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Age-related differences in the incorporation of 3H-arginine into vasopressin in Fischer 344 rats
liv~cpimcntal (;crontolo~,y, Vol. 22, pp. 113 125. 1987
0531-5565/87 $3.00 -} .00 Copyright ' 1987 Pergamon Journals l,td
Printed in the USA. All rights reserved.
AGE-RELATED DIFFERENCES IN THE INCORPORATION OF 3H-ARGININE INTO VASOPRESSIN IN FISCHER 344 RATS
VI.ASTA K . Z B U Z E K , VRATISLAV Z B U Z E K , a n d W E N - T S I E N W u University of Medicine and Dentistry, New Jersey Medical School Department of Aneslhesiology, Newark, NJ Abstract - - We studied the effect of aging on the incorporation of :~H-arginine into
vasopressin (VP) molecule in the hypothalamus (HT) and the rate of its transport into the neurohypophysis (NH) of male Fischer 344 rats of different ages: 2-3 months (young, n - 28), 11-13 months (adult, n = 30) and 30 months (old, n = 27). aHarginine was injected in a single dose through a permanent cannula into the lateral ventricle of an awake animal. The rats were decapitated 1, 6, and 24 h after the injection. VP was extracted from individual HTs and NHs (without pars intermedia), purified in a chromatography column and determined by RIA. Hypot/ta/aml~s: One hour after the injection the all-activity was highest in the young, lower in the adult and lowest in the old rats, whereas the specific activity (cpm/pg VP) was similar in all age groups, reflecting endogenous VP content to be highest in the young, lower in the adult and lowest in the old rats. In the young rats both activities decreased 6 and 24 h after the injection. The adult rats also exhibited declining activities, whereas in the old rats the activities remained unchanged from 1 to 24 h after the injection. Ncurohypophysis: One hour after the injection of radioactive label all-activity and specific activity were significantly lower in the NH of the old than in those of young and adult rats. The activities increased 6 h after the injection in all age groups with the lowest values in the old rats. After 6 h, both activities in the old rats reached only the l-h values t)f the young and adult rats suggesting a delay of at least 6 h in the axonal transport of newly synthetized VP in aged rats. Twenty-four hours after the injection both activities increased in all age groups exhibiting no difference among the age groups, indicating a decreased release of the newly synthetized VP from the NH of aged rats. These data demonstrate an age-related decrease in the rate of incorporation of aH-arginine into VP, in the rate of its axonal transport and in the release of newly synthetized VP from the NH of Fischer 344 rats.
Key
words:
hypothalamus,
vasopressin, 3H-arginine incorporation, neurohypophysis, Fischer 344 rats, aging
vasopressin
transport,
THE LOWER a m o u n t o f n e u r o s e c r e t o r y m a t e r i a l in v a s o p r e s s i n e r g i c n e u r o n e s ( R o d e c k
et al., 1960), the d e c r e a s e d i m m u n o h i s t o c h e m i c a l V P s t a i n i n g i n t e n s i t y ( W a t k i n s a n d C h o y , 1980) a n d l o w e r V P c o n t e n t ( L a n d f i e l d et al., 1980; D o r s a a n d B o t t e m i l l e r , 1982; Correspondence to: Vlasta K. Zbuzek, Department of Anesthesiology, UMD-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103.
(Received 16 May 1986; Accepted 6 November 1986) 113
114
v I<. ZBUZEK et al.
Zbuzek and Wu, 1982; Zbuzek et al., 1983) found in the HT of aged rats of different strains indicated that VP biosynthesis may be reduced during the aging process. On the contrary, reports of other investigators suggested an increased neurosecretory activity thought to be reflecting an increased biosynthetic activity of VP neurons in aged rats and mice (Fliers and Swaab, 1983; Fotheringham et al., 1985; Rogers et al.. 1985). In none of the conflicting studies, however, was a direct technique used to determine the age-related differences in VP biosynthetic activity in the H T - N H system. Labeled amino acids injected into the brain have been employed to study their incorporation into the VP molecule in the HT and axonal transport to the NH in young rats: :~H-tyrosine injected into the cisterna magna was shown to be incorporated into the VP molecule in the NH as early as 1 hour after the injection. The incorporation increased after about 4-6 h, reaching a sustained plateau for at least 6 additional hours (Pickering and Jones, 1971a). :~S-cysteine injected into SON was found 30 min later to be incorporated into proteins in the SON, 1 h later in the median eminence and 2 h later in the NH, where the content of labeled proteins gradually increased up to 12 h and then remained at the same level for 24 h (Gainer et al., 1977). The present study was designed to investigate the effect of aging on the rate of incorporation of 3H-arginine into the precursor molecule of arginine-VP in the HT and its movement into the NH of Fischer 344 rats.
MATERIALS AND METHODS Animals and experimental procedures Male Fischer 344 rats of 3 different ages arbitrarily labeled as young (2-3 months), adult (11-13 months) and old (30 months) were obtained from the National Institute on Aging (NIA) colony. Young and adult animals were maintained by Harlan SpragueDawley Inc. and the old ones by Charles River Breeding-Laboratories under NIA contract. The rats had been kept under barrier-reared conditions from their birth until the delivery to our animal facilities within 24 h and were then kept for 2 weeks in a separate room at a constant temperature of 22 _+ 0.5 °C, 12/12 h light/dark cycle; they received rat Purina Chow diet, and tap water ad libitum. The experiments were carried out during the winter period when the content of VP in the HT and NH is known to be relatively low due to seasonal variations (Zbuzek and Wu, 1979). The animals were anesthetized with sodium pentobarbital (i.p.). Because of agedependent sensitivity to this anesthetic, the doses per kg body weight were adjusted as follows: 70-100 mg for the young, 50-80 mg for the adult and 10-40 mg for the old rats. The head of the rat was fixed in a stereotaxic instrument (David Kopf, Model 900) and a permanent cannula was implanted into the right lateral ventricle. The coordinates (+ 1.5 L, - 4 . 3 V and - 1 . 0 H to bregma) were the same for all three age groups. This was verified, separately, in several animals of each age group by the injection of Evans blue before the experiments were undertaken. L-(2,3,4,5-3H) arginine monohydrochloride in 2% ethanol, 43.5 Ci/mmol, Amersham) was lyophilized and dissolved in saline to obtain 200/zCi per 10/zl. This dose was injected over a period of l min into the lateral ventricle of an awake animal two days after the implantation of the cannula. The HT tissue block (approximately 2 mm from the midline bilaterally at the base of the brain and bordered by the chiasma opticum
VP AND AGING
[
15
6
600
,500
~400
8 oa E 300
,oo
-- 200
O0
lO
20
30
40
50 60 I ml FRACTIONS
70
80
90
I00
FIG. 1.3H-activity (O O) and VP concentration ( © - - --C~) in individual fractions following first chromatography using CM-52 columns. Fractions number 6%80 were pooled, lyophilized and subsequently purified on DEAE columns (See Fig. 2).
arteriorly and corpora mamillaria posteriorly), and the NH (without pars intermedia) were dissected and dried in cold acetone. The health status of the experimental animals was evaluated by gross autopsy with examination for pituitary tumors and splenomegaly (reflecting infectious disease) in order to exclude diseased animals from the study.
Extraction, separation, and purification of VP Acetone-dried tissues were individually homogenized and VP was extracted in 2 ml of 0.25% (v/v) acetic acid, boiled for 2 rain, chilled on ice and centrifuged (3000 rpm for 10 min). The supernatant obtained from the NH and HT was lyophilized with 1 mg of bovine serum albumin (BSA) and dissolved in 0.5 mi or 1 ml, respectively, of 0.002 M ammonium acetate, pH 5.0. Extracted VP from individual glands was chromatographed at room temperature on a 30 × 1 cm column of carboxymethyl cellulose (CM52, Whatman), equilibrated with 0.002 M ammonium acetate, pH 5.0. VP was eluted by an increasing gradient in ionic strength and pH, as described by Pickering and Jones (1971a,b) and 1 ml fractions were collected in a refrigerated chromatography cabinet at 4 °C. The gradient was made by a gradient elution apparatus (Fisher). The elution was initiated with 0.002 M ammonium acetate, pH 5.0 until fraction No. 10, when 0.05 M ammonium acetate, pH 6.9, was introduced to a mixing flask of the gradient former. When the concentration of the eluate reached 0.05 M and pH 6.9 at fraction No. 30, 0.1 M ammonium acetate, pH 6.9, was introduced into the mixing flask. The concentration 0.1 M and pH 6.9 was reached at fraction No. 50 and sustained for the following 50 fractions. Both :~H-activity and VP measured by RIA were determined in each fraction. The peak fractions of '~H-activity matching the peak fractions of radioimmunoassayable VP
V . K . Z B U Z E K et a/.
