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Comparison of calcium effect on in vitro calcitonin and parathyroid hormone release by young and aged thyroparathyroid glands
E.~perimental Gerontology, Vol. 22, pp. 263-269, 1987 Printed in the USA. All rights reserved.
0531-5565/87 $3.00 + .00 1987 Pergamon Journals Ltd
C O M P A R I S O N OF CALCIUM EFFECT ON IN VITRO CALCITONIN AND P A R A T H Y R O I D H O R M O N E RELEASE BY YOUNG AND AGED T H Y R O P A R A T H Y R O I D GLANDS N. WONGSURAWAT and H.J. ARMBRECHT Geriatric Research, Education, and Clinical Center, I11G-JB, Veterans Administration Medical Center, St. Louis, MO 63125 and Departments of Internal Medicine and Biochemistry, St. Louis University School of Medicine, St. Louis, MO 63104
A b s t r a c t - Serum immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) levels are higher in young than aged rats. However, serum calcium concentration does not change with age suggesting that the calcium regulation of PTH and CT secretion may be affected by aging. We compared iPTH and iCT secretion in vitro at low and high calcium concentrations using thyroparathyroid glands removed from young (2-3 months), adult (12-13 months), and old (24-27 months) F-344 male rats fed regular rat chow. Glands from each animal were incubated for 3 h in serum-flee culture media containing 1.0 mM calcium and then transferred to media containing 2.5 mM calcium for another 3 h. lmmunoreactive F F H and iCT concentrations of the media after each incubation period were determined by radioimmunoassay. Immunoreactive PTH and iCT secretion per pair of glands was significantly higher in glands from older animals regardless of calcium concentration. The decrease in iPTH, and increment in iCT, secretion in response to 2.5 mM calcium by glands from old rats was smaller than that observed for glands from young animals. These agerelated changes in the regulation of secretion by calcium may contribute to the increased iPTH and iCT secretion and serum levels seen in older animals.
Key Words: calcium, calcitonin, parathyroid hormone, thyroparathyroid glands, aging
INTRODUCTION SERUM CALCIUM must be maintained within a narrow range for the proper function of almost all organ systems throughout the life span. Although serum calcium itself does not change with age, hormones that regulate calcium metabolism do change markedly with age (Armbrecht, 1984; Wongsurawat et a l . , 1986). Serum immunoreactive parathyroid hormone (iPTH) level is elevated with age in both humans (Wiske et a l . , 1979), and rats (Queener et al., 1980; Kalu et al., 1984). There are also age-related (Received 5 August 1986; Accepted 4 February 1987) 263
264
N. W O N G S U R A W A T A N D H.J. A R M B R E C H T
70
50 40 c v t t--O-
Old Adult
J
60
30 20
~
Young
10 0 0
2
4
6
8
Time (Hours)
Fl(~. I. Effect of age on iPTH secretion by thyroparathyroid gland complexes. Thyroparathyroid gland complexes from three rats of each age group were incubated in serum-free culture media containing 1 mM calcium. After a 1 h preincubation, triplicate samples were removed from the media at the indicated times. The iPTH concentration was determined by radioimmunoassay. Each data point is the mean of three samples, and the average standard error of the mean was 3.8~ of the mean. Lines were determined by linear regression and iPTH concentration was linearly correlated with time for each age group (r > 0.96).
c h a n g e s in c a l c i t o n i n (iCT) l e v e l s in b o t h h u m a n s ( H i l l y a r d et al., 1978; S h a m o n k i et al., 1980) a n d r a t s ( Q u e e n e r et al., 1980; K a l u et al., 1983). H o w e v e r , it is n o t k n o w n if t h e s e a g e - r e l a t e d c h a n g e s in s e r u m i P T H a n d i C T a r e d u e to c h a n g e s in h o r m o n e s e c r e t i o n o r d e g r a d a t i o n o r b o t h . T h e fact t h a t s e r u m i P T H a n d iCT c h a n g e with age but t h a t s e r u m c a l c i u m d o e s not a l s o s u g g e s t s t h a t t h e r e m a y be a l t e r e d r e g u l a t i o n o f i P T H a n d iCT s e c r e t i o n w i t h age. T h e r e f o r e , w e h a v e s t u d i e d e f f e c t o f age on the s e c r e t i o n o f i P T H a n d i C T in v i t r o using rat t h y r o p a r a t h y r o i d g l a n d c o m p l e x e s u n d e r d i f f e r e n t concentrations of calcium MATERIALS
AND METHODS
M a l e F344 rats ( C h a r l e s R i v e r B r e e d i n g L a b o r a t o r i e s , W i l m i n g t o n , M A ) a g e d 2-3 m o n t h s ( y o u n g ) , 12-13 m o n t h s (adult), a n d 24-27 m o n t h s (old) w e r e fed r e g u l a r rat c h o w c o n t a i n i n g 1.2% c a l c i u m for 4 w e e k s p r i o r to e x p e r i m e n t . B l o o d w a s d r a w n f r o m i n f e r i o r v e n a c a v a u n d e r n e m b u t a l a n e s t h e s i a for m e a s u r e m e n t s o f s e r u m c a l c i u m , i P T H a n d iCT. S e r u m i P T H w a s m e a s u r e d b y n o n e q u i l i b r i u m r a d i o i m m u n o a s s a y using c h i c k e n a n t i b o v i n e P T H (CH977) a n t i s e r u m ( C o n a w a y and A n a s t , 1974). S e r u m iCT was m e a s u r e d b y r a d i o i m m u n o a s s a y u s i n g g o a t a n t i b o d y to h u m a n C T ( A n a s t et al., 19751. M e a s u r e m e n t s o f s e r u m i P T H a n d s e r u m iCT w e r e p e r f o r m e d in the l a b o r a t o r y o f Dr. L e o n a r d F o r t e ( V A M e d i c a l C e n t e r , C o l u m b i a , MO). T h y r o p a r a t h y r o i d g l a n d s w e r e s u r g i c a l l y r e m o v e d a n d i n c u b a t e d in 2 ml o f s e r u m free c u l t u r e m e d i a as p r e v i o u s l y d e s c r i b e d ( C o o p e r et al., 1978). G l a n d s w e r e first p r e i n c u b a t e d for 1 h in m e d i a c o n t a i n i n g 1 m M c a l c i u m to a l l o w the g l a n d s to a d j u s t to in v i t r o c o n d i t i o n s . G l a n d s w e r e t h e n t r a n s f e r r e d a n d i n c u b a t e d for 3 h in fresh m e d i a c o n t a i n i n g 1 m M c a l c i u m , and t h e n the g l a n d s w e r e t r a n s f e r r e d to fresh m e d i a c o n t a i n ing 2.5 m M c a l c i u m for a n o t h e r 3 h. in s o m e e x p e r i m e n t s g l a n d s w e r e i n c u b a t e d in the
CALCIUM EFFECT ON HORMONE RELEASE
265
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Thyroparathyroid gland complexes from three rats of each age group were incubated in serum-free culture media containing 2.5 mM calcium. After a 1 h preincubation, triplicate samples were removed from the media at the indicated times. The iCT concentration was determined by radioimmunoassay, and the average standard error of the mean was 2.7~ of the mean. Each data point is the mean of three samples. Lines were determined by linear regression and iCT concentration was linearly correlated with time for each age group Ir > 0.99).
