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.
Highlights
Numerous different transient receptor potential (TRP) nonselective ionic channel subtypes are functionally expressed on nearly every tissue in the human body
The specificity of the LUM, KTN and Alpha-smooth muscle actin (α-SMA) antibodies was verified by showing absence of any IF staining in telomeraseimmortalized human corneal epithelial cells [31]
Cluster of Differentiation 90 (CD90) is a biomarker of mesenchymal stem cells (MSC) derived from keratocytes in vitro
Summary
Numerous different transient receptor potential (TRP) nonselective ionic channel subtypes are functionally expressed on nearly every tissue in the human body. They act as polymodal sensors of environmental stresses and induce both adaptive and maladaptive responses under different conditions [1,2,3]. The 28 mammalian TRP channel subtypes are subdivided into six main subfamilies based on differences in their amino acid sequence homology [8]. They display greater diversity in activation mechanisms and selectivity than any other group of ion channels. TRP vanilloid type 1 (TRPV1; capsaicin receptor) belongs to the vanilloid subfamily and is an archetype for other TRP ion channels that transduce a host of environmental stresses to elicit both adaptive and maladaptive responses [9]
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