Expression of GTP cyclohydrolase I (GTPCH) mRNA in the rat: tissue distribution and effect of reserpine.
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Adrenal Glands↗
Biomedical subjects
G Kapatos
Publications and source records attributed to G Kapatos.
Regulation of tetrahydrobiopterin biosynthesis in cultured hypothalamic and mesencephalic neurons by cyclic AMP dependent GTP cyclohydrolase I gene expression.
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8-Bromo Cyclic Adenosine Monophosphate↗
Differential metabolism of tetrahydrobiopterin in monoamine neurons: a hypothesis based upon clinical and basic research.
This chapter has attempted to describe and integrate some of the clinical and basic research that support our hypothesis that the metabolism of BH4 is normally heterogeneous across different populations of monoamine-containing neurons. Based upon this hypothesis, there may now be reason to support the idea that certain neuropsychiatric illnesses, which are though to be the result (at least in part) of altered monoamine metabolism, might find their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups. Such a specific dysfunction might not be apparent in the rest of the brain or peripheral nervous system, thereby being difficult to detect. Perhaps the application of molecular biological techniques to studies of BH4 metabolism in man will shed new light on these problems.
Animals↗
GABA transporter mRNA: in vitro expression and quantitation in neonatal rat and postmortem human brain.
A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by nuclease protection assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by nuclease protection assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.
Adult↗
Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia.
GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridization. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1.2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2- and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.
Animals↗
Transfected D2 short dopamine receptors inhibit voltage-dependent potassium current in neuroblastoma x glioma hybrid (NG108-15) cells.
Two isoforms of the D2 dopamine receptor exist, termed D2 short (D2s) and D2 long, which differ by the presence or absence of 29 amino acids. To examine the possible coupling of the D2s isoform to voltage-dependent K+ current, NG108-15 cells that were transfected with and stably express this isoform were studied using whole-cell patch-clamp techniques. In transfected, but not untransfected, cells dopamine and quinpirole (QUIN) reduced the normally observed peak outward K+ current, and this effect was abolished by the D2 antagonist sulpiride but not by the alpha 2-adrenergic receptor antagonist idazoxan or the D1 antagonist (R)-(+)-SCH-23380. The D1 receptor agonist SKF 38393 had no effect. QUIN-induced inhibition of K+ current was prevented by loading the cells with the Ca(2+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting a critical role for intracellular Ca2+ mobilization. In contrast, reduction of the concentration of extracellular Ca2+ and inclusion of the Ca2+ channel blocker cobalt did not modify the reduction of K+ current produced by stimulation of D2s receptors. A critical role for intracellular calcium mobilization in the observed effects was further supported by the observation that increases in cytosolic Ca2+ produced by thapsigargin mimicked the effect of QUIN, whereas intracellular ryanodine, which blocks Ca2+ mobilization, abolished the QUIN responsiveness. Finally, the effect of D2S activation on K+ current was not modified by pretreatment of the cells with pertussis toxin. These results suggest that the D2s dopamine receptor expressed in NG108-15 cells inhibits the activity of native K+ current via a mechanism that is dependent upon the mobilization of intracellular Ca2+ and does not involve a pertussis toxin-sensitive G protein.
Calcium↗
Dopamine transporter mRNA content in human substantia nigra decreases precipitously with age.
The dopamine transporter is the primary means of inactivating synaptic dopamine as well as a major site of action for psychostimulants (such as cocaine and amphetamine) and for neurotoxins that induce parkinsonism. In the present study, a human dopamine transporter partial cDNA clone obtained by polymerase chain reaction exhibited 87% and 89% identity at the nucleic acid and amino acid levels, respectively, with transmembrane domains 3-5 of the rat homolog. This clone was used to quantitate human dopamine transporter mRNA by nuclease protection assay. The postmortem content of dopamine transporter mRNA in the substantia nigrae of 18- to 57-yr-old subjects was relatively constant, while in subjects greater than 57 yr old, a precipitous (greater than 95%) decline in substantia nigra dopamine transporter mRNA was evident. In contrast, tyrosine hydroxylase mRNA in the same samples declined in a linear manner with increasing age. In situ hybridization experiments confirmed the profound loss of dopamine transporter gene expression in melanin-positive (presumptive dopamine) nigral neurons. These data may begin to shed light on compensatory changes occurring in human dopamine neurons during normal aging.
Adult↗
Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture.
