10 mM TEA (to inhibit
large-conductance Ca2+-dependent K+ channel), 10 nM iberiotoxin (to
block Ca2+-activated K+ channels), 10 µM nicardipine (to
block N-type Ca2+ channels). During the patch-clamp measurements in the
single-channel mode the activity of the ion channels was first monitored in response to
the potential applied in steps of 10 or 20 mV in the range of – 90 mV to + 60 mV
in order to estimate the channel’s conductance and I-V relationship. After that,
the voltage was clamped at – 60 mV and the channels’ activity was recorded
for several minutes before the drugs were applied.
