GTP Cyclohydrolase I Feedback Regulatory Protein Is Expressed in Serotonin Neurons and Regulates Tetrahydrobiopterin Biosynthesis

Abstract : Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by ~20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L-phenylalanine-induced increase in tetrahydrobiopterin levels. 7,8-Dihydroneopterin, a reduced pteridine capable of inhibiting GTPCH in a GFRP-dependent manner, decreased tetrahydrobiopterin levels in cultures of both serotonin and dopamine neurons. This inhibition was reversed by L-phenylalanine in serotonin but not in dopamine neurons. Our data suggest that GTPCH activity within serotonin neurons is under a tonic inhibitory tone mediated by GFRP and that tetrahydrobiopterin levels are maintained by the balance of intracellular concentrations of tetrahydrobiopterin and L-phenylalanine. In contrast, although tetrahydrobiopterin biosynthesis within dopamine neurons is also feedback-regulated, L-phenylalanine plays no role, and therefore tetrahydrobiopterin may have a direct effect on GTPCH activity.     

Differential In Vivo Regulation of mRNA Encoding the Norepinephrine Transporter and Tyrosine Hydroxylase in Rat Adrenal Medulla and Locus Ceruleus

Abstract: To investigate the regulation of norepinephrine transporter mRNA in vivo, we analyzed the effects of reserpine on its expression in the rat adrenal medulla and locus ceruleus. First, PCR was used to clone a 0.5-kb rat cDNA fragment that exhibits 87% nucleotide identity to the corresponding human norepinephrine transporter cDNA sequence. In situ, the cDNA hybridizes specifically within norepinephrine-secreting cells, but in neither dopamine nor serotonin neurons, suggesting strongly it is a partial rat norepinephrine transporter cDNA. Reserpine, 10 mg/kg administered 24 h premortem, decreased steady-state levels of norepinephrine transporter mRNA in the adrenal medulla by ∼65% and in the locus ceruleus by ∼25%, as determined by quantitative in situ hybridization. Northern analysis confirmed the results of the in situ hybridization analysis in the adrenal medulla but did not detect the smaller changes observed in the locus ceruleus. Both analyses showed that reserpine increased tyrosine hydroxylase expression in the adrenal medulla and locus ceruleus. These results suggest that noradrenergic neurons and adrenal chromaffin cells can coordinate opposing changes in systems mediating catecholamine uptake and synthesis, to compensate for catecholamine depletion.     

Chromogranin A in neurons of the rat cerebellum and spinal cord: quantification and sites of expression.

Chromogranin A (CGA) is an abundant protein of dense-cored secretory vesicles in endocrine and neuronal cells. The present study, for the first time, compares CGA of neurons of the central nervous system with the CGA of adrenal origin. By S1 nucleus protection assay, we found that the 3' part of the CGA mRNA between exons 5-8 of the cerebellum and the spinal cord of the rat is homologous to that of the adrenal. In situ hybridization histochemistry revealed that CGA mRNA in the cerebellar cortex is present in cell bodies of Purkinje cells and in neurons of the deep cerebellar nuclei. The perikarya of these cells also exhibit CGA-like immunoreactivity. CGA mRNA and CGA-like immunoreactivity are also present in the motoneurons of the ventral, lateral, and dorsal horns of the rat spinal cord. The amounts of CGA, as determined by radioimmunoassay in cerebellum and spinal cord, were about one tenth of the amounts detected in the adrenal, adenohypophysis, or the olfactory bulb. The sites of CGA expression suggest that CGA may be involved in signal transduction in the motor system.     

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.     

Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells

New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.     

Four Forms of Tyrosine Hydroxylase Are Present in Human Adrenal Medulla

Alternative splicing can result in up to four forms of human tyrosine hydroxylase (HTH) mRNA (mRNAHTH). In the adrenal gland, a major site of catecholamine biosynthesis, the presence of all four mRNAHTH forms is controversial. In the present study, postmortem human adrenal medullary tissue was analyzed for the presence of multiple forms of HTH protein by blot immunolabeling. Electrophoretic transfers were developed with affinity-purified rabbit anti-HTH antibodies raised against peptides that reproduced the unique amino acid sequences predicted by the four mRNAHTH forms. All four of the predicted HTH protein forms were present in the adrenal glands from a diverse sample population.     

