A highly sensitive fluorometric assay technique was adopted in order to examine the adenylate cyclase activity in the minute right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure (n = 10). Norepinephrine (10-4 M) and adenosine (10-3 M) were incubated for 30 min with 10 μl of membrane preparation (1-2 mg protein/mg) to analyze the extent of the receptor-coupled adenylate cyclase activity. Forskolin (10-4 M) stimulation was used to estimate the maximum adenylate cyclase activity (pmol/mg protein/min, mean ± SE). The new microanalytical cyclic AMP assay involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and fluorometric measurement of NADPH, which is generated in proportion to initial cyclic AMP levels. Basal and forskolin-stimulated maximum adenylate cyclase activities were 75 ± 8 and 123 ± 15, respectively. Norepinephrine increased the adenylate cyclase activity to 107 ± 14, while adenosine tended to decrease it to 65 ± 7. In addition, elimination of adenosine by adenosine deaminase (10 U/ml) slightly increased the adenylate cyclase activity to 82 ± 9. These results indicate that the adenylate cyclase activity can be measured in minute endomyocardial biopsy samples. Use of this new approach shows promise of becoming a new and potentially important way to predict the efficacy of pharmacological treatment.
|Original language||English (US)|
|Number of pages||5|
|Journal||Journal of Clinical Laboratory Analysis|
|State||Published - 2000|
- Congestive heart failure
- Cyclic AMP
- Enzymatic fluorometric assay