Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.16.2 (tyrosine hydroxylase)
 14,760 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cellular localization of carbonic anhydrase (CAH) in the carotid body of the rat was investigated by means of Hansson's cobalt-precipitation technique in cultures of dissociated cells. In both young (2-day-old) and old (77-day-old) cultures, the parenchymal glomus (type-I) cells were selectively stained by this technique, and in addition expressed tyrosine hydroxylase and neuron-specific enolase as revealed by immunofluorescence. Enzymic reaction product of CAH appeared to be predominantly intracellular since staining was more intense and occurred more rapidly following permeabilization of the cell membranes with Triton X-100; its formation was inhibited by the CAH-inhibitor acetazolamide (1-10 microM) or by increasing the pH from 5.8 to 7.5. Cryostat sections of the carotid bifurcation revealed intense CAH-reaction product in cell clusters of the carotid body, in a few cells of the nodose ganglion, and in red blood cells. Neuronal cell bodies of the petrosal ganglion and superior cervical ganglion (SCG) were largely non-reactive. The SCG is known to contain clusters of small intensely fluorescent (SIF) cells, which were also non-reactive when grown in dissociated cell culture. Thus, although glomus and SIF cells are often considered to be similar cell types, functional CAH-activity appears unique to glomus cells, and this may be important for the physiological response of the carotid body to certain chemosensory stimuli.
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PMID:Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat. 197 81

In vitro intracellular recordings were made from neurons in the rat midbrain slice. Two neuronal types could be distinguished in dopamine-containing (DA) midbrain regions based on electrophysiological criteria. One neuron type exhibited short duration action potentials (less than 1.5 msec), could fire at high frequencies (greater than 10 Hz), and exhibited either phasic or burst firing patterns. This neuron did not exhibit tyrosine hydroxylase immunoreactivity. A second neuronal type exhibited a unique set of electrophysiological properties, which included (1) a spontaneous pacemaker-like depolarizing potential, (2) a highly regular firing pattern, (3) long duration (greater than 2 msec) action potentials, and (4) a high (i.e., depolarized) spike threshold. This neuron was consistently double labeled using intracellular staining and immunocytochemical localization of the catecholamine-specific enzyme tyrosine hydroxylase, and thus represented the DA neuronal type. Midbrain DA neurons stained with Lucifer yellow could be separated into 3 classes based on their location and morphology: (1) fusiform neurons with laterally projecting dendrites in the dorsal substantia nigra zona compacta region, (2) multipolar cells with laterally and ventrally projecting dendrites in the ventral substantia nigra zona compacta, and (3) neurons with fusiform and multipolar somata and radially projecting dendrites in the ventral tegmental area. The dendrites also exhibited spine-like protrusions and ended with specialized forked processes. Spontaneously firing DA cells recorded in vitro had a number of distinguishing electrophysiological characteristics in common with those of DA neurons recorded in vivo, such as the presence of a slow depolarizing potential driving spike activity and a characteristic depolarized spike threshold (approximately-36 mV). However, in contrast to that found in vivo, the DA cells characterized here exhibited substantially higher input resistances and fired spontaneously in a very regular pacemaker pattern. Burst firing was not observed. Spike activity was apparently dependent on 4 depolarizing events: (1) a voltage-dependent TTX-sensitive slow depolarization, (2) a cobalt-sensitive low threshold depolarization that was activated during the rebound from brief membrane hyperpolarizations, (3) high threshold dendritic calcium spikes which gave rise to the spike afterhyperpolarization, and (4) a high threshold initial segment sodium spike. These depolarizations were modulated by several processes, including a 4-aminopyridine-insensitive delayed repolarization, an instantaneous and time-dependent anomalous rectifier, and an afterhyperpolarization. Although low threshold depolarizations and rebound action potentials could be triggered by the membrane repolarization following small membrane hyperpolarizations, comparatively larger hyperpolarizations attenuated this rebound activation, thereby suppressing anodal break excitation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. 279 34