s a m e m e d i a for 6 h to e s t a b l i s h the l i n e a r i t y o f i P T H a n d iCT s e c r e t i o n (Figs. 1 a n d 2). I m m u n o r e a c t i v e p a r a t h y r o i d h o r m o n e a n d iCT c o n c e n t r a t i o n s o f the c u l t u r e m e d i a a f t e r e a c h i n c u b a t i o n p e r i o d w e r e d e t e r m i n e d using r a d i o i m m u n o a s s a y kits for rat P T H and human CT (Immuno Nuclear Corp., Stillwater, MN). R e s u l t s a r e g i v e n as the m e a n v a l u e s plus s t a n d a r d e r r o r o f the m e a n ( S E M ) . Stud e n t ' s t w o - t a i l e d t - t e s t for p a i r e d d a t a w a s u s e d for c o m p a r i s o n o f d i f f e r e n c e s . A c o n f i d e n c e level o f 95% o r g r e a t e r w a s c o n s i d e r e d significant. RESULTS S e r u m c a l c i u m , i P T H and iCT w e r e c o m p a r e d in rats o f d i f f e r e n t a g e s ( T a b l e 1). S e r u m c a l c i u m c o n c e n t r a t i o n s w e r e s i m i l a r in all age g r o u p s . O n the o t h e r h a n d , s e r u m l e v e l s o f i P T H a n d i C T s h o w e d s i g n i f i c a n t c h a n g e s w i t h age. S e r u m i P T H a n d iCT w e r e m a r k e d l y h i g h e r in the a d u l t a n d o l d a n i m a l s c o m p a r e d to the y o u n g a n i m a l s . T o e s t a b l i s h the l i n e a r i t y o f h o r m o n e s e c r e t i o n , i P T H and iCT w e r e m e a s u r e d in m e d i a a f t e r i n c u b a t i n g g l a n d s f r o m rats o f the s a m e age g r o u p for 3 a n d for 6 h. I m m u n o r e a c t i v e P T H s e c r e t i o n w a s m a r k e d l y i n c r e a s e d in the a d u l t a n d old rats c o m p a r e d to the y o u n g rats (Fig. l). T h e r e w a s no s i g n i f i c a n t d i f f e r e n c e b e t w e e n the P T H s e c r e t i o n b y a d u l t a n d old r a t s . In all age g r o u p s , i P T H s e c r e t i o n w a s l i n e a r o v e r the 6 h p e r i o d s t u d i e d . I m m u n o r e a c t i v e C T s e c r e t i o n s h o w e d a p r o g r e s s i v e i n c r e a s e with age (Fig. 2). S e c r e t i o n b y o l d rats w a s g r e a t e r t h a n a d u l t rats w h i c h , in turn, w a s g r e a t e r t h a n y o u n g rats. C T s e c r e t i o n w a s a l s o l i n e a r in all age g r o u p s o v e r the 6 h p e r i o d studied. T a b l e 2 s h o w s the e f f e c t o f high a n d l o w c a l c i u m c o n c e n t r a t i o n s on i P T H s e c r e t i o n b y t h y r o p a r a t h y r o i d g l a n d s . P a r a t h y r o i d h o r m o n e s e c r e t i o n w a s h i g h e r in g l a n d s f r o m old r a t s at low ( ! .0 m M ) a n d high (2.5 m M ) c a l c i u m c o n c e n t r a t i o n s . T h e g l a n d s f r o m all age
266
N. WONGSURAWAT AND H.J. ARMBRECHT
T A B L E I. A G E - R E L A T E D CHANGES IN SERUM CA, I P T H A N D I C T
Age Grot¢l?
Serum C a (mg/dl) i P T H (/zl e q / m l ) * * i C T (pg/ml)
Yol/ll,e
A dl/#
Old
10.3 ± 0.6 17 + 11 97 + 17
10.9 + 0.5 95 _+ 29* 754 _+ 83*
10.6 + 0.5 150 ± 20* 687 ± 91"
*Significantly different from Young (/)<0.05, t-test). **"txl e q " refers to ~1 equivalents o f a standard serum pooled from vitamin D
deficient rats. Note: Table entries are the mean _+ S E M o f 8 young rats, 8 adult rats, and 6 old rats.
groups were suppressible by the high calcium concentration by about the same absolute amount. However, the percent decrease induced by the high calcium was significantly less in the adult and old groups compared to the young group. Age-related changes in iCT secretion in response to high and low calcium concentrations are shown in Table 3. Immunoreactive CT secretion was found to correlate positively with age of the animal. This was seen at both concentrations of calcium. Stimulated by increased calcium concentration (from 1 mM to 2.5 mM) in the media, the glands from all age groups had higher levels of calcitonin secretion by about the same absolute amount. However, the percent increment produced by high calcium was significantly less in the adult and old group compared to the young group.