116
2500
-
2000
-
7
o
¢,a
1500
t
-
E o_
1000
6 -& o
-
I
500
-
0
-
- ~
I
I
I
0
2
4
I
I
I
I
6 8 10 12 lrnl FRACTIONS
I
I
14
16
FIG. 2. 3H-activity ( O - - O ) and VP c o n c e n t r a t i o n (© - - © ) in individual fractions following second purification on D E A E columns. Fractions n u m b e r 7-11 were pooled and lyophilized for m e a s u r e m e n t of total cpm and VP concentration.
(Fig. l) were pooled and lyophilized with l mg of B S A . The presence of VP in the lyophilizate was verified by H P L C (performed at T o x i c o l o g y Laboratories, NJMS). To remove impurities, a s e c o n d purification was carried out on a diethylaminoethyl ( D E A E ) c o l u m n (10 × 1 cm) equilibrated with 0.02 M a m m o n i u m acetate, pH 5.0, and TABLE
1. N U M B E R
OF ANIMALS,
BODY WEIGHT, YOUNG,
N u m b e r of animals: Total lh 6h 24 h Body weight (g) N e u r o h y p o p h y s i s (mg) Initial VP content: H y p o t h a l a m u s (ng) N e u r o h y p o p h y s i s (p.g)
AND NH WEIGHT ADULT
AND INITIAL VP CONTENT
OF HT AND NH OF
AND OLD RATS
Young Y (3 months)
Adult A (12 months)
Old 0 (30 months)
28 9 9 l0 236 + 6
30 9 l0 11 381 + 5
27 9 9 9 345 + 6
0.80 _+ 0.02
1.10 -+ 0.03
1.38 + 0.03
73.82 + 4.29
51.21 + 5.26
33.40 + 2.48
1.66 -+ 0.12
2.46 -+ 0.07
2.40 _+ 0.06
Sign(l~cance P < 0.05
Y vs. A; Y vs. O; Avs. O Y vs. A; Y vs. O; Avs. O Y vs. A; Y vs. O; Avs. O Y vs. A; Y vs. 0
VP AND AGING
1 17
7 --
YOUNG
L
AOuLT OLD
-I-
5-
4
--
E>
+ 3 --
2
--
I
--
6
24
HOURS FIG, 3. The incorporation of aH-arginine into VP molecule in the hypothalamus of young, adult and old rats, expressed as mean all-activity (cpm + SE) measuFed 1, 6 and 24 h after the injection of aH-aFginine into the lateral ventricle. Significance is defined as p < 0.05: * compared to young; + compared to 1 h.
used for the elution of VP. Fifteen 1-ml fractions were collected and both all-activity and VP assayed by RIA were determined in each fraction. The peak fractions of allactivity matching the VP peak (Fig. 2) were pooled and lyophilized with 1 mg BSA. The lyophilizate was dissolved in 1 mi of phosphate buffered saline, pH 7.4, containing 0.1% (w/v) BSA, pH 7.4, for the measurement of all-activity and VP by RIA, which was again verified by H P L C . The efficiency of extraction of VP in the H T and N H after both steps of purification was about lfF/k in all three ages.
V P IIl('(ISltl'Ul)lUIll
VP was measured: (1) In single N H s and H T explants following the initial extraction of VP in diluted acetic acid by use of 10/xl portions to obtain the values for the total content of VP in the tissues of the three age groups. This served as a control to verify our" previously reported data on the content of VP in the H T - - N H system in Fischer 344 rats (Zbuzek e t a / . . 1983: 1984). (2) In each fi'action of the eluate obtained from single NHs and HTs following the first purification on a CM52-column and the second on a DEAE-column, 50 p.l (in duplicate) was directly used from the undiluted fractions with low VP concentration, while several parallel dilutions were prepared from those fractions containing high concentrations, to secure optimal conditions for the RIA of VP. (3) In the pooled peak fractions obtained following the second purification on a D E A E column. Several parallel dilutions were prepared of which 50/xl portions (in duplicate) were used for the determination of the total VP concentration in the peak fractions by
] 18
V.K. ZBUZEK
et a/.
~00 YOUNG
90
ADULT
~
OLD
80 70
60 5O F---
o
40
LL ~J
3O 20 //lo
10
6
24
HOURS
FIG. 4. The incorporation of :~H-arginine into VP molecule in the h y p o t h a l a m u s of young, adult and old rats, e x p r e s s e d as m e a n specific activity (cpm/pg VP _+ SE) m e a s u r e d 1,6 and 24 h after the injection of :~H-urginine into the lateral ventricle. Significance is defined as p < 0.05: * c o m p a r e d to young; • compared to adult; + compared to 1 h.
RIA. These values served for the calculation of the specific activity (cpm/pg VP). A double-antibody RIA was employed as described in detail elsewhere (Zbuzek et al., 1984).
Measurement of radioactivity The amount of radioactivity in the fractions was measured in a scintillation spectrometer (Beckman, LS 1800), by use of 5 ml of a high performance pre-mixed liquid scintillation cocktail for aqueous samples (Ready-Solv, HP/b, Beckman). From individual fractions, aliquots of 200/zl from NH (containing lower radioactivity) and 50/zl from HT fractions were counted for 10 min and/or until at least 10,000 counts were obtained. Total radioactivity of pooled peak fractions following DEAE purification was measured in 20 tzl of dissolved lyophilizate (in 4 replicates) to obtain sufficient counting precision at a level of cpm _+ 2 SD error. Calculation The time course of the incorporation of 3H-arginine into the extracted and chromatographed VP, obtained from the HT explants and the NHs (at hours 1, 6, and 24) was expressed as cpm and specific activity (cpm/pg VP) in the HT and NH. The results are presented as mean ___ SE.
VP AND
3.5
1 19
AGING
-
D 3.0-
YOUNG ADULT OLD
2.5 -
2.0E
~.5 -
+ 4 1.0-
0.5
0
I
6 HOURS
24
FIG. 5. VP transport to neurohypophysis of young, adult and old rats expressed as mean :~H-activity (cpm _+ SE) measured 1, 6 and 24 h after the injection of :~H-arginine into the lateral ventricle. Significance is defined as p < 0.05: * compared to young and adult: + compared to 1 h; A compared to 6 h.
Statistical analysis All data were analyzed by one-way analysis of variance. The data obtained for cpm and specific activity were c o m p a r e d among the ages at hours 1,6, 24 after the injection of 3H-arginine and a m o n g the hours 1, 6, 24 for each age group. The differences were defined as p < 0.05 by the Duncan test. RESULTS Three out of 30 old rats had pituitary tumors and were excluded from this study. H o w e v e r , all old rats had testicular tumors which are c o m m o n l y found in aged Fischer 344 rats (Coleman et al., 1977). VP content, measured before purification, reflecting initial endogenous content of the H T , was lowest in the old, higher in the adult and highest in the young rats, whereas the initial VP content in the N H was lower in the young than in the adult and old rats (Table 1).
Hypothalanms :~H-activity (cpm). One hour after the injection of :~H-arginine into the lateral ventricle the young rats incorporated 605,491 +_ 73,884 c p m , the adult 431,206 _+ 89,638 cpm and the old only 275,783 -2-_36,218 c p m , which is a significantly lower amount than that in the young ones; F(2,24) = 5.51, p = 0.0107. Six hours after the injection, :~H-activity declined significantly in the young (294,560 + 37,196 cpm), tended to decline in the adult (290,342 +_ 24,899 cpm) and did not change in the old rats (289,656 _+ 16,465 cpm). There was no difference among the ages; F(2,25) = 0.01, p -- 0.9897. T w e n t y - f o u r hours
120
\ K ZBUZEK c; ~;/
14
Ai',/J, ]
12
--
10
--
E >
8--
L~ 6--
+
o.
+
4f ,6 tg, JF¢!
24
[:l(;. 6. VP transport to neurohypophysis of young, adult a n d o l d r a t s e x p r e s s e d a s m e a n specific activity (cpm/pg VP ~ S E ) m e a s u r e d I, 6 a n d 24 h a t i e r the injection of :~H-arginine into the lateral ventricle. Significance is d e f i n e d a s p ~- 0.05: * c o m p a r e d to young and adult: + compared
to I h: ?,, c o m p a r e d
to 6 h.
after the injection, :~H-activity r e m a i n e d u n c h a n g e d in all three age groups. It w a s 190,237 + 28,563 c p m in the y o u n g , 279,169 +_ 34,184 c p m in the adult and 272,165 +_ 25,873 c p m in the old rats: F(2,27) - 2.66, p - 0.0882. T h e d e c l i n e in :~H-activity fi'om 1 to 24 h after the injection w a s significant in the y o u n g [F(2,25) - 19.18, p - 0.0001]; nonsignificant in the adult [F(2,27) = 2.41, p 0.087] and the old [F(2,24) - 0.11, p = 0.89311 rats (Fig. 3.).
Sp~'~'([~" activity (cpm/p,~, VP). O n e hour after the injection specific activity w a s similar in all three age groups: 66.81 _+ 2.89, 70.89 _+ 8.86 and 73.36 _+ 7.10 c p m / p g VP in the y o u n g , adult and old, r e s p e c t i v e l y ; F(2,24) - 0.27, p - 0.7683. Six hours after the injection, specific activity d e c l i n e d significantly in the y o u n g (40.85 _+ 4.66 c p m / p g VP), tended to d e c l i n e in the adult (59.64 _+ 1.95 c p m / p g VP) and did not c h a n g e in the old rats (75.10 _+ 4.05 c p m / p g VP). There w a s a significant difference a m o n g the ages; F(2,25) - 21.44, p = 0.0001. T w e n t y four hours after the injection a further significant d e c l i n e o c c u r r e d in the y o u n g (29.37 _+ 3.56 c p m / p g VP) and it did not c h a n g e in the adult (56.83 _+ 6.02 c p m / p g VP) and the old rats (61.50 _+ 7.05 c p m / p g VP). The differe n c e a m o n g the ages remained significant; F(2,27) = 9.21, p = 0.0009. T h e d e c l i n e in the specific activity f r o m 1 to 24 h after the injection w a s significant in the y o u n g [F(2,25) = 26.06, p = 0.0001], nonsignificant in the adult [F(2,27) = 1.44, p 0.2555] and old [F(2,24) = 1.45, p - 0.2553] rats (Fig. 4).
VP A N D A G I N G
121
Neurohypophysis all-activity ~cpm). One hour after the injection, all-activity in the N H was significantly higher in the young (44,980 _+ 6,891 cpm) and the adult rats (45,140 +_ 6.233 cpm), than in the old ones (15,156 + 1,651 cpm); F(2,24) = 10.04, p = 0.0007. It increased significantly 6 h after the injection in all age groups. Here again, it was significantly higher in the young (94,967 _+ 5,657 cpm) and the adult (96,420 _+ 5,277 cpm) c o m p a r e d with the old rats (51,696 _+ 3,552 cpm); F(2,25) = 25.91, p = 0.0001. T w e n t y - f o u r hours after the injection the activity increased significantly in all age groups. There was no difference, however, a m o n g the age groups (261,764 _+ 70,735, 201,068 _+ 25,670 and 290,410 +_ 28,783 cpm in the young, adult and old, respectively): F(2,27) = 0.98, p = 0.3880. The increase in :+H-activity from 1 to 24 h was significant in all age groups: F(2,25) = 6.79, p = 0.0044 in the young; F(2,27) = 22.26, p = 0.0001 in the adult, and F(2,24) = 29.45, p = 0.0001 in the old rats (Fig. 5). Specific activity (cpm/pg VP). One hour after the injection, specific activity in the N H was significantly lower in the old (0.52 + 0.04 cpm/pg VP) than in the young (1.79 +_ 0.26 cpm/pg VP) and the adult rats (1.66 _+ 0.23 cpm/pg VP): F(2,24) = 12.02, p = 0.0002. It increased significantly in all age groups 6 h after the injection with significantly lower values in the old (1.66 _+ 0.13 cpm/pg VP) than in the young (3.64 _+ 0.37 cpm/pg VP) and the adult rats (3.80 + 0.37 cpm/pg VP); F(2,25) = 14.02, p = 0.0001. T w e n t y - f o u r hours after the injection the activity further increased significantly in all age groups, exhibiting no difference, h o w e v e r , a m o n g the age groups (11.34 _+ 3.21, 6.94 _+ 0.80 and 9.01 _+ 0.81 cpm/pg VP in the young, adult and old rats, respectively); F(2,27) = 1.31, p = 0.2855. The increase in specific activity from 1 to 24 h was significant in all age groups: F(2,25) = 6.55, p = 0.0051 in the young; F(2,27) = 22.31, p = 0.0001 in the adult and F(2,24) = 95.15, p = 0.0001 in the old rats (Fig. 6). DISCUSSION Accurate m e a s u r e m e n t of the biosynthesis of neuropeptides in vivo encounters inherent problems with the absorption and compartmentalization of the labeled amino acid p r e c u r s o r and competing reactions (Richardson, 1981). Since in our study no attempt was made to determine the specific activity of the amino acid precursor in the intracellular pool, nor that bound to t - R N A , (the immediate source of amino acid for protein synthesis), our results m a y not be a s s u m e d necessarily to be an accurate correlative measure of VP biosynthesis. With this reservation and that of some other age-related determining factors discussed later, we employed the technique of incorporation of radioactive label into the VP molecule in the H T and its transport to the N H , originally described by Pickering and Jones (1971a,b) with the following modifications: T h e y injected 100 ~Ci of aHtyrosine into the cisterna m a g n a of an anesthetized rat and pooled 5 glands to measure the rate of incorporation and the rate of transport of two neuropeptides, VP and oxytocin (OT). Since our primary concern was VP, we e m p l o y e d aH-arginine as a specific label for the newly synthetized VP molecule to eliminate interference with OT. The higher radioactivity (200 ~Ci) e m p l o y e d by us permitted m e a s u r e m e n t of the incorpo-
122
V . K Z B U Z E K ,'t a /
ration in individual H T explants and N H s and thus to detect the variations among the individual animals. The identity of the elution profile of r a d i o i m m u n o a s s a y a b l e VP obtained by separation on the column was verified by H P L C . Finally, the implantation of a permanent cannula into the lateral ventricle allowed us to inject the radioactive label into an awake animal, so as to avoid the effect of anesthesia on VP release (Aziz and Forsling, 1979). To our knowledge, there are no data available in the literature on age-related changes in the rate of the incorporation of labeled amino acid into VP molecule in the H T and in the rate of its transport to the NH. Thus, only the results on the time-course of the incorporation of all-activity obtained in young rats are c o m p a r a b l e with the data of other investigators (Gainer et al.. 1977" Pickering and Jones, 1971a). In the HT, the accumulation of aH-arginine one hour after the injection was twice as high in the young as in the old animals. The intermediate values obtained in the adult rats, lie between those of young and old, and are not significantly different from those of either group, due to a large scatter. Some of the adult rats incorporated amounts of aH-arginine as high as did the young, others as low as did the old rats. Six hours after the injection, :~H-activity declined significantly in the young rats as found by Picketing and Jones (1971a) and remained unchanged up to 24 h. In the adult rats, :~H-activity declined 6 h after the injection but the difference was not significant due to a large scatter of the values at hour 1. Similarly, as in the young rats, the level of all-activity in the adult was not different between 6 to 24 h after the injection. In the old rats, which exhibited the lowest :~H-activity of all age groups, the activity did not change from 1 to 24 h, indicating decreased rate of incorporation of aH-arginine into VP molecule and also slow axonal transport to the N H of aged Fischer 344 rats. Specific activity (expressed as a ratio of cpm per pg of endogenous VP) was similar in all age groups 1 h after the injection, reflecting, in fact, a decremental trend of both all-activity and VP content of HT, from highest in the young to lowest in the old rats. In the N H , the time-course of the incorporation of all-activity exhibited a similar trend in all age groups as did the specific activity. According to Picketing and Jones (1971a) the specific activity of VP in the N H of young rats reached a plateau at 6 h which was sustained up to 12 h after the injection of aH-tyrosine into the cisterna magna. H o w e v e r , their data consist of only 2 m e a s u r e m e n t s of pooled samples at these time intervals and the authors did not investigate the course of incorporation beyond this point. The same authors, (Pickering et el/., 1975), using a more refined technique, showed that a:'S-cysteine, when injected into the 3rd ventricle, was incorporated into the carrier protein of VP, neurophysin, peaking in the N H 12 h after the injection. Gainer, et a/. (1977) similarly reported the incorporation of a:'S-cysteine into the N H peaking at 12 h. Our results, obtained from individual values, (9-10 young animals at each time interval) at 1, 6 and 24 h, are in agreement with these reports. One and 6 hours after the injection, all- and the specific activity are significantly lower in the N H of the old rats, than in the young and adult rats. At 6 h, both activities in old rats reached the l-h values of the young and adult rats suggesting a delay of at least 6 h in the axonal transport of newly synthetized VP in aged rats. T w e n t y - f o u r hours after the injection, there is no difference a m o n g the ages, in both cpm and specific activity, suggesting a decreased release of VP in the old rats. This agrees with our previous data on the impairment of VP release in aged rats studied in the isolated superfused N H (Zbuzek el al., 1984; 1986).
VP AND AGING
123
It could be argued that the age-related differences in the rate of incorporation of the radioactive label are affected by improper administration or by a different rate of diffusion of the label into the tissues of the young and old animals. The following evidence shows that this is unlikely: (a) Several rats of each age group were injected with Evans blue, which in all ages was found to stain evenly the H T - N H area already 1 h after the injection. (b) Similar amounts of radioactivity were recovered in the VP extracts from the HT 1 h after the injection in all age groups. (c) Similar amounts of :~H-activity were recovered in the NH in all age groups 24 h after the injection. The presented data indicate a decrease in the rate of incorporation of :~H-arginine into VP molecule in the HT and in its transport to the NH of aged Fischer 344 rats. They conform with our ~md other authors" previous findings of a low VP content in the HT of aged Sprague-Di~wley (Zbuzek and Wu, 1982), Fischer 344 (Landfield e t a / . . 1980: Zbuzek ct al.. 1983), Long Evans (Dorsa and Bottemiller, 1982), and albino Diisseldorf (Rodeck c t a l . . 1960) strains of rats, measured by specific RIAs and bioassays. Also, a decreased staining intensity of neurosecretory material in the HT magnocellular neurons has been reported in senile albino Di~sseldorf (Rodeck et al., 1960) and old Wistar but not in Sprague-I~awley rats (Kawashima and Kobayashi, 1982). The nonspecific staining techniques employed did not allow to discern whether or not the decreased staining intensity was due solely to the changes of VP content, since several neuropeptides are known to coexist in the magnocellular neurons of the PVN and SON of the HT (for review see Sofroniew et al., 1984). However, other investigators employed specific immunohistochemical techniques to demonstrate a decreased agerelated staining intensity in PVN and SON of Wistar rats (Watkins and Choy, 1980) and in PVN of monkies (Sladek e t a / . . 1978). Interestingly, the staining intensity was not always found to be equally distributed in both the PVN and SON when specific antisera for VP, OT or their respective neurophysins were used. In contrast, recently an abrupt increase of the activity of Golgi apparatus-bound thiamine-pyrophosphatase in the neurons of PVN and SON has been reported (Fliers and Swaab, 1983) in 32-month old male Wistar rats. This finding is difficult to explain, because in the same series of experiments the enzyme activity in 3- up to 26-month old ra{s exhibited an unchanged or slightly declining trend with increasing age. This report belongs to the divergent opinions in the long unresolved dispute, as to whether an activated rather than a decreased neurosecretory activity occurs in the H T - N H system in senescence (Dicker and Nunn, 1958; Morrison and Staroscik, 1964; Frolkis e t a / . , 1982: Fotheringham et al., 1985; Rogers et al., 1985). These conflicting reports based on indirect assessment of age-related VPbiosynthetic activity prompted us to investigate the rate of the incorporation of radioactive label into VP precursor molecule. Our experiments were not designed to reveal to what extent the reported decrease in the incorporation of :~H-arginine into the VP molecule in the aged rats reflects the biosynthetic activity per se. Several possible age-related determining factors might be involved, for example: a decrease in the number of active VP-synthetizing neurons: a shift in the ratio of the number of neuronal/nonneuronal elements (glial cells) present; a difference in the availability of the intracellular free amino acid pool; or, differences in the intracellular degradation of proteins (lysosomal degradation). At present, however, the data available to answer these questions are inconsistent and more evidence on this subject is needed.
124
V.K. ZBUZEKet al.
A c k m m ' l e d g m e n t s - - This research was supported by N1A grant R01 AG02778. We are grateful to Dr. Berta Scharrer of Albert Einstein College of Medicine for her invaluable comments and critique: to Mrs. Annie M. Miller and Ms. Violeta Guerra for their excellent technical assistance; to Mr. Martin Feuerman for the statistical analysis and Ms. Ladonna Levell for typing this manuscript.
REFERENCES Aziz, L. A. and Forsling, M. L. Anaesthesia and vasopressin release in the rat. J. Emlocr. (Lond.) 81, 123P, 1979. Coleman, G. L., Barthold, S. W., Osbaldiston, G. W., Forster, S. J., and Jonas, A. M. Pathological changes during aging in ban'ier-reared Fisher 344 male rats. J. Geront. 32, 258--278, 1977. Dicker, S.E. and Nunn, J. Antidiuresis in adult and old rats. J. Physiol. (Lond.), 141,332-336, 1958. Dorsa, D.M. and Bottemiller, L. Age-related changes of vasopressin content of microdissected areas of rat brain. Brain Res. 242, 151-156, 1982. Fliers, E. and Swaab, D.F. Activation of vasopressinergic and oxytocinergic neurons during aging in the Wistar rat. Pcptides 4, 165-170, 1983. Fothermingham, A., Davies, 1., Goddard, C., Moser, B., and Faragher, B. Age-related changes in water metabolism in the mouse. Abstract, p. 335, 13th International Congr. of Gerontology, New York, NY 1985. Frolkis, V.V., Golovchenko, S.F., Medved, V.I., and Frolkis, R.A. Vasopressin and cardiovascular system in aging. Gerontology 28, 290-302, 1982. Gainer, H., Same, V., and Brownstein, M.J. Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides. J. ('ell. Biol. 73, 366-381, 1977. Kawashima, S. and Kobayashi, Y.J. Morphometric study of the hypothalamo-neurohypophyseal system in aged rats. ,1. Sci. Hiroshima Univ.. Ser. B, Div. l, 30, 229-242, 1982. Landfield, P.W., Sundberg, D.K., Smith, M.S., Eldridge, J.C., and Morris, M. Mammalian aging: Theoretical implications of changes in brain and endocrine systems during mid- and late-life in rats. Peptidcs 1, (Suppl. 1), 185-196, 1980, Morrison, A.B. and Staroscik, R.N. The neurosecretory substance in the neurohypophysis of the rat during maturation and aging. Gerontologia 9, 65-70, 1964. Pickering, B.T. and Jones, C.W. The biosynthesis and intraneural transport of neurohypophysial hormones: preliminary studies in the rat. In: Memoirs q f the Society,lbr Endocrim~logy, Vol. 19: Subcellular Orga;1ization and Functiott in Endocrine Tissues. Heller H. and Ledefis K., (Editors), pp. 337-351, Cambridge Univ. Press, 1971a. Pickering, B.T. and Jones, C.W. Isolation of radioactive oxytocin and vasopressin from the posterior pituitary gland of the rat after the injection of labeled tyrosine into the cerebrospinal fluid. J. Endocr. (Lond.) 49, 93-103, 1971b. Pickering, B.T., Jones, C.W., Burford, G.D,, McPherson, M., Swann, R.W., Heap, P.F., and Morris, J.F. The role of neurophysin proteins: suggestions from the study of their transport and turnover. AttH. N Y Acad. Sci. 248, 15-35, 1975. Richardson, A. The relationship between aging and protein synthesis. In: CR(" H a n d b o o k q f B i o c h e m i s t o ' in Agine. Florini, J.F., (Editor), pp. 7%101, CRC Press, Boca Raton, FL, 1981. Rodeck. H., Lederis, K., and Heller, H. The hypothalamo-neurohypophysial system in old rats. ,I. Emlocr. (Lond.) 21, 225-230, 1960. Rogers, J., Shoemaker, W.J., Morgan, D.G., and Finch, C.E. Senescent change in tissue weight and immunoreactive /3-endorphin, enkephalin and vasopressin in eight regions of C57BL/6J mouse brain and pituitary. Neurobiol. Aging 6, 1-9, 1985. Sladek, J.R. Jr., McConnel, J., and McNeil, T.H. Integrated morphology of neuronal catecholamines and neurophysin in the aged macaque. Adv. k2rp. Med. BioL 113, 241-250, 1978. Sofi'oniew, M.V., Eckenstein, F., Schrell, U., and Cuello, A,C. Evidence for colocalization of neuroactive substances in hypothalamic neurons. In: Coexistence oJ'Neuroactive Substances in Neurons. Chan-Palay, V. and Palay, S.L., (Editors), pp. 73-90, John Wiley and Sons. New York, 1984. Watkins, W.B. and Choy, V.J. The impact of aging on neuronal morphology in the rat hypothalamoneurohypophysial system: An immunohistochemical study. Peptides 1 (Suppl. 1), 239-245, 1980. Zbuzek, V.K. and Wu, W. Seasonal variations in vasopressin secretion in rats. Experientia 35, 1523-1524, 1979.
