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Prostaglandin E2 receptor in the myometrium: Distribution in subcellular fractions
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 212, No. 2, December, pp. 700-704, 1981
Prostaglandin E2 Receptor in the Myometrium: Distribution in Subcellular Fractions’ MARY
E. CARSTEN
AND
JORDAN
D. MILLER
Departmentsof obstetrics and Gyzecdogy,and Anest-, University
of Califonia,
Los Angeles, California
School of Medicins, 3OO.24
Received March 31, 1981, and in revised form July 6, 1981 IjH]Prostaglandin (PG) Es bound specifically to several subcellular fractions from hovine myometrium. The binding was temperature dependent, rapid, and reversible. PGEs and PGE, competed for the yH]pGE, binding site. The PGs inhibited in the following decreasing order: PGE, = PGE, %PGF2, > PGAs > PGFIL) > PG&. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 + 0.14 X lo-‘. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 + 2.3 X lo-” and the sarcoplasmic reticulum contained 11.2 -t 0.8 X 10-l’ mol binding sites/g. The results suggest that there is a high-affinity PGEs receptor present in both plasma membrane and sarcoplasmic reticulum.
Prostaglandins (PG)2 cause uterine contractions and may well be responsible for the onset and maintenance of normal labor. Prostaglandins have been used for induction of labor and termination of pregnancy (1). These actions are thought to be mediated either by a receptor mechanism (2) or ionophore-like effect at the plasma membrane or intracellular level (3). Specific prostaglandin binding sites, demonstrated in whole uterine homogenates and tissue slices (2), are thought to represent binding to receptors. The cellular location of these receptors has not been demonstrated. An intracellular site of action is likely because of intracellular synthesis and low circulating levels of PGs. In support of this, we have previously demonstrated an
effect of PGs on ATP-dependent calcium accumulation and release in a subcellular microsomal fraction (3-5). These findings were in accord with the physiologic contractile action of these PGs. In order to localize these effects, we further purified and fractionated subcellular components (6) and separated plasma membrane from sarcoplasmic reticulum markers. No receptors have been characterized in defined myometrical fractions. Results from an initial study demonstrated specific binding but could not distinguish between one or more high-affinity binding sites. Knowledge of specific binding constants and maximum number of binding sites (6) is required to answer the question of whether specific binding is a property of one or all fractions. This communication attempts to answer this by characterizing the PG receptor in several subcellular fractions from myometrium using Scatchard analysis. The present data demonstrate a single highaffinity binding site that appears to be present in plasma membrane and sarcoplasmic reticulum fractions.
’ This investigation was supported by Grant HD00010 from the National Institute of Child Health and Human Development, United States Public Health Service. ’ Abbreviations used: PG, prostaglandin; EL.., maximum specific binding sites per gram protein; WGA, wheat germ agglutinin. 0003-9861/81/140700-05$02.00/0 Copyright All rights
Q 1981 by Academic Press. Inc. of reproduction in any form reserved.
