Brain Research, 113 (1976) 349-362
© ElsevierScientificPublishingCompany,Amsterdam- Printedin The Netherlands
REGULATION OF RAT ADRENAL DOPAMINE fl-HYDROXYLASE. II. RECEPTOR INTERACTION IN THE REGULATION OF ENZYME SYNTHESIS AND DEGRADATION
ROLANDD. CIARANELLO*,G. FREDERICKWOOTEN** and JULIUSAXELROD Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20014 (U.S.A.)
SUMMARY Rat adrenal gland dopamine fl-hydroxylase is under neuronal regulation from the splanchnic nerve and hormonal control via adrenal cortical glucocorticoids. These regulatory systems act in different ways; neuronal stimuli induce dopamine fl-hydroxylase synthesis while hormonal stimulation inhibits enzyme degradation. Despite these mechanistic differences, both systems require a normally innervated cholinergic receptor to exert their effect. The enzyme response to either neural stimulation or ACTH administration is blocked by splanchnic denervation. Glucocorticoid stimulation of dopamine fl-hydroxylase, however, can occur after adrenal denervation, suggesting that ACTH acts on a receptor which requires splanchnic innervation, but glucocorticoids act distal to the receptor. Similar results were obtained when the effect of these manipulations were studied on phenylethanolamine N-methyltransferase, another enzyme in the catecholamine biosynthetic pathway. A model attempting to unify these and earlier findings is presented, in which the splanchnic nerve is involved in regulating both adrenal cortical glucocortieoidogenesis (by allowing ACTH to act on glucocorticoid synthesis) and adrenal medullary catecholamine biosynthesis (by induction of enzyme synthesis).
INTRODUCTION Dopamine fl-hydroxylase catalyzes the final reaction in noradrenaline biosynthesis. Adrenal medullary dopamine fl-hydroxylase is under neural regulation from the splanchnic nervesS,1° and hormonal control from the pituitary gland and the Present address:Departmentof Psychiatry,StanfordMedicalCenter,Stanford,Calif.94305,U.S.A. ** Present address: Departmentof Neurology,CornellUniversityMedicalCollege,New York, N.Y. 10021, U.S.A. *
350 adrenal cortex5,7,18. Reflex activation of the splanchnic nerves to the adrenal by reserpine administration 5,1° or by direct stimulation with cholinergic agonists 14 results in an increase in dopamine fl-hydroxylase activity. The effects of neural stimulation on dopamine fl-hydroxylase can be blocked by cutting the nerve to the adrenal glandS, 1°. Hypophysectomy results in a marked decline in adrenal medullary dopamine fl-hydroxylaseS,7, TM. Administration of ACTH s,ls or dexamethasone s restores the enzyme levels. While reserpine will induce dopamine fl-hydroxylase above control levels in intact rats, ACTH or dexamethasone restores DBH activity only in hypophysectomized rats. Moreover the enzyme levels are restored only to, but not above, control values by hormone treatment. The contrast between the effects of reserpine and those of ACTH or dexamethasone suggests that the hormonal and neural regulators of dopamine fl-hydroxylase act by different biochemical mechanisms. By combined immunochemical and radiolabeling techniques, we have previously shown that the neural and hormonal controls act by regulating dopamine fl-hydroxylase synthesis and degradation s. Neural stimulation induces dopamine fl-hydroxylase synthesis without altering the rate of enzyme degradation. Hypophysectomy, on the other hand, decreases dopamine fl-hydroxylase levels by accelerating the rate of enzyme degradation without initially affecting the rate of enzyme synthesis. ACTH partially reverses the effects of hypophysectomy on dopamine fl-hydroxylase degradation. Since reserpine increases enzyme levels in the intact rat, we proposed that the failure of ACTH to elevate dopamine fl-hydroxylase in non-hypophysectomized rats might be explained if the degree of inhibition of enzyme degradation by ACTH was already maximal s. In the course of our studies, we became interested in studying the interrelationships between the hormonal and neural regulators of dopamine fl-hydroxylase. We were particularly interested in learning to what extent these two systems interacted. The studies described here show that a cholinergic receptor mediates the transsynaptic induction of dopamine fl-hydroxylase synthesis. The functional activity of this receptor is not dependent on normal adrenal cortical function, since reserpine continues to effectively induce dopamine fl-hydroxylase synthesis even in hypophysectomized rats. Similarly, an intact splanchnic neuronal supply is essential for the functional activity of the receptor which mediates the effects of ACTH on dopamine fl-hydroxylase. ACTH fails to restore dopamine fl-hydroxylase levels in the denervated adrenal of hypophysectomized rats, but is effective in the innervated gland. These results suggest that innervated cholinergic receptors are essential for both the transsynaptic induction of dopamine fl-hydroxylase synthesis and for the ACTH-mediated inhibition of dopamine fl-hydroxylase degradation. A speculative model attempting to unify these findings is presented. MATERIALS A N D METHODS
Animals. Male Sprague-Dawley rats, weighing from 150 to 200 g, were obtained from Zivic-Miller Laboratories (Allison Park, Pa.). Surgical procedures were
351 performed by the supplier when the rats reached 150 g. Adequacy of hypophysectomy was confirmed by inspection of the sella turcica. Dopamine fl-hydroxylase assay. Adrenal glands were homogenized in 5 ml per gland of 5 m M Tris buffer, p H 7.4, containing 150 m M NaC1, 0.2% (v/v), Triton X-100, and 0.2% (w/v) bovine serum albumin. Homogenates were centrifuged at 30,000 × g for 15 min. Dopamine fl-hydroxylase was assayed on 200/~1 portions of the supernatant by a previously described method n, using phenylethylamine as substrate. The enzyme assay was carried out in the presence of 13 # M CuSO 4 to inactivate endogenous tissue inhibitors. Phenylethanolamine N-methyltransferase was purified by a previously published technique a and was used in the second step of the dopamine fl-hydroxylase assay 11. Phenylethanolamine N-methyltransferase levels were assayed as previously described 5 on 50-100/zl portions of the adrenal supernatant. Phenylethanolamine was used as substrate.
Purification of dopamine fl-hydroxylase and preparation of specific antiserum. A specific antiserum to dopamine fl-hydroxylase was obtained by purifying the enzyme from bovine adrenal medullaeS, ~9. Purified dopamine fl-hydroxylase was injected into a male New Zealand rabbit and antiserum was obtained. The isolation methods used and the verification of antiserum specificity have appeared in a previous reportS, 19. -----35
I0' ~ophysectomized ~~hypophysectomized "
./.d antiserum (1:5)
Fig. 1. Immunoprecipitation of dopamin¢ fl-hydroxylascfrom adrenal gland preparations of hypophysectomized and intact rats. Samples were prepared as described in Materials and Methods. Varying volumes of diluted (in 150 mM NaCI) antiserum were added, to constant portions of adrenal supernatants. Equivalence point determinations were made by regression analysis of the linear portion of the curve. The ratio of control enzyme activity to that of the hypophysectomized rats was 2.67, while the ratio of equivalence points was 2.10. These values indicate the correlation between enzyme activity and enzymeprotein content.
Immunotitration of dopamine fl-hydroxylase present in rat adrenal supernatants. Adrenal glands were homogenized and centrifuged as described. One ml portions of the supernatant were transferred to l0 m m ~ 75 m m glass test tubes, and varying amounts of antiserum were added. The tubes were incubated at 37 °C for 60 min and overnight in ice. The immune precipitates were removed by centrifugation at 37,000 g for 30 rain. Enzyme activity remaining in the supernatant fraction was then assayed by the usual methods. A titration curve, such as that shown in Fig. l, was then constructed. The slope of these curves was estimated by linear-regression analysis of the linear portion of the curve. The intersection of this extrapolated line with the abscissa provided an estimate of the amount of antiserum required to completely remove the antigen. This was labeled the equivalence point. Drugs. Reserpine (Serpasil, Ciba) was administered intraperitoneally in a dose of 2.5 mg/kg every other day. A C T H ( A C T H a r gel, Armour) was given subcutaneously, 4 I.U. per day. Dexamethasone phosphate (Decadron, Merck, Sharp and Dohme) was given in a dose of 1 mg/animal/day. RESULTS
Effects of hypophysectomy on dopamine fl-hydroxylase in rat adrenal glands Hypophysectomy causes a profound decrease in rat adrenal dopamine flhydroxylase activity (Table I). This decline is due to a decrease in the number of enzyme molecules. Immunotitration studies carried out on adrenal preparations from hypophysectomized rats show a decrease in the antiserum equivalence point, which closely parallels the decline in enzyme activity (Fg. 1). By measuring incorporation of radioactive amino acids into immunoprecipitable dopamine fl-hydroxylase, we have shown that hypophysectomy reduces dopamine fl-hydroxylase levels by accelerating the rate of enzyme degradation 5. A C T H or dexamethasone treatment restored TABLE l
Effect of hypophyseetomy and drug treatment on adrenaldopamine fl-hydroxylase activtty Rats were hypophysectomized 7 days prior to initiation of drug treatment. Each group contained 8 animals. ACTH, 4 I.U.s.c., and dexamethasone, 5 mg/kg i.p., were ~ven daily for 3 days. Reserpine, 2.5 mg/kg s.c., was administered every other day for 6 days for a total of 3 injections. Animals were killed 24 h after the last injection. Enzyme values are means and standard errors.