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Zbuzek, V.K. and Wu, W. Age-related vasopressin changes in rat plasma and the hypothalamo-hypophyseal system. Exp. Gt, r¢,it. 17, 133-138, 1982. Zbuzek, V.K., Zbuzek, V., and Wu, W. The effect of aging on vasopressin system in Fisher 344 rats. LxT~. G~'rollt. 18, 305-311, 1983. Zbuzek, V., Zbuzek, V.K., and Wu, W. Vasopressin release from individually superfused neurohypophyses decreases in aged rats. N~,ur~Jend~crinoh~gy 39, 538-548, 1984. Zbuzek, V.K., Zbuzek, V., and Wu, W. Decremental vasopressin release after repeated stimulation of superfused neurohypophyses of Fisher 344 rats of different ages. ,/. G~rout. 41, 140-146, 1986.
0531-5565/87 $3.00 -} .00 Copyright ' 1987 Pergamon Journals l,td
Printed in the USA. All rights reserved.
AGE-RELATED DIFFERENCES IN THE INCORPORATION OF 3H-ARGININE INTO VASOPRESSIN IN FISCHER 344 RATS
VI.ASTA K . Z B U Z E K , VRATISLAV Z B U Z E K , a n d W E N - T S I E N W u University of Medicine and Dentistry, New Jersey Medical School Department of Aneslhesiology, Newark, NJ Abstract - - We studied the effect of aging on the incorporation of :~H-arginine into
vasopressin (VP) molecule in the hypothalamus (HT) and the rate of its transport into the neurohypophysis (NH) of male Fischer 344 rats of different ages: 2-3 months (young, n - 28), 11-13 months (adult, n = 30) and 30 months (old, n = 27). aHarginine was injected in a single dose through a permanent cannula into the lateral ventricle of an awake animal. The rats were decapitated 1, 6, and 24 h after the injection. VP was extracted from individual HTs and NHs (without pars intermedia), purified in a chromatography column and determined by RIA. Hypot/ta/aml~s: One hour after the injection the all-activity was highest in the young, lower in the adult and lowest in the old rats, whereas the specific activity (cpm/pg VP) was similar in all age groups, reflecting endogenous VP content to be highest in the young, lower in the adult and lowest in the old rats. In the young rats both activities decreased 6 and 24 h after the injection. The adult rats also exhibited declining activities, whereas in the old rats the activities remained unchanged from 1 to 24 h after the injection. Ncurohypophysis: One hour after the injection of radioactive label all-activity and specific activity were significantly lower in the NH of the old than in those of young and adult rats. The activities increased 6 h after the injection in all age groups with the lowest values in the old rats. After 6 h, both activities in the old rats reached only the l-h values t)f the young and adult rats suggesting a delay of at least 6 h in the axonal transport of newly synthetized VP in aged rats. Twenty-four hours after the injection both activities increased in all age groups exhibiting no difference among the age groups, indicating a decreased release of the newly synthetized VP from the NH of aged rats. These data demonstrate an age-related decrease in the rate of incorporation of aH-arginine into VP, in the rate of its axonal transport and in the release of newly synthetized VP from the NH of Fischer 344 rats.
Key
words:
hypothalamus,
vasopressin, 3H-arginine incorporation, neurohypophysis, Fischer 344 rats, aging
vasopressin
transport,
THE LOWER a m o u n t o f n e u r o s e c r e t o r y m a t e r i a l in v a s o p r e s s i n e r g i c n e u r o n e s ( R o d e c k
et al., 1960), the d e c r e a s e d i m m u n o h i s t o c h e m i c a l V P s t a i n i n g i n t e n s i t y ( W a t k i n s a n d C h o y , 1980) a n d l o w e r V P c o n t e n t ( L a n d f i e l d et al., 1980; D o r s a a n d B o t t e m i l l e r , 1982; Correspondence to: Vlasta K. Zbuzek, Department of Anesthesiology, UMD-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103.
(Received 16 May 1986; Accepted 6 November 1986) 113
114
v I<. ZBUZEK et al.
Zbuzek and Wu, 1982; Zbuzek et al., 1983) found in the HT of aged rats of different strains indicated that VP biosynthesis may be reduced during the aging process. On the contrary, reports of other investigators suggested an increased neurosecretory activity thought to be reflecting an increased biosynthetic activity of VP neurons in aged rats and mice (Fliers and Swaab, 1983; Fotheringham et al., 1985; Rogers et al.. 1985). In none of the conflicting studies, however, was a direct technique used to determine the age-related differences in VP biosynthetic activity in the H T - N H system. Labeled amino acids injected into the brain have been employed to study their incorporation into the VP molecule in the HT and axonal transport to the NH in young rats: :~H-tyrosine injected into the cisterna magna was shown to be incorporated into the VP molecule in the NH as early as 1 hour after the injection. The incorporation increased after about 4-6 h, reaching a sustained plateau for at least 6 additional hours (Pickering and Jones, 1971a). :~S-cysteine injected into SON was found 30 min later to be incorporated into proteins in the SON, 1 h later in the median eminence and 2 h later in the NH, where the content of labeled proteins gradually increased up to 12 h and then remained at the same level for 24 h (Gainer et al., 1977). The present study was designed to investigate the effect of aging on the rate of incorporation of 3H-arginine into the precursor molecule of arginine-VP in the HT and its movement into the NH of Fischer 344 rats.
MATERIALS AND METHODS Animals and experimental procedures Male Fischer 344 rats of 3 different ages arbitrarily labeled as young (2-3 months), adult (11-13 months) and old (30 months) were obtained from the National Institute on Aging (NIA) colony. Young and adult animals were maintained by Harlan SpragueDawley Inc. and the old ones by Charles River Breeding-Laboratories under NIA contract. The rats had been kept under barrier-reared conditions from their birth until the delivery to our animal facilities within 24 h and were then kept for 2 weeks in a separate room at a constant temperature of 22 _+ 0.5 °C, 12/12 h light/dark cycle; they received rat Purina Chow diet, and tap water ad libitum. The experiments were carried out during the winter period when the content of VP in the HT and NH is known to be relatively low due to seasonal variations (Zbuzek and Wu, 1979). The animals were anesthetized with sodium pentobarbital (i.p.). Because of agedependent sensitivity to this anesthetic, the doses per kg body weight were adjusted as follows: 70-100 mg for the young, 50-80 mg for the adult and 10-40 mg for the old rats. The head of the rat was fixed in a stereotaxic instrument (David Kopf, Model 900) and a permanent cannula was implanted into the right lateral ventricle. The coordinates (+ 1.5 L, - 4 . 3 V and - 1 . 0 H to bregma) were the same for all three age groups. This was verified, separately, in several animals of each age group by the injection of Evans blue before the experiments were undertaken. L-(2,3,4,5-3H) arginine monohydrochloride in 2% ethanol, 43.5 Ci/mmol, Amersham) was lyophilized and dissolved in saline to obtain 200/zCi per 10/zl. This dose was injected over a period of l min into the lateral ventricle of an awake animal two days after the implantation of the cannula. The HT tissue block (approximately 2 mm from the midline bilaterally at the base of the brain and bordered by the chiasma opticum
VP AND AGING
[
15
6
600
,500
~400
8 oa E 300
,oo
-- 200
O0
lO
20
30
40
50 60 I ml FRACTIONS
70
80
90
I00
FIG. 1.3H-activity (O O) and VP concentration ( © - - --C~) in individual fractions following first chromatography using CM-52 columns. Fractions number 6%80 were pooled, lyophilized and subsequently purified on DEAE columns (See Fig. 2).
arteriorly and corpora mamillaria posteriorly), and the NH (without pars intermedia) were dissected and dried in cold acetone. The health status of the experimental animals was evaluated by gross autopsy with examination for pituitary tumors and splenomegaly (reflecting infectious disease) in order to exclude diseased animals from the study.
