Marian J. Evinger

Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice

Abstract. 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. MPC lines differ from PC12 cells in that they express both endogenous phenylethanolamine N-methyltransferase (PNMT) and full-length PNMT reporter constructs. PNMT expression is increased by dexamethasone and by cell-cell contact in suspension cultures. Mouse pheochromocytomas are a new tool for studying genes and signaling pathways that regulate cell growth and differentiation in adrenal medullary neoplasms and are a unique model for studying the regulation of PNMT expression.

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. Clonidine also failed to modify the basal and GTP gamma S- or carbachol-stimulated increase in phosphoinositide hydrolysis. However, clonidine increased significantly the production of cyclic GMP (cGMP) as well as the uptake of 45Ca2+. The cGMP response to clonidine was slower (peak at 15 min) and smaller (only about 50% over control) than the response to acetylcholine and was not shared by other agents that bind to imidazole receptors. In contrast, all agents that bind to imidazole receptors increased the influx of 45Ca2+ into chromaffin cells. It is concluded that (a) alpha 2-adrenergic and imidazole receptors are functionally distinct and linked to different signal transduction mechanisms; (b) the classical G-protein coupled soluble second messenger systems are not coupled to imidazole receptors; (c) clonidine may increase cGMP by a non-receptor-mediated intracellular action; and (d) imidazole receptors may regulate intracellular calcium levels through an ion regulating system that may be different from calcium channels.

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 β-hydroxylase (DBH), and phenylethanolamineN-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 2.5-fold elevation of PNMT mRNA. In contrast to depolarizing or cholinergic stimuli, PACAP does not enhance expression of 5′ upstream regions of the PNMT gene transiently transfected into chromaffin cells. Nor does PACAP stimulate the rate of PNMT gene transcription, thereby indicating that the effects of this neuropeptide do not involve enhanced transcription of this gene. However, after 16 h in the presence of transcriptional inhibitors, more PNMT mRNA is present in cultures treated with PACAP relative to control cultures, whereas amounts of TH and DBH mRNAs are not changed. PACAP likely elevates PNMT mRNA levels posttranscriptionally, possibly by stabilizing this message against degradation. Thus, although PACAP is an effective regulator for expression of all three catecholamine enzyme genes, its mechanism of action on PNMT mRNA appears to be distinctive from its effects on TH and DBH gene transcription.

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. The findings demonstrate that pheochromocytomas from adult neurofibromatosis knockout mice express multiple developmentally regulated genes involved in early development of both the CNS and peripheral nervous system. One of the most highly overexpressed genes is receptor tyrosine kinase Ret, which is known to be transiently expressed in the developing adrenal gland, down-regulated in adult adrenals and often overexpressed in human pheochromocytomas. Real-time polymerase chain reaction validated the microarray results and immunoblots confirmed the overexpression of Ret protein. Other highly expressed validated genes include Sox9, which is a neural crest determinant, and Hey 1, which helps to maintain the progenitor status of neural precursors. The findings are consistent with the recently proposed concept that persistent neural progenitors might give rise to pheochromocytomas in adult mouse adrenals and suggest that events predisposing to tumor development might occur before formation of the adrenal medulla or migration of cells from the neural crest. However, the competing possibility that developmentally regulated neural genes arise secondarily to neoplastic transformation cannot be ruled out. In either case, the unique profile of gene expression opens the mouse pheochromocytoma model to new applications pertinent to neural stem cells and suggests potential new targets for treatment of pheochromocytomas or eradication of their precursors.

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 phenylethanolamineN-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. The treatment of cultures with agents that activate signal transduction pathways, forskolin or phorbol esters, dramatically enhanced the expression of both mRNAs. The forskolin-induced increases in the steady-state levels of TH and PNMT mRNAs occurred rapidly and were apparent within 5 hours. These data suggest that the TH and PNMT genes can be regulated by second messengers. In contrast, dexamethasone treatment dramatically increased PNMT mRNA with no change in TH mRNA. The increase in PNMT mRNA was apparent within 6 hours of addition of the drug to the culture medium.

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 19.8 fmol/mg protein and a dissociation constant (KD) of 220 pM in the adrenal medulla, while the M1 selective ligand [3H]telenzepine did not bind detectably. Competition of specific antagonists with [3H]N-methyl-scopolamine for binding to the membranes produced a rank order of potencies with a profile that fitted either the cloned m3 or m4 receptor. In further comparative studies, the adrenal gland of the rat showed the presence of m4 subtype mRNA in addition to the m3 subtype.

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. In situ hybridization analyses also demonstrated the reduction of TH mRNA concentration in surviving dopaminergic neurons in the ipsilateral SN.

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. Sympathetic neurons were unaffected by a similar MDF treatment. Unlike the 2-day critical period for MDF-responsivity in non-CA neurons. CA neurons remained susceptible to MDFs influence over an extended developmental interval (E14-18), suggesting that MDF may be important for TH gene regulation in brain CA neurons even differentiation is complete. Because of these unique properties, MDF may provide a unique opportunity to explore ways in which the TH gene might be directly manipulated in these cell populations in order to correct the CA imbalances that occur in certain neurological diseases and disorders.

Regulation of Phenylethanolamine N-Methyltransferase Gene Expression by Imidazoline Receptors in Adrenal Chromaffin Cells

Abstract: As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of α2‐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. Clonidine also dose‐dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 μM. Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole‐4‐acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I1‐imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotines influence on PNMT mRNA production, diminish clonidines effects on PNMT mRNA. Although 100 μM clonidine diminishes nicotine‐stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.

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. If so, PNMT would appear during development after the initiation of fetal GC synthesis by the adrenal cortex at E18. In this study we examined the time of appearance and the relative level of PNMT mRNA and protein in rat embryos. We found (a) PNMT protein and mRNA are present at E16. Moreover, (b) the proportion of NE and Epi cells is already similar to that of adults and (c) the adult proportion of steady-state PNMT mRNA is also achieved prior to E18. We conclude that the appearance of PNMT is not affected by the surge of fetal GCs. Questions are raised as to the identity of the cues, genetic and/or epigenetic, which determine the differentiation of NE and Epi cells in the adrenal gland.