We sought to determine the ultrastructural localization and the extrinsic sources of the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), in the lateral parabrachial region (PBR) of adult male rats. In the first portion of the study, a rabbit antiserum to TH was immunocytochemically localized in coronal sections through the lateral PBR from acrolein-fixed brains using the peroxidase-antiperoxidase method. Electron-microscopic analysis revealed that perikarya and dendrites with peroxidase immunoreactivity for TH constituted only 17% of the total labeled profiles. Afferents to the TH-labeled perikarya and dendrites usually failed to exhibit immunoreactivity and were thus considered noncatecholaminergic. Somatic synapses were most commonly detected on small immunoreactive perikarya in the central lateral nucleus of the PBR. Other labeled perikarya located in the dorsal lateral or ventral lateral nuclei received few somatic synapses and were morphologically distinct in terms of their larger size, infolded nuclear membrane, and abundance of cytoplasmic organelles. Axons and axon terminals with peroxidase immunoreactivity constituted the remaining labeled profiles in the lateral PBR. These terminals primarily formed symmetric synapses with unlabeled and a few labeled dendrites. The labeled axon terminals were categorized into 2 types: Type I was small (0.3-0.6 micron), contained many small clear vesicles, and exhibited few well-defined synaptic densities. The second type was large (0.8-1.4 micron), contained both small clear and large dense core vesicles, and exhibited well-defined synaptic densities. The 2 types of terminals were morphologically similar to dopaminergic terminals. The location of catecholaminergic neurons contributing to the TH-labeled terminals was determined by combining peroxidase-antiperoxidase immunocytochemistry for TH with retrograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). The tracer was unilaterally injected into the PBR of anesthetized adult rats. Immunocytochemical labeling for TH was seen as a brown reaction product within neurons in known catecholaminergic cell groups. A black granular reaction product formed by a cobalt-intensified and diaminobenzidine-stabilized tetramethyl benzidine reaction for WGA-HRP was evident within many TH-labeled and unlabeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Tyrosine hydroxylase in the rat parabrachial region: ultrastructural localization and extrinsic sources of immunoreactivity. 287 40

The autofluorescent serotonin analogue 5,7-dihydroxytryptamine (5,7-DHT) was used to identify living catecholaminergic neurons in monolayer cultures derived from the embryonic rat mesencephalon. A high correlation between 5,7-DHT accumulation and aldehyde-induced catecholamine fluorescence as well as tyrosine hydroxylase but not dopamine-beta-hydroxylase or phenylethanolamine-N-methyltransferase immunoreactivity was found. This indicates that these cells were dopamine-containing neurons. Whole-cell patch recordings showed that all mesencephalic neurons had resting membrane potentials of -50 mV or greater and input resistances ranging between 200 and 700 M omega and exhibited spontaneous action potentials and postsynaptic potentials. The duration of the action potential of the dopamine-containing neurons was characteristically longer than that of the non-dopamine-containing mesencephalic cells. In some dopamine-containing neurons, repolarization of the action potential was clearly biphasic, and the slow phase of repolarization was reversibly blocked by local application of Cd2+ or Co2+. This "shoulder" in the action potential was never observed in non-dopamine-containing neurons, where Cd2+ or Co2+ application was always without effect. It is concluded that 5,7-DHT can be used to identify living dopamine-containing neurons in dissociated mesencephalic cultures and these neurons express distinct electrical properties.
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PMID:5,7-Dihydroxytryptamine identifies living dopaminergic neurons in mesencephalic cultures. 305 Sep 94

The conversion of tyrosine into dopa [3-(3,4-dihydroxyphenyl)alanine] is the rate limiting step in the biosynthesis of melanins catalysed by tyrosinase. This hydroxylation reaction is characterized by a lag period, the extent of which depends on various parameters, notably the presence of a suitable hydrogen donor such as dopa or tetrahydropterin. We have now found that catalytic amounts of Fe2+ ions have the same effect as dopa in stimulating the tyrosine hydroxylase activity of the enzyme. Kinetic experiments showed that the shortening of the induction time depends on the concentration of the added metal and the nature of the buffer system used and is not suppressed by superoxide dismutase, catalase, formate or mannitol. Notably, Fe3+ ions showed only a small delaying effect on tyrosinase activity. Among the other metals which were tested, Zn2+, Co2+, Cd2+ and Ni2+ had no detectable influence, whereas Cu2+ and Mn2+ exhibited a marked inhibitory effect on the kinetics of tyrosine oxidation. These findings are discussed in the light of the commonly accepted mechanism of action of tyrosinase.
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PMID:Effect of metal ions on the kinetics of tyrosine oxidation catalysed by tyrosinase. 392 96

