Dopamine Synthesis by Non-Dopaminergic Neurons in the Arcuate Nucleus of Rat Fetuses

The aim of the present work was to verify the hypothesis that non-dopaminergic neurons expressing individual complementary dopamine synthesis enzymes can perform the co-located synthesis of dopamine. According to this hypothesis, neurons expressing tyrosine hydroxylase use L-tyrosine for the synthesis of L-dihydroxyphenylalanine (L-DOPA), which then enters neurons expressing aromatic amino acid decarboxylase, which converts L-DOPA to dopamine. Experiments were performed using the mediobasal hypothalamus of rat fetuses, which mostly contains single-enzyme neurons (>99%) and occasional double-enzyme neurons (<1%). Controls were obtained from the fetal substantia nigra, which is enriched with dopaminergic neurons. High-performance liquid chromatography was used to measure levels of dopamine and L-DOPA in cell extracts and the incubation medium after incubation in the presence and absence of exogenous L-tyrosine. Addition of L-tyrosine to the medium led to increases in the level of synthesis and release of L-DOPA in the mediobasal hypothalamus and substantia nigra. In addition, L-tyrosine increased dopamine synthesis in the substantia nigra and decreased dopamine synthesis in the mediobasal hypothalamus. This regional difference in levels of dopamine synthesis is probably due to inhibition of the uptake of L-DOPA from the intercellular medium by neurons in the mediobasal hypothalamus containing aromatic amino acid decarboxylase, due to the competitive binding of the L-DOPA transporter by L-tyrosine. Thus, these results provide the first evidence for the co-located synthesis of dopamine by non-dopaminergic neurons expressing single complementary enzymes involved in the synthesis of this neurotransmitter.__________Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 90, No. 7, pp. 825–832, July, 2004.     

Genetic analysis of the relationships between behavioral and neuroendocrine traits in roman high and low avoidance rat lines

In order to determine whether the coselection observed between the selection trait (active avoidance behavior) of the Roman High Avoidance (RHA) and Roman Low Avoidance (RLA) rat lines and their neuroendocrine characteristics were genetically determined, we analyzed, in nonsegregating (RHA, RLA, and F₁) and segregating (F₂ and the two backcrosses) crosses, the inheritance pattern and the phenotypic correlations among behavioral (shuttle-box behavior), physiological (body, adrenal, and thymus weights), and neuroendocrine (corticosterone and prolactin reactivity, catecholamine enzyme activities) variables. Physiological characteristics and enzyme activities have acrucial role in sex dissociation. Avoidance behavior and prolactin reactivity to novel environment remained associated in segregating crosses despite gene rearrangement. They represented the most important variables to differentiate the Roman lines, perhaps sharing a common regulatory mechanism under genetic control.     

Dopaminergic innervation of the rat globus pallidus characterized by microdialysis and immunohistochemistry

The present study examined the dopaminergic innervation of the rat globus pallidus by in vivo microdialysis and immunohistochemistry in more detail. Using tyrosine hydroxylase immunohistochemistry, two classes of dopaminergic fibers were distinguished morphologically in the globus pallidus. Unilateral infusion of 6-hydroxydopamine into the substantia nigra produced a loss of dopaminergic fiber density in the globus pallidus which was correlated with the nigral extent of the lesion. These findings are in line with the notion that a degenerative loss of nigral dopaminergic cell bodies might also affect the dopamine input of extrastriatal structures such as the globus pallidus. Using in vivo microdialysis, we tested whether dopamine measured in the globus pallidus is of neuronal origin. Perfusion of tetrodotoxin induced a strong and transient decrease of pallidal dopamine. The tetrodotoxin-sensitivity of pallidal dopamine demonstrates the functional significance of the nigropallidal dopaminergic innervation.     

In vitro and in vivo correlations for I65T and M1V mutations at the phenylalanine hydroxylase locus.

