Trisomy 21 (Down's syndrome). Glutathione peroxidase, hexose monophoshate shunt and I.Q.

This work was undertaken to determine the ability of human prostate tissue to synthesize prostaglandin F₂ alpha and to metabolize the F₂ alpha to 15-keto-PGF₂ alpha, 13,14-dihydro-15-keto-PGF₂ alpha and 13,14-dihydro-PGF₂ alpha. Surgically obtained prostate tissue was incubated with either ³H-arachidonic acid or ³H-PGF₂ alpha and incorporation of tritium into PGF₂ alpha or the aforementioned metabolites was determined. The identity of these substances was corroborated by gas chromatography and chemical ionization mass spectrometry. Significant increases above control levels were obtained when testosterone and lactogen were added to the incubation mixture. Maximal biosynthesis and metabolism of the prostaglandin was obtained when androgen and lactogen were present together. It is believed that androgen and lactogen produce a synergistic acceleration of PGF₂ alpha synthesis and metabolism.     

Prostaglandin F2α specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF_2α specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 × 10^?9M and the concentration of binding sites of ～0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF_2α specific binding sites indicates specificity for the 9α-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF_2α analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF_2α binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific ³H-PGE₁ binding could be demonstrated, no high affinity sites could be quantitated. ³H-PGE₁ binding appeared unaffected by changes in temperature or time of incubation, whereas PGF_2α specific binding was significantly modified by both these factors.     

The presence of discrete receptors for prostaglandin F2 alpha in the cell membranes of bovine corpora lutea.

The cell membranes isolated from bovine corpora lutea bound ³H-prostaglandin (PG) F₂α with high affinity and specificity. The specific binding of ³H-PGF₂α was detectable at 10^?10M added ³H-PGF₂α and reached saturation at 10^?7M to 10^?6M. Unlabeled PGF₂α, as low as 10^?9M, inhibited the binding of ³H-PGF₂α with complete inhibition occurring at 10^?6M. The Scatchard analysis of equilibrium binding data revealed that the PGF₂α receptors are heterogeneous: Kd₁?5.1 × 10^?9M, n?289 fmoles/mg protein; Kd₂?1.8 × 10^?8M, n?780 fmoles/mg protein. The relative affinities of various other PGs for binding to PGF₂α receptors were (PGF₂α?100%): PGF₁α?17.5; PGE₁?0.8; PGE₂?22.4; PGA₁?0.007; PGB₁?0.01. The specificity and affinity of ³H-PGF₂α binding is consistent with the possibility that this receptor interaction may reflect an initial event in the action of PGF₂α as a luteolytic agent.     

Properties of [3H] prostaglandin F2α binding to dispersed bovine luteal cells

Intact viable bovine luteal cell suspensions prepared by collagenase digestion of luteal tissue were used in studying the selected properties of [³H] prostaglandin (PG) F_2α binding and compared with those observed in plasma membranes. [³H]PGF_2α specific binding to luteal cells was a rapid (K₁ = 8.4 × 10⁴M^?12αS^?1), reversible (K_?1 = 1.8 × 10^?4S^?1) and saturable process at 24°. There was a single class of receptors with an apparent dissociation constant of 10.6 nM and 1.8 × 10⁵ receptors per cell. The presence of increasing amounts of unlabeled PGs inhibited [³H]PGF_2α binding in a dose-dependent manner. The potency order for this inhibition was: (15S) 15-methyl-PGF_2α methyl ester > ICI-80,996 > PGF_2α > ICI-81,008 > PGF_1α > PGE₂, (15S) 15-methyl-PGE₂ methyl ester > PGF_2α metabolites > other PGs, PGF_1α metabolites and PGE metabolites. Other than the homegeneous nature of binding and a greater association rate in cells, the rest of the [³H]PGF_2α binding properties in cells were in good agreement with those observed in plasma membranes.     

