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Secretion kinetics of prolactin and growth hormone by mouse anterior pituitaries in long term organ cultures
Life Sciences, Vol. 32, pp. 2103-2106 Printed in the U.S.A.
Pergamon Press
SECRETION KINETICS OF PROLACTIN AND GROWTH HORMONE BY MOUSE ANTERIOR PITUITARIES IN LONG TERM ORGAN CULTURES
Joanne Lopez and Frank Talamantes I Department of Biology, Thimann Laboratories, University of California, Santa Cruz, California
(Received
95064
in final form January 31, 1983)
summary Long-term organ cultures of mouse anterior pituitaries provides a system for generating an enriched starting material for the purification of prolactin and growth hormone. Culturing 1 anterior pituitary per ml in serum-free medium for 12 days resulted in a total yield of 40.9 ~g and 6.6 ~g of prolactin and growth hormone, respectively. This yield represented a 1886% and 18% net increase over the control prolactin and growth hormone content in the the unincubated pituitaries. Secondly, culturing of 1-5 anterior pituitaries per ml did not result in a negative feedback autoregulation in the culture system.
Prolactin (PRL) plays a major role in normal (1) and abnormal (2) mammary gland physiology. The laboratory mouse and rat have been extensively used in studies concerning the hormonal control of mammary gland development and differentiation. The availability of large quantities of highly purified mouse prolactin (mPRL) and rat prolactin (rPRL) would allow one the opportunity to use homologous test systems instead of the commonly used heterologous systems whereby one examines the effects of ovine prolactin (oPRL) on rodent mammary tissues. In the absence of inhibitory hypothalamic control, anterior pituitaries (AP's) in culture secrete large quantities of PRL into the medium. Medium from l o n g - t e r m A P organ cultures has been used as starting material for the purification and characterization of chinchilla prolactin (3), mPRL (4, 5, 6), hamster PRL (5, 7), rPRL (8), mouse growth hokmone (mGH)(5) and human growth hormone (hGH)(9). In these previous studies, little attention was paid to the standardization of conditions required to maximize the total PRL yield. For example, in order to insure maximum PRL output by cultured AP's, one should determine whether or not a critical number of AP's in a given volume of culture medium is necessary in order to prevent the possibility of autoregulation or nutrient depletion effecting the total PRL yield. Furthermore, the possibility exists that the medium derived from these long-term cultures could also serve as an enriched hormonal source for the purification of other AP hormones. In our laboratory, we have observed that medium from long-term iRequests for reprints should be addressed to Dr. Frank Talamantes, Laboratories, University of California, Santa Cruz, California 95064.
0024-3205/83/182103-04503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
Thimann
2104
Prolactin
and Growth H o r m o n e
cultures of AP's can also p r o v i d e the p u r i f i c a t i o n of mGH and mPRL.
Secretion
us w i t h a source of
Vol.
starting
32, No.
18,
material
1983
for
In the p r e s e n t study, we report on the s e c r e t i o n k i n e t i c s of m P R L and mGH from l o n g - t e r m cultures of AP's and d e m o n s t r a t e the u s e f u l n e s s of this s y s t e m for p r o v i d i n g an e n r i c h e d starting m a t e r i a l for the p u r i f i c a t i o n of these h o r mones. Materials
and M e t h o d s
