Pergamon Press

Life Sciences, Vol . 22, pp .2043-2050 Printed in the U .S .A .

UTERINE CYCLIC AMP FORMATION : BIPHASIC EFFECT OF ESTROGEN ON CATECHOLAMINE SENSITIVITY Gilbert A . Riaard aad Catherine S . Chew Department of Physiology, Emory University Atlanta, Georgia 30322 (Received in final form April 21, 1978) Summary Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine . 30 seconds later uteri were frozen in situ and assayed for cyclic AMP and glycogen phosphorylase . The AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50~ of that in non-estrogen-treated controls . Catecholamineinduced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls . At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls . Estrogen regulates uterine ß-adrenergic sensitivity but the time courses of estrogen affects on the cyclic AMP and glycogen phosphorylase response changes are different . Catecholamine-induced uterine cyclic AMP formation is biphasic : suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours . Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24-48 hours of estrogen . It is concluded that estrogen has independent effects on the ß-adrenergic-glycogen phosphorylase activation pathway at two different points ; one prior to cyclic AMP formation and another after cyclic AMP formation .

cyclic

The sensitivity of the uterus to adrenergic agents can be altered by treatment with estrogen or progesterone or by changes in endogenous levels of these steroids (for review see references 1,2,3) . This principle was originally estab lished using uterine motility responses and the results have customarily been interpreted in terms of the adrenergic receptor theory originally proposed by Ahlquist (4) ; interaction of the adrenergic agent with a-receptors causes contraction of uterine smooth muscle ; interaction with ß-receptors causes relaxation or inhibition of contractions . It has frequently been concluded that treatment of the animal with estrogen or progesterone alters the relative dominance of one or the other type of receptor thereby changing the mechanical response of the uterine smooth muscle to adrenergic stimuli . More recently it has been shown that adrenergic activation of uterine glycogen phosphorylase (which has been classified as a ß-adrenergic response) is also regulated by ovarian steroids (5,6,7,8) . Robison, Butcher and Sutherland have proposed the general theory that ß-receptors are intimately associated with the enzyme, adenylate cyclase ; that the interaction of an active ß-adrenergic agonist with the receptor stimulates the adenylate cyclase-catalyzed synthesis of adenosine-3',5'-monophosphate (cyclic AMP) which then acts as a second messenger within the cell to stimulate processes which lead to the typical ß-adrenergic response for the tissue in question (9) . 0300-9653/78/0612-2043$02 .00/0 Copyright © 1978 Pergamon Press

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We have shown that treatment of ovariectomized rats with estrogen for 48 hours potentiates catecholamine-stimulation of uterine cyclic AMP accumulation and glycogen phosphorylase activation (7) . These effects of estrogen can be reversed by appropriate progesterone treatment (8) . In the present paper we report additional aspects of the estrogenic regulation of rat uterine cyclic AMP accumulation and glycogen phosphorylase activation in response to catecholamines . METHODS Sexually mature, female rats, albino, outbred Sprague Dawley derived (COBS, CD) from Charles River Breeding Laboratories, Wilmington, Mass ., were used . The rats were ovariectomized and rested 10 to 14 days prior to further treatment . Estrogen (estradiol benzoate, Progynon, Schering) was injected i .p, or s .c . as indicated. Catecholamines were injected via the jugular vein while the rat was under sodium pentobarbital anesthesia (60 mg/Kg) and uteri were frozen, in situ , 30 seconds later as previously described (10) . Tissues were stored at -60° C until extracted . Uterine extracts were prepared, fractionated and assayed for cyclic AMP as previously described (7) using the extract fractionation method of Murad, et al . (11) and the competitive binding assay of Gilman (12) . Glycogen phosphorylâse (a-1, 4-glucan : orthophosphate glycosyltransferase, EC 2 .4 .1 .1 .) was extracted at vary low temperature and assayed as previously described (10,13) . RESULTS Table 1 shows the effects of isoproterenol and epinephrine on~uterine cyclic AMP and glycogen phosphorylase in ovariectomized rats treated with estrogen for 6 and 48 hours . Phosphorylase activation in response to either catecholamine was enhanced by 48 hours of estrogen pretreatment but after 6 hours of estrogen pretreatment the response was no greater than in the estrogen-deficient controls (Table 1) . Cyclic AMP accumulation also was enhanced at 48 hours but at 6 hours of estrogen treatment cyclic AMP accumulation in response to either catecholamine was only 50$ of that in estrogen-deficient controls (Table 1) . TABLE 1 Effects of catecholamines on rat uterine cyclic AMP and phosphorylase at various times after estrogen treatment . A single intravenous injection of catecholamine was given to ovariectomized rats at various times after estrogen injection (50 ug estradiol benzoate per rat, s .c .) . The uteri were frozen in situ 30 seconds after the catecholamine injection . Levels of uterine cyclic AMP and phosphorylase prior to stimulation with isoproterenol have proven to be quite reproducible in our laboratory and have been reported in previous publications at 4-6 pmoles/mg protein for cyclic AMP and 2-3 umoles Pi/min/mg protein for phosphorylase a activity (7) . PART A, Time After Estrogen Cyclic AMP Phosphorylase

ISOPROTERENOL,

0 Hrs 63

1

2

6 ± 1 PART B, EPINEPHRINE,

(1 ug/Kg)

6 Hrs

48 Hrs

28 ± 2

74 ± 3

6 t 0 .4

12 t 1

(3 .3 ug/Kg)

Time After Estrogen

0 Hrs

6 Hrs

48 Hrs

Cyclic AMP

42 ± 4

24 1 1

54 ± 4

7 ± 1

6 t 2

15 t 1

Phosphorylase

Uteriae Cyclic AMP

901 . 22, No . 22, 1978

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Figure 1 shows a more detailed time course of these effects of estrogen pretreatment on uterine catecholamine sensitivity . The magnitude of the phosphorylase activation response to isoproterenol remained at control levels through 12 hours of estrogen pretreatment . However after 24 and especially 48 hours of éstrogen pretreatment the magnitude of phosphorylase activation occurring in response to isoproterenol was much greater than in the estrogen-deficient controls (Figure 1) . In contrast, the magnitude of the cyclic AMP accumulation in response to isoproterenol at 6 through 24 hours of estrogen pretreatment was markedly reduced below the response in estrogen-deficient controls (Figure 1) . By 48 hours of estrogen pretreatment, however, a reversal had occurred and the cyclic AMP response was significantly greater in the estrogen-pretreated rats than in the controls (Figure 1) .

6

c.m

5

m v~ E

N

120

PF~osphorylase Q

100

4

so

2

.. a _ô

40

o. N

20 O

w Q

3

60 Q v .Û Û

w c

L O 6 12 24 48 J Hours after Estrogen

O

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Figure 1 . Effects of isoproterenol on rat uterine cyclic AMP and phosphorylase at various times after estrogen treatment . Experimental conditions similar to those in table 1 . Dose of isoproterenol was 1 ug/Kg . Times of estrogen treatment as indicated .

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Uterine Cyclic AMP

Vol. 22, No . 22, 1978

Figure 2 shows the results of an experiment designed to determine how soon after the beginning of estrogen treatment suppression of the uterine cyclic AMP response to isoproterenol can be shown . The data show no evidence of any lag period and responsiveness appears to decline steadily to minimum values during the first four hours of estrogen treatment. The earliest statistically significant suppression (p

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Pergamon Press Life Sciences, Vol . 22, pp .2043-2050 Printed in the U .S .A . UTERINE CYCLIC AMP FORMATION : BIPHASIC EFFECT OF ESTROGEN ON CATECHO...
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