Region - specific alterations in tyrosine hydroxylase activity in rats exposed to lead

Previous studies from our laboratory showed that subchronic exposure to low levels of Pb resulted in significant decrease in dopamine (DA) content, attenuation of stimulus-induced release of DA in the dopaminergic projection area of nucleus accumbens (NA), and alterations in tyrosine hydroxylase (TH) activity in rat whole brain homogenates. The present study reported here was conducted to assess the functional integrity of DA synthesis in different brain regions of rats subchronically (90-days) exposed to 50 ppm Pb by measuring the activity of the rate limiting enzyme, tyrosine hydroxylase, in seven brain regions. In Pb-exposed rats, TH activity was reduced in two of the seven brain regions investigated, i.e., nucleus accumbens (42% reduction) and frontal cortex (61% reduction) when compared to controls. In contrast, Pb exposure did not affect the TH activity in cerebellum, brainstem, hippocampus, hypothalamus and striatum. The changes in TH activity in nucleus accumbens (NA) and frontal cortex (FC) in Pb-exposed rats were further confirmed by Western blot analysis using TH polyclonal antibody. Collectively, these results indicate that low level subchronic Pb exposure may affect TH protein in these brain regions.     

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Exocrine pancreatic carcinogenesis in the guppy Poecilia reticulata

Exocrine pancreatic neoplasms developed in the guppy Poecilia reticulata following exposure to the direct-acting carcinogen methylazoxymethanol acetate (MAM-Ac). Fish 6 to 10 d old were exposed to nominal, non-toxic concentrations of 4 and 10 mg MAM-Ac l(-1) for 2 h and then transferred to carcinogen-free water for grow-out. Whole specimens were sampled monthly up to 9 mo post-exposure to follow the histologic progression of the lesions. No neoplasms occurred in 119 control specimens examined. Pancreatic acinar cell adenomas and carcinomas occurred in 42 of 243 (17%) of the specimens exposed to MAM-Ac. As in earlier studies, specimens exposed to the low MAM-Ac concentration exhibited a higher pancreatic neoplasm incidence (27.8%) than those exposed to the high concentration (7.8%). Acinar cell adenomas accounted for 27 of the 42 neoplasms. Adenomas exhibited a high degree of acinar cell differentiation and some contained foci of atypical acinar cells that were less differentiated and more basophilic than were surrounding adenoma cells. Carcinomas occurred in 15 specimens and exhibited a range of cellular patterns. Although no distant metastases were found, carcinomas tended to invade neighboring tissues and organs. The occurrence of carcinogen-induced pancreatic neoplasms in guppies strengthens the usefulness of small fish species in carcinogen testing and provides an additional model for studying pancreatic neoplasia.     

Ciliary neurotrophic factor stimulates tyrosine hydroxylase activity

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in norepinephrine synthesis, and its expression and activity are regulated by many factors in sympathetic neurons. Cytokines that act through gp130, such as ciliary neurotrophic factor (CNTF) decrease norepinephrine production in sympathetic neurons by suppressing TH mRNA and stimulating degradation of TH protein, leading to the loss of enzyme. Their effect on the activity of TH is unclear, but recent in vivo observations suggest that cytokines may stimulate TH activity. We investigated this issue by quantifying TH protein levels and activity in cultured sympathetic neurons. We also examined the state of TH phosphorylation on serine 31 and 40, sites known to affect TH activity and degradation. We found that CNTF, acting through gp130, stimulated the rate of l-3,4-dihydroxyphenylalanine production while at the same time decreasing TH enzyme levels, thereby increasing the specific activity of the enzyme. We also found that phosphorylation of TH on Ser31 was increased, and phosphorylation on Ser40 was decreased, after four days of CNTF exposure. Our data are consistent with previous findings that Ser31 phosphorylation stimulates TH activity, whereas Ser40 phosphorylation can target TH for proteasomal degradation.     

Salinity tolerance of the guppy, Poecilia reticulata Peters

Experiments were conducted to determine the adaptability of the guppy to various salt concentrations. The guppy, Poecilia reticulata , (total length 11–40 mm) were subjected to abrupt and gradual changes from fresh water (salinity=0.1%) to various salinities (%). No mortality occurred when the fish were transferred from fresh water to 50% sea water (19.5%). Through gradual adaptation from 50% sea water to 80% of the fish were able to tolerate 100% (39%) sea water for 7 days. After a 7 day stay in sea water, fish were readapted to fresh water during a 3 h period. Through gradual adaptation fish were also able to tolerate salinities ranging from 39.0% (100% sea water) to 58.5%. After a 30 day stay in 150% sea water (58.5%), fish were readapted to fresh water over a 5 h period. The results indicate that they were well able to tolerate the abrupt change from 100 or 150% sea water to fresh water. Females that stayed in 150% sea water (58.5%) for 30 days had embryos in their gonads.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Maternal and embryonic sources of tyrosine hydroxylase during Drosophila embryogenesis

