Cholinergic properties of rat atrial subcellular preparations

Several presynaptic cholinergic markers were measured in subcellular fractions of the rat cardiac atrium. A P2 fraction consisting of isolated terminals, free mitochondria, glycogen and other subcellular organelles was prepared first by sucrose density centrifugation. No intact cells were recovered in this fraction, as determined by electron microscopy. Hemicholinium-3 sensitive choline transport coupled to acetylcholine synthesis was concentrated over 10-fold in the P2 fraction compared to minced atria when expressed per mg protein. Six subfractions were recovered after centrifugation of the P2 fraction over a sucrose-metrizamide density gradient. One of these (fraction 4) consistently contained higher levels of choline acetyltransferase activity and hemicholinium-3 sensitive [3H]acetylcholine synthesis than were present in the P2 fraction of the other five subfractions. Electron microscopy of fraction 4 revealed isolated nerve terminals among other subcellular organelles. Kinetic analysis of total [3H]choline uptake in the P2 fraction revealed two apparent uptake processes, with Kts of approximately 11 microM and 219 microM. [3H]Acetylcholine synthesis was partially sodium-dependent in the P2 fraction, and reached a maximal level between choline concentrations of 100 to 200 microM. Some of the newly synthesized [3H]-acetylcholine in the P2 fraction was released by 50 mM K+ depolarization in a calcium-dependent manner, arguing for a neuronal localization. This depolarization-induced release was attenuated by 10 to 100 microM oxotremorine in an atropine-sensitive manner, but was not affected by 1 microM tetrodotoxin.