Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.
Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.
Rybalkin
Sergei D
SD
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
Rybalkina
Irina G
IG
Feil
Robert
R
Hofmann
Franz
F
Beavo
Joseph A
JA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000906:Antibodies; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D005260:Female; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D010748:Phosphopeptides / Q000737:chemistry / Q000276:immunology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D014590:Uterine Contraction; D014599:Uterus / Q000166:cytology / Q000201:enzymology* / Q000502:physiology
D000906:Antibodies; D010748:Phosphopeptides; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; C517382:Pde5a protein, mouse