Ugrumov M V, Melnikova V I, Lavrentyeva A V, Kudrin V S, Rayevsky K S
Laboratory of Hormonal Regulations, Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov Street, Moscow 117334, Russia.
Neuroscience. 2004;124(3):629-35. doi: 10.1016/j.neuroscience.2004.01.002.
This study was aimed to test our hypothesis about dopamine (DA) synthesis by non-DAergic neurons expressing individual complementary enzymes of the DA synthetic pathway in cooperation, i.e. L-dihydroxyphenylalanine (L-DOPA) synthesized in tyrosine hydroxylase (TH)-expressing neurons is transported to aromatic L-amino acid decarboxylase (AADC)-expressing neurons for conversion to DA. The mediobasal hypothalamus of rats at the 21st embryonic day was used as an experimental model because it contains mainly monoenzymatic TH neurons and AADC neurons (>99%) whereas the fraction of bienzymatic (DAergic) neurons does not exceed 1%. The fetal substantia nigra containing DAergic neurons served as a control. DA and L-DOPA were measured by high performance liquid chromatography in: (1) cell extracts of the cell suspension prepared ex tempora; (2) cell extracts and incubation medium after the static incubation of the cell suspension with, or without exogenous L-tyrosine; (3) effluents of the incubation medium during perifusion of the cell suspension in the presence, or the absence of L-tyrosine. Total amounts of DA and L-DOPA in the incubation medium and cell extracts after the static incubation were considered as the indexes of the rates of their syntheses. L-Tyrosine administration caused the increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra. Moreover, L-tyrosine provoked an increase of DA synthesis in the substantia nigra and its decrease in the mediobasal hypothalamus. This contradiction is most probably explained by the L-tyrosine-induced competitive inhibition of the L-DOPA transport to the monoenzymatic AADC-neurons after its release from the monoenzymatic TH neurons. Thus, this study provides convincing evidence of cooperative DA synthesis by non-DAergic neurons expressing TH or AADC in fetal rats at the end of the intrauterine development.
本研究旨在验证我们的假设，即表达多巴胺（DA）合成途径中单个互补酶的非多巴胺能神经元协同合成DA，也就是说，在表达酪氨酸羟化酶（TH）的神经元中合成的L-二羟基苯丙氨酸（L-DOPA）被转运到表达芳香族L-氨基酸脱羧酶（AADC）的神经元中转化为DA。将胚胎第21天大鼠的中基底下丘脑用作实验模型，因为它主要包含单酶TH神经元和AADC神经元（>99%），而双酶（多巴胺能）神经元的比例不超过1%。含有多巴胺能神经元的胎儿黑质用作对照。通过高效液相色谱法在以下情况中测量DA和L-DOPA：（1）临时制备的细胞悬液的细胞提取物；（2）细胞悬液在有或无外源性L-酪氨酸的情况下静态孵育后的细胞提取物和孵育培养基；（3）在有或无L-酪氨酸的情况下细胞悬液灌流期间孵育培养基的流出物。静态孵育后孵育培养基和细胞提取物中DA和L-DOPA的总量被视为它们合成速率的指标。给予L-酪氨酸导致中基底下丘脑和黑质中L-DOPA合成增加。此外，L-酪氨酸引起黑质中DA合成增加，而中基底下丘脑DA合成减少。这种矛盾很可能是由于L-酪氨酸诱导的L-DOPA从单酶TH神经元释放后向单酶AADC神经元转运的竞争性抑制。因此，本研究为子宫内发育末期胎鼠中表达TH或AADC的非多巴胺能神经元协同合成DA提供了令人信服的证据。
