Expression of the adrenergic phenotype in cultured fetal adrenal medullary cells: role of intrinsic and extrinsic factors

During embryogenesis of the rat the enzymes tryosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH) are first detected by immunocytochemistry or biochemical assay on the 16th day of gestation (E 16). It is not until E 18 that the enzyme phenylethanolamine-N-methyltransferase (PNMT), which is required for biosynthesis of adrenaline, can be detected cytochemically or biochemically. In this study we sought to determine whether the delayed appearance of PNMT is consequent to invasion of the adrenal medulla by E 18 of cells destined to express PNMT, cues provided by the ingrowing splachnic nerves or the action of corticosterone (CS) secreted by the adrenal cortical anlage, a hormone which regulates PNMT in adult rats. When adrenal glands are removed on E 16 and placed in culture, PNMT cannot be detected cyto- or biochemically until 2 days later (E 16 + 2). While CS levels increase 100-fold in vivo between E 16 and E 18, the surge of CS is not necessary for expression of PNMT since (a) adrenals removed on E 16 and cultured in the absence of exogenous ACTH fail to increase CS yet still express PNMT and (b) addition of CS (10^?5M) to the cultures on E 16 does not alter the time of appearance of the enzyme. CS, on the other hand, increases the amount of PNMT protein and activity 3-fold with respect to control at all time points, without any effect on TH. We conclude that (a) it is the cells already present in the adrenal medulla at E 16 which differentiate to express PNMT; (b) the initial expression of PNMT is not controlled by nerves nor by corticosteroids; and (c) corticosteroids have a selective action on regulating the amount of PNMT, once it is expressed, but not TH enzyme protein. It remains to be determined whether the differentiation of PNMT is elicited by genetic or epigenetic signals.     

Differential Regulation of Phenylethanolamine N-Methyltransferase Expression in Two Distinct Subpopulations of Bovine Chromaffin Cells

Abstract: Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were ～20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.     

Catecholamine binding by adrenal medullary protein can interfere with a sensitive radioenzymatic assay for norepinephrine

Norepinephrine (NE) binds extensively to protein that copurifies with phenylethanolamine-N-methyltransferase (PNMT) prepared from bovine adrenal medulla. This binding interferes with a NE assay that employs PNMT to catalyze the transfer of a tritiated methyl group from S-adenosyl-L-methionine to the amine group of NE. It was discovered that the protein binding of endogenous NE is reversed by dialysis at pH 6.0. Preparations of PNMT intended for use in radioenzymatic assays should involve one or more purification steps at pH 6.0.     

The adrenal gland of Triturus carnifex after glucagon administration

The present work was undertaken in order to investigate the influence of endocrine pancreas on the adrenal gland of Triturus carnifex. Our experiments aimed at studying the effects of intraperitoneal injections of glucagon on ultrastructural morphological and morphometrical features of steroidogenic and chromaffin tissues, as well as serum levels of aldosterone, corticosterone, norepinephrine (NE) and epinephrine (E). With regard to steroidogenic tissue, in January and November, glucagon decreased lipid droplet content in steroidogenic cells, that showed clear signs of increased activity. Moreover, increased corticosteroid serum levels were found. With regard to chromaffin tissue, in January glucagon played a stimulatory role on PNMT enzyme, eliciting an increase in the presence of E granules, and a decrease in the presence of NE granules, in the chromaffin cells. Moreover, increased E serum levels and decreased NE serum levels were found. In November, glucagon gave rise to a decrease in the presence of NE and E granules in the cells; E serum levels were strongly increased, whereas NE serum levels did not undergo any significant change. These findings suggest an involvement of the endocrine pancreas of the newt in the modulation of adrenal gland activity.     

Epinephrine: A Potential Neurotransmitter in Retina

Abstract: Dopamine (DA), norepinephrine (NE), and epinephrine (EPI) are present in rat retina. DA is the major catecholamine, whereas NE and EPI represent ∼5% of the DA content. DA is contained in a subpopulation of amacrine cells and has been the subject of numerous studies. We investigated the origin and properties of NE and EPI in retina. Following superior cervical ganglionectomy, there was a decrease in NE content, but no decrease in EPI or phenylethanolamine- N -methyltransferase (PNMT) activity. PNMT in retina has many of the substrate-specificity and inhibitor-sensitivity characteristics of other tissues. Enzyme activity is enhanced in newborn rats by treatment with dexamethasone. Exposure to a lighted environment increases retinal EPI in normal and superior cervical ganglionectomized rats. EPI content increased for more than 2 h in a lighted environment. We conclude that most of the NE is contained within the sympathetic neurons that innervate the eye from the superior cervical ganglion, whereas EPI is contained in retinal elements that are responsive to photic stimulation.     

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

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

Effects of estrogens and progesterone on the catecholaminergic activity of the adrenal medulla in female rats

Some reports in the literature allow to suspect the existence of an effect of sexual steroids on the adrenal catecholamines. To test this possibility, we have examined the catecholaminergic activity in the adrenal medulla of normal cycling rats in three phases of estrous cycle and of ovariectomized (OVX) rats injected with pharmacological doses of estradiol (ES), 2-hydroxyestradiol (HE) and/or progesterone (P). Adrenomedullary content of norepinephrine (NE) was similar during the estrous cycle, while epinephrine (E) content was increased during diestrous. This increase was concomitant with an increased phenylethanolamine-N-methyltransferase (PNMT) activity. Moreover, the monoamine oxidase (MAO) activity was significantly increased during proestrous, while the catechol-O-methyltransferase (COMT) activity was significantly decreased during estrous. In addition to these observations, ovariectomy caused a significant reduction of the E/NE ratio and of COMT and MAO activities. Administration of ES to OVX rats increased the E content, the E/NE ratio and the COMT activity as compared to vehicle-treated OVX rats. Administration of P to OVX animals led also to a significant increase of the E/NE ratio and of the COMT activity but not of the E content, while the administration of this steroid to OVX rats previously treated with ES only increased the COMT activity. Finally, administration of HE caused non-significant changes in NE and E contents and in MAO, COMT and PNMT activities. We can conclude that sexual steroids seem to be able to modify the catecholamine metabolism in the adrenal medulla and, hence, they could alter the ability of this gland to store and release these amines.     

Dopamine in rat adrenal glomerulosa

There is increasing evidence that dopamine (DA) inhibits aldosterone production, but the source of DA for this dopaminergic influence is not known. In the present study we examined the adrenal's zona glomerulosa for the presence of DA. Rats maintained on an intake of regular food were killed by decapitation and the adrenal capsule (containing zona glomerulosa) and the remainder of the gland (containing both cortex and medulla) were examined for their content of DA and also for norepinephrine (NE) and epinephrine (E). DA was found in adrenal glomerulosa in substantial quantity, 1.92 +/- 0.17 (SEM) ng/mg wet weight, representing an approximate concentration of DA of 1-100 microM. DA in adrenal capsule represented 12.2% of the total adrenal content of DA. NE and E were also present in glomerulosa, 3.46 +/- 0.32 and 18.7 +/- 2.1 ng/mg respectively, but, unlike DA, about 98% of the total adrenal content of NE and E was contained in adrenal medulla. The NE/E ratio in capsule and medulla were similar, although slightly higher in adrenal medulla, suggesting that the medulla is the source of the NE and E found in glomerulosa. On the other hand, the DA/E ratio was several-fold higher in glomerulosa than medulla--suggesting that glomerulosa DA was derived at least partially from a source other than adrenal medulla. We also found that short-term culturing of the adrenal reduced DA levels to 1/3 that observed in fresh tissue. This could explain in part why cultured glomerulosa has been shown to be more responsive to administered stimuli. In summary, the findings indicate a significant concentration of DA in adrenal glomerulosa, and suggest that the effects of DA on aldosterone production are mediated locally within the adrenal.     

Activation of brain phenylethanolamine-N-methyltransferase by Triton-X-100: Time related differences in the enzyme activation

The effect of different concentrations of Triton-X-100 (0.2 to 5 %) on the activity of enzyme phenylethanolamine-N-methyltransferase (PNMT) in the brain and adrenal was studied. The addition of 0.2 % Triton-X-100 to the 0.9 % KCl homogenization media resulted in 180 % activation of the brain PNMT. The similar content of this detergent added to the adrenal PNMT preparation had no marked effect on enzyme activity. Rising Triton-X-100 concentrations from 0.2 % to 5 % resulted in higher activation of brain PNMT activity but the adrenal enzyme remained rather stable. An exposure of 15 minutes of brain PNMT preparation to Triton-X-100 was the optimal interval to evoke the maximal increase in enzyme activity. This activation of brain PNMT by Triton-X-100 was observable up to 24 hours after the addition of the detergent.     

Phenylethanolamine N-methyltransferase activity in human brains

The activity of phenylethanolamine N-methyltransferase (PNMT) with noradrenaline and S-adenosylmethionine as substrates was measured in various areas of human brain by high-performance liquid chromatography with electrochemical detection. Commercially available noradrenaline contained about 0.27% adrenaline and was purified for reducing the blank value to increase the sensitivity. Enzymatically formed adrenaline and 3,4-dihydroxybenzylamine (added to the incubation mixture as an internal standard after the reaction) were adsorbed on an aluminum oxide column, eluted with 0.5 m hydrochloric acid and separated by high-performance reversed-phase paired-ion chromatography and measured with electrochemical detection. This assay was very sensitive and PNMT activity was detected in various areas of the human brain including the spinal cord. The enzyme activity was significantly reduced in brain tissues from patients with Parkinson's disease and striatonigral degeneration.     

