Acceleration of choline uptake after depolarization-induced acetylcholine release in rat cortical synaptosomes.

Activation of nerve elements in uivo and in vitro is associated with an increased rate of choline uptake by a Na+-dependent high affinity transport system. Following the methodology of BARKER (1976), rat cortical synaptosomes were depolarized (37"C, 10 min) by 25 mM-KCI in the presence of CaClz (I mM) or other divalent cations. After reisolation by centrifugation, the rate of 3H-choline uptake (1.25 p ~ ) was measured by Millipore filtration. KCl treatment alone failed to accelerate the rate of uptake in the reisolated synaptosomes. CaCI2, BaC12 or SrCI2 (but not MgC12 or MnCI2) were necessary ( l m ~ ) to observe the KCI induced acceleration. Moreover, RbCI, but not LiCl or CsCI, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The conditions mediating the enhanced rate of choline uptake correlated strongly with those associated with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured in response to the various salt treatments. Treatment with KCI (25 mM) and CaCI2 (1 mM), but not KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113 pmol/mg protein. The respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation with 50 pM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system is inversely related to the synaptosomal acetylcholine content. ACETYLCHOLINE biosynthesis is closely linked to acetylcholine release. COLLIER & KATZ (1974), for example, showed that acetylcholine synthesis keeps pace with release in the rat superior cervical sympathetic ganglion. POTTER (1970) has reached similar conclusions using the rat phrenic nerve-diaphragm preparation. In the rat CNS, SIMON et al. (1976) demonstrated that increases or decreases in neuronal activity in specific regions were accompanied by corresponding changes in the Na+ -and temperature-dependent high affinity choline uptake system. Furthermore, BARKER (1976) demonstrated that after K + depolarization of rat brain synaptosomes in uitro, choline uptake was enhanced. He reported an increase in the V,,, of transport, but no change in K,. JENDEN et al. (1976) reported that there was an inverse correlation of choline uptake and acetylcholine content (altered by treatment in uivo) in mouse brain synaptosomes. They suggested that synaptosomal acetylcholine inhibits the high affinity choline uptake system. Following the lead of BARKER (1976) and JENDEN et a!. (1976), the mechanism of the K + mediated increase in the rate of choline uptake was examined. In contrast to BARKER'S results (1976), initial experiments indicated that increased choline uptake occurred under conditions associated with previous acetylcholine release. To confirm this notion, acetylThis work was supported by US. Public Health Service Grant NS-11310. choline content in synaptosomes in response to K + depolarization in the presence or absence of CaZ+ and other agents was measured. Increases in the rate of choline uptake occurred only when acetylcholine depletion was found. MATERIALS AND METHODS Depolarization of synaptosomes. Using male SpragueDawley rats ( 1 5ck2OO g), cerebral cortical synaptosomes were prepared by the procedure of COTMAN et al. (1976). They were suspended in Ringer solution (glucose, IOmM; KCI, 1 mM; MgSO,, 1.2 mM; Na2HP04, 2 . 4 m ~ ; NaCI, 150 m ~ ; Tris-HCI, 10 mM; CaCIZ, 0 mM; all adjusted to pH 7.4) and incubated 10min at 37°C prior to all subsequent manipulations. Unless otherwise noted, the following protocol was used for depolarization. Portions of 2 M-KCI and 0.2 M-CaCI, to give the final specified concentration were added to the synaptosomes (1 mg protein/ml) suspended in Ringer solution at 0". No addition was made to the reference synaptosomes which were carried through all the incubations and centrifugations in parallel with the treated synaptosomes. After the specified addition, the suspensions were incubated for 10min at 37°C. The tubes were centrifuged (5000 #, 10 min, 0°C) and the pellets were resuspended in the original vol of Ringer solution by vortexing and then recentrifuged. The pellets were resuspended in the initial vol of Ringer solution and transport experiments were then performed (pilot studies showed that the vortexing did not produce increased liberation of lactate dehydrogenase into the supernatant). Portions of synaptosomes (90 p1 of 1 mg protein/ml) were pre-incubated at 37°C (5 min) in triplicate. Then 10 pl