CATECHOLAMINES: EFFECTS OF ACTH AND ADRENAL CORTICOIDS Julius Axelrod Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland 20014
Over the past decade, considerable evidence has accumulated showing that ACTH in the pituitary and glucocorticoids in the adrenal cortex have an important influence in regulating the biosynthesis of catecholamines in the adrenal medulla. It had been observed that the ratio of adrenaline to noradrenaline in the adrenal depends on the amount of cortex surrounding the medu1la.l Species having a high ratio of adrenal cortex to medulla have the highest concentration of adrenaline and vice versa. These findings suggested that the adrenal cortex had an influence in the synthesis of catecholamines in the adrenal meduIla. It was also noted that the catecholamine-containing extramedullary chromaffin cells in rats disappeared soon after birth and that the catecholamines in these chromaffin tissues could be maintained after injections of large amounts of glucocorticoids.2 These findings indicated that glucocorticoids secreted from the adrenal cortex into the adrenal medulla somehow controlled the synthesis of catecholamines. With the isolation and characterization of the catecholamine biosynthetic enzymes, it was possible to examine the effects of glucocorticoids and ACTH on the enzymes in the adrenal medulla and other chromaffin tissues.
BIOSYNTHESIS OF CATECHOLAMINES Catecholamines are synthesized from tyrosine by four enzymes3: tyrosine hydroxylase, dopa-decarboxylase, dopamine P-oxidase, and phenylethanolamine 1) . N-methyltransferase (PNMT) (FIGURE Tyrosine hydroxylase converts L-tyrosine to dopa and it is the rate enzyme in catecholamine formation. This enzyme is a mixed function oxidase and requires tetrahydropteridine, 0,, and Fe++as cofactors. Tyrosine hydroxylase is localized in noradrenergic and dopaminergic nerves and in the adrenal medula. Dopa-decarboxylase is somewhat nonspecific. It decarboxylates dopa as well as other aromatic amino acids and it is widely distributed in tissues. Dopamine P-oxidase is a copper-containing mixed-function oxidase and hydroxylates dopamine as well as other phenylethylamines on the fl carbon. It is highly localized in chromaffin granules of the adrenal medulla and in storage vesicles of noradrenergic nerve terminals. The final step in catecholamine biosynthesis is catalyzed by phenylethanolamine N-methyltransferase. This enzyme methylates noradrenaline as well as other P-hydroxylated phenylethanolamine derivatives; S-adenosylmethionine serves as the methyl donor. PNMT Is highly localized in the cytosol of the adrenal medulla, but it is also found in extra-
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Annals New York Academy of Sciences
im NORADRENALINE
ADRENALINE
FIGURE 1. Biosynthetic pathway for catecholamines. medullary chromaffin cells of newborn rats, in certain brain areas of mammals, and in peripheral and central nerves of amphibians.
REGULATION OF PNMT BY GLUCOCORTICOIDS AND ACTH The relationship between glucocorticoids, ACTH, and catecholamine formation was first examined with PNMT.4 This enzyme was chosen because of its high localization in the adrenal medulla, and because a specific and reproducible assay was a ~ a i l a b l e .The ~ corticoid content of the rat adrenal gland was reduced by removal of the pituitary. One week after hypophysectomy there was a profound (80%) fall in PNMT activity in the adrenal medulla (FIGURE 2). A reduction in the adrenaline content of the adrenal was also observed. The administration of ACTH to hypophysectomized rats for 6 days restored 2).l It appeared likely that PNMT activity to almost normal levels (FIGURE ACTH increased PNMT activity in hypophysectomized rats by stimulating
100
1
CNYT
HYDROXVLASE
WIAYINI &ORlDASE.
FIGURE2. Effect of hypophysectomy and ACTH on catecholamine biosynthetic enzymes in the rat adrenal medulla. Rats were killed 17 days after hypophysectomy and PNMT, tyrosine hydroxylase, and dopamine /3-oxidase activity measwed in the adrenal gland. ACTH (4 units) S.C. was given daily for 7 days after hypophysectomy. 0=Normal; LZ4 =Hypox; IIIlIIl= Hypox+ACTH.
Axelrod: Catecholamines
277
secretion of glucocorticoids. This was established by daily injections of large doses of dexamethasone for several days. This potent glucocorticoid restored PNMT activity to hypophysectomized rats. The concentration of glucocorticoid perfusing the adrenal medulla from the portal blood of the cortex is high and apparently sufficient to maintain the levels of PNMT. These experiments clearly indicated that the enzymatic methylation of noradrenaline to adrenaline in the adrenal medulla is regulated by glucocorticoids from the adrenal cortex. Elevation of PNMT by ACTH or glucocorticoids might be due to enzyme activation or synthesis of new protein molecules. The addition of hydrocortisone or dexamethasone to adrenal homogenates had no effect on the activity of PNMT.4 The increase of PNMT activity by dexamethasone in hypophysectomized rats was blocked by protein-synthesis inhibitors, indicating that glucocorticoid increases PNMT activity by stimulating synthesis of new enzyme molecule^.^ The action of glucocorticoids was found to be highly specific; estrogens and testosterone had no effect in restoring enzyme activity in hypophysectomized rats whereas aldosterone had a slight action.6 The repeated administration of ACTH or dexamethasone for several days did not increase PNMT activity in normal rats. However, the enzyme was elevated in rats by persistent elevation in ACTH levels after unilateral adrenalectomy or transplanted ACTH-secreting tumors.X PNMT activity can be increased in adrenals of normal rats after chronic stress, induced by immobili~ation,~ increased salt intake, continuous darkness or light, or injections of insulin and glucagon. It is not clear whether these increases in enzyme activity are due to elevated levels of ACTH and glucocorticoids or to neuronal stimulation. In the case of immobilization-stress it appears that PNMT levels are elevated by both increased activity of the neurons and of the pituitary adrenocortical system. PNMT activity in the adrenal gland can be increased in some strains of mice with intact pituitaries after an injection of corticoids. These results indicate that there is a genetic component to the regulation of PNMT by glucocorticoids. In the fetal rat, adrenaline is first detected in the adrenal gland at 17 days of gestation.1° Just before the initial appearance of PNMT in the fetal adrenal gland there is an increase in steroid synthesis in adrenal cortical cells on day 16 of gestation. The appearance of adrenaline follows the appearance of PNMT in the adrenal gland, and the enzyme then increases rapidly until birth. This suggested that there was a temporal relationship in the fetal rat in the initiation of adrenaline synthesis, PNMT, and corticoids. To establish this association, fetal rats were hypophysectomized by decapitation in vitro at 18 days of gestati0n.l’ PNMT activity was absent in the adrenal medulla and the levels of adrenaline were sharply reduced. The administration of ACTH or corticoids to the mother restored both PNMT activity and adrenaline level to the decapitated fetus. CHROMAFFIN TISSUES GLUCOCORTICOIDS, PNMT AND EXTRA-ADRENAL There is an extensive network of extra-adrenal chromaffin tissue adjacent to the thoracic and abdominal aorta in the coratid body, heart, and sympathetic ganglia, especially in fetal rats. Extra-adrenal chromaffin tissues contains noradrenaline and only trace amounts of PNMT. After the administration of glucocorticoids to newborn rats, chromaffin cells of the abdominal sympathetic
278
Annals New York Academy of Sciences
paraganglia undergo marked hyperplasia and adrenaline appeam2 There is also the appearance of small intensely fluorescent (SIF) cells after an injection of hydrocortisone.13 This suggested that as in the adrenal medulla the corticoids might induce the adrenaline-forming enzyme (PNMT) in extra-chromaffin tissues of newborn rats. Treatment of neonatal rats with dexamethasone caused a sharp elevation of PNMT (FIGURE 3 ) as well as of the small intensely fluorescent catecholamine cells in the abdominal paraganglia and in sympathetic cervical ganglia.l4 The induction of PNMT is accompanied by an increase in the concentration of adrenaline in the ganglia.l5 The elevation of PNMT and the increase of SIF cells was limited to the few days after birth. After the corticoid was discontinued, the enzyme activity fell rapidly and the SIF population decreased, but at a slower rate.14 PNMT was found to be present in a few areas of the brain, especially the brain stem.le Using an antibody directed towards PNMT as a marker, adrenaline-containing nerve tracts were found in the brain.'T Unlike PNMT in the adrenal medulla, the enzyme in the brain is not affected by removal of the pituitary or the adrenal gland. The injection of very large doses of dexamethasone will elevate PNMT in the brain to a small extent.l8 EFFECT OF ACTH
AND DEXAMETHASONE ON DOPAMINE P-OXDASE AND TYROSINE HYDROXYLASE
Two other catecholamine biosynthetic enzymes in the rat adrenal medulla are affected by ACTH and glucocorticoids, tyrosine hydroxylase and dopamine P-0xidase.l After hypophysectomy, tyrosine hydroxylase in the adrenal medulla falls with a half-life of 10 days.lg Denervation of the adrenal gland also causes a fall in tyrosine hydroxylase with a slower half-life of 21 days. The repeated injection of ACTH prevents the decline in adrenal tyrosine hydroxy-
AGE
IN DAYS
FIGURE3. Induction of PNMT in superior cervical ganglia of newborn rats with dexamethasone. Dexamethasone (0.1 p g / g ) was given in 3 daily injections, and the = saline-treated. Each rats were killed 1 day later. B = dexamethasone-treated; bar represents mean+SEM of 5 to 8 animals. *p
Over the past decade, considerable evidence has accumulated showing that ACTH in the pituitary and glucocorticoids in the adrenal cortex have an important influence in regulating the biosynthesis of catecholamines in the adrenal medulla. It had been observed that the ratio of adrenaline to noradrenaline in the adrenal depends on the amount of cortex surrounding the medu1la.l Species having a high ratio of adrenal cortex to medulla have the highest concentration of adrenaline and vice versa. These findings suggested that the adrenal cortex had an influence in the synthesis of catecholamines in the adrenal meduIla. It was also noted that the catecholamine-containing extramedullary chromaffin cells in rats disappeared soon after birth and that the catecholamines in these chromaffin tissues could be maintained after injections of large amounts of glucocorticoids.2 These findings indicated that glucocorticoids secreted from the adrenal cortex into the adrenal medulla somehow controlled the synthesis of catecholamines. With the isolation and characterization of the catecholamine biosynthetic enzymes, it was possible to examine the effects of glucocorticoids and ACTH on the enzymes in the adrenal medulla and other chromaffin tissues.
BIOSYNTHESIS OF CATECHOLAMINES Catecholamines are synthesized from tyrosine by four enzymes3: tyrosine hydroxylase, dopa-decarboxylase, dopamine P-oxidase, and phenylethanolamine 1) . N-methyltransferase (PNMT) (FIGURE Tyrosine hydroxylase converts L-tyrosine to dopa and it is the rate enzyme in catecholamine formation. This enzyme is a mixed function oxidase and requires tetrahydropteridine, 0,, and Fe++as cofactors. Tyrosine hydroxylase is localized in noradrenergic and dopaminergic nerves and in the adrenal medula. Dopa-decarboxylase is somewhat nonspecific. It decarboxylates dopa as well as other aromatic amino acids and it is widely distributed in tissues. Dopamine P-oxidase is a copper-containing mixed-function oxidase and hydroxylates dopamine as well as other phenylethylamines on the fl carbon. It is highly localized in chromaffin granules of the adrenal medulla and in storage vesicles of noradrenergic nerve terminals. The final step in catecholamine biosynthesis is catalyzed by phenylethanolamine N-methyltransferase. This enzyme methylates noradrenaline as well as other P-hydroxylated phenylethanolamine derivatives; S-adenosylmethionine serves as the methyl donor. PNMT Is highly localized in the cytosol of the adrenal medulla, but it is also found in extra-
275
276
Annals New York Academy of Sciences
im NORADRENALINE
ADRENALINE
FIGURE 1. Biosynthetic pathway for catecholamines. medullary chromaffin cells of newborn rats, in certain brain areas of mammals, and in peripheral and central nerves of amphibians.
REGULATION OF PNMT BY GLUCOCORTICOIDS AND ACTH The relationship between glucocorticoids, ACTH, and catecholamine formation was first examined with PNMT.4 This enzyme was chosen because of its high localization in the adrenal medulla, and because a specific and reproducible assay was a ~ a i l a b l e .The ~ corticoid content of the rat adrenal gland was reduced by removal of the pituitary. One week after hypophysectomy there was a profound (80%) fall in PNMT activity in the adrenal medulla (FIGURE 2). A reduction in the adrenaline content of the adrenal was also observed. The administration of ACTH to hypophysectomized rats for 6 days restored 2).l It appeared likely that PNMT activity to almost normal levels (FIGURE ACTH increased PNMT activity in hypophysectomized rats by stimulating
100
1
CNYT
HYDROXVLASE
WIAYINI &ORlDASE.
FIGURE2. Effect of hypophysectomy and ACTH on catecholamine biosynthetic enzymes in the rat adrenal medulla. Rats were killed 17 days after hypophysectomy and PNMT, tyrosine hydroxylase, and dopamine /3-oxidase activity measwed in the adrenal gland. ACTH (4 units) S.C. was given daily for 7 days after hypophysectomy. 0=Normal; LZ4 =Hypox; IIIlIIl= Hypox+ACTH.
Axelrod: Catecholamines
277
secretion of glucocorticoids. This was established by daily injections of large doses of dexamethasone for several days. This potent glucocorticoid restored PNMT activity to hypophysectomized rats. The concentration of glucocorticoid perfusing the adrenal medulla from the portal blood of the cortex is high and apparently sufficient to maintain the levels of PNMT. These experiments clearly indicated that the enzymatic methylation of noradrenaline to adrenaline in the adrenal medulla is regulated by glucocorticoids from the adrenal cortex. Elevation of PNMT by ACTH or glucocorticoids might be due to enzyme activation or synthesis of new protein molecules. The addition of hydrocortisone or dexamethasone to adrenal homogenates had no effect on the activity of PNMT.4 The increase of PNMT activity by dexamethasone in hypophysectomized rats was blocked by protein-synthesis inhibitors, indicating that glucocorticoid increases PNMT activity by stimulating synthesis of new enzyme molecule^.^ The action of glucocorticoids was found to be highly specific; estrogens and testosterone had no effect in restoring enzyme activity in hypophysectomized rats whereas aldosterone had a slight action.6 The repeated administration of ACTH or dexamethasone for several days did not increase PNMT activity in normal rats. However, the enzyme was elevated in rats by persistent elevation in ACTH levels after unilateral adrenalectomy or transplanted ACTH-secreting tumors.X PNMT activity can be increased in adrenals of normal rats after chronic stress, induced by immobili~ation,~ increased salt intake, continuous darkness or light, or injections of insulin and glucagon. It is not clear whether these increases in enzyme activity are due to elevated levels of ACTH and glucocorticoids or to neuronal stimulation. In the case of immobilization-stress it appears that PNMT levels are elevated by both increased activity of the neurons and of the pituitary adrenocortical system. PNMT activity in the adrenal gland can be increased in some strains of mice with intact pituitaries after an injection of corticoids. These results indicate that there is a genetic component to the regulation of PNMT by glucocorticoids. In the fetal rat, adrenaline is first detected in the adrenal gland at 17 days of gestation.1° Just before the initial appearance of PNMT in the fetal adrenal gland there is an increase in steroid synthesis in adrenal cortical cells on day 16 of gestation. The appearance of adrenaline follows the appearance of PNMT in the adrenal gland, and the enzyme then increases rapidly until birth. This suggested that there was a temporal relationship in the fetal rat in the initiation of adrenaline synthesis, PNMT, and corticoids. To establish this association, fetal rats were hypophysectomized by decapitation in vitro at 18 days of gestati0n.l’ PNMT activity was absent in the adrenal medulla and the levels of adrenaline were sharply reduced. The administration of ACTH or corticoids to the mother restored both PNMT activity and adrenaline level to the decapitated fetus. CHROMAFFIN TISSUES GLUCOCORTICOIDS, PNMT AND EXTRA-ADRENAL There is an extensive network of extra-adrenal chromaffin tissue adjacent to the thoracic and abdominal aorta in the coratid body, heart, and sympathetic ganglia, especially in fetal rats. Extra-adrenal chromaffin tissues contains noradrenaline and only trace amounts of PNMT. After the administration of glucocorticoids to newborn rats, chromaffin cells of the abdominal sympathetic
278
Annals New York Academy of Sciences
paraganglia undergo marked hyperplasia and adrenaline appeam2 There is also the appearance of small intensely fluorescent (SIF) cells after an injection of hydrocortisone.13 This suggested that as in the adrenal medulla the corticoids might induce the adrenaline-forming enzyme (PNMT) in extra-chromaffin tissues of newborn rats. Treatment of neonatal rats with dexamethasone caused a sharp elevation of PNMT (FIGURE 3 ) as well as of the small intensely fluorescent catecholamine cells in the abdominal paraganglia and in sympathetic cervical ganglia.l4 The induction of PNMT is accompanied by an increase in the concentration of adrenaline in the ganglia.l5 The elevation of PNMT and the increase of SIF cells was limited to the few days after birth. After the corticoid was discontinued, the enzyme activity fell rapidly and the SIF population decreased, but at a slower rate.14 PNMT was found to be present in a few areas of the brain, especially the brain stem.le Using an antibody directed towards PNMT as a marker, adrenaline-containing nerve tracts were found in the brain.'T Unlike PNMT in the adrenal medulla, the enzyme in the brain is not affected by removal of the pituitary or the adrenal gland. The injection of very large doses of dexamethasone will elevate PNMT in the brain to a small extent.l8 EFFECT OF ACTH
AND DEXAMETHASONE ON DOPAMINE P-OXDASE AND TYROSINE HYDROXYLASE
Two other catecholamine biosynthetic enzymes in the rat adrenal medulla are affected by ACTH and glucocorticoids, tyrosine hydroxylase and dopamine P-0xidase.l After hypophysectomy, tyrosine hydroxylase in the adrenal medulla falls with a half-life of 10 days.lg Denervation of the adrenal gland also causes a fall in tyrosine hydroxylase with a slower half-life of 21 days. The repeated injection of ACTH prevents the decline in adrenal tyrosine hydroxy-
AGE
IN DAYS
FIGURE3. Induction of PNMT in superior cervical ganglia of newborn rats with dexamethasone. Dexamethasone (0.1 p g / g ) was given in 3 daily injections, and the = saline-treated. Each rats were killed 1 day later. B = dexamethasone-treated; bar represents mean+SEM of 5 to 8 animals. *p
