Phenylethanolamine N-methyltransferase Activity Research Articles

Catecholamine synthesizing enzyme activity in brainstem areas from victims of sudden infant death syndrome.

In order to investigate whether central catecholaminergic neurons are altered in sudden infant death syndrome (SIDS), the activities of dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) were determined in nine brainstem areas from 19 victims of totally unexplained SIDS (tSIDS), 9 victims of partially unexplained SIDS (pSIDS) and 8 control infants. The distinction between tSIDS and pSIDS was made according to the results of the autopsy. They were totally negative for the tSIDS victims, while minor pathological alterations were found in pSIDS victims. In both tSIDS and pSIDS victims, the PNMT activity was significantly decreased in the medullary C2m and C21 areas and in the nucleus medullae oblongatae centralis. In the C21 area only DBH activity was decreased. Further, in the nucleus ambiguus PNMT activity was decreased in tSIDS, while DBH was lowered in pSIDS victims. No significant modifications were found in the areas from the dorso-lateral pons (locus coeruleus, Kolliker Fuse and parabrachialis nuclei). Thus, in the two groups of SIDS victims the capacity to synthesize adrenaline was decreased in some medullary areas. It is considered that these biochemical alterations are related to the abnormal central respiratory and arousal control, which is thought to be present in SIDS.

Distribution of phenylethanolamine-N-methyltransferase in the rat heart: effect of 6-hydroxydopamine

The distribution of phenylethanolamine-N-methyltransferase (PNMT), the epinephrine-forming enzyme in the adult rat heart atria and ventricles was studied. The activity of the enzyme in the hearts of adult rats is very low; a significantly higher activity was found in the atria than in the ventricles. A significant increase in PNMT activity was found in all heart areas examined in 6-hydroxydopamine-treated rats compared to vehicle-treated rats. The increased PNMT activity might reflect compensatory or adaptation processes secondary to the destruction of the peripheral sympathetic nerve terminals.

Phenylethanolamine N-methyltransferase activity is decreased in Alzheimer's disease brains.

Phenylethanolamine N-methyltransferase (PNMT) is the rate-limiting enzyme involved in the synthesis of epinephrine and a specific marker for epinephrine neurons. We have previously described the stability of this enzyme in a variety of pre- and postmortem conditions in human brain autopsy specimens and demonstrated its presence in areas of the human brain that are associated with memory and attention. We now report on 5 patients with Alzheimer's disease (AD) who had a decrease in PNMT activity of 37 to 48% in areas of the brain affected by the disease but not in the cerebellum, an area of the brain unaffected by the disease. The degree of decrease in PNMT activity in the hippocampus correlated significantly with the degree of dementia. We have provided direct immunochemical evidence that the decrease in PNMT activity in AD is due to the loss of this specific enzyme protein.

A radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: Application to rat hypothalamic nuclei, pineal gland, and heart

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-[ methyl- 3H]adenosyl- l-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.

Differential effects of nerve growth factor and ciliary neuronotrophic factor on catecholamine storage and catecholamine synthesizing enzymes of cultured rat chromaffin cells.

The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.

ChemInform Abstract: Binding Requirements of Phenolic Phenylethylamines in the Benzonorbornene Skeleton at the Active Site of Phenylethanolamine N‐Methyltransferase.

In order to determine the active site binding orientation of norepinephrine, a series of conformationally defined analogues of the tyramines, in which the ethylamine side chain is held fixed by incorporation into a benzonorbornene skeleton, were prepared and evaluated for phenylethanolamine N-methyltransferase (PNMT) activity. While exo-2-amino-5- and exo-2-amino-8-hydroxybenzonorbornene (7 and 10, respectively) were prepared from 5-methoxybenzonorbornadiene by azidomercuration/demercuration and reduction, it was necessary to employ both normal (inversion of configuration) and abnormal (retention of configuration) Mitsunobu reactions to prepare, stereoselectively, exo-2-amino-6- and exo-2-amino-7-hydroxybenzonorbornene (8 and 9, respectively) from 6- and 7-methoxybenzonorbornen-2-ol. None of the six analogues were substrates. However, exo-2-amino-6-hydroxybenzonorbornene (8) and anti-9-amino-6-hydroxybenzonorbornene (12) displayed significant activity as inhibitors toward PNMT. The greater potency of 8 and 12, as compared to the parent unsubstituted analogues exo-2-amino- and anti-9-amino-benzonorbornene (4 and 5, respectively), indicates the presence of a spatially compact hydrophilic pocket within the aromatic ring binding region of the active site of the enzyme. Furthermore, the greater activity of 12, relative to 8, is consistent with an active site binding preference for molecules in which a more coplanar relationship exists between the aromatic ring and the amine nitrogen. From the findings of this study, it appears that norepinephrine has a different active site binding orientation than most known substrates and competitive inhibitors of PNMT.

Phenylethanolamine N-methyltransferase in the brains of streptozotocin diabetic rats.

Recent evidence suggests that adrenergic neurons in the brainstem and hypothalamus are important for regulation of endocrine and cardiovascular function and the response to stress. Since abnormalities in several of these functions are observed in diabetic subjects, we investigated the effects of streptozotocin-induced diabetes on the activity of the enzyme that converts norepinephrine to epinephrine [phenylethanolamine N-methyltransferase (PNMT)] in the brainstem and hypothalamus of the rat. Enzyme activity was measured in the supernatant of sonicated brain tissues of saline-treated control, diabetic, and insulin-treated diabetic rats. One month after streptozotocin treatment, increases in plasma glucose and food intake occurred; these were restored to control levels by insulin treatment. PNMT activity was approximately 2-fold higher in the brainstem of diabetic rats than in controls (P less than 0.0001), and administration of insulin partially prevented the effects of diabetes on PNMT activity (P less than 0.01 compared to diabetics and P less than 0.05 compared to controls). Brainstem enzyme activity measured in all animals (n = 53) exhibited a significant correlation with plasma glucose concentrations (r = 0.51; P less than 0.001). Diabetes had no apparent effect on PNMT activity in the hypothalamus. These findings are the first to suggest that experimental diabetes alters PNMT activity in the medulla/pons.

Pharmacological analysis of the neurotransmitter mechanisms regulating phenylethanolamine N-methyltransferase in the adrenal gland

The i.p. administration of reserpine daily for 4 days to rats brought about an increase of adrenal phenylethanolamine N-methyltransferase (PNMT) activity. However, the combination of the systemic administration of p-chlorophenylalanine (PCPA) and reserpine for 3 days produced an earlier increase in this adrenal enzyme. The effect was reduced significantly in the denervated gland. Prior administration of 5,7-dihydroxytryptamine (DHT) i.c.v. to rats greatly potentiated the inducing effect of reserpine. On the other hand, the depletion of catecholamines by giving rats alpha-methyl- p-tyrosine (AMPT) i.p. or 6-hydroxydopamine (6-OHDA) i.c.v. did not alter the action of reserpine on adrenal PNMT. PCPA, DHT, AMPT and 6-OHDA did not have any effect by themselves on adrenal PNMT, but the combination of PCPA and AMPT, each given i.p., caused increased adrenal PNMT activity. The administration of dopamine agonists, a treatment that increases adrenal TH, did not modify adrenal PNMT. We conclude that the induction of PNMT by reserpine involves depletion of catecholamines and serotonin, the depletion of serotonin having the more powerful effect. A monoaminergic (serotonergic) inhibitory pathway is involved in the central regulation of adrenal PNMT activity.

