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Detection of Unitary TRPV4 Channel Activity in Primary Human Cerebral Artery Microvascular Endothelial Cells Using TIRF Microscopy
Author(s) -
Sullivan Michelle N,
Pitts Natalie L,
Earley Scott
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1042.22
Subject(s) - chemistry , total internal reflection fluorescence microscope , trpv4 , trpv , ruthenium red , transient receptor potential channel , biophysics , extracellular , endothelium , agonist , medicine , biochemistry , receptor , calcium , membrane , biology , organic chemistry , trpv1
The vanilloid (V) transient receptor potential (TRP) channel TRPV4 is a Ca 2+ ‐permeable cation channel present in the endothelium of cerebral arteries that contributes to the regulation of vascular tone. We sought to develop a method allowing for the detection of unitary TRPV4 channel activity in endothelial cells using Total Internal Reflection Fluorescence Microscopy (TIRFM), a technique that permits observation of fluorescent molecules at or near the plasma membrane. Primary human cerebral artery microvascular endothelial cells (passages 3–5) were loaded with the Ca 2+ indicator dye Fluo‐4 AM and imaged using TIRFM. Under basal conditions, transient Ca 2+ events were recorded with a mean amplitude (F/F o ) of 1.23±0.01, duration of 287±0.04 ms, and frequency of 0.03±0.01 Hz (n =25). Removal of extracellular Ca 2+ abolished these events. Administration of the small molecule TRPV4 agonist GSK1016790A (100 nM) significantly increased the frequency of these events (0.25±0.05 Hz) and decreased the duration (144±0.01 ms), while amplitude remained unchanged (n = 20). In addition, we found that GSK1016790A–induced increases in event frequency were attenuated by the non‐selective TRPV blocker ruthenium red (10 μM). We conclude that Ca 2+ influx events recorded using TIRFM represent unitary TRPV4 activity in primary endothelial cells.