cAMP-activated maxi-Cl⁻ channels in native bovine pigmented ciliary epithelial cells

Abstract

The eye's aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl(-) enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl(-) channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl(-) channels with a unitary conductance of 272 +/- 2 pS (n = 80). Voltage steps from 0 to +/-80 mV, but not to +/-40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl(-) channels. cAMP also activated the maxi-Cl(-) channels in outside-out patches. In both cases, maxi-Cl(-) channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl(-) concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl(-) concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at +/-80 but not at +/-40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl(-) channels of native PE cells that contribute to Cl(-) release particularly from Cl(-)-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl(-) reabsorption into the ciliary stroma.

The maxi-anion channel: a classical channel playing novel roles through an unidentified molecular entity

“…These channels were similar to those observed in cultured cortical astrocytes from rats [4,49,50] and mice [5][6][7]51] as well as in a rat astrocytic RGCN cell line [52]. In epithelia, the urinary bladder [53], gastric [54], pancreatic [55][56][57], colonic [58][59][60], airway [61][62][63], choroid plexus [64], bile duct [65,66], ciliary [67][68][69], renal [9,19,[70][71][72][73][74][75][76][77][78], vestibular [79], placental [80][81][82][83][84][85][86], ruminal [87] and ovarian [84] epithelial cells were also found to express the maxianion channel with properties similar to those of excitable cells. Resembling maxi-anion channels were also found in fibroblasts [14,…”

“…The channel is thought to participate in the Cl -efflux during cell volume regulation in cortical collecting duct epithelium [9,75], pigmented ciliary epithelial cells [68], lymphocytes [102,104], myoblasts [23], placenta [80], neuronal cells [3] and astrocytes [4]. The maxi-anion channel is expressed in the ciliary epithelium, where it may participate in the aqueous humor secretion [67,130], and in the hepatic bile duct, where it could be involved in bile formation [65,66]. Efflux of Cl -ions during apoptotic cell shrinkage is also thought to occur via maxi-anion channels in neuronal cells [46].…”

Regulation of gap junction coupling in bovine ciliary epithelium

“…In contrast to its direct activation of maxi-Cl Ϫ channels of PE cells (24,27), cAMP can lower gap junctional communication indirectly by activating PKA, at least under our experimental conditions. This observation adds to the literature documenting that cAMP exerts actions expected both to stimulate and inhibit net aqueous humor formation (10,21).…”

“…The previously described procedure for cell isolation and preparation (24) was slightly modified to increase the yield of PE-NPE cell couplets. Briefly, fresh bovine eyes were obtained from a local abattoir.…”

Melatonin Receptors Trigger cAMP Production and Inhibit Chloride Movements in Nonpigmented Ciliary Epithelial Cells

“…Also experiments performed using patch clamp techniques have demonstrated that cAMP can activate chloride channels such as the maxi-Cl 2 , although its physiologic role in situ has not been fully elucidated (Mitchell and Civan, 1997;Do et al, 2004b). Small changes in cAMP cytosolic concentrations may produce vast changes in the chloride efflux.…”