Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

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.     

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.     

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.     

Regulatory mechanism of the arginine vasopressin-enhanced green fluorescent protein fusion gene expression in acute and chronic stress

Various kinds of stress cause neuroendocrine responses such as corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP) release from parvocellular division of the paraventricular nucleus (PVN) and activation of the hypothalamo-pituitary adrenal (HPA) axis. We examined the effects of acute and chronic stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using chronic salt loading as an osmotic stimulation, intraperitoneal administration of lipopolysaccharide (LPS) as acute inflammatory stress and adjuvant arthritis (AA) as chronic inflammatory/nociceptive stress. Salt loading caused a marked increase in the eGFP gene expression and eGFP fluorescence in the supraoptic nucleus, magnocellular division of the PVN and internal layer of the median eminence (ME). Administration of LPS caused increased fluorescence in parvocellular division of the PVN and external layer of the ME. AA rats revealed an increased expression of the eGFP gene and eGFP fluorescence in both magnocellular and parvocellular divisions of the PVN and both internal and external layers of the ME. On the other hand, the levels of the CRH gene expression in parvocellular division of the PVN were significantly decreased as AA developed, though plasma concentrations of corticosterone were significantly increased. These results indicate that AVP-eGFP transgenic rats enable the detection of changes in AVP expression more easily than by using procedures such as immunohistochemistry. We propose that AVP-eGFP transgenic rats represent a useful animal model for further understanding of the physiology of AVP expression in the hypothalamo-pituitary system under various physiological conditions, including various kinds of stress.     

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.     

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.     

Effect of vasopressin on the induction of adrenal tyrosine hydroxylase and phenylethanolamine N-methyltransferase

We have assessed the effect of arginine vasopressin (AVP) on adrenal tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) activities. Both enzymes show marked increases after systemic administration of AVP in the range of 66 and 100 micrograms/day. To determine whether the pituitary gland plays a role in these inductions, the effect of AVP (66 micrograms per day, given divided into 3 doses for 4 days) on the adrenal enzymes was studied in hypophysectomized rats. These animals showed induction of TH but not PNMT. This indicates that a pituitary factor(s) mediates the increase in PNMT caused by AVP. Adrenal TH activity was measured after the injection of AVP (1 or 2 micrograms per rat) into the lateral ventricle: there was a statistically significant increase in TH. TH was not induced in the denervated adrenal gland of rats administered AVP systemically. These findings suggest that AVP may act centrally to induce the enzyme. The continuous s.c. infusion of AVP by osmotic minipump at the rate of 1 microgram/day for 6 days led to a striking increase in adrenal TH activity. However, PNMT did not increase significantly. It can be concluded that different mechanisms are involved in the induction of adrenal TH and PNMT caused by AVP. A neural mechanism is involved in TH induction, whereas PNMT induction requires release of a pituitary factor, presumably ACTH, but innervation of the adrenal is not needed for it. Moreover, the inductions of these two enzymes are differentially sensitive to the concentration of circulating AVP.     

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.     

Hypoxic stress‐induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O₂) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl₂ or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal ?893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (?282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.     

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.     

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.     

Epinephrine: a short- and long-term regulator of stress and development of illness : a potential new role for epinephrine in stress

Epinephrine (Epi), which initiates short-term responses to cope with stress, is, in part, stress-regulated via genetic control of its biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT). In rats, immobilization (IMMO) stress activates the PNMT gene in the adrenal medulla via Egr-1 and Sp1 induction. Yet, elevated Epi induced by acute and chronic stress is associated with stress induced, chronic illnesses of cardiovascular, immune, cancerous, and behavioral etiologies. Major sources of Epi include the adrenal medulla and brainstem. Although catecholamines do not cross the blood-brain barrier, circulating Epi from the adrenal medulla may communicate with the central nervous system and stress circuitry by activating vagal nerve β-adrenergic receptors to release norepinephrine, which could then stimulate release of the same from the nucleus tractus solitarius and locus coeruleus. In turn, the basal lateral amygdala (BLA) may activate to stimulate afferents to the hypothalamus, neocortex, hippocampus, caudate nucleus, and other brain regions sequentially. Recently, we have shown that repeated IMMO or force swim stress may evoke stress resiliency, as suggested by changes in expression and extinction of fear memory in the fear-potentiated startle paradigm. However, concomitant adrenergic changes seem stressor dependent. Present studies aim to identify stressful conditions that elicit stress resiliency versus stress sensitivity, with the goal of developing a model to investigate the potential role of Epi in stress-associated illness. If chronic Epi over expression does elicit illness, possibilities for alternative therapeutics exist through regulating stress-induced Epi expression, adrenergic receptor function and/or corticosteroid effects on Epi, adrenergic receptors and the stress axis.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.