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.     

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.     

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.     

Adrenomedullin receptor is found exclusively in noradrenaline-secreting cells of the rat adrenal medulla

Adrenomedullin, originally identified in the adrenal medulla, has binding sites in the adrenal gland; however, its role in the adrenal medulla is unclear. This study was designed to characterise adrenomedullin binding sites in the rat adrenal medulla, using ligand binding studies, immunocytochemistry, and mRNA analysis. A single population of specific adrenomedullin receptors was identified in adrenal medullary homogenates. 125I-Adrenomedullin was displaced only by adrenomedullin1-50 and not by calcitonin gene-related peptide or amylin at concentrations up to 100 nmol/L. The receptor K(D) was 3.64 nmol/L with a receptor density of 570 fmol/mg of protein. Analysis of mRNA revealed that the genes encoding both the putative adrenomedullin receptors, termed calcitonin receptor-like receptor (CRLR) and L1, were expressed in the rat adrenal medulla. Dual-colour indirect-labelled immunofluorescence was used to localise phenylethanolamine N-methyltransferase (PNMT) and the adrenomedullin receptor in the same section. PNMT is the enzyme that converts noradrenaline to adrenaline and is not expressed in noradrenaline-secreting cells. These studies revealed that both CRLR and L1 were expressed only in cells that did not express PNMT, suggesting that adrenomedullin receptors are only found in noradrenaline-secreting cells. Further evidence to support this conclusion was provided by the demonstration of colocalisation of adrenomedullin receptors with dopamine beta-hydroxylase, confirming the presence of the receptors in medullary chromaffin cells. Taken together, these data suggest that adrenomedullin acts through a specific adrenomedullin receptor in the rat adrenal medulla. RT-PCR and northern blot analysis revealed greater abundance of mRNA for L1 than for CRLR, possibly suggesting that L1 may be the major adrenomedullin receptor expressed in this tissue. As it has been reported that adrenomedullin is synthesised predominantly by adrenaline-secreting cells, it appears likely that adrenomedullin is a paracrine regulator in the adrenal medulla.     

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.     

A sensitive radioenzymatic assay for epinephrine forming enzymes

Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces 3H-E from norepinephrine (NE) and 3H-S-adenosylmethionine. The 3H-E is isolated on alumina, 3H-S-adenosylmethionine is precipitated and the 3H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.     

Expression and development of phenylethanolamine N-methyltransferase (PNMT) in rat brain stem: studies with glucocorticoids

To study the differentiation of adrenergic (epinephrine-synthesizing) neurons in brain, the initial appearance and ontogeny of phenylethanolamine N-methyltransferase (PNMT), a specific marker of the adrenergic phenotype, were studied with immunocytochemistry and catalytic assay. The appearance of immunoreactivity to dopamine beta-hydroxylase (DBH-IR), an enzyme common to the noradrenergic and adrenergic phenotypes, was also studied. DBH-IR was initially observed on embryonic Day 13 (E13) in cells located on the ventrolateral floor and wall of the rhombencephalon. A day later (E14), PNMT-IR cells and PNMT catalytic activity were observed in the rhombencephalon suggesting that, as in the adrenal gland, noradrenergic expression precedes adrenergic expression. The PNMT-IR cells were presumed to be precursors of C1 neurons since they were located in the ventrolateral medulla oblongata. Cells located in the wall of the medulla which appeared to be migrating ventrally to the C1 group also contained PNMT-IR. On E15, cells which had PNMT-IR processes coursing through the germinal zone were observed dorsally near the fourth ventricle. Although the location of the C1 cell group was apparent when PNMT was initially expressed, the dorsal C2 and C3 adrenergic cell groups were not evident until late in gestation on E19. Even in the term embryo there appeared to be PNMT-IR cells which had not yet reached their final destination. On E14 and E15, PNMT-IR cells were also observed on the floor of the pons just rostral to the pontine flexure. However, these were not observed in older embryos, suggesting that transient expression of PNMT occurs in brain, as well as in the periphery. To determine whether glucocorticoids regulate brain PNMT, we examined the effects of altered glucocorticoid levels. In contrast to PNMT in the sympathetic nervous system, PNMT activity in medulla oblongata was not affected in neonates or adults by the decrease in glucocorticoids following adrenalectomy or hypophysectomy. Conversely, elevation of glucocorticoids by hormonal treatment did not alter PNMT in neonates. Notably, however, treatment of pregnant rats with dexamethasone on E18-E21, but not earlier, increased PNMT activity in the fetal brain stem. These observations suggest that PNMT expression and development is regulated by different factors in cells derived from neural crest and tube. PNMT is expressed earlier in brain than in adrenal and sympathetic ganglia. Further, the development of PNMT in the periphery, but not in the brain, is dependent on maintenance of physiological levels of glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