Prostaglandin synthesis and binding is increased in regressing NMU mammary carcinoma

Mammary tumors induced in Buffalo rats by treatment with nitrosomethyl urea will regress after oophorectomy. Their ability to synthesize and bind prostaglandins E and F₂α was studied in the growing and regressing states. Prostaglandins present in suspensions of 100,000 xg tumor membranes after 2 hr incubation at 37°C ± 5×10⁻⁴M indomethacin were partially purified by silica gel column chromatography before assay by specific PG RIA. The amounts of PGE and F₂α synthesized rose from 0.13 and 10.5 ng/mg protein in the growing tumors to a maximum of 1.2 and 26.5 ng/mg protein 5 days after oophorectomy. Specific binding of ³H-PGE₂ and ³H-PGF₂α to 15,000 xg tumor membranes was achieved during a 45 min incubation at 23°C ± excess unlabelled PG. Free and bound prostaglandins were separated by filtration. Binding reached equilibrium after 30 min, was saturable and reversible. Scatchard analysis revealed high affinity binding of PGF₂α but only low affinity PGE₂ binding in membranes obtained from growing tumors. A 2–3-fold increase in specific binding of PGE₂ and PGF₂α was noted at 4 days after oophorectomy which represented an increase in the number of PGF₂α receptors. PGE₂ binding retained a low affinity character. The elevated PGF₂α synthesis rates observed in the regressing tumors coupled with a regression-associated increase in receptor number suggests that PGF₂α-plays a significant role in hormone-dependent mammary tumor regression.     

Prostaglandin F2α antagonizes thromboxane A2-induced human platelet aggregation

The effects of prostaglandin F_2α on human blood platelet function were investigated. PGF_2α at 15 μM completely blocked platelet aggregation induced by 500 μM arachidonic acid or 3 μM U46619 but had no effect on aggregatin induced by 7.5 μM ADP. A similar specificity of action was not obtained with either PGI₂ or PGE₂. Thus concentrations of PGI₂ (3 nM) or PGE₂ (20 μ M) which inhibited U46619-induced aggregation by 100% also blocked ADP-stimulated aggregation.The inhibitory properties of PGF_2α were not related to increases in platelet cAMP, since direct measurement of intracellular cAMP revealed that 15 μ M PGF_2α produced no substantial change in cAMP levels. This finding was in direct contrast to results obtained using either PGI₂ or PGE₂. Both PGI₂ (3 nM) and PGE₂ (20 μ M) induced significant increases in platelet cAMP levels.The possibility that PGF_2α directly interacts at the platelet TXA₂/PGH₂ receptor was investigated by measuring [³H]PGF_2α binding to isolated platelet membranes. It was found that [³H] PGF_2α binding reached equilibrium within 30 min at room temperature and could be 90% displaced by addition of 1000 fold excess of unlabelled PGF_2α. Furthermore, when 1000 fold excess of either the TXA₂/PGH₂ “mimetic” U46619 or the TXA₂/PGH₂ antagonist 13-azaprostanoic acid was added, specific [³H] PGF_2α binding was displaced by 95% and 85% respectively. In contrast, the same molar excess of 6-keto-PGF_1α, azo analog 1, or TXB₂, caused displacement of only 15%, 20% or 25% of the [³H] PGF_2α binding. Scatchard analysis indicated that [³H] PGF_2α has two binding sites; i.e., a high affinity binding site with an apparent Kd of 50 nM and a low affinity binding site with apparent Kd of 320 nM. These results suggest that the selective inhibition by PGF_2α of AA or U46619-induced aggregation may be mediated through interaction at the platelet TXA₂/PGH₂ receptor.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Prostaglandin E1 and F2α specific binding in bovine corpora lutea: Comparison with luteolytic effects

Preliminary characterization indicated the presence of separate prostaglandin (PG)E₁ and (PG)F_2α binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 × 10⁻⁹M and 2.1 × 10⁻⁸M for PGE₁ and PGF_2α, respectively. Competition of several natural prostaglandins for the PGE₁ and PGF_2α bovine luteal specific binding sites indicates specificity for the 9-keto or 9α-hydroxyl moiety, respectively. Differences in relative ability to inhibit ³H-PG binding were found due to sensitivity to the absence or presence of the 5,6-cis-double bond as well.Bovine luteal function was affected following treatment of heifers with 25 mg PGF_2α as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contrast, treatment with 25 mg PGE₁ resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE₁ treatment compared to pretreatment values. In so far as data obtained in vitro on PGF_2α relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF_2α induced luteolysis in the bovine, PGF_2α relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE₁ relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

