多巴胺对大鼠背角WDR神经元的抑制不被酚妥拉明和纳洛酮所拮抗

本实验用微电极细胞外记录方法研究了脊髓局部应用多巴胺对大鼠背角WDR神经元的抑制作用。实验在43只SD大鼠上共记录到54个WDR神经元。多巴胺的剂量从0.26×10~(-6)mol/kg-1.58×10~(-6)mol/kg逐步增加,其对伤害性经皮电刺激诱发的背角神经元后串放电的抑制作用也随之加强;0.52×10~(-6)mol/kg多巴胺的作用在给药后5min即出现,15min达高峰并在此后的25min基本维持在同一水平,这个作用可被静注多巴胺能受体拮抗剂氟哌啶(0.66×10~(-6)mol/kg)完全翻转,而不受静注酚妥拉明(2.65×10~(-6)mol/kg)和纳洛酮(1.37×10~(-6)mol/kg)影响。上述结果表明,多巴胺可能是参与脊髓水平伤害性信息传递调控的另一单胺类神经递质。     

多巴胺受体拮抗剂对缢蛏多巴胺D2类受体的影响

为探究多巴胺受体拮抗剂对缢蛏多巴胺D2类受体的影响,实验以一龄缢蛏作为研究对象,以3种多巴胺受体拮抗剂多潘立酮(domperidone, Domp)、舒必利(sulpiride, Sulp)、盐酸氯丙臻(chlorprothixene hydrochloride, Chlo)为受试物,设置2 h和6 h两个时间点,探究在三种作用浓度(10~(-2)mol/L, 10~(-3)mol/L和10~(-5)mol/L)下每种受试物对缢蛏两个多巴胺D2类受体(ScDopR2-1和ScDopR2-2)表达量的抑制效果。荧光定量检测多巴胺D2类受体的表达量,并检测c AMP的含量变化。检测结果表明,10-2mol/L浓度下,Chlo组别两个多巴胺受体基因相比对照组在2 h表达量均下降,而cAMP相比对照组含量上升,因此选取Chlo作为缢蛏多巴胺D2类受体拮抗剂,浓度选为10~(-2)mol/L,本实验为进一步研究缢蛏多巴胺D2类受体的功能提供了药理学基础。     

尖镰孢青霉素V酰化酶的纯化及性质

通过硫酸铵沉淀、硅藻土吸附、DEAD-纤维素离子交换层析和Sephadex G-200凝胶过滤,由尖镰孢(Fusarium oxysporum)FP941培养滤液中得到了聚丙烯酰胺凝胶电泳均一的青霉素V酰化酶。酶作用最适pH为7.0,最适温度为50℃。酶在pH6.0—8.0和42℃以下稳定。酶作用青霉素V的米氏常数Km为4.65×10~(-3)mol/L;苯氧乙酸是酶的竞争性抑制剂,抑制常数Ki为23.87×10~(-3)mol/L;6-氨基青霉烷酸是酶的非竞争性抑制剂,抑制常数Ki为30.01×10~(-3)mol/L。某些金属离子对酶有抑制作用,Fe~(2+)最强,其次是Hg~(2+)和Cu~(2+)。用SDS凝胶电泳测定酶亚基分子量为77600;用分子筛测定自然酶分子量为148000。     

D-葡萄糖苷酶抑制剂的研究

用高灵敏度的荧光法研究了13种糖类化合物对α或β-D-葡萄糖苷酶的抑制作用．实验结果表明：5-氨基-5-脱氧-D-吡喃葡萄糖-1-磺酸铵盐（1）、5-氨基-5-脱氧-D-吡喃葡萄糖-1-磺酸（2）、5-氨基-5-脱氧-D-葡萄糖-1．亚硫酸加成物（3）等三种氮杂糖对α或β-D-葡萄糖苷酶均呈现竞争性抑制作用．化合物（1）的Kiα＝372μmol／L;Kiβ＝6．38μmol/L．化合物（2）的Kiα＝34．4μmol／L;Kiβ＝6．84μmol／L．化合物（3）的Kiα＝47．2μmol/L;Kiβ＝11．9μmol/L．上述三个化合物对β-D-葡萄糖苷酶的抑制作用都强子α-D-葡萄糖苷酶．尤其对化合物（1）,其Kiα／Kiβ＝58．3,表明其抑制糖苷酶活性有较好的选择性．     

