EFFECTS OF DEXAMETHASONE ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE AND ADRENALINE IN THE BRAINS AND SUPERIOR CERVICAL GANGLIA OF ADULT AND NEONATAL RATS

Abstract— Phenylethanolamine N -methyltransferase (PNMT) activity assayed by a sensitive radiochemical method was found to be distributed unevenly in the adult rat brain. Highest activities of this enzyme were located in the medulla and the hypothalamus. Small amounts of adrenaline were identified in the hypothalamus using a sensitive enzymatic radiochemical assay procedure, whereas in the medulla and other brain regions the values for adrenaline were at the limits of the sensitivity of the assay for this amine. The daily administration of dexamethasone (1 mg/kg) to adult rats for 13 days significantly increased PNMT activity in medulla and hypothalamus and also increased the adrenaline content of the hypothalamus. Five daily injections of dexamethasone (0·1 mg/kg) to newborn rats did not alter the PNMT activity or the catecholamine content of the brain, but markedly increased the PNMT activity and adrenaline content of superior cervical ganglia. Higher doses of dexamethasone given to newborn rats (6 daily injections of 1 mg/kg) increased PNMT activity both in the medulla and in the hypothalamus.     

EFFECT OF NGF AND DEXAMETHASONE ON PHENYL-ETHANOLAMINE-N-METHYL TRANSFERASE (PNMT) ACTIVITY IN NEONATAL RAT SUPERIOR CERVICAL GANGLIA

Abstract— Nerve-growth factor (NGF) or dexamethasone administered to newborn rats result in increased levels of phenylethanolamine-N-methyl transferase (PNMT) activity in the superior cervical ganglia. Treatment with dexamethasone, but not NGF, also results in increased levels of epinephrine which parallel the increased PNMT in the ganglia. Dexamethasone slows the decrease in PNMT levels after cessation of NGF treatment, suggesting that dexamethasone decreases the rate of PNMT destruction. Examination of the ganglia by histofluorescence indicates that NGF increases the bundles of fibres while dexamethasone increases the number of small intensely fluorescent cells.     

EFFECTS OF DEXAMETHASONE AND NERVE GROWTH FACTOR ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE AND ADRENALINE IN ORGAN CULTURES OF NEWBORN RAT SUPERIOR CERVICAL GANGLION

Abstract— Phenylethanolamine N -methyltransferase (PNMT) and adrenaline (A) have been studied in organ cultures of neonatal rat sympathetic ganglia. Organ culture for 2 days without added nerve growth factor (NGF) caused a fall in noradrenaline (NA) and PNMT contents but there was no change in dopamine (DA) or A contents compared to controls. However, in the presence of dexamethasone, there was a marked increase in both PNMT activity and A content, but no change in NA or DA content. Addition of NGF to cultures stimulated with dexamethasone caused no further significant change in PNMT activity or A content, whereas both NA and DA were increased. Prolonged culture without NGF, in the presence of dexamethasone resulted in reductions in both NA and DA content, but the high levels of PNMT activity and A content were sustained. The results are consistent with the hypothesis that both PNMT and A are not contained in the noradrenergic cell bodies but are located chiefly within the small intensely fluorescent cells in sympathetic ganglia.     

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.     

EFFECT OF DEXAMETHASONE ON PHENYLETHANOLAMINE N-METHYLTRANSFERASE IN CHROMAFFIN TISSUE OF THE NEONATAL RAT

Abstract— Treatment of neonatal rats with dexamethasone resulted in the appearance of phenylethanolamine- N -methyltransferase (PNMT) and numerous small, intensely fluorescent (SIF) cells in abdominal paraganglia and in sympathetic paravertebral ganglia. These cells may be derived from a primitive stem cell precursor, but because of their unusual anatomical features, origin from ganglion cells cannot be altogether ruled out. Associated with the proliferation of the cells was a marked increase in the PNMT activity of the tissues. The PNMT response to the glucocorticoid was limited to the first few days of life, as was the SIF cell response. After discontinuance of dexamethasone, the enzyme activity fell very rapidly, while the number of cells declined at a slower rate.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase (PNMT) in organ culture of superior cervical ganglia

Glucocorticoid regulation of the adrenergic enzyme, phenylethanolamine N-methyltransferase (PNMT) was studied in organ cultures of the superior cervical ganglion (SCG) from newborn rats. Although PNMT catalytic activity was present in control ganglia, enzyme levels were too low to allow visualization of PNMT immunofluorescent cells. Addition of dexamethasone (DEX) or corticosterone to the medium resulted in a large increase in PNMT activity and bright PNMT immunoreactive (PNMT-IR) staining in cells resembling small, intensely fluorescent (SIF) cells. Addition of non-glucocorticoid steroids was ineffective. Exposure to a brief, 2-hr pulse of DEX (10(-6) M) in vitro elicited the same increase in PNMT as continual exposure to DEX. Studies using metabolic inhibitors demonstrated that the steroid-dependent increase in PNMT activity required both protein and RNA synthesis. Furthermore, the increase was inhibited by cytochalasin B and by the glucocorticoid receptor antagonists, DEX 21-mesylate and cortisol 21-mesylate. These observations suggest that glucocorticoids increase PNMT protein in SIF cells by interacting with specific steroid receptors that undergo translocation to the nucleus.     

CATECHOLAMINES IN SYMPATHETIC GANGLIA OF RAT: EFFECTS OF DEXAMETHASONE AND RESERPINE1

The superior cervical sympathetic ganglion of rats contains a finite amount of epinephrine all of which is of ganglionic origin. Treatment of new-born rats with dexamethasone for 8 days results in a 112-fold increase in the epinephrine concentration, whereas the norepinephrine and dopamine are increased by only 1·4- and 1·9-fold respectively. This epinephrine increase in newborn rats is reversible if treatment is discontinued, and it fails to occur in adult animals. The epinephrine store of normal and dexamethasone treated animals is resistant to the depletion by reserpine. There is no increase in the epinephrine content in organs innervated by axons emanating from the ganglion. The data presented support the localization of epinephrine in small intensely fluorescent cells in the ganglion and we propose that epinephrine may be released from these cells and function as a modulator of ganglionic transmission.     

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.     

A COMPARISON OF THE NEURAL REGULATION OF TYROSINE HYDROXYLASE ACTIVITY IN SYMPATHETIc GANGLIA OF ADULT MICE AND RATS

Surgical decentralization of the superior cervical ganglion (SCG) in rats and mice led to a fall in ganglionic tyrosine hydroxylase (T-OH) activity, and a loss of more than 90 per cent of the preganglionic neurone marker, choline acetyl transferase. T-OH activity was reduced by more than 50 per cent in mice SCG ten days after surgery, but fell by only 25 per cent in rat SCG after 21 days. The surgical procedure did not cause obvious histo-logical damage or loss of SCG cells in either species. Both T-OH and choline acetyl transferase activities in rat and mouse SCG recovered to normal three months after surgery. Reserpine treatment was more effective in rats in causing increased ganglionic T-OH activity than in mice. Neither decentralization nor reserpine treatment caused any changes in DOPA-decarboxylase or monoamine oxidase activities in rat SCG. These results demonstrate that T-OH activity in SCG is subject to trans-synaptic regulation in both rats and mice; this regulation does not apply to DOPA-decarboxylase or monoamine oxidase. Differences in basal sympathetic tone may explain the different results obtained in mice and rats.     

ACTH increases adrenal medullary PNMT activity in neonatal rats

Levels of plasma corticosterone and the activities of adrenomedullary dopamine-beta-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.     

Autoradiographic study of 3H-DOPA uptake by superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

The functional state of sympathetic ganglia in spontaneously hypertensive rats (SHR) was compared with that of ganglia in normotensive Wistar Kyoto rats (WKY) by examining catecholamine synthetic activity by light microscopic autoradiography 3H-L-dihydroxyphenyl alanine (3H-DOPA). The number of silver grains over the perikarya of ganglion cells in the superior cervical (SCG) and stellate ganglia (SG) of newborn, 10-day-old and 30-day-old animals was counted on photographic enlargements. There were significantly more silver grains over ganglion cells in SHR compared with those in age-matched WKY at almost all incorporation times at all ages examined in SCG, at all incorporation times in newborn rats, and at incorporation times of 15 and 60 min in SG of 10-day-old rats. The increased incorporation of the label by both sympathetic ganglia was more marked in newborn than in 30-day-old animals. These results indicate that catecholamine synthetic activity in these ganglion cells is increased in SHR from the newborn stage, suggesting that a congenital hyperfunction of sympathetic ganglia occurs in SHR.     

Regeneration of Cholinesterases in the Stellate and Normal and Denervated Superior Cervical Ganglia of the Cat Following Inactivation by Sarin

Abstract: Experiments were designed to test the hypothesis that ganglionic butyrylcholinesterase (BuChE) is derived from acetylcholinesterase (AChE). At 5 to 8 days following preganglionic denervation of the right superior cervical ganglion (SCG), cats were given sarin, 2.0 μmol/kg, i.v. At intervals of 1 h and 1, 2, 3, 6, 11, and 22 days later, they were killed, and the AChE and BuChE contents of both SCG and both stellate ganglia (StG) were assayed. The regeneration of AChE in the normal ganglia occurred in two phases: an initial rapid phase, to 25-40% of control activity in 1 day, and a slow phase, to approximately 70% of control activity in 22 days. BuChE reached approximately 85% of control activity in normal SCG and StG at 22 days. In the denervated SCG, AChE activity reached a maximum of approximately 17% of normal at 1 day, the value prior to the administration of sarin, and did not increase appreciably above this subsequently. BuChE activity in the denervated SCG reached approximately 50% of normal ganglia at 22 days. At each interval, its activity approached 55% of that of the contralateral normal SCG, the value found in the denervated SCG prior to the administration of sarin. Hence, the regeneration of BuChE appears to be independent of the presence of AChE in the neuropil. The origin of ganglionic BuChE remains obscure.     

