Identification of phenylethanolamine N-methyltransferase gene expression in stellate ganglia and its modulation by stress

Phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is the terminal enzyme of the catecholaminergic pathway converting noradrenaline to adrenaline. Although preferentially localized in adrenal medulla, evidence exists that PNMT activity and gene expression are also present in the rat heart, kidney, spleen, lung, skeletal muscle, thymus, retina and different parts of the brain. However, data concerning PNMT gene expression in sympathetic ganglia are still missing. In this study, our effort was focused on identification of PNMT mRNA and/or protein in stellate ganglia and, if present, testing the effect of stress on PNMT mRNA and protein levels in this type of ganglia. We identified both PNMT mRNA and protein in stellate ganglia of rats and mice, although in much smaller amounts compared with adrenal medulla. PNMT gene expression and protein levels were also increased after repeated stress exposure in stellate ganglia of rats and wild-type mice. Similarly to adrenal medulla, the immobilization-induced increase was probably regulated by glucocorticoids, as determined indirectly using corticotropin-releasing hormone knockout mice, where immobilization-induced increase of PNMT mRNA was suppressed. Thus, glucocorticoids might play an important role in regulation of PNMT gene expression in stellate ganglia under stress conditions.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase (PNMT) in organ culture of superior cervical ganglia

Glucocorticoid regulation of the adrenergic enzyme, phenylethanolamine N-methyltransferase (PNMT) was studied in organ cultures of the superior cervical ganglion (SCG) from newborn rats. Although PNMT catalytic activity was present in control ganglia, enzyme levels were too low to allow visualization of PNMT immunofluorescent cells. Addition of dexamethasone (DEX) or corticosterone to the medium resulted in a large increase in PNMT activity and bright PNMT immunoreactive (PNMT-IR) staining in cells resembling small, intensely fluorescent (SIF) cells. Addition of non-glucocorticoid steroids was ineffective. Exposure to a brief, 2-hr pulse of DEX (10(-6) M) in vitro elicited the same increase in PNMT as continual exposure to DEX. Studies using metabolic inhibitors demonstrated that the steroid-dependent increase in PNMT activity required both protein and RNA synthesis. Furthermore, the increase was inhibited by cytochalasin B and by the glucocorticoid receptor antagonists, DEX 21-mesylate and cortisol 21-mesylate. These observations suggest that glucocorticoids increase PNMT protein in SIF cells by interacting with specific steroid receptors that undergo translocation to the nucleus.     

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 μ M . 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 nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 μ M 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.     

Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.     

Tissue-specific regulation of glucocorticoid receptor mRNA by dexamethasone

The effect of glucocorticoids on tissue-specific regulation of glucocorticoid receptor mRNA was studied in intact and adrenalectomized rats. Glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot blot hybridization using a glucocorticoid cRNA probe. Glucocorticoid receptor mRNA was greatest in the lung with the relative levels in other tissues as follows: spleen, 70%; brain, 55%; liver, 50%; kidney, 43%; heart, 35%; adrenal, 13%; and testis only 8%. A tissue-specific difference in glucocorticoid receptor mRNA accumulation was found after adrenalectomy. There was little change in glucocorticoid receptor mRNA levels in liver and lung, but the brain and kidney demonstrated a 40 and 80% increase in mRNA, respectively. In contrast, dexamethasone treatment resulted in a consistent decrease of 40-60% in the accumulation of glucocorticoid receptor mRNA in all tissues studied. These results provide in vivo evidence for the autoregulation of the glucocorticoid receptor by its homologous ligand and demonstrate the existence of tissue-specific regulation of the glucocorticoid receptor mRNA levels in states of glucocorticoid excess and depletion.     

Relationship between apolipoprotein E mRNA expression and tissue cholesterol content in rat adrenal gland.

