CORRELATION OF PROSTAGLANDIN E, RECEPTOR BINDING WITH EVOKED UTERINE CO@.CRACTION:

MODIFICATION BY DISULFIDE REDUCTION 1

M. Johnson, R. Jessup, S. Jessup and P .W. Ramwell

Department of Physiology & Biophysics Georgetown University

Washington, D , C . 20007

ABSTRACT

Both reversible (K3= 0.72 x lo* l/mole) and irreversible (K = 0.62 x lo* mole) binding of PGEI-H was observed in rat uterus and was correlated with induced myometrial contraction. Each binding process consisted of a fast and slow component. In the binding that occurred prior to the onset of the uterine response (~60 set) , the fast reversible component (T u 5 set) represented N 80%, the slow reversible component (T v 27 sei) represented 15% and the remaining 5% was associated with the corn 8. ined fast and slow irreversible components (Tt * 24 afd 155 set respectively), Reversible and irreversible binding were PGEl-H concentration-dependent, sensitive to competition by PGEl and modified by inhibitors of prostaglandin-induced uterine contraction. Thij modification was reflected as a significant reduction in the velocity of PGEI-H binding. Only the above characteristics asso- ciated with reversible PGE I binding fully satisfied the physiological require- ments of receptor interaction. In addition, a critical role for disulfide groups in the receptor binding site was indicated.

’ Supported by Office of Naval Research Contract # 3287-888.

Accepted ?Iny 10

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INTRODUCTION

Prostaglandin binding has been demonstrated in uterus (1.2, 2a) and in other tissues (3-9). However, the physiological significance of this binding is not apparent due to a lack of correlation with induced responses in the same cell type. Prostaglandin-induced contraction of the isolated rat uterus is characterized by (i) a rapid onset (6 60 se@, (ii) a rapid recovery upon washing and (iii) a reproducible and physio- logical dose-response relationship, Logically, the prostaglandin receptor interaction directly associated with uterine contraction must reflect all of these properties. This communication correlates prostaglandin binding to uterine contraction and elucidates the mode of action of some inhibitors.

MATERIALS AND METHODS

Radioactive prostaglandin El (PGEl-5.6 H3, 59 Ci/mmol; Amersham Searle) was verified by thin layer chromatography (10) and smooth muscle bioassay, Non-radioactive prostaglandins were a gift from the Upjohn Company. ‘I-oxa-prostynoic acid was generously supplied by Dr. J. Fried, University of Chicago. Dithiothreitol, 5,5l dithiobis-(2-nitrobenzoic acid) and p-hydroxymercuribenzoic acid were obtained from the Sigma Chemical Company. Solutions of thio compounds were prepared fresh daily in De Jalon’s Solution (11) containing 10 pg/mI indomethacin (1) (D-J-I medium). The sodium salt of DTNB was prepared by reacting the acid with an equivalent amount of NaHC03. A low pressure mercury discharge lamp (Pen Ray #llSC-2) was used as the source of U/V light (253.7 nm).

Female virgin Sprague-Dawley rats (120-140 g) tween 9-11 a.m., 14 days after bilateral ovariectomy . excised, trimmed and immersed in D-J-I medium.

Uterine Contraction

were sacrificed be- Uterine horns were

A 1 cm segment was suspended in a lucite bath containing 0.5 ml of aerated D-J-I medium, pH 7.4 at 21°C as previously described (11). Tissues were subjected to a tension of 300 mg and contractions were measured isoto- nically with a linear motion transducer (Phipps and Bird. > Uterine horns were cycled at 5 min intervals which included an agonist contact time of 90 sec. Indomethacin does not modify uterine responses to prostaglandins (lla> .

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ANALYSIS OF BINDING

The following terms as used in this manuscript are defined:

1. PGE,-H3 receptor binding: Binding that is sensitive to competition by unlabeled PGEl and can be directly correlated with subsequent evoked uterine contraction.

