John P. Perkins

Methods for the Study of Cyclic AMP Metabolism in Intact Cells

The concentration of adenosine 3′:5′-monophosphate (cAMP) in cells is determined predominantly by the relative rates of synthesis by adenylate cyclase and degradation by phosphodiesterase; although other contributing factors may exist. For example, a significant portion of the cAMP content under basal conditions may exist in a protein-bound form that is not susceptible to hydrolysis by phosphodiesterase activity (1). Also, in certain cases the rate of secretion of cAMP from hormonally-stimulated cells can be a major factor in determining the intracellular content (2, 3). It is usually assumed that hormones elicit a rise in cellular cAMP levels by activation of adenylate cyclase. However, if some degree of turnover of cAMP occurs under basal conditions, then theoretically, the steady state level of cAMP could be raised by either an increase in its rate of synthesis or a decrease in its rate of degradation.

ONTOGENETIC DEVELOPMENT OF THE CATECHOLAMINE-SENSITIVE ADENYL CYCLASE ACTIVITY OF RAT CEREBRAL CORTEX**This work was supported in part by Grant NS 10233 from the National Institute of Neurological Diseases and Stroke.

Publisher Summary This chapter discusses a study which analyzes the ontogenetic development of catecholamine sensitive adenyl cyclase activity of rat cerebral cortex. Also, because adenosine is known to interact synergistically with norepinephrine (NE) to cause marked increases in the adenosine 3′,5′-monophosphate (cAMP) content of brain slices, the development of responsiveness to adenosine and to the combination of adenosine and NE is examined. The formation of 14C-cAMP in response to NE (30 μM), adenosine (100 μM), or 30 μM NE plus 100 μM adenosine was determined in slices of cerebral cortex from rats from birth to 45 days of age. The procedure involved incubation of slices with 14C-adenine to label ATP pools followed by isolation of the 14C-cAMP formed during incubation of slices with the two agonists using ion-exchange chromatography. The validity of this procedure was verified by conducting comparison assays, on the same samples, using the method of Gilman (1970). It is observed that responsiveness to NE develops abruptly between the 10th and 12th days after birth. Responsiveness to adenosine is first detected on day 5 and then it gradually increases to a maximum by day 15.