Acute Hypoxia Induces Enkephalin Production and Release in an Adrenergic Cell Line Model of Neonatal Chromaffin Cell Responses to Hypoxic Stress.

Objective: Prior to maturation of the human sympathetic nervous system, the neonatal adrenal medulla senses and responds to hypoxia. In addition to catecholamine release, the adrenal medulla synthesizes and stores opioid peptides, notably enkephalin (ENK). However, it is not known whether acute hypoxia evokes adrenal ENK production and release, as seen in the central nervous system (CNS).

Pheochromocytomas in Nf1 knockout mice express a neural progenitor gene expression profile.

Pheochromocytomas are adrenal medullary tumors that typically occur in adult patients, with increased frequency in multiple endocrine neoplasia type 2, von Hippel-Lindau disease, familial paraganglioma syndromes and neurofibromatosis type 1 (NF1). Pheochromocytomas arise in adult mice with a heterozygous knockout mutation of exon 31 of the murine Nf1 gene, providing a mouse model for pheochromocytoma development in NF1. We performed a microarray-based gene expression profiling study comparing mouse pheochromocytoma tissue to normal adult mouse adrenal medulla to develop a basis for studying the pathobiology of these tumors.

Nicotine stimulates expression of the PNMT gene through a novel promoter sequence.

Transcription of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT, E.C. 2.

Hypoxia activates multiple transcriptional pathways in mouse pheochromocytoma cells.

Mouse pheochromocytoma cells (MPCs) provide an excellent model system for investigating the effects of hypoxia on catecholamine enzyme genes and on transcription factors mediating stress responses. RT-PCR detects rapid, transient increases in PNMT mRNA in hypoxic MPC 712 cells. Additionally, elevation of mRNAs encoding transcription factors hypoxia inducible factor 1 (HIF-1) alpha subunit and Egr-1 are evident within 60 min incubation in anoxia.

Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice.

Transplantable tumors and cell lines have been developed from pheochromocytomas arising in mice with a heterozygous knockout mutation of the neurofibromatosis gene, Nf1. Nf1 encodes a ras-GTPase-activating protein, neurofibromin, and mouse pheochromocytoma (MPC) cells in primary cultures typically show extensive spontaneous neuronal differentiation that may result from the loss of the remaining wild-type allele and defective regulation of ras signaling. However, all MPC cell lines express neurofibromin, suggesting that preservation of the wild-type allele may be required to permit the propagation of MPC cells in vitro.

Neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression in bovine chromaffin cells differs from other catecholamine enzyme genes.

Expression of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) is regulated by hormonal and neural stimuli. Because the 5'-upstream regions of the PNMT do not contain sequences analogous to those demonstrated to convey neural regulation to the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) catecholamine-synthesizing enzyme genes, functional and biochemical analyses have been utilized to characterize PNMT promoter responses to cholinergic and depolarizing agents. In primary cultures of bovine adrenal medullary chromaffin cells, reporter gene expression from transiently transfected 3- and 0.

Pituitary adenylate cyclase activating polypeptide (PACAP) regulates expression of catecholamine biosynthetic enzyme genes in bovine adrenal chromaffin cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) elevates levels of the mRNAs encoding the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in primary cultures of bovine adrenal chromaffin cells. PACAP potently (in nanomolar concentrations) increases the amount of mRNA for each of the three catecholamine biosynthetic enzymes. At 10 nM PACAP, TH and DBH mRNA levels increase approx 10-fold; 1 nM PACAP produces an approx 2.

Regulation of PNMT gene promoter constructs transfected into the TE 671 human medulloblastoma cell line.

The human medulloblastoma TE 671 cell line has been evaluated as a model for studying expression of transiently transfected phenylethanolamine N-methyltransferase (PNMT) promoter-fusion gene constructs. Because TE 671 cells are one of few continuous lines exhibiting a neuronal phenotype, possess both nicotinic and muscarinic ligand binding properties, and express PNMT mRNA, they represent a likely candidate system for study of cholinergic-regulated PNMT gene expression. When transfected with constructs containing from 0.