DISCUSSION These studies demonstrate that iPTH and iCT secretion by the thyroparathyroid complex is altered with aging. In each case, there is increased secretion of the hormone with age. The changes in iPTH secretion occur during maturation (young to adult), but
T A B L E 2. A G E - R E L A T E D CHANGES IN I P T H
iPTH Secretion (ng/h/rat) Age Group Young Adult Old
SECRETION
(1.0 mM ('a)
(2.5 mM Ca)
% Decrease induced by 2.5 mM ('a
12.5 _+ 0.7 14.5 ± 1.2 15.6 _+ 1.0
2.8 ± 0.2* 5.3 ± 0.3* 6.6 _+ 0.6*
78 _+ 2 63 ± 2** 58 _+ 4**
*Significantly different from iPTH secretion at 1.0 m M C a o f same age group (/) < 0 . 0 5 , t-test). **Significantly different from percent decrease of young group (p < 0 . 0 5 , t-test). Note: Table entries are the mean + S E M o f 16 young rats, 16 adult rats, and 10 old rats.
267
CALCIUM EFFECT ON HORMONE RELEASE TABLE 3. AGE-RELATED CHANGES IN ICT SECRETION
iCT Secretion (pg/h) Age Group Young Adult Old
(I.0 mM Ca)
(2.5 m M Ca)
% Increase induced by 2.5 m M Ca
151 ± 39 364 ± 67 523 ± 57
375 ± 50* 550 ± 55* 731 ± 13"
148 ± 33 51 ± 15"* 40 + 2**
* Significantly different from iCT secretion at 1.0 mM Ca of the same age group (p < 0.05, t-test). ** Significantly different from percent increase of young group (p < 0.05, t-test). Note: Table entries are the mean ± SEM of 6 rats.
the changes in iCT secretion take place during both maturation and aging (adult to old). In addition, the percent responsiveness of the adult and old glands to calcium decreases relative to the young glands. The decreased responsiveness of the thyroparathyroid complex to calcium may be due to age-related changes in the regulation of the secretory pathway. In the case of iPTH secretion, there may be age-related changes in the effect of calcium on release of stored iPTH, degradation of iPTH, or modification of adenylate cyclase activity (Brown, 1982). Other tissues also show decreased responsiveness with age to stimuli that involve calcium as an intermediary. These include alpha-adrenergic stimulation of parotid cell electrolytes secretion (Ito et al., 1982) and lectin stimulation of lymphocyte proliferation (Wu et al., 1985). It is also of interest that the response of the parathyroid gland to calcium is altered in very young animals. In neonatal calves, iPTH secretion is not suppressed by serum calcium to the same degree as it is in older calves (Keaton et al., 1978). Thus, the sensitivity of the parathyroid gland to calcium is not fixed but may change during early development, aging, and some disease states (Brown, 1982). In the case of iCT secretion, it has been reported that old buffalo rats show greater increment than young rats to calcium infusion in vivo (Queener et al., 1980). This finding suggests that factors other than iCT secretion alone influence serum iCT levels in vivo. The increased secretion of iPTH and iCT with age could be due to several factors. One possibility is that there is an increase in the number of cells secreting these hormones with age. In the case of iCT, this possibility is suspected by the fact that the total iCT content of the thyroid gland increases with age (Queener et al., 1980). However, the increased secretion of iCT is probably not due to medullary thyroid carcinoma. Although a high incidence of this carcinoma has been reported in some strains of rats (Boorman et al., 1972), the incidence in F344 rats is very low (Coleman et al., 1977). The second possibility is that the number of cells does not change but that the regulation of hormone secretion by these cells is altered with age. In the case of iPTH secretion, increased secretion could be due to decreased sensitivity to calcium with age (Table 2). In addition, there may be age-related changes in levels of, or responsiveness to, other factors which modulate iPTH secretion. These factors include 1,25dihydroxyvitamin D (Wongsurawat et al., 1986) the alpha- and beta-adrenergic catecholamines, somatostatin, and prostaglandins (Brown, 1982). In the case of iCT secre-
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N. WONGSURAWATAND H.J. ARMBRECHT
tion, decreased calcium sensitivity does not explain the increased iCT secretion with a g e ( T a b l e 3), s i n c e c a l c i u m e n h a n c e s i C T s e c r e t i o n . T h e r e f o r e , o t h e r f a c t o r s m u s t be i n v o l v e d in t h e i n c r e a s e in i C T s e c r e t i o n w i t h a g e . In s u m m a r y , t h e r e is i n c r e a s e d s e c r e t i o n o f i P T H a n d i C T b y t h e t h y r o p a r a t h y r o i d g l a n d c o m p l e x w i t h a g e . T h i s i n c r e a s e d s e c r e t i o n m a y c o n t r i b u t e to t h e rise in s e r u m i P T H a n d i C T w i t h a g e ( T a b l e 1). H o w e v e r + h o r m o n e l e v e l s m a y a l s o b e m o d u l a t e d by t h e r a t e o f h o r m o n e d e g r a d a t i o n a n d e x c r e t i o n , a n d t h e s e p a r a m e t e r s n e e d to be m e a s u r e d b e f o r e a c o m p l e t e e x p l a n a t i o n o f s e r u m l e v e l s c a n b e g i v e n . In a d d i t i o n to t h e a g e - r e l a t e d c h a n g e s in s e r u m l e v e l s , t a r g e t t i s s u e r e s p o n s i v e n e s s to P T H a n d C T a l s o d e c r e a s e s w i t h a g e . O l d e r rats s h o w d e c r e a s e d c a l c e m i c r e s p o n s e ( K a l u e t a l . , 1982) a n d d e c r e a s e d r e n a l 1 , 2 5 - d i h y d r o x y v i t a m i n D p r o d u c t i o n ( A r m b r e c h t e t a l . , 19821 c o m p a r e d to y o u n g rats. T h e c a p a c i t y o f C T to l o w e r s e r u m c a l c i u m a l s o d e c l i n e s w i t h a g e ( C o p p a n d K u c z e r p a , 1968). F u r t h e r w o r k is n e e d e d to d e t e r m i n e w h e t h e r t h e c h a n g e s in h o r m o n e l e v e l s a r e r e s p o n s i b l e f o r o r r e s u l t f r o m t h e a g e - r e l a t e d c h a n g e s in t a r g e t tissue responsiveness.
Acknowh'd,~,ments - - This work was supported by USPHS Grant AM 32158 and by the Veterans Adminis-
tration. The authors thank Dr. Keonard R. Forte, VA Medical Center, Columbia, MO for the measurement of serum iPTH and iCT. The authors also thank Monica Boltz and Susan Porter lot technical assistance und Sandy Melliere and Debbie Sell ti)r secretarial assistance.
REFERENCES Anast, C.S., David, L., Winnacker, J., Glass, L., Baskin, W., Brubaker, L.+ and Burns, T. Serum calcitoninlowering effect of magnesium in patients with medullary carcinoma of the thyroid..l ('lin. lnvem~t. 56+ 1615-1621, 1975. Armbrecht, H.J. In: Nutritional lnterveHtion in the A~,in~,, Proce,~,~, Armbrecht, H.J.. Prendergast, J.M., and Coe, R.M., (Editors), pp. 69-83, Springer-Verlag, New York, 1984. Armbrecht, H.J., Wongsurawat, N., Zenser, T.V., and Davis. B.B. Differential effects of parathyroid hormone on the renal 1,25-dihydroxyvitamin D3 and 24+25-dihydroxyvitamin D3 production of young and adult rats. Endocrinolo~,y 111, 1339-1344, 1982. Boorman. G.A., van Noord+ M.J.+ and Hollander. C.F. Naturally occurring medullary thyroid carcinoma in the rat. Arch. Path. 94, 35-41, 1972. Brown, E.M. PTH secretion in vivo and in vitro. Regulation by calcium and other secretagogues. Mim'ral Electrolytes Metah. 8, 13(I-15(I, 1982. Coleman, G.L., Barthold, S.W., and Osbaldiston, G.W. Pathologicul changes during aging in barrier-reared Fischer 344 male rats. J. Gerontol. 32, 258-278, 1977. Conaway, H.H. and Anast, C.S. Double-antibody radioimmunoassay for parathyroid hormone..I, l+ah. ('liH. Meal. 83, 129-138+ 1974. Cooper+ C.W., Ramp, W.K.. Ross. A.J., Ill and Wells, S.J. Jr. Concurrent secretion of calcitonin und parathyroid hormone in vitro from the rat thyroparathyroid complex. Proc. Soc. E+vp. Biol. Med. 158, 299-303, 1978. Copp, D.H. and Kuczerpa, A.V. A new bioassay lot+calcitonin and effect of age and dietary calcium on response. In: Cah'itonin+ Taylor, S., tEditor), Springer-Vetlag, New York, 1968. Hillyard, C.J., Stevenson, J.C. and Mclntyre, 1. Relative deficiency of plasma calcitonin in normal women. Lam+et I, 961-962, 1978. lto, H., Baum, B.J., Uchida, T., Hoopes, M.T., Bodner, L. and Roth, G.S. Modulation of rat parotid cell alpha-adrenergic responsiveness tit a step subsequent to receptor activation..I. Biol. Chem. 257, 9532-9538, 1982. Kalu. D.N., Hardin, R.R., Murata, 1., Huber+ M.B., and Roos, B.A. Age-dependent modulation of parathyroid hormone action. A~,e 5, 25-29, 1982.
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Kalu, D.N., Cockerham, R., Yu, B.P., and Ross, B.A. Lifelong dietary modulation of calcitonin levels in rats. Endocrinolo~,,y 113, 2010-2016, 1983. Kalu, D.N., Hardin, R.H., Cockerham, R., and Yu, B.P. Aging and dietary modulation of rat skeleton and parathyroid hormone. Endocrinolo~,,y 115, 123%1244, 1984. Keaton, J.A., Banto. J.A., Moore, M.P., Gruel, J.B., and Mayer, G.P. Altered parathyroid response to calcium in hypercalcemic neonatal calves. Endocrinolo~,,y 103, 2161-2167, 1978. Queener, S.F., Bell, N.H., Larson, S.M., Henry. D.P., and Slatopolsky, E. Comparison of the regulation of calcitonin in serum of old and young buffalo rats. J. Endocrinolo:,,y 87, 73-80, 1980. Shamonki, I.M., Frumar, A.M,, Tataryn, I.V., Meldrum, D.R., Davidson, B.H., Parthmore, J.G., Judd, H.L., and Deftos, L.J. Age-related changes of calcitonin secretion in females. J. Clin. Endocrim~l. Metah. 50, 437-439, 1980. Wiske, P.S., Epstein, S., Bell, N.H., Queeener, S.F., Edmondson, J., and Johnston, C.C. Increase in immunoreactive parathyroid hormone with age. N. En~,,I. J. Med. 300, 141%1421, 1979. Wongsurawat, N., Siegel, N.A., and Armbrecht, H.J. Aging and vitamin D metabolism. In: Current Re,~earch on Calcium-Ref,,ulatin~,, Hormones. Cooper, C.W. (Editor), University of Texas Press, Texas, 1986. Wu, W., Pahlavani, M., Richardson. A., and Cheung, H.T. Effect of maturation and age on lymphocyte proliferation induced by A23187 through an interleukin-independent pathway. J. Leukocyte Biol. 38, 531540. 1985.