AP Organ Cultures. Organ culture was c a r r i e d out in a - M E M m e d i u m (GIBCO) supplemented with 0.29 m g / m l L-glutamine, 0.035 m g / m l P e n i c i l l i n G and 0.05 mg/ml streptomycin. AP's from C3H/He female mice, 6-8 wks of age, were removed immediately following d e c a p i t a t i o n and e x t e n s i v e l y w a s h e d in culture m e d i a prior to culture. AP's were then t r a n s f e r r e d to 15 × 15 mm D a c r o n rafts supported on stainless steel grids in the center wells of 60 × 15 mm organ culture dishes. C u l t u r e dishes c o n t a i n e d 1-5 AP's per 1 ml of medium. Cultures were m a i n t a i n e d in a w a t e r - j a c k e t e d i n c u b a t o r at 3 7 ° C in an a t m o s p h e r e of 95% 0 2 / 5 % CO 2 for 12 days. Fresh m e d i u m was supplied e v e r y 24 hrs and collected m e d i u m was stored at - 4 ° C until analyzed by r a d i o i m m u n o a s s a y (RIA). U n i n c u b a t e d AP's were h o m o g e n i z e d in 1.0 ml of h o m o g e n i z a t i o n b u f f e r containing 0.15 M NaCl, i0 m M s o d i u m p h o s p h a t e (pH 7.4), 1% T r i t o n X-100, 0.5% s o d i u m d e o x y c h o l a t e and 0.01% s o d i u m azide and then a n a l y z e d b y RIA for PRL and GH content. RIA's. mGH in culture m e d i u m was m e a s u r e d by a h o m o l o g o u s double antibody RIA (i0). m P R L was m e a s u r e d a c c o r d i n g to a m o d i f i c a t i o n of a h o m o l o g o u s double a n t i b o d y RIA p r e v i o u s l y d e v e l o p e d in this l a b o r a t o r y (ii). mPRL and mGH were iodinated by a m o d i f i c a t i o n of the l a c t o p e r o x i d a s e m e t h o d (12). Five ~g of m P R L d i s s o l v e d in i0 ~i of 50 mM sodium p h o s p h a t e (pH 7.5), 0.5 ~ g lact o p e r o x i d a s e in 20 ~i of 0.5 M sodium p h o s p h a t e (pH 7.5) and I00 ng of freshly d i l u t e d H 2 0 2 were added to 1 mCi of carrier free Na125I (Amersham Searle) and allowed to react for 3 m i n u t e s at room temperature, after which the r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 300 ~ i of 0.01 M p h o s p h a t e buffered saline (PBS; pH 7.6) containing 0.15 M EDTA, 0.15 M NaCI and 0.01% sodium azide. Iodinated m P R L and mGH were p u r i f i e d from the reaction mixture as p r e v i o u s l y d e s c r i b e d (ii). Statistics. t w o - w a y analysis
Significant of variance.
d i f f e r e n c e s b e t w e e n groups were determined by A level of P < 0.05 was c o n s i d e r e d significant. Results
C u l t u r i n g 1 A P / m l for 12 days in s e r u m - f r e e m e d i u m results in a total yield of 40.9 ~g and 6.6 ~g of m P R L and mGH, r e s p e c t i v e l y (Table 1). This yield (40.9 ~g) r e p r e s e n t s a 1023% net increase over the initial average PRL content (4 ~g) per u n i n c u b a t e d AP. However, when 5 A P ' s / m l are c u l t u r e d for 12 days, the total PRL yield (377.2 ~g) r e p r e s e n t s a 1886% net increase over the initial average P R L content (20 ~g) in the u n i n c u b a t e d AP's. In contrast to the total PRL yield, the total mGH yield (6.6 ~g) after 12 days result in a net increase of o n l y 18% over the initial mGH content (38 ~g) per u n i n c u b a t e d AP. To d e t e r m i n e if a u t o r e g u l a t i o n was i n f l u e n c i n g the output of P R L / G H by the cultured AP's, an e s t i m a t e d E x p e c t e d V a l u e (EV) was calculated. The E V was d e r i v e d b y m u l t i p l y i n g the o b s e r v e d total P R L / G H y i e l d p r o d u c e d from one AP by the number of AP's/ml. To d e t e r m i n e w h e t h e r the OV was s i g n i f i c a n t l y greater than the EV, d a t a d e r i v e d from c u l t u r i n g 2, S, 4 and 5 AP's were normalized by d i v i d i n g the amount of P R L or GH s e c r e t e d b y the numbers of incubated AP's. If the EV was equal to the O b s e r v e d V a l u e (OV), m a x i m u m hormonal output occurred. A greater O V would indicate e n h a n c e d h o r m o n a l s e c r e t i o n w h e r e a s a s m a l l e r OV w o u l d indicate a d e p r e s s e d h o r m o n a l p r o d u c t i o n under our culture conditions. No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d b e t w e e n P R L or
Vol.
32, No.
