Prostaglandin E2 Enhances TRPV1 Channel Activity Through Protein Kinase a Stimulation

Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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 10^th 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).

Conclusions: : 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.

Keywords: cornea: epithelium • ion channels • signal transduction: pharmacology/physiology