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H. Yang, F. Zhang, Z. Wang, Z. Pan, P. S. Reinach; Prostaglandin E2 Enhances TRPV1 Channel Activity Through Protein Kinase a Stimulation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2621. doi: https://doi.org/.
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Transient receptor potential vanilloid type-1 (TRPV1) channel activation induces proinflammatory cytokine release (i.e. IL-6 and IL-8) and edema in the cornea. Prostaglandin E2 (PGE2) is another edema mediator and increases cAMP formation. Accordingly, we determined in human corneal epithelial cells (HCEC) whether PGE2 augments TRPV1 channel activation through protein kinase A stimulation.
Ca2+ transients were monitored in fura2-loaded HCEC with a single cell fluorescence imaging system. TRPV1 agonists, capsaicin (CAP), anandamide (AEA) and resiniferatoxin (RTX) along with its antagonist, capsazepine (CPZ) validated TRPV1 function. PGE2 and its cognate receptor antagonist, AH-6809; cAMP analog, 8-(4-Chlorophenylthio)-adenosine-3',5'-cyclic monophosphate (CPT-cAMP); a phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX) and a PKA inhibitor, Rp-Adenosine 3’,5’-cyclic monophosphorothioate triethylammonium salt hydrate (Rp-cAMPS); assessed PKA involvement in mediating PGE2 enhancement of TRPV1 activity. Time dependent effects of either CAP, AEA, RTX, interleukin-6 (IL-6) were determined on transepithelial electrical resistance (TEER) of HCEC cultured on Transwell inserts.
CAP (5 µM) alone induced an increase in the F340/380 nm ratio (i.e 0.22 ± 0.2, n = 4 coverslips). Thirty min preincubation with 1µM PGE2, followed by 5 µM CAP nearly doubled this response (Δ ratio = 0.41 ± 0.1, n = 4; p< 0.02). Similar to PGE2, 30 min preincubation with either 10 µM IBMX or 10 µM CPT-cAMP enhanced the CAP (5µM) -induced increase in this ratio (i.e 0.44 ± 0.2, n = 6 coverslips). Rp-cAMPS (10µM) blocked the response to CAP by 75%. AH-6809 (10µM) blocked the PGE2-induced augmentation by 94% whereas 10 µM CPZ suppressed it by 74%. On the 10th day after HCEC seeding, the TEER reached 430 ± 42 ohm.cm2 (n = 12). After 3 h of exposure to either AEA (10µM), CAP (5µM), RTX (10µM) or IL-6 (500 ng/ml), the respective declines were 20%, 19%, 16% and 19%. After 18 h, AEA and IL-6 caused the TEER to fall by 64% and 26%, respectively, whereas with CAP and RTX the TEER was unchanged. Similar declines occurred 3 and 18 hours after exposure to a 375 mOsm hypertonic medium (i.e. 37% and 47%, respectively).
PGE2 enhances TRPV1 channel activation by CAP through PKA pathway stimulation. Losses in HCEC barrier function resulting from TRPV1 activation may account for edema in the intact cornea.
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