700
PROSTAGLANDIN MATERIALS
RECEPTOR IN MYOMETRIUM
AND METHODS
A microsomal fraction was obtained from pregnant bovine uterus by a procedure previously described (3), with the exception that all solutions contained 10 ag/ml indomethacin. In summary, a uterine homogenate was centrifuged at 25OOgX 20 min, at 15,OOOgx 20 min. followed by centrifugation of the supernatant at 4O,OOOg., X 90 min. The pellet was suspended in 0.08 M NaCl, 0.005 M sodiun oxalate, and used for sucrose density gradient centrifugation. Discontinuous sucrose density gradients were either (A) of 35,45, and 55% (w/v) sucrose or (B) of 24,28, 33, and 45% (w/v) sucrose. The light-weight fraction of gradient A ranging from 25 to 45% sucrose is equivalent to the combined 24-28,28-33, and 33-451 sucrose fractions of gradient B. The light-weight microsomes (25-45% sucrose) from gradient A were used in all situations, unless otherwise indicated. Microsomal layers, formed at the interfaces, were removed with a syringe and protein concentration determined by the method of Lowry (7). PGE2 binding to various fractions was carried out. Aliquots of protein were incubated in a buffer containing 0.25 M sucrose, 0.5 @dCaC12,0.02 M phosphate, pH 7.4, indomethacin 10 pg/ml, and prostaglandin. Ten levels of rH]PGE2 (from 2.5 to 172 X lo-” M) in the absence and presence of unlabeled PGEa (1.72 X 10e7M) were used. Protein concentration was approximately 0.25 mg/ml. The incubations were carried out in a final volume of 400 ~1 for 1 h at 37°C. Free PG was removed by the addition of 1 ml of 2.5% Norite A in 0.1% gelatin, 0.02 M phosphate buffer, pH 7.4 (2). The tubes were centrifuged for 15 min at 2200g at 4°C and an aliquot of the supernatant was counted for tritium in a liquid scintillation system. Specific binding was computed as the difference between rH]PGEs bound (total binding) and rH]PGE* bound in the presence of excess unlabeled PGEe (nonspecific binding); the result was corrected for protein concentration. Analysis was by the Scatchard method (8). The best straight line was calculated by linear regression. The dissociation constant (&) was computed at l/-slope. Maximum specific binding sites per gram protein (B,.,) is the X intercept. A single experiment was performed on human term-pregnant uterus obtained at Cesarean hysterectomy. The protocol was approved by the Human Subject Protection Committee and written consent from the patient was obtained. Preparative and experimental procedures were identical with those used in the bovine experiments. Competition of various PGs and oxytocin for PGEa binding sites was carried out in the same buffer by incubating protein simultaneously with 2 X 10m9M [3H]PGEz in the absence and presence of the unlabeled test compounds for 1 h at 37°C. These compounds were tested at six levels (1 X lo-* to 2 X 10m5 M) except that PGE compounds were used at concen-
701
trations between 0.3 X low9 and 2 X 10m5M. PGE compounds were originally dissolved in absolute ethanol (25 mg PGE in 1 ml) and diluted with buffer. Comparable amounts of ethanol were added to the incubation medium of the controls. All other eompounds were dissolved in the buffer, as specified earlier. The incubation and experimental procedure was as above. The difference in counts bound in the presence and absence of 2 X 10m5M cold PGEs was the maximum specific binding. The percentage of control was the difference in counts bound in the presence and absence of a competitor, divided by the maximum specific binding in the same experiments, times 100. To demonstrate the reversibility of [sI-DPGEr binding, three sets of tubes were set up, two (A, B) to assess total binding (containing [aH]pGEs) and the other (C) to assess nonspecific binding (containing pH]PGE* + PGEa) as before. After 1 h of incubation at 37°C with 3.5 X lo-’ M WPGE, an excess PGEs (0.1 ml, 1.5 X 10s6 M) was added to one set of total binding tubes (B), 0.1 ml buffer to the other sets. Norite-gelatin was added as above to tubes A, B, C, at each time interval, from 0 to 4 h. The specifically bound [sI-HPGE2was calculated at each time as: (B, - C,)/(A, - C,) X 100 = % of control at time t, where t varies from 0 to 4 h. This method eliminates errors caused by changes in nonspecific binding. [3H]PGEz (100-200 Ci/mmol) was obtained from New England Nuclear, unlabeled PGs courtesy of the Upjohn Company. Oxytocin was from CalBiochem, indomethacin from Sigma. RESULTS
