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LA Voloboueva, YQ Chen, F Wang, TN Litvin, J Buck, LR Levin, SS Miller; Soluble Adenylyl Cyclase in Native Bovine and Human RPE: Expression and Function . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2427.
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© ARVO (1962-2015); The Authors (2016-present)
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 (JV), transepithelial potential (TEP), and resistance (Rt). 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 Rt by 10-25 Ω×cm2, and increased JV 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, Rt, and Jv. 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 Rt that were 50% lower than the changes observed in control HCO3/CO2 Ringer; no change in JV was observed. Conclusion: Bovine and human RPE cells express sAC. In bovine, sAC inhibitors altered TEP/Rt and increased JV. The JV 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.
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