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Developmental changes in rat adrenal tyrosine hydroxylase, dopamine-β-hydroxylase and catecholamine levels: Effect of denervation
DEVELOPMENTAL
BIOLOGY 29,
204-213
(1972)
Developmental Changes in Rat Adrenal Tyrosine Hydroxylase, Dopamine-,6- Hydroxylase and Catecholamine Levels: ROBERT Department
of Biochemistry,
Effect of Denervation
L. PATRICK’
AND
Duke
Medical
University Accepted
March
NORMAN Center,
KIRSHNER* Durham,
North
Carolina
27710
27, 1972
Developmental changes in rat adrenal tyrosine hydroxylase, dopamine-@-hydroxylase, and catecholamine levels: effect of denervation. Levels of adrenal tyrosine hydroxylase, dopamineP-hydroxylase, and catecholamines were found to be dependent upon both the age and the innervation of the adrenal gland. During growth from 8 to 15 wk of age, tyrosine hydroxylase, dopamine$-hydroxylase, and catecholamines increased by 51, 33, and 57%, respectively, in intact adrenal glands. Denervating the left adrenal gland at 8 wk of age completely prevented the increases seen in the innervated glands. In younger animals, during growth from 4 to 12 wk of age, tyrosine hydroxylase activity increased 2- to S-fold, dopamine-@-hydroxylase activity increased about 2-fold, and catecholamine levels increased 3. to 5-fold in intact adrenal glands. Denervation of the adrenal gland at 23 days of age markedly depressed the increase in enzyme activities and catecholamines to approximately one half that seen in innervated glands.
that intravenous injection of acetylcholine will increase the levels of tyrosine hyLevels of adrenal tyrosine hydroxylase droxylase in denervated rat adrenals indihave been shown to increase following experimental conditions which increase cating that the neurotransmitter itself is splanchnic nerve activity. These include the agent which initiates the events leadinsulin-induced hypoglycemia (Patrick and ing to a rise in enzyme activity. Adrenal Kirshner, 1971a; Viveros et al., 1969b), dopamine-P-hydroxylase activity has also treatment with reserpine (Mueller et al., been shown to increase after neural stimu1969b), 6-hydroxydopamine (Thoenen et lation, usually following an initial decline al., 1969), phenoxybenzamine (Mueller et in activity (Molinoff et al., 1970; Patrick and Kirshner, 1971a; Viveros et al., 1969b). al., 1969a) and immobilization (Kvetnansky Adrenal catecholamines decline in both et al., 1970) or cold stress (Thoenen, 1971). Denervation of the adrenal gland, denervated and intact glands following as well as inhibitors of protein synthesis reserpine administration, but the recovery to control levels is more rapid in the intact such as actinomycin D and cycloheximide gland even though it is depleted to a abolish the increases in tyrosine hydroxylase activity, suggesting that the rise in greater extent than the denervated gland enzyme activity is due to the release of a (Patrick and Kirshner, 1971b). The reneurogenic factor which either directly or covery of the denervated gland following indirectly induces protein synthesis. reserpine treatment can be stimulated by treatment (Patrick and Patrick and Kirshner (1971a) have shown acetylcholine Kirshner, 1971b). ’ Present address: Department of Psychiatry, Because of these effects of stimulation Stanford University Medical School, Stanford, California 94305. upon adrenal catecholamine-synthesizing *This work was supported by a grant from the capabilities and stores, it became of inNational Institutes of Health, AM 05427. The authors terest to determine the effect of denervawish to thank Mrs. Nannie Jordan and Mrs. Dorothy Leathers for their excellent technical assistance. tion upon the development of adrenal INTRODUCTION
204 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form resewed.
PATRICK
AND
KIRSHNER
Developmental
tyrosine hydroxylase, dopamine- ,@hydroxylase, and catecholamine levels. METHODS
Male Sprague-Dawley rats were used in all studies. Both denervated and unoperated animals were obtained from ZivicMiller Laboratories, Allison Park, Pennsylvania. Two groups of denervated animals were employed: (1) those with their left adrenals denervated at 23 days of age, and (2) those with their left adrenals denervated at 8 wk of age. Animals receiving insulin were fasted for 24 hr and then injected with 5 international units/kg body weight, via the tail vein, and sacrificed 4 hr later. Animals were housed 2 per cage in a quiet room on a 12-hr light-dark cycle. Animals were killed between 10 AM and 12 noon. Preparation of homogenates. Bats were killed by a blow on the base of the skull and bled at the neck. Each of the adrenal glands was separately homogenized in 2.5 ml of ice-cold 0.3 M sucrose in a PotterElvehjem all-glass homogenizer. The left and right adrenals of unoperated animals were homogenized and assayed separately to check for possible intrinsic differences during development. No differences were observed in the unoperated animals, and the values for these animals are expressed as an average of the two glands, i.e., as that value that would be obtained if both glands were homogenized and assayed together. Aliquots were removed for catecholamine and dopamine-/3-hydroxylase determinations, and the remainder of the homogenate was centrifuged at 26,000 g for 20 mins. The supernatant fraction was assayed for tyrosine hydroxylase activity and protein content. Assay of dopamine-P-hydrorylase. Dopamine-fl-hydroxylase was assayed as described previously (Viveros et al., 1969a). The reaction mixture contained potassium phosphate buffer, pH 5.8, 200
Changes
in Rat Adrenals
205
mM, fumarate, pH 5.8, 120 mM; ascorbate, 2.0 mM, adenosine-5-triphosphate, 0.1 mM; tranylcypramine, 1 mM; 3Htyramine, generally labeled (7.3 Ci/mmole) 0.01 mM; catalase, 600 units; Triton X100, 0.5%; and 0.4 ml of the sucrose homogenate in a final volume of 1.0 ml. In addition, each reaction mixture contained a final concentration of 4 mM p-hydroxymercuribenzoate to inactivate endogenous inhibitors. The mixtures were incubated in air at 37°C for 30 min, and the reactions were stopped by the addition of 1 ml of 7% perchloric acid. After centrifugation, 1 ml of the supernatant fluid was assayed by the periodate oxidation method of Friedman and Kaufman (1965) for the amount of octopamine formed. Five percent glycerol was used to stop the periodate oxidation instead of 10% sodium bisulfite. Dopamine-&hydroxylase activity was linear with tissue concentration and time. Assay of tyrosine hydroxylase. Tyrosine hydroxylase activity was assayed as described by Nagatsu et al. (1964). The reaction mixture contained sodium acetate buffer, pH 6.0, 200 mM; ferrous ammonium sulfate, 0.5 mM; tranylcypramine, 0.1 mM; 2-amino-6,7-dimethyl-4-hydroxy5,6,7,8-tetrahydropteridine hydrochloride, 2 mM; 2-mercaptoethanol, 20 mM; 3,53H-tyrosine, 0.02 mM (specific activity 31.6 Ci/mmole, and 0.4 ml of the 26,000 g sucrose supernatant fraction in a final volume of 1 ml. The reaction mixtures were incubated in air at 37” for 15 min, and the reaction was stopped by the addition of 0.05 ml of glacial acetic acid. Activity was linear with tissue concentration and time. Since the tyrosine concentration in most rat tissues is approximately 10e4 M (Schurr et al., 1950; Udenfriend, 1966) and we dilute the adrenals approximately 250-fold during the preparation of the enzyme, the amount of tyrosine contributed by the tissue is about 2% of the total amount used in the assay. Therefore, it is highly unlikely that changes in endogenous
