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A specific increase in intraneuronal monoamine oxidase activity in the rat vas deferens following adrenalectomy
Life Sciences Vol . 11, Part I, pp . 1037-1048, 1972. Printed in Great Britain
Pergamon Press
A SPECIFIC INCREASE IN INTRANEURONAL MONOAMINE OXIDASE ACTIVITY IN THE RAT VAS DEFERENS FOLLOWING ADRENALECTOMY Sumathy S . Sampath and David E . Clarke Department of Pharmacology, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15213
(Received 19 July 1972; in final form 24 August 1972) Sumna~ Bilateral adrenalectomy, but not adrenal demedullation, resulted in a significant increase (4rNX) in the monoamine oxidase (MAO) activity of the rat vas deferens . This increase was completely prevented by surgical denervation and also by daily steroid replacement therapy with dexamethasone . Hypophysectomy, adrenocorticotropic hormone (ACTH) or hypophysectomy plus ACTH, failed to affect MAO activity . It is concluded that bilateral adrenalectomy induces a specific increase in the intraneuronal MAO activity of the vas deferens, as a result of adrenal steroid insufficiency . Furthermore, the effects of steroid lack are not mediated indirectly through an enhanced secretion of ACTH from the pituitary gland . Rat heart monoamine oxidase (MAO) activity is markedly elevated following bilateral adrenalectomy (1-6) .
Avakian and Callingham (1),
who first reported this phenomenon, also showed that this increase in cardiac MAO activity could be prevented by daily injections of hydrocortisone .
Subsequent studies have shown that bilateral adrenal-
ectomy not only affects cardiac MAO, but also increases the MAO activity in certain other organs of the rat (2,6), particularly those possessing a fairly rich sympathetic innervation (6) .
This latter
observation prompted the present authors to investigate the possibility that bilateral adrenalectomy might increase selectively the activity of MAO located inside sympathetic nerves, rather than that which is situated extraneuronally .
We have tested this hypothesis using the
innervated and surgically denervated rat vas deferens, since about 50X of the MAO activity in this organ resides within postganglionic
1037
1038
MAO Activity and Adrenalectomy
sympathetic fibers
(7) .
Vol. 11, No . 22
In addition, we studied the influence of
hypophysectomy and adrenocorticotropic hormone (ACTH)
on the MAO
activity of the vas deferens, since it seemed reasonable to spectulate that the effects of adrenal steroid lack might be mediated through a resulting hypersecretion of ACTH . Methods The experiments were performed on male, albino Sprague-Dawley rats, weighing between 175 and 200 grams . Unilateral denervation of the left vas deferens was performed in vivo under sodium pentobarbital anesthesia
(60 mg/kg, i .p .),
according to the method described by Birmingham (8) .
The right vas
deferens was gently pulled from the scrotum and replaced, in order to serve as the contra lateral sham-operated control .
Bilateral
adrenalectomy and sham-operations were performed 48 hours later . All operated rats were immediately supplied with 0 .9% w/v NaCI solution as the sole drinking fluid and the animals were sacrificed 10 days later .
Bilateral adrenal demedullation was performed by the method
of Shromblad (9) .
This group of animals and the sham-operated controls
were maintained on NaCI as described above . In some of the foregoing experiments, dexamethasone sodium phosphate (Decadron, Merck Sharp b Dohme), (2 x 30 u9/rat/day for 10 days), was administered intraperitoneally. Hypophysectomized and sham-operated control rats were obtained from Zivic Miller Laboratories, Inc ., Pittsburgh, Pa . were maintained at 26-27 °C on a normal
The animals
laboratory diet supplemented
with pears and were given a drinking fluid containing 0.9% w/v NaCI
in a 5% w/v sucrose solution .
days following surgery. of two groups :
The animals were sacrificed lr1
The sham-operated control rats consisted
Group I was of equivalent age to the hypophysectomized
MAO Activity and Adrenalectomy
Vol. 11, No. 22
1039
animals, whereas Group II was younger, so as to be of an equal mean weight to the hypophysectomized rats at the time of sacrifice .
In
some experiments, purified ACTH in gelatin (Cortrophin Gel, Organon) was administered subcutaneously to both hypophysectomized and control groups in a dose of 4 units/rat/day for 10 days . ACTH treatment and the surgical
The adequacy of the
procedures was assessed by the result-
ing change in adrenal weight . MAO activity was assayed, on whole tissue
homogenates, by
determining the indole acetic acid (IAA) formed from tryptamine in the presence of excess aldehyde dehydrogenase, as described by Lovenberg, et al . (10) .