2. PGE,-H3 non-receptor binding Binding which cannot be correlated with evoked uterine contraction and is not sensitive to competition by unlabeled PGE 1.

3. Reversible binding: Binding which may be reversed by simple dilution (washing),

4. Irreversible binding: Binding which is resistant to prolonged washing.

Reversible Binding in Intact Uterus

Intact uterine horns from the same animal were preincubated for 60 min at room temperature (21’C) in D-J-I medium (1.0 ml) in the presence or absence of inhibitors. Uteri were then immersed in 200 ~1 of D-J-I medium containing PGE all readily avai able binding sites. 4

-H3 (0.16 pCi/ml) for 60 min. This results in saturation of One horn was superfused (5 ml D-J-I

medium at 1 ml/min) to remove any reversibly-bound prostaglandin, and expose for relabeling those sites associated with reversible binding. Both horns were blotjed dry and immersed in 200 pl of D-J-I medium (21OC) con- taining PGEl-H (1.0 - 5.0 ng/ml) for varying time intervals (5-120 set) to relabel the exposed sites. Following removal of the tissue, the degree of PGEl-H3 binding was determined by analysis of the prostaglandin concentration in the medium, Errors due to diffusion and/or entrappment are minimized by this technique,and the extracellular water space in ovariectomized uteri under these conditions remains constant, Differences between the binding of PGEl-H3 by superfused (washed) and unwashed tissues represents the reversibly bound prostaglandin. Estimations using this technique were repeated several times on the same tissue. Radioactive samples were counted in Bray’s solution in a MK II Nuclear Chicago scintillation counter.

Irreversible Binding in Uterine Strips

Uteri were slit longitudinally and rinsed in cold D-J-I medium (4OC). Strips were either left intact, or scraped to remove the endometrium and sliced laterally into pieces weighing 5 mg , Tissues were preincubated for 60 min at room temperature in 2.5 ml of stirred D-J-I medium in the absence or pre- sence of inhibitors. Tissues slices (-20 mg) were then incubated for varying

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time intervals (5-120 set) in 250 pl of D-J-I medium (21’C) containing PGEl-H3 (1.0 - 5.0 ng/ml) with or without a lOO-fold excess of non-radioactive PGEl., Incubation was terminated by ra id freezing and tissues were washed 3 times in 0.5 ml of cold D-J-I medium (4 8 C) for a total of 30 min. This re- moves all reversib

P- bound prostaglandin, and the difference between the

binding of PGEl-H in the absence or presence of non-radioactive PGEl (12), then represents irreversibly-bound prostaglandin. The tissue strips were blotted dry, solubilized in 250 pl of 0.5 N-NaOH and neutralized with BBS-2 (Beckman) before counting in a toluene-liquifluor mixture to determine irre- versible binding.

RESULTS

Incubation of the intact uterus with PGEI-H3 for 5-120 set resulted in a dose-dependent rate of reversible binding. This rate, for a11 concentrations of PGEl studied, was most rapid within 5 set , decaying exponentially to minimal levels and equilibrium after 120 set (Figure lA) . Similar results were obtained using both uterine and myometrial strips where irreversible binding was monitored. From the data shown in Table I, it can be seen that reversible and irreversible binding of PGEl-H3 were comprised of fast and slow compo- nents, which differed in their respective rate constants. Within the time required to elicit a pharmacolo+ ical response in the uterus ( < 60 set), the majority (-80%) of PGEl-H bound was directly associated with the fast component of reversible binding, This component had a half time (T ) of

3-l -1 approximately 5 set and an association rate constant (k) of 56 fg , mg . sec. , The degree of binding at 5 set was routinely examined as an index of the fast reversible component since the half times for the other components were all >24 sec. We have shown that this time interval, although sensitive to some

error in timing, may be reproduced with statistical accuracy (Figures 4-7). Irreversible binding was also monitored at 5 set as a direct comparison with reversible binding.

