PNMT Activity Research Articles

Genetic regulation of catecholamine synthesis, storage and secretion in the spontaneously hypertensive rat

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

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.

Neural regulation of phenyethanolamine N-methyltransferase in vivo: transcriptional and translational changes

The hypothesis that neural regulation of rat adrenal medullary phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) occurs through transcriptional control is examined by following temporal changes in PNMT mRNA expression using paradigms of acute and chronic reserpine treatment. Concommitant changes in PNMT activity and protein were also measured to determine if reserpine induced changes depend solely on gene activation. Further, changes in adrenal corticosterone were measured to examine whether mRNA and enzyme responses might be mediated via reserpine induced changes in ACTH, and hence, corticosterone. Steady-state levels of PNMT mRNA peaked at ∼ 8-fold normal by 6 h after a single reserpine injection (10 mg/kg i.p.), and then declined to control values. With continued treatment, a second, slower rise occurred after three alternate day injections (∼ 3-fold basal levels). Enzyme activity and protein rose simultaneously but were attenuated in magnitude and time course by comparison to message. With both acute and chronic treatment, activity increased 2.0-fold, peaking at 12 h after a single dose of reserpine and again after four doses of the drug. Protein, as measured by immunotitration, was elevated 1.2-and 1.4-fold, respectively. Adrenal corticosterone rose ∼ 8-fold at 6 h, declined slightly at 12 h, rose again, and remained elevated thereafter. Comparison of the time courses for the various indices demonstrated that the early parallel bursts in PNMT mRNA and corticosterone are consistent with an increase in transcriptional activity. However, subsequent discordant expression in these two indices and concomittant magnitudinal differences in enzyme activity and protein suggest that other regulatory controls may limit the transcriptional activation of PNMT and the expression of active PNMT enzyme. Moreover, these latter controls appear to determine the ultimate limitation on PNMT expression and hence, adrenergic function.

Adrenergic expression in the rat adrenal gland: multiple developmental regulatory mechanisms

Phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) has been used as a marker to examine development of adrenergic expression in the rat adrenal gland and the putative role of glucocorticoids in this process. PNMT enzymatic activity increases 7–10-fold from birth to postnatal day 35. Immunotitration studies show that PNMT protein only increases 4–5-fold during this same time period. Moreover, the slopes from the immunotitration curves decrease with increasing age. Thus, a more active enzyme with lower affinity for the antiserum appears to be present in the older animals. Quantitative solution hybridization shows that PNMT mRNA increases 2.5-fold from birth through postnatal day 11. Thereafter, it declines, and eventually plateaus at values insignificantly different from birth by postnatal day 25. Northern analysis further shows that two forms of PNMT mRNA are expressed. Adrenal corticosterone remains low from birth through postnatal day 11, but then increases nearly 10-fold by adulthood. The lack of concordance between changes in PNMT activity, protein, and mRNA suggests that adrenergic expression is developmentally regulated at multiple levels; the above provides evidence for both transcriptional and post-transcriptional controls, since changes in PNMT mRNA may differ in both magnitude and direction from changes in PNMT activity and protein during the developmental window examined. These developmental regulatory mechanisms may be in part glucocortocoid-mediated, but corticosteroid control of PNMT gene expression does not appear to be the predominant mechanism of control.

Epinephrine in mammalian brain

1. 1. Epinephrine is widely distributed in brains of various species throughout phylogeny but maintains its localization to hypothalamus and brainstem/medulla in all species studied. 2. 2. A general decrease in brain epinephrine content is observed phylogenetically beyond fishes with wide variation within species. 3. 3. The cellular localization of epinephrine forming enzyme is dissociated from epinephrine stores in hypothalamus where epinephrine appears to be primarily a hormone. 4. 4. Three proposed functional pools of epinephrine are described. Synthesis of a hormonal pool and a second, perhaps nonfunctional, pool co-stored in noradrenergic terminals in the forebrain occurs extraneuronally and is probably inhibited acutely in the presence of high corticosteroids due to inhibition of uptake 2. Synthesis of epinephrine in the neuronal pool found primarily in the medulla may be enhanced due to increased PNMT activity in the presence of elevated corticosteroids. 5. 5. Phylogenetic and pharmacological data suggest that epinephrine may play an important role in tonic regulation of the level of arousal, reward and sensitivity to environmental stimuli in mammals.