Biochemical evidence for an interaction between adrenaline and noradrenaline neurons in the rat brainstem

In this study, we sought to determine if there was an interaction between the C 2 adrenaline-containing (A) neurons of the rat medulla oblongata and the noradrenaline-containing (NA) cell bodies of the locus coeruleus (LC). For this purpose, the biochemical response of the NA cell bodies of the LC after a lesion of the C 2 region was studied by using as markers the in vitro activities of the catecholamine synthesizing enzymes: tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT). An increase in TH activity, not associated with any change in DBH or PNMT activity, was found in the LC (+104%, P < 0.001) 4 days after a bilateral electrolytic lesion (3 mA for 5 s) of the C 2 region. Conversely, the electrolytic lesioning of the neighboring A 2 region of NA neurons did not modify the TH activity of the LC. These results suggest the existence of an ascending adrenergic inhibitory control of the NA cell bodies of the LC.

Destruction of the preganglionic nerves by β-bungarotoxin does not interfere with normal embryonic development of the rat adrenal medulla

Using β-bungarotoxin (β-BTX) as a tool to eliminate the preganglionic cholinergic nerve supply to the embryonic rat adrenal gland, we have investigated whether or not these nerves affect the differentiation of embryonic chromaffin cells (pheochromoblasts). Rat fetuses received a single injection of 1 or 2 μg β-BTX or an identical volume of saline at embryonic day (E) 17 and were taken for morphological and biochemical analyses at E 21. Administration of β-BTX caused a 15 to 20% reduction in body weight, crown-rump-length and adrenal weight. Spinal cord development was reduced and acetylcholinesterase-positive cells in ventral and lateral columns were virtually absent in toxin-treated animals. In adrenal glands, a decrease of choline acetyltransferase activity to 13% of control levels and a concomitant decrease of ultrastructurally identifiable nerve fibers and axon terminals revealed that application of 2 μg β-BTX effectively reduced the neuronal input to E 21 adrenal glands. Values for total adrenal catecholamines, relative amounts of adrenaline and noradrenaline, tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities were unaltered. All ultrastructural features of pheochromoblasts (except the lack of synapse-like axon terminals) were inconspicuous. Corticosterone levels in adrenals and plasma were identical to controls. These data strongly suggest that normal embryonic development of adrenal chromaffin cells does not require an intact nerve supply.

Binding requirements of phenolic phenylethylamines in the benzonorbornene skeleton at the active site of phenylethanolamine N-methyltransferase.

In order to determine the active site binding orientation of norepinephrine, a series of conformationally defined analogues of the tyramines, in which the ethylamine side chain is held fixed by incorporation into a benzonorbornene skeleton, were prepared and evaluated for phenylethanolamine N-methyltransferase (PNMT) activity. While exo-2-amino-5- and exo-2-amino-8-hydroxybenzonorbornene (7 and 10, respectively) were prepared from 5-methoxybenzonorbornadiene by azidomercuration/demercuration and reduction, it was necessary to employ both normal (inversion of configuration) and abnormal (retention of configuration) Mitsunobu reactions to prepare, stereoselectively, exo-2-amino-6- and exo-2-amino-7-hydroxybenzonorbornene (8 and 9, respectively) from 6- and 7-methoxybenzonorbornen-2-ol. None of the six analogues were substrates. However, exo-2-amino-6-hydroxybenzonorbornene (8) and anti-9-amino-6-hydroxybenzonorbornene (12) displayed significant activity as inhibitors toward PNMT. The greater potency of 8 and 12, as compared to the parent unsubstituted analogues exo-2-amino- and anti-9-amino-benzonorbornene (4 and 5, respectively), indicates the presence of a spatially compact hydrophilic pocket within the aromatic ring binding region of the active site of the enzyme. Furthermore, the greater activity of 12, relative to 8, is consistent with an active site binding preference for molecules in which a more coplanar relationship exists between the aromatic ring and the amine nitrogen. From the findings of this study, it appears that norepinephrine has a different active site binding orientation than most known substrates and competitive inhibitors of PNMT.

