Regulation Of Phenylethanolamine N-methyltransferase Research Articles

Fetal programming of adrenal PNMT and hypertension by glucocorticoids in WKY rats is dose and sex-dependent.

Biochemical changes in utero may alter normal fetal development, resulting in disease later in life, a phenomenon known as fetal programming. Recent epidemiological studies link fetal programming to negative health outcomes, such as low birth weight and hypertension in adulthood. Here, we used a WKY rat model and studied the molecular changes triggered by prenatal glucocorticoid (GC) exposure on the development of hypertension, and on the regulation of phenylethanolamine N-methyl transferase (PNMT), the enzyme responsible for biosynthesis of epinephrine, and a candidate gene linked to hypertension. Clinically, high doses of the synthetic GC dexamethasone (DEX) are used to treat infant respiratory distress syndrome. Elevated maternal GCs have been correlated with fetal programming of hypertension. The aim of this study was to determine if lower doses of DEX would not lead to detrimental fetal programming effects such as hypertension. Our data suggests that prenatal stress programs for increased expression of PNMT and altered regulation of PNMT in males and females. Importantly, we identified that DEX mediated programming was more apparent in the male rats, and the lower dose 10μg/kg/day of DEX did not lead to changes in blood pressure (BP) in female rats suggesting that this dose is below the threshold for programming of hypertension. Furthermore, sex-specific differences were observed in regards to programming mechanisms that may account for hypertension in males.

Double stable isotope ultra performance liquid chromatographic-tandem mass spectrometric quantification of tissue content and activity of phenylethanolamine N-methyltransferase, the crucial enzyme responsible for synthesis of epinephrine

Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-L-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.

Role of reactive oxygen species in the neural and hormonal regulation of the PNMT gene in PC12 cells

In adrenergic cells norepinephrine is N‐methylated by the enzyme phenylethanolamine N‐methyltransferase (PNMT) to produce epinephrine. Prior studies have demonstrated that the PNMT gene in adrenal chromaffin cells may be regulated hormonally via the hypothalamic‐pituitary‐adrenal axis and neurally via stimulation of the splanchnic nerve. Further, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2 on the hormonal and neural stimulation of PNMT. RT‐PCR analyses show inductions of the PNMT intron‐retaining and intronless mRNA splice variants by CoCl2 (3.0‐ and 1.76‐fold respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and dexamethasone show increased promoter activity (18.5‐fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2‐induced ROS on the neural stimulation of PNMT. Our findings demonstrate that CoCl2‐induced ROS have synergistic effects on the hormonal and neural activation of the PNMT promoter and suggest post‐transcriptional regulation of PNMT following hormonal or neural activation during hypoxia. This study was funded by the Natural Sciences and Engineering Research Council of Canada.

Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

The stress hormone, epinephrine, is produced predominantly by adrenal chromaffin cells and its biosynthesis is regulated by the enzyme phenylethanolamine N-methyltransferase (PNMT). Studies have demonstrated that PNMT may be regulated hormonally via the hypothalamic-pituitary-adrenal axis and neurally via the stimulation of the splanchnic nerve. Additionally, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2, on the hormonal and neural stimulation of PNMT in an in vitro cell culture model, utilizing the rat pheochromocytoma (PC12) cell line. RT-PCR analyses show inductions of the PNMT intron-retaining and intronless mRNA splice variants by CoCl2 (3.0- and 1.76-fold, respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and the synthetic glucocorticoid, dexamethasone, show increased promoter activity (18.5-fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2-induced ROS on the neural stimulation of PNMT via forskolin. Our findings demonstrate that CoCl2-induced ROS have synergistic effects on hormonal and neural activation of the PNMT promoter.

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.

Neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression in bovine chromaffin cells differs from other catecholamine enzyme genes.

Expression of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) is regulated by hormonal and neural stimuli. Because the 5'-upstream regions of the PNMT do not contain sequences analogous to those demonstrated to convey neural regulation to the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) catecholamine-synthesizing enzyme genes, functional and biochemical analyses have been utilized to characterize PNMT promoter responses to cholinergic and depolarizing agents. In primary cultures of bovine adrenal medullary chromaffin cells, reporter gene expression from transiently transfected 3- and 0.9-kb-containing PNMT promoter constructs is stimulated approximately twofold by nicotine and muscarine. Depolarizing concentrations of K+ produce fourfold increases in expression. These responses are not detected with constructs containing the proximal 0.3-kb promoter, indicating that the regions between -273 and -877 bp convey neural responsiveness for the PNMT gene in bovine chromaffin cells. Electrophoretic mobility shift assays (EMSAs) with oligonucleotides encoding these regions of the PNMT promoter revealed distinctions in migration of nuclear protein complexes formed following treatment of chromaffin cells with nicotine, muscarine, or 50 mM K+. Thus, the PNMT promoter between 0.3 and 0.9 kb contains sequences capable of responding to cholinergic and depolarization stimuli. Moreover, these treatments influence the interactions of specific nuclear proteins with this region of the PNMT promoter.

Regulation of the adrenomedullary catecholaminergic system after mild, acute stress.

The time course of regulation of rat adrenomedullary phenylethanolamine N-methyltransferase (PNMT) and tyrosine hydroxylase (TH) activity was studied after a single episode of 20-min restraint stress. Significant increases in PNMT and TH activity were observed 18 h after the beginning of the stress. The time course of acute stress-induced regulation of PNMT and TH was examined for the influence of neural and hormonal input. Unilateral denervation was performed, and the animals were exposed to a single episode of restraint. PNMT activity increased similarly in both the innervated and denervated adrenals, with a significant increase observed at 36 h after the stress. TH activity was similar in both denervated and innervated adrenals, with a significant increase observed at 24 and 36 h after stress. Finally, suppression of endogenous corticosterone with dexamethasone delayed the stress-induced increase in activity of PNMT but not TH. The present study indicates that increases in catecholamine biosynthetic enzymes can be observed after a single episode of mild, acute stress. In addition, glucocorticoids appear to be important in the time course of the stress-induced increase in PNMT but not TH activity, whereas neural input does not seem to affect the time course of these increases.

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.

Phenylethanolamine N-methyltransferase-containing neurons in rat retina: immunohistochemistry, immunochemistry, and molecular biology

We sought to characterize in detail neurons in rat retina that contain phenylethanolamine N-methyltransferase (PNMT), the epinephrine biosynthetic enzyme. Cell bodies and processes of PNMT-containing neurons in retina were identified by immunohistochemistry. The coexistence of other catecholamine biosynthetic enzymes in the same cells was also investigated. Biochemical, molecular biological and immunochemical methods were applied to determine whether retinal PNMT is similar to the adrenal enzyme, since regulation of PNMT in retina and adrenal appears to be different. The results show that there are two types of PNMT-containing cells: those containing PNMT exclusively and those containing PNMT with two other catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), but not dopamine beta-hydroxylase (DBH). PNMT-only cell bodies are localized in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Their processes are observed in outer and inner strata of the inner plexiform layer (IPL). Only a small fraction of PNMT neurons in INL also contain TH and AADC. These cells send their processes to the adjacent stratum of the IPL. Antibodies to bovine adrenal DBH, however, fail to localize DBH in any rat retinal cells. Immunochemical titration shows that PNMT from both retina and adrenal gland has the same immunoreactivity. Furthermore, a PNMT-cDNA probe hybridizes equally with PNMT-mRNA isolated from both the retina and the adrenal gland. These results indicate that PNMT is identical in these tissues.

Tetrahydroisoquinolinecarboxylic acids and regulation of phenylethanolamine N-methyltransferase in cultured adrenal medulla

Explants of rat adrenal medullae, cultured for up to 48 hr in the presence of 3′,4′-deoxynorlaudanosolinecarboxylic acid (DNLCA) or α-methyltyrosine, exhibited a 69% increase in phenylethanolamine N-methyltransferase (PNMT) activity as measured in dialyzed homogenates. A related tetrahydroisoquinoline, norlaudanosolinecarboxylic acid (NLCA), when added to the medium did not elevate PNMT activity. No increase in the amount of PNMT was detected by immunochemical titration of homogenates from DNLCA-treated cultured medulla, nor were there changes in rates of synthesis or degradation of the enzyme. Although DNLCA is an inhibitor of tyrosine 3-monooxygenase, it had no effect on PNMT activity when added directly to an incubation mixture in vitro. Kinetic analyses of dialyzed homogenates from explants cultured in the presence of DNLCA revealed that the V max of PNMT was higher than that of control tissue. There was no decrease in K m after DNLCA treatment. The increase in PNMT activity appears to be a compensatory response to depletion of medullary catecholamines by DNLCA or α-methyltyrosine.

Regulation of phenylethanolamine N-methyltransferase

Publisher Summary This chapter discusses regulation of phenylethanolamine N -methyltransferase (PNMT). PNMT is localized almost exclusively to the adrenal medulla, although traces of activity have been reported in heart, brain, in extra-adrenal chromaffin tissue and in sympathetic ganglia. PNMT catalyzes the N -methylation of a number of primary and secondary β-hydroxyphenylethylamine derivatives. The requirement of the enzyme for β-hydroxylated phenylethylamine substrates appears to vary with species: PNMT from monkey, rabbit, rat and human with N -methylate only phenylethylamine substrates bearing a β-hydroxyl group. PNMTs from the rat and the frog share common substrate specificities, but virtually every other biochemical parameter of these enzymes differs. These include pH and thermal optima, thermal stability and starch-block migration. When these studies are compared with those of previous findings a striking uniformity of substrate specificity is observed, coupled with an equally striking heterogeneity of electrophoretic migration. These findings suggest that the evolution of PNMT has permitted amino acid substitutions which affect charge, molecular weight and stability of the enzyme, but has not tolerated alterations at the catalytic site of the molecule, which has remained relatively constant.

Regulation of phenylethanolamine N-methyl transferase

Phenylethanolamine N-methyl transferase (PNMT) catalyzes the methylation of norepinephrine, forming epinephrine in the adrenal medulla. In vitro, PNMT accepts a variety of phenylethanolamines and phenylethylenediamines as substrates. It is inhibited by phenethylamines and other compounds structurally similar to its substrates, as well as by agents that bind to sulfhydryl groups. Specific inhibitors of PNMT may be useful pharmacologic tools and potentially drugs. A group of benzylamines, including 2,3-dichloro-α-methylbenzylamine, are among the most potent inhibitors presently known. In vivo, maintenance of PNMT levels in the adrenal medulla is dependent upon an adequate supply of glucocorticoids from the adrenal cortex. Neural input to the medulla may also influence the enzyme level. The possibility that product inhibition may influence PNMT activity in vivo requires further study.