In all protein fractions [3H]PGE2-specific binding was found to be saturable, as previously reported for one of the fractions (9). Nonspecific binding was found to be linear with respect to free [‘H]PGEa concentration. Preliminary experiments showed that PG binding was rapid. Halfmaximum binding occurred at 15 min and was essentially complete by 1 h at 37°C. At lower temperatures decreased rate of specific binding occurred. Nonspecific binding increased with time and temperature. There was no specific PGEa binding in boiled preparations, though nonspecific binding was increased (results not shown). Figure 1 shows the dissociation of specifically bound rH]PGEa indicating the reversibility of the reaction. The rate of dissociation decreased with time. Quantitation of the PGEa receptor was
702
CARSTEN AND MILLER
carried out by Scatchard analysis. Comparison was then made of the receptor in the various subfractions obtained on sucrose density gradient B. Results were normalized for protein concentration. This method was validated by showing that a 2.5-fold increase of protein in the incubation mixture did not change the calculated Kd or maximum binding sites per gram of protein. The results of the Scatchard analysis for the sarcoplasmic reticulum fraction (C) and two plasma membrane fractions (D and E) for all experiments are tabulated in Table 1. Kd values were not significantly different between C, D, and E. The maximum number of binding sites, however, is significantly greater in D than in either C or E. Figure 2 presents Scatchard plots from the single human experiment. As can be seen, a single straight line fits the points, thus demonstrating only one kind of high-affinity binding site with a single Kd. The single human experiment falls within the range of Kd and maximum
i io 6b
Ii0
240
Minutes
FIG. 1. Reversibility of [aHJPGE* binding in the presence of excess unlabeled PGE, in the lightweight microsomal fraction from gradient A(3545% sucrose); average of three experiments. [dHJPGEswas allowed to bind for 60 min at 37’C. Excess of unlabeled PGEB was added and allowed to compete for the time indicated. Each point represents the difference in total [%lpGEr binding in the presence and absence of excess PGEo added at 0 time, divided by control which is total binding for 60 min plus the time indicated minus nonspecific binding for the same time interval.
TABLE I SPECIFIC PGEp BINDING CONSTANTS FOR SUBCELLULAR FRACTIONS
C D E
2.50 i
4
B I"
(W
(molk)
Number of experiments
11.2 * 0.8 x 10-I’ 21.4 k 2.3 X lo-” 12.2 + 1.0 x 10-I’
11 8 8
0.19 x 10-L
3.78 + 0.30 X 10-O 3.05 * 0.26 x 10-s Average
2.74 * 0.14 X 10”
PC C-D6 D-Eb C-Eb
“8 ns “S
0.0005 0.002 ns
‘Mean + SEM. b By paired t test in eight
experiments.
number of binding sites presented in Table I. The largest number of binding sites was fond in fraction D, a plasma membrane fraction. From our data, fractions C and E appear to have the same number of binding sites. However, fraction E is a plasma membrane fraction, and C is a sarcoplasmic reticulum fraction. When increasing amounts of unlabeled PGs were incubated with a constant amount of rH]PGE,, dose-dependent inhibition of binding occurred as seen in Fig. 3. For PGEz, half-maximal inhibition occurred at approximately 5.2 X lo-’ M, for PGEi, at 3.8 X lo-’ M. Other PGs required between 500 and 3300 X lo-‘M for halfmaximal inhibition. The order of potency for inhibition was PGE2 = PGEi %PGF& > PGA2 > PGF,, > PGB2. Oxytocin did not inhibit PH]PGEz binding. DISCUSSION
Specific binding of hormones to membrane preparations has been associated with hormone receptors in a number of tissues. Specific binding implies that there is a finite number of binding sites, hence the fraction bound decreases as the total concentration of hormone increases (10). We have demonstrated this relationship in a previous publication (9). A Scatchard plot of the data yields binding constants and maximum number of binding sites. A prerequisite of this analysis is that the
PROSTAGLANDIN
703
RECEPTOR IN MYOMETRIUM
0.06 F 005 I 2 I x 004 I; f 0.03 QJ g
002
2 m
0.01
-
-
1.
. - .
