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C.W. Do, K. Peterson-Yantorno, C.H. Mitchell, M.M. Civan; Cyclic AMP Activates Maxi-Cl- Channels in Freshly Dissociated Bovine Pigmented Ciliary Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3452.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ATP stimulates whole-cell cAMP-activated Cl- currents and reduces volume of immortalized bovine pigmented ciliary epithelial (PE) cells. We investigated the effects of cAMP on single-channel activity and volume of freshly dissociated bovine PE cells. Methods: Single-channel activity was studied with excised inside-out patches of native bovine PE cells. Volume was monitored by measuring cell area with calcein fluorescence. Results: Maxi-Cl- channels were detected in excised inside-out patches of native bovine PE cells with mean conductance of 268 ± 3 pS (N = 42). Open channel probability (Po) was measured at transmembrane voltages clamped at -80 to +80 mV in 20-mV steps. In symmetrical K+-free solutions containing 130 mM NaCl, the Po was 0.057 ± 0.026 at -80 mV (cytoplasmic surface negative to bath, N = 38). Cyclic AMP caused a concentration-dependent increase in Po without changing unitary conductance over a range of concentrations from 30 µM to 500 µM when applied to the cytoplasmic side of the patches. At -80 mV, the addition of 500 µM cAMP increased Po from 0.049 ± 0.042 to 0.687 ± 0.042 (N = 22). The cAMP-triggered activation was completely reversible. The Cl- channel blockers SITS (1 mM) and NPPB (100 µM and 500 µM) significantly and reversibly inhibited the cAMP-activated maxi-Cl- channels. Application of the membrane-permeable cAMP analogue 8-Br-cAMP triggered shrinkage of intact PE cells. The shrinkage was mediated by enhanced Cl- release because NPPB completely blocked the cAMP-induced volume change. Conclusions: The current results establish that cAMP activates Cl- channels in native PE cells in addition to immortalized cells. The cAMP indeed acts directly on its target, the maxi-Cl- channels, and not through protein kinase A. The cAMP-activation provides a possible pathway for reducing the rate of net aqueous humor secretion by enhancing Cl- reabsorption back into the stroma.
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