Dopamine (DA)-containing neurons in primary dissociated cell cultures derived from the embryonic mouse mesencephalon (day E13) were studied by histochemical and electrophysiological techniques. DA neurons exhibited two distinct morphologies, fusiform and multipolar, tended to reside in groups and organize dendrites into common fascicles. While these neurons expressed the cell-surface marker acetylcholinesterase, the presence of this enzyme could not be used to identify DA neurons unequivocally, since it was also observed in nondopaminergic cells. Neurons were therefore identified as DA by their distinct morphology, and this identification was validated with a double-labeling procedure that entailed the intracellular deposition of a fluorescent dye (Lucifer yellow or ethidium bromide), followed by processing for tyrosine hydroxylase immunocytochemistry. DA neurons identified in this manner were observed to have resting membrane potentials between -50 and -75 mV, input resistances of 50-360 M omega, and membrane time constants of 4.1-14.1 msec. Forty-seven percent of these cells displayed spontaneous activity that was irregular in nature and often contained bursts (burst length was between two and six action potentials). The DA neurons displayed a variety of ionic conductances, including (1) a Na+ conductance (gNa) that underlies the action potential, (2) Ca2+ conductances (gCa) that mediate the nonsomatic low- and high-threshold spikes observed, and (3) at least three K+ conductances (gK). Voltage-clamp analysis revealed several distinct transmembrane ionic currents, including (1) a large, rapidly inactivating tetrodotoxin-sensitive inward Na+ current (INa), (2) a 4-aminopyridine-sensitive, transient early outward K+ current that required a conditioning hyperpolarization of the membrane to be activated by a subsequent depolarization (A-current, IA), (3) a slowly developing inward current that was seen only after a conditioning hyperpolarization of the membrane and that was dependent on the presence of external Ca2+ ions (ICa), and (4) a late-onset, noninactivating K+ current. Between 25% and 54% of the late-onset K+ current was Ca(2+)-dependent and was not affected by tetraethylammonium ions. This current was termed IAHP. The remaining current was not sensitive to changes in the extracellular Ca2+ concentration but was blocked by external tetraethylammonium. This current was termed IK. The direct pressure application of DA (1-200 microM) onto the soma dose-dependently hyperpolarized these neurons; this effect was potentiated by the presence of the catecholamine reuptake blocker cocaine hydrochloride (10-200 microM). Under voltage-clamp conditions, DA was observed to increase IK significantly and had little effect on IAHP.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholinesterase↗
Non-specific binding of normal human IgG, including F(ab')2 and Fc fragments, to embryonic rat brain neurons and human cortex synaptosomes.
Binding of normal human IgG to embryonic rat brain neurons was quantitated by flow cytometry. IgG binding was linear between 0.05 and 1.5 mg/ml; slight binding was detectable even at normal cerebrospinal fluid concentrations. Similar binding curves were obtained for purified Fc and F(ab')2 fragments from normal human IgG. Normal human IgG also bound to synaptosomes (resealed nerve terminals) from human cerebral cortex. However, competition assays utilizing 125I-IgG showed no evidence for specific binding. This study indicates that the specificity of putative anti-neuronal antibodies should be confirmed by competition assays as for other receptor-ligand binding.
Animals↗
Quantitation of rat dopamine transporter mRNA: effects of cocaine treatment and withdrawal.
Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.
Animals↗
Tetrahydrobiopterin turnover in cultured rat sympathetic neurons: developmental profile, pharmacologic sensitivity, and relationship to norepinephrine synthesis.
We have examined the turnover of 5,6,7,8-tetrahydrobiopterin (BH4) and the effect of decreasing BH4 levels on in situ tyrosine hydroxylase (TH) activity and norepinephrine (NE) content in a homogeneous population of NE-containing neurons derived from the superior cervical ganglion (SCG) of the neonatal rat and maintained in tissue culture. Initial studies indicated that the level of BH4 within SCG cultures increased fourfold between 5 and 37 days in vitro (DIV). This increase in BH4 levels was determined to result from an increase in the rate of BH4 biosynthesis without a change in the rate of degradation. Regardless of culture age, the BH4 content of SCG neurons was observed to turn over with a half-life of approximately 2.5 h. BH4 synthesis by SCG neurons was found to be five times more sensitive to inhibition by 2,4-diamino-6-hydroxypyrimidine (DAHP) and 25 times less sensitive to inhibition by N-acetylserotonin than was previously reported for CNS neurons in culture. Under basal conditions, the rates of in situ TH activity and BH4 biosynthesis were similar. In response to inhibition of BH4 biosynthesis by DAHP and a 90-95% decrease in BH4 levels, in situ TH activity declined by 75%. NE levels declined by 30% following a 24-h period of inhibition of BH4 synthesis. After 2 days of BH4 synthesis inhibition, the level of NE was decreased by 47%. On treatment days 3 and 4, the decline in NE content plateaued at 24% of control levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Characterization of the distribution of G alpha o in rat striatal synaptosomes and its colocalization with tyrosine hydroxylase.