β3肾上腺素受体介导多巴胺对大鼠远端结肠运动的抑制作用

目的研究多巴胺（DA）对大鼠结肠运动影响的机制。方法采用离体组织灌流方法记录大鼠远端结肠自发性节律运动,观察DA的作用以及阻断剂的影响,再用反转录实时多聚酶链反应（real time RT-PCR）检测受体基因的表达。结果DA（≥1.0×10-5mol/L）对结肠远端（紧接肛门淋巴结近端）离体纵行肌条（2.0 mm×10 mm）的运动具有抑制作用,多巴胺受体阻断剂（D1受体阻断剂SCH23390,1.0×10-7mol/L,D2受体阻断剂Sulpide,1.0×10-7mol/L）不能阻断多巴胺的抑制效应,但加入β3受体抑制剂cyanopindolol（7.5×10-7mol/L）,DA的抑制作用显著减弱。real time RT-PCR检测发现β1、β2、β3受体mRNA在远端结肠均有表达。结论DA可通过β3受体发挥对远端结肠运动的抑制作用。     

酪氨酸对人离体滋养层细胞孕酮与hCG分泌的影响

本文观察三种剂量(2×10~(-5)mol/L,2×10~(-4)mol/L和2×10~(-3)mol/L)的酪氮酸对离体培养的滋养层细胞孕酮及hCG分泌的影响,并对其抑制效应的机理作了初步探讨。实验结果表明,三种剂量的酪氨酸均可抑制滋养层细胞孕酮分泌(P<0.01),但是,在孕酮分泌受酪氨酸抑制的同时,未见对hCG分泌发生影响(P>0.05),进一步观察了酪氨酸对滋养层细胞3β-羟甾脱氢酶活性的影响,结果表明,酪氨酸能显著抑制3β-羟甾脱氢酶活性,提示酪氨酸对滋养层细胞孕酮生成的抑制作用与抑制3β-羟甾脱氢酶活性有关。     

多巴胺、雌二醇及睾酮对虎纹蛙离体脑垂体促性腺激素分泌的影响 EFFECTS OF DOPAMINE，ESTRADIOL AND TESTOSTERONE ON GONADOT ROPIN RELEASE FROM THE PITUITARYFRAGMENTS OF Rana rugulosa

为了解虎纹蛙促性腺激素分泌的调节机理，用离体静态培育系统和放射免疫测定法，研究了多巴胺(DA)、雌二醇(E[2])和睾酮(T)对雌性虎纹蛙离体脑垂体薄片促黄体激素(LH)和促卵泡激素(FSH)分泌活动的影响。结果表明：0.1-10 μmol/L的DA对成熟前期和冬眠期虎纹蛙离体脑垂体薄片的LH及FSH的释放都有抑制作用，并且随着DA浓度的增加，抑制作用逐渐增强。1和10 μmol/L的E[2]显著刺激成熟前期蛙LH的释放，而0.1-10 μmol/L的E2显著抑制其FSH的释放；T对其FSH的释放无显著影响，但10 μmol/L的T显著抑制其LH释放。0.1-100 μmol/L 的E[2]或T对冬眠期蛙LH和FSH的释放均无显著影响。这些结果说明，多巴胺和性类固醇激素在脑垂体水平上对虎纹蛙LH和FSH的释放有直接的调节作用，而性类固醇激素的作用可能与性腺的发育阶段(季节)有关。     