0531-5565/87 $3.00 + .00 1987 Pergamon Journals Ltd
C O M P A R I S O N OF CALCIUM EFFECT ON IN VITRO CALCITONIN AND P A R A T H Y R O I D H O R M O N E RELEASE BY YOUNG AND AGED T H Y R O P A R A T H Y R O I D GLANDS N. WONGSURAWAT and H.J. ARMBRECHT Geriatric Research, Education, and Clinical Center, I11G-JB, Veterans Administration Medical Center, St. Louis, MO 63125 and Departments of Internal Medicine and Biochemistry, St. Louis University School of Medicine, St. Louis, MO 63104
A b s t r a c t - Serum immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) levels are higher in young than aged rats. However, serum calcium concentration does not change with age suggesting that the calcium regulation of PTH and CT secretion may be affected by aging. We compared iPTH and iCT secretion in vitro at low and high calcium concentrations using thyroparathyroid glands removed from young (2-3 months), adult (12-13 months), and old (24-27 months) F-344 male rats fed regular rat chow. Glands from each animal were incubated for 3 h in serum-flee culture media containing 1.0 mM calcium and then transferred to media containing 2.5 mM calcium for another 3 h. lmmunoreactive F F H and iCT concentrations of the media after each incubation period were determined by radioimmunoassay. Immunoreactive PTH and iCT secretion per pair of glands was significantly higher in glands from older animals regardless of calcium concentration. The decrease in iPTH, and increment in iCT, secretion in response to 2.5 mM calcium by glands from old rats was smaller than that observed for glands from young animals. These agerelated changes in the regulation of secretion by calcium may contribute to the increased iPTH and iCT secretion and serum levels seen in older animals.
Key Words: calcium, calcitonin, parathyroid hormone, thyroparathyroid glands, aging
INTRODUCTION SERUM CALCIUM must be maintained within a narrow range for the proper function of almost all organ systems throughout the life span. Although serum calcium itself does not change with age, hormones that regulate calcium metabolism do change markedly with age (Armbrecht, 1984; Wongsurawat et a l . , 1986). Serum immunoreactive parathyroid hormone (iPTH) level is elevated with age in both humans (Wiske et a l . , 1979), and rats (Queener et al., 1980; Kalu et al., 1984). There are also age-related (Received 5 August 1986; Accepted 4 February 1987) 263
264
N. W O N G S U R A W A T A N D H.J. A R M B R E C H T
70
50 40 c v t t--O-
Old Adult
J
60
30 20
~
Young
10 0 0
2
4
6
8
Time (Hours)
Fl(~. I. Effect of age on iPTH secretion by thyroparathyroid gland complexes. Thyroparathyroid gland complexes from three rats of each age group were incubated in serum-free culture media containing 1 mM calcium. After a 1 h preincubation, triplicate samples were removed from the media at the indicated times. The iPTH concentration was determined by radioimmunoassay. Each data point is the mean of three samples, and the average standard error of the mean was 3.8~ of the mean. Lines were determined by linear regression and iPTH concentration was linearly correlated with time for each age group (r > 0.96).
c h a n g e s in c a l c i t o n i n (iCT) l e v e l s in b o t h h u m a n s ( H i l l y a r d et al., 1978; S h a m o n k i et al., 1980) a n d r a t s ( Q u e e n e r et al., 1980; K a l u et al., 1983). H o w e v e r , it is n o t k n o w n if t h e s e a g e - r e l a t e d c h a n g e s in s e r u m i P T H a n d i C T a r e d u e to c h a n g e s in h o r m o n e s e c r e t i o n o r d e g r a d a t i o n o r b o t h . T h e fact t h a t s e r u m i P T H a n d iCT c h a n g e with age but t h a t s e r u m c a l c i u m d o e s not a l s o s u g g e s t s t h a t t h e r e m a y be a l t e r e d r e g u l a t i o n o f i P T H a n d iCT s e c r e t i o n w i t h age. T h e r e f o r e , w e h a v e s t u d i e d e f f e c t o f age on the s e c r e t i o n o f i P T H a n d i C T in v i t r o using rat t h y r o p a r a t h y r o i d g l a n d c o m p l e x e s u n d e r d i f f e r e n t concentrations of calcium MATERIALS
AND METHODS
M a l e F344 rats ( C h a r l e s R i v e r B r e e d i n g L a b o r a t o r i e s , W i l m i n g t o n , M A ) a g e d 2-3 m o n t h s ( y o u n g ) , 12-13 m o n t h s (adult), a n d 24-27 m o n t h s (old) w e r e fed r e g u l a r rat c h o w c o n t a i n i n g 1.2% c a l c i u m for 4 w e e k s p r i o r to e x p e r i m e n t . B l o o d w a s d r a w n f r o m i n f e r i o r v e n a c a v a u n d e r n e m b u t a l a n e s t h e s i a for m e a s u r e m e n t s o f s e r u m c a l c i u m , i P T H a n d iCT. S e r u m i P T H w a s m e a s u r e d b y n o n e q u i l i b r i u m r a d i o i m m u n o a s s a y using c h i c k e n a n t i b o v i n e P T H (CH977) a n t i s e r u m ( C o n a w a y and A n a s t , 1974). S e r u m iCT was m e a s u r e d b y r a d i o i m m u n o a s s a y u s i n g g o a t a n t i b o d y to h u m a n C T ( A n a s t et al., 19751. M e a s u r e m e n t s o f s e r u m i P T H a n d s e r u m iCT w e r e p e r f o r m e d in the l a b o r a t o r y o f Dr. L e o n a r d F o r t e ( V A M e d i c a l C e n t e r , C o l u m b i a , MO). T h y r o p a r a t h y r o i d g l a n d s w e r e s u r g i c a l l y r e m o v e d a n d i n c u b a t e d in 2 ml o f s e r u m free c u l t u r e m e d i a as p r e v i o u s l y d e s c r i b e d ( C o o p e r et al., 1978). G l a n d s w e r e first p r e i n c u b a t e d for 1 h in m e d i a c o n t a i n i n g 1 m M c a l c i u m to a l l o w the g l a n d s to a d j u s t to in v i t r o c o n d i t i o n s . G l a n d s w e r e t h e n t r a n s f e r r e d a n d i n c u b a t e d for 3 h in fresh m e d i a c o n t a i n i n g 1 m M c a l c i u m , and t h e n the g l a n d s w e r e t r a n s f e r r e d to fresh m e d i a c o n t a i n ing 2.5 m M c a l c i u m for a n o t h e r 3 h. in s o m e e x p e r i m e n t s g l a n d s w e r e i n c u b a t e d in the
CALCIUM EFFECT ON HORMONE RELEASE
265
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Thyroparathyroid gland complexes from three rats of each age group were incubated in serum-free culture media containing 2.5 mM calcium. After a 1 h preincubation, triplicate samples were removed from the media at the indicated times. The iCT concentration was determined by radioimmunoassay, and the average standard error of the mean was 2.7~ of the mean. Each data point is the mean of three samples. Lines were determined by linear regression and iCT concentration was linearly correlated with time for each age group Ir > 0.99).