Mutations at the phenylalanine hydroxylase (PAH) locus are the major cause of hyperphenylalaninemia. We have previously described four mutations (M1V, IVS12nt1, R408W, and S349P) at the PAH locus in French Canadians with ancestry in eastern Quebec. Here we report (1) identification of another mutation, on a haplotype 9 chromosome, which converts codon 65 from isoleucine (ATT) to threonine (ACT), (2) expression analysis of the I65T mutation in COS cells demonstrating 75% loss of both immunoreactive protein and enzyme activity, and (3) expression analysis of the most prevalent PKU allele (M1V) in eastern Quebec, showing nondetectable levels of PAH protein and activity, a finding compatible with a mutation in the translation initiation codon. Homozygosity for M1V and codominant inheritance of I65T/R408W were both associated with classical phenylketonuria.     

三个新的PAH基因短串联重复序列多态性及其在苯丙酮尿症产前诊断中的应用

目的提高经典型苯丙酮尿症的产前诊断的成功率。方法在苯丙氨酸羟化酶（phenylalanine hydroxylase，PAH）基因附近选择了3个新的短串联重复序列（short tandem repeat，STR）位点（PAH26、PAH32和PAH9），进行扩增长度片段多态性分析，确定它们在中国人群的分布及在诊断中的应用价值。结果3个新的STR位点的多态信息含量分别为0．518（PAH26）、0．413（PAH32）和0．362（PAH9）。这3个位点之间，PAH9与第3内含子中的STR位点（TCTA）n之间存在连锁不平衡，其他位点组合不存在连锁不平衡。联合第3内含子中的STR位点（TCTA）n和2个新的位点，可以对家系中突变基因标记进行95％N断，并成功地用于4例产前诊断中。结论选择性地应用PAH基因中的3个STR位点组合，可以95％地区分经典型苯丙酮尿症家系中父母的两个基因，从而准确地进行快速产前诊断。     

The distribution of terminal sympathetic nerve fibers in bundle branches and false tendons of the bovine heart. An immunohistochemical and catecholamine histofluorescence study

Summary The sympathetic innervation in false tendons as a whole and the distribution of the terminal sympathetic nerve fibers in the conduction tissue in the bundle branches is unclear. Therefore, in the present study, false tendons and bundle branch regions of the bovine heart were examined using tyrosine hydroxylase (TH) immunohistochemistry and the glyoxylic acid induced catecholamine (CA) fluorescence method for demonstration of sympathetic nerve fibers. Acetylcholinesterase (AChE) histochemistry was also applied. Some of the nerve fascicles in the false tendons were found to contain large numers of sympathetic nerve fibers and such nerve fibers formed plexuses in the walls of arteries and arterioles in these structures. In both false tendons and bundle branches sympathetic nerve fibers 1) were non-homogeneously distributed in the conduction tissue, most regularly occurring in the channels of extracellular space that are present within the bundles of Purkinje fibres. and 2) showed the same pattern of distribution in relation to Purkinje fibre bundle surfaces as the AChE-positive nerve branches. The observations show that there is a substantial sympathetic innervation in false tendons. The final distribution of the nerve fibers in these structures and in the bundle branches are discussed in relation to what is known of tissue morphology and the occurrence of sympathetic nerve influences in these regions. In the present study, previous CA-fluorescence observations of a marked sympathetic innervation in bundle branch regions, in terms of the presence of sympathetic nerve fibers in nerve fascicles and vessel walls, were also corroborated by the application of TH-immunohistochemistry.     

原发性高血压患者红细胞[Ca2+]i浓度变化及影响因素

目的：观察原发性高血压患者红细胞[Ca^2 ]i,多巴胺β羟化酶及ATP含量变化并分析其结果。方法：测定35例高血压患者的红细[Ca^2 ]i、ATP、血清多巴胺β羟化酶活性,血糖及血浆胰岛素含量,并以30例健康成年人为对照。结果：高血压患者的红细胞[Ca^2 ]i、ATP、多巴胺β羟化酶均明显高于对照组（P＜0．05,P＜0．01）,但血糖与胰岛素未见明显变化。结论：高血压患者血清多巴胺β羟化酶活性增强伴随ATP与[Ca^2 ]i升高。     

The mycotoxin ochratoxin A is a substrate for phenylalanine hydroxylase in isolated rat hepatocytes and in vivo