A novel effect of indomethacin on prostaglandin F2 alpha synthesis and metabolism by the human prostate

Human prostate tissue has been found to be an excellent organ for synthesizing and metabolizing prostaglandin F₂ alpha. Employing ³H-arachidonic acid as substrate, tritium incorporation into PGF₂ alpha and three different metabolites, namely, 15-keto-PGF₂ alpha, 13, 14-dihydro-PGFP₂ alpha, and 13, 14-dihydro-15-keto-PGF₂ alpha, has been shown. The identity of these substances was corroborated by gas chromatography and chemical ionization mass spectrometry. Indomethacin has been widely reported as an inhibitor of PGF₂ alpha synthesis. In our experiments, upon adding indomethacin, slight to no inhibition of PGF₂ alpha synthesis was seen. Instead, we found a highly significant increase in the incorporation of the tritium of arachidonic acid into 15-keto-PGF₂ alpha. We interpret these findings as evidence that the indomethacin may block not PGF₂ alpha synthetase, but the Δ13-reductase, thus causing the accumulation of this enzyme's substrate, 15-keto-PGF₂ alpha.     

Cationic dependency of high affinity prostaglandin F2α receptors in bovine corpus luteum cell membranes

The specific binding of [³H] Prostaglandin (PG) F₂α to bovine corpus luteum cell membranes prepared in homogenizing buffer containing either 1 mM EDTA (H-EDTA) or 1 mM Ca²⁺ (HCa²⁺) was examined. The membranes prepared in H-EDTA buffer bound less [³H] PGF₂α and had a single class of PGF₂α receptors with an apparent dissociation constant (Kd) of 2.7 × 10^?8M. The addition of Ca²⁺ to these membranes resulted in increased binding with the appearance of new PGF₂α receptors of Kd = 4.3 × 10^?9M. The membranes prepared in HCa²⁺ buffer contained two classes of receptors with Kds = 2.9 × 10^?9M and 2.9 × 10^?8M. The removal of Ca²⁺ from these membranes resulted in lower binding as well as a complete disappearance of receptors of Kd = 2.9 × 10^?9M. These results suggest the dependency of high affinity PGF₂α receptors, in bovine corpus luteum cell membranes, on cations.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Specific binding of a stable prostacyclin analogue (Iloprost) to rat oxyntic mucosa

It has been reported that prostacyclin (PGI₂) is the predominant species of prostanoid in rat oxyntic mucosa. However since PGI₂ is inactivated under physiological conditions it has not been possible to demonstrate specific PGI₂ binding to the rat stomach. Therefore a stable PGI₂ analogue, Iloprost, was chosen as ligand in this study. Binding of labelled Iloprost to the 20,000 xg homogenate fraction of rat oxyntic mucosa was specific, dissociable, saturable and dependent upon the temperature and time of incubation. Neither tritiated PGE₂ nor 6 keto PGF_1α displayed any significant specific binding to rat stomach. A Scatchard plot of the equilibrium binding data for Iloprost was curvilinear and could be resolved into at least two binding sites. The average parameters determined from Scatchard analysis were: dissociation constants of 1.8 × 10⁻¹¹ M and 7.1 × 10⁻⁸ M and corresponding binding site concentrations of 12.0 pmole/mg and 4800 pmoles/mg protein respectively. PGI₂ was less potent than unlabelled Iloprost in displacing ³H-Iloprost from its binding site. The addition of PGE₂ to the incubation medium resulted in an increase in ³H-Iloprost binding. It is concluded that rat oxyntic mucosa has specific binding sites for PGI₂-like agents but not for either PGE₂ or 6 keto PGF_1α.     

3H] prostaglandins binding to dispersed bovine luteal cells: evidence for discrete prostaglandin receptors.

Suspensions of dispersed bovine luteal cells prepared by collagenase digestion of luteal tissue specifically bound [³H]Prostaglandin (PG) E₁ and [³H]PGF_2α. While the number of sites per cell (～ 1.8 × 10⁵) were about the same for both [³H]PGs, the apparent Kds were different: [³H]PGE₁ ? 2.4 nM; [³H]PGF_2α ? 11 nM. The [³H]PGs binding was inhibited in a dose-dependent manner in the presence of increasing concentrations of unlabeled PGs. Potency order for inhibition of [³H]PGE₁ binding was: PGE₂ > PGE₁ > PGF_2α > PGF_1α. The corresponding data for [³H]PGF_2α was: PGF_2α > PGF_1α > PGE₂ > PGE₁. While [³H]PGE₁ and [³H]PGF_2α bind to their own receptors with high affinity, their affinities for each other's binding were extremely low. Thus, these results demonstrate that luteal cells, like plasma membranes isolated from luteal tissue, contain receptors for PGEs and PGF_2α which are discrete with respect to specificity and affinity.     

Lack of negative cooperativity among binding sites for prostaglandin F2alpha in bovine corpus luteum cell membranes.

The Scatchard analysis of equilibrium prostaglandin (PG) F₂α binding revealed that the binding was heterogeneous. The Hill plot of the same data had a slope of 0.68. This suggested that the heterogeneous nature of [³H] PGF₂α binding was either due to the presence of negative cooperativity or to the presence of two groups of independent binding sites. The kinetic experiments revealed that the presence of excess unlabeled PGF₂α in a diluting medium had no effect on dissociation rates at 25 fold dilution and it even retarded dissociation at higher dilutions. Furthermore, the observations that the low affinity PGF₂α binding sites can exist in the absence of high affinity binding sites and high affinity binding sites can be selectively abolished by treatment with N-ethylmaleimide suggest that negative cooperativity was not responsible for heterogeneous [³H] PGF₂α binding.     

Characterization of prostaglandin F2α receptor of mouse 3T3 fibroblasts and its functional expression in Xenopus laevis oocytes

Prostaglandin (PG) F_2α increased [³H]thymidine incorporation into quiescent NIH 3T3 cells, stimulated phosphoinositide breakdown, and raised intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner with ED₅₀ values of 2.0 × 10^?8 M, 4.6 × 10^?8 M, and 7.5 × 10^?8 M, respectively. The increase in [³H]thymidine incorporation with PGF_2α was additive with that seen with epidermal growth factor (EGF) or insulin. The peak [Ca²⁺]_i increase with PGF_2α was still obvious in the absence of extracellular Ca²⁺ and was insensitive to islet activating protein (IAP) pretreatment. Membranes prepared from NIH 3T3 cells exhibited a specific binding for PGF_2α, which was sensitive to GTP_γS but not sensitive to IAP pretreatment. Xenopus laevis oocytes injected with NIH 3T3 cell mRNA between 18S and 28S rRNA fractionated by sucrose gradient, expressed a PGF_2α-specific Cl^? current when examined by voltage clamp. This Cl^? current was also insensitive to IAP pretreatment and not affected by extracellular Ca²⁺ concentration ([Ca²⁺]_o). These results indicate 1) that the NIH 3T3 cells expressed a specific PGF_2α receptor which is linked to phosphoinositide-specific phospholipase C (PLC) activation and to mobilization of Ca²⁺ via an IAP-insensitive G-proteins(s), 2) that this PGF_2α receptor may play an active role in the proliferation of NIH 3T3 cells, and 3) that this PGF_2α receptor can be expressed in the oocyte system. © 1993 Wiley-Liss, Inc.     

Receptors for prostaglandins and gonadotropins in the cell membranes of bovine corpus luteum

The cell membranes exhibited specific binding to ³H-prostaglandin E₁ (³H-PGE₁) and ¹²⁵I-human chorionic gonadotropin (¹²⁵I-HCG). Unlabeled PGE₁,PGE₂ (1.4 × 10^?7M), PGF_1α and PGF_2α (1.4 × 10^?5M) decreased ³H-PGE₁ binding by more than 80%. The binding of ¹²⁵I-HCG was completely inhibited by 5 × 10^?8M unlabeled HCG. However, the unlabeled PGE₁ (1.15 × 10^?6M) and HCG (8.4 × 10^?7M) had no effect on ¹²⁵I-HCG and ³H-PGE₁ binding respectively. A PG antagonist, 7-oxa-13-prostynoic acid, inhibited only ³H-PGE₁ binding but not ¹²⁵I-HCG binding. These results suggest the presence of specific receptors for PGE₁ and HCG in the cell membranes and that the binding occurs either at two different sites on the same receptor or that each binds to a “different” receptor molecule.     

Effect of in vivo prostaglandin treatment on 3H-PGF2α uptake in hamster corpora lutea

Pregnant hamsters were administered (SC) prostaglandin or vehicle on the morning of the 4th day of pregnancy. Serum progesterone was significantly depressed (p<.01) at 0.5, 2, and 6 hours after treatment with 100 μg PGF_2α. Serum progesterone levels were unchanged 2 hours and 6 hours after treatment with 100 μg PGF_2β and 2 hours after treatment with 1 mg PGF_2β. Progesterone levels were depressed to less than 1 ng/ml 6 hours after treatment with 1 mg PGF_2β. The specific uptake of ³H-PGF_2α in whole hamster corpora lutea was significantly depressed 2 hours and 6 hours following 100 μg PGF_2α treatment. A 15% depression in specific uptake occurred 0.5 hour post-treatment. Treatment with 100 μg PGF_2β resulted in no change. Administration of 1 mg PGF_2β resulted in depressed ³H-PGF_2α uptake at both 2 and 6 hours post-treatment.Prostacyclin (PGI₂) treatment resulted in no change in either ³H-PGF_2α specific uptake or serum progesterone 2 hours after 100 μg treatment SC. These parameters were both reduced approximately 30% 6 hours post-treatment. Treatment with 6-keto-PGF_1α resulted in a complete lack of measurable ³H-PGF_2α uptake and serum progesterone levels less than 1 ng/ml at both 2 and 6 hours after treatment with 1 mg SC.     

PGF2α-induced loss of corpus luteum gonadotrophin receptors

In experiments and we have previously shown that PGF_2α directly antagonized the action of gonadotrophins on the corpus luteum. To determine if this action of PGF_2α may occur as a consequence of an induced loss of gonadotrophin receptors, binding of hCG to rat luteal tissue was measured following PGF_2α treatment . In immature rats which were treated with exogenous gonadotrophin to luteinize the gonads, PGF_2α produced a marked and highly significant decrease in circulating progesterone when administered 24 hours before sacrifice. Although the affinity constant (Ka; 1.2-2 × 10¹⁰ L/M) of the luteal receptor to hCG was not affected, PGF_2α treatment produced a marked fall in the binding capacity of the luteal tissue to hCG. This response was absent, however, when PGF_2α was incubated directly with luteal receptor or administered during early pseudopregnancy when corpora lutea are more resistant to luteolysis. Experiments are in progress to determine if the decrease in capacity of luteal receptors to bind hCG is the mechanism or a consequence of luteolysis produced by PGF_2α.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Prostaglandin E2 receptor in the myometrium: distribution in subcellular fractions

[³H]Prostaglandin (PG) E₂ bound specifically to several subcellular fractions from bovine myometrium. The binding was temperature dependent, rapid, and reversible. PGE₂ and PGE₁ competed for the [³H]PGE₂ binding site. The PGs inhibited in the following decreasing order: PGE₂ = PGE₁ ? PGF_2α > PGA₂ > PGF_1β > PGB₂. 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 × 10^?9. Quantitative differences in the maximum number of binding sites were observed between fractions. One plasma membrane fraction contained 21.4 ± 2.3 × 10^?11 and the sarcoplasmic reticulum contained 11.2 ± 0.8 × 10^?11 mol binding sites/g. The results suggest that there is a high-affinity PGE₂ receptor present in both plasma membrane and sarcoplasmic reticulum.