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At least 289 records · Page 16Linked to original sources
PubMed1984

A simple method to determine whole cell uptake of radiolabelled oestrogen and progesterone and their subcellular localization in breast cancer cell lines in monolayer culture.

Specific uptake of tritiated 17 beta-oestradiol and R5020, a synthetic progestin, in breast cancer cell lines ( MCF7 and T47D) growing in monolayer culture in multiwell plates has been shown. Binding characteristics, calculated by Scatchard analysis, indicate the presence of steroid receptors of similar affinities and capacities to those already obtained with broken cell preparations. Lysis of the cells by treatment with a hypotonic buffer reveals the subcellular localization of the receptors so the method can be used to study receptor dynamics such as nuclear translocation and processing. Cell growth can be measured by DNA determination directly in the multiwell plates. Thus, the method provides a convenient way of studying the effects of steroid hormones (or any antihormone or chemotherapeutic agent) on growth and receptor content of breast cancer cells in monolayer culture.

Breast Neoplasms↗
PubMed1985

Oestradiol and progesterone receptors in the pig oviduct during the oestrous cycle.

The concentrations of the tissue receptors for oestradiol (E) and progesterone (P) in the porcine oviduct at different stages on the oestrous cycle have been investigated by in vitro binding and exchange methods. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The concentrations of ERc and ERn were two-fold higher in the ampulla as compared to the isthmus. The amount of ERc in the isthmic portion of the oviduct did not vary throughout the oestrous cycle. However, the ampullar ERc concentrations increased during prooestrus, showed a maximum at standing oestrus, thereafter decreasing. Significant variations in the amount of oviductal ERn were observed. Despite the differences in ERn amounts between segments, the concentration of ERn increased significantly during late prooestrus, attaining a three-fold elevation and remaining elevated during the period of standing oestrous and early luteal phase (days 3-4), thereafter returning to basal levels. No significant variations in the amount of isthmic PRc were found throughout the period studied. The ampulla, however, showed a significant increase in PRc concentrations during standing oestrus, thereafter decreasing. The concentrations of PRn in isthmus and ampulla were of about the same magnitude and varied significantly during the oestrous cycle, increasing in concentration from standing oestrous onwards. The temporal relationships between the variations in levels of oestradiol and progesterone receptors in oviductal tissues and those of the circulating plasma levels were established. The data obtained in this study suggest a relationship between the changes in the levels of oestradiol and progesterone oviductal binding during the first days of the oestrous cycle, and the gamete and embryo transport throughout the oviduct in the porcine species.

Animals↗
PubMed1985

Use of a new radioactive ligand, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H]19-nortestosterone for the estimation of androgen receptors in rat liver cytosol.

The use of the testosterone derivative, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H] 19-nortestosterone for the estimation of cytosolic androgen receptors in male rat liver is described. Use of this compound demonstrates binding which has a similar dissociation constant, maximum binding and steroid specificity to that seen with other synthetic testosterone derivatives. In contrast to previous data significant binding to the progesterone receptor also occurs and future studies with this ligand should employ triamcinolone acetonide to block such binding.

Animals↗
PubMed1985

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486.

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.

Affinity Labels↗
PubMed1986

Structural analyses of progesterone receptors.

Progesterone receptors of T47Dco human breast cancer cells consist of two equimolar hormone-binding proteins of mol. wt approximately 85,000 (A protein) and 115,000 (B protein). Both proteins can be demonstrated in intact cells by in situ photoaffinity labeling; that is, in cells treated with the synthetic progestin [3H]R5020, irradiated 2 min with 300 nm u.v., solubilized directly in SDS and subjected to electrophoresis under denaturing conditions. These proteins are 6000-10,000 dalton heavier than the corresponding proteins of chick oviducts. This difference has been measured by direct comparison of photolabeled chick and human receptors on SDS-PAGE and by immunoblotting with the 9G10 antibody prepared against chick protein B. The antibody binds to a protein of mol. wt approximately 106,000 in human cells that is smaller than the hormone-bound B protein and larger than the hormone-bound A. In T47Dco cells, in situ photolabeled, untransformed receptors, as well as transformed nuclear-bound receptors, have equimolar amounts of A and B proteins. This ratio remains stable during a 1 h 37 degrees C in vitro incubation. Analysis of the in situ labeled receptors on gradient gels shows that the untransformed B protein exists as a doublet of mol. wt approximately 115,000 and 119,000 while the A protein is a singlet. After [3H]R5020 treatment, nuclear receptors change further: during the first 30 min in the nucleus the B protein shifts entirely to the heavier, mol. wt = 119,000 form. Between 30 and 60 min after nuclear binding, the A protein first becomes a doublet of 85,000 and 89,000 dalton then shifts entirely to the 4000 dalton heavier form. Later, nuclear processing leads to the simultaneous disappearance of both proteins without generation of smaller molecular weight fragments. Cleveland mapping studies show that the A and B proteins are closely related; despite the initial difference in the molecular weight of A and B, digestion with S. aureaus V8 protease yields identical fragmentation patterns for each, with sequential peptides of mol. wt approximately 49,000, 39,000, 26,000 and 14,000.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗
PubMed1986

Assessment of progestin receptor polymorphism by various synthetic ligands using HPLC.