The tissue was checked for linearity with respect to
both tissue concentration and incubation time, so as to be sure that the initial velocity rate of the reaction was being measured . Endogenous norepinephrine was isolated from perchloric acid tissue extracts by alumina adsorption at pH 8 .6, followed by elution with 0 .2 N acetic acid .
The norepinephrine was then oxidized according
to the method of Chang (11) and the fluorescence read on an AmincoBowman spectraphotofluor~ometer .
The values were corrected for a
mean norepinephrine recovery of 8D ± 1 .8% . Wet/dry tissue weight ratios were determined by heating the tissue to a constant weight at 85°C . Results Figure 1 shows that the MAO activity of the intact vas deferens was significantly higher 10 days after bilateral adrenalectomy than in the sham-operated controls .
Surgical denervation of the vas
deferens significantly reduced (p
< 0 .01) the MAO activity in sham-
adrenalectomized rats by about 50% and also completely abolished the increase in MAO activity resulting from bilateral adrenalectomy . The completeness of surgical denervation was validated by measuring endogenous norepinephrine .
Table 1 shows that no detectable
1040
MAO Activity and Adrenalectomy
Vol . 11, No. 22
~ 20.0 = 18 .0 W
N
/ INTACT ® DENERVATED I
16.0 14.0
E 12 .0 a `~
W
10.0 8.0 6 .0 4.0 2 .0 SHAM
AX
Figure 1 . The effect of bilateral adrenalectomy (AX) on the MAO activity in the intact and denervated rat vas deferens . Each column represents the mean + the standard error from 6 animals . Statistical differences : shâm intact vis AX intact, p < 0 .05 ; sham intact vis sham denervated, p <0 .01 ; AX intact vis AX den ervated, p < 0 .001 ; sham denervated vis AX denervated, not significant at p = 0 .05 .
TABLE 1 The effect of various conditions on the norepinephrine (NE) content of the vas deferens . Each value represents the mean of six determinations .
CONDITION
NE ug/9 wet weight ± S .E . 6 .70 + 0 .83
Innervated Denervated
not detectable
Adrenalectomized
7 .03 + 0 .89
Sham-operated
6 .66 + 0.71
(None of the values are significantly different from each other at p = 0 .05) norepinephrine was found in the denervated vas deferens compared to 7 ug/9 in the contralateral intact organ .
Furthermore, despite
the increased MAO activity in adrenalectomized rats, the endogenous
1041
MAO Activity and Adrenalectomy
Vol . 11, No. 22
norepinephrine content of the vas deferens remained unchanged . The water content of the innervated vas deferens following bilateral adrenalectorgy (84 .04 ± 0 .790 was statistically higher (p < 0 .01) than after sham-adrenalectomy (78 .33 + 1 .720 . The effect of steroid administration in conjunction with bilateral adrenalectomy was studied utilizing the potent glucocorticoid, dexamethasone .
Figure 2 shows that dexamethasone completely
prevented the rise in MAO activity due to bilateral adrenalectomy (p < 0 .01), whereas it failed to alter enzymatic activity in shamadrenalectomized rats .
Adrenal demedullation alone, or in
combination with dexamethasone, failed to produce any significant change in MAO activity .
10 9 W
8
â â
H 4 N W 3 O
2
3 4
8
2 I
Figure 2 . The effect of dexamethasone (DEX . 2 x 30 ug/rat/day, i .p .) on the MAO activity of the rat vas deferens after bilateral adrenalectorgy (AX) and demedullation (DM) . Column (1) sham untreated, (2) sham operated plus DEX, (3) AX untreated, (4) AX plus DEX, (5) DM untreated, (6) DM plus DEX . Each column represents the mean + the standard error from 6 animals . Column (3) is significantly different l'rcm all other columns, p < 0.01 .
104 2
MAO Activity and Adrenalectomy
Vol. 11, No . 22
Figure 3 shows that the HMO activity in the vas deferens is not significantly altered by hypophysectomy, ACTH or hypophysectomy plus ACTH .
Furthermore,
the MAO activity of the two sham-operated
control groups did not differ significantly from each other .
This
result strongly indicates that the MAO activity, or the enzyme s) itself, did not change over the experimental period as a result of continued growth and maturity .
WEIGHT MATCHED
AGE MATCHED
x W N N_ F
E v~ ç â ri
W
O i
Figure 3 . The effect of hypophysectomy (2), ACTH (3) and hypophysectomy plus ACTH (4) on the MAO activity of the rat vas Each deferens . Columns (1) refer to the respective control groups . column represents the mean + the standard error from 10 animals . None of the columns are significantly different from each other at p = 0 .05. Table 2 illustrates the expected effects resulting from ACTH treatment and hypophysectomy on the weight of the adrenal glands .
