Estrogenic regulation of uterine cyclic amp metabolism

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Biochimica et Biophysica Acta, 362 (1974) 493--500 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27506

ESTROGENIC R E G U L A T I O N OF UTERINE CYCLIC AMP METABOLISM*

CATHERINE S. CHEW and GILBERT A. R I N A R D * *

Department of Physiology, Emory University, Atlanta, Ga. 30322 (U.S.A.) (Received April 16th, 1974)

Summary Ovariectomized and ovariectomized, estrogen-treated (48 h) rats were injected intravenously with increasing doses of epinephrine. Uteri were frozen in situ 30 s later. Estrogen pre-treatment significantly increased the sensitivity of both cyclic AMP and phosphorylase to epinephrine. The cyclic AMP response to intravenous injection of the pure ~-agonist, isoproterenol, was enhanced by estrogen pre-treatment (48 h) and the cyclic AMP response of isolated uteri treated with epinephrine in vitro was also enhanced by in vivo estrogen pre-treatment (48 h). Other groups of ovariectomized rats were treated with estrogen and cyclic AMP levels were estimated at various times after estrogen treatment. 6 h after intraperitoneal injection and 48 h after subcutaneous injection, estrogen caused 20 and 30% increases in cyclic AMP. Estrogen had no effect on cyclic AMP 30 s after intravenous injection or 15 min after intraperitoneal injection. There was also no change in uterine catecholamine sensitivity 30 s after intravenous estrogen injection. The uterine site(s) at which estrogen acts to alter uterine cyclic AMP metabolism could be uterine fl-adrenergic receptors, adenyl cyclase, and/or phosphodiesterase.

Introduction Leonard and Crandall [1] and Diamond and Brody [2] have shown that epinephrine rapidly activates uterine phosphorylase (a-l, 4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1) and that the response is greater in estrogen-treated tissues than in ovariectomized, untreated controls. Dobbs and Robison [3] and Polacek et al. [4] have shown that epinephrine increases

* Publication No. 1205, Division of Basic Health Sciences, E m o r y University. ** Please address reprint requests to: Dr Gilbert A. Rinard, Department of Physiology, E m o r y University, Atlanta, Georgia 30322, U.S.A.

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uterine cyclic AMP and that the response is blocked by fi-adrenergic blocking agents [5]. Epinephrine-induced activation of uterine phosphorylase is also considered to be a fi-adrenergic effect [6]. Acute estrogen treatment, without epinephrine treatment, has also been reported to increase uterine cyclic AMP [7] and adenyl cyclase activity [8]. These reports have led some investigators to postulate that estrogen action is mediated directly by cyclic AMP [9], indirectly by cyclic AMP subsequent to the release and action of catecholamines [8,10], or by a mechanism which does not involve cyclic AMP [11,12]. The present study offers evidence that estrogen does have effects on uterine cyclic AMP metabolism. However, we agree with reports by Sanborn et al. [11] and Zor et al. [12] that estrogen does not cause an acute elevation of cyclic AMP as reported by Szego and Davis [7] and others [8]. Instead, we find that long term estrogen action induces a small but reproducible elevation in basal cyclic AMP levels. In addition, estrogen sensitizes the uterus to epinephrine stimulation in such a way that epinephrine-induced increases in both cyclic AMP and phosphorylase a are greater after long-term estrogen treatment. Materials and Methods Sexually mature, female rats, purchased from Charles River Breeding Laboratories, Wilmington, Mass., were ovariectomized prior to hormone treatment. Hormones used included progynon benzoate (Schering), epinephrine (adrenalin chloride, Parke-Davis), isoproterenol (isuprel. HC1, Winthrop Labs), and free estradiol (Schering). Estradiol solutions for intravenous injections were prepared according to the method of Roberts and Szego [13]. Rats were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally). Epinephrine and isoproterenol were injected intravenously into the jugular vein of anesthetized animals. Uteri were rapidly frozen in situ as described previously [14] and stored at --60°C until extraction. For in vitro experiments, uteri were rapidly excised and hung in an oxygenated (O: ,--CO: ; , 95%: 5%) equilibration chamber containing Krebs-Ringer bicarbonate buffer with 200 mg % glucose, pH 7.4, 37 ° C. Before uterine excision, loops of surgical silk (DeKnatel, 3--0) were sutured to the tips of the uterine horns and glass beads weighing approximately 1.1 g were sutured to each horn just above the uterine birufication. Uterine horns were hung on glass hooks and suspended in the equilibration chamber for 30 min. Each uterine horn was then transferred to a smaller oxygenated chamber containing the same Krebs--Ringer bicarbonate buffer at 37°C and allowed to re-equilibrate for 5 min before epinephrine treatment. 2 min after epinephrine was added to the chamber, the uterine horn was removed, immediately frozen between aluminum tongs pre-cooled in liquid N2, and stored at --60°C until extraction. Cyclic AMP was assayed by the Gilman competitive protein binding method [15]. Binding protein was prepared from fresh New Zealand rabbit skeletal muscle by the method of Walsh et al. [16], except that two peaks were eluted from the DEAE-cellulose column with 0.03 M and 0.1 M phosphate buffers. The second peak was used as the source of cyclic AMP binding protein. Frozen uterine tissue was prepared for cyclic AMP assay as follows. The tissue