吗啡和血管紧张素Ⅱ对大鼠脑突触小体Ca~(2 )摄取的拮抗效应

行为实验已多次证明,脑室注射血管紧张素Ⅱ(AⅡ)可以对抗吗啡的镇痛作用,但机制不明。吗啡阻止神经末梢钙摄取被认为是其镇痛的机理之一,因此本工作研究了AⅡ和吗啡对大鼠脑突触小体~(45)Ca摄取的作用及相互关系。结果表明,吗啡(10~(-8)—10~(-6)mol/L)对~(45)Ca摄取有明显的抑制作用,10~(-7)mol/L时抑制41％(P<0.001),该效应可被吗啡受体阻断剂纳洛酮(10~(-6)mol/L)完全翻转。与吗啡的作用相反,AⅡ(10~(-8)—110~(-6)mol/L)可促进突触小体对~(45)Ca的摄取,10~(-7)mol/L时增加75％(P<0.001),该效应可被AⅡ受体阻断剂Saralasin(10~(-6)mol/L)完全翻转。将不同剂量的AⅡ(10~(-8)—10~(-6)mol/L)和10~(-8)mol/L吗啡与突触小体共同孵育,则吗啡抑制~(45)Ca摄取的作用被完全翻转。以上结果表明,AⅡ促进脑突触小体Ca~(2 )摄取,对抗了吗啡抑制Ca~(2 )摄取的作用,可能是AⅡ抗吗啡镇痛的机制之一。     

溶酶体抑制剂对小鼠行为学与黑质多巴胺神经元的影响

目的初步探讨溶酶体抑制剂对小鼠行为学及多巴胺神经元功能的影响。方法于小鼠右侧中脑黑质（SN）区,立体定向微量注入溶酶体抑制剂和蛋白酶体抑制剂。观察阿朴吗啡诱导的小鼠旋转行为改变以及行为学变化;检测黑质区酪氨酸羟化酶（TH）阳性细胞数。结果阿朴吗啡未能诱导出小鼠旋转行为。蛋白酶体抑制剂组为10～15圈。多巴胺损害程度与溶酶体的剂量相关。磷酸氯喹25μmol/L时多巴胺神经元几乎没有损害作用;50μmol/L时局部区域多巴胺神经元有轻微损害;100μmol/L时损害明显;200μmol/L时黑质区注射局部神经元损害最严重,黑质注射区未见有神经元存在。1～4周呈现出较为明显逐渐恢复的特点。结论溶酶体功能抑制与多巴胺神经元凋亡相关,不同剂量溶酶体抑制剂在不同时间对多巴胺神经功能的损害不同。     

天冬酰胺酶及PEG_2-天冬酰胺酶对L-天冬酰胺、谷氨酰胺亲和性的研究

本文报导了天冬酰胺酶及PEG_2-天冬酰胺酶对废物L-天冬酰胺、谷氨酰胺亲和性的研究,结果表明:PEG_2-天冬酰胺酶对谷氨酰胺的亲和性明显强于天冬酰胺酶(Km值分别为7.35×10~(-3)mol/L和7.14×10~(-2)mol/L),对天冬酰胺的亲和性略强于天冬酰胺酶(Km值分别为2.9×10~(-5)mol/L和4.0×10~(-5)mol/L)。天冬酰胺酶和PEG_2-天冬酰胺酶的CD光谱表明:天冬酰胺和谷氨酰胺对天冬酰胺酶和PEG_2-天冬酰胺酶的构象影响较大,但天冬酰胺酶和PEG_2-天冬酰胺酶的构象变化趋势有明显的不同。     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Temporal and Spatial Disparity in cFOS Expression and Dopamine Phenotypic Differentiation in the Neonatal Mouse Olfactory Bulb