Limited depletion of central adrenaline stores following administration of adrenaline synthesis inhibitors in rats

The aim of the present study was to maximise the selective depletion of adrenaline stores in the rat brainstem and spinal cord, obtained by administering inhibitors of adrenaline synthesis. Partial depletions of adrenaline in the hypothalamus and medulla were observed after a single injection of either LY134046 or SKF 64139 (40 mg/kg i.p.). The rate and extent of depletion seen in the hypothalamus 3 or 6 h after a single dose of LY134046 could be increased by simultaneous exposure to cold or by lowering blood pressure, but not by prior administration of the synthesis inhibitor at 12 h intervals for 3 days. None of the treatments used were able to significantly lower spinal cord adrenaline levels, despite at least 80% inhibition of spinal cord PNMT activity. These results suggest that the ability to reduce central adrenaline stores by synthesis inhibition is limited, especially in the spinal cord.

A highly sensitive assay for phenylethanolamine N-methyltransferase in human brain.

A rapid, highly sensitive assay for phenylethanolamine N-methyltransferase in brain using the natural substrate, norepinephrine, is described. The method is based on the selective adsorption and elution of the reaction product, epinephrine, from alumina. A small but important further lowering of blanks and increase in sensitivity is attained by removal of the radiolabeled substrate, [methyl-3H]-S-adenosylmethionine by precipitation as the reineckate prior to adsorption of norepinephrine to alumina. The assay has a sensitivity of 30 fmole and the PNMT activity could be measured in as little as 1 mg (wet wt) of human locus coeruleus tissue. The sensitivity is enhanced by homogenizing tissue in small volumes and removing potential inhibitors by dialysis. We report for the first time PNMT activity in specific regions of the human cerebral and cerebellar cortex.

Effects of Sinoaortic Baroreceptor Denervation on Blood Pressure and PNMT Activity in Medulla Oblongata and Spinal Cord of Normotensive and Genetically Hypertensive Rats

Effects of Sinoaortic Baroreceptor Denervation on Blood Pressure and PNMT Activity in Medulla Oblongata and Spinal Cord of Normotensive and Genetically Hypertensive Rats

ENDOGENOUS EPINEPHRINE (E) SECRETION REGULATES SURFACTANT RELEASE

Adrenergic mechanisms are important in the regulation of pulmonary surfactant metabolism. We gave SKF29661, a specific inhibitor of adrenal epinephrine synthesis to 15 pregnant New Zealand White rabbits to study the role of fetal adrenal epinephrine in the regulation of alveolar surfactant (AS) content in the immediate neonatal period. Does were given 0, 25, 50 and 100 mg/kg/d starting at 20 days gestation. Fetuses were delivered at 31 days by C-section (term:31 days). One-half of each litter was sacrificed immediately and the remainder were allowed to breathe for one hour and then sacrificed. After sacrifice, the adrenals were immediately removed; one was assayed for catecholamine content, and the other for PNMT activity. A thorough alveolar wash was then performed to recover the AS. Total AS phosphatidylcholine (PC) was determined by thin layer chromatography. SKF 29661 caused significant reduction in adrenal E content at each dosage (p<.001); maximal effect was 57% reduction at 100 mg/kg/day. There was a progressive decrease in alveolar PC with increasing dosage of SKF29661 (p<.01). Furthermore, adrenal E in both treated and control animals correlated positively with alveolar PC (r=0.450, p<.001). Conclusions: 1) SKF29661 crosses the placenta and inhibits fetal adrenal E production; 2) reduced fetal adrenal E is associated with decreased alveolar PC. These results suggest that in the neonatal period, endogenous E release plays an important role in the regulation of AS possibly by increasing secretion.

Brain epinephrine systems: detailed comparison of adrenergic and noradrenergic metabolism, receptor number and in vitro regulation, in two inbred rat strains

Epinephrine content and PNMT activity in medulla pons and hypothalamus of F344 inbred rats is from 3- to 8-fold higher than that of Buf inbred rats. These strain-dependent differences in brain adrenergic neurons are reciprocally related to altered α 1- and α 2-adrenergic receptor density in PNMT-containing brain regions. Radioligand binding indices related to α 2-receptor function reveal that receptors may be ‘desensitized’ as well as ‘down-regulated’ in the strain with high PNMT activity (F344), and may be ‘supersensitive’ as well as ‘up-regulated’ in the strain with low PNMT activity (Buf). The association between epinephrine-containing neurons and α-adrenergic receptor regulation appears specific, since α-adrenergic receptor density and regulation in brain regions devoid of PNMT and epinephrine is similar in F344 and Buf rats. While noradrenergic metabolism in F344 rats is greater than that in Buf rats, this difference is generalized throughout the brain and, thus, bears no apparent relationship to the localized alterations in α-adrenergic receptor density. Moreover, ß-adrenergic receptor density in the 2 strains is similar in all brain regions. These data suggest that a significant proportion of α-adrenergic receptors in medulla-pons and hypothalamus are intimately related to and regulated by epinephrine-containing nerve endings.

Adrenaline and noradrenaline neurons in rat lower brain stem: anatomical and pharmacological neurochemistry.

Since there was no study available on the comparative anatomical neurochemistry of the noradrenaline (NA) and adrenaline (A) containing neurons of the lower brain stem, we studied the distribution of the activities of the three major catecholamines (CA)-synthesizing enzymes in coronal sections of the rat medulla oblongata dissected into microcubes. In the dorso-medial region, there was a 1500 micron rostro-caudal difference in the localization of the peak of PNMT activity compared with the peaks of TH and DBH activities. This result led to a new microdissection technique allowing the preferential microdissection of the C2 A neurons versus the A2 NA neurons. The response of these two populations of CA neurons was then studied after a sustained decrease in blood pressure induced in young SHR by a 14 days dihydralazine treatment. The C2 adrenergic region exhibited an overall increase in TH, DBH and PNMT activity (+69%, +45% and +33%; p less than 0.01 respectively) while the A2 noradrenergic region was unaffected. Thus, the NA and A neurons of the rat dorso-medial lower brain stem do not seem to exhibit the same biochemical response after a prolonged hypotension. This preliminary result favors the hypothesis of a different functional role for the neighboring A2 and C2 neurons in central control of blood pressure.

Dissociation between hypothalamic catecholamine levels and epinephrine-forming enzyme activity after midbrain hemitransections in the rat

We found decreased catecholamine (norepinephrine and epinephrine) levels in selective hypothalamic nuclei of the rat after complete midbrain hemitransections. In contrast, the activity of the epinephrine-forming enzyme (phenylethanolamine-N-methyltransferase, PNMT, EC 2.1.1.28) was unchanged in the hypothalamus after the lesions. Our results demonstrate a dissociation between hypothalamic catecholamine levels and PNMT activity, and suggest that hypothalamic PNMT may not be localized in catecholaminergic axons originating in the brainstem.

Catecholamines and phenylethanolamine N-methyltransferase in selected brain nuclei and in the pineal gland of neurogenically hypertensive rats

Catecholamines (dopamine, norepinephrine and epinephrine) and the epinephrine-forming enzyme, phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) were measured in selective brainstem and hypothalamic nuclei and in the pineal gland of acutely (3 days after operation) and chronically (2 weeks after operation) neurogenically hypertensive (sinoaortically denervated, SAD) rats. Alterations in catecholamine levels and in PNMT activity were restricted to a few brain nuclei and the pineal gland, and were dependent on the time elapsed since SAD. Our results suggest a participation of discrete hypothalamic norepinephrine and brainstem epinephrine neurons in the development and maintenance of neurogenic hypertension.

Distribution of tyrosine hydroxylase, dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem

The activities of the three major catecholamines synthesizing enzymes were determined on supernatants from microcubes dissected out from 500 μm thick coronal sections of the rat lower brainstem. The microcubes exhibiting significant or high enzymatic activities represented only one-third of the microcubes analyzed and were generally localized either in the dorso-medial or in the ventro-lateral region of the lower brainstem. In the ventro-lateral region, referred to as the A 1-C 1 region, the rostro-caudal distribution of the enzymatic activities exhibited a small difference between the maximal TH and DBH activities and the maximum PNMT activity located 500 μm more rostrally. Conversely, in the dorso-medoal region, referred to as the A 2-C 2 region, there was a much larger rostro-caudal difference (about 1500 μm) between the peaks of TH and DBH activities and the more rostral peak of PNMT activity. Such a distribution suggests a separation between the adrenaline (A) and the noradrenaline (NA) containing neurons within the dorso-medial region. According to the results of this study, a preferential microdissection of the A versus NA neurons seems possible in the A 2–C 2 region of the rat medulla oblongata.

Regional changes in phenylethanolamine-N-methyltransferase of rat brain during development.

The changes of phenylethanolamine-N-methyltransferase (PNMT, EC 2.1.1.28), the enzyme that catalyzes the final step in the biosynthesis of adrenaline, were studied during the development of several regions of rat brain. PNMT is present in medulla oblongata-pons, hypothalamus, cerebellum, and midbrain five days before birth, and a progressive increase in the enzyme activity is observed during development. The adult levels are attained between 15 and 20 days, depending on the region. The increases in PNMT activity in the rostral regions are higher than in the caudal regions. PNMT attains adult levels earlier than tyrosine hydroxylase and dopamine-beta-hydroxylase. The apparatus for adrenaline synthesis seems to be mature at three weeks after birth in the medulla oblongata-pons, which contains the cell bodies of adrenaline-containing neurons. In the other regions the adult levels of enzyme activity are attained at 15 days after birth.

Distribution of PNMT and Epinephrine in the medulla oblongata of normotensive and spontaneous hypertensive rats

The distribution of the Epinephrine forming enzyme (PNMT) activity and Epinephrine (E) levels was investigated in the medulla oblongata of spontaneous hypertensive rats (SH-rats) and in two normotensive strains, namely Wistar Kyoto rats (WK-rats) and Wistar rats. The PNMT activity increases progressively from the caudal to rostral parts in the C1 and C2 regions of the medulla oblongata. The enzyme activity and the E levels are in all parts of the C1 and C2 regions higher in Wistar rats than in WK-rats. The PNMT activity in all parts of the C2 region (with the exception of the caudal region), and in the middle part of the C1 region is higher in SH-rats than in WK-rats. The E levels in the SH-rats are higher than in WK-rats in the mediocaudal parts of the C2 and C1 regions.

Catecholamine synthesis in the chromaffin tissue of the African lungfish, Protopterus aethiopicus

The activities of dopamine-beta-hydroxylase (DBH, E.C. 1.14.17.1) and phenylethanol-N-methyl transferase (PNMT, E.C.1.1.1.10) were determined in tissue homogenates from the chromaffin tissue containing atrium and intercostal arteries from the African lungfish. Clearly measurable activities of DBH were found in both kinds of tissues, while PNMT activity was ascertained only in the intercostal arteries. The activity of DBH in the atrium indicates a possibility for local synthesis of the large amounts of catecholamines, especially noradrenaline, found in this tissue. Catecholamines synthetized in and released from the chromaffin tissues of Protopterus, may be of great importance for the adrenergic control in this animal in the absence of a well developed sympathetic nervous system.

Neonatal corticosteroid permanently alters brain activity of epinephrine-synthesizing enzyme in stressed rats

(Accepted December 14th, 1978) The enzyme responsible for the conversion of norepinephrine to epinephrine, phenylethanolamine N-methyltransferase (PNMT; E. C. 2.1.1.28), is present in the adrenal medulla 1, other chromaffin tissues, sympathetic ganglia 4, and the central nervous systemlL Immunofluorescence and radioenzymatic assays have localized PNMT in discrete brain regions including the hypothalamus 10,15. Cell bodies contai- ning the enzyme are restricted to two cell groups in the rostral medulla of the brain stem, the ventral C1 region and the dorsal Cz region, areas which have established roles in cardiovascular regulation. The activity of non-specific N-methyltransferase is negligible in the brain stem 14. Epinephrine levels are reported to vary directly with PNMT activity in both adrenal and brainlZ, 19. Glucocorticoids are required for maximal activity of adrenal PNMT. In intact rats the physiological level of circulating corticosteroid appears to be virtually optimally effective, since supernormal levels of exogenous steroid do not result in increased activity of PNMT 3,~0. In perinatal rats, treatment with the potent synthetic glucocorticoid, dexameth- asone, produces a marked rise in PNMT activity in the superior cervical ganglion and other ganglia 8,11. This increased is transitory, with enzyme activity falling rapidly after cessation of treatment. Neonatal dexamethasone administration has also been shown to increase PNMT activity in both hypothalamus and brain stem medullalL In brain, the effect of glucocorticoid on PNMT activity has been examined only acutely, at the termination of steroid treatment. We wished to determine whether the neonatal enhancement of brain PNMT activity by dexamethasone might be a permanent effect. Such a finding would suggest that perinatal stress, with its concomitant rise in circulating glucocorticoids, as well as exposure to exogenous corticoids, can affect the level of synthesis of epinephrine in the adult animal. Pregnant Sprague-Dawley rats from Charles River were received in mid- gestation. We pooled the pups on the day following parturition, and randomly * To whom all correspondence should be sent at the following address: Departments of Psychology and Biology, Virginia Polytechnic Institute and State University, Blacksburg, Va. 24061, U.S.A.

Adrenaline levels in brain stem nuclei and effects of a PNMT inhibitor on spontaneously hypertensive rats

The adrenaline content of specific brain stem areas was decreased in young (4 week-old) but not in adult (14 week-old) SHR. Some of these areas also showed increased PNMT activity, indicating a possible enhanced release or metabolism of adrenaline early in the development of the hypertension. Dopamine and noradrenaline levels, on the contrary, were not changed either in young or adult animals. Administration of a PNMT inhibitor to adult SHR resulted in a decrease of the blood pressure in SHR to control levels. These observations support the hypothesis of a participation of adrenergic mechanisms in the development of genetic hypertension, and indicate the possibility for the use of PNMT inhibitors as hypotensive agents.

Central elevation of phenylethanolamine N-methyltransferase activity following stress

Phenylethanolamine N-methyltransferase (PNMT, E.C.2.1.1.28) is the terminal enzyme in the synthesis of adrenaline ill the adrenal medulla 1, other chromaffin tissues, and in the mammalian brain iv. Changes in the level of adrenal and hypothalamic PNM T activity have been correlated with changes in the tissue content of adrenaline 13,15,1s,2°. It is well established that PNMT requires glucocorticoids for maximal activity. The synthetic glucocorticoid, dexamethasone, has been shown to induce adrenal PNMT in hypophysectomized rats ~9 and to stimulate the enzyme in extra-adrenal chromaffin tissue of neonatal rats~, 6 and brain of adult rats 13. Induction occurs over a period of days and is presumed to be due to an increase in the rates of enzyme synthesisS, 20. The dose of dexamethasone employed to produce PNMT stimulation has been very high, generally 0.5-1.0 mg/kg. The physiological role of glucocorticoids in the regulation of PNMT in the intact animal is less clear. Adrenal PNMT activity may be regulated by ACTH (indirectly acting through adrenal glucocorticoid synthesis), by circulating glucocorticoids directly, and also by neuronal activity via the splanchnic nerves in different strains of mice 5 and rats H. Short-term treatment with dexamethasone has been reported to be without effect on adrenal PNMT in the intact animaPg, z0, although the activity of the adrenal enzyme is increased 40 days after unilateral adrenalectomy 3 and the brain enzyme after 13 days of dexamethasone treatment lz. The half-life of adrenal PNMT, estimated without blockade of protein synthesis, is reported to be quite long (6 days) 4. Based on these considerations, Ciaranello and Black 4 have suggested that PNMT finds its bio- logical role only in chronic stress situations. Nothing is known regarding the effect of stress on the activity of PNMT in brain tissue. The neuroanatomical positions of the adrenergic cell bodies in the rostral medulla oblongata of the brain stem ~0 is suggestive of a relationship to stress. The ventral adren- ergic cell group (C1) coincides in part with the vasomotor center 9 and is thought to project to the spinal cord, the hypothalamus, and other forebrain regions known to mediate aspects of the stress response 10. The dorsal cell group (C2) is in part coincident with the cardioinhibitory center (the dorsal motor nucleus of the vagus) 9 which re- ceives input from baroreceptors.