495 was pulverized with a percussion m o r t a r at liquid N2 t e m p e r a t u r e [ 1 4 ] , weighed in a --10°C freezer and rapidly homogenized in 3 ml of cold 5% fluorometric grade trichloroacetic acid (Harleco) which had been previously labeled with a 0.5 nM c o n c e n t r a t i o n of 3 H-labeled cyclic AMP (Schwarz, 28 Ci/mmole). The labeled h o m o g e n a t e was centrifuged at 36 000 X g for 10 min. The precipitate was saved for protein analysis [17] and the supernatant was brought to 0.1 M HC1, e xt r act ed eight times with twice its volume of anesthesia-grade water saturated diethyl ether (Mallinckrodt), then" evaporated to dryness at 40°C on an Evapomix (Buchler Instruments, Fort Lee, N.J.). The evaporated e x t r a c t was brought to a final c on cent rat i on of 100 mg tissue wet weight equivalent/ml with 0.05 M sodium acetate buffer (pH 4.0). Recovery of added label at this poi nt was 75--85%. Part o f each extract was f ur t her purified on a 0.5 cm X 3 cm Dowex AG 1-X 8 f o r mate (200--400 mesh) column (Bio-Rad) by the m e t h o d of Murad et al. [ 1 8 ] . Each fractionated ext r a c t was evaporated and again brought to a c o n c en tr atio n of 100 mg tissue wet weight equivalent/ml with 0.05 M sodium acetate buffer. Comparison of fractionated and unfractionated extract with paper c h r o m a t o g r a p h y [19] dem ons t r a t e d that fractionation removed all detectable nucleotide contaminants except 5'-AMP which has been shown n o t to interfere with the cyclic AMP assay ( [ 1 5 ] , and Chew, C.S. and Rinard, G.A., unpublished). Labeled cyclic AMP was purified before use on Dowex 1-X 8 formate columns because it was f o u n d to contain adenosine and adenine. Overall recovery from homogenization through col um n purification was

70--85%. In routine assays, each tube contained, in a final volume of 0.2 ml, 1 pmole 3 H-labeled cyclic AMP, sufficient cyclic AMP binding protein to bind approximately 40% of total counts, and i00 pl or 25 pl (catecholamine-stimufated) aliquots of fractionated tissue extract. All components except binding protein were made up in 50 mM sodium acetate buffer, pH 4.0. Standard curves were obtained by linear regression. Results