The mammalian olfactory bulb (OB) is among the few regions in adult brain which generates interneurons. A subpopulation of these phenotypically diverse interneurons is dopaminergic (DA) periglomerular cells. Full phenotypic development as indicated by expression of tyrosine hydroxylase (TH), the first enzyme in DA biosynthesis, requires afferent activity or equivalent depolarizing conditions. To investigate the hypothesis that cFOS regulates TH expression, this study analyzed OB slice cultures obtained from neonatal transgenic mice expressing 9 kb of TH promoter directing expression of green fluorescent protein (TH/GFP). Cultures were depolarized with 50 mM potassium chloride (KCl), the calcium channel blocker, nifedipine (10 μM) with KCl, or an equimolar concentration of sodium chloride (NaCl). Depolarization increased cFOS expression 6-fold peaking at about 3 h. Staining decreased rapidly returning to control, NaCl, levels by 48 h post-stimulation when TH/GFP expression was highest. Nifedipine blocked the increase in TH and cFOS suggesting that similar signal transduction pathways mediate both responses. Special issue dedicated to John P. Blass.     

Dopamine Synthesis Precedes Dopamine Uptake in Embryonic Rat Mesencephalic Neurons

We have measured [3H]dopamine ([3H]DA) uptake and tyrosine hydroxylase-immunopositive immunostaining in cells acutely dissociated from the embryonic ventral mesencephalon (MSC). DA and its metabolites as well as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) activities were determined in homogenates taken from the MSC and striatum (STR). In the embryonic ventral MSC measurable DA and tyrosine hydroxylase (TH) immunostaining were present as early as embryonic day (E) 12.5. At E14 the number of TH+ neurons was about 50% of the values at E18. In the MSC, DA concentration increased sharply at E16 and reached a plateau before birth that was 10-fold lower than adult values. In the STR, DA was first detected at E16, suggesting that DA fibers reach the STR at this embryonic stage. High-affinity DA uptake appeared in the MSC only at E16, concomitantly with the arrival of DA fibers in the STR, increased sharply between E16 and E18, and reached a plateau before birth. This uptake mechanism was not selective for catecholamine uptake inhibitors. Thus, DA synthesis in the MSC preceded the onset of high-affinity uptake mechanism, which could be correlated to the beginning of striatal DA innervation. Measurable MAO and COMT activities were detected as early as E13 (MSC) and E15 (STR), but not DA metabolites, which appeared later. We conclude that the high-affinity DA uptake mechanism in MSC DA neurons develops coincident with the arrival of DA fibers to the STR. The sharp increase of DA uptake between E16 and E18 is due only in part to an increase in the number of TH+ cells. These results support the hypothesis that in vivo the target STR neurons regulate the maturation of MSC DA cells.     

Short-Term Inhibitory Effect of Estradiol on Tyrosine Hydroxylase Activity in Tuberoinfundibular Dopaminergic Neurons In Vitro

Abstract: The short-term inhibition by estradiol of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by L-3,4-di-hydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a 30–40% decrease within 1 h of incubation with estradiol. To determine whether a dephosphorylation process was involved in this decline in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition: In controls, we observed that two kinetically different forms of TH coexisted, with one exhibiting a K_l(DA) of 26.4 ± 2 μM the other being ∼ 10-fold more sensitive to DA inhibition, with a [k_1{DA)] of 2.56 ± 0.17 μM. likely corresponding to a phosphorylated and active form and to a non-phosphorylated and poorly active form, respectively. Conversely. after estradiol treatment all TH molecules exhibited the same K_1(DA) of 2.5 ± 0.3 μM. This effect was stereospecific, because 17α-estradiol could not promote it. whereas with 17β-estradiol. it could be observed at only 10⁻¹¹M and after a short delay (30 min). Finally, this decrease in the K_1(DA) of the purported active form of TH could be prevented by okadaic acid (an inhibitor of protein phosphatases). These results suggest that estradiol can act directly on the mediobasal hypothalamus to trigger a rapid decline in TH activity and that this action may involve a decrease in TH phosphorylation.     