Extraction, separation, and purification of VP Acetone-dried tissues were individually homogenized and VP was extracted in 2 ml of 0.25% (v/v) acetic acid, boiled for 2 rain, chilled on ice and centrifuged (3000 rpm for 10 min). The supernatant obtained from the NH and HT was lyophilized with 1 mg of bovine serum albumin (BSA) and dissolved in 0.5 mi or 1 ml, respectively, of 0.002 M ammonium acetate, pH 5.0. Extracted VP from individual glands was chromatographed at room temperature on a 30 × 1 cm column of carboxymethyl cellulose (CM52, Whatman), equilibrated with 0.002 M ammonium acetate, pH 5.0. VP was eluted by an increasing gradient in ionic strength and pH, as described by Pickering and Jones (1971a,b) and 1 ml fractions were collected in a refrigerated chromatography cabinet at 4 °C. The gradient was made by a gradient elution apparatus (Fisher). The elution was initiated with 0.002 M ammonium acetate, pH 5.0 until fraction No. 10, when 0.05 M ammonium acetate, pH 6.9, was introduced to a mixing flask of the gradient former. When the concentration of the eluate reached 0.05 M and pH 6.9 at fraction No. 30, 0.1 M ammonium acetate, pH 6.9, was introduced into the mixing flask. The concentration 0.1 M and pH 6.9 was reached at fraction No. 50 and sustained for the following 50 fractions. Both :~H-activity and VP measured by RIA were determined in each fraction. The peak fractions of '~H-activity matching the peak fractions of radioimmunoassayable VP
V . K . Z B U Z E K et a/.
116
2500
-
2000
-
7
o
¢,a
1500
t
-
E o_
1000
6 -& o
-
I
500
-
0
-
- ~
I
I
I
0
2
4
I
I
I
I
6 8 10 12 lrnl FRACTIONS
I
I
14
16
FIG. 2. 3H-activity ( O - - O ) and VP c o n c e n t r a t i o n (© - - © ) in individual fractions following second purification on D E A E columns. Fractions n u m b e r 7-11 were pooled and lyophilized for m e a s u r e m e n t of total cpm and VP concentration.
(Fig. l) were pooled and lyophilized with l mg of B S A . The presence of VP in the lyophilizate was verified by H P L C (performed at T o x i c o l o g y Laboratories, NJMS). To remove impurities, a s e c o n d purification was carried out on a diethylaminoethyl ( D E A E ) c o l u m n (10 × 1 cm) equilibrated with 0.02 M a m m o n i u m acetate, pH 5.0, and TABLE
1. N U M B E R
OF ANIMALS,
BODY WEIGHT, YOUNG,
N u m b e r of animals: Total lh 6h 24 h Body weight (g) N e u r o h y p o p h y s i s (mg) Initial VP content: H y p o t h a l a m u s (ng) N e u r o h y p o p h y s i s (p.g)
AND NH WEIGHT ADULT
AND INITIAL VP CONTENT
OF HT AND NH OF
AND OLD RATS
Young Y (3 months)
Adult A (12 months)
Old 0 (30 months)
28 9 9 l0 236 + 6
30 9 l0 11 381 + 5
27 9 9 9 345 + 6
0.80 _+ 0.02
1.10 -+ 0.03
1.38 + 0.03
73.82 + 4.29
51.21 + 5.26
33.40 + 2.48
1.66 -+ 0.12
2.46 -+ 0.07
2.40 _+ 0.06
Sign(l~cance P < 0.05
Y vs. A; Y vs. O; Avs. O Y vs. A; Y vs. O; Avs. O Y vs. A; Y vs. O; Avs. O Y vs. A; Y vs. 0
VP AND AGING
1 17
7 --
YOUNG
L
AOuLT OLD
-I-
5-
4
--
E>
+ 3 --
2
--
I
--
6
24
HOURS FIG, 3. The incorporation of aH-arginine into VP molecule in the hypothalamus of young, adult and old rats, expressed as mean all-activity (cpm + SE) measuFed 1, 6 and 24 h after the injection of aH-aFginine into the lateral ventricle. Significance is defined as p < 0.05: * compared to young; + compared to 1 h.
used for the elution of VP. Fifteen 1-ml fractions were collected and both all-activity and VP assayed by RIA were determined in each fraction. The peak fractions of allactivity matching the VP peak (Fig. 2) were pooled and lyophilized with 1 mg BSA. The lyophilizate was dissolved in 1 mi of phosphate buffered saline, pH 7.4, containing 0.1% (w/v) BSA, pH 7.4, for the measurement of all-activity and VP by RIA, which was again verified by H P L C . The efficiency of extraction of VP in the H T and N H after both steps of purification was about lfF/k in all three ages.
V P IIl('(ISltl'Ul)lUIll
VP was measured: (1) In single N H s and H T explants following the initial extraction of VP in diluted acetic acid by use of 10/xl portions to obtain the values for the total content of VP in the tissues of the three age groups. This served as a control to verify our" previously reported data on the content of VP in the H T - - N H system in Fischer 344 rats (Zbuzek e t a / . . 1983: 1984). (2) In each fi'action of the eluate obtained from single NHs and HTs following the first purification on a CM52-column and the second on a DEAE-column, 50 p.l (in duplicate) was directly used from the undiluted fractions with low VP concentration, while several parallel dilutions were prepared from those fractions containing high concentrations, to secure optimal conditions for the RIA of VP. (3) In the pooled peak fractions obtained following the second purification on a D E A E column. Several parallel dilutions were prepared of which 50/xl portions (in duplicate) were used for the determination of the total VP concentration in the peak fractions by
] 18
V.K. ZBUZEK
et a/.
~00 YOUNG
90
ADULT
~
OLD
80 70
60 5O F---
o
40
LL ~J
3O 20 //lo
10
6
24
HOURS
FIG. 4. The incorporation of :~H-arginine into VP molecule in the h y p o t h a l a m u s of young, adult and old rats, e x p r e s s e d as m e a n specific activity (cpm/pg VP _+ SE) m e a s u r e d 1,6 and 24 h after the injection of :~H-urginine into the lateral ventricle. Significance is defined as p < 0.05: * c o m p a r e d to young; • compared to adult; + compared to 1 h.
RIA. These values served for the calculation of the specific activity (cpm/pg VP). A double-antibody RIA was employed as described in detail elsewhere (Zbuzek et al., 1984).
Measurement of radioactivity The amount of radioactivity in the fractions was measured in a scintillation spectrometer (Beckman, LS 1800), by use of 5 ml of a high performance pre-mixed liquid scintillation cocktail for aqueous samples (Ready-Solv, HP/b, Beckman). From individual fractions, aliquots of 200/zl from NH (containing lower radioactivity) and 50/zl from HT fractions were counted for 10 min and/or until at least 10,000 counts were obtained. Total radioactivity of pooled peak fractions following DEAE purification was measured in 20 tzl of dissolved lyophilizate (in 4 replicates) to obtain sufficient counting precision at a level of cpm _+ 2 SD error. Calculation The time course of the incorporation of 3H-arginine into the extracted and chromatographed VP, obtained from the HT explants and the NHs (at hours 1, 6, and 24) was expressed as cpm and specific activity (cpm/pg VP) in the HT and NH. The results are presented as mean ___ SE.
