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.     

Gene expression of the phenylethanolamine N-methyltransferase is differently modulated in cardiac atria and ventricles

Phenylethanolamine N-methyltransferase (PNMT) is a final enzyme in catecholamine synthesizing cascade that converts noradrenaline to adrenaline. Although most profuse in adrenal medulla, PNMT is expressed also in the heart, particularly in cardiac atria and ventricles. In atria, the PNMT mRNA is much more abundant compared to ventricles. In present study we aimed to find out whether there is a difference in modulation of the PNMT gene expression in cardiac atria and ventricles. We used three methodological approaches: cold as a model of mild stress, hypoxia as a model of cardiac ischemic injury, and transgenic rats (TGR) with incorporated mouse renin gene (mREN-2)27, to determine involvement of renin-angiotensin pathway in the PNMT gene expression. We have found that PNMT gene expression was modulated differently in cardiac atria and ventricles. In atria, PNMT mRNA levels were increased by hypoxia, while cold stress decreased PNMT mRNA levels. In ventricles, no significant changes were observed by cold or hypoxia. On the other hand, angiotensin II elevated PNMT gene expression in ventricles, but not in atria. These results suggest that PNMT gene expression is modulated differently in cardiac atria and ventricles and might result in different physiological consequences.     

Induction of Gene Expression of the Catecholamine-Synthesizing Enzymes by Insulin-Like Growth Factor-I

Abstract: The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N -methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 ± 0.4-fold after 48 h, DBH by 5.1 ± 0.3-fold in 24 h, and PNMT by 2.8 ± 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.     

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.     

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR.In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization.This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.     

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.     

Parallel Up-Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time-dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and dopamine γ-hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μ M ) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0-, 2.4-, and 7.0-fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6-, 1.8-, and 8.0-fold increases, respectively. Low, but detectable, phenylethanolamine N -methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6-fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up-regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.     

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.     

Long-term hypoxia modulates expression of key genes regulating adrenomedullary function in the late gestation ovine fetus

We previously communicated that long-term hypoxia (LTH) resulted in a selective reduction in plasma epinephrine following acute stress in fetal sheep. The present study tested the hypothesis that LTH selectively reduces adrenomedullary expression of phenylethanolamine-N-methyltransferase (PNMT), the rate-limiting enzyme for epinephrine synthesis. We also examined the effect of LTH on adrenomedullary nicotinic, muscarinic, and glucocorticoid receptor (GR) expression. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dGA); adrenomedullary tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dGA. Contrary to our hypothesis, in addition to PNMT, adrenomedullary expression (mRNA, protein) of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) were reduced in the LTH fetus. Immunocytochemistry indicated that TH and DBH expression was lower throughout the medulla, while PNMT appeared to reflect a reduction in PNMT-expressing cells. Nicotinic receptor alpha 1, 2, 3, 5, 6, 7, beta 1, 2, and 4 subunits were expressed in the medulla of LTH and control fetuses. Messenger RNA for alpha 1 and 7 and beta 1 and 2 subunits was lower in LTH fetuses. Muscarinic receptors M1, M2, and M3 as well as the GR were also expressed, and no differences were noted between groups. In summary, LTH in fetal sheep has a profound effect on expression of key enzymes mediating adrenomedullary catecholamine synthesis. Further, LTH impacts nicotinic receptor subunit expression potentially altering cholinergic neurotransmission within the medulla. These findings have important implications regarding fetal cardiovascular and metabolic responses to stress in the LTH fetus.     

Altered Function of Peripheral Organ Systems in Rats Exposed to Chronic Mild Stress Model of Depression

1. In depression, psychiatric symptoms are frequently associated with impaired cardiovascular function and perhaps also increased risk for cancer diseases. Pathophysiological basis of this comorbidity is not clearly understood. Molecular events involved, particularly factors modified by chronic stress exposure, may only be evaluated in animal models of depression.2. Present experiments were aimed to study parameters related to cardiovascular system (tyrosine hydroxylase (TH) gene expression in adrenal glands) and carcinogenesis (retinoic acid receptors in the liver) in the chronic mild stress model of depression.3. Chronic mild stress induced a rise in adrenal TH gene expression in both male and female rats. Gender dependent changes were found in retinoic acid receptor binding with stress-induced activation in females but not males. Ovariectomized animals exhibited higher retinoic acid receptor binding, slightly elevated TH mRNA levels and failed to respond to chronic mild stress exposure with further increase in TH mRNA levels. Similarly, chronic mild stress induced an anhedonic state manifested by decreased sucrose preference in control but not ovariectomized rats.4. Presented data document that central neurochemical and behavioral changes in animals exposed to chronic mild stress model of depression are associated with changes in adrenal TH gene expression and with gender dependent changes in retinoic acid receptor status in the liver. Such alterations may participate in the development of pathological changes and could participate on increased risk for cardiovascular and oncologic comorbidity in depressive patients.     