‘h 0
4
8 Speuflc
BIndIng
(mol/gxlO-“I
FIG. 2. Scatchard plots of rH]PGE2 binding to several membrane fractions from human myometrium. The results are corrected for nonspecific binding and normalized for protein concentration. (c) K,j = 2.75, B,,, = 9.35; (D) & = 3.63, B,,,, = 19.9; (E) & = 3.33, B,, = 13.6; (Kd (M X lo-‘), B,., (mol/g X lo-“)). C is enriched in sarcoplasmic reticulum, D and E are enriched in plasma membrane-(6).
reaction is reversible. Though exchange of bound TH]PGE, for PGEz is limited to 65% in 4 h, the initial rate is very high. The best interpretation is that irreversible changes related to prolonged incubation at 3’7°C are responsible for this observation. The fact that the rate of specific binding increases with increasing temperature, and that specific binding is absent in boiled preparations, is suggestive of the protein nature of the “receptor.” After correction for nonspecific binding (10) our Scatchard plots demonstrate a single affinity of this site over the concentration tested. While we cannot eliminate a specific binding site of much lower affinity, such a site would probably not be a candidate for a receptor. Our Kd fits with Kd for PG binding in other systems (11, 12) and is in the range of biological activity reported (13). Like other investigators, we have demonstrated that PGs of lower uterine contractile activity exhibit lower competitive effects on specific PGEz binding. The relatively low competitive effect of PGFti agrees with findings of other investigators (12) and suggests the existence of separate receptors for PGEz and PGF&. There was no competition of oxytocin for the PGEz binding site. Oxytocin, a polypeptide contractile agent, is thought to work through a different receptor, because of major structural differences.
Previous work showing a partial purification of subcellular components encouraged us to look for localization of the PG receptor. These fractions, obtained after sucrose density gradient centrifugation, were previously characterized (6). To verify the distribution of subcellular components in the preparations used in the present work, periodic checks were made using 5’-nucleotidase as a marker. No difference in the distribution was found from that previously reported. The subfractions obtained were enriched in sarcoplasmic re100
/
3
30 Unlabeled
\.‘.,. 300 Competttor(M
3poo
20,000
x 10-T
FIG. 3. The specificity of rHjPGE* binding in the light-weight microsomal fraction from gradient A (25-45% sucrose). The percentage of control is calculated as the difference in counts bound in the presence and absence of a competitor, divided by the difference in counts bound in the presence and absence of 2 X 10m5M cold PGE2, times 166.
704
CARSTEN
ticulum (C, 2423% sucrose) and plasma membrane (D, 28-33% sucrose, and E, 3345% sucrose). D and E are plasma membrane fractions as demonstrated by high and approximately equal levels of 5’-nucleotidase activity, ouabain inhibition of Na,K-ATPase, and WGA binding. They differ from one another in as much as ATP-dependent calcium accumulation is more than twice as high in D as in E. Onepoint analyses of PG-specific binding in these fractions were done previously, but interpretation had to wait for complete Scatchard analysis. These are presented in the present paper. The ATP-dependent calcium accumulation correlates with the number of specific PGEz binding sites in plasma membrane fractions D and E. However, when comparing fraction C (sarcoplasmic reticulum) with either plasma membrane fraction, this relationship does not hold. Though there is an approximate twofold greater ATP-dependent calcium accumulation in Fraction C than in plasma membrane fraction D, the number of PGE:! binding sites is only approximately half of that found in D. These results are best explained by assuming that both ATP-dependent calcium accumulation and specific PG binding are intrinsic properties
AND MILLER
of both sarcoplasmic reticulum and plasma membrane. Alternative hypotheses, such as cross-contamination, cannot account for all of these results taken together. REFERENCES 1. WIQVIST, N., AND WILHELMSSON, L. (1979) @need Obst& Invest lO, l-8. 2. KIMBALL, F. A., KIRTON, K. T., SPILUL, C. H., AND WYNGARDEN, L. J., (1975) Bill Reprod 13,
482-489. 3. CARSTEN,M. E., AND MILLER, J. D. (1977) J. Bid C&m. 252, 1576-1531. 4. CARSTEN, M. E. (1973) Gyzecd Invest 4,95-105. 5. CARSTEN, M. E. (1974) Prostaglandins 5,33-40. 6. CARSTEN, M. E., AND MILLER, J. D. (1930) Arch,
Bicchem Biophys. 294,404-412. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FAR& A. L., AND RANDALL, R. J. (1951) J. Bid Chem 193, 265-275. 8. SCATCHARD, G. (1949) Ann N. Z Acud SC%51, 660-679. 9. CARSTEN, M. E., AND MILLER, J. D. (1980) Adwan
Prostaglandin Thrombvxam Rex 6,401-405. 10. CHAMNESS, G. C., AND MCGUIRE, W. L. (1975) steroids 26,538~542. 11. RAO, C. V. (1974) .I. Bid Chem 249,7203-7209. 12. KIMBALL, F. A., KIRTON, K. T., AND WYNGARDEN, L. J. (1975) Pro&an&m&s 10,853-864. 13. KIRTON, K. T., PHARRISS, B. B., AND FORBES, A. D. (1970) Biol Reprod 3,163-168.