18, 1983
Prolactin
and G r o w t h H o r m o n e
Secretion
2105
TABLE I PRL and GH Y i e l d
from C u l t u r e d Mouse A n t e r i o r P i t u i t a r i e s
Total Y i e l d / 1 2 (~g/ml)
Days
PRL Pits/ml
1 2 3 4 5
OV a
40.9 123.6 200.7 229.9 377.2
(n-7)
Aver. Y i e l d / P i t / D a y (~g/ml )
GH E~
81.8 122.7 163.6 204.5
OV
6.6 17.9 26.8 34.3 43.2
EV
13.2 19.8 26.4 33.0
a.
Observed Value
b.
E x p e c t e d Value (EV) d e t e r m i n e d by m u l t i p l y i n g by the number of AP's/ml.
c.
V a l u e s are m e a n ± S.E.M.
PRL c
GH
3.4±0.61 5.2±0.67 5.6±0.60 4.6±0.60 6.3±0.59
0.6±0.15 0.8±0.13 0.7±0.12 0.7±0.12 0.7±0.10
(OV) d e t e r m i n e d b y RIA. the OV p r o d u c e d
from
c u l t u r e conditions. No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d b e t w e e n GH s e c r e t i o n and the number of c u l t u r e d AP's (Table I).
1
AP
PRL
or
Discussion Two salient findings emerge from the p r e s e n t studies. First, long-term culture of mouse AP's p r o v i d e s a s y s t e m for g e n e r a t i n g an e n r i c h e d s t a r t i n g m a t e r i a l for the p u r i f i c a t i o n s of m P R L and to a lesser extent for the purification of mGH. Secondly, c u l t u r i n g of 1-5 AP's/ml did not result in a negative feedback a u t o r e g u l a t i o n in our culture system. The c u l t u r e m e d i u m g e n e r a t e d from l o n g - t e r m cultures of AP's has previously p r o v i d e d us w i t h e x c e l l e n t raw m a t e r i a l for s u b s e q u e n t p u r i f i c a t i o n of a v a r i e t y of secreted rodent PRL's. This system is relatively inexpensive, reliable and p r o v i d e s one w i t h large q u a n t i t i e s of the secreted form of PRL. A l t h o u g h we h a v e found that submerged mouse AP's still p r o d u c e large quantities of mPRL, it has b e e n p r e v i o u s l y d e m o n s t r a t e d that rat A P ' s p r o d u c e more PRL if t h e y are c u l t u r e d on a p l a t f o r m - t y p e of s y s t e m in contrast to c u l t u r i n g them in a submerged culture s y s t e m (13). At the p r e s e n t time, we h a v e not examined the e f f e c t s of d i f f e r e n t types of c u l t u r e m e d i a on m P R L production. Our use of u - M E M m e d i u m was selected on the basis of its p o s i t i v e e f f e c t s on PRLproduction by rat a n t e r i o r p i t u i t a r y cells (14). We h a v e r e c e n t l y o b s e r v e d that in contrast to the P R L yield (40 ~ g / p i t / 1 2 days) from AP's of mice 6-8 wks of age, the yield of P R L is d o u b l e d (80 ~ g / p i t / 1 2 days) when one uses A P ' s from mice of 8 m o n t h s of age. The q u e s t i o n of w h e t h e r P R L is capable of d i r e c t l y inhibiting its own secretion has not yet b e e n d e f i n i t i v e l y answered (15, 16, 17, 18). For example, in vitro p e r f u s i o n of rat p i t u i t a r i e s b y I00 or 200 ng/ml of rat prolactin, have demonstrated inhibition of PRL by the p e r f u s e d p i t u i t a r i e s (17). Furthermore, after the exposure to ovine prolactin, PRL secretion was
2106
P r o l a c t i n and G r o w t h H o r m o n e S e c r e t i o n
Vol.
32, No.
