Cooke L J, Rylett R J
Department of Physiology, The University of Western Ontario, London, Canada.
Brain Res. 1997 Mar 21;751(2):232-8. doi: 10.1016/s0006-8993(96)01183-3.
The present investigation examines the effects of phosphatase inhibition on short-term regulation of cholinergic function, with particular emphasis on choline acetyltransferase, the enzyme which synthesizes acetylcholine. Rat hippocampal synaptosomes were treated with either okadaic acid (10 nM) or calyculin-A (50 nM) to inhibit protein phosphatases 1 and 2A for 20 min prior to subfractionation of nerve terminals and measurement of choline acetyltransferase activity, or quantification of high-affinity choline transport and acetylcholine synthesis. Inhibition of synaptosomal phosphatases did not alter total or salt-soluble choline acetyltransferase activity, but membrane-bound and water-soluble forms of the enzyme were selectively increased in okadaic acid-treated nerve terminals to 129 +/- 11% and 137 +/- 10% of control, respectively. High-affinity choline transport was reduced to 77 +/- 6% and 76 +/- 7% of control in calyculin-A- and okadaic acid-treated nerve terminals, respectively. Acetylcholine synthesis was reduced to 73 +/- 6% of control in calyculin-A-treated synaptosomes only; acetylcholine synthesis was at control levels in okadaic acid-treated cultures correlating with enhanced choline acetyltransferase activity in the water-soluble and nonionically membrane-bound fractions. These investigations indicate a role for phosphoprotein phosphatases in the regulation of acetylcholine synthesis in the cholinergic nerve terminal. The observed increases in choline acetyltransferase activity in two subcellular fractions appears to compensate for decreased choline precursor availability, allowing acetylcholine synthesis to be maintained at control levels. The uncoupling of choline transport and acetylcholine synthesis in this situation represents a unique functional role for a subfraction of choline acetyltransferase.
本研究考察了磷酸酶抑制对胆碱能功能短期调节的影响，尤其着重于胆碱乙酰转移酶，即合成乙酰胆碱的酶。在对神经末梢进行亚分级分离并测量胆碱乙酰转移酶活性，或对高亲和力胆碱转运及乙酰胆碱合成进行定量分析之前，用冈田酸（10 nM）或花萼海绵诱癌素A（50 nM）处理大鼠海马突触体20分钟，以抑制蛋白磷酸酶1和2A。突触体磷酸酶的抑制并未改变总胆碱乙酰转移酶活性或盐溶性胆碱乙酰转移酶活性，但在经冈田酸处理的神经末梢中，该酶的膜结合形式和水溶性形式分别选择性增加至对照的129±11%和137±10%。在经花萼海绵诱癌素A和冈田酸处理的神经末梢中，高亲和力胆碱转运分别降至对照的77±6%和76±7%。仅在经花萼海绵诱癌素A处理的突触体中，乙酰胆碱合成降至对照的73±6%；在经冈田酸处理的培养物中，乙酰胆碱合成处于对照水平，这与水溶性和非离子膜结合部分中胆碱乙酰转移酶活性增强相关。这些研究表明磷蛋白磷酸酶在胆碱能神经末梢乙酰胆碱合成的调节中发挥作用。在两个亚细胞部分中观察到的胆碱乙酰转移酶活性增加似乎补偿了胆碱前体可用性的降低，从而使乙酰胆碱合成得以维持在对照水平。在这种情况下，胆碱转运与乙酰胆碱合成的解偶联代表了胆碱乙酰转移酶一个亚分级部分的独特功能作用。
