December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
The Effect of PKA in the Circadian Rhythm of Ciliary Epithelium
Author Affiliations & Notes
  • Y Li
    Ophthalmology Yale University New Haven CT
  • D Lu
    New Haven CT
  • M Sears
    New Haven CT
  • Footnotes
    Commercial Relationships   Y. Li, None; D. Lu , None; M. Sears , None.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 1367. doi:
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      Y Li, D Lu, M Sears; The Effect of PKA in the Circadian Rhythm of Ciliary Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1367.

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Abstract

Abstract: : Purpose: Intraocular pressure and aqueous flow of rabbit have a circadian rhythm, we hypothesis that there are differentially expressed factors in the circadian cycle to regulate aqueous humor formation in the ciliary epithelium. The aim of this study is to screen differential display genes in ciliary epithelium in dark time and light time and testify the differential displaied gene variation style in the rabbit housed in 12 light and 12 dark circadian cycle. Method: Total RNA from pure ciliary epithelium of rabbits at light time (CT1) and Dark time(CT13) were compared by differential display reverse transcription-polymerase chain reaction (DD RT-PCR), a differential expressed band choose as a probe to screen the pure ciliary epithelium cDNA library constructed by ourselves. The differentially expressed in dark time was identified as PKA catalytic. Specific primers designed from reporter genes of PKA regulatory subunits to amplify PKA subunits. PKA level were evaluated with western bolt for protein and semi-quantitative RT PCR and Northern blot for mRNA. The location of the PKA subunits were got from in situ hybridization. PKA-CL is cloned from the ciliary epithelium, to testify its interaction with PKA, two mammalian expression vector pCMV-PKACL and pcDNA3-PKACalpha were contransfected into HEK 293 cells, fusion protein were detected with Co Immunoprecipitin. Result: Rabbit PKA Calpha, RI alpha, RIIalpha and RII beta has a high homology to human, rat, mouse and bovine. The densitometry of PKA subunits band in the western blot show a circadian cycle, just like the circadian cycle of aqueous flow and cAMP, the peak comes up at CT 19 and then decrease. The mRNA detected with Northen blot show the same circadian cycle. The location dectected with in situ hybridization demonstrate that PKA C alpha disper at the membranes of PE and NPE; PKA RI alpha located at the gap junction of PE and NPE; RII alpha and RII beta can be been at the membranes of PE and NPE. A positive band of fusion protein were found with COIP. Conclusion: PKA Diurnal expression of PKA Calpha, RII alpha, RII alphaand RII beta sugest that PKA play an important role in regulating of aqueous flow. This effective function could be make with regulating chloride channel.

Keywords: 349 circadian rhythms • 348 ciliary body 
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