Prostaglandin E2 Receptor in the Myometrium: Distribution in Subcellular Fractions’ MARY
E. CARSTEN
AND
JORDAN
D. MILLER
Departmentsof obstetrics and Gyzecdogy,and Anest-, University
of Califonia,
Los Angeles, California
School of Medicins, 3OO.24
Received March 31, 1981, and in revised form July 6, 1981 IjH]Prostaglandin (PG) Es bound specifically to several subcellular fractions from hovine myometrium. The binding was temperature dependent, rapid, and reversible. PGEs and PGE, competed for the yH]pGE, binding site. The PGs inhibited in the following decreasing order: PGE, = PGE, %PGF2, > PGAs > PGFIL) > PG&. No competitive effect could be found for oxytocin. Scatchard analysis of the binding data were interpreted as showing a single high-affinity binding constant. There was no difference in the binding constant between the various fractions. The average molar dissociation constant was 2.74 + 0.14 X lo-‘. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 + 2.3 X lo-” and the sarcoplasmic reticulum contained 11.2 -t 0.8 X 10-l’ mol binding sites/g. The results suggest that there is a high-affinity PGEs receptor present in both plasma membrane and sarcoplasmic reticulum.
Prostaglandins (PG)2 cause uterine contractions and may well be responsible for the onset and maintenance of normal labor. Prostaglandins have been used for induction of labor and termination of pregnancy (1). These actions are thought to be mediated either by a receptor mechanism (2) or ionophore-like effect at the plasma membrane or intracellular level (3). Specific prostaglandin binding sites, demonstrated in whole uterine homogenates and tissue slices (2), are thought to represent binding to receptors. The cellular location of these receptors has not been demonstrated. An intracellular site of action is likely because of intracellular synthesis and low circulating levels of PGs. In support of this, we have previously demonstrated an
effect of PGs on ATP-dependent calcium accumulation and release in a subcellular microsomal fraction (3-5). These findings were in accord with the physiologic contractile action of these PGs. In order to localize these effects, we further purified and fractionated subcellular components (6) and separated plasma membrane from sarcoplasmic reticulum markers. No receptors have been characterized in defined myometrical fractions. Results from an initial study demonstrated specific binding but could not distinguish between one or more high-affinity binding sites. Knowledge of specific binding constants and maximum number of binding sites (6) is required to answer the question of whether specific binding is a property of one or all fractions. This communication attempts to answer this by characterizing the PG receptor in several subcellular fractions from myometrium using Scatchard analysis. The present data demonstrate a single highaffinity binding site that appears to be present in plasma membrane and sarcoplasmic reticulum fractions.
’ This investigation was supported by Grant HD00010 from the National Institute of Child Health and Human Development, United States Public Health Service. ’ Abbreviations used: PG, prostaglandin; EL.., maximum specific binding sites per gram protein; WGA, wheat germ agglutinin. 0003-9861/81/140700-05$02.00/0 Copyright All rights
Q 1981 by Academic Press. Inc. of reproduction in any form reserved.