Developmental profiles of catecholaminergic enzymes in adrenals of perinatal rats: effect of a hypoxic environment

Fetal and early neonatal development of adrenal catecholaminergic enzymes was studied in rats maintained under normal (normoxic) and high-altitude, 3800 m, 13% PO2 (hypoxic) conditions. In adrenals of normoxic fetuses, tyrosinehydroxylase (TH), DOPA-decarboxylase (DDC), phenylethanolamine-N-methyltransferase (PNMT), catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) showed rapid increases in activity from day 19 to day 21 of gestation. The activities of all enzymes but TH were higher at day 1 postpartum compared to fetal values: TH was equiactive just before and after birth. In animals conceived, born and raised at high altitude, several changes indicative of impaired adrenal development occurred. The activities of the synthesizing enzymes, TH, DDC and PNMT, were variably affected at some time during the perinatal period. The activities of the catabolizing enzymes, MAO and COMT, at high altitude were increased on the last days of gestation but depressed after birth, compared to control levels. Catecholamine content in high-altitude adrenals was altered on day 19 of gestation when epinephrine was lower, and again on day 1 postpartum when both norepinephrine and epinephrine were higher than in control adrenals at sea level. Normal developmental changes and high-altitude-induced disturbances in adrenal catecholaminergic enzymes are discussed with reference to differences observed in adrenal cortical function between sea-level and high-altitude animals.     

Effect of hydrocortisone on catecholamines and the enzymes synthesizing them in the developing sympathetic ganglion

Summary Newborn rats were daily injected with 0.2 mg hydrocortisone acetate for seven days. They were killed 1, 7 or 21 days after the last injection, together with untreated controls. Hydrocortisone caused a great increase in the number of the small, intensely fluorescent (SIF) cells and the appearance of similar small cells with intense immunohistochemical reactions for tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine (noradrenaline)N-methyltransferase (PNMT) in the superior cervical ganglion. At the same time, the adrenaline content and the PNMT activity of the ganglion greatly increased, while no significant changes were observed in the dopamine or noradrenaline content or TH or DBH activity. All these changes essentially disappeared after a recovery period of seven or 21 days.It is concluded that hydrocortisone causes a temporary increase in the number of SIF cells by causing a synthesis of TH, DBH and PNMT in previously existing small, non-fluorescent cells, which start to synthesize and store adrenaline, thus becoming intensely fluorescent SIF cells. These SIF cells are different from the normal SIF cells of the same ganglion, most of which appear at a later stage of postnatal development when response to hydrocortisone is lost, which contain TH but neither DBH nor PNMT, and which permanently remain in the ganglion.     

Influence of cold exposure and thyroid hormones on regulation of adrenal catecholamines.

Since thyroid hormones influence urinary excretion of catecholamines after exposure to cold, the effects of hyper- and hypo-thyroidism on adrenal tyrosine hydroxylase (TH) (EC 1.14.16.2), phenylethanolamine-N-methyl transferase (PNMT) (EC 2.1.1.28), and serum dopamine-beta-hydroxylase (DbetaH) (EC 1.14.17.1) of rats of 23 and 4 degrees C were studied. TH changes resembled the urinary excretion pattern at 4 degrees C in being higher after 8 days than after 1 day of exposure, and in declining as acclimation occurred. At 23 degrees C, TH activity of hypothyroid rats was significantly higher than in euthyroid or hyperthyroid animals, and after 1 day at 4 degrees C the value increased even more. While in the hypothyroid animals at 4 degrees C the concentration of adrenal catecholamines was less, the epinephrine to norepinephrine ratio was higher than at 23 degrees C. Very high TH activity with a decline in catecholamine concentration suggests that the capacity of TH had been exceeded. PNMT activity was significantly elevated in this group. TH activity was not decreased in the hyperthyroid group at 23 degrees C, and was increased after 8 days at 4 degrees C, suggesting that circulating thyroid hormones have no direct inhibitory effect on TH. Serum DbetaH was elevated after exposure to 4 degrees C, regardless of thyroid hormonal status. The activation of adrenal TH in hypothyroid rats at 23 degrees C and of TH, PNMT, and serum DbetaH at 4 degrees C is probably the result of increased activity of the sympathetic nervous system.     

Alterations in γ-aminobutyric acid content in the rat superior cervical ganglion and pineal gland

The endogenous γ-aminobutyric acid (GABA) content of the rat pineal gland and superior cervical ganglion (SCG) was measured by high pressure liquid chromatography. It was found that GABA levels in both tissues increased after decapitation of the animals. The GABA content of tissues frozen within 20 seconds after decapitation was the same as that of tissues removed from animals killed by microwave irradiation. Amino-oxyacetic acid, a GABA-transaminase inhibitor, increased the endogenous GABA content of both of these tissues. Dimethylphenylpiperizinium or isoniazid administration did not alter GABA levels in these tissues. Isoproterenol increased the GABA content of the SCG but did not change the pineal gland GABA levels. The ability of the pineal gland to take up and accumulate ³H-GABA was significantly reduced in rats that had been ganglionectomized. A fluctuation in endogenous GABA levels in the pineal gland was seen to occur when measures were taken at different times of the day. These results tend to suggest that GABA may have some functional role in the pineal gland and the superior cervical ganglion.     

BIOCHEMICAL AND MORPHOLOGICAL EFFECTS OF TESTOSTERONE TREATMENT ON DEVELOPING SYMPATHETIC NEURONS

Abstract— Neonatal rats were treated with testosterone propionate (TP) or isoproterenol (ISO) to determine whether (1) increases in salivary gland mass are associated with alteration of developing sympathetic neurons and (2) whether effects on neuron growth are secondary to altered target mass itself or to increases in salivary growth factors. TP treatment is known to result in salivary tubule hypertrophy and elevated nerve growth factor (NGF) content whereas IS0 treatment results in acinar hypertrophy and no known alteration in NGF. TP treatment increased submaxillary gland weight as well as tyrosine hydroxylase (T-OH) activity, adrenergic neuron numbers and total protein in the innervating superior cervical ganglion (SCG). Unilateral sialectomy prevented the increase in T-OH activity in the SCG, suggesting that the salivary glands were necessary for this effect. T-OH activity and total protein were elevated in the distant sixth lumbar sympathetic ganglion after TP treatment, suggesting that sympathetic development as a whole was affected and that humoral factors may be involved. Salivary gland weight was also elevated following ISO administration, but T-OH activity in the SCG was not affected. These observations suggest that TP treatment increases T-OH activity and sympathetic neuron numbers by alteration of specific salivary humoral growth factor(s).     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.     

Hormone mediated changes in monoamine stores and regulation of enzymes of biosynthesis and metabolism in the rat adrenal gland. Influence of progesterone, estradiol, ACTH and testosterone administration

The influence of daily injections, for 7 days, of 17-hydroxyprogesterone caproate (8 mg/100 g body weight pro die), of estradiol-17 beta 17-undecylate (1.2 mg/100 g body weight pro die) of testosterone enanthate (2 mg/100 g body weight pro die) and of ACTH (4 IU/100 g body weight pro die) to male Wistar rats, weighing 200 g, were investigated. Monamine storage and regulation of enzymes phenylethanolamine N-methyl transferase (PNMT), monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) were studied in the adrenal glands. Results from testosterone treated rats were especially significant : the hormone administration decreased the stores of dopamine and norepinephrine (epinephrine precursors), stimulated PNMT and decreased metabolism of epinephrine by MAO and COMT. However, the adrenal content of epinephrine remained in the normal range. These findings suggest that uptake of epinephrine is accelerated in peripheral tissues by testosterone.     

Morphometric study of the superior cervical and stellate ganglia of spontaneously hypertensive rats during the prehypertensive stage

To compare the functional state of the superior cervical (SCG) and stellate sympathetic ganglia (SG) of spontaneously hypertensive rats (SHR) with those of age-matched normotensive Wistar Kyoto rats (WKY), ganglion cell volume and area occupied by ganglion cells relative to each whole ganglionic area were morphometrically examined using the Texture Analyse System (TAS) in rats at 0, 10 and 30 days of age. The weight of each ganglion relative to animal weight was also measured. The ganglion cell volume and the relative area of ganglionic cells in both ganglia of SHR were significantly larger (P<0.05) than those of age-matched WKY at ages 0 and 10 days after birth. The relative ganglionic weights of SHR were significantly larger (P<0.01) compared with those of WKY at all ages examined, except for SG at 0 days after birth. These results show that the relative volume of sympathetic ganglion cells is greater in both SCG and SG of SHR than that of WKY, suggesting that hyperfunction of sympathetic ganglia occurs at the prehypertensive stage as a primary factor in the development of hypertension in SHR.