Inactivation of atrial natriuretic factor-stimulated cyclic guanosine 3',5'-monophosphate (cGMP) in UMR-106 osteoblast-like cells.
Previous studies have suggested a role of cyclic guanosine 3', 5'-monophosphate (cGMP) in the differentiation and proliferation of osteoblasts. We studied the effect of ANF (atrial natriuretic factor) on intracellular cGMP accumulation, cGMP efflux, and cGMP-phosphodiesterase (PDE) activity in UMR-106 osteoblast-like cells. ANF rapidly increased both intracellular cGMP and cGMP efflux. ANF-stimulated intracellular cGMP peaked at 2 min in the absence and at 10 min in the presence of 0.25 mM 3-isobutyl-1-methylxanthine. Probenecid, an antagonist of anion transport, blocked the efflux of cGMP (IC(50) = 0.1 mM), ruling out simple diffusion as a mechanism of the efflux. cGMP-PDE activity was increased threefold in crude homogenates from ANF-treated cells (IC(50) = 23 nM). ANF-evoked stimulation of cGMP-PDE activity was reached simultaneously with the peak in intracellular cGMP. Separation of the PDEs by Q-Sepharose chromatography revealed three cGMP-hydrolyzing peaks. The first peak was sensitive to the PDE5 (cGMP-specific PDE) isoenzyme-selective inhibitor zaprinast (IC(50) = 0.45 microM). The second peak was stimulated fourfold by the addition of calcium/calmodulin, indicating the presence of PDE1. The third peak was sensitive to the PDE2 (cGMP-stimulated PDE) isoenzyme-selective inhibitor 9-[2-hydroxy-3-nonyl]adenine (EHNA) (IC(50) = 3 microM), and was activated by over 300% in the presence of 4 microM cGMP. Our results show that ANF-stimulated cGMP is released from UMR-106 cells by a probenecid-sensitive mechanism. ANF also stimulates cGMP hydrolysis by activating cGMP-PDE activity. Three distinct cGMP-hydrolyzing PDEs, namely PDE5, PDE1, and PDE2, are present in the studied cells.
Ahlström
M
M
Department of Applied Chemistry and Microbiology, P.O. Box 27, University of Helsinki, 00014, Helsinki, Finland.
Lamberg-Allardt
C
C
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006152:Cyclic GMP / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism; D010641:Phenotype; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D009320:Atrial Natriuretic Factor; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP