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.     

Effects of Cold Exposure on Rat Adrenal Tyrosine Hydroxylase: An Analysis of RNA, Protein, Enzyme Activity, and Cofactor Levels

Long-term cold exposure (5-7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3-6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48-72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold-induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre- and posttranslational regulation. Our findings also suggest that cold stress-induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.     

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.     

Vagotomy Affects Lipopolysaccharide-Induced Changes of Urocortin 2 Gene Expression in the Brain and on the Periphery

The corticotropin-releasing hormone family of peptides is involved in regulating the neuroendocrine stress response. Also, the vagus nerve plays an important role in the transmission of immune system-related signals to brain structures, thereby orchestrating the neuroendocrine stress response. Therefore, we investigated gene expression of urocortin 2 (Ucn2) and c-fos, a markers of neuronal activity, within the hypothalamic paraventricular nucleus (PVN), a brain structure involved in neuroendocrine and neuroimmune responses, as well as in the adrenal medulla and spleen in vagotomized rats exposed to immune challenge. In addition, markers of neuroendocrine stress response activity were investigated in the adrenal medulla, spleen, and plasma. Intraperitoneal administration of lipopolysaccharide (LPS) induced a significant increase of c-fos and Ucn2 gene expression in the PVN, and adrenal medulla as well as increases of plasma corticosterone levels. In addition, LPS administration induced a significant increase in the gene expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla. In the spleen, LPS administration increased gene expression of c-fos, while gene expression of TH and PNMT was significantly reduced, and gene expression of Ucn2 was not affected. Subdiaphragmatic vagotomy significantly attenuated the LPS-induced increases of gene expression of c-fos and Ucn2 in the PVN and Ucn2 in the adrenal medulla. Our data has shown that Ucn2 may be involved in regulation of the HPA axis in response to immune challenge. In addition, our findings indicate that the effect of immune challenge on gene expression of Ucn2 is mediated by vagal pathways.

     

Delayed increase of adrenal TH adn PNMT activities following cold stress in the mouse.

The activities of the adrenal enzymes tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) were found to be elevated when mice were subjected to 4°C ambient temperature. Only a single hr of cold exposure is required to achieve increased activity, provided that the measurements are made 12 hr after the cold exposure is initiated. After the cold stress is terminated, PNMT activity remain elevated for 12 hr. TH demonstrates a biphasic response to cold exposure, as the enzyme activity shows a second increase 12 hr after the stress has ended. The data indicates that short periods of stress result in demonstrated biochemical changes that persist long after the stress has ended.     

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.     

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)     

Differential Effects of Increasing Gestational Age and Placental Restriction on Tyrosine Hydroxylase, Phenylethanolamine N-Methyltransferase, and Proenkephalin A mRNA Levels in the Fetal Sheep Adrenal

Abstract: We have demonstrated that there are differential changes in the levels of tyrosine hydroxylase (TH), phenylethanolamine N -methyltransferase (PNMT), and proenkephalin A (Pro Enk A) mRNA in the fetal sheep adrenal during late gestation. Adrenal TH mRNA:18S rRNA ratios increased between gestational days 100 (0.98 ± 0.13; n = 6) and 125 (1.40 ± 0.15; n = 6) and then decreased, whereas adrenal PNMT mRNA:18S rRNA ratios increased regularly between gestational days 100 (0.08 ± 0.01) and 146 (0.17 ± 0.03). The ratio of adrenal Pro Enk A mRNA to 18S rRNA was higher at gestational day 125 (0.085 ± 0.005) than at either 80–100 days (0.038 ± 0.007) or 140–146 days of gestation (0.055 ± 0.013). In 12 ewes, the growth and development of the placenta were restricted (placental restriction group) from conception. The ratio of adrenal PNMT mRNA to 18S rRNA was significantly reduced in the placental restriction group of fetal sheep (0.003 ± 0.002) compared with controls (0.011 ± 0.002), and there was a significant correlation between the ratio of adrenal PNMT mRNA to 18S rRNA and the mean arterial P o ₂ ( r = 0.88, p < 0.0005). In contrast, TH mRNA and Pro Enk mRNA were unaffected by placental restriction. Adrenaline and nonadrenaline syntheses are therefore differentially regulated in the adrenal during late gestation and in response to chronic intrauterine hypoxemia.     

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.     

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.     

Semi-quantitative RT-PCR analysis of environmental influence on P450scc and PNMT mRNA expression in rat adrenal glands.

Environmental influence on brain function, particularly spatial learning and memory, has been extensively investigated, but little is known about the influence of environmental conditions on the functions of peripheral organs. In the present study, the effects of different housing conditions on the steady-state levels of mRNAs encoding cholesterol side-chain cleavage enzyme (cytochrome P450scc) and phenylethanolamine N-methyltransferase (PNMT) in adrenal glands was examined to investigate the environmental influence on both adrenocortical and adrenomedullary functions. Behavioral changes of the animals housed in different conditions were first examined to assess the relevance of environmental manipulation used. In consistent with previous findings, housing of the animals in enriched conditions resulted in the significant reduction of spontaneous motor activity (locomotor activity and rearing) in comparison with housing in isolated conditions, thus indicating the relevance of housing conditions used in this work for investigating the environmental influence on adrenal function. Then, the effects of these housing conditions on P450scc and PNMT mRNA levels in adrenal glands were examined using semi-quantitative RT-PCR method. In comparison with the isolated group, the enriched group showed significantly higher levels of P450scc mRNA. In contrast, PNMT mRNA levels in the enriched group were significantly lower than those in the isolated group. These results propose the possibility that the environmental conditions may cause differential alterations in adrenocortical and adrenomedullary functions, although their possible association with behavioral changes still remains to be elucidated.     

ACTH increases adrenal medullary PNMT activity in neonatal rats

Levels of plasma corticosterone and the activities of adrenomedullary dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) were measured in the 7-day-old rat following the administration of adrenocorticotropic hormone (ACTH) for 7 consecutive days beginning with day 1. ACTH led to significant adrenal hypertrophy and a concomitant elevation (10 to 15 fold) of plasma corticosterone concentration. Whereas DBH activity remained unchanged, adrenal PNMT activity was increased significantly following ACTH-induced elevation of plasma corticosterone levels. These results indicate that the pituitary-adreno-cortical-adrenomedullary axis is functional in the neonatal rat. Furthermore, since the transsynaptic control mechanisms are known to be non-functional or immature in the 7-day-old rat, our data suggest that neonatal rat adrenal catecholamine biosynthesis may be largely controlled by the pituitary-adrenocortical axis via glucocorticoids.     

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.     

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.