Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Enzymes Related to Monoamine Transmitter Metabolism in Brain Microvessels

The activities of tyrosine hydroxylase, aromatic L-aminoacid decarboxylase, monoamine oxidase, and catechol-O-methyltransferase were measured in microvessel (capillaries and venules), parenchymal arterioles, and pial vessels from rat brains, and the decarboxylase activity was compared in brain microvessels from rabbit, cat, dog, pig, cow, baboon, and man. Cranial sympathectomy was performed to estimate the neuronal contribution to the enzyme activities. All vascular regions had substantial activities of the various enzymes studied. The activity of aromatic L-aminoacid decarboxylase in cerebral microvessels was high in rat, dog, pig, cow, and man; intermediate in rabbit and cat; and low in baboon. In addition to this enzyme, cerebral microvessels also contained tyrosine hydroxylase and monoamine oxidase. Aromatic aminoacid decarboxylase and monoamine oxidase serve an enzymatic barrier function at the microvascular level, whereas the main function of tyrosine hydroxylase is probably to synthesize monoamines within nerve terminals that remain in close association with microvessels under the conditions used for preparation of the microvascular fraction. In larger intracerebral and pial vessels monoamine oxidase was present both in the wall itself and in perivascular sympathetic nerves; the remaining two enzymes had a primarily neuronal localization. The latter types of vessels also contained catechol-O-methyltransferase in their walls.     

Comparison of rat and guinea pig as sources of the S9 fraction in the salmonella/mammalian microsome mutagenicity test

A highly significant enhancement of mutagenicity occurs with 11 polycyclic aromatic hydrocarbons when 3-methylcholanthrene-induced guinea pig liver S9 is substituted for Aroclor-induced rat liver S9 in the Ames test. The use of MC-induced guinea pig liver S9 is particularly valuable for detecting the weak mutagenicity of benz[c]acridine, which is barely positive in a standard Ames assay. However, anthracene and phenanthrene, which are generally considered not to be carcinogens, remain non-mutagenic for strain TA100. This enhancement of mutagenicity does not correlate with arylhydrocarbon hydroxylase activities of the various liver preparations and does not apply to certain other non-PAH mutagens, including β-naphthylamine, aflatoxin B₁ and 4-dimethylaminoazobenzene.     

Developmental regulation of phenylalanine hydroxylase activity in Drosophila melanogaster

Herein we demonstrate that Drosophila larvae possess a synthetic activity capable of converting phenylalanine to tyrosine. This system is readily extractable and displays many characteristics of phenylalanine hydroxylase systems described in other organisms, the most notable being that a tetrahydropteridine is required for full expression of activity. The level of phenylalanine hydroxylase activity present in the organism varies with the stage of development: from an undetected level of activity at the first larval instar, there is a rapid increase in phenylalanine hydroxylase activity which reaches a peak at the time of puparium formation, after which there is a rapid decrease again to an undetected level.     

Regulation of the binding of 7,12-dimethylbenz[a]-anthracene to DNA of cultured murine epidermal cells at metabolic level: competition of the binding by polycyclic aromatic hydrocarbons and by other precarcinogens.

The binding of labeled carcinogen [3H]DMBA to murine epidermal cells (MEC) DNA in culture has been studied. The influence of unlabeled noncarcinogenic and carcinogenic polycyclic aromatic hydrocarbons (PAH), several PAH metablites, and various directly and indirectly acting non-PAH carcinogens on the binding of [3H]DMBA to MEC DNA has been examined. All the carcinogenic PAH and some of non-carcinogenic PAH effectively inhibit the binding of [3H]DMBA to MEC DNA. The non-PAH chemical carcinogens requiring metabolic activation also reduce the binding of labeled DMBA to MEC DNA; however, a higher concentration of these compounds is required for 50% inhibition of binding than the concentrations of PAH for the same degree of inhibition of binding of [3H]DMBA to MEC DNA. The directly acting carcinogens do not significantly inhibit the binding of [3H]DMBA to DNA. The relationship between structures of PAH and their ability to inhibit the binding of [3H]DMBA to MEC DNA is also discussed. Thus, it appears that the binding of DMBA to cellular DNA is primarily controlled at a level of metabolism and to some extent at the level of binding of reactive metabolites to DNA.     

Sensitivity of cultured rat hepatocytes to aflatoxin B1 and benzo(a)pyrene

Summary We have tested the sensitivity of a cloned rat hepatocyte line, RL-PR-C, to aflatoxin B₁ and benzo(a)pyrene as a function of population-doubling level. The cells were much more sensitive to the cytotoxic action of these agents subsequent to 230 population doublings. This sensitivity corresponded to the enhanced inducibility of arylhydrocarbon hydroxylase activity by 3-methylcholanthrene. Supported by Grants CA 21258 and CA 12056 from the National Cancer Institute.     

Inhibition of 3,4-dihydroxy-l-phenylalanine decarboxylase in rat striatal synaptosomes by amino acids interacting with substrate transport

Dopamine synthesis from 3,4-dihydroxy-l-phenylalanine in rat striatal synaptosomes was inhibited by a number of amino acids with aromatic or large aliphatic side chains. Inhibition was not seen when aromatic amino acid decarboxylase activity was measured in disrupted synaptosomes. Similarly, inhibition of dopamine synthesis from tyrosine was seen in the presence of leucine. The inhibition most likely results from interactions of the amino acids with substrate transport across the synaptosome plasma membrane, rather than directly with the catalytic enzymes. The kinetic data obtained are used to infer information about the relevant transport process; they suggest the potential importance of amino acid efflux as a regulatory step.     

Uncovering the evolutionary origin of blue anthocyanins in cereal grains

Functional divergence after gene duplication plays a central role in plant evolution. Among cereals, only Hordeum vulgare (barley), Triticum aestivum (wheat) and Secale cereale (rye) accumulate delphinidin‐derived (blue) anthocyanins in the aleurone layer of grains, whereas Oryza sativa (rice), Zea mays (maize) and Sorghum bicolor (sorghum) do not. The underlying genetic basis for this natural occurrence remains elusive. Here, we mapped the barley Blx1 locus involved in blue aleurone to an approximately 1.13 Mb genetic interval on chromosome 4HL, thus identifying a trigenic cluster named MbHF35 (containing HvMYB4H, HvMYC4H and HvF35H). Sequence and expression data supported the role of these genes in conferring blue‐coloured (blue aleurone) grains. Synteny analyses across monocot species showed that MbHF35 has only evolved within distinct Triticeae lineages, as a result of dispersed gene duplication. Phylogeny analyses revealed a shared evolution pattern for MbHF35 in Triticeae, suggesting that these genes have co‐evolved together. We also identified a Pooideae‐specific flavonoid 3′,5′‐hydroxylase (F3′5′H) lineage, termed here Mo_F35H2, which has a higher amino acid similarity with eudicot F3′5′Hs, demonstrating a scenario of convergent evolution. Indeed, selection tests identified 13 amino acid residues in Mo_F35H2 that underwent positive selection, possibly driven by protein thermostablility selection. Furthermore, through the interrogation of barley germplasm there is evidence that HvMYB4H and HvMYC4H have undergone human selection. Collectively, our study favours blue aleurone as a recently evolved trait resulting from environmental adaptation. Our findings provide an evolutionary explanation for the absence of blue anthocyanins in other cereals and highlight the importance of gene functional divergence for plant diversity and environmental adaptation.