Dopaminergic striatal synaptosomes can be detected and isolated with a fluorescence-activated cell sorter (FACS). In the present study, two antigens were detected simultaneously with primary antisera raised in different species and species-specific fluorescent secondary antibodies with different emission spectra. Double-label FACS analysis was used to determine whether tyrosine hydroxylase (TH) and the alpha subunit of Go (G alpha o) are colocalized in striatal synaptosomes. Rabbit antibodies generated against a synthetic fragment of G alpha o (corresponding to amino acids 22-35) combined with fluorescein-conjugated secondary antibodies were used to detect G alpha o-containing striatal synaptosomes. Preadsorption of G alpha o antiserum with the synthetic peptide antigen reduced labeling to the level obtained with preimmune serum. Approximately 65-75% of striatal synaptosomes were specifically labeled by G alpha o antiserum. Tyrosine hydroxylase-containing synaptosomes were detected with a mouse monoclonal antibody to TH and R-phycoerythrin-conjugated secondary antibody. They comprised 15-17% of total striatal synaptosomes. Double-label studies indicated that at least 50% of TH-containing synaptosomes also contained G alpha o. These findings suggest that G alpha o may not be a protein component of all striatal nerve terminals, and provide a basis for a role for G alpha o in signal transduction within subpopulations of intrinsic and afferent nerve terminals, including those of nigrostriatal dopamine neurons.
Animals↗
Effect of aging on tyrosine hydroxylase protein content and the relative number of dopamine nerve terminals in human caudate.
This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence-activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH-containing nerve terminals or the concentration of TH protein were found in subjects 15-63 years of age. In light of previous studies showing an age-related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH-containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age-related differences in the relative abundance of these forms were found.
Aging↗
Expression of a human cocaine-sensitive dopamine transporter in Xenopus laevis oocytes.
The human dopamine transporter was expressed in Xenopus laevis oocytes following injection of mRNA isolated from human brain substantia nigra. The specific accumulation of [3H]dopamine into these oocytes was time and Na+ dependent. Furthermore, [3H]dopamine accumulation was prevented by coincubation of oocytes with dopamine (100 microM) or with the dopamine uptake inhibitors GBR 12909 (1 microM) or cocaine (3 microM). In contrast, oocyte injection of mRNA isolated from human globus pallidus, an area devoid of dopamine neuron perikarya, did not elicit expression of the dopamine transporter. Oocyte expression of the human dopamine transporter can be used for the further characterization and cloning of this low-abundance membrane protein.
Animals↗
Immunological evidence for the requirement of sepiapterin reductase for tetrahydrobiopterin biosynthesis in brain.
Specific antibodies to sepiapterin reductase were used to investigate its involvement in de novo (6R)-5,6,7,8-tetrahydrobiopterin (BH4) biosynthesis in rat brain. Antisepiapterin reductase (anti-SR) serum totally inhibited NADPH-dependent sepiapterin reductase activity in supernatants from discrete rat brain areas and liver. The anti-SR serum also inhibited the conversion of 7,8-dihydroneopterin triphosphate to BH4 in rat brain extracts. The inhibition was accompanied by a concentration-dependent increase in the formation of 6-lactoyltetrahydropterin (6LPH4), a proposed intermediate in BH4 biosynthesis. In addition, anti-SR serum was used to characterize the distribution and molecular properties of sepiapterin reductase in rat tissues. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting indicated that there was a single polypeptide with the same molecular weight (28,000) as that of the subunit of pure sepiapterin reductase present in all tissues examined except for liver, where an immunoreactive protein of higher molecular weight (30,500) also was detected. Two-dimensional gel electrophoresis of rat striatum and liver demonstrated that the isoelectric point of sepiapterin reductase from both tissues was 6.16 and that the higher molecular weight immunoreactive material in liver had an isoelectric point of 7.06. Our studies with specific anti-SR serum confirmed the results of previous studies using chemical inhibitors of sepiapterin reductase, which suggested that sepiapterin reductase activity was essential for BH4 biosynthesis in the CNS and that 6LPH4 could be a precursor of BH4.
Alcohol Oxidoreductases↗
Developmental expression of Go in neuronal cultures from rat mesencephalon and hypothalamus.