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

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-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 (less than 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 Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters 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 less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.     

Role of glucocorticoids in expression of the adrenergic phenotype in rat embryonic adrenal gland

Differentiation of the noradrenergic and adrenergic phenotypes was documented in rat embryonic adrenal chromaffin cells in vivo from 12.5 days of gestation (E12.5) to term. The initial appearance of three enzymes in the catecholaminergic pathway, tyrosine hydroxylase (T-OH), dopamine-β-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) as well as endogenous catecholamines (CA), was followed by immunohistochemistry and histofluorescence. T-OH and DBH, were employed as indices of noradrenergic expression, whereas PNMT, the epinephrine-synthesizing enzyme, was used as an index of adrenergic expression. At E12.5, T-OH, DBH, and CA were present in cells of the sympathetic ganglia at the level of the adrenal anlage. By 13.5 days, cells containing T-OH, DBH, and CA, were observed between the sympathetic ganglia and developing adrenal, and within the adrenal itself. While T-OH, DBH, and CA were present in adrenal medullary cells from the earliest stages of adrenal development, PNMT, in contrast, was undetectable in ganglion primordia, migrating cells, or within the adrenal before 17 days. PNMT initially appeared at E17 in small clusters of cells scattered throughout the adrenal. The number of cells containing PNMT and the intensity of staining increased dramatically from E17 to term.A number of experimental manipulations were employed in vivo to investigate the role of glucocorticoids in differentiation of the adrenergic phenotype. Chronic or acute treatment of mothers and/or embryos with various glucocorticoids, adrenocorticotrophic hormone (ACTH), or S-adenosylmethionine (SAM) did not result in precocious appearance of PNMT. Moreover, the initial expression of PNMT was not prevented or delayed by embryonic hypophysectomy or by treatment with inhibitors of adrenocortical function. Consequently, the initial expression of PNMT on E17.0 is not dependent on normal glucocorticoid levels, cannot be induced prematurely by glucocorticoids, and is independent of the pituitary-adrenal axis. However, the ontogenetic increase in PNMT levels after initial expression has occurred does require intact pituitary-adrenal function. Our observations suggest that different mechanisms regulate initial expression and subsequent modulation of neurotransmitter phenotype.     

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

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

Gene expression of the phenylethanolamine N-methyltransferase is differently modulated in cardiac atria and ventricles

Phenylethanolamine N-methyltransferase (PNMT) is a final enzyme in catecholamine synthesizing cascade that converts noradrenaline to adrenaline. Although most profuse in adrenal medulla, PNMT is expressed also in the heart, particularly in cardiac atria and ventricles. In atria, the PNMT mRNA is much more abundant compared to ventricles. In present study we aimed to find out whether there is a difference in modulation of the PNMT gene expression in cardiac atria and ventricles. We used three methodological approaches: cold as a model of mild stress, hypoxia as a model of cardiac ischemic injury, and transgenic rats (TGR) with incorporated mouse renin gene (mREN-2)27, to determine involvement of renin-angiotensin pathway in the PNMT gene expression. We have found that PNMT gene expression was modulated differently in cardiac atria and ventricles. In atria, PNMT mRNA levels were increased by hypoxia, while cold stress decreased PNMT mRNA levels. In ventricles, no significant changes were observed by cold or hypoxia. On the other hand, angiotensin II elevated PNMT gene expression in ventricles, but not in atria. These results suggest that PNMT gene expression is modulated differently in cardiac atria and ventricles and might result in different physiological consequences.     

Effect of vasopressin on the induction of adrenal tyrosine hydroxylase and phenylethanolamine N-methyltransferase

We have assessed the effect of arginine vasopressin (AVP) on adrenal tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) activities. Both enzymes show marked increases after systemic administration of AVP in the range of 66 and 100 micrograms/day. To determine whether the pituitary gland plays a role in these inductions, the effect of AVP (66 micrograms per day, given divided into 3 doses for 4 days) on the adrenal enzymes was studied in hypophysectomized rats. These animals showed induction of TH but not PNMT. This indicates that a pituitary factor(s) mediates the increase in PNMT caused by AVP. Adrenal TH activity was measured after the injection of AVP (1 or 2 micrograms per rat) into the lateral ventricle: there was a statistically significant increase in TH. TH was not induced in the denervated adrenal gland of rats administered AVP systemically. These findings suggest that AVP may act centrally to induce the enzyme. The continuous s.c. infusion of AVP by osmotic minipump at the rate of 1 microgram/day for 6 days led to a striking increase in adrenal TH activity. However, PNMT did not increase significantly. It can be concluded that different mechanisms are involved in the induction of adrenal TH and PNMT caused by AVP. A neural mechanism is involved in TH induction, whereas PNMT induction requires release of a pituitary factor, presumably ACTH, but innervation of the adrenal is not needed for it. Moreover, the inductions of these two enzymes are differentially sensitive to the concentration of circulating AVP.     

Chromogranin A biosynthetic cell populations in bovine endocrine and neuronal tissues: detection by in situ hybridization histochemistry

Chromogranin A is a highly acidic protein that is found in the secretory granules of many endocrine and neuronal cells. To localize bovine cell populations involved in chromogranin A biosynthesis, the distribution of the mRNA encoding this protein was determined with in situ hybridization histochemistry. In the adrenal gland, the mRNA was found in the chromaffin cells of the medulla but was absent from the cortex. The distribution of the mRNA in the medulla was uneven; cells located at the periphery were more heavily labeled than those in the center of the gland. Because the adrenal medulla is composed of several cell types, the chromogranin A-containing cells were further characterized for the presence of neuropeptide and adrenergic markers. Adjacent sections were examined for the mRNAs encoding enkephalin and phenylethanolamine N-methyltransferase (PNMT), the enzyme that catalyzes the formation of epinephrine from norepinephrine. Both mRNAs were present in a narrow band of cells at the periphery of the medulla. However, in contrast to the distribution of chromogranin A mRNA, the enkephalin and PNMT mRNAs were detected in only a small number of cells in the inner medullary region. The difference in the distribution of the enkephalin and PNMT mRNAs from that of chromogranin A suggests that the expression of these genes is differentially regulated. In addition to the adrenal gland, chromogranin A mRNA is expressed by many other tissues. In the parathyroid gland, which is rich in the mRNA but exhibits little chromogranin A-like immunoreactivity, the message was present in most cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of corticotropin-releasing factor on adrenal DBH and PNMT activity

The continuous administration of CRF (corticotropin-releasing factor) by the intraventricular route, 100 ng/day, to rats over a period of 7 days, results in significant increases of DBH (dopamine beta-hydroxylase) and PNMT (phenethanol N-methyltransferase) activities in the adrenal glands. The pattern of increase in DBH response to various doses of CRF does not correspond to the effects observed on plasma corticosterone, a result that suggests that CRF is acting to increase the adrenal enzyme by means other than through the pituitary-adrenal axis. In contrast, PNMT responds to CRF in a manner indicating a correlation with glucocorticoid availability. Moreover, PNMT induction by CRF persists even after adrenal denervation; it also occurs when CRF is given subcutaneously for 3 days, in a dose of 60 ng/day. Injection of reserpine did not potentiate the effect of intraventricularly administered CRF.     

Epinephrine and dopamine colocalization with norepinephrine in various peripheral tissues: guanethidine effects

Chemical sympathectomy with guanethidine (Gnt) selectively destroys the postganglionic noradrenergic neurons, whereas dopaminergic fibers and nonneural catecholamine-secreting cells are spared. As a result, the relative proportions of norepinephrine (NE), epinephrine (E), and dopamine (DA) in tissues can be differentially affected. This study was done to show the possible differences in the relative amount of catecholamines in some organs and tissues that might indicate the nature of the secretory cells from which they originate. The contents of NE, E, and DA were assessed in rats neonatally treated with Gnt. Gnt-treated rats showed significantly lower levels of NE (P < 0.01) in all tissues except the adrenal gland and paraganglia. Epinephrine was present in all tissues with mean levels below 25 ng/g, with the exception of the adrenal gland (700 microg/gland) and paraganglia (100 ng/g). Only the heart showed lower values in Gnt-treated rats. Mean DA levels were also very high in paraganglia (530 ng/g). In the Gnt-treated rats, DA levels fell practically to zero except in the duodenum, mesentery, and adrenal, whereas there were high levels in the paraganglia, which were significantly different from controls. The results suggest that the three catecholamines are contained mainly in noradrenergic sympathetic fibers of muscle, white adipose tissue, heart, liver, pancreas, and spleen. The duodenum and mesentery may have dopaminergic fibers or E- and DA-containing nonneural cells. Hepatic-vagus paraganglia contain all the catecholamines in relatively high amounts in nonneural cells, and Gnt treatment raises DA levels without affecting the other amines.     

Inheritance of Adrenal Phenylethanolamine N-Methyltransferase Activity in the Rat

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