Isozymes of cyclic-3',5'-nucleotide phosphodiesterases in renal epithelial LLC-PK1 cells.
Metabolism of cAMP and cGMP by the major types (families) of cyclic-3',5'-nucleotide phosphodiesterases (PDE) was studied in confluent renal epithelial LLC-PK1 cells grown in vitro. LLC-PK1 cells mainly contain the cAMP-specific rolipram-sensitive PDE type-IV (PDE-IV), the Ca(2+)-calmodulin dependent PDE type-I and cGMP-specific PDE type-V; all these PDEs are mainly localized in cytosol. Analysis of PDE activities in soluble extract of LLC-PK1 cell homogenate by FPLC ionex chromatography on Mono-Q column also disclosed the presence of low activities of cGMP-stimulated PDE-II and PDE-III. Moreover, activity of PDE-IV was resolved into four distinct chromatographic peaks. The increase of cAMP level in response to incubation of intact LLC-PK1 cells with vasopressin (AVP) was markedly enhanced in the presence of rolipram, but not in the presence of other PDE isozyme-specific inhibitors. Incubation with AVP and atriopeptin (ANP) together resulted in increase in cGMP and a small decrease of cAMP accumulation in LLC-PK1 cells. Results of these studies first show that the LLC-PK1 cells contain all five major types of PDE isozymes where PDE-IV, PDE-I and PDE-V are quantitatively predominant. The rolipram-sensitive PDE-IV, present in several chromatographically distinct forms, appears to be the key PDE isozyme involved in control of cAMP generated in response to stimulation by AVP in LLC-PK1 cells.
Division of Nephrology, Mayo Clinic, Rochester, Minnesota 55905.
M E
ME
Rassier
S J
SJ
McIntyre
M
M
Yamaki
S
S
Takeda
J T
JT
Lin
T P
TP
Dousa
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004848:Epithelium / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D008566:Membranes / Q000201:enzymology
D007527:Isoenzymes; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP