Regulation of phenylethanolamine N-methyltransferase (PNMT) mRNA in the rat adrenal medulla by corticosterone

In the adrenal medulla of adult rat, physiological levels of glucocorticoid hormones are required to maintain the catalytic activity of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). The present study was undertaken to determine whether glucocorticoid regulation of PNMT occurs at the level of mRNA coding for PNMT. Adult male Sprague-Dawley rats were hypophysectomized (HPX) and killed after 2 weeks; pellets of corticosterone were implanted for 1, 3 or 7 days prior to killing. Determinations were made of plasma corticosterone levels, adrenal PNMT activity and PNMT mRNA levels by Northern gel analysis. HPX resulted in a decrease in plasma corticosterone to undetectable levels, and decreases in PNMT activity and PNMT mRNA levels to 1 and 18% of the levels observed in sham rats, respectively. Corticosterone replacement produced high prolonged plasma levels of corticosterone which were 10 times those of sham rats, and significantly increased levels of PNMT activity and mRNA. However, corticosterone replacement failed to restore PNMT activity and mRNA levels fully. These results suggest that the maintenance of PNMT mRNA levels is dependent on maintaining corticosterone levels and supports the hypothesis that PNMT gene expression in the adrenal medulla is directly regulated by glucocorticoids produced by the adrenal cortex. However, the results also suggest that in the chronically HPX rat, factors in addition to naturally produced glucocorticoids are required for full restoration of PNMT mRNA levels.     

Co-localization of RNAs coding for phenylethanolamine N-methyltransferase and proenkephalin A in bovine and ovine adrenals

A 29-mer oligodeoxyribonucleotide probe, complementary to the coding region of bovine phenylethanolamine N-methyltransferase (PNMT) mRNA was synthesized. Characterization of this probe by Northern blot hybridization showed that it hybridized to a single band in RNA extracted from bovine and ovine adrenal medullae. The molecular size of this hybridized band was approximately 1.0-1.2 kb which is consistent with recently reported data on the molecular weight of bovine PNMT mRNA. In situ hybridization histochemistry was carried out with this probe on bovine and ovine adrenal sections and results compared on adjacent sections with a probe against proenkephalin A (ProEnk A) mRNA synthesized previously. Both showed a similar localization to the outer margin of cells in the adrenal medulla. The results of this study provide strong evidence at the level of mRNA expression that ProEnk A mRNA is expressed preferentially in the adrenaline synthesizing cells within the adrenal medulla. Further, it demonstrates the usefulness of a synthetic oligodeoxyribonucleotide probe for the study of PNMT gene expression.     

Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyltransferase and cold-induced alterations in adrenal PNMT mRNA and protein

Cold stress is known to increase the synthesis and release of catecholamines in the sympathoadrenal system. Previously, we have demonstrated that cold exposure results in a 3- to 4-fold increase in adrenomedullary tyrosine hydroxylase (TH) activity, which is mediated by concomitant alterations in TH mRNA and protein levels. To further investigate the effects of stress on the expression of the catecholamine biosynthetic enzymes, we have isolated a rat cDNA clone encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT). The cDNA clone is 905 nucleotides in length and contains a single open reading frame corresponding to 270 amino acids. The amino acid sequence predicted from this nearly full-length cDNA is 89% and 86% identical to that of bovine and human PNMT, respectively. Using the rat PNMT cDNA as a hybridization probe, we have measured the effects of cold stress on the relative abundance of adrenomedullary PNMT mRNA. Levels of PNMT protein were also estimated using an immunoblot analysis. As in the case of TH, cold exposure resulted in a rapid and prolonged increase in PNMT mRNA abundance, followed by concomitant increases in PNMT immunoreactivity. However, there appear to be quantitative and qualitative differences in the adaptive response of TH and PNMT to cold stress.     

Developmental expression of proenkephalin mRNA and peptides in rat striatum

The development of proenkephalin (PE) gene expression in the rat striatum was examined at the mRNA and peptide levels. Immunocytochemistry was performed with antisera generated to the PE-specific peptide product Met-enkephalin-Arg-Gly-Leu (MERGL). The distribution of immunostaining was compared with the distribution of PE mRNA, determined by in situ hybridization with an oligonucleotide probe. PE mRNA first appeared at E16 in the caudal ventrolateral striatum, followed at E17-18 by the appearance of MERGL immunoreactivity in a similar distribution. The anatomical gradients of PE gene expression were similar to the pattern of histogenesis of striatal neurons, suggesting that the timing of PE gene expression is related to the time of neuronal withdrawal from the mitotic cycle. The relation of the development of PE gene expression to the known patterns of striatal histogenesis, neurochemical compartmentalization and dopaminergic innervation is discussed.     

Distribution of neurons containing phenylethanolamine N-methyltransferase in medulla and hypothalamus of rat

Neurons immunocytochemically labeled with the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase were mapped in the brain of rat pretreated with colchicine. In medulla, immunoreactive cells in the C1 and C2 groups were distributed in a more complex manner than described previously. C1 neurons were identified in the reticular formation of ventrolateral medulla and were organized into two populations: (1) a cell column extending throughout the ventrolateral medulla, and lying ventral to the ambiguus cell group and either dorsal to the precerebellar lateral reticular nucleus or interposed between its two subdivisions; (2) a rostral cell cluster forming medial to the column at caudal levels and enlarging close to and in parallel with the ventral surface of the rostral ventrolateral medulla. A large proportion of cells and processes of the rostral cell group were oriented medially and ventromedially. processes of C1 neurons were traced dorsally toward the nucleus tractus solitarii, dorsal motor nucleus, and principal tegmental adrenergic bundle, ventrally toward the ventral surface, laterally toward the trigeminal complex, and medially or ventromedially toward the raphe. C2 neurons were located in the dorsomedial medulla and were subdivided into four distinct populations: (1) neurons in the rostral nucleus paragigantocellularis pars dorsalis (NGCd) and medial longitudinal fasciculus (MLF) were contiguous and similar in size and shape, with their long diameters oriented horizontally or diagonally along several axes; (2) neurons of the periventricular gray were located in a cytoarchitecturally undefined area dorsal to the MLF; these cells were ovoid, smaller, and organized more compactly than those in the NGCd-MLF; (3) a cell group in the rostromedial nucleus tractus solitarii (NTS) and dorsal motor nucleus overflowed caudally into the intermediate thirds of both structures; and (4) a parvicellular group in the NTS was compactly organized in the dorsolateral NTS and was best developed at the level of the area postrema. Processes of C2 neurons were generally directed sagitally, medially, and laterally along the ventricular floor and ventrally or medially toward the raphe; other fibers arborized and terminated within the NTS and dorsal motor nucleus. In the medulla, local processes were traced from C1 and C2 neurons directly into respective ventral and dorsal parts of the medullary raphe and surrounding intraparenchymal blood vessels. Fibers from these neurons were also followed, respectively, onto the ventral subpial surface and the floor of the fourth ventricle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Developmental expression of proenkephalin mRNA in rat striatum and in striatal cultures

Proenkephalin mRNA shows a biphasic developmental profile in rat striatum, with an initial peak at postnatal day 2, a decline to embryonic levels by day 7, and a second increase to adult levels over the course of the second to 4th week after birth. The same 4-fold increase is seen in cultured striatal neurons, over the same time course but without a biphasic response. Cultured fetal glia also contain proenkephalin mRNA.     

Strain differences in distribution of phenylethanolamine N-methyltransferase activity from rat brain and adrenal gland

Phenylethanolamine N-methyltransferase (PNMT) activity was measured in adrenal glands and medulla oblongata from 4 inbred rat strains, Fischer 344, Buffalo, Lewis and Sprague-Dawley rats. Adrenal enzyme activity was markedly different among the strains with the highest in Fischer, followed by Sprague-Dawley, Lewis and Buffalo rats in decreasing order. In medulla oblongata, the PNMT activity of Buffalo rat was the lowest being about one half of that of the other strains. Despite differences in the enzymes activity, immunotitration results indicate that there is no immunochemical difference between adrenal or medulla oblongata PNMT among the strains. Furthermore, the strain differences in the activity are not due to presence of an inactive enzyme, but to the amount of the enzyme. Our preliminary findings by dot blot hybridization, using a 32P-labeled cDNA probe for PNMT suggest that differences in adrenal and medulla oblongata PNMT activity between Fischer and Buffalo rats are partially due to differences in the amount of PNMT mRNA present.     

Glucocorticoid effects on phenylethanolamine N-methyltransferase (PNMT) in explants of embryonic rat medulla oblongata

Although glucocorticoid hormones have important roles in the development of neurotransmitter systems in cells derived from the neural crest, it is not known whether they have parallel effects on neuronal development in the brain. To address this issue, we have established an in vitro system of fetal medulla oblongata (MO) to follow development of the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT). Embryonic MO was explanted from E13 or E18 embryos and maintained for up to 3 weeks. Successful culture of adrenergic neurons was possible only in explants taken from young embryos, since E18 explants failed to develop. In E13 explants, immunoreactivity to both PNMT and tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis, was observed. PNMT catalytic activity which was barely detectable at the time of explanation increased markedly during the first week in vitro. To study the effects of glucocorticoids on PNMT development in central neurons, MO explants were grown in glucocorticoid deficient medium in which rat serum from adrenalectomized rats was substituted for human placental serum. Addition of natural glucocorticoids, cortisol or corticosterone, or the mineralcorticoid, deoxycorticosterone, during the third culture week had no effect on PNMT activity. Dexamethasone (DEX), a synthetic glucocorticoid, also had no effect on PNMT during the first or second weeks in culture. However, addition of DEX during the third culture week resulted in a doubling of PNMT activity. However, attempts to block the DEX effect during the third week or to block the increase in PNMT activity during the first week in control cultures with the glucocorticoid receptor antagonist, dexamethasone 21-mesylate, were unsuccessful. These results suggest that PNMT in central neurons does not require glucocorticoids for ontogeny during the embryonic period. This is in contrast to PNMT in adrenal medulla which requires glucocorticoids for normal development during both the embryonic and postnatal periods. More generally, these studies suggest that development of the same neurotransmitter phenotype in brain and periphery may be differentially regulated.     

Development of phenylethanolamine N-methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata.

The adrenergic phenotypic marker, phenylethanolamine N-methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons.

Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT-immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT-IR neurons were observed in cultures prepared only from embryos of 46–51 somites (embryonic day E13–13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16. In cultures of the E13 rat, PNMT was found to be catalytically active at 4 days in vitro and the levels of PNMT activity per neuron, as estimated from the number of PNMT-IR neurons in sister cultures, increased 2-fold in cultures grown for 8 days in the presence of 10% fetal bovine serum and 1.5-fold in defined, serum-free conditions. Treatment of cultures with either corticosterone or dexamethasone had no effect on PNMT activity, supporting previous studies suggesting that PNMT in central neurons is not regulated by glucocorticoids.

These studies provide a convenient and defined in vitro system for studying factors that influence the development of central adrenergic neurons. The differential development of PNMT-IR and TH-IR neurons in these cultures suggests that the factors involved in adrenergic neuron survival and/or differentiation are not common to catecholaminergic neurons in general.     

Strain-specific differences in levels of the mRNA for the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase

The activity of the epinephrine biosynthetic enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is 3- to 8-fold greater in rats of the Fischer 344 and Buffalo strains. The biochemical basis for the strain differences has been analyzed at the level of PNMT protein and messenger RNA production. Fischer rat adrenals possess approximately 5-fold more PNMT protein than those of the Buffalo rat as established by Western blotting and immunoprecipitation of adrenal gland homogenates. Poly(A)+ RNAs purified from adrenal glands of each strain were translated in a reticulocyte lysate system, immunoprecipitated with antibody to PNMT and fractionated by SDS-PAGE. A 35S-labelled protein of Mr = 34,000 was immunoprecipitated from adrenals of Fischer and Buffalo rats, indicating that the molecular weights of PNMT do not differ in these strains prior to post-translational processing. Hybridization of a 740 base pair (bp) cDNA for PNMT indicated that the mRNAs for PNMT are the same size in the adrenals of both strains. However, the adrenals of Fischer rats contain 2- to 4-fold more PNMT mRNA than Buffalo rats, as established by quantitative dot blot hybridization and Northern blot analysis. The medulla oblongata, the site of cell bodies of central adrenergic neurons, also contains approximately 2-fold more PNMT mRNA in Fischer rats. The strain specificity in the production of PNMT reflects differences in the expression of the gene for PNMT. Thus, an inherited capacity for PNMT expression may in fact provide the intrinsic determinants responsible for neurotransmitter production. These data provide a direct link between regulation of catecholamine enzyme biosynthesis at the genomic level and the availability of specific catecholamines for neurotransmitter and hormonal functions.     

Metyrapone-induced glucocorticoid depletion modulates tyrosine hydroxylase and phenylethanolamine N-methyltransferase gene expression in the rat adrenal gland by a noncholinergic transsynaptic activation

The hypothalamic corticotropin-releasing hormone system and the sympathetic nervous system are anatomically and functionally interconnected and hormones of the hypothalamic-pituitary-adrenocortical axis contribute to the regulation of catecholaminergic systems. To investigate the role of glucocorticoids on activity of the adrenal gland, we analysed plasma and adrenal catecholamines, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA expression in rats injected with metyrapone or dexamethasone. Metyrapone-treated rats had significantly lower epinephrine and higher norepinephrine production than control rats. Metyrapone increased TH protein synthesis and TH mRNA expression whereas its administration did not affect PNMT mRNA expression. Dexamethasone restored plasma and adrenal epinephrine concentrations and increased PNMT mRNA levels, which is consistent with an absolute requirement of glucocorticoids for PNMT expression. Adrenal denervation completely abolished the metyrapone-induced TH mRNA expression. Blockage of cholinergic neurotransmission by nicotinic or muscarinic receptor antagonists did not prevent the metyrapone-induced rise in TH mRNA. Finally, pituitary adenylate cyclase activating polypeptide (PACAP) adrenal content was not affected by metyrapone. These results provide evidence that metyrapone-induced corticosterone depletion elicits transsynaptic TH activation, implying noncholinergic neurotransmission. This may involve neuropeptides other than PACAP.     

Regulation of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA levels in the sympathoadrenal system by the pituitary-adrenocortical axis

The pituitary-adrenocortical axis plays a complex role in the regulation of the levels of enzymes of the catecholamine biosynthetic pathway. In this report we have explored molecular mechanisms of these regulations, by examining the effects of hypophysectomy (HPX) and dexamethasone (DEX) on tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels in the adrenal medulla (AM) and superior cervical ganglia (SCG). Three weeks after hypophysectomy weights (-48%), total RNA (-49%), and DNA (-22%) contents in AM were significantly reduced, when compared to sham-operated animals (SO). In SCG decreases in weight (-23%) and in the ratio of RNA/DNA (-25%) were also found. TH mRNA contents paralleled decreases in total RNA levels and no significant change in the relative abundance of TH mRNA was found. When HPX rats were injected for 5 days with DEX (1 mg/kg, i.p.), TH mRNA levels in the SCG (+51%) and in the AM (+74%) were significantly increased when compared to saline-treated HPX animals. DEX given to SO rats increased TH mRNA in SCG (+49%); a 27% increase in TH mRNA in the AM was also observed. The relative abundance of PNMT mRNA in the AM was reduced after hypophysectomy (-64%). This decrease was completely reversed by DEX. In contrast, DEX did not affect PNMT mRNA levels in the AM of SO rats. PNMT mRNA was not detected in SCG of saline- or DEX-treated rats. In conclusion, our findings suggest that the pituitary-adrenocortical axis is involved in the regulation of the steady-state levels of TH and PNMT mRNAs. This regulation involves: (1) induction of TH mRNA contents in AM and SCG by increased plasma glucocorticoid levels; and (2) maintenance of the steady-state levels of PNMT mRNA in AM by glucocorticoid-dependent mechanisms.     

Coordinate and differential regulation of proenkephalin A and PNMT mRNA expression in cultured bovine adrenal chromaffin cells: responses to secretory stimuli.