Acute Stimulatory Effect of Estradiol on Striatal Dopamine Synthesis

Abstract: The acute effect of physiological doses of estradiol (E2) on the dopaminergic activity in the striatum was studied. In a first series of experiments, ovariectomized rats were injected with 17α or 17β E2 (125, 250, or 500 ng/kg of body weight, s.c.), and in situ tyrosine hydroxylase (TH) activity (determined by DOPA accumulation in the striatum after intraperitoneal administration of NSD 1015) was quantified. A dose-dependent increase in striatal TH activity was observed within minutes after 17β (but not 17α) E2 treatment. To examine whether E2 acts directly on the striatum, in a second series of experiments, anesthetized rats were implanted in the striatum with a push-pull cannula supplied with an artificial CSF containing [³H]tyrosine. The extracellular concentrations of total and tritiated dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured at 20-min intervals. Addition of 10^?9M 17β (but not 17α) E2 to the superfusing fluid immediately evoked an ～50% increase in [³H]DA and [³H]DOPAC extracellular concentrations, but total DA and DOPAC concentrations remained constant. This selective increase in the newly synthesized DA and DOPAC release suggested that E2 affects DA synthesis rather than DA release. Finally, to determine whether this rapid E2-induced stimulation of DA synthesis was a consequence of an increase in TH level of phosphorylation, the enzyme constant of inhibition by DA (K_{i DA}) was calculated. Incubation of striatal slices in the presence of 10^?9M 17β (but not 17α) E2 indeed evoked an approximate twofold increase in the K_{i DA} of one form of the enzyme. It is concluded that physiological levels of E2 can act directly on striatal tissue to stimulate DA synthesis. This stimulation appears to be mediated, at least in part, by a decrease in TH susceptibility to end-product inhibition, presumably due to phosphorylation of the enzyme. The rapid onset of this effect, and the fact that the striatum does not contain detectable nuclear E2 receptors, suggest a nongenomic action of the steroid.     

Inhibition of dihydropteridine reductase by catecholamines and related compounds

Catecholamines and related compounds, such as dopamine, 5- or 6-hydroxydopamine, N-methyldopamine, tyramine, octopamine, norepinephrine and epinephrine, inhibit human liver dihydropteridine reductase (NADH:6,7-dihydropteridine oxidoreductase, EC 1.6.99.10) noncompetitively with Ki values ranging from 7.0 X 10(-6) - 1.9 X 10(-4)M (I50 values = 2.0 X 10(-5) - 2.0 X 10(-4)M). The tyrosine analogs alpha-methyltyrosine and 3-iodotyrosine are weak inhibitors of this enzyme (I50 greater than 10(-3)M). The inhibitory effect of catecholamines is slightly decreased by O-methylation of one hydroxyl group, but is essentially abolished by total methylation. The inhibitory strength of the catecholamines and related compounds tested against this enzyme can be arranged in the following order: dopamine, 6-hydroxydopamine, 5-hydroxydopamine, N-methyldopamine greater than tyramine, 3-O-methyldopamine, 4-O-methyldopamine much greater than epinephrine, 3-O-methylepinephrine, norepinephrine, octopamine less than tyrosine much less than alpha-methyltyrosine, 3-iodotyrosine much less than homoveratrylamine. These results suggest that dopamine, norepinephrine and epinephrine may serve as physiological regulators of mammalian dihydropteridine reductase.     

Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal Dopaminergic Terminals

The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor-dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4-dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under "normal" conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7-dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99), 7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), Trans-(-)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-2H-pyrazolo-3,4- quinoline, and 3-(3-hydroxyphenyl)-N-n-propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol-containing drugs apomorphine and TL-99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under "normal" conditions. In contrast, when tissues were incubated in a K+ -enriched (60 mM) medium, (-)-sulpiride and other DA antagonists enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because (-)-sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d-amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of TH normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+ -induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7-OH-DPAT or the antagonist (-)-sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP-dependent phosphorylation of the enzyme.     