Among extrahepatic tissues the adrenal gland has one of the highest concentrations of apoE mRNA and the highest rate of apoE synthesis. In the present investigation several previously described in vivo treatments were used to assess the relationship between apoE expression and cellular cholesterol in the rat adrenal gland. Treatment of rats with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) to lower serum cholesterol concentration and deplete adrenal gland cholesterol content decreased adrenal gland apoE mRNA concentration. These adrenal responses were blocked by dexamethasone (DEX) suggesting that the effect of 4-APP occurred indirectly via stimulation of the adrenal gland by endogenous adrenocorticotrophic (ACTH). Relative to control rats, DEX treatment increased both adrenal gland cholesterol content and apoE mRNA concentration. Concurrent ACTH and DEX administration reduced both adrenal gland cholesterol content and apoE mRNA concentration relative to DEX-treated rats. ACTH administration also rapidly decreased adrenal gland apoE mRNA concentration and cholesterol content in rats pretreated with DEX. In all the above experiments, adrenal gland cholesterol content and apoE mRNA concentration were positively correlated (r = 0.78, P = 0.0001). In contrast, aminoglutethimide treatment, which blocks adrenal gland steroidogenesis and greatly increases adrenal gland cholesterol content, was without effect on apoE mRNA concentration. ACTH administration to rats treated with DEX + aminoglutethimide resulted in decreased adrenal apoE mRNA despite greatly increased adrenal cholesterol content. This uncoupling of adrenal gland cholesterol content and apoE mRNA concentration suggests that apoE mRNA expression and cellular cholesterol are regulated independently by ACTH.     

Dexamethasone treatment increases neuropeptide Y levels in rat hypothalamic neurones

Immunoreactive glucocorticoid receptors (GR) have previously been demonstrated in neuropeptide Y (NPY) neurones of the rat hypothalamus. To determine whether NPY synthesis is influenced by glucocorticoids, the effect of dexamethasone (DEX) on the levels of immunoreactive NPY in rat hypothalamic neurones was investigated in vivo and in vitro. Daily injections of DEX (0.1 mg/day) for 5 days increased the NPY content of the mediobasal hypothalamus in female rats by 117% (p less than 0.002). Primary cultures of hypothalamic neurones were also sensitive to the effect of glucocorticoids. Intracellular NPY levels were significantly increased (p less than 0.001) compared to control values by 151%, 222% and 268% when cultures were maintained in a defined serum free medium containing DEX 10(-9), 10(-8) and 10(-7) M respectively.     

Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites

The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.     

Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone

The regulation of angiotensinogen gene expression in response to adrenalectomy and dexamethasone treatment was examined in multiple rat tissues. Angiotensinogen mRNA as quantitated by slot blot hybridization utilizing an angiotensinogen cRNA probe was most abundant in the liver with levels in the brain, kidney, and adrenal of 50, 25, and 10%, respectively. No angiotensinogen mRNA was detected in testes or heart. Although no change in the quantity of angiotensinogen mRNA was found following adrenalectomy and maintenance on 0.9% saline, dexamethasone treatment of both normal and adrenalectomized rats resulted in a time-dependent and tissue-specific accumulation of angiotensinogen mRNA. In normal animals, the hepatic response to treatment was a 4.5-fold increase in angiotensinogen mRNA by 8 h which remained 2.4-fold above basal levels by 24 h. Angiotensinogen mRNA levels in the brains of normal rats treated with dexamethasone increased only 60% by 6 h and returned to basal levels by 24 h. In contrast to the increases seen in brain and liver, angiotensinogen mRNA derived from kidney did not significantly change following dexamethasone treatment. In adrenalectomized animals, the hepatic response to dexamethasone was similar to normal animals with a 3.7-fold increase by 6 h. The accumulation in brain was greater in these animals compared to normals and increased 3-fold by 8 h. Finally, dexamethasone did not significantly increase levels in the kidney. These results clearly demonstrate glucocorticoid regulation of angiotensinogen mRNA levels in liver and brain. In contrast, the kidney, an organ known to contain glucocorticoid receptors, does not respond with increased angiotensinogen mRNA levels following glucocorticoid stimulation. These studies provide the first evidence for tissue-specific differences in the control of angiotensinogen mRNA.     