700
PROSTAGLANDIN MATERIALS
RECEPTOR IN MYOMETRIUM
AND METHODS
A microsomal fraction was obtained from pregnant bovine uterus by a procedure previously described (3), with the exception that all solutions contained 10 ag/ml indomethacin. In summary, a uterine homogenate was centrifuged at 25OOgX 20 min, at 15,OOOgx 20 min. followed by centrifugation of the supernatant at 4O,OOOg., X 90 min. The pellet was suspended in 0.08 M NaCl, 0.005 M sodiun oxalate, and used for sucrose density gradient centrifugation. Discontinuous sucrose density gradients were either (A) of 35,45, and 55% (w/v) sucrose or (B) of 24,28, 33, and 45% (w/v) sucrose. The light-weight fraction of gradient A ranging from 25 to 45% sucrose is equivalent to the combined 24-28,28-33, and 33-451 sucrose fractions of gradient B. The light-weight microsomes (25-45% sucrose) from gradient A were used in all situations, unless otherwise indicated. Microsomal layers, formed at the interfaces, were removed with a syringe and protein concentration determined by the method of Lowry (7). PGE2 binding to various fractions was carried out. Aliquots of protein were incubated in a buffer containing 0.25 M sucrose, 0.5 @dCaC12,0.02 M phosphate, pH 7.4, indomethacin 10 pg/ml, and prostaglandin. Ten levels of rH]PGE2 (from 2.5 to 172 X lo-” M) in the absence and presence of unlabeled PGEa (1.72 X 10e7M) were used. Protein concentration was approximately 0.25 mg/ml. The incubations were carried out in a final volume of 400 ~1 for 1 h at 37°C. Free PG was removed by the addition of 1 ml of 2.5% Norite A in 0.1% gelatin, 0.02 M phosphate buffer, pH 7.4 (2). The tubes were centrifuged for 15 min at 2200g at 4°C and an aliquot of the supernatant was counted for tritium in a liquid scintillation system. Specific binding was computed as the difference between rH]PGEs bound (total binding) and rH]PGE* bound in the presence of excess unlabeled PGEe (nonspecific binding); the result was corrected for protein concentration. Analysis was by the Scatchard method (8). The best straight line was calculated by linear regression. The dissociation constant (&) was computed at l/-slope. Maximum specific binding sites per gram protein (B,.,) is the X intercept. A single experiment was performed on human term-pregnant uterus obtained at Cesarean hysterectomy. The protocol was approved by the Human Subject Protection Committee and written consent from the patient was obtained. Preparative and experimental procedures were identical with those used in the bovine experiments. Competition of various PGs and oxytocin for PGEa binding sites was carried out in the same buffer by incubating protein simultaneously with 2 X 10m9M [3H]PGEz in the absence and presence of the unlabeled test compounds for 1 h at 37°C. These compounds were tested at six levels (1 X lo-* to 2 X 10m5 M) except that PGE compounds were used at concen-
701
trations between 0.3 X low9 and 2 X 10m5M. PGE compounds were originally dissolved in absolute ethanol (25 mg PGE in 1 ml) and diluted with buffer. Comparable amounts of ethanol were added to the incubation medium of the controls. All other eompounds were dissolved in the buffer, as specified earlier. The incubation and experimental procedure was as above. The difference in counts bound in the presence and absence of 2 X 10m5M cold PGEs was the maximum specific binding. The percentage of control was the difference in counts bound in the presence and absence of a competitor, divided by the maximum specific binding in the same experiments, times 100. To demonstrate the reversibility of [sI-DPGEr binding, three sets of tubes were set up, two (A, B) to assess total binding (containing [aH]pGEs) and the other (C) to assess nonspecific binding (containing pH]PGE* + PGEa) as before. After 1 h of incubation at 37°C with 3.5 X lo-’ M WPGE, an excess PGEs (0.1 ml, 1.5 X 10s6 M) was added to one set of total binding tubes (B), 0.1 ml buffer to the other sets. Norite-gelatin was added as above to tubes A, B, C, at each time interval, from 0 to 4 h. The specifically bound [sI-HPGE2was calculated at each time as: (B, - C,)/(A, - C,) X 100 = % of control at time t, where t varies from 0 to 4 h. This method eliminates errors caused by changes in nonspecific binding. [3H]PGEz (100-200 Ci/mmol) was obtained from New England Nuclear, unlabeled PGs courtesy of the Upjohn Company. Oxytocin was from CalBiochem, indomethacin from Sigma. RESULTS
