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Renin-angiotensin system expression and secretory function in cultured human ciliary body non-pigmented epithelium

¹ Wellcome Trust, Cellular Physiology Research Unit, Department of Physiology, National University of Ireland, Cork, Ireland
² Department of Physiology, Chinese University of Hong Kong, Shatin NT, Hong Kong
³ Department of Visual Science, Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA

Correspondence to:
Correspondence to:
Dr Anthony Cullinane, Wellcome Trust Cellular Physiology Research Unit, Department of Physiology, National University of Ireland, Cork, Ireland;
abcullinane{at}hotmail.com

Background: Renin-angiotensin system (RAS) components have been identified in human ciliary body and aqueous humour, pointing to a role for the RAS in the regulation of aqueous humour dynamics. Here, the authors examine the functional expression of a RAS and the effects of angiotensin II (AII) on a signal transduction pathway and ion secretion mechanism in cultured human ciliary body non-pigmented epithelium (HNPE).

Methods: RAS expression was examined in cultured HNPE cells using polymerase chain reaction (PCR) analysis. Secretory function was determined using spectrofluorescence imaging microscopy to measure cell calcium (Ca²⁺_I) and volume responses. Single channel patch clamp techniques were employed to investigate ion channel activity.

Results: PCR analysis demonstrated the expression of angiotensinogen and the AT_1b receptor in HNPE cells. A large conductance potassium (BK) channel (mean 190 (SEM 5.6) pS, n = 22 cells), was observed in plasma membrane patches. This channel was calcium sensitive with channel open probability (Po) increasing with increasing Ca²⁺_I (K_0.5 10.79 (0.44) µM Ca²⁺, Hill coefficient of 1.04 (0.04)). AII (100 nM) increased the number (N) of active BK channels in HNPE cells and also the probability of channel opening (Po). N.P_o increased from 0.008 (0.002) to 1.38 (0.4) following the addition of AII (p=0.0064). AII also induced a rapid rise in Ca²⁺_I from resting values of 112 (14) nM to a peak of 992 (106) nM (p<10^-4). A simultaneous cell volume reduction of 24.70% (3.34%) (p<10^-4) occurred during this calcium signal. Losartan (1 µM) significantly blocked the AII induced BK channel activation (p=0.0131), the Ca²⁺_I response (p<10^-4), and the AII induced volume effect (p=0.0046).

Conclusion: It was demonstrated that AII activates a Ca²⁺_I signalling system which subsequently increases potassium ion channel activity. These effects are accompanied simultaneously by cell volume loss, indicating that AII acts as receptor operated secretagogue in HNPE cells. The ability of an AT₁ receptor antagonist to inhibit these processes may thus offer a new family of pharmaceutical agents to the current armamentarium in the treatment of glaucoma.

Keywords: renin-angiotensin system; secretory function; ciliary body; epithelium

Abbreviations: ACE, angiotensin converting enzyme; AII, angiotensin II; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HNPE, human ciliary body non-pigmented epithelium; PCR, polymerase chain reaction; RAS, renin-angiotensin system

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