Existence of cardiac PNMT mRNA in adult rats: elevation by stress in a glucocorticoid-dependent manner

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that synthesizes epinephrine from norepinephrine. The aim of this study was to determine potential PNMT gene expression in the cardiac atria and ventricles of adult rats and to examine whether the gene expression of this enzyme is affected by immobilization stress. PNMT mRNA levels were detected in all four parts of the heart, with the highest level in the left atrium. Both Southern blot and sequencing verified the specificity of PNMT detected by RT-PCR. Single immobilization for 2 h increased gene expression of PNMT in both atria and ventricles. In atria, this effect was clearly modulated by glucocorticoids, because either adrenalectomy or hypophysectomy prevented the increase in PNMT mRNA levels in response to immobilization stimulus. This study establishes, for the first time, that PNMT gene expression occurs in cardiac atria and also, to a small extent, in ventricles of adult rats. Immobilization stress increases gene expression in atria and ventricles. This increase requires an intact hypothalamus-pituitary-adrenocortical axis, indicating the involvement of glucocorticoids.     

Role of glucocorticoids in expression of the adrenergic phenotype in rat embryonic adrenal gland

Differentiation of the noradrenergic and adrenergic phenotypes was documented in rat embryonic adrenal chromaffin cells in vivo from 12.5 days of gestation (E12.5) to term. The initial appearance of three enzymes in the catecholaminergic pathway, tyrosine hydroxylase (T-OH), dopamine-β-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) as well as endogenous catecholamines (CA), was followed by immunohistochemistry and histofluorescence. T-OH and DBH, were employed as indices of noradrenergic expression, whereas PNMT, the epinephrine-synthesizing enzyme, was used as an index of adrenergic expression. At E12.5, T-OH, DBH, and CA were present in cells of the sympathetic ganglia at the level of the adrenal anlage. By 13.5 days, cells containing T-OH, DBH, and CA, were observed between the sympathetic ganglia and developing adrenal, and within the adrenal itself. While T-OH, DBH, and CA were present in adrenal medullary cells from the earliest stages of adrenal development, PNMT, in contrast, was undetectable in ganglion primordia, migrating cells, or within the adrenal before 17 days. PNMT initially appeared at E17 in small clusters of cells scattered throughout the adrenal. The number of cells containing PNMT and the intensity of staining increased dramatically from E17 to term.A number of experimental manipulations were employed in vivo to investigate the role of glucocorticoids in differentiation of the adrenergic phenotype. Chronic or acute treatment of mothers and/or embryos with various glucocorticoids, adrenocorticotrophic hormone (ACTH), or S-adenosylmethionine (SAM) did not result in precocious appearance of PNMT. Moreover, the initial expression of PNMT was not prevented or delayed by embryonic hypophysectomy or by treatment with inhibitors of adrenocortical function. Consequently, the initial expression of PNMT on E17.0 is not dependent on normal glucocorticoid levels, cannot be induced prematurely by glucocorticoids, and is independent of the pituitary-adrenal axis. However, the ontogenetic increase in PNMT levels after initial expression has occurred does require intact pituitary-adrenal function. Our observations suggest that different mechanisms regulate initial expression and subsequent modulation of neurotransmitter phenotype.     

Effects of Dexamethasone In Vivo and In Vitro on Hexose Transport in Brain Microvasculature

Glucocorticoids induce hyperinsulinemia, hyperglycemia, and depress glucose transport by aortic endothelium. High glucocorticoid doses are used for many diseases, but with unknown effects on brain glucose transport or metabolism. This study tested the hypothesis that glucocorticoids affect glucose transport or metabolism by brain microvascular endothelium. Male rats received dexamethasone (DEX) sc with sucrose feeding for up to seven days. Cerebral microvessels from rats treated with DEX/sucrose demonstrated increased GLUT1 and brain glucose extraction compared to controls. Glucose transport in vivo correlated with hyperinsulinemia. Pre-treatment with low doses of strep-tozotocin blunted hyperinsulinemia and prevented increased glucose extraction induced by DEX. In contrast, isolated brain microvessels exposed to DEX in vitro demonstrated suppression of 2-deox-yglucose uptake and glucose oxidation. We conclude that DEX/sucrose treatment in vivo increases blood-brain glucose transport in a manner that requires the effects of chronic hyperinsulinemia. These effects override any direct inhibitory effects of either hyperglycemia or DEX.