In all protein fractions [3H]PGE2-specific binding was found to be saturable, as previously reported for one of the fractions (9). Nonspecific binding was found to be linear with respect to free [‘H]PGEa concentration. Preliminary experiments showed that PG binding was rapid. Halfmaximum binding occurred at 15 min and was essentially complete by 1 h at 37°C. At lower temperatures decreased rate of specific binding occurred. Nonspecific binding increased with time and temperature. There was no specific PGEa binding in boiled preparations, though nonspecific binding was increased (results not shown). Figure 1 shows the dissociation of specifically bound rH]PGEa indicating the reversibility of the reaction. The rate of dissociation decreased with time. Quantitation of the PGEa receptor was
702
CARSTEN AND MILLER
carried out by Scatchard analysis. Comparison was then made of the receptor in the various subfractions obtained on sucrose density gradient B. Results were normalized for protein concentration. This method was validated by showing that a 2.5-fold increase of protein in the incubation mixture did not change the calculated Kd or maximum binding sites per gram of protein. The results of the Scatchard analysis for the sarcoplasmic reticulum fraction (C) and two plasma membrane fractions (D and E) for all experiments are tabulated in Table 1. Kd values were not significantly different between C, D, and E. The maximum number of binding sites, however, is significantly greater in D than in either C or E. Figure 2 presents Scatchard plots from the single human experiment. As can be seen, a single straight line fits the points, thus demonstrating only one kind of high-affinity binding site with a single Kd. The single human experiment falls within the range of Kd and maximum
i io 6b
Ii0
240
Minutes
FIG. 1. Reversibility of [aHJPGE* binding in the presence of excess unlabeled PGE, in the lightweight microsomal fraction from gradient A(3545% sucrose); average of three experiments. [dHJPGEswas allowed to bind for 60 min at 37’C. Excess of unlabeled PGEB was added and allowed to compete for the time indicated. Each point represents the difference in total [%lpGEr binding in the presence and absence of excess PGEo added at 0 time, divided by control which is total binding for 60 min plus the time indicated minus nonspecific binding for the same time interval.
TABLE I SPECIFIC PGEp BINDING CONSTANTS FOR SUBCELLULAR FRACTIONS
C D E
2.50 i
4
B I"
(W
(molk)
Number of experiments
11.2 * 0.8 x 10-I’ 21.4 k 2.3 X lo-” 12.2 + 1.0 x 10-I’
11 8 8
0.19 x 10-L
3.78 + 0.30 X 10-O 3.05 * 0.26 x 10-s Average
2.74 * 0.14 X 10”
PC C-D6 D-Eb C-Eb
“8 ns “S
0.0005 0.002 ns
‘Mean + SEM. b By paired t test in eight
experiments.
number of binding sites presented in Table I. The largest number of binding sites was fond in fraction D, a plasma membrane fraction. From our data, fractions C and E appear to have the same number of binding sites. However, fraction E is a plasma membrane fraction, and C is a sarcoplasmic reticulum fraction. When increasing amounts of unlabeled PGs were incubated with a constant amount of rH]PGE,, dose-dependent inhibition of binding occurred as seen in Fig. 3. For PGEz, half-maximal inhibition occurred at approximately 5.2 X lo-’ M, for PGEi, at 3.8 X lo-’ M. Other PGs required between 500 and 3300 X lo-‘M for halfmaximal inhibition. The order of potency for inhibition was PGE2 = PGEi %PGF& > PGA2 > PGF,, > PGB2. Oxytocin did not inhibit PH]PGEz binding. DISCUSSION
Specific binding of hormones to membrane preparations has been associated with hormone receptors in a number of tissues. Specific binding implies that there is a finite number of binding sites, hence the fraction bound decreases as the total concentration of hormone increases (10). We have demonstrated this relationship in a previous publication (9). A Scatchard plot of the data yields binding constants and maximum number of binding sites. A prerequisite of this analysis is that the
PROSTAGLANDIN
703
RECEPTOR IN MYOMETRIUM
0.06 F 005 I 2 I x 004 I; f 0.03 QJ g
002
2 m
0.01
-
-
1.
