Properties of a 3H-adenine prelabeled vesicular preparation from guinea pig cerebral cortex. Optimization of incubation conditions for basal- and histamine-stimulated 3H-cyclic AMP levels.

The effects of several variables related to incubation conditions were studied on both the basal and the histamine-stimulated 3H-cyclic AMP levels in a 3H-adenine prelabeled vesicular preparation of guinea pig cerebral cortex. Varying the preincubation time (i.e., the duration of incubation before labeling) caused pronounced differences in the postlabeling time course of 3H-cyclic AMP levels. A preincubation time of 45 min was found to minimize time-related postlabeling declines in 3H-cyclic AMP basal activity. Labeling of the homogenate for an entire experiment in one vessel ("bulk labeling") also contributed to time-related declines in activity; these changes were minimized by labeling aliquots of homogenate individually. Rapid sample mixing during incubation was found to be necessary to ensure adequate suspension of the preparation and to achieve a stable basal activity. Such mixing altered the pH of the samples; maintenance of the pH at 7.4 required a decrease in the bicarbonate concentration and/or use of supplementary buffers. Several parameters related to histamine concentration-response curves were found to be affected by various combinations of buffers and labeling methods. Individual labeling in Krebs-ringer-bicarbonate (15 mM) buffer gave the most stable and reproducible responses to histamine. This method permits detailed pharmacological characterization of transmitter-stimulated cyclic AMP changes in a cell-free system that retains many characteristics of brain slices.