Tyrosine hydroxylase (TH), the enzyme which catalyzes the conversion of tyrosine to L-DOPA and is rate limiting in catecholamine biosynthesis, is biochemically expressed in late stage wild-type Drosophila oocytes as well as in early embryogenesis. Null mutant alleles of TH (pale) are embryonic lethals with death occurring in the late embryonic or early larval periods of development. Staging of embryos demonstrated that inhibition of the enzymatic activity of TH by alpha-methyl-p-tyrosine (alphaMT) retards the progression of embryos primarily during the organogenesis stages of embryonic development, with lesser effects on earlier and later stages. On the other hand, time of gene action studies with a conditional temperature sensitive pale mutant (ple(ts1)) at its restrictive temperature (29 degrees C) indicate an onset of tyrosine hydroxylase gene action beginning in the oocyte stage of development. Thus, maternal as well as embryonic effects on the secretion and/or functionality of this enzyme may play roles in the early developmental program of the organism.     

Comparison of cDNA and genomic forms of tyrosine hydroxylase gene therapy of the brain with Trojan horse liposomes

BACKGROUND: The present study examines whether chromosomal derived forms of therapeutic genes can be delivered to brain following intravenous administration. The brain expression of a rat tyrosine hydroxylase (TH) cDNA is compared to the brain expression of a plasmid DNA encoding the 18 kb rat TH gene. METHODS: TH gene expression is measured in cell culture and in vivo in brain in experimental Parkinson's disease (PD). A total of four eukaryotic expression plasmids encoding rat TH were engineered wherein the size of the TH expression cassette ranged from 1.5 kb, in the case of the cDNA form of the gene, to 17.5 kb, in the case of the largest size genomic construct. The TH expression plasmids were delivered to either cultured cells or to rat brain in vivo with Trojan horse liposomes (THLs), which target the non-viral plasmid DNA to cells via cell membrane receptors. RESULTS: The pattern of TH gene expression in cell culture and in vivo was similar: the cDNA form of the TH gene was fast-acting with short duration of action, and the genomic form of the TH gene was slow-acting with longer duration of action. The most sustained replacement of striatal TH enzyme activity in experimental PD was produced by combination gene therapy where both the cDNA and the genomic forms of the TH gene were administered simultaneously. CONCLUSIONS: Eukaryotic expression plasmids encoding genomic forms of therapeutic genes, as large as 18 kb, can be successfully incorporated in THLs and delivered to brain following intravenous administration.     

Neurobiological consequences of acute footshock stress: effects on tyrosine hydroxylase phosphorylation and activation in the rat brain and adrenal medulla

Stress activates selected neuronal systems in the brain and this leads to activation of a range of effector systems. Our aim was to investigate some of the relationships between these systems under basal conditions and over a 40‐min period in response to footshock stress. Specifically, we investigated catecholaminergic neurons in the locus coeruleus (LC), ventral tegmental area and medial prefrontal cortex (mPFC) in the brain, by measuring tyrosine hydroxylase (TH) protein, TH phosphorylation and TH activation. We also measured the effector responses by measuring plasma adrenocorticotrophic hormone, corticosterone, glucose and body temperature as well as activation of adrenal medulla protein kinases, TH protein, TH phosphorylation and TH activation. The LC, ventral tegmental area and adrenal medulla all had higher basal levels of Ser19 phosphorylation and lower basal levels of Ser31 phosphorylation than the mPFC, presumably because of their cell body versus nerve terminal location, while the adrenal medulla had the highest basal levels of Ser40 phosphorylation. Ser31 phosphorylation was increased in the LC at 20 and 40 min and in the mPFC at 40 min; TH activity was increased at 40 min in both tissues. There were significant increases in body temperature between 10 and 40 min, as well as increases in plasma adrenocorticotropic hormone at 20 min and corticosterone and glucose at 20 and 40 min. The adrenal medulla extracellular signal‐regulated kinase 2 was increased between 10 and 40 min and Ser31 phosphorylation was increased at 20 min and 40 min. Protein kinase A and Ser40 phosphorylation were increased only at 40 min. TH activity was increased between 20 and 40 min. TH protein and Ser19 phosphorylation levels were not altered in any of the brain regions or adrenal medulla over the first 40 min. These findings indicate that acute footshock stress leads to activation of TH in the LC, pre‐synaptic terminals in the mPFC and adrenal medullary chromaffin cells, as well as changes in activity of the hypothalamic‐pituitary‐adrenal axis.

     

Phenotypic correlates of spermatozoon quality in the guppy, Poecilia reticulata

The phenotype-linked fertility hypothesis suggests that a femalecan benefit directly from mate choice when the cues she usesindicate the quantity and/or quality of his spermatozoa. Wetested the link between sperm quality and male body size andcoloration in the resource-free mating system of the guppy,a tropical fish characterized by strong female choice. Largermales possessed larger testes and are therefore predicted toproduce larger numbers of spermatozoa than smaller males. Largermale guppies also produced longer spermatozoa than smaller males.Degree of carotenoid coloration did not predict either the quantityor the quality of a male's spermatozoa. These results are consistentwith a previous study that showed that female guppies in thestudy population prefer larger males to brightly colored males.The male-size directed increase in spermatozoon size may bethe result of interplay between sperm competition and the coevolutionof spermatozoon traits with the female reproductive tract.     

Mechanisms underlying shoal composition in the Trinidadian guppy, Poecilia reticulata

Free-ranging groups are frequently assorted by phenotypic characters. However, very little is known about the underlying processes that determine this structuring. In this study, we investigate the mechanisms underlying the phenotypic composition of shoals of guppies ( Poecilia reticulata ) in a high-predation stream in Trinidad's Northern Mountain Range. We collected 57 entire wild shoals, which were strongly assorted by body length. Shoal encounters staged within an experimental arena showed shoal fission (but not fusion) events to be an important mechanism in generating phenotypic assortment. In the wild, fission and fusion between guppy shoals occurred extremely frequently and thus are unlikely to constrain the opportunities for shoal assortment. However, fission and fusion processes occur under the restrictions imposed by the distribution of individuals within the environment. We observed size specific segregation within the habitat in three dimensions, providing a passive mechanism that contributes to the maintenance of the observed homogeneity of group composition. Furthermore sex differences were found in social behaviour. Individual male guppies switched between shoals more frequently than females and left a shoal more often than females. We argue that shoal composition is determined by habitat segregation on a medium spatial scale and by fission/fusion processes on a small spatial scale (with sex-specific shoal dynamics adding a additional layer of complexity).     

Dominant lethal effects of triethylenemelamine in the guppy Poecilia reticulata.

The need to screen potential chemical pollutants for mutagenicity has increased with the increasing volume of such materials being introduced into natural water. The present study demonstrates the utility of a fish, the guppy (Poecilia reticulata), as a model test system in which water-borne chemical mutagens may be assayed for dominant lethal effects. Mature male guppies were injected with three doses of triethylenemelamine (0.1, 0.2 and 0.4 mg/kg) in addition to a control sham treatment. Each male was subsequently mated to virgin females. In an alternative test, male fish were allowed to swim in triethylenemelamine solutions of known concentration for a period of 24 h prior to being mated to virgin female guppies. 10 days following matings, females were dissected, and numbers of live and dead embryos were recorded. Significant dose effects were demonstrated by analysis of variance techniques in both the injection and the emersion tests with the results showing higher percentages of dead embryos and lower total number of embryos with increasing doses of TEM.     

Geographical variation in allometry in the guppy (Poecilia reticulata)

Variation in static allometry, the power relationship between character size and body size among individuals at similar developmental stages, remains poorly understood. We tested whether predation or other ecological factors could affect static allometry by comparing the allometry between the caudal fin length and the body length in adult male guppies (Poecilia reticulata) among populations from different geographical areas, exposed to different predation pressures. Neither the allometric slopes nor the allometric elevations (intercept at constant slope) changed with predation pressure. However, populations from the Northern Range in Trinidad showed allometry with similar slopes but lower intercepts than populations from the Caroni and the Oropouche drainages. Because most of these populations are exposed to predation by the prawn Macrobrachium crenulatum, we speculated that the specific selection pressures exerted by this predator generated this change in relative caudal fin size, although effects of other environmental factors could not be ruled out. This study further suggests that the allometric elevation is more variable than the allometric slope.     

Nicotine induces tyrosine hydroxylase plasticity in the neurodegenerating striatum

It has been shown that nicotine prevents the loss of dopamine (DA) in the corpus striatum (CS) after 6-hydroxydopamine injection in the substantia nigra. To study the role of the enzyme tyrosine hydroxylase (TH; EC 1.14.16.2) in this experimental paradigm, we have examined its activity by assessing the accumulation of l-3,4-dihydroxyphenylalanine after inhibiting the subsequent enzyme in the DA synthetic pathway, aromatic l-amino acid decarboxylase, with 3-hydroxybenzylhydrazine. In addition the amount of TH protein was assessed by western blotting and its distribution in the CS was examined using immunohistochemical methods. 6-hydroxydopamine injection produced a significant decrease in DA levels and l-3,4-dihydroxyphenylalanine accumulation, as well as decreases in TH protein and TH immunoreactive fibres in the CS. After nicotine treatment, the decrease in TH protein in the CS was significantly reduced, with a concomitant preservation of TH activity, but nicotine did not alter the number of TH immunoreactive fibres. The activity and amount of TH did not change in the contralateral (intact) CS. Thus, nicotine induces long lasting TH plasticity in the degenerating CS. A synergistic action of nicotine-activated and lesion-originated signals appears necessary for the expression of this neuronal molecular plasticity.     

