Enzymatic fluorometric assay for tissue cAMP

✍ Scribed by Atsushi Sugiyama; Phi Wiegn; Scott McKnite; Keith G. Lurie

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 581 KB
• Volume: 8
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860080616

✦ Synopsis

cAMP is commonly measured using either immunoassay or high-performance liquid chromatography. The current methods are sensitive but may lack versatility and be expensive; also, radioactivity is potentially harmful to the operator and environment. Given these concerns, we developed a highly sensitive enzymatic fluorometric assay for CAMP. The method consists of five steps: (1) destruction of interfering compounds with apyrase, 5' nucleotidase, adenosine deaminase, and alkaline phosphatase; (2) conversion of cAMP toAMP; (3) conversion ofAMP to ATP; (4) amplification ofATP by ATP-ADP cycling; and (5) fluorometric measurement of resultant NADPH. cAMP was measured in male Sprague Dawley rats anesthetized with pentobarbital. Stimulated rats (n=4) received isoproterenol (16 pg/kg, s.q.) and aminophylline (20 mgkg, s.q.), whereas con-trols (n=4) received no additional drug. With the enzymatic fluorometric assay, cAMP content in heart, liver, and kidney (pmol/mg wet wt, mean f SEM) was 0.34 0.03,0.33 f 0.03, and 0.92 f 0.11 in the control group and 0.77 f 0.10, 0.66 2 0.04, and 1.53 f 0.12 in the stimulated group, respectively. The total assay duration including sample reading procedure varied at 4.5-9.5 hr, depending on its sensitivity. cAMP from the same samples was measured using a commercially available enzyme immunoassay kit and was found to be very similar to the enzymatic fluorometric assay. We conclude that this new assay is sensitive, safe, versatile, and inexpensive and can be used to measure cAMP in multiple types of tissue, including biopsy samples weighing <200 pg.