Soluble Adenylyl Cyclase in Native Bovine and Human RPE: Expression and Function

Abstract

Abstract: : Purpose: In the present study we determined the presence of HCO₃-regulated soluble adenylyl cyclase (sAC) in RPE and the changes in RPE physiology caused by known sAC inhibitors. Methods: Total RNA was isolated from human fetal eyes and reverse transcripted with oligo(dT)_12-18 and random primers. Pooled cDNAs were used for PCR with sAC primers. Western blots and adenylyl cyclase activity were measured using bovine eye tissues. Intact monolayers of bovine RPE-choroid were used to measure fluid transport (J_V), transepithelial potential (TEP), and resistance (R_t). Results: Message for sAC was detected with two different primer sets in native human fetal RPE, retina and choroid, and cultured human fetal RPE by RT-PCR. Sequencing of cloned PCR products confirmed the identity of sAC. Western blots confirmed the presence of sAC in bovine RPE and retina. In bovine RPE, cAMP levels were increased 3-fold by addition of 25 mM HCO₃. In control-HCO₃ Ringer, two sAC inhibitors, 2-hydroxyestradiol and 2-hydroxyestrone, decreased TEP by 1-3 mV, increased R_t by 10-25 Ω×cm₂, and increased J_V by 0.5-1.4 µl×cm_-2×hr_-1. The parent estrogens, which do not affect sAC activity, elicited only non-specific changes in TEP, R_t, and J_v. Changes in fluid absorption across RPE seemed to be dependent upon bicarbonate regulation of sAC activity. In bicarbonate/CO₂-free HEPES Ringer with 3 mM acetazolamide, the two sAC inhibitors, as well as the inactive parent estrogens, caused only non-specific changes in TEP and R_t that were 50% lower than the changes observed in control HCO₃/CO₂ Ringer; no change in J_V was observed. Conclusion: Bovine and human RPE cells express sAC. In bovine, sAC inhibitors altered TEP/R_t and increased J_V. The J_V increase was completely blocked in HEPES Ringer. These results suggest the presence of a HCO₃ - regulated enzyme (sAC) whose inhibition alters RPE electrical activity and increases fluid absorption across the epithelium.

Keywords: 394 electrophysiology: non-clinical • 399 enzymes/enzyme inhibitors • 567 retinal pigment epithelium