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

Abstract

Background: Renin-angiotensin system (RAS) components have been identified in human ciliary body and aqueaus humour, painting to a role for the RAS in the regulation of aqueaus humour dynamics. Here, the authors examine the functional expression of a RAS and the effects af angiotensin II (AII) an 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. Secretary function was determined using spectrafluorescence imaging microscopy to measure cell calcium (Ca2+1) 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 AT1b 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 (Pa) increasing with increasing Ca2+1 (K0.5 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 (Pa). N.Po 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+1 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+1 response (p<10-4), and the AII induced volume effect (p=0.0046). Conclusion: It was demonstrated that AII activates a Ca2+1 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 armomentarium in the treatment of glaucoma.