Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma

Expression of the noradrenaline transporter (NAT) was examined in normal human adrenal medulla and phaeochromocytoma by using immunohistochemistry and confocal microscopy. The enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were used as catecholamine biosynthetic markers and chromogranin A (CGA) as a marker for secretory granules. Catecholamine content was measured by using high performance liquid chromatography (HPLC). In normal human adrenal medulla (n=5), all chromaffin cells demonstrated strong TH, PNMT and NAT immunoreactivity. NAT was co-localized with PNMT and was located within the cytoplasm with a punctate appearance. Human phaeochromocytomas demonstrated strong TH expression (n=20 samples tested) but variable NAT and PNMT expression (n=24). NAT immunoreactivity ranged from absent (n=3) to weak (n=10) and strong (n=11) and, in some cases, occupied an apparent nuclear location. Unlike the expression seen in normal human adrenal medullary tissue, NAT expression was not consistently co-localized with PNMT. PNMT also showed highly variable expression that was poorly correlated with tumour adrenaline content. Immunoreactivity for CGA was colocalized with NAT within the cytoplasm of normal human chromaffin cells (n=4). This co-localization was not consistent in phaeochromocytoma tumour cells (n=7). The altered pattern of expression for both NAT and PNMT in phaeochromocytoma indicates a significant disruption in the regulation and possibly in the function of these proteins in adrenal medullary tumours.     

Expression of the adrenergic phenotype in cultured fetal adrenal medullary cells: role of intrinsic and extrinsic factors

During embryogenesis of the rat the enzymes tryosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) are first detected by immunocytochemistry or biochemical assay on the 16th day of gestation (E 16). It is not until E 18 that the enzyme phenylethanolamine-N-methyltransferase (PNMT), which is required for biosynthesis of adrenaline, can be detected cytochemically or biochemically. In this study we sought to determine whether the delayed appearance of PNMT is consequent to invasion of the adrenal medulla by E 18 of cells destined to express PNMT, cues provided by the ingrowing splachnic nerves or the action of corticosterone (CS) secreted by the adrenal cortical anlage, a hormone which regulates PNMT in adult rats. When adrenal glands are removed on E 16 and placed in culture, PNMT cannot be detected cyto- or biochemically until 2 days later (E 16 + 2). While CS levels increase 100-fold in vivo between E 16 and E 18, the surge of CS is not necessary for expression of PNMT since (a) adrenals removed on E 16 and cultured in the absence of exogenous ACTH fail to increase CS yet still express PNMT and (b) addition of CS (10^?5M) to the cultures on E 16 does not alter the time of appearance of the enzyme. CS, on the other hand, increases the amount of PNMT protein and activity 3-fold with respect to control at all time points, without any effect on TH. We conclude that (a) it is the cells already present in the adrenal medulla at E 16 which differentiate to express PNMT; (b) the initial expression of PNMT is not controlled by nerves nor by corticosteroids; and (c) corticosteroids have a selective action on regulating the amount of PNMT, once it is expressed, but not TH enzyme protein. It remains to be determined whether the differentiation of PNMT is elicited by genetic or epigenetic signals.     

Immunohistochemical absence of adrenergic neurons in the dorsal part of the solitary tract nucleus in sudden infant death

The immunohistochemical distribution of TH and PNMT containing neuronal elements was investigated utilizing peroxidase anti-peroxidase methods in newborn control and sudden infant death syndrome (SIDS) brainstems. The TH immunoreactive neurons, within the medulla oblongata, displayed a similar distribution in both control and SIDS tissue. However, PNMT immunoreactive neurons seen in the dorsal part of the nucleus of tractus solitarius in control tissue were not observed in SIDS tissue. This alteration of adrenergic neurons in the dorsal part of NTS (region reported to be implicated in the control of blood pressure and respiration) could explain the cardiorespiratory disorders in SIDS.     

Identification of the prolactin-releasing peptide-producing cell in the rat adrenal gland

Prolactin-releasing peptide (PrRP) is a novel peptide found in bovine hypothalamus as an endogenous ligand of an orphan G-protein-coupled receptor (hGR3). It is known that PrRP is widely distributed and plays roles in the central nervous system (CNS). In particular, PrRP acts as a neurotransmitter that mediates stress and activates the hypothalamo-pituitary-adrenal axis. On the other hand, only a few studies have so far been performed on PrRP in peripheral tissues. Among peripheral tissues, appreciable levels of PrRP are found only in the adrenal gland; however, the PrRP-producing cells in the adrenal gland have not been identified. In this study, we detected PrRP mRNA in the rat adrenal medulla. So, we tried to identify the PrRP-producing cells in primary culture cells of the adrenal medulla. We found immunopositive PrRP cells among the cultured cells from the adrenal gland, but not in the adrenal gland tissue, by means of immunocytochemistry. The PrRP immunopositive cells were double positive for tyrosine hydroxylase (TH) and for phenylethanolamine N-methyltransferase (PNMT), which indicates that PrRP may be produced in a part of the adrenaline cells in the adrenal gland. This is the first report that PrRP is produced in the adrenaline-containing cells of the adrenal gland.     