18,
1983
d a t a do not s u p p o r t the h y p o t h e s i s that P R L is c a p a b l e of i n h i b i t i n g its own secretion at the p i t u i t a r y level. In a c c o r d a n c e w i t h our data, o t h e r s t u d i e s h a v e not d e m o n s t r a t e d a P R L f e e d b a c k at the p i t u i t a r y level (8). However, we feel that this issue is not an all or none p h e n o m e n a . F o r example, one m u s t take into a c c o u n t the type of e x p e r i m e n t a l s y s t e m used (e.g., organ culture v e r s u s t i s s u e culture; s t a t i c v e r s u s p e r f u s i o n c u l t u r e s ) and the p h y s i o l o g i c a l state of the a n i m a l s (e.g., p r o e s t r u s v e r s u s estrus; early pregnancy versus late p r e g n a n c y ) that serve as the d o n o r of the AP's. Acknowledgements This w o r k was s u p p o r t e d b y N I H g r a n t s RR08132 and HD14966 to Dr. F. Talamantes. We t h a n k Dr. Y. S i n h a for the s u p p l y of m G H and a n t i s e r u m to mGH. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.
13. 14.
15. 16. 17. 18.
J.J. ELIAS, H o r m o n a l P r o t e i n s and Pe~tides, C.H. Li, p. 37, Academic Press, Inc., N e w Y o r k (1980). C.W. W E L S C H and H. NAGASAWA, C a n c e r Res. 37 9 5 1 - 9 6 3 (1977). M. J I B S O N and F. T A L A M A N T E S , Gen. Comp. Endocrinol. 3_44 4 0 2 - 4 0 7 (1978). L.F. SHOER, N.R. S H I N E and F. T A L A M A N T E S , Biochim. Biophys. A c t a 537 336347 (1978). P. C O L O S I and F. TAI2tMANTES, Arch. Biochem. B i o p h y s . 21__227 5 9 - 7 6 1 (1981). T.A. BEWLEY, P. C O L O S I and F. T A L A M A N T E S , B i o c h e m i s t r y (in p r e s s ) (1982). P. COLOSI, E. M A R K O F F , A. LEVY, L. OGREN, N. SHINE and F. TALAMANTES, E n d o c r i n o l o g y 108 8 5 0 - 8 5 4 (1981). S. FARMER, T.A. BEWLEY, S.M. R U S S E L L and C.S. NICOLL, Biochim. Biophys. A c t a 437 5 6 2 - 5 7 0 (1976). F. T A L A M A N T E S , J. LOPEZ, U.J. L E W I S and C.B. WILSON, A c t a Endocrinologica 98 8-13 (1981). Y.N. SINHA, F.W. SELBY, U.J. L E W I S and W.P. V A N D E R L A A N , Endocrinology 9_~1 7 8 4 - 7 9 2 (1972). E. M A R K O F F , P. C O L O S I and F. T A L A M A N T E S , L i f e Sci. 28 2 0 3 - 2 1 1 (1981). J.I. T H O R E L L and B . G . J O H A N N S O N , Biochim. Biophys. A c t a 251 3 6 3 - 3 6 9 (1971). R.R. GALA, A c t a E n d o c r i n o l . 65 4 6 6 - 4 7 6 (1970). W.W. W I L F I N G E R , J.A. DAVIS, E.C. A U G U S T I N E and W.C. HYMER, Endocrinology 105 5 3 0 - 5 3 6 (1979). D.C. HERBERT, H. I S H I K A W A and E.G. RENNELS, Endocrinology 104 97-100 (1979). S. MELMED, H.E. CARLSON, J. B R I G G S and J.M. HERSHMAN, Horm. Res. 1__2 340344 (1980). H.A. ZACUR, W.E. MITCH, J.E. TYSON, P.T. O S T R O W and G.V. FOSTER, Am. J. Physiol. 242 E 2 2 6 - E 2 3 3 (1982). L. VICIAN, M.E. L I E B E R M A N and J. GORSKI, E n d o c r i n o l o g y ii0 7 2 2 - 7 2 6 (1982).
Pergamon Press
SECRETION KINETICS OF PROLACTIN AND GROWTH HORMONE BY MOUSE ANTERIOR PITUITARIES IN LONG TERM ORGAN CULTURES
Joanne Lopez and Frank Talamantes I Department of Biology, Thimann Laboratories, University of California, Santa Cruz, California
(Received
95064
in final form January 31, 1983)
summary Long-term organ cultures of mouse anterior pituitaries provides a system for generating an enriched starting material for the purification of prolactin and growth hormone. Culturing 1 anterior pituitary per ml in serum-free medium for 12 days resulted in a total yield of 40.9 ~g and 6.6 ~g of prolactin and growth hormone, respectively. This yield represented a 1886% and 18% net increase over the control prolactin and growth hormone content in the the unincubated pituitaries. Secondly, culturing of 1-5 anterior pituitaries per ml did not result in a negative feedback autoregulation in the culture system.
