Regulation of gap junction coupling in bovine ciliary epithelium.
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.
Wang
Zhao
Z
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Do
Chi Wai
CW
Valiunas
Virginijus
V
Leung
Chi Ting
CT
Cheng
Angela K W
AK
Clark
Abbott F
AF
Wax
Martin B
MB
Chatterton
Jon E
JE
Civan
Mortimer M
MM
D000818:Animals; D001082:Aqueous Humor / Q000378:metabolism; D003994:Bucladesine / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D019850:Heptanol / Q000378:metabolism; D007546:Isoquinolines / Q000378:metabolism; D018408:Patch-Clamp Techniques; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D000097003:Gap Junction alpha-5 Protein
D003994:Bucladesine; D018031:Connexin 43; D017630:Connexins; D017868:Cyclic AMP-Dependent Protein Kinases; D005456:Fluorescent Dyes; D019850:Heptanol; D007546:Isoquinolines; D034741:RNA, Small Interfering; D000097003:Gap Junction alpha-5 Protein; C017475:lucifer yellow