Vol. 11, No . 22
MAO Activity and Adrenalectomy
TABLE 2 The effect of various treatments and procedures on the mean weight of rat adrenal glands . Each group contained six animals .
CONDITION
ADRENAL WEIC~iT (mg/pair + S .E .)
1
Weight matched control
45 .70 ± 0 .89
2
Weight matched control + ACTH
54,47 + 1 .20a
3
Age matched control
68 .92 ± 2 .00
4
Age matched control + ACTH
75 .50 ± 0.9ßb
5
Hypophysectomized
17 .67 ± 0.72c
6
Hypophysectomized + .ACTH
23 .75 ± 0.51d
a-significantly b-significantly c-significantly d-significantly
different different different different
from from from from
1, p 3, p 1 and 5, p
< 0 .001 < 0 .05 3, p < 0.001 < 0.001
Discussion Bilateral adrenalectomy produced a clear rise (40%) in the MAO activity of the innervated vas deferens .
This elevation was not due
to enzyme concentration consequential to an increased tissue mass to water ratio, since the organs from adrenalectomized rats contained about 6% more water than those from the sham-operated animals . Thus, the actual elevation in MAO activity has been slightly underestimated when expressed on a tissue-weight basis . Although previous studies (2,6) have shown that bilateral adrenalectomy increases the MAO activity of the rat vas deferens, they have not determined the possible mechanism and the specific localization of the increase, within the structures of this organ .
The present
data strongly indicate that the increase is located solely within the sympathetic innervation, since bilateral adrenalectomy failed to
Vol. 11, No . 22
MAO Activity and Adrenalectomy
1044
alter MAO activity following surgical possibility is that the fall
denervation .
An alternative
in enzymatic activity after denervation
might result from a postsynaptic loss of enzyme which is sensitive to the effects of adrenalectomy .
Such a loss might result from
the absence of neuroch~nical transmission .
However, this possibility
seems far less plausible in view of the elegant rebuttal
by Jarrott
and Iversen (7) against the existence of any such tropic neuronal fluence .
in-
These authors, and Jarrott (12), have presented excellent
evidence that the diminution in the rat vas deferens MAO activity following surgical
denervation represents that portion of the total
enzyme which is intraneuronally located .
This conclusion applies
to the present study which employed the same denervation technique. Also we obtained about the same decrease in MAO activity (50%) as the above mentioned authors .
Furthermore,
this occurred at a time
when the endogenous norepinephrine level was depleted to below measurable limits, thus attesting to the completeness of our denervation technique .
It seems fairly certain therefore, that the rise
in MAO activity following bilateral adrenalectomy was indeed intraneuronally located .
The question then arises as to whether this
increase derives from existing sympathetic nerves or from an creased sympathetic innervation to the vas deferens .
in-
Muntzing (13)
has reported just such an effect in the rat prostate under conditions of androgenic insufficiency . chemical
On the basis of biochemical and histo-
evidence he concluded that part of the resulting elevation
in MAO activity was attributable to the increased density of the sympathetic innervation .
Our studies tend to rule out this
possibility in the vas deferens, since the norepinephrine content was not significantly altered following bilateral adrenalectomy . It seems extremely unlikely that exactly the same amount of norepinephrine, as found in the vas deferens from sham-operated rats, would
Vol. 11, No. 22
1045
MAO Activity and Adrenalectomy
now be distributed within an increased innervation .
The finding
of an unaltered endogenous level of norepinephrine, in the presence of an elevated intraneuronal MAO activity, points to a functional increase in the synthesis rate of norepinephrine in order to maintain normal storage levels .
In this respect, Westfall and Osada (4) have shown
that the turnover rate of cardiac norepinephrine is increased markedly in adrenalectomized rats . Caesar, et al . (2) observed a significant increase in mitochondrial. and microsomal MAO activity of the rat vas deferens following adrenalectomy .
In view of the present data it seems likely that the MAO
in the sympathetic nerves of this particular organ is situated in association with both of these subcellular structures .
This observation provides
further indirect evidence for the growing claim that part of the MAO in sympathetic neurons is integral with the microsomes (14, 15, 16) . Furthermore, the increase in deaminated catecholamine metabolites recovered in the urine of adrenalectomized rats (2), must now be considered to result from an enhanced intraneuronal metabolism, rather than through an increased deamination at extraneuronal sites .
This is
especially so, since both this phenomenon (2) and the rise in heart (1) and vas deferens MAO activity, is prevented by steroid replacement therapy. Bhagat (5) found that adrenal demedullation had no significant effect on rat heart MAO activity .
