During stimulation of a central AChergic nerve terminal, the neurotransmitter ACh is released and diffuses across the synaptic cleft to act on a postsynaptic receptor site before it is broken down to choline and acetate. It is well established that the choline in the synaptic cleft is returned to the presynaptic nerve terminal by a transport process to form releasable ACh. Recently reported results from our laboratory suggest that in rat hippocampal tissue, extracellular acetate is also recycled to from ACh. Acetate reuptake appears to occur in a much different way that does choline reuptake. Acetate reuptake occurs independently of sodium and appears to be tightly coupled to the activity of ACS, that enzyme which converts acetate to acetyl CoA when ATP and CoA are present. The long range hypothesis being tested is that ACS mediates the uptake of extracellular acetate into hippocampal AChergic nerve terminals by metabolizing accumulated acetate to acetyl CoA and thereby creating a diffusion gradient which drives extracellular acetate inward. The immediate goal is to ascertain whether ACS is essential to the accumulation of extracellular acetate by the mammalian cell. To accomplish this, the principal investigator will isolate the rat cDNA encoding ACS. Once this has been accomplished, the principal investigator will express the rat ACS cDNA in COS and CHO cells so that the uptake of extracellular acetate can be tested. Accumulation of extracellular acetate by transfected, untransfected, and mock transfected COS and CHO cells will be determined and compared. Knowledge that acetate reuptake into hippocampal AChergic nerve terminal occurs physiologically and is mediated by ACS will help improve our understanding of presynaptic ACh metabolism and may lead to the development of therapeutically useful drugs. Knowledge of these molecular techniques will help the principal investigator test other important aspects of presynaptic ACh metabolism in the future such as whether all of the choline-O-acetyltransferase in a central AChergic nerve terminal is soluble or whether some is also membrane-bound.