SARS冠状病毒主蛋白酶抑制剂的筛选及抑制动力学研究

对SARS冠状病毒主蛋白酶(SARS-CoV M^pro)进行异源重组表达与提纯,并以其为靶点,利用基于荧光共振能量转移(FRET)技术的体外药物筛选模型,对蛋白酶抑制剂聚焦库96种化合物进行了体外抑制活性的评价,并从动力学的角度探讨筛选出的阳性化合物对SARS-CoV M^pro的抑制能力与机制。结果表明:通过筛选获得抑制率>80%、淬灭率<20%的化合物5种,为P-1-08、P-1-19、P-2-24、P-2-28、P-2-54,其半数有效抑制浓度(IC₅₀)分别为:0.69±0.05μmol/L、1.19±0.41μmol/L、0.14±0.01μmol/L、1.36±0.07μmol/L、0.36±0.03μmol/L。其中化合物P-1-08、P-1-19、P-2-24、P-2-54对SARS冠状病毒主蛋白酶的抑制作用为不可逆抑制,化合物P-2-28的抑制作用为可逆抑制。根据Lineweaver-Burk图和Dixon图的研究,发现化合物P-2-28对SARS冠状病毒主蛋白酶呈竞争性抑制,抑制常数Ki为0.81μmol/L。通过对底物浓度,IC₅₀值及Ki值关系的研究,进一步验证了P-2-28的抑制作用为竞争性抑制。该抑制剂的发现为SARS冠状病毒主蛋白酶抑制剂的研究打下基础,为抗SARS病毒药物开发提供了先导化合物。     

Evidence for Protein Kinase C Involvement in the Short-Term Activation by Prolactin of Tyrosine Hydroxylase in Tuberoinfundibular Dopaminergic Neurons

Abstract: The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a K _1(DA) value of 29.92 ± 0.49 μ M , the other being × 15-fold more sensitive to DA inhibition with a K _1(DA) of 1.96 ± 0.09 μ M , likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low K _1(DA) remained unaffected, whereas the K _1(DA) of the purported active form of TH increased to 62.6 ± 0.8 μ M , suggesting an increase in the enzyme phosphorylation. This increase in the K _I(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12- O -tetradecan-oylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.     

Acidic Fibroblast Growth Factor and Catecholamines Synergistically Up-Regulate Tyrosine Hydroxylase Activity in Developing and Damaged Dopamine Neurons in Culture

Abstract: Our previous studies indicate that, in certain non-catecholamine (CA) neurons, expression of the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH) can be initiated by the obligatory interaction of acidic fibroblast growth factor (aFGF) and a CA activator. In this study, we sought to determine whether these same differentiation factors also play a role in regulating existing TH expression in CA neurons. Thus, the effects of exogenous aFGF and CAs on TH were studied in developing or toxin-damaged dopamine (DA) neurons from the embryonic day 15 rat ventral midbrain, where it was likely to be at physiologically low levels. Cultures were incubated with various concentrations of aFGF, DA, or aFGF and DA. Some cultures were first damaged with 2.5 µ M 1-methyl-4-phenylpyridinium. In developing DA neurons, an 80% increase in TH activity was found only after cotreatment with aFGF (100 ng/ml) and DA (1 µ M ) or other monoamines. Likewise, in damaged DA neurons, aFGF and DA reversed the 50% loss in TH activity caused by toxin. This was observed within 4 h of treatment and was not associated with changes in the number or appearance of DA neurons, suggesting a biochemical rather than a trophic effect. Pretreatment with protein or RNA synthesis inhibitors eliminated the increase. In PC12 cells, where TH is highly expressed, activity was unaltered by treatment. We conclude that the aFGF and CAs may be involved in not only the initiation but also the regulation of TH.