Determination of Ca2+/calmodulin-stimulated phosphodiesterase activity in intact cells.
Ca(2+)/calmodulin (CaM)-stimulated phosphodiesterases (PDEs) constitute a large family (PDE1 family) of enzymes. All members of the PDE1 family can be stimulated by Ca(2+) in the presence of CaM in vitro. It has been shown that the Ca(2+)/CaM-stimulated PDE activity present in the vessel wall or vascular smooth muscle cells can be stimulated in vivo by contracting reagents that increase intracellular Ca(2+) concentrations. We describe in detail a technique used to estimate the extent of PDE1 activation in vivo by measuring in vitro the PDE activity that represents the extent of association between Ca(2+)-CaM and PDE1 in vivo. The technique involves the extraction and rapid assay of enzyme activity at a low temperature and in the presence of trifluoperazine to minimize the changes in association between Ca(2+)-CaM and the PDE1 family member during cell lysis and assaying activity. This technique can be used to measure Ca(2+)/CaM-stimulated PDE activity in cultured cells or tissues.
Yan
Chen
C
Pulmonary Critical Care Branch, NHLBI, NIH, Bethesda, MD, USA.
D000818:Animals; D001011:Aorta / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002147:Calmodulin / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D032389:Myocytes, Smooth Muscle / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D014268:Trifluoperazine / Q000737:chemistry
D002147:Calmodulin; D014268:Trifluoperazine; D010727:Phosphoric Diester Hydrolases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D002118:Calcium