Nicotine dependence in a prospective population-based study of adolescents : the protective role of a functional tyrosine hydroxylase polymorphism

Dopamine is a key neurotransmitter of the mesolimbic reward pathway in the human brain, and tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. Consequently, the gene encoding TH is a strong candidate for involvement in the genetic component of addiction. The importance of this gene in nicotine dependence is supported by many studies showing a link between nicotine administration and TH expression. A functional tetranucleotide repeat polymorphism within intron 1 of the TH gene (HUMTH01-VNTR) has been shown to modify tobacco use in two independent Caucasian samples from the USA and Australia. Using information drawn from an eight-wave Australian population-based longitudinal study of adolescent health, we tested the effect of the HUMTH01-VNTR on nicotine dependence. Comparisons were made between dependent smokers and non-dependent smokers. These data provide further support for a protective association between the K4 allele and dependent smoking (odds ratio 0.54, 95% confidence interval 0.28-1.0). No associations were observed at any of three other common TH polymorphisms (rs6356, rs6357 and HUMTH01-PstI). Including these data, three independent studies, two of which use identical phenotypes, have now identified a protective relationship between the K4 allele of the functional HUMTH01-VNTR polymorphism and high-level smoking.

Association between dependent smoking and a polymorphism in the tyrosine hydroxylase gene in a prospective population-based study of adolescent health

This study reports pilot data on an association between tobacco dependence and a five-allele tetranucleotide repeat polymorphism in the first intron of the tyrosine hydroxylase (TH) gene. One hundred and twenty-six Australian adolescents who had participated in the Health in Transition Study (1993–1997), and who showed patterns of either dependent or nondependent smoking across four waves of data collection, consented to participation in the pilot study. The smoking status of those recruited was confirmed using a telephone-administered drug use questionnaire during 2000. Tobacco dependence was defined as smoking more than 6 days per week and more than 10 cigarettes per day during wave 5 (year 2000) and at lfeast one prior wave (n = 58). A second, more stringent phenotype included smoking within an hour of waking (n = 37). The control group comprised adolescents who had used tobacco but had remained low-level social smokers across each wave of data (n = 56). DNA was collected using a mouthwash procedure. Using the more strictly defined tobacco dependence phenotype, and after adjusting for sex, a significant protective association was found between the K4 allele and tobacco dependence (OR 0.27, 95% confidence interval [CI] 0.09, 0.82). No association was found using the liberal criteria of tobacco dependence (OR 0.51, 95% confidence interval [CI] 0.23, 1.2). These preliminary results replicate a previous association between tobacco use and the K4 allele of the TH gene (Lerman et al., 1997). The potential significance of including time to first cigarette in definitions of tobacco dependence and the possible role that these TH variants might play in tobacco dependence are discussed.

The effect of social defeat on tyrosine hydroxylase phosphorylation in the rat brain and adrenal gland

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated acutely by protein phosphorylation and chronically by protein synthesis. No studies have systematically investigated the phosphorylation of these sites in vivo in response to stressors. We specifically investigated the phosphorylation of TH occurring within the first 24 h in response to the social defeat stress in the rat adrenal, the locus coeruleus, substantia nigra and ventral tegmental area. Five groups were investigated; home cage control (HCC), two groups that underwent social defeat (SD+) which were sacrificed either 10 min or 24 h after the end of the protocol and two groups that were put into the cage without the resident being present (SD−) which were sacrificed at time points identical to the SD+. We found at 10 min there were significant increases in serine 40 and 31 phosphorylation levels in the locus coeruleus in SD+ compared to HCC and increases in serine 40 phosphorylation levels in the substantia nigra in SD+ compared to SD−. We found at 24 h there were significant increases in serine 19 phosphorylation levels in the ventral tegmental area in SD+ compared to HCC and decreases in serine 40 phosphorylation levels in the adrenal in SD+ compared to SD−. These findings suggest that the regulation of TH phosphorylation in different catecholamine-producing cells varies considerably and is dependent on both the nature of the stressor and the time at which the response is analysed.