Ochratoxin A (OTA), is a mycotoxin contaminating food and feed stuffs, consisting of a chlorinated dihydroisocoumarin linked through a 7-carboxyl group tol-phenylalanine by an amide bond. When OTA (0.12–1.4 mM) is incubated with freshly isolated rat hepatocytes, it inhibits both the hydroxylation of phenylalanine (0.05 mM) to tyrosine, catalyzed by phenylalanine hydroxylase and the subsequent metabolism of tyrosine as measured by homogentisate oxidation. The IC₅₀ of OTA for phenylalanine hydroxylation is 0.43 mM. OT, (0.5–1.0 mM), the dihydroisocoumarin moiety of OTA, does not inhibit phenylalanine hydroxylase activity under these conditions. During incubations of hepatocytes with uniformly labelled [³H]-OTA and unlabelled phenylalanine, tyrosine-ochratoxin A is formed (up to 6% of the total mycotoxin added), indicating that ochratoxin can act as a substrate for phenylalanine hydroxylase. In vivo tyrosine-OTA is also found in liver of poisoned animals.     

Identification of amino-terminal sequences contributing to tryptophan hydroxylase tetramer formation

Tryptophan hydroxylase (TPH) catalyzes the rate-limiting step in the biosynthesis of serotonin. In the rabbit, TPH exists as a tetramer of four identical 51-kDa subunits comprised of 444 amino acids each. The enzyme consists of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Previous studies demonstrated that within the carboxyl-terminus of TPH, there resides an intersubunit binding domain (a leucine zipper) that is essential for tetramer formation. However, it is hypothesized that a 4,3-hydrophobic repeat identified within the regulatory domain of TPH (residues 21–41) may also be involved in macromolecular assembly. To test this hypothesis, a series of amino-terminal deletions (NΔ15, 30, 41, and 90) were created and assessed for macromolecular structure using size-exclusion chromatography. The amino-terminal deletion NΔ15, upstream from the 4,3-hydrophobic repeat, was capable of forming tetramers. However, when a portion of the 4,3-hydrophobic repeat was deleted (NΔ30), a heterogeneous elution pattern of tetramers, dimers, and monomers was observed. Complete removal of the 4,3-hydrophobic repeat (NΔ41) rendered the enzyme incapable of forming tetramers; a monomeric form predominated. In addition, a double-point mutation (V28R-L31R) was created in the hydrophobic region of the enzyme. The introduction of two arginines (R) at positions 28 and 31 respectively, in the helix disrupted the native tetrameric state of TPH. According to size-exclusion chromatography analysis, the double-point mutant (V28R-L31R) formed dimers of 127 kDa. Thus, it is concluded that there is information within the amino-terminus that is necessary for tetramer formation of TPH. This additional intersubunit binding domain in the amino-terminus is similar to that found in the carboxyl-terminus.     

DNA strand breaks in Alzheimer's disease

The shell and core of the nucleus accumbens exhibit different vulnerabilities to neurotoxins. Calcium binding proteins are reported to offer some neuroprotection against excitotoxicity by suppressing or buffering intracellular calcium. Differences in the distributions of the calbindin-D 28kD (CB) and calretinin (CR) might be related to the different vulnerabilities to neurotoxins of dopaminergic neurons in the ventral mesencephalon that project to the core and medial shell of the nucleus accumbens. To address this possibility, Fluoro-Gold (FG) was injected into accumbens subterritories and numbers of retrogradely labeled neurons in the ventral tegmental area containing CB and CR immunoreactivities (ir) were expressed as a percentage of total numbers of labeled neurons. The perikaryal diameters and lengths of the immunoreactive dendrites of FG labeled neurons were also measured. About 70% and 35% of retrogradely labeled cells observed following core and medial shell injections, respectively, exhibited CB immunoreactivity. Differences were not observed in the percentages of FG labeled cells exhibiting CR immunoreactivity following medial shell (13%) and core (15%) injections. The mean perikaryal diameters and median summed lengths of dendrites of retrogradely labeled neurons containing CB were smaller than in labeled neurons lacking CB following injections in both core and medial shell of the nucleus accumbens. The data indicate that the different 6-hydroxydopamine (6-OHDA) vulnerabilities of ventral mesencephalic dopaminergic neurons are not obviously related to the presence of CB and CR.