Increase in ACh release from the rat brain cortex slices by 3',5'-cyclic adenosine monophosphate.

It is known that activities of adenylate cyclase and cyclic nucleotide phosphodiesterase are higher in brain tissue than in other tissues (1-3), and regulatory roles of 3',5'-cyclic adenosine monophosphate (cyclic AMP) on brain function have been reported by several workers (4-11). On the other hand, it has been observed that secretion of many kinds of hormones (12-17) and enzymes (18, 19) is stimulated by intracellular cyclic AMP, and such is known to be stored in subcellular granules. Acetylcholine (ACh) is also known to be stored in granules, the so-called synaptic vesicles in the nerve endings (20), and released by nerve stimulation. Accordingly, it is considered that release of ACh may be stimulated by cyclic AMP. The assumption has been supported (21-24) or questioned (25-27) by electro physiological experiments done on the neuromuscular junction or guinea pig ileum. We carried out studies on brain cortex slices by estimation of the amount of ACh released into the medium. Adult healthy Sprague Dawley rats were decapitated and the brains were immediately removed. The cortex slices were then prepared using a Stadie-Rigg's slicer and were weighed on a torsion balance. About 80 mg of the brain cortex slices was incubated with shaking in 2 ml of Ringer's solution containing 127.2 mM NaCl, 5.13 mM KCI, 2.75 mM CaCI2, 1.28 mM MgSO4, 2.5 mM Na2HPO4-HCI buffer (pH 7.4), 19.25 mM Tris-HCI buffer (pH 7.4), 10 mM glucose and 0.05 mM eserine sulfate. After obtaining a steady state by pre incubation of the slices for 15 min at 37"C, the medium was changed and the slices were then incubated for 15 min with or without effectors. The amount of ACh released into the medium during incubation for 15 min was estimated with eserinzed abdominal muscle of Rana Papiens according to the method of Chang and Gaddum (28). Dibutyryl cyclic AMP, norepinephrine, theophylline or histamine, all had no significant effect on contraction of the frog abdominal muscle by ACh under our conditions, but 35 mM KC1 did cause a contraction of the muscle as is generally known. Thus, the amount of ACh in the high K+ medium was estimated by comparison with known concentrations of ACh in frog Ringer's containing 11.7 mM KC1 after dilution of the experimental materials with 2 volumes of K-free frog Ringer's solution. As shown in Table 1 Exp. A, amount of ACh released from the rat brain cortex slices