Reversible and irreversible binding by intact uterus and myometrial strips respectively, exhibited a qualitative but not a quantitative similarity with respect to PGE -H3 concentration (Figure 1B). (k = 0.83 fq.mgJ1s&.-’

Irreversible binding > was approximately l-3% of reversible binding (k=

56 fg.mg. sec. ‘), However, following superfusion of intact uterus to remove reversibly bound PGEl-H3, the level of irreversible binding was 12 fg/mg and was now comparable to that in myometrium (14 fg/mg). This indicates that the major component (>97%) of binding at 5 set in intact uterus, was in fact reversible, and agrees well with that projected from the respective rate constants (Table 1). The reversibly-bound prostaglandin removed by superfusion was identified as authentic PGEl by both chromatography (10) and bioassay (11).

The similarity between irreversible binding after superfusion and that after rapid freezing indicates that such binding was impervious to the freezing technique (see Methods). Both reversible and irreversible binding of PGEl-H3 were also sensitive to competition by unlabeled PGEl, a reported index of

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Figure 1 A. Prostaglandin E ,-H3 reversible binding by intact uterus.

A single uterine horn was preincubated for 1 hr in 200 t.11 D-J-I medium, containing 1 ng/ml PGEI-H3 at 21°C. It was superfused and then suspended in 200 ~1 of D-J-I medium containing 1.0 (& , 2.0 (0) and 5.0 (0) ng/ml PGE1-H3 for the time intervals shown. The tissue was superfused between each immersion to remove reversibly bound PGEl. The figure represents a characteristic result of 10 experiments. Binding is expressed as PGE 1-H3 uptake in pg/mg/sec.

B. Dose-dependent reversible binding by intact uterus (e) and irrever- sible binding by myometrial strips (m> of PGE,-HJ at 5 sec. Irreversible binding (m> represents the difference (x20) between the

binding in the presence or absence of lOO-fold excess of unlabeled PGEl at each concentration.

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PGE.-H’ BINDING IN ISOLATED RAT UTERUS

TABLE 1

IALF TIME CT+) (set>

1SSOCIATION RATE CONSTANT

[k = fg.mg.-lsec.-l)

Li OF TOTAL BINDING IN 60 sec.

Reversible Binding

Fast Slow Component Component

5.0

56.0

79.8

26.5

2.1

15.6

Irreversible Binding

.._-

Fast Slow omponent Component

24.0 155.0

0.83

4.0

0.18

1.0

The data was calculated from a plot of B-Bt versus time, where Bt .is the amount of PGEl-H3 bound at a given time (t), and B is the amount bound at equilibrium, in the presence of 1 ng/ml PGEl-H3. Reversible and irreversible binding were estimated as described in the text.

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binding specificity (12). Scatchard plots of reversible and irreversible binding yielded straight lines, indicating that single classes of binding sites were involved and that fast and slow binding take place at the same

$ inding

site. An apparent affinity constant (K) of 0.72 x lo8 l/mole PGEl-H was obtained from the reversible component in intact uterus (Figure 2A). Irrever- sible binding in myometrium yielded a similar affinity constant of 0.62 x lo8 l/mole. These binding constants are in agreement with those reported in beef thyroid membranes (6) and consistent with effective pharmacological concentra- tions of prostaglandins in the isolated uterus. The maximum amounts of PGEl-H3 bound for the two processes when extrapolated to infinite substrate concen- tration were 4407 fg/mg (r.eversible) and 20.2 fg/mg (irreversible) respectively.

A critical requirement for PGE -receptor binding is the correlation of that binding with an induced respon s&. in the same cell type. Reversible binding of PGEl-H3 (Table 1) and the onset of prostaglandin-induced contraction in the isolated rat uterus both occur within 60 sec. The correlation of PGEl-H3 rever- sible binding with the subsequent uterine response is shown in Figure 2B. This was then extended to those cases where uterine tachyphylaxis was evident (13). Repeated application of PGEl (1 ng/ml) resulted in a successive and parallel decrease in both the contractile response and the rate of PGEl-H3 reversible binding (Figure 3A). Indeed phylaxis exhibited in PGE1-H 9

there was a linear relationship between the tachy- binding and in uterine response (Figure 3B).

The physiological basis for tachyph laxis is unknown. However, we have demonstrated that 12 fg/mg PGEl-H Y remains irreversibly bound after a single exposure for 5 set to PGEl (1 ng/ml) , Decreased responsiveness (tachyphylaxis) may, therefore, reflect progressive accumulation of PGE at the receptor site. Adamson et al (13) have reported that there is no cross- esensitization between a. the prostaTl=dins in the tachyphylactic uterus and this accumulation may, there- fore, not influence the binding of the other prostaglandins .

We have previously shown that prostaglandin-induced contractions of the rat uterus are inhibited by agents which modify membrane thio groups (14,15). It has been reported, however, that sulfhydryl inhibitors have minimal effects on PGEl binding (6). Under conditions which result in inhibition of uterine contraction, dithiothreitol (DTT , O.lmM) and ultraviolet irradiation (U/V) maximally inhibited (e 50%) the rate of reversible binding of PGE l-H3 (Figures 4 & 5). This inhibition was correlated directly with the subsequent diminution of the evoked response. Kinetic studiy indJ$ated a significant decrease in the velocity of binding (kl = 16.5 fg ,mg . sec. ) with no effect on the apparent affinity constant. The data further suggest that the initial rapid binding of PGEl-H3 determines the magnitude of the induced physiological event.

The effect of these thio inhibitors on the irreversible binding of PGEl-H3 was also studied, In contrast to the observation on reversible binding (see

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Figures 4 & 5). DTT and U/V induced 90-1008 inhibition of PGEl-H3 irre- versible binding in myometrium (Figure 6). In addition, there was no appa- rent relationship between the inhibition of irreversible binding and the modi- fication of uterine contraction. U/V and DTT-induced inhibition of prostaglan- din action may be prevented or reversed with sulfhydryl-oxidizing agents such as 5,5’-dithiobis- (2-nitrobenzoic acid), DTNB (14,15). Figure 7 illus- trates that U/V significantly decreased irreversible binding of PGEl-H3 in rat myometrium , It had little effect on the irreversible non-receptor component but significantly inhibited receptor binding. The effect of U/V was fully reversed by subsequent exposure to DTNB (O.lmM) , suggesting modification of those groups involved in PGEl-H3 binding, DTNB alone had no effect on PGE 1 binding.

7-oxa-13-prostynoic acid (‘loxa), a reported prostaglandin antagonist (17) also inhibited irreversible binding of PGEl-H3 in the myometrium (Figure 6). This inhibition (~100%) is comparable with that reported in other systems (3,6), However, 7oxa (10 pg/ml) resulted in only-308 inhibitions of both reversible PGE l-H3 binding and uterine contraction.

DISCUSSION

Previous reports on PGEl-H3 binding are characterized by the use of incubation times which are unrelated to evoked cellular events, and also by a lack of correlation with physiological responses in the cell type (l-9). The only reported correlation has been between PGEl binding in a rat adipocyte preparation and cyclic AMP accumulation in mouse ovary (3). In addition, receptor binding has been defined in terms of the “specific” binding remaining after prolonged washing (1,2,4,8).

The characteristics of prostaglandin-induced uterine contraction indicate both rapid and reversible prostaglandin receptor interaction. Evidence to support this hypothesis was afforded by the following experimental observations:

1. The rate of binding of PGEl-H3 in uterine tissues was maximal (~80% occurring within 10 set) at a time prior to the onset of the evoked response.

2. A major component (-95%) of the binding was readily reversible. 3. This rate of binding was prostaglandin dose-dependent. 4. A direct correlation with the response of both the normal and tachy-

phylactic uterus was shown, 5. Changes in the rate of binding induced by a variety of inhibitors was

reflected in and was correlated with subsequent modified uterine

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contraction, 6. An affinity constant of 0.62 - 0.72 x lOa l/mole for this rapid binding

process is consistent with pharmacological levels of prostaglandin .

The mass of endometrium in 14-day ovariectomized rats does not signifi- cantly contribute to the overall uterine weight (<l%) . Indeed, binding rates obtained in the presence (intact uterine strips) or absence of the endometrium (myometrial strips) were identical, indicating that binding of PGE -H3 in the endometrium under these conditions was negligible. We feel it is t I, erefore justified, to compare directly the binding in myometrium with that in intact uterus. Irreversible binding represented ... 5% of the binding which occurred prior to the onset of the induced response. The fast-irreversible component was kinetically similar to the slow-reversible component (Table 1) , and was correlated with evoked uterine contraction. It was also sensitive to inhibitors of prostaglandin action. This would suggest that irreversible binding occurs at the same site as the reversible component. Indeed, the apparent affinity constants for the two processes were equivalent, However, the observed correlation between the inhibitions by DTT , U/V and 7oxa of reversible binding and uterine contraction contrasts with that seen in irreversible binding. This may indicate that the contribution of irreversible binding to physiological events is minimal. We conclude, that only the initial, rapid (Tf = 5 set) and reversible binding of prostaglandin which precedes and reflects the induced physiological response, fully satisfies the requirements for prostaglandin-receptor interaction. In addition, the inhibition of prostaglandin binding by U/V and lipophobic thio reagents would support the hypothesis (3,5,6) that such binding occurs at the membrane level.

A direct relationship between inhibition of reversible prostaglandin receptor binding and subsequent uterine responses was seen with DTT , U/V and 7oxa. This inhibition was only evident when the prostaglandin receptor was exposed and was prevented by prior protection of that receptor with PGEl. DTT and U/V reduce membrane disulfides in prostaglandin-responsive systems (14, 15) resulting in an increase in the number of titratable membrane sulfhydryl groups (16). 7oxa is a reported competitive prostaglandin antagonist (3). The resultant of these diverse effects is an inhibition of uterine contraction. We suggest therefore that those disulfides , critical to prostaglandin action and sensi- tive to reduction by DTT and U/V, are located at or near the prostaglandin recep- tor binding site.

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CPGE, Ii%-

Figure 2 A. PGEl-H3 reversible binding (pg/mg) by intact UterUS at 5 set expressed as a function of the concentration of PGEl (pg/ml) available.

B. Correlation of PGE1-H3 reversible binding at 5 sec. with PGE1- induced contraction in the isolated uterus.

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Figure 3 A. Correlation of PGE1-H3 reversible binding with induced contraction during tachyphylaxis. Both uterine horns from the same animal were repeat- edly exposed to 1 ng/ml PGE1-H3 or to PGE , and binding at 5 set (0) and uterine contraction at 90 set (0) respective y were monitored. Tissues were 1 superfused for 5 min between each application as described previously. The lines represent linear regression analysis of the data.

B. PGE 1-induced uterine contraction expressed as a function of the rever- sible binding at 5 set during tachyphylaxis. The initial contraction and rate of uptake are expressed as 100% in each case.

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5 IO 30 60

Time Csec)

Figure 4 The effect of DTT (0.1 mM) on PGE,-H3 reversible binding in intact uterus. Paired uterine horns from the same animal were preincubated for 1 hr at Zl°C in either 1 .O ml of D-J-I medium containing DTT (0) or in medium alone (0). Tissues3were then preincubated (1 hr at 21oC) in medium containing 1 ng/ml

and reversible binding was determined as previously Each point represents the mean of six estimations and the bars the

standard error about the mean, pd 0.025 where indicated.