. - .
‘h 0
4
8 Speuflc
BIndIng
(mol/gxlO-“I
FIG. 2. Scatchard plots of rH]PGE2 binding to several membrane fractions from human myometrium. The results are corrected for nonspecific binding and normalized for protein concentration. (c) K,j = 2.75, B,,, = 9.35; (D) & = 3.63, B,,,, = 19.9; (E) & = 3.33, B,, = 13.6; (Kd (M X lo-‘), B,., (mol/g X lo-“)). C is enriched in sarcoplasmic reticulum, D and E are enriched in plasma membrane-(6).
reaction is reversible. Though exchange of bound TH]PGE, for PGEz is limited to 65% in 4 h, the initial rate is very high. The best interpretation is that irreversible changes related to prolonged incubation at 3’7°C are responsible for this observation. The fact that the rate of specific binding increases with increasing temperature, and that specific binding is absent in boiled preparations, is suggestive of the protein nature of the “receptor.” After correction for nonspecific binding (10) our Scatchard plots demonstrate a single affinity of this site over the concentration tested. While we cannot eliminate a specific binding site of much lower affinity, such a site would probably not be a candidate for a receptor. Our Kd fits with Kd for PG binding in other systems (11, 12) and is in the range of biological activity reported (13). Like other investigators, we have demonstrated that PGs of lower uterine contractile activity exhibit lower competitive effects on specific PGEz binding. The relatively low competitive effect of PGFti agrees with findings of other investigators (12) and suggests the existence of separate receptors for PGEz and PGF&. There was no competition of oxytocin for the PGEz binding site. Oxytocin, a polypeptide contractile agent, is thought to work through a different receptor, because of major structural differences.
Previous work showing a partial purification of subcellular components encouraged us to look for localization of the PG receptor. These fractions, obtained after sucrose density gradient centrifugation, were previously characterized (6). To verify the distribution of subcellular components in the preparations used in the present work, periodic checks were made using 5’-nucleotidase as a marker. No difference in the distribution was found from that previously reported. The subfractions obtained were enriched in sarcoplasmic re100
/
3
30 Unlabeled
\.‘.,. 300 Competttor(M
3poo
20,000
x 10-T
FIG. 3. The specificity of rHjPGE* binding in the light-weight microsomal fraction from gradient A (25-45% sucrose). The percentage of control is calculated as the difference in counts bound in the presence and absence of a competitor, divided by the difference in counts bound in the presence and absence of 2 X 10m5M cold PGE2, times 166.
704
CARSTEN
ticulum (C, 2423% sucrose) and plasma membrane (D, 28-33% sucrose, and E, 3345% sucrose). D and E are plasma membrane fractions as demonstrated by high and approximately equal levels of 5’-nucleotidase activity, ouabain inhibition of Na,K-ATPase, and WGA binding. They differ from one another in as much as ATP-dependent calcium accumulation is more than twice as high in D as in E. Onepoint analyses of PG-specific binding in these fractions were done previously, but interpretation had to wait for complete Scatchard analysis. These are presented in the present paper. The ATP-dependent calcium accumulation correlates with the number of specific PGEz binding sites in plasma membrane fractions D and E. However, when comparing fraction C (sarcoplasmic reticulum) with either plasma membrane fraction, this relationship does not hold. Though there is an approximate twofold greater ATP-dependent calcium accumulation in Fraction C than in plasma membrane fraction D, the number of PGE:! binding sites is only approximately half of that found in D. These results are best explained by assuming that both ATP-dependent calcium accumulation and specific PG binding are intrinsic properties
AND MILLER
of both sarcoplasmic reticulum and plasma membrane. Alternative hypotheses, such as cross-contamination, cannot account for all of these results taken together. REFERENCES 1. WIQVIST, N., AND WILHELMSSON, L. (1979) @need Obst& Invest lO, l-8. 2. KIMBALL, F. A., KIRTON, K. T., SPILUL, C. H., AND WYNGARDEN, L. J., (1975) Bill Reprod 13,
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