The developmental expression of the alpha-subunit of Go was examined in neuronal cultures derived from rat mesencephalon (MES) and hypothalamus (HYP). These cultures were essentially free of contaminating glia and were maintained as a stable population for periods up to 3 weeks. Immunoblotting utilizing specific antisera against Go indicated that in neurons from both brain regions, membrane concentrations of Go increased dramatically during the first 2 weeks in vitro. Thereafter, increases in the amount of Go per neuron kept pace with increasing process (axons and dendrites) formation. Multiple forms of immunoreactive Go were detected in MES and HYP neurons, and the proportions of these forms changed between 4 and 14 days in culture. Finally, increasing neuron density significantly increased membrane levels of Go in MES but not HYP cultures.
Animals↗
Tetrahydrobiopterin synthesis rate and turnover time in neuronal cultures from embryonic rat mesencephalon and hypothalamus.
6-(R)-(L-erythro-1',2'-Dihydroxypropyl)-2-amino- 4-hydroxy-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin, BH4) synthesis rate and turnover time were estimated in cultures derived from the embryonic rat mesencephalon (MES) and hypothalamus (HYP) by following the decline in BH4 levels after blockade of BH4 biosynthesis by N-acetylserotonin (NAS) or 2,4-diamino-6-hydroxypyrimidine (DAHP). BH4 content of both culture systems decreased by 75% following an 8-h incubation with maximally effective concentrations of NAS (200 microM) or DAHP (10 mM). Parameters describing BH4 metabolism were calculated from steady-state levels of BH4 and first-order rate constants determined by a nonlinear regression analysis of the exponential BH4 decline. These parameters were confirmed using an alternative procedure that examined the first-order rate of recovery of BH4 following termination of BH4 synthesis inhibition. Steady-state levels of BH4 in HYP cultures (70.3 +/- 9.4 pg/culture) were significantly greater than that for MES (46.5 +/- 2.8 pg/culture). The average fractional rate constants of BH4 loss for MES (0.153 +/- 0.015/h) and HYP (0.159 +/- 0.014/h) were equivalent. The calculated rate of BH4 synthesis was significantly greater for HYP (11.29 +/- 2.13 pg/culture/h) than for MES (7.11 +/- 0.85 pg/culture/h), owing to the greater steady-state concentration of BH4. BH4 turnover time for MES (6.68 +/- 0.67 h) and HYP (6.40 +/- 0.62 h) and half-life for MES (4.63 +/- 0.46 h) and HYP (4.44 +/- 0.43 h) did not differ. The turnover of the cofactor is thus rapid enough that alterations in its synthesis or degradation could acutely modify the rate of monoamine biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Stimulation of D2 dopamine receptors decreases intracellular calcium levels in rat anterior pituitary cells but not striatal synaptosomes: a flow cytometric study using indo-1.
An important question is whether all D2 dopamine (DA) receptors employ the same signal transduction mechanisms. Anterior pituitary cells and striatal synaptosomes, which possess pharmacologically similar D2 DA receptors, were compared with respect to the effect of D2 DA receptor stimulation on free intracellular Ca2+ levels [( Ca2+]i). Flow cytometry, in combination with either the fluorescent calcium indicator indo-1 or fluorescent voltage-sensitive dyes, was used to measure [Ca2+]i and to detect changes in membrane potential. In subpopulations of anterior pituitary cells, increases in [Ca2+]i were produced by elevated K+, veratridine, thyrotropin-releasing hormone, and BAY K 8644. These increases were blocked by nifedipine, suggesting the involvement of L-type voltage-sensitive calcium channels (VSCC's). In 10-15% of the cells, D2 agonists decreased resting [Ca2+]i, reversed stimulus-induced increases in [Ca2+]i, and caused a hyperpolarization. In striatal synaptosomes, elevated K+ and veratridine also increased [Ca2+]i. However, the K+-induced increase was eliminated if choline was substituted for Na+ in the medium, suggesting that Ca2+ entry in response to sustained K+ depolarization resulted from reversal of Na+/Ca2+ exchange. Nifedipine and verapamil inhibited K+-induced increases in [Ca2+]i only at concentrations greater than 10 microM, while omega-conotoxin had no effect. D2 agonists had no effect on resting or stimulated [Ca2+]i but did hyperpolarize 10-20% of the synaptosomes, indicating that D2 DA receptors are functional in this preparation. The ability of pituitary but not striatal D2 DA receptors to modulate [Ca2+]i may reflect the fact that the two systems differ with respect to pathways for Ca2+ influx.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