Vasodilator mechanisms in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus (Rajiformes; Rhinobatidae)

This study investigated the nature of previous termvasodilator mechanismsnext term in the dorsal aorta of the giant shovelnose ray, Rhinobatus typus. Anatomical techniques found no evidence for an endothelial nitric oxide synthase, but neural nitric oxide synthase was found to be present in the perivascular nerve fibres of the dorsal aorta and other arteries and veins using both NADPH-diaphorase staining and immunohistochemistry with a specific neural NOS antibody. Arteries and veins both contained large nNOS-positive nerve trunks from which smaller nNOS-positive bundles branched and formed a plexus in the vessel wall. Single, varicose nNOS-positive nerve fibres were present in both arteries and veins. Within the large bundles of both arteries and veins, groups of nNOS-positive cell bodies forming microganglia were observed. Double-labelling immunohistochemistry using an antibody to tyrosine hydroxylase showed that nearly all the NOS nerves were not sympathetic. Acetylcholine always caused constriction of isolated rings of the dorsal aorta and the nitric oxide donor, sodium nitroprusside, did not mediate any dilation. Addition of nicotine (3×10−4 M) to preconstricted rings caused a vasodilation that was not affected by the nitric oxide synthase inhibitor, Image -NNA (10−4 M), nor the soluble guanylyl cyclase inhibitor, ODQ (10−5 M). This nicotine-mediated vasodilation was, therefore, not due to the synthesis and release of NO. Disruption of the endothelium significantly reduced or eliminated the nicotine-mediated vasodilation. In addition, indomethacin (10−5 M), an inhibitor of cyclooxygenases, significantly increased the time period to maximal dilation and reduced, but did not completely inhibit the nicotine-mediated vasodilation. These data support the hypothesis that a prostaglandin is released from the vascular endothelium of a batoid ray, as has been described previously in other groups of fishes. The function of the nitrergic innervation of the blood vessels is not known because nitric oxide does not appear to regulate vascular tone.

Catecholamine and oxytocin cells respond to hypovolaemia as well as hypotension

Medullary catecholamine and hypothalamic neurosecretory oxytocin cells are activated by hypotension, but previous studies have provided uncertain outcomes concerning their ability to respond to a purely hypovolaemic stimulus. In the present study, injections of PEG/water and pentolinium were used to elicit non-hypotensive, isosmotic hypovolaemia and isovolaemic, isosmotic hypotension, respectively, in conscious rats. Animals were sacrificed 2 h after treatment. Immunolabelling for Fos, tyrosine hydroxylase and oxytocin established that these two stimuli activate almost identical populations of catecholamine neurons in the ventrolateral and dorsomedial medulla, and very similar populations of oxytocin cells in the supraoptic and paraventricular nuclei of the hypothalamus.

Purity and enrichment of laser-microdissected midbrain dopamine neurons

The ability to microdissect individual cells from the nervous system has enormous potential, as it can allow for the study of gene expression in phenotypically identified cells. However, if the resultant gene expression profiles are to be accurately ascribed, it is necessary to determine the extent of contamination by nontarget cells in the microdissected sample. Here, we show that midbrain dopamine neurons can be laser-microdissected to a high degree of enrichment and purity. The average enrichment for tyrosine hydroxylase (TH) gene expression in the microdissected sample relative to midbrain sections was approximately 200-fold. For the dopamine transporter (DAT) and the vesicular monoamine transporter type 2 (Vmat2), average enrichments were approximately 100- and 60-fold, respectively. Glutamic acid decarboxylase (Gad65) expression, a marker for GABAergic neurons, was several hundredfold lower than dopamine neuron-specific genes. Glial cell and glutamatergic neuron gene expression were not detected in microdissected samples. Additionally, SN and VTA dopamine neurons had significantly different expression levels of dopamine neuron-specific genes, which likely reflects functional differences between the two cell groups. This study demonstrates that it is possible to laser-microdissect dopamine neurons to a high degree of cell purity. Therefore gene expression profiles can be precisely attributed to the targeted microdissected cells.

Steroid 21-hydroxylase expression in cultured rat astrocytes

Over the past decade or so it has become widely recognised that the brain is a significant steroidogenic organ. Many publications have highlighted the ability of the brain to synthesise and interconvert a large number of steroid products including cholesterol, progesterone and testosterone. In this study, in vitro experiments were performed to determine if 21-hydroxylation of steroids is undertaken by rat brain astrocytes in culture. This is a common reaction that occurs in the adrenal gland and other organs in mammals, catalysing the conversion of pregn-4-ene-3,20-dione (progesterone) to 21-hydroxypregn-4-ene-3,20-dione (deoxycorticosterone).