VP AND
3.5
1 19
AGING
-
D 3.0-
YOUNG ADULT OLD
2.5 -
2.0E
~.5 -
+ 4 1.0-
0.5
0
I
6 HOURS
24
FIG. 5. VP transport to neurohypophysis of young, adult and old rats expressed as mean :~H-activity (cpm _+ SE) measured 1, 6 and 24 h after the injection of :~H-arginine into the lateral ventricle. Significance is defined as p < 0.05: * compared to young and adult: + compared to 1 h; A compared to 6 h.
Statistical analysis All data were analyzed by one-way analysis of variance. The data obtained for cpm and specific activity were c o m p a r e d among the ages at hours 1,6, 24 after the injection of 3H-arginine and a m o n g the hours 1, 6, 24 for each age group. The differences were defined as p < 0.05 by the Duncan test. RESULTS Three out of 30 old rats had pituitary tumors and were excluded from this study. H o w e v e r , all old rats had testicular tumors which are c o m m o n l y found in aged Fischer 344 rats (Coleman et al., 1977). VP content, measured before purification, reflecting initial endogenous content of the H T , was lowest in the old, higher in the adult and highest in the young rats, whereas the initial VP content in the N H was lower in the young than in the adult and old rats (Table 1).
Hypothalanms :~H-activity (cpm). One hour after the injection of :~H-arginine into the lateral ventricle the young rats incorporated 605,491 +_ 73,884 c p m , the adult 431,206 _+ 89,638 cpm and the old only 275,783 -2-_36,218 c p m , which is a significantly lower amount than that in the young ones; F(2,24) = 5.51, p = 0.0107. Six hours after the injection, :~H-activity declined significantly in the young (294,560 + 37,196 cpm), tended to decline in the adult (290,342 +_ 24,899 cpm) and did not change in the old rats (289,656 _+ 16,465 cpm). There was no difference among the ages; F(2,25) = 0.01, p -- 0.9897. T w e n t y - f o u r hours
120
\ K ZBUZEK c; ~;/
14
Ai',/J, ]
12
--
10
--
E >
8--
L~ 6--
+
o.
+
4f ,6 tg, JF¢!
24
[:l(;. 6. VP transport to neurohypophysis of young, adult a n d o l d r a t s e x p r e s s e d a s m e a n specific activity (cpm/pg VP ~ S E ) m e a s u r e d I, 6 a n d 24 h a t i e r the injection of :~H-arginine into the lateral ventricle. Significance is d e f i n e d a s p ~- 0.05: * c o m p a r e d to young and adult: + compared
to I h: ?,, c o m p a r e d
to 6 h.
after the injection, :~H-activity r e m a i n e d u n c h a n g e d in all three age groups. It w a s 190,237 + 28,563 c p m in the y o u n g , 279,169 +_ 34,184 c p m in the adult and 272,165 +_ 25,873 c p m in the old rats: F(2,27) - 2.66, p - 0.0882. T h e d e c l i n e in :~H-activity fi'om 1 to 24 h after the injection w a s significant in the y o u n g [F(2,25) - 19.18, p - 0.0001]; nonsignificant in the adult [F(2,27) = 2.41, p 0.087] and the old [F(2,24) - 0.11, p = 0.89311 rats (Fig. 3.).
Sp~'~'([~" activity (cpm/p,~, VP). O n e hour after the injection specific activity w a s similar in all three age groups: 66.81 _+ 2.89, 70.89 _+ 8.86 and 73.36 _+ 7.10 c p m / p g VP in the y o u n g , adult and old, r e s p e c t i v e l y ; F(2,24) - 0.27, p - 0.7683. Six hours after the injection, specific activity d e c l i n e d significantly in the y o u n g (40.85 _+ 4.66 c p m / p g VP), tended to d e c l i n e in the adult (59.64 _+ 1.95 c p m / p g VP) and did not c h a n g e in the old rats (75.10 _+ 4.05 c p m / p g VP). There w a s a significant difference a m o n g the ages; F(2,25) - 21.44, p = 0.0001. T w e n t y four hours after the injection a further significant d e c l i n e o c c u r r e d in the y o u n g (29.37 _+ 3.56 c p m / p g VP) and it did not c h a n g e in the adult (56.83 _+ 6.02 c p m / p g VP) and the old rats (61.50 _+ 7.05 c p m / p g VP). The differe n c e a m o n g the ages remained significant; F(2,27) = 9.21, p = 0.0009. T h e d e c l i n e in the specific activity f r o m 1 to 24 h after the injection w a s significant in the y o u n g [F(2,25) = 26.06, p = 0.0001], nonsignificant in the adult [F(2,27) = 1.44, p 0.2555] and old [F(2,24) = 1.45, p - 0.2553] rats (Fig. 4).
VP A N D A G I N G
121
Neurohypophysis all-activity ~cpm). One hour after the injection, all-activity in the N H was significantly higher in the young (44,980 _+ 6,891 cpm) and the adult rats (45,140 +_ 6.233 cpm), than in the old ones (15,156 + 1,651 cpm); F(2,24) = 10.04, p = 0.0007. It increased significantly 6 h after the injection in all age groups. Here again, it was significantly higher in the young (94,967 _+ 5,657 cpm) and the adult (96,420 _+ 5,277 cpm) c o m p a r e d with the old rats (51,696 _+ 3,552 cpm); F(2,25) = 25.91, p = 0.0001. T w e n t y - f o u r hours after the injection the activity increased significantly in all age groups. There was no difference, however, a m o n g the age groups (261,764 _+ 70,735, 201,068 _+ 25,670 and 290,410 +_ 28,783 cpm in the young, adult and old, respectively): F(2,27) = 0.98, p = 0.3880. The increase in :+H-activity from 1 to 24 h was significant in all age groups: F(2,25) = 6.79, p = 0.0044 in the young; F(2,27) = 22.26, p = 0.0001 in the adult, and F(2,24) = 29.45, p = 0.0001 in the old rats (Fig. 5). Specific activity (cpm/pg VP). One hour after the injection, specific activity in the N H was significantly lower in the old (0.52 + 0.04 cpm/pg VP) than in the young (1.79 +_ 0.26 cpm/pg VP) and the adult rats (1.66 _+ 0.23 cpm/pg VP): F(2,24) = 12.02, p = 0.0002. It increased significantly in all age groups 6 h after the injection with significantly lower values in the old (1.66 _+ 0.13 cpm/pg VP) than in the young (3.64 _+ 0.37 cpm/pg VP) and the adult rats (3.80 + 0.37 cpm/pg VP); F(2,25) = 14.02, p = 0.0001. T w e n t y - f o u r hours after the injection the activity further increased significantly in all age groups, exhibiting no difference, h o w e v e r , a m o n g the age groups (11.34 _+ 3.21, 6.94 _+ 0.80 and 9.01 _+ 0.81 cpm/pg VP in the young, adult and old rats, respectively); F(2,27) = 1.31, p = 0.2855. The increase in specific activity from 1 to 24 h was significant in all age groups: F(2,25) = 6.55, p = 0.0051 in the young; F(2,27) = 22.31, p = 0.0001 in the adult and F(2,24) = 95.15, p = 0.0001 in the old rats (Fig. 6). DISCUSSION Accurate m e a s u r e m e n t of the biosynthesis of neuropeptides in vivo encounters inherent problems with the absorption and compartmentalization of the labeled amino acid p r e c u r s o r and competing reactions (Richardson, 1981). Since in our study no attempt was made to determine the specific activity of the amino acid precursor in the intracellular pool, nor that bound to t - R N A , (the immediate source of amino acid for protein synthesis), our results m a y not be a s s u m e d necessarily to be an accurate correlative measure of VP biosynthesis. With this reservation and that of some other age-related determining factors discussed later, we employed the technique of incorporation of radioactive label into the VP molecule in the H T and its transport to the N H , originally described by Pickering and Jones (1971a,b) with the following modifications: T h e y injected 100 ~Ci of aHtyrosine into the cisterna m a g n a of an anesthetized rat and pooled 5 glands to measure the rate of incorporation and the rate of transport of two neuropeptides, VP and oxytocin (OT). Since our primary concern was VP, we e m p l o y e d aH-arginine as a specific label for the newly synthetized VP molecule to eliminate interference with OT. The higher radioactivity (200 ~Ci) e m p l o y e d by us permitted m e a s u r e m e n t of the incorpo-
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V . K Z B U Z E K ,'t a /