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Figure 5 The effect of U/V on the reversible binding of PGE,-H3 in intact uterus. Paired uterine horns from the same animal were immersed in 1.0 ml of D-J-I medium and preincubated for 1 hr at 21°C in the absence or presence of U/V irradiation (242 microwatts per cm2). Tissues were then incubated (1 hr at 210C) in 1 .O ml D-J-I medium containing 1 ng/ml PGEl-H3, superfused and reversible binding was determined as previously described. The effect of U/V is expressed as a % of the control rate of binding at each time interval. The mean of eighteen estimations and the standard error about the mean is indicated, pc where shown.

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1

A

L

- 0

I

C

6 Figure Irreversible binding of PGEl-H3 (1 ng/ml) at 5 set in myometriai strips after exposure to (A) U/V irradiation (242 microwatts per cm2), (B) 7 oxa (10 pg/ml) , (C) DTT (0.1 mM) and (D) PHMB (0.1 mM) for 60 min at 21°C, Data is expressed as % inhibition of control binding, determined after 1 hr preincubation in D-J-I medium alone. The mean ;t S .E. of six estimations is illustrated.

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A

0 I-Ii . . . . . . . . . B C

l-

1 0 :: :: . . . . . . l . I

Figure 7 The inhibition of myometrial irreversible binding of PGE,-H3 (1 ng/ml) by U/V irradiation and reversal by DTNB. The components of binding at 5 set are expressed as total (8), non-rece tor (0) , and receptor (8). The c:ffect of exposure to U/V (242 microwatts per cm ! ) for 60 min in the (B) absence and (C) presence of DTNB (0.1 mM) are compared to the control response (A). Values represent the mean + S .E. of twelve estimations p< 0.001 where indicated (0).

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2a. Wakeling, A.E. and L .J . Wyngarden. In vitro studies on the nature of prostaglandin El binding in the hamster uterus. Prostaglandins 5: 291, 1974.

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6. Moore, W .V. and J . Wolff. Binding of prostaglandin El to beef thyroid membranes. J . Biol. Chem. 248: 5705, 1973.

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9. Attallah, A .A. and J .B , Lee. Specific binding sites in the rabbit kidney for prostaglandin A. Prostaglandins 4: 703, 1973.

10. G&en, K. and B. Samuelsson. Thin-layer chromatography of the prostaglandins . J . Lipid Res . 5: 117, 1964.

11. Ramwell, P .W., 3 .E. Shaw and R. Jessup. Spontaneous and evoked release of prostaglandins from frog spinal cord. Am. J . Physiol . 211: 998. 1966.

lla. Vane, J.R. and K.I. Williams. The contribution of prostaglandin production to contractions of the isolated uterus of the rat. Br . J . Pharmac. 48: 629, 1973.

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12. Baxter, G . D . and G . M . Tomkins . Specific cytoplasmic glucocorticoid hormone receptors in hepatoma tissue culture cells, Proc . Nat. Acad. Sci. 68: 932, 1971.

13. Adamson, U . , R. Eliasson and B. Wiklund. Tachyphylaxis in rat uterus to some prostaglandins. Acta. Physiol. Stand. 70: 451, 1967.

14. Johnson, M., R , Jessup and P . W . Ramwell. Ultraviolet light modifi- cation of the prostaglandin receptor. Prostaglandins 4: 593, 1973.

15. Johnson, M. , R. Jessup and P .W. Ramwell. The significance of protein disulfide and sulfhydryl groups in prostaglandin action. Prostaglandins . 5: 125, 1974.

16. Johnson, M. and R. Jessup . Unpublished observations, 1974.

17. Fried, J. T .S. Santhanakrishnan, J. Himizu, C .H. Lin, S .H. Ford, B . Rubin and E .O. Grigas. Prostaglandin antagonists: synthesis and smooth muscle activity. Nature 223: 208, 1969.

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