s a m e m e d i a for 6 h to e s t a b l i s h the l i n e a r i t y o f i P T H a n d iCT s e c r e t i o n (Figs. 1 a n d 2). I m m u n o r e a c t i v e p a r a t h y r o i d h o r m o n e a n d iCT c o n c e n t r a t i o n s o f the c u l t u r e m e d i a a f t e r e a c h i n c u b a t i o n p e r i o d w e r e d e t e r m i n e d using r a d i o i m m u n o a s s a y kits for rat P T H and human CT (Immuno Nuclear Corp., Stillwater, MN). R e s u l t s a r e g i v e n as the m e a n v a l u e s plus s t a n d a r d e r r o r o f the m e a n ( S E M ) . Stud e n t ' s t w o - t a i l e d t - t e s t for p a i r e d d a t a w a s u s e d for c o m p a r i s o n o f d i f f e r e n c e s . A c o n f i d e n c e level o f 95% o r g r e a t e r w a s c o n s i d e r e d significant. RESULTS S e r u m c a l c i u m , i P T H and iCT w e r e c o m p a r e d in rats o f d i f f e r e n t a g e s ( T a b l e 1). S e r u m c a l c i u m c o n c e n t r a t i o n s w e r e s i m i l a r in all age g r o u p s . O n the o t h e r h a n d , s e r u m l e v e l s o f i P T H a n d i C T s h o w e d s i g n i f i c a n t c h a n g e s w i t h age. S e r u m i P T H a n d iCT w e r e m a r k e d l y h i g h e r in the a d u l t a n d o l d a n i m a l s c o m p a r e d to the y o u n g a n i m a l s . T o e s t a b l i s h the l i n e a r i t y o f h o r m o n e s e c r e t i o n , i P T H and iCT w e r e m e a s u r e d in m e d i a a f t e r i n c u b a t i n g g l a n d s f r o m rats o f the s a m e age g r o u p for 3 a n d for 6 h. I m m u n o r e a c t i v e P T H s e c r e t i o n w a s m a r k e d l y i n c r e a s e d in the a d u l t a n d old rats c o m p a r e d to the y o u n g rats (Fig. l). T h e r e w a s no s i g n i f i c a n t d i f f e r e n c e b e t w e e n the P T H s e c r e t i o n b y a d u l t a n d old r a t s . In all age g r o u p s , i P T H s e c r e t i o n w a s l i n e a r o v e r the 6 h p e r i o d s t u d i e d . I m m u n o r e a c t i v e C T s e c r e t i o n s h o w e d a p r o g r e s s i v e i n c r e a s e with age (Fig. 2). S e c r e t i o n b y o l d rats w a s g r e a t e r t h a n a d u l t rats w h i c h , in turn, w a s g r e a t e r t h a n y o u n g rats. C T s e c r e t i o n w a s a l s o l i n e a r in all age g r o u p s o v e r the 6 h p e r i o d studied. T a b l e 2 s h o w s the e f f e c t o f high a n d l o w c a l c i u m c o n c e n t r a t i o n s on i P T H s e c r e t i o n b y t h y r o p a r a t h y r o i d g l a n d s . P a r a t h y r o i d h o r m o n e s e c r e t i o n w a s h i g h e r in g l a n d s f r o m old r a t s at low ( ! .0 m M ) a n d high (2.5 m M ) c a l c i u m c o n c e n t r a t i o n s . T h e g l a n d s f r o m all age
266
N. WONGSURAWAT AND H.J. ARMBRECHT
T A B L E I. A G E - R E L A T E D CHANGES IN SERUM CA, I P T H A N D I C T
Age Grot¢l?
Serum C a (mg/dl) i P T H (/zl e q / m l ) * * i C T (pg/ml)
Yol/ll,e
A dl/#
Old
10.3 ± 0.6 17 + 11 97 + 17
10.9 + 0.5 95 _+ 29* 754 _+ 83*
10.6 + 0.5 150 ± 20* 687 ± 91"
*Significantly different from Young (/)<0.05, t-test). **"txl e q " refers to ~1 equivalents o f a standard serum pooled from vitamin D
deficient rats. Note: Table entries are the mean _+ S E M o f 8 young rats, 8 adult rats, and 6 old rats.
groups were suppressible by the high calcium concentration by about the same absolute amount. However, the percent decrease induced by the high calcium was significantly less in the adult and old groups compared to the young group. Age-related changes in iCT secretion in response to high and low calcium concentrations are shown in Table 3. Immunoreactive CT secretion was found to correlate positively with age of the animal. This was seen at both concentrations of calcium. Stimulated by increased calcium concentration (from 1 mM to 2.5 mM) in the media, the glands from all age groups had higher levels of calcitonin secretion by about the same absolute amount. However, the percent increment produced by high calcium was significantly less in the adult and old group compared to the young group.
