Measurement of both cyclic [3H]AMP and cyclic [3H]GMP in cultured vascular smooth muscle cells labeled with [3H]hypoxanthine: use in studies of cardiovascular drugs.
We describe a method for prelabeling cultured vascular smooth muscle cells that permits rapid and accurate measurements of changes in the amounts of cyclic AMP and of cyclic GMP in 5 x 10(5) cells. This procedure utilizes [3H]hypoxanthine to radiolabel both the adenine and guanine nucleotide pools and simple column chromatographic steps to isolate and separate the 3H-labeled cyclic nucleotides. The application of the method to studies of the actions of cardiovascular drugs on vascular smooth muscle cells is illustrated by measurements of the effects of isoproterenol, nitroprusside, and inhibitors of cyclic AMP phosphodiesterases on the cyclic nucleotide levels in these cells. If required, the mass amounts of cyclic AMP and cyclic GMP present could be determined by measurement of the specific radioactivities of the precursor [3H]ATP and [3H]GTP, respectively. The cyclic nucleotide values calculated by the latter method were almost identical to those obtained with larger numbers of cells using commercially available radioimmunoassays, thus validating the prelabeling assays. The method described should be applicable to any type of cultured cell that can utilize [3H]hypoxanthine to replenish its ATP and GTP pools.
Maurice
D H
DH
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Lee
R M
RM
Haslam
R J
RJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002317:Cardiovascular Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis* / Q000378:metabolism; D006152:Cyclic GMP / Q000032:analysis* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006160:Guanosine Triphosphate / Q000378:metabolism; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D009131:Muscle, Smooth, Vascular / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D011863:Radioimmunoassay; D051381:Rats; D011921:Rats, Inbred WKY; D015203:Reproducibility of Results; D014316:Tritium
D002317:Cardiovascular Agents; D007042:Hypoxanthines; D014316:Tritium; D009599:Nitroprusside; D019271:Hypoxanthine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D007545:Isoproterenol