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Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTCI) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Re) in recipient to donor cell was 0.47 ± 0.09 (n = 11)10 mm after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R, by ∼60% to 0.20 ± 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by -∼60%, reducing Rïfrom 0.41 ± 0.05 (n = 15) to 0.17 ± 0.05 (n = 20) after 10 mm. Junctional currents were lowered by ∼50% (n = 6) after 10-mm perfusion with 500 p.M dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication. [ABSTRACT FROM AUTHOR]

Intraocular pressure (TOP) is regulated by the resistance to outflow of the eye's aqueous humor. Elevated resistance raises TOP and can cause glaucoma. Despite the importance of outflow resistance, its site and regulation are unclear. The small size, complex geometry, and relative inaccessibility of the outflow pathway have limited study to whole animal, whole eye, or anterior-segment preparations, or isolated cells. We now report measuring elemental contents of the heterogeneous cell types within the intact human trabecular outflow pathway using electron-probe X-ray microanalysis. Baseline contents of Nat, K[sup+] Cl[sup-], and P and volume (monitored as Na+K contents) were comparable to those of epithelial cells previously studied. Elemental contents and volume were altered by ouabain to block Na[sup+]-K[sup+]-activated ATPase and by hypotonicity to trigger a regulatory volume decrease (RVD). Previous results with isolated trabecular meshwork (TM) cells had disagreed whether TM cells express an RVD. In the intact tissue, we found that all cells, including TM cells, displayed a regulatory solute release consistent with an RVD. Selective agonists of A[sub1] and A[sub2] adenosine receptors (ARs), which exert opposite effects on lop, produced similar effects on juxtacanalicular (JCT) cells, previously inaccessible to functional study, but not on Schlemm's canal cells that adjoin the JCT. The results obtained with hypotonicity and AR agonists indicate the potential of this approach to dissect physiological mechanisms in an area that is extremely difficult to study functionally and demonstrate the utility of electron microprobe analysis in studying the cellular physiology of the human trabecular outflow pathway in situ. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]