Regulation of catecholamine-synthesising enzymes and beta-adrenoceptors gene expression in ventricles of stressed rats

Stress exposure activates the sympathoneural system, resulting in catecholamine release. Chronic stress is associated with development of numerous disorders, including cardiovascular diseases. Here we investigated the expression of mRNAs for catecholamine biosynthetic enzymes tyrosine-hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine N-methyl-transferase, and for beta(1)- and beta(2)-adrenoceptors in the right and left ventricles of rats exposed to chronic unpredictable mild stress. The tyrosine-hydroxylase and dopamine-beta-hydroxylase mRNA levels were not affected by stress, whereas the phenylethanolamine N-methyltransferase mRNA levels significantly increased in both right and left ventricles. No changes in beta(1)-adrenoceptor mRNA levels in either right or left ventricles were observed. At the same time, stress produced a significant increase of beta(2)-adrenoceptor mRNA levels in left ventricles. These results suggest that elevated expression of phenylethanolamine N-methyltransferase in both ventricules and beta(2)-adrenoceptor genes in left ventricles could provide a molecular mechanism that leads to altered physiological response, which is important for the organism coping with stress.     

Cold-induced increases in phenylethanolamine N-methyltransferase (PNMT) mRNA are mediated by non-cholinergic mechanisms in the rat adrenal gland

Previously, we reported that cold stress induces a rapid increase in adrenomedullary PNMT mRNA levels, followed by concomitant increases in PNMT immunoreactivity (10). In the present study, the extracellular signals mediating this adaptive response to stress were investigated using northern analysis and RNA slot-blot hybridization. Although adrenal denervation significantly diminished cold-induced increments in adrenomedullary PNMT mRNA levels, it did not completely abolish the cold stress response. In contrast to these results, splanchnectomy completely inhibited cold-induced increments in TH mRNAs in the same tissue samples. These findings indicate that the effects of cold exposure on PNMT mRNA levels are mediated by both neural and non-neural mechanisms, and that adrenal PNMT and TH are differentially regulated in response to cold stress. Surprisingly, the neural component of the PNMT stress response could not be attenuated by peripheral administration of chlorisondamine, a powerful nicotinic ganglionic blocking agent. In contrast, chlorisondamine was effective in inhibiting sympathetic neural activity, as judged by the drug's ability to completely block increases in blood pressure, heart rate, and plasma catecholamines resulting from spinal cord stimulation in pithed rats. The administration of atropine, a muscarinic receptor antagonist, also failed to inhibit cold-induced alterations in adrenal PNMT mRNA. These results suggest that the trans-synaptic induction of adrenal PNMT mRNA involves a non-cholinergic component, and that cold-induced increases in PNMT mRNA are not coupled to acetylcholine-mediated adrenal catecholamine release.     

Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Differentiation of adrenomedullary catecholamine synthesizing enzyme responses to repeated immobilization in hybrid rats

The response of adrenomedullary catecholamine synthesizing enzymes to repeated immobilization was studied in hybrid (F₁) offspring of 2 inbred rat strains (LEW and F344). Immobilization-induced increases in tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyl-transferase (PNMT) activities in one of the parental strains (F344) previously were shown to be dependent upon intact adrenal gland innervation but independent of the pituitary gland, while responses in the other parental strain (LEW) were independent of adrenal innervation but dependent upon pituitary function. Factors determining immobilization-induced increases in adrenal enzymes of F₁ offspring were enzyme-specific. Increased PNMT activity was pituitary dependent in F₁ rats, whereas increased TH and DBH activities after immobilization were dependent upon an intact adrenal gland innervation. These results suggest that the factor(s) regulating PNMT responses are differentiable from those regulating TH and DBH responses. The results also indicate that analysis of PNMT responses to immobilization in backcross populations is feasible, and could indicate whether strain-specific response mechanisms are heritable.     

Induction of Adrenal Tyrosine Hydroxylase mRNA by Single Immobilization Stress Occurs Even After Splanchnic Transection and in the Presence of Cholinergic Antagonists

Abstract: Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.