Vagotomy Affects Lipopolysaccharide-Induced Changes of Urocortin 2 Gene Expression in the Brain and on the Periphery

The corticotropin-releasing hormone family of peptides is involved in regulating the neuroendocrine stress response. Also, the vagus nerve plays an important role in the transmission of immune system-related signals to brain structures, thereby orchestrating the neuroendocrine stress response. Therefore, we investigated gene expression of urocortin 2 (Ucn2) and c-fos, a markers of neuronal activity, within the hypothalamic paraventricular nucleus (PVN), a brain structure involved in neuroendocrine and neuroimmune responses, as well as in the adrenal medulla and spleen in vagotomized rats exposed to immune challenge. In addition, markers of neuroendocrine stress response activity were investigated in the adrenal medulla, spleen, and plasma. Intraperitoneal administration of lipopolysaccharide (LPS) induced a significant increase of c-fos and Ucn2 gene expression in the PVN, and adrenal medulla as well as increases of plasma corticosterone levels. In addition, LPS administration induced a significant increase in the gene expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla. In the spleen, LPS administration increased gene expression of c-fos, while gene expression of TH and PNMT was significantly reduced, and gene expression of Ucn2 was not affected. Subdiaphragmatic vagotomy significantly attenuated the LPS-induced increases of gene expression of c-fos and Ucn2 in the PVN and Ucn2 in the adrenal medulla. Our data has shown that Ucn2 may be involved in regulation of the HPA axis in response to immune challenge. In addition, our findings indicate that the effect of immune challenge on gene expression of Ucn2 is mediated by vagal pathways.

     

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Structure and expression of mouse apolipoprotein E gene

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.     

Structure and expression of canary myc family genes.

We found that the canary N-myc gene is highly related to mammalian N-myc genes in both the protein-coding region and the long 3' untranslated region. Examined coding regions of the canary c-myc gene were also highly related to their mammalian counterparts, but in contrast to N-myc, the canary and mammalian c-myc genes were quite divergent in their 3' untranslated regions. We readily detected N-myc and c-myc expression in the adult canary brain and found N-myc expression both at sites of proliferating neuronal precursors and in mature neurons.     

Expression patterns of the novel imprinted genes Nap1l5 and Peg13 and their non-imprinted host genes in the adult mouse brain

Recent work has implicated imprinted gene functioning in neurodevelopment and behaviour and defining the expression patterns of these genes in brain tissue has become a key prerequisite to establishing function. In this work we report on the expression patterns of two novel imprinted loci, Nap1l5 and Peg13, in adult mouse brain using in situ hybridisation methods. Nap1l5 and Peg13 are located, respectively, within the introns of the non-imprinted genes Herc3 and the Tularik1 (T1)/KIAA1882 homologue in two separate microimprinted domains on mouse chromosomes 6 and 15. These 'host' genes are highly expressed in brain and consequently we were interested in assessing their expression patterns in parallel to the imprinted genes. The brain expression of all four genes appeared to be mainly neuronal. The detailed expression profiles of Nap1l5 and Peg13 were generally similar with widespread expression that was relatively high in the septal and hypothalamic regions, the hippocampus and the cerebral cortex. In contrast, there was some degree of dissociation between the imprinted genes and their non-imprinted hosts, in that, whilst there was again widespread expression of Herc3 and the T1/KIAA1882 homologue, these genes were also particularly highly expressed in Purkinje neurons and piriform cortex. We also examined expression of the novel imprinted genes in the adrenal glands. Nap1l5 expression was localised mainly to the adrenal medulla, whilst Peg13 expression was observed more generally throughout the adrenal medulla and the outer cortical layers.     

Changes in levels of mRNA coding for catecholamine synthesizing enzymes and neuropeptide Y in the adrenal medulla of the newborn rat.

We have measured levels of mRNA coding for the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), phenylethanolamine N-methyltransferase (PNMT) and for neuropeptide Y (NPY) in rat adrenal medulla by using in situ hybridization histochemistry. Ages of one day before birth (E21), 12 h, 24 h, 2 days and 4 days after birth and in adults were studied. TH, D beta H and NPY mRNA levels increased markedly postnatally. Twelve hours after birth the levels of mRNA for TH, D beta H and NPY were, respectively, 512 +/- 18%, 370 +/- 24% and 253 +/- 21% of E21 levels. At 24 h of age NPY mRNA level was 437 +/- 73% of fetal value. In contrast, the levels of mRNA coding for PNMT increased more slowly and reached 196 +/- 9% of E21 level on postnatal day four and was further increased in adult rats.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) mRNAs in pheochromocytomas. Methods: The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. Results: mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH (p=0.0018) and PNMT (p=0.05) mRNA expression, as well as epinephrine (p=0.0342) levels in 16 intra-adrenal pheochromocytomas. Conclusion: Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.     