The acetone precipitation of a partially purified tyrosine 3-monooxygenase (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) resulted in the complete loss of enzymatic activity. The enzymatic activity was restored by incubation with iron and dithiothreitol. The restoration of the activity was a pH-, temperature- and time-dependent reaction. Since cobalt, nickel, copper, zinc, manganese, cadmium, magnesium calcium and barium ions were all ineffective in restoring activity, iron ion appeared to be specifically required in the restoration of the enzyme activity. Dithiothreitol could be partially replaced in the restoration step by glutathione, 2-mercaptoethanol or cysteine.
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PMID:Inactivation of tyrosine 3-monooxygenase by acetone precipitation and its restoration by incubation with a sulfhydryl agent and iron. 611 46

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.
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PMID:Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing. 751 27

1. Intracellular microelectrode and whole-cell patch-clamp recordings were obtained from adult guinea pig celiac ganglion neurons grown in tissue culture for 7-14 days. Over 90% of neurons showed phasic-type action-potential discharge with the use of either type of recording electrode; they stained immunohistochemically for catecholamines, tyrosine hydroxylase, and neuropeptide Y. Input resistance (140 M omega) and action-potential amplitude (103 mV) were significantly greater with whole-cell than with microelectrode recordings, but other passive electrical properties were similar. 2. Five potassium currents were characterized: an apamin-sensitive after hyperpolarizing current (IAHP), an apamin and tetraethylammonium-insensitive slow IAHP, an M-like current, a transient outward IA current, and a delayed rectifier IK current. A hyperpolarization-activated cationic Ih current was also present. The first three currents were not observed with whole-cell recordings. 3. Cadmium (200 microM), cobalt (1 mM), lanthanum (30 microM), or a low calcium/high magnesium solution blocked both IAHPS and the M-like current; barium (1 mM) also blocked these currents. 4. Kinetics of the M-like current were best described by a double exponential fit to deactivating tail currents with time constants of 50 and 390 ms at -50 mV. The apamin-sensitive and slow IAHP decayed exponentially with time constants of 145 ms and 3.5 s, respectively. There was no correlation between occurrence of M-like current (95% of neurons) and slow IAHP (40% of neurons), nor any correlation between magnitude of M-like current and IAHP in those cells exhibiting both currents. 5. Muscarine and substance P (SP) caused depolarizations or inward currents (under voltage clamp) at the resting potential (-55 mV) associated with a decreased membrane conductance. The slow IAHP and the M-like current, but not the apamin-sensitive IAHP nor the IA, were blocked by muscarine and SP (IC50 3 microM and 100 nM, respectively). Muscarine and SP also decreased a "leak" potassium current. 6. We conclude that celiac neurons express two calcium-dependent IAHP currents and a calcium-dependent M-current; these are seen by fine-tipped intracellular microelectrodes but not by whole-cell patch electrodes. These currents are not required for spike frequency accommodation. Muscarine and SP reduce these currents, as well as voltage-independent leakage potassium current.
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PMID:Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia. 768 76

