L Zhou1, W J Thompson, D E Potter. 1. Department of Pharmacology and Toxicology, Morehouse School of Medicine, Atlanta, Georgia 30310, USA.
Abstract
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 detected. 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.
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 detected. 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.