In the present experiments the same
procedure also failed to alter this enzyme in the vas deferens . Therefore, a lack of medullary catecholamine secretion is not a potent stimulus for elevating intraneuronal MAO . adrenal steroid lack is extremely potent .
On the other hand,
Our experiments show that
the intraneuronal rise in the MAO activity of the vas deferens is dependent upon the absence of adrenal glucocorticoid activity .
This conclusion
is based on the fact that the potent glucocorticoid, dexamethasone,
1046
Vol. il, No . 22
MAO Activity and Adrenalectomy
completely prevented the MAO increase following adrenalectomy . However, excess glucocorticoid activity, as might be expected to result from treating intact rats with dexamethasone, failed to affect MAO activity .
This result is consistent with the similar in-
effectiveness of ACTH treatment on the enzymatic activity of MAO in intact rats .
Thus a possible hypersecretion of corticosterone,
the predominant steroid released from the rat adrenal (17), also fails to affect MAO activity in the vas deferens . The mechanism whereby adrenal steroid lack results in an increase in MAO activity has not been resolved, but it is known not to be an indirect apparent increase consequential to mitochondria) permeability changes (3) .
In addition, the present experiments have shown that the
increase in MAO activity is not mediated through an enhanced secretion of ACTH from the pituitary gland,
since hypophysectomy, ACTH and hypo-
physectomy plus ACTH, failed to affect the enzyme .
The failure of these
procedures to influence MAO cannot be explained on the grounds of inadequate dosage or incomplete surgery, since the adrenal weights revealed the expected alterations, similar to those reported by Weinshilboum and Axelrod (8) .
Hypophysectomy arrests growth, thus retarding maturity of
the hypophysectomized rats compared with the sham-operated controls . Cardiac MAO activity is known to increase as a function of heart weight (19), and such a relationship might have applied in the vas deferens, resulting in spurious differences between the sham and hypophysectomized groups .
However,
this possibility was refuted by the failure to obtain a
significant difference between the sham-operated age-matched and weightmatched control groups .
Furthermore, neither of these two control groups
differed significantly from any hypophysectomized grôup .
However the failure
of hypophysectomy to enhance MAO activity is somewhat surprising, since the adrenal corticosterone content and in vitro synthesis rate were markedly
Vol. 11, No. 22
MAO Activity and Adrenalectomy
attenuated by this procedure (6, unpublished observations) .
1047
Even so,
hypophysectomy would not be expected to lower plasma steroid levels to the same extent as bilateral adrenalectomy and such a difference might be the crucial precipitating factor .
On the other hand,
a severe defficiency of glucocorticoid activity may not be the only factor involved .
Thus, the failure of hypophysectomy to raise MAO
activity might suggest a vital role of another pituitary hormone or hormones, other than ACTH . Açknowled~ments The excellent surgical skill of Mr . T-M. Shih is gratefully appreciated .
This investigation was supported by U.S .P .H .S . Grant
GM-1217 . References V . M. AVAKIAN and B . A. CALLINGHAM,
Br . J . Pharmac . , 33, 211P (1968) .
2.
P . M. CAESAR, G. G . S . COLLINS and M. SANDLER, 19, 921 (1970) .
3.
B . A. CALLINGHAM, and L . D . CORTE,
4.
T. C . WESTFALL and H . OSADA,
5.
B . BHAGAT,
6.
S . S . SAMPATH, T-M . SHIH and D . E . CLARKE, Fedn . Proc . , 31, 544 (1972) .
7.
B. JARROTT and L. L . IVERSON,
8.
A . T . BIRMINGFIAM,
9.
B . C . R. STROMBLAD and M. NICKERSON, 154 (1961) .
Br . J . Pharmac . , 41, 292P (1971) .
J . Pharmac . exp . Ther . 167, 300 (1969) .
Br . J . Pharmac . , 37, 34 (1969) .
J . Neurochem. , 18, 1 (1971) .
J . Physiol ., Lond . , 206, 645 (1970) . J . Pharmac . exp . Ther, 134,
W . LOVENBERG, R . J . LEVINE and A . SJOERDSMA, 135, 7 (1962) . 11 . C . C . CHANG, 12 . B. JARROTT, 13 . J . MUNTZING,
Biochem . Pharmac . ,
J . Pharmac . exp . Ther .
Int. J . Neuropharmac . 3, 643 (1964) . J . Neurochem. 18, 7 (1971) . Acta pharmac . tox . , 30, 208 (1971) .
14 . R . H . ROTH and L . STJARNE,
Acta physi ol . stand.