Previous reports have indicated that 21-hydroxylation occurs within the rat brain, however, the precise identity of the cells expressing 21-hydroxylase has not yet been determined. Several metabolites, such as 5α-pregnan-3α-ol-20-one (tetrahydroprogesterone) and 3α,21-dihydroxy-5-pregnan-20-one (tetrahydrodeoxycorticosterone) were of particular interest because of their modulatory role in neuronal function, such as their agonist activity at γ-aminobutyric acid (GABA_A) receptors.

Evidence was obtained for the expression of peripheral 21-hydroxylase enzyme (P450c21) in cultured rat brain astrocytes by a combination of mass spectroscopy and molecular biology techniques. This is a significant finding as expression of 21-hydroxylase within astrocytes may be indicative of a wider role for these cells in modulating neuronal behaviour.

Tyrosine residues of the granulocyte colony-stimulating factor transmit proliferation and differentiation signals in murine bone marrow cells

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of granulopoiesis and acts through binding to its specific receptor (G-CSF-R) on neutrophilic granulocytes. Previous studies of signaling from the 4 G-CSF-R cytoplasmic tyrosine residues used model cell lines that may have idiosyncratic, nonphysiological responses. This study aimed to identify specific signals transmitted by the receptor tyrosine residues in primary myeloid cells. To bypass the presence of endogenous G-CSF-R, a chimeric receptor containing the extracellular domain of the epidermal growth factor receptor in place of the entire extracellular domain of the G-CSF-R was used. A series of chimeric receptors containing tyrosine mutations to phenylalanine, either individually or collectively, was constructed and expressed in primary bone marrow cells from G-CSF-deficient mice. Proliferation and differentiation responses of receptor-expressing bone marrow cells stimulated by epidermal growth factor were measured. An increased 50% effective concentration to stimulus of the receptor Ynull mutant indicated that specific signals from tyrosine residues were required for cell proliferation, particularly at low concentrations of stimulus. Impaired responses by mutant receptors implicated G-CSF-R Y764 in cell proliferation and Y729 in granulocyte differentiation signaling. In addition, different sensitivities to ligand stimulation between mutant receptors indicated that G-CSF-R Y744 and possibly Y729 have an inhibitory role in cell proliferation. STAT activation was not affected by tyrosine mutations, whereas ERK activation appeared to depend, at least in part, on Y764. These observations have suggested novel roles for the G-CSF-R tyrosine residues in primary cells that were not observed previously in studies in cell lines.

Dimerisation of N-acetyl-L-tyrosine ethyl ester and Aß peptides via formation of dityrosine

Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid β-peptide (Aβ). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H2O2). The generation of H2O2 has been linked with Aβ neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Aβ. This residue has also been shown to be important to Aβ aggregation and amyloid formation. It is possible that the formation of an Aβ tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Aβ aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Aβ dimers containing dityrosine. Here we report the use of horseradish peroxidase and H2O2 to dimerise N-acetyl-l-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Aβ peptides. We also report a simple fluorescent plate reader method for monitoring Aβ dimerisation via dityrosine formation.

Metal catalyzed oxidation of tyrosine residues by different oxidation systems of copper/hydrogen peroxide

Metal-catalysed oxidation (MCO) reactions result in the formation of reactive oxygen species (ROS) in biological systems. These ROS cause oxidative stress that contributes to a number of pathological processes leading to a variety of diseases. Tyrosine is one residue that is very susceptible to oxidative modification and the formation of dityrosine (DT) and 3,4-dihydroxyphenylalanine (DOPA) have been widely reported in a number of diseases. However, the mechanisms of MCO of tyrosine in biological systems are poorly understood and require further investigation. In this study we investigated the mechanism of DT and DOPA formation by MCO using N-acetyl tyrosine ethyl ester as a model for tyrosine in proteins and peptides. The results showed that DT formation could be observed upon Cu²⁺/H₂O₂ oxidation at pH 7.4. Our results indicate that it is unlikely to be via Fenton chemistry since Cu⁺/H₂O₂ oxidative conditions did not lead to the formation of DT.