The expression of proenkephalin A (ProEnk A) mRNA and phenylethanolamine N-methyltransferase (PNMT) mRNA in response to nicotine and to a number of secretagogues was examined in cultured bovine adrenal chromaffin cells. Prolonged incubation with nicotine (10 microM) resulted in a 2-fold increase in ProEnk A mRNA but had no significant effect on the level of PNMT mRNA. Similarly, prolonged stimulation with high K+ (56 mM) induced a time-dependent elevation in the level of ProEnk A mRNA reaching 4-fold basal level after 24 h incubation. By contrast, the level of PNMT mRNA was not changed by treatment with high K+. The increase in the level of ProEnk A mRNA by high K+ was abolished by the presence of 10 microM D600, a calcium channel blocker. Unlike the effects of high K+, treatment of the cells with the sodium channel activator veratridine significantly elevated the levels of both ProEnk A and PNMT mRNA. This increase in ProEnk A and PNMT mRNA levels was however less affected by D600. Stimulation of the cells with Ba2+ (1.1 mM) also stimulated the levels of ProEnk A and PNMT mRNA and this action required the presence of extracellular Ca2+. This was in contrast to the effect of Ba2+ in stimulating catecholamine secretion, which was inhibited by Ca2+ and enhanced in Ca2(+)-free buffer. The results of the present study indicate that membrane depolarization and entry of extracellular Ca2+ play an important role on the regulation of ProEnk A and PNMT mRNAs, in addition to their well-known actions on hormone secretion. Furthermore, these results suggest that the expression of ProEnk A mRNA and PNMT mRNA are under independent regulation in response to secretory stimulation.     

Differential effects of placental restriction on IGF-II, ACTH receptor and steroidogenic enzyme mRNA levels in the foetal sheep adrenal

We have investigated the effects of restriction of placental growth on foetal adrenal growth and adrenal expression of mRNAs for Insulin-like Growth Factor II (IGF-II), the IGF binding protein IGFBP-2, Steroidogenic Factor 1 (SF-1) and adrenocorticotrophic hormone (ACTH) receptor (ACTH-R) and the steroidogenic cytochrome P-450 enzymes: cholesterol side chain cleavage (CYP11A1), 17alpha-hydroxylase (CYP17) and 21-hydroxylase (CYP21A1); and 3beta-hydroxysteroid dehydrogenase/Delta5Delta4 isomerase (3betaHSD). Endometrial caruncles were removed from non-pregnant ewes before mating (placental restriction group; PR). The total adrenal: foetal weight ratio was higher in PR (n=6 foetuses) than in control foetuses (n=6 foetuses). There was no difference in plasma ACTH concentrations between the PR and control foetuses between 130 and 140 days gestation. Adrenal IGF-II mRNA levels were lower (P<0.05) in the PR group, however, adrenal IGFBP-2 mRNA levels were not different between the PR and control groups. Adrenal ACTH-R mRNA levels were also lower whilst CYP11A1 mRNA levels were increased (P<0.005) in the PR group. We conclude that foetal adrenal growth and steroidogenesis are stimulated as a consequence of foetal growth restriction and that factors other than ACTH are important in foetal adrenal activation during chronic, sustained hypoxaemia.     

Developmental changes in neurophysiological taste response from the medulla in sheep

To determine whether functional characteristics of the taste system change during development, electrophysiological taste responses were recorded from neurons in the solitary complex (nucleus and tractus solitarius) in the medulla of fetal, newborn and adult sheep. Taste stimuli included NH₄Cl, KCl, NaCl, LiCl, citric acid, and HCl, applied to the anterior tongue. Fetal neurons at all ages (84–137 days of gestation) responded to stimulation of the tongue with NH₄Cl and KCl, but responses to NaCl and LiCl were only obtained in older fetuses (after 114 days of gestation), lambs and adults. Responses to citric were obtained at all ages; however, HCl responses were only infrequently obtained in young fetuses. Other developmental changes included a progressive decrease in latency of the responses to NH₄Cl, KCl, citric acid and HCl, and an increase in the duration of the neural response discharge as a function of gestational age. Since taste buds do not acquire the structural characteristics of the adult until the last third of gestation ( 100–147 days), these functional changes in taste response characteristics take place concurrently with structural development. Mammalian fetuses swallow amniotic fluid in utero, and therefore, the fetal taste system is stimulated during structural and functional development. Thus, there is an opportunity for fetal gustatory experience to influence the developing taste system.