206
DEVELOPMENTAL
BIOLOGY
tyrosine concentrations will appreciably affect the tyrosine hydroxylase assay. Cutecholamines. Aliquots of the adrenal fractions (0.1 ml) were added to 2 ml of 3.5% perchloric acid. The mixture was centrifuged, and the supernatant fluid was decanted, diluted, and assayed for catecholamine content without further treatment, as described previously (Viveros et al., 196913). Proteins. Proteins were determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Statistical methods. Results are expressed on a per gland basis. Student’s t-test was used to determine statistical significance. Materials. 3H-tyramine and 3H-tyrosine were obtained from New England Nuclear Corporation, and insulin was obtained from Squibb. RESULTS
Tyrosine
Hydroxylase
In the first group of experiments, adult rats were used. The left adrenal glands
VOLUME
29, 1972
were denervated at 8 wk of age (259 g), and observations on the tyrosine hydroxylase activity were made from 2 to 49 days after denervation (Fig. 1). Throughoui this period the tyrosine hydroxylase activity in the denervated gland showed no change while the tyrosine hydroxylase activity of the intact right gland showed a 55% increase. Twenty-eight days after denervation the activity in the denervated gland was significantly less than the activity of the intact gland. The increase in activity of the right gland from operated animals was the same as that found in glands from unoperated animals. In the second group of animals, the left adrenal gland was denervated at 23 days of age, shortly after weaning, and observations on the tyrosine hydroxylase activity were made at intervals from 6 to 60 days after denervation (Fig. 2). In contrast to glands denervated at the older age, the denervated glands from the younger animals showed a steady increase in the tyrosine hydroxylase activity to levels at 60 days post-denervation that were ap-
20-
----
Denervoted
Adrenals
lntoct Adrenals of Operated Animals ._.-.-
8-
of Animals
(6) I 2
I 246*3
FIG. ber of of the intact
Adrenals Unoperoted
1 7
t 14 I 306=
I 21
I2
I 28 I 38959
I t 42 49 Doys Denervoted I I 465223 480*-18 8ody Weights
I 63
I
487512
1. Development of adrenal tyrosine hydroxylase activity during growth from 8 to 15 wk of age. The numanimals in each group is shown in parentheses. Values are expressed as the means + the standard errors means. Adrenal denervation was performed at 8 wk of age. Denervated glands differ significantly from right glands at 28 days (p < 0.05), 42 days (p < 0.05), and 49 days (p < 0.01) post denervation.
PATRICK
AND
Deuelopmental
KIRSHNER
Changes
in Rat Adrenals
207
16 -
6-
----
Denervoted
__
lntoct Adrenals of Operated Anmols Adrenols Unoperoted
._.-.I 6
2
I 09*2
I 13 I 147f3
I 27 I 24024
Adrenals
of Animals
I 60 Doys
Denervoted 1 426+6
Body
Weqhts
FIG. 2. Development of adrenal tyrosine hydroxylase activity during growth from number of animals in each group is shown in parentheses. Values are expressed as the errors of the means. Adrenal denervation was performed at 23 days of age. Denervated cantly from intact right glands at 13 days (p < O.OP, 27 days (p < 0.021, and 60 days (p tion. Denervated glands 60 days post denervation differ significantly from denervated nervation (p < 0.001).
proximately twice those at 6 days after denervation. However, the intact right gland and the glands from unoperated controls showed larger increases; 27 and 60 days after denervation the tyrosine hydroxylase activity of these glands was significantly higher than the activity in the denervated glands. Dopamine-@-hydroxylase In the group of animals denervated at 8 weeks of age, there were no significant changes in the dopamine-P-hydroxylase activity of the denervated gland. The intact right gland as well as glands from unoperated rats showed increases in dopamine-P-hydroxylase (Fig. 3). The changes in dopamine-P-hydroxylase activity in the animals denervated at 23 days of age were qualitatively similar to the changes in tyrosine hydroxylase (Fig. 4). The activity in the denervated gland 60 days after denervation was approxi-
4 to 12 wk of means * the glands differ < 0.001) post glands 6 days
age. The standard signifidenervapost de-
mately twice that at 6 days while the activities in the intact right gland and in the unoperated animals showed much larger increases. At 27 and 60 days the denervated glands had significantly lower activity than both sets of innervated glands. Catecholamines In both the older and younger animals the changes in catecholamine content were qualitatively similar to changes in tyrosine hydroxylase and dopamine-(5 hydroxylase. In the group denervated at 8 wk of age the catecholamine content of the denervated glands showed no changes while the innervated glands of both operated and unoperated animals showed similar increases (Fig. 5). In the animals denervated at 23 days of age, the catecholamine content of the denervated glands increased, at 60 days, 136% above the levels at 6 days while the innervated glands had much larger increases. At 27
208
DEVELOPMENTAL
BIOLOGY
VOLUME
29.
1972
(8) _.-.-.-.1
----
Denervoted
__
Intact Adrenals of Operated Animals
_ _ _ Adrenals Unoperated II
I 7
2
I 14
I 21
I 28
I I 42 49 Denervated
Days I 246*
I
3
306+12
I
38929
I
Adrenals
of Animals
I 63
I
I
465223 480’18 Body Weights
487fl2
FIG. 3. Development of adrenal dopamine-8-hydroxylase activity during growth from 8 to 15 wk of age. The number of animals in each group is shown in parentheses. Values are expressed as the mean + the standard errors of the means. Adrenal denervation was performed at 8 wk of age. Denervated glands differ significantly from intact right glands 49 days post denervation (p < 0.002).
Denervoted ___
Adrenals Unoperoted
--L
I 6 I e9*2
I I3
27
I 147*3
I 24824
I
of Animals
I
Days
Denervated
60 I 428+8
Body
FIG. number errors cantly glands
Adrenals
lnracr Adrenals of Operated Animals
Weights
4. Development of adrenal dopamine-ghydroxylase activity during growth from 4 to 12 wk of age. The of animals in each group is shown in parentheses. Values are expressed as the means l the standard of the means. Adrenal denervation was performed at 23 days of age. Denervated glands differ signififrom intact right glands at 27 days (p < 0.01) and 60 days (p < 0.01) post denervation. Denervated 60 days post denervation differ significantly from denervated glands 6 days post denervation (p < 0.02).
PATRICK
AND
Developmental
KIRSHNER
Changes
----
Denervated
--
lntacl Adrenals of ODerated Animals
.8I 2
I 7
I
I
246)
I 14
3
306212
I 21
I 28 I 38929
Days
I 42 Denervated I
(Ia I 49 I
465+23 480+18 Body Weights
in Rat Adrenals
- - Adrenals Unoperated
209
Adrenals
of Animals
I 63 I
487+12
FIG 5. Development of adrenal catecholamine levels during growth from 8 to 15 wk of age. The number of animals in each group is shown in parentheses. Values are expressed as the means * the standard errors of the means. Adrenal denervation was performed at 8 weeks of age. Denervated glands differ significantly from intact right glands at 42 days (p < 0.05) and 49 days (p < 0.001) post denervation.
and 60 days after denervation, the catecholamine levels of the intact glands were significantly greater than those of denervated glands (Fig. 6). Supernatant
Protein
The protein content of the 26,000 g supernatant fraction was determined in the adrenal glands of rats denervated at 23 days of age and in a corresponding age group of unoperated animals. There was an increase in protein content during the experimental period but no marked differences between the intact and denervated glands were observed (Fig. 7). At 60 days the intact right gland showed a small (16%) significant increase over the denervated gland, but the protein content of the denervated glands was not different from that of the unoperated controls. Effect of Denervation Gland Weight
on Rat Adrenal
In the group of animals denervated at 8 weeks of age no differences were ob-
served between adrenal weight of intact and denervated glands for up to 28 days postoperatively (Table l), a time at which intact adrenal tyrosine hydroxylase activity was significantly higher than that of the denervated adrenal (Fig. 1). Response
to Insulin
Treatment
Because the data indicated a distinct difference between the effects of denervation at 23 days of age (inhibition of development) and that of denervation at 8 wk of age (complete prevention of development) the possibility of nerve regeneration or incomplete initial denervation in the animals denervated at the younger age had to be considered. Our previous experiments with the animals denervated at 8 weeks of age indicated that only the intact right gland would respond to insulin with a depletion of catecholamines, followed by an increase in tyrosine hydroxylase activity (Patrick and Kirshner, 1971a). In the present experiments, rats denervated at 23 days of age were treated with insulin at
210
DEVELOPMENTAL
gi "
IO .I
BIOLOGY
VOLUME
29, 1972
/
.x
0
Denervaled
(6) ,,.' ,
6 v----
(5) 19)
T;P 4
I.' f -------x
Adrenals
Intact Adrenals of Operated Anmals
02)
---
i -6
Adrenals Unoperoted
of Animals
(61
-t
I 09f2
I 14753
DOYS Denervated
I 240f
4
Body
Wetights
-7k I 42050
FIG. 6. Development of adrenal catecholamine levels during growth from 4 to 12 wk of age. The number of animals in each group is shown in parentheses. Values are expressed as the means * the standard errors of the means. Adrenal denervation was performed at 23 days of age. Denervated glands differ significantly from intact right glands at 27 days (p < 0,002) and 60 days (p c 0.001) post denervation. Denervated glands 60 days post denervation differ significantly from denervated glands 6 days post denervation (p < 0.001).
1600
-
Denervoted
Adrenals
Intact Adrenals of Operoted Animals Adrenals Unoperated
---
I 6
I 13
I 27
I 147f3
I 248+4
Denervoted I 428%
Body
FE. ber of of the on the glands animals vated
Ammals
I 60 Days
I 0922
of
Weights
7. Development of adrenal supernatant protein content during growth from 4 to 12 wk of age. The numanimals in each group is shown in parentheses. Values are expressed as the means * the standard errors means. Adrenal denervation was performed at 23 days of age. Protein determinations were performed 26,000 g supernatant fraction. Denervated glands not significantly different from unoperated intact 27 and 60 days post denervation, and also not significantly different from intact right glands of operated 27 days post denervation. Intact glands of operated animals show a small (16%) increase over denerglands (p < 0.005).
PATRICK
Developmental
AND KIRCHNER
various times after denervation and the catecholamine content of the gland was determined 4 hr after insulin injection (Table 2). Rats given insulin 6, 27, and 60 days after denervation showed catecholamine depletions in the intact adrenals of 73%, 73%, and 68%, respectively (p less than 0.001 from the control right glands at all three time periods after denervation). The denervated glands showed no signs of depletion after insulin treatment, thus indicating that they were initially fully denervated, and remained so throughout the course of the experiment.
EFFECT
TABLE 1 OF DENERVATION UPON RAT ADRENAL GLAND WEIGHT’ Adrenal
Days
weight
(mg/gland)
denervated Intact 7
21.5
14
* 1.3 (4) 25.2 i 1.9
21
23.5
Denervated 25.2
* 2.7 (4) 25.5 * 1.4
(8) zt 1.3
(8) 26.3
(6) 28
23.2
i 1.0 (4)
i
1.6
(6) 25.2
ztz 2.3 (4)
a Left adrenal denervation was performed at 8 wk of age. Values are the means * SEM. The number of animals in each group is in parentheses. None of the differences is significant.
EFFECT OF INSULIN
TREATMENT
6 27 60
DISCUSSION
Rat adrenal tyrosine hydroxylase, dopamine-fl-oxidase, and catecholamines undergo age-dependent increases (Figs. l-6); the agent(s) responsible for the increases has not yet been identified. The effects of denervation upon these increases were dependent upon the age at which the denervations were performed; denervation at 8 wk of age completely prevented any further increases in enzyme and catecholamine levels, whereas denervation at 23 days of age markedly inhibited, but did no.? completely prevent the increases (Fig. l-6). Insulin treatment of the animals denervated at 23 days revealed no neurogenitally induced decline in catecholamine levels of the denervated glands when tested 6, 27, and 60 days post-operatively, while the intact glands responded with 70% depletion (Table 2). These data argue against incomplete initial denervation, or possible nerve regeneration as the explanation for the increases seen in animals denervated at 23 days of age. Differences in circulating hormone levels are also an unlikely explanation, since between 8 and 12 wk of age, animals denervated at 8 wk show no increases in catecholamine-synthesizing enzymes and stores, while animals denervated at 23
TABLE 2 ON ADRENAL CATECHOLAMINE LEVELS (~G/GLAND AT 23 DAYS OF AGE Untreated
Days denervated
Left 4.5
Insulin Right
zt 0.6 (9) 5.2 h 0.5
6.2 zt 0.7 (11) 8.4 * 0.7
(12)
(12)
10.6
211
Changes in Rat Adrenals
Left 4.3
zt 0.6b
4.3
(5) * 0.3”
(6)
OF RATS DENERVATED
treated Right 1.7
* 0.2” (5) 2.3 A 0.1”
(6)
i 0.6 20.9 * 1.2 9.4 zt 0.6b 6.6 * 1.0” (13) (13) (7) (7) The number of animals in each group is shown in parentheses. Values are expressed as the means + the standard errors of the means. Left adrenal denervation was performed at 23 days of age. Animals were killed 4 hr after insulin treatment (5 international units/kg iv). The letters above the standard errors refer to the following p values (Students t test): “p < 0.001 compared to control right gland; %ot significantly different from control left gland.
212
DEVELOPMENTAL
BIOLOGY
days do, It would thus appear that the older animals have developed more of a “nerve dependency” for the age-dependent increases. An example of an apparent “nerve dependency” can be seen in salamander forelimb regeneration (Guth, 1969). When the forelimb of the salamander is amputated, the scar tissue breaks down the tissue grows and differentiates into a new forelimb. Regeneration does not occur if the spinal nerves are cut at the time of amputation. If denervation is performed during the early stages of regeneration, the process stops and proceeds further only after the nerves grow back. An intact nerve supply is not required for the embryonic development of the limb, however, and in fact, the aneurogenic limb, produced by removal of the appropriate portion of the spinal cord and neural crest at an early stage of development, is capable of regenerating an amputated forelimb. When an aneurogenic limb is grafted to a normal host and is then amputated, regeneration occurs. Furthermore, when the grafted aneurogenic limb becomes innervated by the host’s nerves, it retains its ability to regenerate when denervation and amputation are performed within 10 days after host innervation, but loses the ability to regenerate when denervation and amputation are performed 13 days after host innervation. It would thus appear that the limb acquires a “nerve dependency” after a certain number of days of innervation. A similar dependency might be operative in the rat adrenal for increasing its catecholamine-synthesizing enzymes and stores. An alternate explanation could be that the adrenals have an inherent ability to achieve certain levels of enzymes or catecholamines regardless of the state of their nerve supply. Increases beyond this value would be completely nerve dependent. In fact, the levels of catecholamine-synthesizing enzymes and stores of animals denervated at 23 days of
VOLUME
29. 1972
age did not increase above those of animals denervated at 8 wk of age (Figs. l-6). However, this may be the result of an insufficient period of study. The data presented here suggest that the tyrosine hydroxylase, dopamine-phydroxylase, and catecholamine levels of adrenal glands are dependent upon both the age of the animal and upon the neuronal input. These observations are entirely consistent with previous reports that treatments which increase splanchnic stimulation of the adrenal gland result, within 24 hr, in increased levels of tyrosine hydroxylase in intact adrenal glands but not in denervated glands (Kvetnansky et al., 1970; Thoenen et al., 1969; Patrick and Kirshner, 197la), and are also consistent with the report that intravenous injection of acetylcholine will result in increased tyrosine hydroxylase activities in denervated glands (Patrick and Kirshner, 1971a). The increased levels of enzymes and catecholamines in innervated glands indicate that these are adaptive changes to neuronal stimulation, and that, even under ordinary housing conditions, neuronal stimulation is sufficient to result in significant increases in enzyme and catecholamine content. Axelrod et al. (1970) have shown that environmental stimulation can result in increased levels of tyrosine hydroxylase in mice. The neuronal-dependent increases observed here may be due, to some degree, to environmental stimulation but may also reflect a physiological requirement for increased adrenal medullary secretion to compensate for increased body size. A decline, with a half-life of approximately 21 days, in adrenal tyrosine hydroxylase following denervation has been reported (Mueller et al., 1970), but our studies show no such changes. Leastsquare plots of the effect of denervation at 8 wk of age do show a slightly negative slope for tyrosine hydroxylase, dopamineP-hydroxylase, and catecholamines, but
PATRICK AND KIRSHNER
Developmental
Changes in Rat Adrenals
213
the slopes are not significantly different in dopamine-B-hydroxylase activity. Proc. Nat. Acad. Sci. U. S. 66,453-458. from zero. An explanation for this apparent discrepancy may be the manner in MUELLER, R. A., THOENEN, H., and AXELROD, J. (1969a). Adrenal tyrosine hydroxylase: Compensawhich the animals are maintained prior to tory increase in activity after chemical sympathecdenervation. Twenty-four hours after tomy. Science 163, 468-469. treatment with reserpine on three suc- MUELLER, R. A., THOENEN, H., and AXELROD, J. (1969b). Increase in tyrosine hydroxylase activity cessive days, activity is increased 3-fold after reserpine administration. J. Pharmacol. Erp. and requires about 10 days to return to Z’her. 169, 74-79. normal levels (Patrick and Kirshner, MULLER, R. A., THOENEN, H. and AXELROD, J. 1971b). Thus, if the animals were sub(1970). Effect of pituitary and ACTH on the mainjected to conditions which stimulate tenance of basal tyrosine hydroxylase activity in the rat adrenal gland. Endocrinology 86,751-755. splanchnic discharge such as transportation from suppliers, new or crowded sur- NAGATSU, T., LEVI’IT, M., and UDENFRIEND,S. (1964). A rapid and simple radioassay for tyrosine hyroundings, the levels of tyrosine hydroxyldroxylase activity. A&. Biochem. 9, 122-126. ase may be elevated and would show a PATRICK, R. L., and KIRSHNER, N. (1971a). Effect of decline to basal levels upon transection of stimulation on the levels of tyrosine hydroxylase, dopamine-/3-hydroxylase, and catecholamines in the nerve supply. On the other hand, if intact and denervated rat adrenal glands. Mol. animals were maintained for l-2 wk under Pharmacol. 7, 87-96. conditions of low or minimal neuronal in- PATRICK, R. L., and KIRSHNER, N. (1971b). Acetylput, basal levels of tyrosine hydroxylase choline-induced stimulation of catecholamine remay be established and show no further covery in denervated rat adrenals after reserpineinduced depletion. Mol. Phurmacol. 7, 389-396. decrease upon denervation. SCHURR, REFERENCES
AXELROD, J., MUELLER, R. A., HENRY, J. P., and STEPHENS, P. M. (1970). Changes in enzymes involved in the biosynthesis and metabolism of noradrenaline and adrenaline after psychosocial stimulation. Nature (London) 225,1059-1060. FRIEDMAN, S., and KAUFMAN, S. (1965). 3,4-Dihydroxyphenylethylamine-fl-hydroxylase. J. Biol. Chem. 240, 4763-4773. GUTH, L. (1969). “Trophic” effects of Vertebrate Neurons. Neurosci. Res. Bull. 7, l-73. KVETNANSKY, R., WEISE, V. K., and KOPIN, I. 3. (1970). Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-N-methyl transferase by repeated immobilization of rats. Endocrinology 87, 744-749. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MOLINOFF, P., BRIMIJOIN, S., WEINSHILBOUM,R., and AXELROD, J. (1970). Neurally mediated increase
P. E., THOMPSON,
H. T., HENDERSON,
L. M.,
and ELVEHJEM, C. A. (1950). A method for the determination of free amino acids in rat organs and tissues. J. Biol. Chem. 182, 29-37. THOENEN, H. (1971). Induction of tyrosine hydroxylase in peripheral and central adrenergic neurons by cold-exposure of rats. Nature (London) 228, 861862. THOENEN, H., MUELLER, R. A., and AXELROD, J. (1969). Increased tyrosine hydroxylase activity after drug-induced alteration of sympathetic transmission. Nature (London) 221, 1264. THOENEN, H., MUELLER, R. A., and AXELROD, J. (1969). Trans-synaptic induction of adrenal tyrosine hydroxylase. J. Pharmacol. Exp. 7’her. 169, 244254. UDENFRIEND, S. (1966). Tyrosine hydroxylase. Pharmacol. VIVEROS, 0. KIRSHNER,
Reu.
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H., ARQUEROS, L., CONNETT, R. J., and N. (1969a). Mechanism of secretion from the adrenal medulla III. Mol. Phurmacol. 5,60-6&X VIVEROS, 0. H., ARQUEROS, L., CONNETT, R. J., and KIRSHNER, N. (196913). Mechanism of secretion from the adrenal medulla IV. Mol. Pharmacol. 5, 69-82.
BIOLOGY 29,
204-213
(1972)
Developmental Changes in Rat Adrenal Tyrosine Hydroxylase, Dopamine-,6- Hydroxylase and Catecholamine Levels: ROBERT Department
of Biochemistry,
Effect of Denervation
L. PATRICK’
AND
Duke
Medical
University Accepted
March
NORMAN Center,
KIRSHNER* Durham,
North
Carolina
27710
27, 1972
Developmental changes in rat adrenal tyrosine hydroxylase, dopamine-@-hydroxylase, and catecholamine levels: effect of denervation. Levels of adrenal tyrosine hydroxylase, dopamineP-hydroxylase, and catecholamines were found to be dependent upon both the age and the innervation of the adrenal gland. During growth from 8 to 15 wk of age, tyrosine hydroxylase, dopamine$-hydroxylase, and catecholamines increased by 51, 33, and 57%, respectively, in intact adrenal glands. Denervating the left adrenal gland at 8 wk of age completely prevented the increases seen in the innervated glands. In younger animals, during growth from 4 to 12 wk of age, tyrosine hydroxylase activity increased 2- to S-fold, dopamine-@-hydroxylase activity increased about 2-fold, and catecholamine levels increased 3. to 5-fold in intact adrenal glands. Denervation of the adrenal gland at 23 days of age markedly depressed the increase in enzyme activities and catecholamines to approximately one half that seen in innervated glands.
that intravenous injection of acetylcholine will increase the levels of tyrosine hyLevels of adrenal tyrosine hydroxylase droxylase in denervated rat adrenals indihave been shown to increase following experimental conditions which increase cating that the neurotransmitter itself is splanchnic nerve activity. These include the agent which initiates the events leadinsulin-induced hypoglycemia (Patrick and ing to a rise in enzyme activity. Adrenal Kirshner, 1971a; Viveros et al., 1969b), dopamine-P-hydroxylase activity has also treatment with reserpine (Mueller et al., been shown to increase after neural stimu1969b), 6-hydroxydopamine (Thoenen et lation, usually following an initial decline al., 1969), phenoxybenzamine (Mueller et in activity (Molinoff et al., 1970; Patrick and Kirshner, 1971a; Viveros et al., 1969b). al., 1969a) and immobilization (Kvetnansky Adrenal catecholamines decline in both et al., 1970) or cold stress (Thoenen, 1971). Denervation of the adrenal gland, denervated and intact glands following as well as inhibitors of protein synthesis reserpine administration, but the recovery to control levels is more rapid in the intact such as actinomycin D and cycloheximide gland even though it is depleted to a abolish the increases in tyrosine hydroxylase activity, suggesting that the rise in greater extent than the denervated gland enzyme activity is due to the release of a (Patrick and Kirshner, 1971b). The reneurogenic factor which either directly or covery of the denervated gland following indirectly induces protein synthesis. reserpine treatment can be stimulated by treatment (Patrick and Patrick and Kirshner (1971a) have shown acetylcholine Kirshner, 1971b). ’ Present address: Department of Psychiatry, Because of these effects of stimulation Stanford University Medical School, Stanford, California 94305. upon adrenal catecholamine-synthesizing *This work was supported by a grant from the capabilities and stores, it became of inNational Institutes of Health, AM 05427. The authors terest to determine the effect of denervawish to thank Mrs. Nannie Jordan and Mrs. Dorothy Leathers for their excellent technical assistance. tion upon the development of adrenal INTRODUCTION
204 Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form resewed.
PATRICK
AND
KIRSHNER
Developmental
tyrosine hydroxylase, dopamine- ,@hydroxylase, and catecholamine levels. METHODS
Male Sprague-Dawley rats were used in all studies. Both denervated and unoperated animals were obtained from ZivicMiller Laboratories, Allison Park, Pennsylvania. Two groups of denervated animals were employed: (1) those with their left adrenals denervated at 23 days of age, and (2) those with their left adrenals denervated at 8 wk of age. Animals receiving insulin were fasted for 24 hr and then injected with 5 international units/kg body weight, via the tail vein, and sacrificed 4 hr later. Animals were housed 2 per cage in a quiet room on a 12-hr light-dark cycle. Animals were killed between 10 AM and 12 noon. Preparation of homogenates. Bats were killed by a blow on the base of the skull and bled at the neck. Each of the adrenal glands was separately homogenized in 2.5 ml of ice-cold 0.3 M sucrose in a PotterElvehjem all-glass homogenizer. The left and right adrenals of unoperated animals were homogenized and assayed separately to check for possible intrinsic differences during development. No differences were observed in the unoperated animals, and the values for these animals are expressed as an average of the two glands, i.e., as that value that would be obtained if both glands were homogenized and assayed together. Aliquots were removed for catecholamine and dopamine-/3-hydroxylase determinations, and the remainder of the homogenate was centrifuged at 26,000 g for 20 mins. The supernatant fraction was assayed for tyrosine hydroxylase activity and protein content. Assay of dopamine-P-hydrorylase. Dopamine-fl-hydroxylase was assayed as described previously (Viveros et al., 1969a). The reaction mixture contained potassium phosphate buffer, pH 5.8, 200
Changes
in Rat Adrenals
205
mM, fumarate, pH 5.8, 120 mM; ascorbate, 2.0 mM, adenosine-5-triphosphate, 0.1 mM; tranylcypramine, 1 mM; 3Htyramine, generally labeled (7.3 Ci/mmole) 0.01 mM; catalase, 600 units; Triton X100, 0.5%; and 0.4 ml of the sucrose homogenate in a final volume of 1.0 ml. In addition, each reaction mixture contained a final concentration of 4 mM p-hydroxymercuribenzoate to inactivate endogenous inhibitors. The mixtures were incubated in air at 37°C for 30 min, and the reactions were stopped by the addition of 1 ml of 7% perchloric acid. After centrifugation, 1 ml of the supernatant fluid was assayed by the periodate oxidation method of Friedman and Kaufman (1965) for the amount of octopamine formed. Five percent glycerol was used to stop the periodate oxidation instead of 10% sodium bisulfite. Dopamine-&hydroxylase activity was linear with tissue concentration and time. Assay of tyrosine hydroxylase. Tyrosine hydroxylase activity was assayed as described by Nagatsu et al. (1964). The reaction mixture contained sodium acetate buffer, pH 6.0, 200 mM; ferrous ammonium sulfate, 0.5 mM; tranylcypramine, 0.1 mM; 2-amino-6,7-dimethyl-4-hydroxy5,6,7,8-tetrahydropteridine hydrochloride, 2 mM; 2-mercaptoethanol, 20 mM; 3,53H-tyrosine, 0.02 mM (specific activity 31.6 Ci/mmole, and 0.4 ml of the 26,000 g sucrose supernatant fraction in a final volume of 1 ml. The reaction mixtures were incubated in air at 37” for 15 min, and the reaction was stopped by the addition of 0.05 ml of glacial acetic acid. Activity was linear with tissue concentration and time. Since the tyrosine concentration in most rat tissues is approximately 10e4 M (Schurr et al., 1950; Udenfriend, 1966) and we dilute the adrenals approximately 250-fold during the preparation of the enzyme, the amount of tyrosine contributed by the tissue is about 2% of the total amount used in the assay. Therefore, it is highly unlikely that changes in endogenous
206
DEVELOPMENTAL
BIOLOGY
tyrosine concentrations will appreciably affect the tyrosine hydroxylase assay. Cutecholamines. Aliquots of the adrenal fractions (0.1 ml) were added to 2 ml of 3.5% perchloric acid. The mixture was centrifuged, and the supernatant fluid was decanted, diluted, and assayed for catecholamine content without further treatment, as described previously (Viveros et al., 196913). Proteins. Proteins were determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Statistical methods. Results are expressed on a per gland basis. Student’s t-test was used to determine statistical significance. Materials. 3H-tyramine and 3H-tyrosine were obtained from New England Nuclear Corporation, and insulin was obtained from Squibb. RESULTS
Tyrosine
Hydroxylase
In the first group of experiments, adult rats were used. The left adrenal glands
VOLUME
29, 1972
were denervated at 8 wk of age (259 g), and observations on the tyrosine hydroxylase activity were made from 2 to 49 days after denervation (Fig. 1). Throughoui this period the tyrosine hydroxylase activity in the denervated gland showed no change while the tyrosine hydroxylase activity of the intact right gland showed a 55% increase. Twenty-eight days after denervation the activity in the denervated gland was significantly less than the activity of the intact gland. The increase in activity of the right gland from operated animals was the same as that found in glands from unoperated animals. In the second group of animals, the left adrenal gland was denervated at 23 days of age, shortly after weaning, and observations on the tyrosine hydroxylase activity were made at intervals from 6 to 60 days after denervation (Fig. 2). In contrast to glands denervated at the older age, the denervated glands from the younger animals showed a steady increase in the tyrosine hydroxylase activity to levels at 60 days post-denervation that were ap-
20-
----
Denervoted
Adrenals
lntoct Adrenals of Operated Animals ._.-.-
8-
of Animals
(6) I 2
I 246*3
FIG. ber of of the intact
Adrenals Unoperoted
1 7
t 14 I 306=
I 21
I2
I 28 I 38959
I t 42 49 Doys Denervoted I I 465223 480*-18 8ody Weights
I 63
I
487512
1. Development of adrenal tyrosine hydroxylase activity during growth from 8 to 15 wk of age. The numanimals in each group is shown in parentheses. Values are expressed as the means + the standard errors means. Adrenal denervation was performed at 8 wk of age. Denervated glands differ significantly from right glands at 28 days (p < 0.05), 42 days (p < 0.05), and 49 days (p < 0.01) post denervation.
PATRICK
AND
Deuelopmental
KIRSHNER
Changes
in Rat Adrenals
207
16 -
6-
----
Denervoted
__
lntoct Adrenals of Operated Anmols Adrenols Unoperoted
._.-.I 6
2
I 09*2
I 13 I 147f3
I 27 I 24024
Adrenals
of Animals
I 60 Doys
Denervoted 1 426+6
Body
Weqhts
FIG. 2. Development of adrenal tyrosine hydroxylase activity during growth from number of animals in each group is shown in parentheses. Values are expressed as the errors of the means. Adrenal denervation was performed at 23 days of age. Denervated cantly from intact right glands at 13 days (p < O.OP, 27 days (p < 0.021, and 60 days (p tion. Denervated glands 60 days post denervation differ significantly from denervated nervation (p < 0.001).
proximately twice those at 6 days after denervation. However, the intact right gland and the glands from unoperated controls showed larger increases; 27 and 60 days after denervation the tyrosine hydroxylase activity of these glands was significantly higher than the activity in the denervated glands. Dopamine-@-hydroxylase In the group of animals denervated at 8 weeks of age, there were no significant changes in the dopamine-P-hydroxylase activity of the denervated gland. The intact right gland as well as glands from unoperated rats showed increases in dopamine-P-hydroxylase (Fig. 3). The changes in dopamine-P-hydroxylase activity in the animals denervated at 23 days of age were qualitatively similar to the changes in tyrosine hydroxylase (Fig. 4). The activity in the denervated gland 60 days after denervation was approxi-
4 to 12 wk of means * the glands differ < 0.001) post glands 6 days
age. The standard signifidenervapost de-
mately twice that at 6 days while the activities in the intact right gland and in the unoperated animals showed much larger increases. At 27 and 60 days the denervated glands had significantly lower activity than both sets of innervated glands. Catecholamines In both the older and younger animals the changes in catecholamine content were qualitatively similar to changes in tyrosine hydroxylase and dopamine-(5 hydroxylase. In the group denervated at 8 wk of age the catecholamine content of the denervated glands showed no changes while the innervated glands of both operated and unoperated animals showed similar increases (Fig. 5). In the animals denervated at 23 days of age, the catecholamine content of the denervated glands increased, at 60 days, 136% above the levels at 6 days while the innervated glands had much larger increases. At 27
208
DEVELOPMENTAL
BIOLOGY
VOLUME
29.
1972
(8) _.-.-.-.1
----
Denervoted
__
Intact Adrenals of Operated Animals
_ _ _ Adrenals Unoperated II
I 7
2
I 14
I 21
I 28
I I 42 49 Denervated
Days I 246*
I
3
306+12
I
38929
I
Adrenals
of Animals
I 63
I
I
465223 480’18 Body Weights
487fl2
FIG. 3. Development of adrenal dopamine-8-hydroxylase activity during growth from 8 to 15 wk of age. The number of animals in each group is shown in parentheses. Values are expressed as the mean + the standard errors of the means. Adrenal denervation was performed at 8 wk of age. Denervated glands differ significantly from intact right glands 49 days post denervation (p < 0.002).
Denervoted ___
Adrenals Unoperoted
--L
I 6 I e9*2
I I3
27
I 147*3
I 24824
I
of Animals
I
Days
Denervated
60 I 428+8
Body
FIG. number errors cantly glands
Adrenals
lnracr Adrenals of Operated Animals
Weights
4. Development of adrenal dopamine-ghydroxylase activity during growth from 4 to 12 wk of age. The of animals in each group is shown in parentheses. Values are expressed as the means l the standard of the means. Adrenal denervation was performed at 23 days of age. Denervated glands differ signififrom intact right glands at 27 days (p < 0.01) and 60 days (p < 0.01) post denervation. Denervated 60 days post denervation differ significantly from denervated glands 6 days post denervation (p < 0.02).
PATRICK
AND
Developmental
KIRSHNER
Changes
----
Denervated
--
lntacl Adrenals of ODerated Animals
.8I 2
I 7
I
I
246)
I 14
3
306212
I 21
I 28 I 38929
Days
I 42 Denervated I
(Ia I 49 I
465+23 480+18 Body Weights
in Rat Adrenals
- - Adrenals Unoperated
209
Adrenals
of Animals
I 63 I
487+12
FIG 5. Development of adrenal catecholamine levels during growth from 8 to 15 wk of age. The number of animals in each group is shown in parentheses. Values are expressed as the means * the standard errors of the means. Adrenal denervation was performed at 8 weeks of age. Denervated glands differ significantly from intact right glands at 42 days (p < 0.05) and 49 days (p < 0.001) post denervation.
and 60 days after denervation, the catecholamine levels of the intact glands were significantly greater than those of denervated glands (Fig. 6). Supernatant
Protein
The protein content of the 26,000 g supernatant fraction was determined in the adrenal glands of rats denervated at 23 days of age and in a corresponding age group of unoperated animals. There was an increase in protein content during the experimental period but no marked differences between the intact and denervated glands were observed (Fig. 7). At 60 days the intact right gland showed a small (16%) significant increase over the denervated gland, but the protein content of the denervated glands was not different from that of the unoperated controls. Effect of Denervation Gland Weight
on Rat Adrenal
In the group of animals denervated at 8 weeks of age no differences were ob-
served between adrenal weight of intact and denervated glands for up to 28 days postoperatively (Table l), a time at which intact adrenal tyrosine hydroxylase activity was significantly higher than that of the denervated adrenal (Fig. 1). Response
to Insulin
Treatment
Because the data indicated a distinct difference between the effects of denervation at 23 days of age (inhibition of development) and that of denervation at 8 wk of age (complete prevention of development) the possibility of nerve regeneration or incomplete initial denervation in the animals denervated at the younger age had to be considered. Our previous experiments with the animals denervated at 8 weeks of age indicated that only the intact right gland would respond to insulin with a depletion of catecholamines, followed by an increase in tyrosine hydroxylase activity (Patrick and Kirshner, 1971a). In the present experiments, rats denervated at 23 days of age were treated with insulin at
210
DEVELOPMENTAL
gi "
IO .I
BIOLOGY
VOLUME
29, 1972
/
.x
0
Denervaled
(6) ,,.' ,
6 v----
(5) 19)
T;P 4
I.' f -------x
Adrenals
Intact Adrenals of Operated Anmals
02)
---
i -6
Adrenals Unoperoted
of Animals
(61
-t
I 09f2
I 14753
DOYS Denervated
I 240f
4
Body
Wetights
-7k I 42050
FIG. 6. Development of adrenal catecholamine levels during growth from 4 to 12 wk of age. The number of animals in each group is shown in parentheses. Values are expressed as the means * the standard errors of the means. Adrenal denervation was performed at 23 days of age. Denervated glands differ significantly from intact right glands at 27 days (p < 0,002) and 60 days (p c 0.001) post denervation. Denervated glands 60 days post denervation differ significantly from denervated glands 6 days post denervation (p < 0.001).
1600
-
Denervoted
Adrenals
Intact Adrenals of Operoted Animals Adrenals Unoperated
---
I 6
I 13
I 27
I 147f3
I 248+4
Denervoted I 428%
Body
FE. ber of of the on the glands animals vated
Ammals
I 60 Days
I 0922
of
Weights
7. Development of adrenal supernatant protein content during growth from 4 to 12 wk of age. The numanimals in each group is shown in parentheses. Values are expressed as the means * the standard errors means. Adrenal denervation was performed at 23 days of age. Protein determinations were performed 26,000 g supernatant fraction. Denervated glands not significantly different from unoperated intact 27 and 60 days post denervation, and also not significantly different from intact right glands of operated 27 days post denervation. Intact glands of operated animals show a small (16%) increase over denerglands (p < 0.005).
PATRICK
Developmental
AND KIRCHNER
various times after denervation and the catecholamine content of the gland was determined 4 hr after insulin injection (Table 2). Rats given insulin 6, 27, and 60 days after denervation showed catecholamine depletions in the intact adrenals of 73%, 73%, and 68%, respectively (p less than 0.001 from the control right glands at all three time periods after denervation). The denervated glands showed no signs of depletion after insulin treatment, thus indicating that they were initially fully denervated, and remained so throughout the course of the experiment.
EFFECT
TABLE 1 OF DENERVATION UPON RAT ADRENAL GLAND WEIGHT’ Adrenal
Days
weight
(mg/gland)
denervated Intact 7
21.5
14
* 1.3 (4) 25.2 i 1.9
21
23.5
Denervated 25.2
* 2.7 (4) 25.5 * 1.4
(8) zt 1.3
(8) 26.3
(6) 28
23.2
i 1.0 (4)
i
1.6
(6) 25.2
ztz 2.3 (4)
a Left adrenal denervation was performed at 8 wk of age. Values are the means * SEM. The number of animals in each group is in parentheses. None of the differences is significant.
EFFECT OF INSULIN
TREATMENT
6 27 60
DISCUSSION
Rat adrenal tyrosine hydroxylase, dopamine-fl-oxidase, and catecholamines undergo age-dependent increases (Figs. l-6); the agent(s) responsible for the increases has not yet been identified. The effects of denervation upon these increases were dependent upon the age at which the denervations were performed; denervation at 8 wk of age completely prevented any further increases in enzyme and catecholamine levels, whereas denervation at 23 days of age markedly inhibited, but did no.? completely prevent the increases (Fig. l-6). Insulin treatment of the animals denervated at 23 days revealed no neurogenitally induced decline in catecholamine levels of the denervated glands when tested 6, 27, and 60 days post-operatively, while the intact glands responded with 70% depletion (Table 2). These data argue against incomplete initial denervation, or possible nerve regeneration as the explanation for the increases seen in animals denervated at 23 days of age. Differences in circulating hormone levels are also an unlikely explanation, since between 8 and 12 wk of age, animals denervated at 8 wk show no increases in catecholamine-synthesizing enzymes and stores, while animals denervated at 23
TABLE 2 ON ADRENAL CATECHOLAMINE LEVELS (~G/GLAND AT 23 DAYS OF AGE Untreated
Days denervated
Left 4.5
Insulin Right
zt 0.6 (9) 5.2 h 0.5
6.2 zt 0.7 (11) 8.4 * 0.7
(12)
(12)
10.6
211
Changes in Rat Adrenals
Left 4.3
zt 0.6b
4.3
(5) * 0.3”
(6)
OF RATS DENERVATED
treated Right 1.7
* 0.2” (5) 2.3 A 0.1”
(6)
i 0.6 20.9 * 1.2 9.4 zt 0.6b 6.6 * 1.0” (13) (13) (7) (7) The number of animals in each group is shown in parentheses. Values are expressed as the means + the standard errors of the means. Left adrenal denervation was performed at 23 days of age. Animals were killed 4 hr after insulin treatment (5 international units/kg iv). The letters above the standard errors refer to the following p values (Students t test): “p < 0.001 compared to control right gland; %ot significantly different from control left gland.
212
DEVELOPMENTAL
BIOLOGY
days do, It would thus appear that the older animals have developed more of a “nerve dependency” for the age-dependent increases. An example of an apparent “nerve dependency” can be seen in salamander forelimb regeneration (Guth, 1969). When the forelimb of the salamander is amputated, the scar tissue breaks down the tissue grows and differentiates into a new forelimb. Regeneration does not occur if the spinal nerves are cut at the time of amputation. If denervation is performed during the early stages of regeneration, the process stops and proceeds further only after the nerves grow back. An intact nerve supply is not required for the embryonic development of the limb, however, and in fact, the aneurogenic limb, produced by removal of the appropriate portion of the spinal cord and neural crest at an early stage of development, is capable of regenerating an amputated forelimb. When an aneurogenic limb is grafted to a normal host and is then amputated, regeneration occurs. Furthermore, when the grafted aneurogenic limb becomes innervated by the host’s nerves, it retains its ability to regenerate when denervation and amputation are performed within 10 days after host innervation, but loses the ability to regenerate when denervation and amputation are performed 13 days after host innervation. It would thus appear that the limb acquires a “nerve dependency” after a certain number of days of innervation. A similar dependency might be operative in the rat adrenal for increasing its catecholamine-synthesizing enzymes and stores. An alternate explanation could be that the adrenals have an inherent ability to achieve certain levels of enzymes or catecholamines regardless of the state of their nerve supply. Increases beyond this value would be completely nerve dependent. In fact, the levels of catecholamine-synthesizing enzymes and stores of animals denervated at 23 days of
VOLUME
29. 1972
age did not increase above those of animals denervated at 8 wk of age (Figs. l-6). However, this may be the result of an insufficient period of study. The data presented here suggest that the tyrosine hydroxylase, dopamine-phydroxylase, and catecholamine levels of adrenal glands are dependent upon both the age of the animal and upon the neuronal input. These observations are entirely consistent with previous reports that treatments which increase splanchnic stimulation of the adrenal gland result, within 24 hr, in increased levels of tyrosine hydroxylase in intact adrenal glands but not in denervated glands (Kvetnansky et al., 1970; Thoenen et al., 1969; Patrick and Kirshner, 197la), and are also consistent with the report that intravenous injection of acetylcholine will result in increased tyrosine hydroxylase activities in denervated glands (Patrick and Kirshner, 1971a). The increased levels of enzymes and catecholamines in innervated glands indicate that these are adaptive changes to neuronal stimulation, and that, even under ordinary housing conditions, neuronal stimulation is sufficient to result in significant increases in enzyme and catecholamine content. Axelrod et al. (1970) have shown that environmental stimulation can result in increased levels of tyrosine hydroxylase in mice. The neuronal-dependent increases observed here may be due, to some degree, to environmental stimulation but may also reflect a physiological requirement for increased adrenal medullary secretion to compensate for increased body size. A decline, with a half-life of approximately 21 days, in adrenal tyrosine hydroxylase following denervation has been reported (Mueller et al., 1970), but our studies show no such changes. Leastsquare plots of the effect of denervation at 8 wk of age do show a slightly negative slope for tyrosine hydroxylase, dopamineP-hydroxylase, and catecholamines, but
PATRICK AND KIRSHNER
Developmental
Changes in Rat Adrenals
213
the slopes are not significantly different in dopamine-B-hydroxylase activity. Proc. Nat. Acad. Sci. U. S. 66,453-458. from zero. An explanation for this apparent discrepancy may be the manner in MUELLER, R. A., THOENEN, H., and AXELROD, J. (1969a). Adrenal tyrosine hydroxylase: Compensawhich the animals are maintained prior to tory increase in activity after chemical sympathecdenervation. Twenty-four hours after tomy. Science 163, 468-469. treatment with reserpine on three suc- MUELLER, R. A., THOENEN, H., and AXELROD, J. (1969b). Increase in tyrosine hydroxylase activity cessive days, activity is increased 3-fold after reserpine administration. J. Pharmacol. Erp. and requires about 10 days to return to Z’her. 169, 74-79. normal levels (Patrick and Kirshner, MULLER, R. A., THOENEN, H. and AXELROD, J. 1971b). Thus, if the animals were sub(1970). Effect of pituitary and ACTH on the mainjected to conditions which stimulate tenance of basal tyrosine hydroxylase activity in the rat adrenal gland. Endocrinology 86,751-755. splanchnic discharge such as transportation from suppliers, new or crowded sur- NAGATSU, T., LEVI’IT, M., and UDENFRIEND,S. (1964). A rapid and simple radioassay for tyrosine hyroundings, the levels of tyrosine hydroxyldroxylase activity. A&. Biochem. 9, 122-126. ase may be elevated and would show a PATRICK, R. L., and KIRSHNER, N. (1971a). Effect of decline to basal levels upon transection of stimulation on the levels of tyrosine hydroxylase, dopamine-/3-hydroxylase, and catecholamines in the nerve supply. On the other hand, if intact and denervated rat adrenal glands. Mol. animals were maintained for l-2 wk under Pharmacol. 7, 87-96. conditions of low or minimal neuronal in- PATRICK, R. L., and KIRSHNER, N. (1971b). Acetylput, basal levels of tyrosine hydroxylase choline-induced stimulation of catecholamine remay be established and show no further covery in denervated rat adrenals after reserpineinduced depletion. Mol. Phurmacol. 7, 389-396. decrease upon denervation. SCHURR, REFERENCES
AXELROD, J., MUELLER, R. A., HENRY, J. P., and STEPHENS, P. M. (1970). Changes in enzymes involved in the biosynthesis and metabolism of noradrenaline and adrenaline after psychosocial stimulation. Nature (London) 225,1059-1060. FRIEDMAN, S., and KAUFMAN, S. (1965). 3,4-Dihydroxyphenylethylamine-fl-hydroxylase. J. Biol. Chem. 240, 4763-4773. GUTH, L. (1969). “Trophic” effects of Vertebrate Neurons. Neurosci. Res. Bull. 7, l-73. KVETNANSKY, R., WEISE, V. K., and KOPIN, I. 3. (1970). Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-N-methyl transferase by repeated immobilization of rats. Endocrinology 87, 744-749. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MOLINOFF, P., BRIMIJOIN, S., WEINSHILBOUM,R., and AXELROD, J. (1970). Neurally mediated increase
P. E., THOMPSON,
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and ELVEHJEM, C. A. (1950). A method for the determination of free amino acids in rat organs and tissues. J. Biol. Chem. 182, 29-37. THOENEN, H. (1971). Induction of tyrosine hydroxylase in peripheral and central adrenergic neurons by cold-exposure of rats. Nature (London) 228, 861862. THOENEN, H., MUELLER, R. A., and AXELROD, J. (1969). Increased tyrosine hydroxylase activity after drug-induced alteration of sympathetic transmission. Nature (London) 221, 1264. THOENEN, H., MUELLER, R. A., and AXELROD, J. (1969). Trans-synaptic induction of adrenal tyrosine hydroxylase. J. Pharmacol. Exp. 7’her. 169, 244254. UDENFRIEND, S. (1966). Tyrosine hydroxylase. Pharmacol. VIVEROS, 0. KIRSHNER,
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H., ARQUEROS, L., CONNETT, R. J., and N. (1969a). Mechanism of secretion from the adrenal medulla III. Mol. Phurmacol. 5,60-6&X VIVEROS, 0. H., ARQUEROS, L., CONNETT, R. J., and KIRSHNER, N. (196913). Mechanism of secretion from the adrenal medulla IV. Mol. Pharmacol. 5, 69-82.