The effect of tetrahydrobiopterin on the in situ phosphorylation of tyrosine hydroxylase in rat striatal synaptosomes

Tetrahydrobiopterin (BH₄), the obligatory cofactor of the aromatic amino acid hydroxylases, decreased the in situ³²P-phosphorylation of tyrosine hydroxylase (TH) in rat striatal synaptosomes. Incubation of pre-³²P-labeled synaptosomes with BH₄ in the presence of a permeant analogue of cAMP decreased the cAMP-stimulated level of³²P label incorporation into TH by about 50%, as determined by immunoprecipitation and autoradiography of SDS-polyacrylamide gels. The extent of inhibition mirrored changes in intrasynaptosomal BH₄ levels and varied both as a function of BH₄ concentration and length of incubation. A similar decrease in the amount of TH³²P-labeling was observed with the precursor of BH₄, sepiapterin. This effect, in turn, was reversed by the inhibitor of sepiapterin reductase, N-acetyl-serotonin. Finally, exposure of pre-³²P-labeled synaptosomes to the inhibitor of protein phosphatase 2A, okadaic acid, blocked the response to BH₄. Collectively, the data suggest that BH₄ stimulates the dephosphorylation of TH in situ and thus may play a dual role both as a cofactor for catalysis and a regulator of hydroxylase activity.Special issue dedicated to Dr. Bernard W. Agranoff.     

Schooling preferences for familiar fish in the guppy, Poecilia reticulata

Although schooling behaviour is an important antipredator response of guppies ( Poecilia reticulata ) in Trinidad it is not known whether fish elect to school with particular individuals. Familiarity is a simple mechanism that could lead to non-random associations within schools. In this paper we test the idea that prior experience of particular individuals influences the choice of partners during schooling. We show (in a laboratory study) that female guppies prefer to associate with familiar individuals. However, we are unable to detect any schooling preference for unfamiliar females from the same population.     

Substrate-mediated enhancement of phosphorylated tyrosine hydroxylase in nigrostriatal dopamine neurons: evidence for a role of alpha-synuclein

Tyrosine hydroxylase (TH) protein, phosphorylated at serine-40, serine-31 and serine-19, and enzyme catalytic activity were compared under basal conditions and in activated nigrostriatal dopamine (NSDA) neurons of wild-type and homozygous alpha-synuclein knockout mice. Mice were injected with the D2 antagonist raclopride to stimulate NSDA neuronal activity in the presence or absence of supplemental l-tyrosine. There was no difference in phosphorylated TH levels or TH catalytic activity between wild-type and alpha-synuclein knockout mice under basal conditions or following raclopride-induced acceleration of NSDA activity. In wild-type animals, tyrosine administration potentiated the raclopride-induced increase in phosphorylated TH and enzyme activity. However, tyrosine administration did not enhance phosphorylated TH levels or enzyme catalytic activity in raclopride-stimulated NSDA neurons in alpha-synuclein knockout mice. These findings suggest that alpha-synuclein plays a role in the ability of tyrosine to either enhance TH phosphorylation or hinder TH inactivation during accelerated neuronal activity. The present study supports the hypothesis that alpha-synuclein functions as a molecular chaperone protein that regulates the phosphorylation state of TH in a substrate and activity-dependent manner.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Inheritance of the snakeskin color pattern in the guppy, Poecilia reticulata

We made single-pair reciprocal crosses between the Green Snakeskin and Yellow Snakeskin domesticated strains of the guppy, Poecilia reticulata. The two snakeskin strains differ by a single autosomal gene, with the Green Snakeskin strain having the wild-type background coloration caused by the dominant gene (B), whereas the Yellow Snakeskin is homozygous for the recessive blond allele (bb). The snakeskin body and tail patterns characterizing males of these two strains are determined by two genes--Ssb and Sst--that are closely linked on the Y chromosome. The greenish-yellow tail color of the Green Snakeskin strain is mediated by an X-linked dominant gene, Grt. The recessive wild-type allele, Grt+, gives the hyaline tail color. In the Yellow Snakeskin strain, the Grt gene is expressed as a golden-yellow color as a result of the presence of the bb homozygous condition. The putative genotypes of the males and females of the Green Snakeskin strain are BB XGrt YSsb,Sst and BB XGrt XGrt, respectively. Males and females of the Yellow Snakeskin strain have the putative genotypes bb XGrt YSsb,Sst and bb XGrtXGrt, respectively. As a result of crossing over between the X and Y chromosomes, a few males and females of these two snakeskin strains may carry one or both snakeskin pattern genes (Ssb and Sst) on the X chromosome.