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.

     

Expression of phenylethanolamine N-methyltransferase in rat sympathetic ganglia and extra-adrenal chromaffin tissue

To determine whether similar mechanisms regulate adrenergic phenotypic expression in different cellular populations, the superior cervical sympathetic ganglion (SCG) and extra-adrenal chromaffin tissue were studied in the fetal and neonatal rat; results were compared to those previously obtained with the adrenal medulla. Phenylethanolamine N-methyltransferase (PNMT), the enzyme which converts norepinephrine to epinephrine, was used as an index of adrenergic expression. PNMT catalytic activity was initially detectable in the SCG of normal, untreated fetuses at 17.0 days of gestation (E17.0), and increased three- to fourfold until postnatal day 2. Thereafter activity decreased precipitously, and was undetectable 2 weeks after birth. Immunohistochemical studies, using specific antisera to PNMT, were employed to localize the enzyme. Immunoreactivity (PNMT-IR) was undetectable in sympathetic ganglia of control animals, suggesting that this method is less sensitive than the catalytic assay. Following glucocorticoid treatment, cells heavily stained for PNMT-IR were observed in paravertebral sympathetic ganglia, including the SCG, and in the organ of Zuckerkandl. In the SCG, PNMT-IR was present in small cells presumed to be small, intensely fluorescent (SIF) cells and was never observed in principal ganglion neurons. The increase in PNMT-IR after steroid treatment was strikingly age dependent: initiation of treatment at progressively older ages during the first week of life resulted in fewer and fewer PNMT-IR cells. No response was apparent after 1 week. Moreover, treatment of pregnant rats was associated with appearance of PNMT-IR at E18.5, but not at E16.5. After treatment from days 0 to 6 of life, PNMT-IR gradually disappeared. However, retreatment on days 24–30 caused the reappearance of PNMT-IR, suggesting that exposure to steroids at birth causes (a) an immediate increase in PNMT-IR and (b) responsiveness to steroids during adulthood. Consequently, the disappearance of PNMT-IR after exposure to steroids at birth, is not simply due to death of SIF cells. We conclude that proximity to the adrenal cortex is not necessary for initial expression of PNMT. More generally, the expression of PNMT by ganglion SIF cells parallels that in adrenal chromaffin cells since initial expression was not dependent on high local concentrations of glucocorticoids, whereas subsequent development did require high levels of the hormones. Our observations suggest that similar mechanisms regulate expression and development of the adrenergic phenotype in adrenal and sympathetic ganglia.     

Partial expression of catecholaminergic traits in cholinergic chick ciliary ganglia: Studies in vivo and in vitro

