Regulation of gap junction coupling in bovine ciliary epithelium

Abstract

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 (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R(f)) in recipient to donor cell was 0.47 +/- 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R(f) by approximately 60% to 0.20 +/- 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 microM) also reduced LY dye transfer by approximately 60%, reducing R(f) from 0.41 +/- 0.05 (n = 15) to 0.17 +/- 0.05 (n = 20) after 10 min. Junctional currents were lowered by approximately 50% (n = 6) after 10-min perfusion with 500 microM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 microM) 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.

Fig. 1.

Pathways for aqueous humor formation. For clarity, only the major transporters subserving secretion are indicated. Mechanisms that could permit ionic recycling at the basolateral membranes have been omitted. Na⁺, Cl⁻, and water from the stroma enter pigmented epithelial (PE) cells through electroneutral symports and antiports, cross gap junctions (GJ) into the nonpigmented epithelial (NPE) cells, and are released through Na⁺-K⁺-activated ATPase, Cl⁻ channels, and AQP1 and AQP4 aquaporin water channels, respectively. Aquaporins have not yet been identified in PE cells. Tight junctions (TJ) connect neighboring NPE cells.

Fig. 2.

Effect of heptanol on junctional currents of a PE-NPE cell couplet. Following the voltage protocol displayed at bottom, voltages applied to the stepped cell (cell 1) produced junctional currents recorded in the recipient cell (cell 2) (A). Currents in both cells were stable when measured under control conditions at points during the subsequent 16 min (B–D). Perfusion with 4.3 mM heptanol for 2.5 min abolished the junctional currents (E), but currents in both the stepped and recipient cells were restored after subsequent washout for 7.5 min (F). t, Time.

Fig. 3.

Effects of dibutyryl-cAMP, H-89, and heptanol on junctional currents of PE-NPE cell couplets. Currents in the stepped cell (cell 1; A–F, bottom) and in the recipient cell (cell 2; A–F, top) were recorded during the course of perfusing cAMP (500 μM dibutyryl-cAMP) and heptanol (4.3 mM) sequentially with or without H-89 (2 μM) in separate couplets. H-89 prevented the inhibitory effect of cAMP.

Fig. 4.

Summary of effects of dibutyryl-cAMP, with (n = 6) or without (n = 6) the PKA blocker H-89, on junctional currents of PE-NPE cell couplets. Values were calculated at voltages of +10 mV (A) and −10 mV (B). In the absence of H-89, dibutyryl-cAMP inhibited the junctional currents by ∼50% (*P < 0.01).

Fig. 5.

Transfer of Lucifer Yellow dye through gap junctions of a PE-NPE cell couplet. Transfer of the fluorescent dye from the donor to the recipient cell was observed as early as 1 min after break-in. A bright-field photomicrograph of the PE-NPE couplet taken at the conclusion of the experiment is included at bottom.

Fig. 6.

Effects of dibutyryl-cAMP and heptanol on Lucifer Yellow (LY) transfer through PE-NPE gap junctions. In comparison to control couplets (n = 15), cAMP (n = 20) and heptanol (n = 6) reduced gap junctional permeability (*P = 0.002, ANOVA) in parallel experiments. In this figure and Fig. 10, the dye transfer has been quantified as the mean intensity ratio (± SE) in the recipient to the donor cell.

Fig. 7.

Relative quantification of message for connexin 43 (Cx43) and Cx40 by quantitative RT-PCR (qRT-PCR). Values are presented as means ± SD. RNA was prepared from duplicate samples extracted from bovine kidney and brain and from a single harvest of bovine ciliary epithelial cells. Each value was averaged from four real-time qRT-PCR measurements. Message detected in ciliary epithelial cells for Cx43 was two orders of magnitude larger than that for Cx40. Message for the widely distributed Cx43 was also detected in bovine kidney and brain. However, as expected, Cx40 was substantially expressed in kidney, but not in brain.

Fig. 8.

Effect of small interfering RNA (siRNA) knockdown on relative quantification of message for Cx43 by qRT-PCR. Values are presented as means ± SD. Control cells were nontreated (control), treated with nontargeting control siRNA (NTC1), or treated with lipofectant alone (−siRNA). Relative to NTC1, transfection with siRNA directed against Cx43 (siCx43) reduced the message after 24 h (n = 4, *P < 0.001).

Fig. 9.

Effect of siRNA knockdown on Western immunoblot of Cx43. Expression of the Cx43 protein product was substantially reduced by the Cx43 siRNA in comparison to the control lanes 48 h after transfection. Tubulin served as the housekeeping control. Similar results were obtained in a replicate immunoblot, and the mean band intensities are provided in the text.

Fig. 10.

Effect of siRNA knockdown of Cx43 on Lucifer Yellow dye transfer across PE-NPE gap junctions. Values are means ± SE. Incubation with Cx43 siRNA for 48 h (n = 13) inhibited dye transfer observed in control cells (n = 11) by ∼60% (P < 0.02).