DISCUSSION These studies demonstrate that iPTH and iCT secretion by the thyroparathyroid complex is altered with aging. In each case, there is increased secretion of the hormone with age. The changes in iPTH secretion occur during maturation (young to adult), but
T A B L E 2. A G E - R E L A T E D CHANGES IN I P T H
iPTH Secretion (ng/h/rat) Age Group Young Adult Old
SECRETION
(1.0 mM ('a)
(2.5 mM Ca)
% Decrease induced by 2.5 mM ('a
12.5 _+ 0.7 14.5 ± 1.2 15.6 _+ 1.0
2.8 ± 0.2* 5.3 ± 0.3* 6.6 _+ 0.6*
78 _+ 2 63 ± 2** 58 _+ 4**
*Significantly different from iPTH secretion at 1.0 m M C a o f same age group (/) < 0 . 0 5 , t-test). **Significantly different from percent decrease of young group (p < 0 . 0 5 , t-test). Note: Table entries are the mean + S E M o f 16 young rats, 16 adult rats, and 10 old rats.
267
CALCIUM EFFECT ON HORMONE RELEASE TABLE 3. AGE-RELATED CHANGES IN ICT SECRETION
iCT Secretion (pg/h) Age Group Young Adult Old
(I.0 mM Ca)
(2.5 m M Ca)
% Increase induced by 2.5 m M Ca
151 ± 39 364 ± 67 523 ± 57
375 ± 50* 550 ± 55* 731 ± 13"
148 ± 33 51 ± 15"* 40 + 2**
* Significantly different from iCT secretion at 1.0 mM Ca of the same age group (p < 0.05, t-test). ** Significantly different from percent increase of young group (p < 0.05, t-test). Note: Table entries are the mean ± SEM of 6 rats.
the changes in iCT secretion take place during both maturation and aging (adult to old). In addition, the percent responsiveness of the adult and old glands to calcium decreases relative to the young glands. The decreased responsiveness of the thyroparathyroid complex to calcium may be due to age-related changes in the regulation of the secretory pathway. In the case of iPTH secretion, there may be age-related changes in the effect of calcium on release of stored iPTH, degradation of iPTH, or modification of adenylate cyclase activity (Brown, 1982). Other tissues also show decreased responsiveness with age to stimuli that involve calcium as an intermediary. These include alpha-adrenergic stimulation of parotid cell electrolytes secretion (Ito et al., 1982) and lectin stimulation of lymphocyte proliferation (Wu et al., 1985). It is also of interest that the response of the parathyroid gland to calcium is altered in very young animals. In neonatal calves, iPTH secretion is not suppressed by serum calcium to the same degree as it is in older calves (Keaton et al., 1978). Thus, the sensitivity of the parathyroid gland to calcium is not fixed but may change during early development, aging, and some disease states (Brown, 1982). In the case of iCT secretion, it has been reported that old buffalo rats show greater increment than young rats to calcium infusion in vivo (Queener et al., 1980). This finding suggests that factors other than iCT secretion alone influence serum iCT levels in vivo. The increased secretion of iPTH and iCT with age could be due to several factors. One possibility is that there is an increase in the number of cells secreting these hormones with age. In the case of iCT, this possibility is suspected by the fact that the total iCT content of the thyroid gland increases with age (Queener et al., 1980). However, the increased secretion of iCT is probably not due to medullary thyroid carcinoma. Although a high incidence of this carcinoma has been reported in some strains of rats (Boorman et al., 1972), the incidence in F344 rats is very low (Coleman et al., 1977). The second possibility is that the number of cells does not change but that the regulation of hormone secretion by these cells is altered with age. In the case of iPTH secretion, increased secretion could be due to decreased sensitivity to calcium with age (Table 2). In addition, there may be age-related changes in levels of, or responsiveness to, other factors which modulate iPTH secretion. These factors include 1,25dihydroxyvitamin D (Wongsurawat et al., 1986) the alpha- and beta-adrenergic catecholamines, somatostatin, and prostaglandins (Brown, 1982). In the case of iCT secre-
268
N. WONGSURAWATAND H.J. ARMBRECHT
tion, decreased calcium sensitivity does not explain the increased iCT secretion with a g e ( T a b l e 3), s i n c e c a l c i u m e n h a n c e s i C T s e c r e t i o n . T h e r e f o r e , o t h e r f a c t o r s m u s t be i n v o l v e d in t h e i n c r e a s e in i C T s e c r e t i o n w i t h a g e . In s u m m a r y , t h e r e is i n c r e a s e d s e c r e t i o n o f i P T H a n d i C T b y t h e t h y r o p a r a t h y r o i d g l a n d c o m p l e x w i t h a g e . T h i s i n c r e a s e d s e c r e t i o n m a y c o n t r i b u t e to t h e rise in s e r u m i P T H a n d i C T w i t h a g e ( T a b l e 1). H o w e v e r + h o r m o n e l e v e l s m a y a l s o b e m o d u l a t e d by t h e r a t e o f h o r m o n e d e g r a d a t i o n a n d e x c r e t i o n , a n d t h e s e p a r a m e t e r s n e e d to be m e a s u r e d b e f o r e a c o m p l e t e e x p l a n a t i o n o f s e r u m l e v e l s c a n b e g i v e n . In a d d i t i o n to t h e a g e - r e l a t e d c h a n g e s in s e r u m l e v e l s , t a r g e t t i s s u e r e s p o n s i v e n e s s to P T H a n d C T a l s o d e c r e a s e s w i t h a g e . O l d e r rats s h o w d e c r e a s e d c a l c e m i c r e s p o n s e ( K a l u e t a l . , 1982) a n d d e c r e a s e d r e n a l 1 , 2 5 - d i h y d r o x y v i t a m i n D p r o d u c t i o n ( A r m b r e c h t e t a l . , 19821 c o m p a r e d to y o u n g rats. T h e c a p a c i t y o f C T to l o w e r s e r u m c a l c i u m a l s o d e c l i n e s w i t h a g e ( C o p p a n d K u c z e r p a , 1968). F u r t h e r w o r k is n e e d e d to d e t e r m i n e w h e t h e r t h e c h a n g e s in h o r m o n e l e v e l s a r e r e s p o n s i b l e f o r o r r e s u l t f r o m t h e a g e - r e l a t e d c h a n g e s in t a r g e t tissue responsiveness.