68, 342 (1966) .
1048
MAO Activity and Adrenalectomy
15 . J . de CHAMPLAIN, R . A. MUELLER and J . AXELROD, Ther . , 166, 339 (1969) . 16 . L . STJARNE, R . H . ROTH and N . J. GIARMAN, 2008 (1968) . 17 . I . E . BUSH,
Vol. 11, No . 22
J . Pharmac . exp .
Biochem . Pharmac . _17,
J . Endocrinology , 9, 95 (1953) .
18 . R . WEINSHILBOUM and J . AXELROD,
Endoçrinology, 87, 894 (1970) .
19 . J . de CHAMPLAIN, L . R . KRAKOFF and J . AXELROD, 23, 361 (1968) .
Circulation Res . ,
Pergamon Press
A SPECIFIC INCREASE IN INTRANEURONAL MONOAMINE OXIDASE ACTIVITY IN THE RAT VAS DEFERENS FOLLOWING ADRENALECTOMY Sumathy S . Sampath and David E . Clarke Department of Pharmacology, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania 15213
(Received 19 July 1972; in final form 24 August 1972) Sumna~ Bilateral adrenalectomy, but not adrenal demedullation, resulted in a significant increase (4rNX) in the monoamine oxidase (MAO) activity of the rat vas deferens . This increase was completely prevented by surgical denervation and also by daily steroid replacement therapy with dexamethasone . Hypophysectomy, adrenocorticotropic hormone (ACTH) or hypophysectomy plus ACTH, failed to affect MAO activity . It is concluded that bilateral adrenalectomy induces a specific increase in the intraneuronal MAO activity of the vas deferens, as a result of adrenal steroid insufficiency . Furthermore, the effects of steroid lack are not mediated indirectly through an enhanced secretion of ACTH from the pituitary gland . Rat heart monoamine oxidase (MAO) activity is markedly elevated following bilateral adrenalectomy (1-6) .
Avakian and Callingham (1),
who first reported this phenomenon, also showed that this increase in cardiac MAO activity could be prevented by daily injections of hydrocortisone .
Subsequent studies have shown that bilateral adrenal-
ectomy not only affects cardiac MAO, but also increases the MAO activity in certain other organs of the rat (2,6), particularly those possessing a fairly rich sympathetic innervation (6) .
This latter
observation prompted the present authors to investigate the possibility that bilateral adrenalectomy might increase selectively the activity of MAO located inside sympathetic nerves, rather than that which is situated extraneuronally .
We have tested this hypothesis using the
innervated and surgically denervated rat vas deferens, since about 50X of the MAO activity in this organ resides within postganglionic
1037
1038
MAO Activity and Adrenalectomy
sympathetic fibers
(7) .
Vol. 11, No . 22
In addition, we studied the influence of
hypophysectomy and adrenocorticotropic hormone (ACTH)
on the MAO
activity of the vas deferens, since it seemed reasonable to spectulate that the effects of adrenal steroid lack might be mediated through a resulting hypersecretion of ACTH . Methods The experiments were performed on male, albino Sprague-Dawley rats, weighing between 175 and 200 grams . Unilateral denervation of the left vas deferens was performed in vivo under sodium pentobarbital anesthesia
(60 mg/kg, i .p .),
according to the method described by Birmingham (8) .
The right vas
deferens was gently pulled from the scrotum and replaced, in order to serve as the contra lateral sham-operated control .
Bilateral
adrenalectomy and sham-operations were performed 48 hours later . All operated rats were immediately supplied with 0 .9% w/v NaCI solution as the sole drinking fluid and the animals were sacrificed 10 days later .
Bilateral adrenal demedullation was performed by the method
of Shromblad (9) .
This group of animals and the sham-operated controls
were maintained on NaCI as described above . In some of the foregoing experiments, dexamethasone sodium phosphate (Decadron, Merck Sharp b Dohme), (2 x 30 u9/rat/day for 10 days), was administered intraperitoneally. Hypophysectomized and sham-operated control rats were obtained from Zivic Miller Laboratories, Inc ., Pittsburgh, Pa . were maintained at 26-27 °C on a normal
The animals
laboratory diet supplemented
with pears and were given a drinking fluid containing 0.9% w/v NaCI
in a 5% w/v sucrose solution .
days following surgery. of two groups :
The animals were sacrificed lr1
The sham-operated control rats consisted
Group I was of equivalent age to the hypophysectomized
MAO Activity and Adrenalectomy
Vol. 11, No. 22
1039
animals, whereas Group II was younger, so as to be of an equal mean weight to the hypophysectomized rats at the time of sacrifice .