None ACTH Dexarnethasone Reserpine
Dopamme t~-hydroxylase (units/adrenalgland) Sham hypophysectomy
27.6 ± 32.1 ~ 35.1 t 74.0 ±
7.9 ± 1.4"* 20.0 :t: 1.3"** 30.3 :t: 5.9*** 45.5 :k: 4.7***
3.7 2.2 4.5 9.2*
* P < 0.001, greater than sham hypophysectomizcd control. ** P < 0.001, less than sham hypophysectomizcd control. *** P < 0.001, greater than hypophysectomizcd control.
!o0 \ ~=~ "
~\ ~ o n t r o J
20 30 40 ~1 antiserum (I : 2)
Fig. 2. Immunoprecipitation of dopamine fl-hydroxylase from adrenal glands of reserpine-treated and control rats. Samples were prepared as described in Materials and Methods. Antiserum was diluted 1:2 in 150 mM NaC1. The ratio of enzyme activity between reserpine-treated and control animals was 2.65; the ratio of equivalence points was 2.50.
dopamine fl-hydroxylase activity (Table I) and levels5 and partially inhibited the in vivo degradation of dopamine fl-hydroxylase5.
Effects of reserpine on dopamine fl-hydroxylase Reserpine causes a marked elevation in dopamine fl-hydroxylase activity in the adrenal glands (Table I). This is accompanied by a parallel increase in the number of immunoprecipitable enzyme molecules (Fig. 2). Unilateral adrenal denervation blocked the reserpine induction of dopamine fl-hydroxylases. Labeled amino acid incorporation studies showed that reserpine induced dopamine fl-hydroxylase synthesis without affecting the rate of enzyme degradation5.
Effects of reserpine in hypophysectomizedrats We next performed experiments to study interactions between the hormonal and neuronal systems regulating dopamine fl-hydroxylase. To determine if an intact pituitary-adrenal system was required for reserpine induction of the enzyme, rats which had been previously hypophysectomized were treated with this drug. Although hypophysectomy caused a reduction of dopamine fl-hydroxylase to one-fourth that of control levels, reserpine administration induced the enzyme 38 enzyme units, an amount nearly equal to that seen in intact animals (Table I). Thus the reserpine induction of dopamine fl-hydroxylase does not depend on the presence of the pituitary gland or on a normal adrenal cortex.
354 TABLE 11
Effect~ o] hormone treatment m hypophysectonuzed, adrenal denervated rat.~ Each group consisted of 4-6 ammals. Rats which had been hypophysectom~zed or sham hypophysectomlzed were subjected to umlateral adrenal denervatlon 4 days later. One week after the second surgery treatment with ACTH, 4 1 U., or dexamethasone, 1 rag/rat dady for 3 days was begun. The ammals were killed 24 h after the last rejection. Enzyme actwlty is expressed as mean and standard error of the mean
Hypophysectomy Hypophysectomy Hypophysectomy Sham hypophysectomy Shamhypophysectomy Shamhypophysectomy
None Dexamethasone ACTH None Dexamethasone ACTH
Dopamine fl-hydroxylase (units/adrenalgland) Denervated
23 ± 44 ± 32 ± 106 :k 87 i 104 ~-
33 _~: 3§ 50 ~: 3*, 9§ 60 ± 7", ***, 9§§ 135 ± 11 109 ± 4 133 i 15
3 4**.9§ 3§§§ 8 4 15
* P < 0.01, differs from lpsllateral adrenal, untreated hypophysectomlzed. ** P < 0.05, differs from ipsilateral adrenal, untreated hypophysectomized. *** P < 0.01, differs from contralateral adrenal. § P < 0.001, differs from ipsilateral adrenal, untreated sham hypophysectomized. §9 p < 0.001, differs fromipsilateral adrenal, shamhypophysectomized, treated with dexamethasone. 999 p < 0.001, differs from ipsilateral adrenal, sham hypophysectomized, treated with ACTH.
Effects of A C T H or dexamethasone in rats subjected to hypophysectomy and unilateral adrenal denervation The following experiments were designed to determine if an intact preganglionic neuron was required for h o r m o n e treatment to reverse the effects o f h y p o p h y s e c t o m y on dopamine fl-hydroxylase degradation. Rats were hypophysectomized and 4 days later were subjected to unilateral adrenal denervation. One week after this latter surgery, treatment with A C T H or dexamethasone was begun. H y p o p h y s e c t o m y caused a decline in dopamine fl-hydroxylase levels to about 25% of control values (Table II). A C T H administration caused a doubling o f dopamine fl-hydroxylase levels in the innervated adrenal, but had no significant effect on the enzyme in the denervated gland. Dexamethasone, on the other hand, was effective in both the innervated and denervated glands. These findings indicate that A C T H regulation o f dopamine fl-hydroxylase requires an intact splanchnic neuron, while dexamethasone acts at a step distal to the postsynaptic receptor. These results were confirmed in two additional experiments o f the same design. Neural and endocrine interrelationships in the regulation o f phenylethanolamine Nmethyltransferase Phenylethanolamine N-methyltransferase catalyzes the terminal step in adrenaline biosynthesis 1. The regulation o f this enzyme is qualitatively similar to that o f dopamine fl-hydroxylase and tyrosine hydroxylase 2. Reserpine administration induces enzyme levels above control values, but at a slower rate than the induction o f either
355 tyrosine hydroxylase or dopamine fl-hydroxylases. Hypophysectomy reduces phenylethanolamine N-methyltransferase levels profoundly; ACTH or dexamethasone will restore the enzyme to control levels~°. To determine in what other ways the neural and hormonal regulation of phenylethanolamine N-methyltransferase and dopamine fl-hydroxylase were similar, the experiments described above were repeated and phenylethanolamine N-methyltransferase levels were determined. Effects o f reserpine in intact and hypophysectomized rats
Reserpine increases phenylethanolamine N-methyltransferase levels in adrenals of intact rats a,l°. When reserpine was administered to rats with intact or denervated adrenals, phenylethanolamine N-methyltransferase levels rose only in the innervated gland (Table III). These results suggest that the transsynaptic stimulation of phenylethanolamine N-methyltransferase, like that of tyrosine hydroxylase and dopamine fl-hydroxylase, requires an intact splanchnic nerve. Hypophysectomy causes a marked decrease in phenylethanolamine N-methyltransferase levels (Table IV). When reserpine was administered to hypophysectomized rats, enzyme activity was significantly restored. These results indicate that reserpine is effective in increasing phenylethanolamine N-methyltransferase levels in the absence of the pituitary gland. Studies on hypophysectomized, unilaterally denervated rats
Rats were subjected to hypophysectomy, followed 6 days later by unilateral adrenal denervation. ACTH or dexamethasone treatment was begun 4 days alter denervation. Hypophysectomy resulted in a reduction of enzyme levels to 40~o of control values. ACTH administration partially restored enzyme activity in the innervated adrenal (Table IV), but, in the denervated gland, the effect of ACTH was abolished (Table V). When dexamethasone was administered instead of ACTH, an increase in phenylethanolamine N-methyltransferase activity was seen in both adrenals, indicating that the glucocorticoid effect on the enzyme does not depend on TABLE III Effect of reserpine on phenylethanolamine N-methyltransferase in unilateral adrenal denervated rats
Each group contained 6 animals, which were treated with 2.5 mg/kg reserpine on alternate days for 2 injections and killed 24 h after the last dose. Reserpine treatment was begun 14 days after the surgery. In the intact controls, only the fight adrenal was taken for assay. Surgical treatment
None None Unilateral denervation Unilateral denervation
None Reserpine None Reserpine
* P < 0.01, differsfrom untreated control.
Phenyethanolamine N-methyltransferase (units /adrenalgland) Denervated
0.892 4- 0.019 0.893 4- 0.045
0.750 4- 0.016 1.240 4- 0.052* 0.797 4- 0.055 1.088 4- 0.073*
356 TABLE IV Effect of reserpme or A C T H on phenylethanolamine N-methyltransferase m hypophyseetomtzed rats
Numbers in parentheses are group sizes. Animals were hypophysectomized 14 days before Inmatlon of either ACTH (4 I.U. daily for 3 days) or reserpine (2.5 mg/kg, one rejection). Animals were killed 8 (ACTH) or 48 (reserpine) h after the last injection of drug. Surgical treatment
Phenylethanolamine N-methyltransferase (units/adrenal gland)
Sham hypophysectomy Hypophysectomized Hypophysectomized Hypophysectomized
None None ACTH Reserpine
0.708 ± 0.06 0.281 -5=0.014 0.347 ± 0.006 0.412 i 0.065
(6) (5)* (6)*,** (4)***
* P ,( 0.001, differsfrom sham hypophysectomlzexl. ** P