We have previously demonstrated that at embryonic Day (E) 8, some cells of the chick ciliary ganglion (CG) contain the catecholaminergic (CA) enzyme tyrosine hydroxylase (TH), but not phenylethanolamine-N-methyltransferase (PNMT); and that in culture essentially all cells express both enzymes. In the present study, we sought to determine, first, whether the expression of adrenergic traits in the CG in vivo is transient or permanent in the CG. To do so, CGs were removed from E5 to postnatal Day 5, fixed, and processed for the immunocytochemical localization of the CA enzymes: TH, L-amino acid decarboxylase (AADC), and PNMT. At all stages examined, some CG neurons expressed TH immunoreactivity (TH-IR) and all contained AADC-IR. However, none stained with PNMT antibodies, indicating that these cells stably express some, but not all, of the CA enzymes. Second, we examined whether CG neurons in culture expressed other CA markers. CG neurons did not contain detectable levels of TH enzyme activity nor did they transport and store exogenously supplied monoamines. These results indicate that some but not all traits necessary for adrenergic function are present in CG neurons in vitro. Third, we sought to establish whether CA expression in CG neurons is affected by modification in culture conditions. Cultures of CG neurons continued to express TH-IR even when grown in the presence of either 50% HCM or 20 mM KCl for 5 days. Finally, the expression of the cholinergic enzyme, choline acetyltransferase (CAT) was assessed in CG cultures by biochemical assay. CAT activity increased five-fold between 5 and 17 days in vitro, irrespective of the presence of TH-IR in 100% of the CG neurons of sister cultures. These data suggest that at least a subpopulation of CG neurons express both TH and CAT in culture. We conclude that the postmitotic neurons of the CG are able to express some but not all of the traits characteristic of a CA phenotype while maintaining cholinergic expression. These findings suggest that (1) the appearance of the full complement of adrenergic properties is not coordinated and may be regulated by different environmental cues and (2) parasympathetic neurons can express both adrenergic and cholinergic traits simultaneously.     

Development of adrenal chromaffin cells is largely normal in mice lacking the receptor tyrosine kinase c-Ret

c-Ret encodes a receptor tyrosine kinase that is essential for normal development of the kidney as well as enteric and sympathetic neurons. Since sympathetic neurons and neuroendocrine chromaffin cells originate from a common progenitor cell, we have examined the relevance of c-Ret for the development of adrenal chromaffin cells by analyzing mouse mutants lacking c-Ret. Adrenal chromaffin cells express c-Ret mRNA at embryonic day (E) 12.5 and 13.5, yet levels of expression decline at later embryonic and postnatal ages. Adrenal medullae of c-Ret deficient mice show normal numbers of tyrosine hydroxylase (TH)-immunoreactive cells at E13.5 and at birth. Ultrastructurally, adrenal chromaffin cells of c-Ret(-/-) mice appear unaltered: chromaffin cells develop typical secretory chromaffin granules, the morphological hallmark of chromaffin cells, and synaptic terminals appear normal. However, adrenaline levels and numbers of chromaffin cells immunoreactive for the adrenaline synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) are reduced by about 30% in c-Ret-deficient mice arguing for a direct or indirect role of c-Ret in the regulation of PNMT. Thus, despite expression of c-Ret, adrenal chromaffin cells develop largely normal in mice lacking c-Ret. We therefore conclude that sympathetic neurons and neuroendocrine chromaffin cells profoundly differ in their requirement for c-Ret signaling during development.     

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)     

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.     

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.     

Inheritance of Adrenal Phenylethanolamine N-Methyltransferase Activity in the Rat

Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that catalyzes the S-adenosyl-L-methionine-dependent methylation of (-)norepinephrine to (-)epinephrine in the adrenal medulla. Adrenal PNMT activity is markedly different in two highly inbred rat strains; enzyme activity in the F344 strain is more than fivefold greater than that in the Buf strain. Initial characterization of the enzyme in the two inbred strains reveals evidence for catalytic and structural differences, as reflected in dissimilar Km values for the cosubstrate (S-adenosyl-L-methionine) and prominent differences in thermal inactivation curves. To assess adrenal PNMT activity in an F344 X Buf pedigree, we employed a statistical procedure to test for one- and two-locus hypotheses in the presence of within-class correlations due to cage or litter effects. The PNMT data in the pedigree are best accounted for by segregation at a simple major locus superimposed upon a polygenic background; data obtained from the biochemical studies suggest that the major locus is a structural gene locus.