Vascular Endothelial Growth Factor (VEGF) Induced Downstream Responses to Transient Receptor Potential Vanilloid 1 (TRPV1) and 3-Iodothyronamine (3-T1AM) in Human Corneal Keratocytes

Abstract

This study was undertaken to determine if crosstalk among the transient receptor potential (TRP) melastatin 8 (TRPM8), TRP vanilloid 1 (TRPV1), and vascular endothelial growth factor (VEGF) receptor triad modulates VEGF-induced Ca2+ signaling in human corneal keratocytes. Using RT-PCR, qPCR and immunohistochemistry, we determined TRPV1 and TRPM8 gene and protein coexpression in a human corneal keratocyte cell line (HCK) and human corneal cross sections. Fluorescence Ca2+ imaging using both a photomultiplier and a single cell digital imaging system as well as planar patch-clamping measured relative intracellular Ca2+ levels and underlying whole-cell currents. The TRPV1 agonist capsaicin increased both intracellular Ca2+ levels and whole-cell currents, while the antagonist capsazepine (CPZ) inhibited them. VEGF-induced Ca2+ transients and rises in whole-cell currents were suppressed by CPZ, whereas a selective TRPM8 antagonist, AMTB, increased VEGF signaling. In contrast, an endogenous thyroid hormone-derived metabolite 3-Iodothyronamine (3-T1AM) suppressed increases in the VEGF-induced current. The TRPM8 agonist menthol increased the currents, while AMTB suppressed this response. The VEGF-induced increases in Ca2+ influx and their underlying ionic currents stem from crosstalk between VEGFR and TRPV1, which can be impeded by 3-T1AM-induced TRPM8 activation. Such suppression in turn blocks VEGF-induced TRPV1 activation. Therefore, crosstalk between TRPM8 and TRPV1 inhibits VEGFR-induced activation of TRPV1.

Transient Cell Membrane Disruptions induce Calcium Waves in Corneal Keratocytes

“…TRPV1 and TRPM8 are the only calcium transporters identified in keratocytes to date 18,19 , and no studies have examined cell to cell calcium signaling in keratocytes. In order to accurately analyze TPMD-induced calcium signaling, we chose to examine several parameters in human and mouse cultured stromal cells, and in keratocytes within human corneal rims.…”

“…While many corneal keratocyte signaling pathways have been previously described (e.g. reviewed in 16,17 ), the only keratocyte calcium transport pathways described to date are through the TRPV1 and TRPM8 channels 18,19 . The role of these two channels in keratocyte TPMD-induced calcium wave propagation was examined in the current study.…”

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells

“… 45 , 62 , 63 Although the function of cytosolic TRPV4-ir puncta is less clear, total internal reflection microscopy studies have linked such puncta to stimulus- and development-dependent trafficking of endosomes, membrane translocation, insertion, and/or internalization. 64 – 68 Given that TRPV4 is expressed in the CE, 12 , 14 afferents, 15 fibroblasts, 69 stromal keratocytes, 70 and the endothelium ( Fig. 1 ) it probably functions as a pan-corneal regulator of sensory inputs.…”

Bedeutung von transienten Rezeptorpotenzialkanälen für die okuläre Oberfläche: Einblick und Ausblick – von der Laborforschung zur klinischen Praxis

“…B. Borneol) sind vielversprechend [49,50]. Hier wäre auch eine Anwendung von 3-T 1 AM denkbar [10,43]. Außerdem gibt es Studien, die zeigen, dass über siRNA-Techniken (siRNA: small interfering oligonucleotide of RNA) die Genexpression von TRPV1 unterdrückt werden kann (Tivanisiran) und in weiteren klinischen Studien getestet werden könnte [51].…”

“…Adrenerge Rezeptoren sind an G-Proteine gekoppelt und können über 3-T 1 AM aktiviert werden[52]. Neuere Untersuchungen deuten auf eine Assoziation zwischen VEGF-Rezeptoren und TRPV1-Kanälen hin[10] (Zeichnung S. Mergler).…”