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Ling Zhou, William J. Thompson, David E. Potter; Multiple Cyclic Nucleotide Phosphodiesterases in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1745-1752.
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purpose. To characterize cyclic nucleotide phosphodiesterase isozyme activities
in human trabecular meshwork cells and primary cultures of porcine
trabecular meshwork cells.
methods. Radioimmunoassay of acetylated acid extracts was used to determine
changes in cyclic adenosine monophosphate (cAMP) and cyclic quanosine
monophosphate (cGMP) in human trabecular meshwork cells treated with
phosphodiesterase isoform selective inhibitors. Cyclic nucleotide
phosphodiesterase activities were measured using the two-step
radioisotope procedure (Thompson). Enzyme activities in the supernatant
of human cells were fractionated using anion-exchange chromatography.
Additionally, human and porcine trabecular meshwork cell transcripts of
phosphodiesterase family–specific isoforms were studied by reverse
transcription-polymerase chain reaction and nucleotide sequencing.
results. In intact human cells, selective inhibitors for phosphodiesterase 4
(rolipram) and 5 (E4021) gene families were effective in augmenting
cyclic nucleotide accumulation in response to isoproterenol or sodium
nitroprusside, respectively. cAMP and cGMP hydrolytic activities,
resolved using Trisacryl M anion-exchange chromatography, showed a cAMP
phosphodiesterase peak that was minimally sensitivity to cGMP but
modestly inhibited by rolipram and a cGMP phosphodiesterase peak that
was sensitive to inhibition by E4021. Further evaluation of the cGMP
phosphodiesterase demonstrated Michaelis-Menten kinetics and
competitive inhibition by E4021. Messenger RNA transcripts for
phosphodiesterase 4, 5, and 7 isozymes were isolated in human
trabecular meshwork cells. However, in porcine trabecular meshwork
cells only isozymes for phosphodiesterase 4 and 5 isozymes were
conclusions. Human trabecular meshwork cells express phosphodiesterase 4, 5, and 7
gene family isoforms and enzyme activities, suggesting that selective
isoform inhibitors could be used to augment the actions of antiglaucoma
drugs that use cyclic nucleotides as second messengers.
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