In
some experiments, purified ACTH in gelatin (Cortrophin Gel, Organon) was administered subcutaneously to both hypophysectomized and control groups in a dose of 4 units/rat/day for 10 days . ACTH treatment and the surgical
The adequacy of the
procedures was assessed by the result-
ing change in adrenal weight . MAO activity was assayed, on whole tissue
homogenates, by
determining the indole acetic acid (IAA) formed from tryptamine in the presence of excess aldehyde dehydrogenase, as described by Lovenberg, et al . (10) .
The tissue was checked for linearity with respect to
both tissue concentration and incubation time, so as to be sure that the initial velocity rate of the reaction was being measured . Endogenous norepinephrine was isolated from perchloric acid tissue extracts by alumina adsorption at pH 8 .6, followed by elution with 0 .2 N acetic acid .
The norepinephrine was then oxidized according
to the method of Chang (11) and the fluorescence read on an AmincoBowman spectraphotofluor~ometer .
The values were corrected for a
mean norepinephrine recovery of 8D ± 1 .8% . Wet/dry tissue weight ratios were determined by heating the tissue to a constant weight at 85°C . Results Figure 1 shows that the MAO activity of the intact vas deferens was significantly higher 10 days after bilateral adrenalectomy than in the sham-operated controls .
Surgical denervation of the vas
deferens significantly reduced (p
< 0 .01) the MAO activity in sham-
adrenalectomized rats by about 50% and also completely abolished the increase in MAO activity resulting from bilateral adrenalectomy . The completeness of surgical denervation was validated by measuring endogenous norepinephrine .
Table 1 shows that no detectable
1040
MAO Activity and Adrenalectomy
Vol . 11, No. 22
~ 20.0 = 18 .0 W
N
/ INTACT ® DENERVATED I
16.0 14.0
E 12 .0 a `~
W
10.0 8.0 6 .0 4.0 2 .0 SHAM
AX
Figure 1 . The effect of bilateral adrenalectomy (AX) on the MAO activity in the intact and denervated rat vas deferens . Each column represents the mean + the standard error from 6 animals . Statistical differences : shâm intact vis AX intact, p < 0 .05 ; sham intact vis sham denervated, p <0 .01 ; AX intact vis AX den ervated, p < 0 .001 ; sham denervated vis AX denervated, not significant at p = 0 .05 .
TABLE 1 The effect of various conditions on the norepinephrine (NE) content of the vas deferens . Each value represents the mean of six determinations .
CONDITION
NE ug/9 wet weight ± S .E . 6 .70 + 0 .83
Innervated Denervated
not detectable
Adrenalectomized
7 .03 + 0 .89
Sham-operated
6 .66 + 0.71
(None of the values are significantly different from each other at p = 0 .05) norepinephrine was found in the denervated vas deferens compared to 7 ug/9 in the contralateral intact organ .
Furthermore, despite
the increased MAO activity in adrenalectomized rats, the endogenous
1041
MAO Activity and Adrenalectomy
Vol . 11, No. 22
norepinephrine content of the vas deferens remained unchanged . The water content of the innervated vas deferens following bilateral adrenalectorgy (84 .04 ± 0 .790 was statistically higher (p < 0 .01) than after sham-adrenalectomy (78 .33 + 1 .720 . The effect of steroid administration in conjunction with bilateral adrenalectomy was studied utilizing the potent glucocorticoid, dexamethasone .
Figure 2 shows that dexamethasone completely
prevented the rise in MAO activity due to bilateral adrenalectomy (p < 0 .01), whereas it failed to alter enzymatic activity in shamadrenalectomized rats .
Adrenal demedullation alone, or in
combination with dexamethasone, failed to produce any significant change in MAO activity .
10 9 W
8
â â
H 4 N W 3 O
2
3 4
8
2 I
Figure 2 . The effect of dexamethasone (DEX . 2 x 30 ug/rat/day, i .p .) on the MAO activity of the rat vas deferens after bilateral adrenalectorgy (AX) and demedullation (DM) . Column (1) sham untreated, (2) sham operated plus DEX, (3) AX untreated, (4) AX plus DEX, (5) DM untreated, (6) DM plus DEX . Each column represents the mean + the standard error from 6 animals . Column (3) is significantly different l'rcm all other columns, p < 0.01 .
104 2
MAO Activity and Adrenalectomy
Vol. 11, No . 22
Figure 3 shows that the HMO activity in the vas deferens is not significantly altered by hypophysectomy, ACTH or hypophysectomy plus ACTH .
Furthermore,
the MAO activity of the two sham-operated
control groups did not differ significantly from each other .
