Ca(2+)/calmodulin-dependent cyclic nucleotide phosphodiesterase in cGMP metabolism in rabbit parotid acinar cells.
Muscarinic cholinergic receptor activation provokes cGMP formation in parotid acinar cells. We investigated the involvement of Ca(2+)/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) in cGMP breakdown in rabbit parotid acinar cells. The muscarinic agonist carbachol stimulated cGMP formation in the cells. The carbachol-induced cGMP formation was enhanced in the presence of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MM-IBMX), a PDE1 inhibitor. cGMPPDE activity in rabbit parotid acinar cells was reduced by about 25% in the absence of Ca(2+)/ calmodulin or in the presence of MM-IBMX. Ca(2+)/calmodulin-dependent cGMP-PDE in rabbit parotid acinar cells was purified using Calmodulin-Sepharose 4B and Mono Q ion-exchange column chromatography. Two dominant fractions with cGMP-PDE activity, referred to as the P-1 and P-2 fractions, were eluted from the Mono Q ion-exchange column. The Km values for cGMP of PDE in the P-1 and P-2 fractions were 0.82 microM and 0.40 microM, respectively, which were much lower than that for cAMP. The EC(50) for Ca(2+) and calmodulin of PDEs in the P-1 and P-2 fractions were 458 nM and 426 nM, respectively, and 32 nM and 137 nM, respectively. Protein bands that crossreacted with anti-PDE1A antibody were detected. These results suggest that Ca(2+)/calmodulin-dependent PDE, PDE1A, is involved in cGMP breakdown in rabbit parotid acinar cells.
Department of Anesthesiology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan.
Nakayasu
N
Sairenji
Keitaro
K
Satoh
Hiroshi
H
Sugiya
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D006152:Cyclic GMP / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D010306:Parotid Gland / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology; D014970:Xanthines / Q000494:pharmacology
C119531:8-methoxymethyl-3-isobutyl-1-methylxanthine; D002147:Calmodulin; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D014970:Xanthines; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP; D002118:Calcium