Regulation of phenylethanolamine N-methyltransferase gene expression by imidazoline receptors in adrenal chromaffin cells.

As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of alpha 2-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine.

Mammalian adrenal chromaffin cells coexpress the epinephrine-synthesizing enzyme and neuronal properties in vivo and in vitro.

Adrenal chromaffin cells and neurons of the sympathetic ganglia are derived from common precursors in the neural crest. The phenotype of the sympathoadrenal progenitor cell is unknown, but adult chromaffin cells are distinguished by the expression of phenylethanolamine-N-methyltransferase (PNMT) and the lack of neurofilament (NF) and neuritic processes. Mature neurons have processes and express NF, but are PNMT-.

A single transmitter regulates gene expression through two separate mechanisms: cholinergic regulation of phenylethanolamine N-methyltransferase mRNA via nicotinic and muscarinic pathways.

ACh regulates the gene encoding phenylethanolamine N-methyltransferase (PNMT) in bovine adrenal chromaffin cells. In addition to stimulating catecholamine release from these cells, cholinergic agents elevate transcription of the PNMT gene. Carbachol, which activates both nicotinic and muscarinic receptors, produces 12-19-fold increases in PNMT mRNA and a 22-fold increase in epinephrine release.

Muscle-derived differentiation factor increases expression of the tyrosine hydroxylase gene and enzyme activity in cultured dopamine neurons from the rat midbrain.

Our earlier work demonstrated that certain populations of brain neurons which do not synthesize catecholamine (CA) neurotransmitters in vivo, will, when grown in culture with muscle-derived differentiation factor (MDF), unexpectedly express the gene for the CA biosynthetic enzyme tyrosine hydroxylase (TH). In this paper, we sought to determine whether MDF could also regulate TH expression in those neurons which normally synthesize CA neurotransmitters. Incubation of cultured dopamine neurons from the ventral midbrain with MDF elevated the levels of TH mRNA and TH enzyme activity 5- to 40-fold higher than that measured in control cultures.

Lesions of nigrostriatal pathway reduce expression of tyrosine hydroxylase gene in residual dopaminergic neurons of substantia nigra.

The effects of unilateral mechanical transection of the nigrostriatal bundle of rat brain on the level of tyrosine hydroxylase (TH) mRNA and on the activity of TH enzyme in the substantia nigra (SN) were examined. Lesions resulted, by 14 days, in reductions of TH mRNA level to 10% of control and of TH enzyme activity to 39% of control in the ipsilateral SN. The percentage of TH mRNA is lower than either the percentage of surviving dopaminergic neurons or the remaining TH enzyme activity.

Glucocorticoids stimulate transcription of the rat phenylethanolamine N-methyltransferase (PNMT) gene in vivo and in vitro.

1. Phenylethanolamine N-methyltransferase (PNMT) is regulated by glucocorticoid hormones. This study investigates the ability of glucocorticoids to modulate transcription of the rat PNMT gene in vivo and in vitro.

Effects of clonidine and other imidazole-receptor binding agents on second messenger systems and calcium influx in bovine adrenal chromaffin cells.

Clonidine and related imidazoline compounds bind to alpha 2-adrenergic as well as to newly described non-adrenergic imidazole/imidazoline receptors in brain and peripheral tissues. The present study was undertaken to identify the signal transduction mechanism coupled to this new class of receptors (imidazole receptors) using bovine adrenal chromaffin cells. Clonidine did not modify the basal or forskolin-stimulated production of cyclic AMP (cAMP), suggesting the absence of functionally active alpha 2-adrenergic receptors in adrenal chromaffin cells.

The presence of an M4 subtype muscarinic receptor in the bovine adrenal medulla revealed by mRNA and receptor binding analyses.

The muscarinic receptor subtype present in the bovine adrenal medulla was characterized. Hybridization of RNA to highly specific m1-m5 muscarinic receptor cDNA probes detected the presence of only m4 subtype mRNA in this tissue. Muscarinic receptor binding studies using the non-selective ligand [3H]N-methyl-scopolamine showed a single class of binding sites with a maximum density of 19.

