Optical Recording Reveals Novel Properties of GSK1016790A-Induced Vanilloid Transient Receptor Potential Channel TRPV4 Activity in Primary Human Endothelial Cells

Abstract

Critical functions of the vascular endothelium are regulated by changes in intracellular [Ca 2ϩ ]. Endothelial dysfunction is tightly associated with cardiovascular disease, and improved understanding of Ca 2ϩ entry pathways in these cells will have a significant impact on human health. However, much about Ca 2ϩ influx channels in endothelial cells remains unknown because they are difficult to study using conventional patchclamp electrophysiology. Here we describe a novel, highly efficient method for recording and analyzing Ca 2ϩ -permeable channel activity in primary human endothelial cells using a unique combination of total internal reflection fluorescence microscopy (TIRFM), custom software-based detection, and selective pharmacology. Our findings indicate that activity of the vanilloid (V) transient receptor potential (TRP) channel TRPV4 can be rapidly recorded and characterized at the singlechannel level using this method, providing novel insight into channel function. Using this method, we show that although TRPV4 protein is evenly distributed throughout the plasma membrane, most channels are silent even during maximal stimulation with the potent TRPV4 agonist N-. Furthermore, our findings indicate that GSK1016790A acts by recruiting previously inactive channels, rather than through increasing elevation of basal activity.

Transient Receptor Potential Channels as Drug Targets: From the Science of Basic Research to the Art of Medicine

“…The TRPV4 agonist GSK1016790A, which seems to act by recruiting inactive TRPV4 channels from intracellular depots (Sullivan et al, 2012), can produce dramatic deformation of cultured human endothelial cells (Willette et al, 2008), but it is only part of the story. It was recently shown that TRPV4 channels expressed on alveolar macrophages can play a critical role in ventilatorinduced injury of mouse isolated lungs (Hamanaka et al, 2010).…”

Role of TRPV channels in regulating various pancreatic β-cell functions: Lessons from <i>in vitro</i> studies

“…This effects of hIAPP was supressed by addition of gadolinium a non-specific TRP channel inhibitor. Another experiments showed that the hIAPPinduced intracellular Ca 2+ rise in min6 cells can be also suppressed by ruthenium red, which is a noncompetitive pan inhibitor of TRPs, in particularly TRPV channels including TRPV4 (and also TRPV1) (56,57 (47). Consistent with the incapability of capsaicin to affect calcium levels in rat primary β-cells, others reported that rat pancreatic islets lack TRPV1 (49).…”

TRP channel Ca2+ sparklets: fundamental signals underlying endothelium-dependent hyperpolarization

“…Like L-type Ca 2ϩ channel sparklets, unitary Ca 2ϩ influx events through TRP channels are called "TRP channel Ca 2ϩ sparklets," and the molecular identity of the responsible TRP channel is specified (78,82). For example, single-channel Ca 2ϩ influx events through TRPV4 are called TRPV4 Ca 2ϩ sparklets.…”

“…For example, single-channel Ca 2ϩ influx events through TRPV4 are called TRPV4 Ca 2ϩ sparklets. TRPV4 Ca 2ϩ sparklets were recently characterized in primary human microvascular endothelial cells (82). In this study, endothelial cells were loaded with the fast Ca 2ϩ indicator dye fluo 4-AM, and Ca 2ϩ sparklets at the plasma membrane, indicated by transient fluorescent signals, were recorded using TIRF microscopy ( Fig.…”

“…Several TRP channels are known to be involved in endothelial cell Ca 2ϩ influx and regulation of vascular tone (Table 1) (18,19,75,105). Recently, optical recording methods were used to describe Ca 2ϩ influx events through unitary transient receptor potential vanilloid (TRPV4) channels, termed "TRPV4 Ca 2ϩ sparklets," in vascular endothelial cells (78,82). More interestingly, analysis of TRPV4 Ca 2ϩ sparklets in the intact endothelium indicated that a small number of sparklets can evoke a near-maximal endothelium-dependent vasodilation in pressurized arteries (78).…”