Sequence complementarity between the 5'-terminal regions of mRNAs for rat mitochondrial cytochrome P-450c27/25 and a growth hormone-inducible serine protease inhibitor. A possible gene overlap.

Recently we reported that a P-450c27/25 cDNA probe hybridizes to two RNA species of about 1.9 and 2.3-2.4 kilobase pairs (kb) in some rat tissues. To understand the molecular relationship between the two mRNAs, we have isolated and characterized a cDNA for the larger, previously uncharacterized 2.3-kb mRNA species. The 2.3-kb cDNA is identical to the previously reported 1.9-kb P-450c27/25 cDNA excepting a 400-nucleotide-long 5' extension. The terminal 291 nucleotides of this extension exhibit 100% complementarity with the 5'-translated region of the mRNA belonging to a family of growth hormone-inducible serine protease inhibitors (SPI). Northern blot analysis, using strand-specific probes, and S1 nuclease protection revealed the presence of the 2.3-kb mRNA exhibiting the sequence characteristics of the larger cDNA. These results were further confirmed by polymerase chain reaction amplification of reverse transcribed RNA. Expression of the 2.3-kb cDNA in COS cells resulted in the correct mitochondrial targeting of a 52-kDa protein exhibiting the properties of P-450c27/25. Furthermore, both the 1.9- and 2.3-kb mRNAs appear to direct the synthesis of a similarly sized 55-kDa precursor protein in a reticulocyte lysate system. Restriction mapping, polymerase chain reaction amplification and partial sequencing of a 25-kb genomic DNA clone suggest the proximal location of the SPI and the P-450c27/25 protein coding regions in the rat genome on either side of a common overlap region. The results also show that the P-450c27/25 mRNAs are regulated by growth hormone in parallel to the SPI mRNAs. These results collectively suggest that a growth hormone-inducible SPI family mRNA and the P-450c27/25 mRNA are encoded by two closely linked, possibly overlapping genes.     

Tissue-specific and high-level expression of the human tyrosine hydroxylase gene in transgenic mice.

Transgenic mice carrying multiple copies of the human tyrosine hydroxylase (TH) gene have been produced. The transgenes were transcribed correctly and expressed specifically in brain and adrenal gland. The level of human TH mRNA in brain was about 50-fold higher than that of endogenous mouse TH mRNA. In situ hybridization demonstrated an enormous region-specific expression of the transgene in substantia nigra and ventral tegmental area. TH immunoreactivity in these regions, though not comparable to the increment of the mRNA, was definitely increased in transgenic mice. This observation was also supported by Western blot analysis and TH activity measurements. However, catecholamine levels in transgenics were not significantly different from those in nontransgenics. These results suggest unknown regulatory mechanisms for human TH gene expression and for the catecholamine levels in transgenic mice.     

Characterization of a cDNA clone for a rare mRNA modulated by ovariectomy in mammary carcinomas

A complementary DNA (cDNA) clone (p13) for a rare mRNA was isolated from a cDNA library generated from total polyA+ RNA of 14-day lactating rat mammary gland. In vitro translation of the positively selected mRNA from p13 cDNA revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a polypeptide of 24 kDa. The p13 cDNA clone hybridized on northern blots predominantly to approximately 1100 base size RNA and weakly to approximately 3800 base size RNA from lactating mammary gland. It hybridized only to approximately 3800 base size RNA from rat liver. Southern blot analysis of genomic DNA showed differences in gene organization in mammary gland and liver. The mRNA level for the 24 kDa polypeptide was higher in 7-12 DMBA-induced tumor and lower in the MTW9 carcinoma as compared to lactating mammary gland. After ovariectomy, the mRNA level in mid pregnant gland increased but was reduced in the 7-12 DMBA tumors.