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Articles by A. F Clark
Total Records ( 2 ) for A. F Clark
  Z Wang , C. W Do , V Valiunas , C. T Leung , A. K. W Cheng , A. F Clark , M. B Wax , J. E Chatterton and M. M. Civan

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 (Rf) 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 Rf by ~60% to 0.20 ± 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 µM) also reduced LY dye transfer by ~60%, reducing Rf from 0.41 ± 0.05 (n = 15) to 0.17 ± 0.05 (n = 20) after 10 min. Junctional currents were lowered by ~50% (n = 6) after 10-min perfusion with 500 µM 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.

  X Liu , C. O. C Bellamy , M. A Bailey , L. J Mullins , D. R Dunbar , C. J Kenyon , G Brooker , S Kantachuvesiri , K Maratou , A Ashek , A. F Clark , S Fleming and J. J. Mullins

Severe forms of hypertension are characterized by high blood pressure combined with end organ damage. Through the development and refinement of a transgenic rat model of malignant hypertension incorporating the mouse renin gene, we previously identified a quantitative trait locus on chromosome 10, which affects malignant hypertension severity and morbidity. We next generated an inducible malignant hypertensive model where the timing, severity, and duration of hypertension was placed under the control of the researcher, allowing development of and recovery from end organ damage to be investigated. We have now generated novel consomic Lewis and Fischer rat strains with inducible hypertension and additional strains that are reciprocally congenic for the refined chromosome 10 quantitative trait locus. We have captured a modifier of end organ damage within the congenic region and, using a range of bioinformatic, biochemical and molecular biological techniques, have identified angiotensin-converting enzyme as the modifier of hypertension-induced tissue microvascular injury. Reciprocal differences between angiotensin-converting enzyme and the anti-inflammatory tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro in the kidney, a tissue susceptible to end organ damage, suggest a mechanism for the amelioration of hypertension-dependent damage.

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