This
result strongly indicates that the MAO activity, or the enzyme s) itself, did not change over the experimental period as a result of continued growth and maturity .
WEIGHT MATCHED
AGE MATCHED
x W N N_ F
E v~ ç â ri
W
O i
Figure 3 . The effect of hypophysectomy (2), ACTH (3) and hypophysectomy plus ACTH (4) on the MAO activity of the rat vas Each deferens . Columns (1) refer to the respective control groups . column represents the mean + the standard error from 10 animals . None of the columns are significantly different from each other at p = 0 .05. Table 2 illustrates the expected effects resulting from ACTH treatment and hypophysectomy on the weight of the adrenal glands .
Vol. 11, No . 22
MAO Activity and Adrenalectomy
TABLE 2 The effect of various treatments and procedures on the mean weight of rat adrenal glands . Each group contained six animals .
CONDITION
ADRENAL WEIC~iT (mg/pair + S .E .)
1
Weight matched control
45 .70 ± 0 .89
2
Weight matched control + ACTH
54,47 + 1 .20a
3
Age matched control
68 .92 ± 2 .00
4
Age matched control + ACTH
75 .50 ± 0.9ßb
5
Hypophysectomized
17 .67 ± 0.72c
6
Hypophysectomized + .ACTH
23 .75 ± 0.51d
a-significantly b-significantly c-significantly d-significantly
different different different different
from from from from
1, p 3, p 1 and 5, p
< 0 .001 < 0 .05 3, p < 0.001 < 0.001
Discussion Bilateral adrenalectomy produced a clear rise (40%) in the MAO activity of the innervated vas deferens .
This elevation was not due
to enzyme concentration consequential to an increased tissue mass to water ratio, since the organs from adrenalectomized rats contained about 6% more water than those from the sham-operated animals . Thus, the actual elevation in MAO activity has been slightly underestimated when expressed on a tissue-weight basis . Although previous studies (2,6) have shown that bilateral adrenalectomy increases the MAO activity of the rat vas deferens, they have not determined the possible mechanism and the specific localization of the increase, within the structures of this organ .
The present
data strongly indicate that the increase is located solely within the sympathetic innervation, since bilateral adrenalectomy failed to
Vol. 11, No . 22
MAO Activity and Adrenalectomy
1044
alter MAO activity following surgical possibility is that the fall
denervation .
An alternative
in enzymatic activity after denervation
might result from a postsynaptic loss of enzyme which is sensitive to the effects of adrenalectomy .
Such a loss might result from
the absence of neuroch~nical transmission .
However, this possibility
seems far less plausible in view of the elegant rebuttal
by Jarrott
and Iversen (7) against the existence of any such tropic neuronal fluence .
in-
These authors, and Jarrott (12), have presented excellent
evidence that the diminution in the rat vas deferens MAO activity following surgical
denervation represents that portion of the total
enzyme which is intraneuronally located .
This conclusion applies
to the present study which employed the same denervation technique. Also we obtained about the same decrease in MAO activity (50%) as the above mentioned authors .
Furthermore,
this occurred at a time
when the endogenous norepinephrine level was depleted to below measurable limits, thus attesting to the completeness of our denervation technique .
It seems fairly certain therefore, that the rise
in MAO activity following bilateral adrenalectomy was indeed intraneuronally located .
The question then arises as to whether this
increase derives from existing sympathetic nerves or from an creased sympathetic innervation to the vas deferens .
in-
Muntzing (13)
has reported just such an effect in the rat prostate under conditions of androgenic insufficiency . chemical
On the basis of biochemical and histo-
evidence he concluded that part of the resulting elevation
in MAO activity was attributable to the increased density of the sympathetic innervation .
Our studies tend to rule out this
possibility in the vas deferens, since the norepinephrine content was not significantly altered following bilateral adrenalectomy . It seems extremely unlikely that exactly the same amount of norepinephrine, as found in the vas deferens from sham-operated rats, would
Vol. 11, No. 22
1045
MAO Activity and Adrenalectomy
now be distributed within an increased innervation .
The finding
of an unaltered endogenous level of norepinephrine, in the presence of an elevated intraneuronal MAO activity, points to a functional increase in the synthesis rate of norepinephrine in order to maintain normal storage levels .
In this respect, Westfall and Osada (4) have shown
that the turnover rate of cardiac norepinephrine is increased markedly in adrenalectomized rats . Caesar, et al . (2) observed a significant increase in mitochondrial. and microsomal MAO activity of the rat vas deferens following adrenalectomy .
In view of the present data it seems likely that the MAO
in the sympathetic nerves of this particular organ is situated in association with both of these subcellular structures .