Recombinant human tyrosine hydroxylase has been purified as a metal-free apoenzyme (apo-hTH1) which tightly binds one Fe2+, Co2+, or Zn2+ per subunit with activation only by Fe2+ and competitive inhibition by the other cations. L-tyrosine and L-phenylalanine are alternative substrates for this enzyme, giving similar Vmax values, although the KM value for phenylalanine is about 8-fold greater than for tyrosine. Apo-hTH1 enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 values of the protons of enzyme-bound phenylalanine both in the presence and in the absence of the oxidized form of the cofactor L-erythro-7,8-dihydrobiopterin (BH2), which was used as an inactive analog of the natural cofactor (6R)-1-erythro-tetrahydrobiopterin (BH4). No effects of hTH1-Zn2+ on 1/T1 or 1/T2 are found. From paramagnetic effects of hTH1-Co2+ on 1/T1 of phenylalanine protons at 250 and 600 MHz, in the presence of BH2, a correlation time (tau c) of 1.8 +/- 0.1 ps was found. Using this tau c value, and assuming that only one proton of the pairs H3,H5, and H2,H6 is experiencing the total paramagnetic effect (asymmetric limiting case), distances from enzyme-bound Co2+ to phenylalanine (+/- 1.2 A) of 6.1 A (H3 or H5), 6.3 A (H2 or H6), 7.0 A (H4), 7.3 A (H alpha), > or = 7.4 A (H beta-pro-S), and > or = 7.6 A (H beta-pro-R) were calculated. The distances to H3 or H5 and to H2 or H6 are slightly increased to 6.8 and 7.0 A, respectively, if each proton of both degenerate pairs equally experiences the paramagnetic effect of Co2+ (symmetric limiting case). These distances place the aromatic ring of phenylalanine in the second coordination sphere of the metal, which would permit an Fe-bound oxy or peroxy species to approach molecular contact with C3/C4, suggesting a direct role of Fe2+ in the hydroxylation reaction. The same correlation time and similar distances were found in the absence of BH2 with H4 of phenylalanine slightly closer to the metal. In the ternary hTH1-Zn(2+).BH2.phenylalanine complex, eight interproton distances in the enzyme-bound phenylalanine were determined by NOESY spectra at 600 MHz at 35-, 50-, and 75-ms mixing times. The conformation of enzyme-bound phenylalanine, consistent with the six Co(2+)-proton distances and the eight interproton distances, is partially extended with torsional angles chi 1 = 97 degrees +/- 3 degrees and chi 2 = -78 degrees +/- 2 degrees.
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PMID:Conformation and interaction of phenylalanine with the divalent cation at the active site of human recombinant tyrosine hydroxylase as determined by proton NMR. 810 Jan 48

Activation of neurons in the ventromedial medulla by electrical stimulation or by microinjection of opioid or cholinergic agonists produces antinociception that is mediated in part by spinally-projecting noradrenergic neurons. Several lines of evidence indicate that these noradrenergic neurons are located in the pontine A7 catecholamine cell group. For example, anatomical studies have demonstrated that neurons in the ventromedial medulla project to the noradrenergic neurons in the A7 catecholamine cell group that provide the major noradrenergic innervation of the spinal cord dorsal horn. In addition, electrical and chemical stimulation of A7 neurons produces antinociception that can be reduced by intrathecal injection of alpha2-adrenoceptor antagonists. The present studies provide more direct evidence that activation of neurons in the ventromedial medulla produces antinociception by activating noradrenergic neurons in the A7 cell group. Neurons in the ventromedial medulla were stimulated by microinjecting the cholinergic agonist carbachol (5 microg) into sites in the nucleus raphe magnus or the nucleus gigantocellularis pars alpha of pentobarbital anesthetized Sprague-Dawley rats. In some experiments, the local anesthetic tetracaine (10 microg) was then microinjected near the A7 cell group to inactivate the spinally-projecting noradrenergic neurons. In other experiments, cobalt chloride (100 mM) was microinjected near the A7 cell group to block synaptic activation of spinally-projecting noradrenergic neurons. Microinjection of carbachol into sites in the ventromedial medulla produced antinociception, assessed using the tail flick test, that lasted more than 60 min. However, the effects of carbachol were attenuated by microinjection of either tetracaine or cobalt into sites near the A7 cell group neurons identified by tyrosine hydroxylase-immunoreactivity. Similar injections of tetracaine or cobalt more than 500 microm from the A7 neurons did not alter the antinociceptive effect of carbachol. These results support the conclusion that the antinociception produced by activating neurons in the ventromedial medulla is mediated in part by the subsequent activation of spinally-projecting noradrenergic neurons in the A7 cell group.
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PMID:The antinociception produced by microinjection of a cholinergic agonist in the ventromedial medulla is mediated by noradrenergic neurons in the A7 catecholamine cell group. 1008 77


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