Cyclic AMP assay validation Because o f the presence of an inhibitor in the diethyl ether washed trichloroacetic acid-treated uterine extracts, it was necessary to fractionate the extracts on Dowex AG 1-X 8 formate columns (see Materials and Methods). When extracts were treated in this manner, a linear relationship over a concentration range o f 5--30 mg tissue wet weight equivalent per assay tube was obtained. Recovery of cyclic AMP added to tissue extracts was 99 + 6% over this c o n c e n t r a t i o n range. The coefficient of variation (standard deviation expressed as a percentage of the mean) between assays for a 100 pm ol e/ m l tissue e x t r act was 7%. The within assay coefficient of variation for the same extract randomized in f o u r places t h r o u g h o u t the assay was 6%. Sensitivity, defined as two standard deviations (S.D.) greater than zero, was 0.05--0.2 pmoles. Precision, defined as h = S.D. X 100/slope was usually 0.1 with a range of 0.05--0.2. All apparent cyclic AMP activity was dest royed by incubation with cyclic nucleotide phosphodiesterase. A phosphodiesterase reaction mixture containing

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tissue cyclic AMP activity (150 pmole cyclic AMP/ml, 300 mg tissue equivalent/ml) and phosphodiesterase (1 mg/ml, Sigma Co.) was incubated for 4 h at 30°C in 0.08 M Tris, 1.3 mM MgC12, pH 7.5 and then boiled for 10 min. When aliquots (25 pl) of this phosphodiesterase-treated extract were added to each point of the assay standard curve, the standard curve was not altered in any way. Increasing the assay concentration of phosphodiesterase-treated extracts from 2.5 mg per assay tube to 10 mg per assay tube had no effect on these results.

Effect of estrogen on epinephrine stimulation of cyclic AMP and phosphorylase An experiment was designed to study the effect of varying doses of epinephrine on uterine cyclic AMP and phosphorylase in ovariectomized and ovariectomized, estrogen-treated rats. A group of 2 5 0 - - 3 0 0 g Charles River rats were ovariectomized and allowed to recover for 10 days. Half the animals were injected subcutaneously with 50 pg estradiol benzoate (0.1 ml in peanut oil). 48 h later the rats were anesthetized and injected intravenously with epinephrine. 30 s after that uteri were rapidly frozen in situ as described previously [141. Fig. 1 shows that the administration of epinephrine to estrogen-treated animals caused a greater increase in cyclic AMP than was observed in ovariectomized controls treated with epinephrine alone. Fig. 2 demonstrates that uterine phosphorylase activation in response to epinephrine was also greater after estrogen pre-treatment. Total phosphorylase activity was not significantly altered under these conditions. Estrogen treatment increased the sensitivity of both cyclic AMP and phosphorylase a to epinephrine. In addition to the in vivo experiment, an in vitro experiment was performed to rule out the possibility that alterations in uterine blood flow caused by estrogen and epinephrine were responsible for our observation that prior

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497

TABLE I EFFECT OF CATECHOLAMINES TROL ANIMALS

ON UTERINE CYCLIC AMP IN ESTROGEN-TREATED

AND CON-

H a l f of t w o g r o u p s o f o v a r i e c t o m i z e d r a t s w e r e i n j e c t e d s u b c u t a n e o u s l y w i t h e s t r a d i o l b e n z o a t e ( 5 0 p g / r a t , s u b c u t a n e o u s l y ) . 48 h later all t h e a n i m a l s in t h e first g r o u p w e r e i n j e c t e d w i t h i s o p r o t e r e n o l (1 .ug/ k g , i n t r a v e o u s l y ) a n d t h e u t e r i w e r e f r o z e n in s i t u 30 s later. A n i m a l s in t h e s e c o n d g r o u p w e r e a n e s t h e t i z e d 48 h a f t e r e s t r o g e n t r e a t m e n t . T h e u t e r i w e r e e x c i s e d , a l l o w e d to e q u i l i b r a t e in o x y g e n a t e d K r e b s - R i n g e r b i c a r b o n a t e b u f f e r f o r 30 m i n , t r a n s f e r r e d to a n o x y g e n a t e d t r e a t m e n t c h a m b e r c o n t a i n i n g t h e s a m e b u f f e r a n d a l l o w e d to r e - e q u i l i b r a t e f o r 5 rain. E p i n e p h r i n e (3 X 10 -7 M) w a s a d d e d to t h e c h a m b e r . 2 m i n l a t e r t h e u t e r i w e r e r e m o v e d a n d r a p i d l y f r o z e n w i t h a l u m i n u m t o n g s p r e - c o o l e d in l i q u i d N2. Uterine cyclic AMP ( p m o l e s / m g protein)*

I s o p r o t e r e n o l , in vivo E p i n e p h r i n e , in v i t r o

Control

Estrogen

26.6 + 2.36 (11) 2 7 . 4 -+ 5 . 1 5 (4)

4 2 . 6 + 2 . 3 3 * * (11) 57.9 +- 8.01 t (4)

* M e a n -+ S.E., n u m b e r o f o b s e r v a t i o n s i n d i c a t e d w i t h i n p a r e n t h e s e s . ** P < 0 . 0 0 1 , E s t r o g e n s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l . ¢ P < 0.05, Estrogen significantly different from control

treatment with estrogen enhances epinephrine-stimulated increases in cyclic AMP. Half of a group of ovariectomized rats were treated with estrogen (50 pg/rat, subcutaneously). 48 h Later the rats were anesthetized, the uteri were excised and treated as described in Materials and Methods. Table I shows that estrogen pre-treatment also enhances the increases in cyclic AMP which occur after in vitro epinephrine stimulation. Because an acute elevation of uterine cyclic AMP within 30 s after intravenous injection of estrogen has been reported [ 7 ] , another experiment was performed to determine whether estrogen acutely enhances epinephrineinduced cyclic AMP formation and phosphorylase activation. Ovariectomized rats were anesthetized and injected intravenously with either estradiol (5 pg/kg) or vehicle. Epinephrine (4 pg/kg) was administered intravenously 30 s after estradiol. There was a large increase (10 X ) in uterine cyclic AMP over baseline

T A B L E II EFFECT OF ESTROGEN ALONE ON UTERINE CYCLIC AMP O v a r i e c t o m i z e d r a t s w e r e i n j e c t e d i n t r a v e n o u s l y w i t h e s t r a d i o l (5 p g / r a t ) or v e h i c l e f o r t h e 30 s e x p e r i m e n t , i n t r a p e r i t o n e a l l y w i t h e s t r a d i o l (5 p g / r a t ) or vehicle f o r t h e 15 m i n a n d 6 h e x p e r i m e n t s , a n d subc u t a n e o u s l y w i t h e s t r a d i o l b e n z o a t e ( 5 0 p g / r a t ) f o r t h e 48 h e x p e r i m e n t . U t e r i w e r e f r o z e n in s i t u a f t e r the appropriate interval of time. Time after estrogen

Uterine cyclic AMP ( p m o l e s / m g protein)* Control

30 s 15 m i n 6h 48 h

4.26 4.24 4.23 4.13

-+ 0 . 5 2 -+ 0 . 3 0 +- 0 , 3 2 +- 0 . 2 7

Estrogen (6) (7) (15) (8)

* M e a n -+ S.E., n u m b e r o f o b s e r v a t i o n s i n d i c a t e d w i t h i n p a r e n t h e s e s . ** P < 0 . 0 5 , e s t r o g e n s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l . t P < 0.01, estrogen significantly different from control.

4.26 4.54 5.29 5.39

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498 levels observed in other experiments (Table II). However, there was no difference in cyclic AMP accumulation between estrogen-treated animals and controls (41.0 + 3.6 and 39.3 + 2.5 pmoles/mg protein, respectively). Phosphorylase a and total phosphorylase activities were also the same in estrogentreated and control animals.

Effect of estrogen on isoproterenol-induced cyclic AMP accumulation Since epinephrine is known to have both a- and ~-adrenergic action, the effect of the pure ~-agonist, isoproterenol, on uterine cyclic AMP was examined. Half of a group of ovariectomized rats were treated with estradiol benzoate (50 pg/rat, subcutaneously). 48 h later, all rats were treated with isoproterenol (1 pg/kg, intravenously). Table I indicates that estrogen significantly enhanced the cyclic AMP response to isoproterenol. Effect of estrogen alone on cyclic AMP Since acute estrogen treatment failed to potentiate epinephrine-stimulated increases in cyclic AMP, the effect of acute estrogen in the absence of exogenous epinephrine, on uterine cyclic AMP accumulation was re-investigated. The effect of long-term estrogen action on cyclic AMP was also investigated. Table II shows that estrogen did not cause an acute elevation of cyclic AMP. 30 s after estradiol (5 pg/rat, intravenously) cyclic AMP levels were unaltered. Likewise, 15 min after estradiol (5 pg/rat, intraperitoneally) there was no change in cyclic AMP. Estrogen did, however, cause an increase in cyclic AMP after a longer period of time. 6 and 48 h after treatment with estradiol (5 pg/rat, intraperitoneally) and estradiol benzoate (50 pg/rat, subcutaneously) respectively, small but significant increases in cyclic AMP were observed (Table II). Discussion These results show that estrogen enhances catecholamine-induced increases in uterine cyclic AMP and phosphorylase, both in vivo and in vitro. In the absence of exogenous catecholamines, estrogen increases uterine cyclic AMP and phosphorylase activity [20]. Hormones other than estrogen have been reported to alter tissue sensitivity to catecholamines. H y p o p h y s e c t o m y caused adipose tissue phosphorylase to become insensitive to epinephrine stimulation. Sensitivity was almost completely restored by a combination of tri-iodothyronine and growth hormone [21]. Similarly, phosphorylase activation by epinephrine and glucagon was impaired in isolated, perfused livers obtained from adrenalectomized rats. Glucocorticoid treatment restored the activation of phosphorylase to normal. Similar responses were seen when gluconeogenesis was studied. Stimulation of lipolysis by epinephrine was also impaired in adipose tissue from adrenalectomized rats [22] and hypophysectomized rats [21]. In these cases, the impaired catecholamine sensitivity was associated with a decreased sensitivity to cyclic AMP rather than a decreased ability to form cyclic AMP. Therefore, these observations on the mechanism by which hypophyseal and adrenal hormones alter the sensitivity of liver and adipose tissue to catecholamines are different

499

from the observations we have made on uterine tissue. In the rat uterus, estrogen alters the catecholamine sensitivity of some part of the system which controls cyclic AMP formation. When the uterus is under estrogen domination, epinephrine administered in vivo or in vitro stimulates greater uterine cyclic AMP formation and phosphorylase activation than it does in uteri from ovariectomized animals. Although estrogen causes only small increases in cyclic AMP in comparison to the many-fold increases induced by some other hormones, maximal physiological responses to certain of these hormones have been associated with 20--40% increases in cyclic AMP [23]. It is possible that there is a physiologically inactive " p o o l " of cyclic AMP in uterine tissue which contributes to the high control value. The small increases in cyclic AMP which are caused by estrogen could be a result of a stimulation of the production of a free, active form of the nucleotide. A small increase in uterine phosphorylase activity after long-term action of estrogen has also been shown to occur [20]. This observation can be taken as supporting evidence that estrogen causes increases in cyclic AMP. In addition, Thomas et al. [9] have shown that 4 h of estrogen treatment significantly stimulated uterine conversion of [3 H] adenosine to 3H-labeled cyclic AMP. This suggests that uterine cyclic AMP may be elevated as early as 4 h after estrogen treatment. Although only whole uteri were used in these experiments, the changes in uterine cyclic AMP caused by estrogen (and catecholamine) administration most likely occur in myometrial tissue because: (1), the non-pregnant rat m y o m e t r i u m makes up only a small fraction of the whole uterus [24] and (2), Bhalla et al. [25] have shown that isoproterenol-stimulated increases in cyclic AMP are the same in isolated myometrial strips from ovariectomized rats as they are in whole castrate uteri. Acknowledgements We are grateful to Ann Hunter Adams for her technical assistance. This research was supported by National Institutes of Health Grant HD 06489 and N.I.H. Contract No. 69-2130. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