Differential and coordinate regulation of TH and PNMT mRNAs in chromaffin cell cultures by second messenger system activation and steroid treatment.

Primary cultures of chromaffin cells were prepared from bovine adrenal medullae and the levels of mRNA for tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) determined. The cells expressed moderate levels of TH mRNA and low levels of PNMT mRNA. The latter appeared to be more sensitive than TH mRNA to variations in the culture medium.

Identification of a functional glucocorticoid response element in the phenylethanolamine N-methyltransferase promoter using fusion genes introduced into chromaffin cells in primary culture.

The rat gene encoding phenylethanolamine N-methyltransferase (PNMT) was cloned and a consensus sequence for a glucocorticoid response element (GRE) was found at -513 bp, 5' to the transcriptional start site. In order to define the function of this element, fusion genes containing the PNMT promoter and a chloramphenicol acetyltransferase (CAT) reporter gene were constructed. These constructs did not express after transfection into any of 7 continuous cell lines, none of which endogenously produce PNMT.

Do glucocorticoids induce adrenergic differentiation in adrenal cells of neural crest origin?

The chromaffin cells of the adrenal medulla originate in the neural crest and migrate to populate the emerging adrenal gland. When differentiated, the adrenal medulla is formed by two populations of cells: the norepinephrine (NE) cells, which contain the first 3 enzymes of the catecholamine pathway, and the epinephrine (Epi) cells, which contain all 4 enzymes. It has been suggested that in rat, the last enzyme, phenylethanolamine-N-methyltransferase (PNMT), appears in NE cells that are exposed to very high levels of fetal glucocorticoids (GCs), such as those present in the adrenal gland.

A muscle-derived factor(s) induces expression of a catecholamine phenotype in neurons of cultured rat cerebral cortex.

We sought to determine the source of the signal(s) that promotes expression of the catecholamine (CA) enzyme tyrosine hydroxylase (TH) in cultured neurons of embryonic rat cerebral cortex, a tissue which is not thought to contain CA cells in vivo. Cortical neurons were cultured with their non-neuronal constituents and 48 hr later immunostained for TH. Fibroblasts or glia had no effects, however, blood vessels increased the numbers of TH neurons nearly 4-fold.

Strain-specific differences in transcription of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase.

Phenylethanolamine N-methyltransferase (PNMT), the enzyme which synthesizes the catecholamine epinephrine (adrenaline), may be regulated at many levels of expression. This study examines one level, the production of PNMT hnRNA, by measuring its rate of transcription in the rat adrenal gland and bovine adrenal medulla using an in vitro nuclear transcription run-on assay. Furthermore, when the transcriptional rate is compared in strains of rat known to possess distinctive levels of epinephrine and PNMT enzyme, the rate of PNMT transcription in Fischer rats is greater than in Buffalo or Sprague-Dawley rats: relative ratios are 0.

Complex transcriptional regulation by glucocorticoids and corticotropin-releasing hormone of proopiomelanocortin gene expression in rat pituitary cultures.

Proopiomelanocortin (POMC) peptide secretion from rat anterior pituitary corticotrophs and intermediate pituitary melanotrophs is stimulated by corticotropin-releasing hormone (CRH). CRH-stimulated secretion in the corticotrophs is inhibited by glucocorticoids in a complex fashion, involving both a fast, direct blockade of POMC secretion (minutes to hours) and a longer inhibitory action (hours to days) that decreases the amount of POMC peptide available for release. The current studies tested the ability of CRH to stimulate beta-endorphin (a peptide derived from POMC) secretion and POMC gene transcription in cultured anterior and neurointermediate lobe pituitary cells, and examined interactions between CRH and glucocorticoids in regulating POMC gene expression using an in vitro nuclear transcription run-on assay.

Strain-specific differences in levels of the mRNA for the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase.

The activity of the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.