Evidence for involvement of protein kinase in the activation by adenosine 3':5'-monophosphate of brain tyrosine 3-monooxygenase.

Addition of adenosine 3':5'-monophosphate (cAMP) to high speed supernatant preparations obtained from rat brain caused a 3- to 4-fold increase in tyrosine 3-monooxygenase (tyrosine hydroxylase) activity. The tyrosine 3-monooxygenase remained in an activated state upon removal of the cAMP by passing the enzyme through a Sephadex G-25 column. Substances which inhibit cAMP-dependent protein kinase, namely, EDTA, ADP, and adenosine, and protein kinase modulator, each antagonized the activation of tyrosine 3-monooxygenase produced by cAMP. Furthermore, addition of partially purified brain cAMP-dependent protein kinase caused a several-fold increase in tyrosin 3-monooxygenase activity. The activation of tyrosine 3-monooxygenase by added cAMP and protein kinase required the presence of ATP and Mg-2+. These data suggests that the cAMP activation of tyrosine 3-monooxygenase may be mediated by a cAMP-dependent protein kinase.     

Properties of N-Acetylhistamine Deacetylase in Mammalian Brain

Properties of N-acetylhistamine deacetylase in rat brain were studied, utilizing a sensitive coupled radioenzymatic assay. The Km for N-acetylhistamine for this deacetylase was 660 microM and its Vmax was 330 pmol/h/mg protein. N-Acetylhistamine deacetylase activity increased 80% in the presence of 1 mM Mn2+. The Km of Mn2+ was 40 microM. The enzyme is primarily a soluble enzyme with a relatively uniform regional distribution, unlike the distribution for histamine and histidine decarboxylase. Neonatal activity of this enzyme in rat brain is higher than in adult brain. alpha-Fluoromethylhistidine does not affect the activity of N-acetylhistamine, indicating that deacetylation probably does not play a regulatory role in the synthesis of brain histamine.     

Partial characterization of histidine decarboxylase in hamster and rat brain employing a new method

Histidine decarboxylase activity in hamster and rat brains were studied using a newly developed sensitive, direct radioenzymatic microassay. For our assay conditions, we determined aK _m forl-histidine of 320 M and aV _max for histidine decarboxylase of 110 pmol histamine/hr/mg protein in rat hypothalamus. The regional distributions of both histidine decarboxylase and histamine levels were similar in the hamster and rat with the most activity in hypothalamus. Most of the histidine decarboxylase activity in rat hypothalamus was in the cytosol fraction. The developmental pattern of histidine decarboxylase in the fetal rat did not reveal a prenatal spike in activity. Histidine decarboxylase activity in rat brain reached adult levels by four weeks. Alpha-fluoromethylhistidine inhibited histidine decarboxylase activity almost totally in vitro at 10 M and about 80% in vivo after six days of infusion (100mg/kg/day) in all brain regions except the cerebellum. Likewise, histamine levels were depleted about 75% in all brain regions except the cerebellum.     

Variations of brain histamine levels in germ-free and nephrectomized rats

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.     

Biochemical characterization of histamine-sensitive adenylate cyclase in mammalian brain.

Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (K_a, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the K_m (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg²⁺ stimulated enzymatic activity; the kinetic properties of this activation by Mg²⁺ suggest the existence of a Mg²⁺ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg²⁺. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg²⁺, low concentrations of Ca²⁺ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca²⁺. Other divalent cations, including Zn²⁺, Cu²⁺, and Cd²⁺, were also inhibitory. Of the divalent cations tested, only Co²⁺ and Mn²⁺ could replace Mg²⁺ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F^?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed.     

Regulation of tyrosine hydroxylase from human pheochromocytoma, bovine adrenal and rat striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum can be activated by Mg²⁺, ATP and cyclic AMP. In pheochromocytoma, this activation is due to a decreased Km for the pterin cofactor, whereas in adrenal medulla, it is a result of an increase in the Vmax. Norepinephrine increases the Km for pterin cofactor for tyrosine hydroxylase from both of these tissues. The Ki for norepinephrine is not altered by the presence of Mg²⁺, ATP and cyclic AMP with enzyme from pheochromocytoma or adrenal medulla. On the other hand, striatal tyrosine hydroxylase shows a two-fold increase in the Ki for dopamine after exposure to Mg²⁺, ATP and cyclic AMP.     

Changes in tissue histamine during the estrous cycle, pregnancy and pseudopregnancy in the golden hamster

The histamine content of reproductive tissues and skeletal muscle was determined in the golden hamster during the estrous cycle, pregnancy, and pseudopregnancy. Histidine decarboxylase activity was measured in uterine implantation sites and intersites from Day 4 to Day 10 of pregnancy. Histidine decarboxylase was also measured in mesometria and placentas on selected days of gestation. During the estrous cycle, uterine and skeletal muscle histamine levels were highest on Day 2 and lowest on Day 4 of the cycle. The ovarian histamine content did not change significantly among the different stages of the cycle. While the histamine content of uterine implantation sites of attachment was high on Days 4 and measurable on Days 5 and 6 of pregnancy, the levels were below the limits of detection by Day 7. On the other hand, the highest levels of histamine were in the uterine interimplantation sites on Days 8 and 9. The ovarian levels of histamine were highest on Day 13 of pregnancy. Histamine in skeletal muscle did not change significantly during pregnancy. The histidine decarboxylase activity in the implantation sites began rising on Day 9 and increased dramatically on Day 10. Placental histidine decarboxylase activity was very high on Days 13 and 15. Overall, we observed changes in uterine and skeletal muscle histamine during the estrous cycle that may be explainable in light of previously reported changes in mast cell numbers and circulating estrogens. During pregnancy, histamine levels of implantation sites and implantation intersites varied, as did the histamine content of ovarian tissue. Histidine decarboxylase activity rises in the uterus and placental tissue after the formation of the placenta.     

Histidine Transport into Rat Brain Synaptosomes

Abstract: Histidine transport and metabolism in rat brain synptosomes were investigated to study the possible role of histidine uptake in the synthesis of the putative neurotransmitter histamine (HA). Histidine uptake was found to be regionally distributed and temperature sensitive and was not totally independent of sodium or possium ions. Transport was inhibited by metabolic inhibitors, as well as by promethazine and quinacrine. A number of other HA-related agents and several histidine metabolites had no effect. Kinetic analyses of histidine transport revealed the presence of both high- and lowaffinity systems in cerebral cortex. Histidine uptake increased following preexposure of synaptosomes to depolarizing concentrations of potassium. This effect was dependent on the presence of calcium ions during the preincubation. No newly formed [³H]HA was detectable in rat brain synaptosomes following [³H]histidine transport. Lesions of the medial forebrain bundle did not alter histidine uptake in the hippocampus or cerebral cortex. Ontogenic studies indicated that the histidine uptake system developed rapidly and reached a peak during postnatal days 12–17. Overall, the present findings do not support a role for histidine transport in the regulation or maintenance of neurotransmitter pools of HA in rat brain.     

A time-saving thin-layer chromatography plate-scraping system

A system for efficiently scraping thin-layer chromatography plates is described. A height-adjustable rack for holding the thin-layer chromatography plate is constructed of transparent acrylic plate. The base provides a surface on which to slide vial holders and also gives stability to the rack. The plate is scraped with a single-edge razor blade cut to an appropriate width and the scrapings fall into a polypropylene funnel which sits on a vial that is in either a test tube rack or a cassette from a scintillation counter. The plate-scraping system reduces scraping time by more than 60% and increases the accuracy of results.