Co-localization of tyrosine hydroxylase and phenylethanolamine-N-methyltransferase immunoreactivity in the rat retina: a re-examination using double labeling on semi-thin sections

The precise morphology and distribution of phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive cells has been observed and compared with the previously described tyrosine hydroxylase (TH)-immunoreactive cells, in the rat retina. The PNMT-positive cells are small amacrine neurons, located either in the inner nuclear layer or in the ganglion-cell layer. They send processes mainly to the middle sublayer and to a lesser extent to the outermost sublayer of the inner plexiform layer. They resemble the small TH-positive bouquet cells described previously. In order to ascertain the relationship between PNMT-positive cells and TH-positive bouquet cells, a double immunohistochemical labeling technique, using anti-TH and anti-PNMT antisera, has been developed on semi-thin sections. The result of these experiments clearly indicate that the populations of TH-positive cells and PNMT-positive (presumably epinephrinergic) cells are separated. The absence of TH enzyme in the PNMT-positive cells raises the question of the enzymatic activity of PNMT, which appears to be different from the classical pathway of catecholamine biosynthesis in the retina.

Kainic acid depletes retinal phenylethanolamine-N-methyltransferase activity of rats.

Kainic acid, an excitotoxic agent to the retina as well as to neuronal cell bodies in the brain, was administered intraocularly to rats in order to study the sensitivity of phenylethanolamine-N-Methyltransferase (PNMT) containing amacrine cells to this agent. Results show that these cells are very sensitive to the toxic effects of kainate. A dose of 5 nmoles caused a significant reduction in retinal PNMT activity. Higher doses further depleted enzyme activity.

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 difference 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.

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 enzyme 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.

An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol 0-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (<0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronateagarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was <0.001%. Norepinephrine can be directly quantified in 25 μl of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 ± 21.7 pg/ml) was significantly lower than venous plasma NE (367 ± 32.7 pg/ml) in all subjects (p <0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity 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 β-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. Our studies also elucidate three similarities in central and peripheral PNMT development: (1) in both adrenal and brain, adrenergic expression follows noradrenergic expression, (2) in both brain and periphery, PNMT is transiently expressed in some cells, and (3) glucocorticoid treatment of pregnant rats increases PNMT in the embryonic brain and sympathetic nervous system. Finally, we have observed a critical period during which glucocorticoid treatment increases PNMT activity in the brain.

ACTH increases adrenal medullary PNMT activity in neonatal rats

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

Myocardial ischemia stimulates catecholamine synthesis and catabolism in the dog adrenal medulla.

The effects of regional ischemia of the myocardium upon metabolism and catabolism of catecholamines were studied in the dog adrenal medulla. Regional ischemia was induced by ligating a small branch of the left anterior descending coronary artery for 1 or 24 hours. Sham-operated controls were run in parallel. Coronary ligation for 24 hours resulted in a decrease in the level of epinephrine and an increase in the level of dopamine in the adrenal medulla, and also resulted in an increase in the activities of the phenylethanolamine-N-methyltransferase (PNMT), monoamine oxidase, and catechol-O-methyltransferase. The results after coronary ligation for 1 hour were essentially the same as those after coronary ligation for 24 hours, except that the activity of PNMT decreased. These results suggest that both synthesis and catabolism of catecholamines in the adrenal medulla are accelerated during coronary ligation for a period of 1 to 24 hours.

Adrenal phenylethanolamine-N-methyltransferase activity in the young rat: influence of thyroid hormones and insulin-induced hypoglycemia.

The postnatal evolution of adrenal phenylethanolamine-N-methyltransferase (PNMT) activity is slightly accelerated by hypothyroidism and slowed by hyperthyroidism. In 14-day-old rats a single stimulation of the adrenals by insulin-induced hypoglycemia does not lead to any change in enzymatic activity. If the stimulation is repeated for 4 days, it leads to a net increase in adrenal PNMT activity in the control rats, while no increase occurs in hypo- or hyperthyroid animals.