Acknowh'd,~,ments - - This work was supported by USPHS Grant AM 32158 and by the Veterans Adminis-
tration. The authors thank Dr. Keonard R. Forte, VA Medical Center, Columbia, MO for the measurement of serum iPTH and iCT. The authors also thank Monica Boltz and Susan Porter lot technical assistance und Sandy Melliere and Debbie Sell ti)r secretarial assistance.
REFERENCES Anast, C.S., David, L., Winnacker, J., Glass, L., Baskin, W., Brubaker, L.+ and Burns, T. Serum calcitoninlowering effect of magnesium in patients with medullary carcinoma of the thyroid..l ('lin. lnvem~t. 56+ 1615-1621, 1975. Armbrecht, H.J. In: Nutritional lnterveHtion in the A~,in~,, Proce,~,~, Armbrecht, H.J.. Prendergast, J.M., and Coe, R.M., (Editors), pp. 69-83, Springer-Verlag, New York, 1984. Armbrecht, H.J., Wongsurawat, N., Zenser, T.V., and Davis. B.B. Differential effects of parathyroid hormone on the renal 1,25-dihydroxyvitamin D3 and 24+25-dihydroxyvitamin D3 production of young and adult rats. Endocrinolo~,y 111, 1339-1344, 1982. Boorman. G.A., van Noord+ M.J.+ and Hollander. C.F. Naturally occurring medullary thyroid carcinoma in the rat. Arch. Path. 94, 35-41, 1972. Brown, E.M. PTH secretion in vivo and in vitro. Regulation by calcium and other secretagogues. Mim'ral Electrolytes Metah. 8, 13(I-15(I, 1982. Coleman, G.L., Barthold, S.W., and Osbaldiston, G.W. Pathologicul changes during aging in barrier-reared Fischer 344 male rats. J. Gerontol. 32, 258-278, 1977. Conaway, H.H. and Anast, C.S. Double-antibody radioimmunoassay for parathyroid hormone..I, l+ah. ('liH. Meal. 83, 129-138+ 1974. Cooper+ C.W., Ramp, W.K.. Ross. A.J., Ill and Wells, S.J. Jr. Concurrent secretion of calcitonin und parathyroid hormone in vitro from the rat thyroparathyroid complex. Proc. Soc. E+vp. Biol. Med. 158, 299-303, 1978. Copp, D.H. and Kuczerpa, A.V. A new bioassay lot+calcitonin and effect of age and dietary calcium on response. In: Cah'itonin+ Taylor, S., tEditor), Springer-Vetlag, New York, 1968. Hillyard, C.J., Stevenson, J.C. and Mclntyre, 1. Relative deficiency of plasma calcitonin in normal women. Lam+et I, 961-962, 1978. lto, H., Baum, B.J., Uchida, T., Hoopes, M.T., Bodner, L. and Roth, G.S. Modulation of rat parotid cell alpha-adrenergic responsiveness tit a step subsequent to receptor activation..I. Biol. Chem. 257, 9532-9538, 1982. Kalu. D.N., Hardin, R.R., Murata, 1., Huber+ M.B., and Roos, B.A. Age-dependent modulation of parathyroid hormone action. A~,e 5, 25-29, 1982.
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Kalu, D.N., Cockerham, R., Yu, B.P., and Ross, B.A. Lifelong dietary modulation of calcitonin levels in rats. Endocrinolo~,,y 113, 2010-2016, 1983. Kalu, D.N., Hardin, R.H., Cockerham, R., and Yu, B.P. Aging and dietary modulation of rat skeleton and parathyroid hormone. Endocrinolo~,,y 115, 123%1244, 1984. Keaton, J.A., Banto. J.A., Moore, M.P., Gruel, J.B., and Mayer, G.P. Altered parathyroid response to calcium in hypercalcemic neonatal calves. Endocrinolo~,,y 103, 2161-2167, 1978. Queener, S.F., Bell, N.H., Larson, S.M., Henry. D.P., and Slatopolsky, E. Comparison of the regulation of calcitonin in serum of old and young buffalo rats. J. Endocrinolo:,,y 87, 73-80, 1980. Shamonki, I.M., Frumar, A.M,, Tataryn, I.V., Meldrum, D.R., Davidson, B.H., Parthmore, J.G., Judd, H.L., and Deftos, L.J. Age-related changes of calcitonin secretion in females. J. Clin. Endocrim~l. Metah. 50, 437-439, 1980. Wiske, P.S., Epstein, S., Bell, N.H., Queeener, S.F., Edmondson, J., and Johnston, C.C. Increase in immunoreactive parathyroid hormone with age. N. En~,,I. J. Med. 300, 141%1421, 1979. Wongsurawat, N., Siegel, N.A., and Armbrecht, H.J. Aging and vitamin D metabolism. In: Current Re,~earch on Calcium-Ref,,ulatin~,, Hormones. Cooper, C.W. (Editor), University of Texas Press, Texas, 1986. Wu, W., Pahlavani, M., Richardson. A., and Cheung, H.T. Effect of maturation and age on lymphocyte proliferation induced by A23187 through an interleukin-independent pathway. J. Leukocyte Biol. 38, 531540. 1985.