L e o n a r d , S.L. a n d C r a n d a l l , M. ( 1 9 6 3 ) E n d o c r i n o l o g y 73, 8 0 7 - - 8 1 5 D i a m o n d , J . a n d B r o d y , T.M. ( 1 9 6 5 ) B i o c h e m . P h a r m a e o l . 14, 7 - - 1 6 Dobbs, J.M. and Robison, G.A. (1968) Fed. Proc. 27, 352 P o l a c e k , I., B o l a n , J. a n d D a n i e l , E.E. ( 1 9 7 1 ) C a n . J. P h y s i o l . P h a x m a c o l . 4 9 , 9 9 9 - - 1 0 0 4 P o l a c e k , I. a n d D a n i e l , E.E. ( 1 9 7 1 ) C a n . J. P h y s i o l . P h a x m a c o l . 4 9 , 9 8 8 - - 9 9 8 D i a m o n d , J. a n d B r o d y , T.M. ( 1 9 6 5 ) J . P h a r m a c o l . E x p . T h e r . 1 5 2 , 2 0 2 - - 2 1 1 S z e g o , C.M. a n d Davis, J . S . ( 1 9 6 8 ) P r o c . N a t l . A c a d . Sci. U.S. 58, 1 7 1 1 - - 1 7 1 8 R o s e n f e l d , M.G. a n d O ' M a l l e y , B.W. (l 9 7 0 ) S c i e n c e 1 6 8 , 2 5 3 - - 2 5 5 T h o m a s , J . A . , C z a p , B., L i n g , C.M. a n d S i n g h a l , R . L . ( 1 9 7 2 ) H o r m . M e t a b . Res. 4, 3 1 3 - - 3 1 4 S z e g o , C.M. a n d Davis, J . S . ( 1 9 6 9 ) Molec. P h a r m a c o l . 5, 4 7 0 - - 4 8 0 S a n b o r n , B.M., BhaUa, R . C . a n d K o r e n m a n , S.G. ( 1 9 7 3 ) E n d o c r i n o l o g y 9 2 , 4 9 4 - - 4 9 9 Z o r , U., K o c h , Y., L a m p r e c h t , S . A . , A u s h e r , J. a n d L i n d e r , H . R . ( 1 9 7 3 ) J. E n d o c r i n o l . 58, 5 2 5 - - 5 3 3 R o b e r t s , S. a n d S z e g o , C.M. ( 1 9 4 7 ) E n d o c r i n o l o g y 4 0 , 7 3 - - 8 5 Rinard, G.A. (1972) Biochim. Biophys. Acta 286,416--425 G i l m a n , A . G . ( 1 9 7 0 ) P r o c . N a t l . A c a d . Sci. U.S. 6 7 , 3 0 5 - - 3 1 2

500 16 17 18 19 20 21 22 23

Walsh, D.A., Perkins, J.P. and Krebs, E.G. (1968) J. Biol. Chem. 243, 3763--3765 Lowry, O.H., Rosehrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 Murad, F., Manganieno, V. and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S. 68, 736--739 Osnes, J., Christofferson, T., MCreland, J. and Oye, I. (1972) J. Chromatogr. 67, 139--147 Rinard, G.A. and Chew, C.S. (1974) Endocrinology 94, 1621--1627 Hellman, D.E.W., Eisen, H.J. and Goodman, H.M. (1971) Horm. Metab. Res. 3, 331--335 Friedmann, N., Exton, J.H. and Park, C.R. (1967) Biochem. Biophys. Res. Commun. 29, 113--119 Robison, G.A., Butcher, R.W. and Sutherland, E.W. (1971) Cyclic AMP, p. 31, Academic Press, New York 24 Allen, W.M. (1931) Anat. Rec. 48~ 65--91 25 Bhalla, R.C., Sanborn~ B.M. and Korenman, S.G. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3761--3764