Determination of levels of cyclic AMP in the myenteric plexus of guinea-pig small intestine.

Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to investigate changes in levels of cyclic 3',5'-adenosine monophosphate (cAMP) in response to stimulation of adenylate cyclase by forskolin and inhibition of phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX). A linear relation with a positive correlation coefficient greater than 0.98 was found between: (1) amount of cAMP and number of ganglia; (2) amount of protein and number of ganglia; (3) amount of DNA and amount of protein; (4) amount of DNA and number of ganglia. Basal levels of cAMP were 2.25 +/- 0.21 fmol per ganglion for 900 ganglia. Forskolin stimulated a dose-dependent increase in cAMP over a concentration range of 0.05 to 50 microM, with a level of 18.6 +/- 4.9 fmol/ganglion at 50 microM forskolin. The inactive forskolin analog 1,9-dideoxyforskolin did not elevate cAMP. Addition of IBMX to the incubation medium stimulated a dose-dependent increase in cAMP over a concentration range of 0.1-1000 microM, with a level of 17.58 +/- 3.38 fmol/ganglion at 1000 microM IBMX. Application of 1 mM IBMX strongly potentiated the stimulating action of forskolin on cAMP levels. Our results derived from direct determination of cAMP changes in small intestinal myenteric ganglia are consistent with existing electrophysiological evidence for second messenger function of cAMP in slow synaptic modulation of excitability in AH/Type 2 neurons of the enteric nervous system.