ration in individual H T explants and N H s and thus to detect the variations among the individual animals. The identity of the elution profile of r a d i o i m m u n o a s s a y a b l e VP obtained by separation on the column was verified by H P L C . Finally, the implantation of a permanent cannula into the lateral ventricle allowed us to inject the radioactive label into an awake animal, so as to avoid the effect of anesthesia on VP release (Aziz and Forsling, 1979). To our knowledge, there are no data available in the literature on age-related changes in the rate of the incorporation of labeled amino acid into VP molecule in the H T and in the rate of its transport to the NH. Thus, only the results on the time-course of the incorporation of all-activity obtained in young rats are c o m p a r a b l e with the data of other investigators (Gainer et al.. 1977" Pickering and Jones, 1971a). In the HT, the accumulation of aH-arginine one hour after the injection was twice as high in the young as in the old animals. The intermediate values obtained in the adult rats, lie between those of young and old, and are not significantly different from those of either group, due to a large scatter. Some of the adult rats incorporated amounts of aH-arginine as high as did the young, others as low as did the old rats. Six hours after the injection, :~H-activity declined significantly in the young rats as found by Picketing and Jones (1971a) and remained unchanged up to 24 h. In the adult rats, :~H-activity declined 6 h after the injection but the difference was not significant due to a large scatter of the values at hour 1. Similarly, as in the young rats, the level of all-activity in the adult was not different between 6 to 24 h after the injection. In the old rats, which exhibited the lowest :~H-activity of all age groups, the activity did not change from 1 to 24 h, indicating decreased rate of incorporation of aH-arginine into VP molecule and also slow axonal transport to the N H of aged Fischer 344 rats. Specific activity (expressed as a ratio of cpm per pg of endogenous VP) was similar in all age groups 1 h after the injection, reflecting, in fact, a decremental trend of both all-activity and VP content of HT, from highest in the young to lowest in the old rats. In the N H , the time-course of the incorporation of all-activity exhibited a similar trend in all age groups as did the specific activity. According to Picketing and Jones (1971a) the specific activity of VP in the N H of young rats reached a plateau at 6 h which was sustained up to 12 h after the injection of aH-tyrosine into the cisterna magna. H o w e v e r , their data consist of only 2 m e a s u r e m e n t s of pooled samples at these time intervals and the authors did not investigate the course of incorporation beyond this point. The same authors, (Pickering et el/., 1975), using a more refined technique, showed that a:'S-cysteine, when injected into the 3rd ventricle, was incorporated into the carrier protein of VP, neurophysin, peaking in the N H 12 h after the injection. Gainer, et a/. (1977) similarly reported the incorporation of a:'S-cysteine into the N H peaking at 12 h. Our results, obtained from individual values, (9-10 young animals at each time interval) at 1, 6 and 24 h, are in agreement with these reports. One and 6 hours after the injection, all- and the specific activity are significantly lower in the N H of the old rats, than in the young and adult rats. At 6 h, both activities in old rats reached the l-h values of the young and adult rats suggesting a delay of at least 6 h in the axonal transport of newly synthetized VP in aged rats. T w e n t y - f o u r hours after the injection, there is no difference a m o n g the ages, in both cpm and specific activity, suggesting a decreased release of VP in the old rats. This agrees with our previous data on the impairment of VP release in aged rats studied in the isolated superfused N H (Zbuzek el al., 1984; 1986).
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It could be argued that the age-related differences in the rate of incorporation of the radioactive label are affected by improper administration or by a different rate of diffusion of the label into the tissues of the young and old animals. The following evidence shows that this is unlikely: (a) Several rats of each age group were injected with Evans blue, which in all ages was found to stain evenly the H T - N H area already 1 h after the injection. (b) Similar amounts of radioactivity were recovered in the VP extracts from the HT 1 h after the injection in all age groups. (c) Similar amounts of :~H-activity were recovered in the NH in all age groups 24 h after the injection. The presented data indicate a decrease in the rate of incorporation of :~H-arginine into VP molecule in the HT and in its transport to the NH of aged Fischer 344 rats. They conform with our ~md other authors" previous findings of a low VP content in the HT of aged Sprague-Di~wley (Zbuzek and Wu, 1982), Fischer 344 (Landfield e t a / . . 1980: Zbuzek ct al.. 1983), Long Evans (Dorsa and Bottemiller, 1982), and albino Diisseldorf (Rodeck c t a l . . 1960) strains of rats, measured by specific RIAs and bioassays. Also, a decreased staining intensity of neurosecretory material in the HT magnocellular neurons has been reported in senile albino Di~sseldorf (Rodeck et al., 1960) and old Wistar but not in Sprague-I~awley rats (Kawashima and Kobayashi, 1982). The nonspecific staining techniques employed did not allow to discern whether or not the decreased staining intensity was due solely to the changes of VP content, since several neuropeptides are known to coexist in the magnocellular neurons of the PVN and SON of the HT (for review see Sofroniew et al., 1984). However, other investigators employed specific immunohistochemical techniques to demonstrate a decreased agerelated staining intensity in PVN and SON of Wistar rats (Watkins and Choy, 1980) and in PVN of monkies (Sladek e t a / . . 1978). Interestingly, the staining intensity was not always found to be equally distributed in both the PVN and SON when specific antisera for VP, OT or their respective neurophysins were used. In contrast, recently an abrupt increase of the activity of Golgi apparatus-bound thiamine-pyrophosphatase in the neurons of PVN and SON has been reported (Fliers and Swaab, 1983) in 32-month old male Wistar rats. This finding is difficult to explain, because in the same series of experiments the enzyme activity in 3- up to 26-month old ra{s exhibited an unchanged or slightly declining trend with increasing age. This report belongs to the divergent opinions in the long unresolved dispute, as to whether an activated rather than a decreased neurosecretory activity occurs in the H T - N H system in senescence (Dicker and Nunn, 1958; Morrison and Staroscik, 1964; Frolkis e t a / . , 1982: Fotheringham et al., 1985; Rogers et al., 1985). These conflicting reports based on indirect assessment of age-related VPbiosynthetic activity prompted us to investigate the rate of the incorporation of radioactive label into VP precursor molecule. Our experiments were not designed to reveal to what extent the reported decrease in the incorporation of :~H-arginine into the VP molecule in the aged rats reflects the biosynthetic activity per se. Several possible age-related determining factors might be involved, for example: a decrease in the number of active VP-synthetizing neurons: a shift in the ratio of the number of neuronal/nonneuronal elements (glial cells) present; a difference in the availability of the intracellular free amino acid pool; or, differences in the intracellular degradation of proteins (lysosomal degradation). At present, however, the data available to answer these questions are inconsistent and more evidence on this subject is needed.
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A c k m m ' l e d g m e n t s - - This research was supported by N1A grant R01 AG02778. We are grateful to Dr. Berta Scharrer of Albert Einstein College of Medicine for her invaluable comments and critique: to Mrs. Annie M. Miller and Ms. Violeta Guerra for their excellent technical assistance; to Mr. Martin Feuerman for the statistical analysis and Ms. Ladonna Levell for typing this manuscript.
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Zbuzek, V.K. and Wu, W. Age-related vasopressin changes in rat plasma and the hypothalamo-hypophyseal system. Exp. Gt, r¢,it. 17, 133-138, 1982. Zbuzek, V.K., Zbuzek, V., and Wu, W. The effect of aging on vasopressin system in Fisher 344 rats. LxT~. G~'rollt. 18, 305-311, 1983. Zbuzek, V., Zbuzek, V.K., and Wu, W. Vasopressin release from individually superfused neurohypophyses decreases in aged rats. N~,ur~Jend~crinoh~gy 39, 538-548, 1984. Zbuzek, V.K., Zbuzek, V., and Wu, W. Decremental vasopressin release after repeated stimulation of superfused neurohypophyses of Fisher 344 rats of different ages. ,/. G~rout. 41, 140-146, 1986.