This observation provides
further indirect evidence for the growing claim that part of the MAO in sympathetic neurons is integral with the microsomes (14, 15, 16) . Furthermore, the increase in deaminated catecholamine metabolites recovered in the urine of adrenalectomized rats (2), must now be considered to result from an enhanced intraneuronal metabolism, rather than through an increased deamination at extraneuronal sites .
This is
especially so, since both this phenomenon (2) and the rise in heart (1) and vas deferens MAO activity, is prevented by steroid replacement therapy. Bhagat (5) found that adrenal demedullation had no significant effect on rat heart MAO activity .
In the present experiments the same
procedure also failed to alter this enzyme in the vas deferens . Therefore, a lack of medullary catecholamine secretion is not a potent stimulus for elevating intraneuronal MAO . adrenal steroid lack is extremely potent .
On the other hand,
Our experiments show that
the intraneuronal rise in the MAO activity of the vas deferens is dependent upon the absence of adrenal glucocorticoid activity .
This conclusion
is based on the fact that the potent glucocorticoid, dexamethasone,
1046
Vol. il, No . 22
MAO Activity and Adrenalectomy
completely prevented the MAO increase following adrenalectomy . However, excess glucocorticoid activity, as might be expected to result from treating intact rats with dexamethasone, failed to affect MAO activity .
This result is consistent with the similar in-
effectiveness of ACTH treatment on the enzymatic activity of MAO in intact rats .
Thus a possible hypersecretion of corticosterone,
the predominant steroid released from the rat adrenal (17), also fails to affect MAO activity in the vas deferens . The mechanism whereby adrenal steroid lack results in an increase in MAO activity has not been resolved, but it is known not to be an indirect apparent increase consequential to mitochondria) permeability changes (3) .
In addition, the present experiments have shown that the
increase in MAO activity is not mediated through an enhanced secretion of ACTH from the pituitary gland,
since hypophysectomy, ACTH and hypo-
physectomy plus ACTH, failed to affect the enzyme .
The failure of these
procedures to influence MAO cannot be explained on the grounds of inadequate dosage or incomplete surgery, since the adrenal weights revealed the expected alterations, similar to those reported by Weinshilboum and Axelrod (8) .
Hypophysectomy arrests growth, thus retarding maturity of
the hypophysectomized rats compared with the sham-operated controls . Cardiac MAO activity is known to increase as a function of heart weight (19), and such a relationship might have applied in the vas deferens, resulting in spurious differences between the sham and hypophysectomized groups .
However,
this possibility was refuted by the failure to obtain a
significant difference between the sham-operated age-matched and weightmatched control groups .
Furthermore, neither of these two control groups
differed significantly from any hypophysectomized grôup .
However the failure
of hypophysectomy to enhance MAO activity is somewhat surprising, since the adrenal corticosterone content and in vitro synthesis rate were markedly
Vol. 11, No. 22
MAO Activity and Adrenalectomy
attenuated by this procedure (6, unpublished observations) .
1047
Even so,
hypophysectomy would not be expected to lower plasma steroid levels to the same extent as bilateral adrenalectomy and such a difference might be the crucial precipitating factor .
On the other hand,
a severe defficiency of glucocorticoid activity may not be the only factor involved .
Thus, the failure of hypophysectomy to raise MAO
activity might suggest a vital role of another pituitary hormone or hormones, other than ACTH . Açknowled~ments The excellent surgical skill of Mr . T-M. Shih is gratefully appreciated .
This investigation was supported by U.S .P .H .S . Grant
GM-1217 . References V . M. AVAKIAN and B . A. CALLINGHAM,
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2.
P . M. CAESAR, G. G . S . COLLINS and M. SANDLER, 19, 921 (1970) .
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B . A. CALLINGHAM, and L . D . CORTE,
4.
T. C . WESTFALL and H . OSADA,
5.
B . BHAGAT,
6.
S . S . SAMPATH, T-M . SHIH and D . E . CLARKE, Fedn . Proc . , 31, 544 (1972) .
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B. JARROTT and L. L . IVERSON,
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A . T . BIRMINGFIAM,
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B . C . R. STROMBLAD and M. NICKERSON, 154 (1961) .
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J . Pharmac . exp . Ther . 167, 300 (1969) .
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Biochem . Pharmac . ,
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MAO Activity and Adrenalectomy
15 . J . de CHAMPLAIN, R . A. MUELLER and J . AXELROD, Ther . , 166, 339 (1969) . 16 . L . STJARNE, R . H . ROTH and N . J. GIARMAN, 2008 (1968) . 17 . I . E . BUSH,
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