Expression and development of phenylethanolamine N-methyltransferase (PNMT) in rat brain stem: studies with glucocorticoids

To study the differentiation of adrenergic (epinephrine-synthesizing) neurons in brain, the initial appearance and ontogeny of phenylethanolamine N-methyltransferase (PNMT), a specific marker of the adrenergic phenotype, were studied with immunocytochemistry and catalytic assay. The appearance of immunoreactivity to dopamine beta-hydroxylase (DBH-IR), an enzyme common to the noradrenergic and adrenergic phenotypes, was also studied. DBH-IR was initially observed on embryonic Day 13 (E13) in cells located on the ventrolateral floor and wall of the rhombencephalon. A day later (E14), PNMT-IR cells and PNMT catalytic activity were observed in the rhombencephalon suggesting that, as in the adrenal gland, noradrenergic expression precedes adrenergic expression. The PNMT-IR cells were presumed to be precursors of C1 neurons since they were located in the ventrolateral medulla oblongata. Cells located in the wall of the medulla which appeared to be migrating ventrally to the C1 group also contained PNMT-IR. On E15, cells which had PNMT-IR processes coursing through the germinal zone were observed dorsally near the fourth ventricle. Although the location of the C1 cell group was apparent when PNMT was initially expressed, the dorsal C2 and C3 adrenergic cell groups were not evident until late in gestation on E19. Even in the term embryo there appeared to be PNMT-IR cells which had not yet reached their final destination. On E14 and E15, PNMT-IR cells were also observed on the floor of the pons just rostral to the pontine flexure. However, these were not observed in older embryos, suggesting that transient expression of PNMT occurs in brain, as well as in the periphery. To determine whether glucocorticoids regulate brain PNMT, we examined the effects of altered glucocorticoid levels. In contrast to PNMT in the sympathetic nervous system, PNMT activity in medulla oblongata was not affected in neonates or adults by the decrease in glucocorticoids following adrenalectomy or hypophysectomy. Conversely, elevation of glucocorticoids by hormonal treatment did not alter PNMT in neonates. Notably, however, treatment of pregnant rats with dexamethasone on E18-E21, but not earlier, increased PNMT activity in the fetal brain stem. These observations suggest that PNMT expression and development is regulated by different factors in cells derived from neural crest and tube. PNMT is expressed earlier in brain than in adrenal and sympathetic ganglia. Further, the development of PNMT in the periphery, but not in the brain, is dependent on maintenance of physiological levels of glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ectopic expression of Thy-1 in the kidneys of transgenic mice induces functional and proliferative abnormalities

Hybrid human--mouse Thy-1.1 genes were injected into pronuclei of Thy-1.2 mice to produce transgenic animals. A hybrid gene composed of the 5' part of the mouse Thy-1.1 gene combined with the 3' human untranslated regions was expressed abnormally in the kidney podocytes, which resulted in severe protein-uria and subsequent death in several founder mice. A hybrid Thy-1 gene composed of the human coding region with the 5' and 3' flanking regions of the mouse gene was expressed abnormally in a different part of the kidney (the tubular epithelia), which resulted in a proliferative kidney disorder. In addition, a neoplasm was found in the brain of one of these mice. These results show that the Thy-1 protein can play an important role in the activation, proliferation, and differentiation of many different cell types.     

Catecholamine Metabolism in Paraganglioma and Pheochromocytoma: Similar Tumors in Different Sites?

Pheochromocytoma (PHEO) and paraganglioma (PGL) are catecholamine-producing neuroendocrine tumors that arise respectively inside or outside the adrenal medulla. Several reports have shown that adrenal glucocorticoids (GC) play an important regulatory role on the genes encoding the main enzymes involved in catecholamine (CAT) synthesis i.e. tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). To assess the influence of tumor location on CAT metabolism, 66 tissue samples (53 PHEO, 13 PGL) and 73 plasma samples (50 PHEO, 23 PGL) were studied. Western blot and qPCR were performed for TH, DBH and PNMT expression. We found a significantly lower intra-tumoral concentration of CAT and metanephrines (MNs) in PGL along with a downregulation of TH and PNMT at both mRNA and protein level compared with PHEO. However, when PHEO were partitioned into noradrenergic (NorAd) and mixed tumors based on an intra-tumoral CAT ratio (NE/E >90%), PGL and NorAd PHEO sustained similar TH, DBH and PNMT gene and protein expression. CAT concentration and composition were also similar between NorAd PHEO and PGL, excluding the use of CAT or MNs to discriminate between PGL and PHEO on the basis of biochemical tests. We observed an increase of TH mRNA concentration without correlation with TH protein expression in primary cell culture of PHEO and PGL incubated with dexamethasone during 24 hours; no changes were monitored for PNMT and DBH at both mRNA and protein level in PHEO and PGL. Altogether, these results indicate that long term CAT synthesis is not driven by the close environment where the tumor develops and suggest that GC alone is not sufficient to regulate CAT synthesis pathway in PHEO/PGL.