Prolactin (PRL) plays a major role in normal (1) and abnormal (2) mammary gland physiology. The laboratory mouse and rat have been extensively used in studies concerning the hormonal control of mammary gland development and differentiation. The availability of large quantities of highly purified mouse prolactin (mPRL) and rat prolactin (rPRL) would allow one the opportunity to use homologous test systems instead of the commonly used heterologous systems whereby one examines the effects of ovine prolactin (oPRL) on rodent mammary tissues. In the absence of inhibitory hypothalamic control, anterior pituitaries (AP's) in culture secrete large quantities of PRL into the medium. Medium from l o n g - t e r m A P organ cultures has been used as starting material for the purification and characterization of chinchilla prolactin (3), mPRL (4, 5, 6), hamster PRL (5, 7), rPRL (8), mouse growth hokmone (mGH)(5) and human growth hormone (hGH)(9). In these previous studies, little attention was paid to the standardization of conditions required to maximize the total PRL yield. For example, in order to insure maximum PRL output by cultured AP's, one should determine whether or not a critical number of AP's in a given volume of culture medium is necessary in order to prevent the possibility of autoregulation or nutrient depletion effecting the total PRL yield. Furthermore, the possibility exists that the medium derived from these long-term cultures could also serve as an enriched hormonal source for the purification of other AP hormones. In our laboratory, we have observed that medium from long-term iRequests for reprints should be addressed to Dr. Frank Talamantes, Laboratories, University of California, Santa Cruz, California 95064.
0024-3205/83/182103-04503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
Thimann
2104
Prolactin
and Growth H o r m o n e
cultures of AP's can also p r o v i d e the p u r i f i c a t i o n of mGH and mPRL.
Secretion
us w i t h a source of
Vol.
starting
32, No.
18,
material
1983
for
In the p r e s e n t study, we report on the s e c r e t i o n k i n e t i c s of m P R L and mGH from l o n g - t e r m cultures of AP's and d e m o n s t r a t e the u s e f u l n e s s of this s y s t e m for p r o v i d i n g an e n r i c h e d starting m a t e r i a l for the p u r i f i c a t i o n of these h o r mones. Materials
and M e t h o d s
AP Organ Cultures. Organ culture was c a r r i e d out in a - M E M m e d i u m (GIBCO) supplemented with 0.29 m g / m l L-glutamine, 0.035 m g / m l P e n i c i l l i n G and 0.05 mg/ml streptomycin. AP's from C3H/He female mice, 6-8 wks of age, were removed immediately following d e c a p i t a t i o n and e x t e n s i v e l y w a s h e d in culture m e d i a prior to culture. AP's were then t r a n s f e r r e d to 15 × 15 mm D a c r o n rafts supported on stainless steel grids in the center wells of 60 × 15 mm organ culture dishes. C u l t u r e dishes c o n t a i n e d 1-5 AP's per 1 ml of medium. Cultures were m a i n t a i n e d in a w a t e r - j a c k e t e d i n c u b a t o r at 3 7 ° C in an a t m o s p h e r e of 95% 0 2 / 5 % CO 2 for 12 days. Fresh m e d i u m was supplied e v e r y 24 hrs and collected m e d i u m was stored at - 4 ° C until analyzed by r a d i o i m m u n o a s s a y (RIA). U n i n c u b a t e d AP's were h o m o g e n i z e d in 1.0 ml of h o m o g e n i z a t i o n b u f f e r containing 0.15 M NaCl, i0 m M s o d i u m p h o s p h a t e (pH 7.4), 1% T r i t o n X-100, 0.5% s o d i u m d e o x y c h o l a t e and 0.01% s o d i u m azide and then a n a l y z e d b y RIA for PRL and GH content. RIA's. mGH in culture m e d i u m was m e a s u r e d by a h o m o l o g o u s double antibody RIA (i0). m P R L was m e a s u r e d a c c o r d i n g to a m o d i f i c a t i o n of a h o m o l o g o u s double a n t i b o d y RIA p r e v i o u s l y d e v e l o p e d in this l a b o r a t o r y (ii). mPRL and mGH were iodinated by a m o d i f i c a t i o n of the l a c t o p e r o x i d a s e m e t h o d (12). Five ~g of m P R L d i s s o l v e d in i0 ~i of 50 mM sodium p h o s p h a t e (pH 7.5), 0.5 ~ g lact o p e r o x i d a s e in 20 ~i of 0.5 M sodium p h o s p h a t e (pH 7.5) and I00 ng of freshly d i l u t e d H 2 0 2 were added to 1 mCi of carrier free Na125I (Amersham Searle) and allowed to react for 3 m i n u t e s at room temperature, after which the r e a c t i o n was t e r m i n a t e d by the a d d i t i o n of 300 ~ i of 0.01 M p h o s p h a t e buffered saline (PBS; pH 7.6) containing 0.15 M EDTA, 0.15 M NaCI and 0.01% sodium azide. Iodinated m P R L and mGH were p u r i f i e d from the reaction mixture as p r e v i o u s l y d e s c r i b e d (ii). Statistics. t w o - w a y analysis
Significant of variance.
d i f f e r e n c e s b e t w e e n groups were determined by A level of P < 0.05 was c o n s i d e r e d significant. Results
C u l t u r i n g 1 A P / m l for 12 days in s e r u m - f r e e m e d i u m results in a total yield of 40.9 ~g and 6.6 ~g of m P R L and mGH, r e s p e c t i v e l y (Table 1). This yield (40.9 ~g) r e p r e s e n t s a 1023% net increase over the initial average PRL content (4 ~g) per u n i n c u b a t e d AP. However, when 5 A P ' s / m l are c u l t u r e d for 12 days, the total PRL yield (377.2 ~g) r e p r e s e n t s a 1886% net increase over the initial average P R L content (20 ~g) in the u n i n c u b a t e d AP's. In contrast to the total PRL yield, the total mGH yield (6.6 ~g) after 12 days result in a net increase of o n l y 18% over the initial mGH content (38 ~g) per u n i n c u b a t e d AP. To d e t e r m i n e if a u t o r e g u l a t i o n was i n f l u e n c i n g the output of P R L / G H by the cultured AP's, an e s t i m a t e d E x p e c t e d V a l u e (EV) was calculated. The E V was d e r i v e d b y m u l t i p l y i n g the o b s e r v e d total P R L / G H y i e l d p r o d u c e d from one AP by the number of AP's/ml. To d e t e r m i n e w h e t h e r the OV was s i g n i f i c a n t l y greater than the EV, d a t a d e r i v e d from c u l t u r i n g 2, S, 4 and 5 AP's were normalized by d i v i d i n g the amount of P R L or GH s e c r e t e d b y the numbers of incubated AP's. If the EV was equal to the O b s e r v e d V a l u e (OV), m a x i m u m hormonal output occurred. A greater O V would indicate e n h a n c e d h o r m o n a l s e c r e t i o n w h e r e a s a s m a l l e r OV w o u l d indicate a d e p r e s s e d h o r m o n a l p r o d u c t i o n under our culture conditions. No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d b e t w e e n P R L or
Vol.
32, No.
18, 1983
Prolactin
and G r o w t h H o r m o n e
Secretion
2105
TABLE I PRL and GH Y i e l d
from C u l t u r e d Mouse A n t e r i o r P i t u i t a r i e s
Total Y i e l d / 1 2 (~g/ml)
Days
PRL Pits/ml
1 2 3 4 5
OV a
40.9 123.6 200.7 229.9 377.2
(n-7)
Aver. Y i e l d / P i t / D a y (~g/ml )
GH E~
81.8 122.7 163.6 204.5
OV
6.6 17.9 26.8 34.3 43.2
EV
13.2 19.8 26.4 33.0
a.
Observed Value
b.
E x p e c t e d Value (EV) d e t e r m i n e d by m u l t i p l y i n g by the number of AP's/ml.
c.
V a l u e s are m e a n ± S.E.M.
PRL c
GH
3.4±0.61 5.2±0.67 5.6±0.60 4.6±0.60 6.3±0.59
0.6±0.15 0.8±0.13 0.7±0.12 0.7±0.12 0.7±0.10
(OV) d e t e r m i n e d b y RIA. the OV p r o d u c e d
from
c u l t u r e conditions. No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d b e t w e e n GH s e c r e t i o n and the number of c u l t u r e d AP's (Table I).
1
AP
PRL
or
Discussion Two salient findings emerge from the p r e s e n t studies. First, long-term culture of mouse AP's p r o v i d e s a s y s t e m for g e n e r a t i n g an e n r i c h e d s t a r t i n g m a t e r i a l for the p u r i f i c a t i o n s of m P R L and to a lesser extent for the purification of mGH. Secondly, c u l t u r i n g of 1-5 AP's/ml did not result in a negative feedback a u t o r e g u l a t i o n in our culture system. The c u l t u r e m e d i u m g e n e r a t e d from l o n g - t e r m cultures of AP's has previously p r o v i d e d us w i t h e x c e l l e n t raw m a t e r i a l for s u b s e q u e n t p u r i f i c a t i o n of a v a r i e t y of secreted rodent PRL's. This system is relatively inexpensive, reliable and p r o v i d e s one w i t h large q u a n t i t i e s of the secreted form of PRL. A l t h o u g h we h a v e found that submerged mouse AP's still p r o d u c e large quantities of mPRL, it has b e e n p r e v i o u s l y d e m o n s t r a t e d that rat A P ' s p r o d u c e more PRL if t h e y are c u l t u r e d on a p l a t f o r m - t y p e of s y s t e m in contrast to c u l t u r i n g them in a submerged culture s y s t e m (13). At the p r e s e n t time, we h a v e not examined the e f f e c t s of d i f f e r e n t types of c u l t u r e m e d i a on m P R L production. Our use of u - M E M m e d i u m was selected on the basis of its p o s i t i v e e f f e c t s on PRLproduction by rat a n t e r i o r p i t u i t a r y cells (14). We h a v e r e c e n t l y o b s e r v e d that in contrast to the P R L yield (40 ~ g / p i t / 1 2 days) from AP's of mice 6-8 wks of age, the yield of P R L is d o u b l e d (80 ~ g / p i t / 1 2 days) when one uses A P ' s from mice of 8 m o n t h s of age. The q u e s t i o n of w h e t h e r P R L is capable of d i r e c t l y inhibiting its own secretion has not yet b e e n d e f i n i t i v e l y answered (15, 16, 17, 18). For example, in vitro p e r f u s i o n of rat p i t u i t a r i e s b y I00 or 200 ng/ml of rat prolactin, have demonstrated inhibition of PRL by the p e r f u s e d p i t u i t a r i e s (17). Furthermore, after the exposure to ovine prolactin, PRL secretion was
2106
P r o l a c t i n and G r o w t h H o r m o n e S e c r e t i o n
Vol.
32, No.
18,
1983
d a t a do not s u p p o r t the h y p o t h e s i s that P R L is c a p a b l e of i n h i b i t i n g its own secretion at the p i t u i t a r y level. In a c c o r d a n c e w i t h our data, o t h e r s t u d i e s h a v e not d e m o n s t r a t e d a P R L f e e d b a c k at the p i t u i t a r y level (8). However, we feel that this issue is not an all or none p h e n o m e n a . F o r example, one m u s t take into a c c o u n t the type of e x p e r i m e n t a l s y s t e m used (e.g., organ culture v e r s u s t i s s u e culture; s t a t i c v e r s u s p e r f u s i o n c u l t u r e s ) and the p h y s i o l o g i c a l state of the a n i m a l s (e.g., p r o e s t r u s v e r s u s estrus; early pregnancy versus late p r e g n a n c y ) that serve as the d o n o r of the AP's. Acknowledgements This w o r k was s u p p o r t e d b y N I H g r a n t s RR08132 and HD14966 to Dr. F. Talamantes. We t h a n k Dr. Y. S i n h a for the s u p p l y of m G H and a n t i s e r u m to mGH. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12.
13. 14.
15. 16. 17. 18.
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