Tritiated R5020 and [3H]ORG-2058 were utilized to investigate apparent polymorphism of progestin receptors by vertical-tube gradient centrifugation and HPLC in size exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Rapid centrifugation (3 h) following molybdate stabilization (1 h) showed mainly 8-9S receptor species with 90-96% recovery. [3H]R5020 appeared to associate with a receptor isoform sedimenting faster than that bound to [3H]ORG-2058. Excess unlabeled R5020 did not eliminate all of the [3H]R5020 binding by the 8-9S component suggesting some nonspecific association while excess unlabeled ORG-2058 suppressed this binding by either ligand. Separate labeling of cytosol with each ligand and mixing prior to gradient separation showed at least two receptor species isoforms sedimenting in the 8-9S region with mol. wt of 190,000 and 173,000 D. Sephacryl S-300 chromatography revealed two radioactive peaks with either ligand but with slight molecular weight differences. HPSEC confirmed the presence of isoforms with different molecular size and shape as a function of the radioactive ligand employed. HPIEC showed the presence of two labeled receptor species irrespective of the ligand used. The first peak appeared at the void volume of the column (10 mM), co-eluted with free ligand, indicating the possibility of ligand stripping by the column. The second peak bound both steroids specifically and eluted with 100 mM phosphate. HPCF identified a single specific receptor eluting at a pH of 5.6-6.1, but with free steroid in the void volume irrespective of the ligand used. [3H]ORG-2058 appeared to be less prone to the stripping phenomenon than was [3H]R5020. These data suggest these ligands either bind to different progestin receptor species or they modify receptor characteristics in a fashion that allows separation based upon size and shape.

Centrifugation, Density Gradient↗
PubMed1986

Progestin and antiprogestin effects on progesterone receptor transformation.

Transformation of the rabbit uterine progesterone receptor following binding to several synthetic steroids was studied. Cytosolic receptors were prepared with and without 10 mM sodium molybdate. Following incubation with the 3H-ligands the cytosols were chromatographed on phosphocellulose minicolumns. The rank order of the compounds to promote transformation in the absence of molybdate was: medroxyprogesterone acetate (MPA) greater than 17 alpha, 21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) greater than progesterone much greater than deoxycorticosterone (DOC) much greater than 20 alpha-hydroxyprogesterone (20 alpha OH-P). The rank order was the same in the presence of molybdate, but the amount of transformation was reduced by 35-90%. Molybdate inhibited transformation to a greater extent when the receptor was bound to progesterone, DOC and 20 alpha OH-P than when bound to MPA or R5020. The antiprogestin, 11 beta-[4-(dimethylamino)phenyl]-17 beta-hydroxy-17-(1-propynyl)-4,9-estradiene-3-one (RU38486, synthesized by The Upjohn Company and designated U-66990), promoted approximately twice as much receptor transformation as did progesterone. MPA, R5020 and U-66990 all dissociated from the progesterone receptor much more slowly than did progesterone. In all cases dissociation was faster in the presence of molybdate than in its absence. These data demonstrate that potent progestins (MPA and R5020) promote a greater amount of receptor transformation than does progesterone, and that steroids with little progestin bioactivity (DOC and 20 alpha OH-P) promote very little transformation. In addition, the antiprogestin activity of U-66990 cannot be attributed to a lack of progesterone receptor transformation nor to a rapid rate of dissociation from the receptor.

Animals↗
PubMed1986

Estrogenic feminization of the LH response to orchidectomy: association with prolonged nuclear estradiol receptor retention and induction of cytoplasmic progestin receptors in brain and pituitary.

A sex difference in the LH rise after gonadectomy is clearly observable in the rat. While male rats respond with an early (10-12 h) increase in LH after orchidectomy, a delayed response (2-3 days) is recorded after ovariectomy. In this study we tested the hypothesis that the delayed response to gonadectomy in the E2-treated males is due to a more prolonged retention of E2 (when compared with the corresponding male feedback signal, testosterone) within specific central nuclear receptor sites. Orchidectomized (ORDX) animals implanted with either empty or E2-filled Silastic capsules were sacrificed at 0, 24, 48 and 72 h after ORDX or E2-capsule removal. LH levels in ORDX rats rose several-fold by 24 h, whereas E2-treated, ORDX rats, showed no changes in peripheral LH levels until 72 h after E2-capsule removal. At the time of E2-capsule removal (0 h) large increases in nuclear estradiol receptor (NER) levels were seen in anterior pituitary, preoptic area, and hypothalamus (HYP). Twenty-four hours after E2-capsule removal, NER levels were still high in the 3 areas, and by 48 h NER values had returned to control (ORDX) levels, with the exception of HYP where they were slightly but significantly elevated. The increase in NER, as well as the subsequent decline after E2-capsule removal was paralleled by similar changes in cytosolic progestin receptor (CPR) levels in all three regions. Cytosolic testosterone levels were not changed by the E2-treatment. The results indicate that the feminized response to orchidectomy observed in E2-implanted males is related to a prolonged retention of the E2-receptor in nuclear sites. Further, they indicate that E2-treatment in males, as is the case in females, can induce a marked increase in progestin receptor levels within specific brain regions as well as in the pituitary. The reduction in NER and CPR levels to castrate values precedes the first detectable increase in peripheral LH levels. In conclusion, the pattern of LH rise after gonadectomy in the rat is dependent upon the steroidal milieu at the time of removal of the gonads.

Animals↗
PubMed1986

Analysis of the relation between receptor binding affinity and antagonist efficacy of antiglucocorticoids.

The biological potencies of four antiglucocorticoids, RU486 (RU), dexamethasone-oxetanone (DOX), R5020, and progesterone have been studied with respect to dexamethasone induction of tyrosine aminotransferase (TAT) in rat hepatoma tissue culture (HTC) cells. Their inhibitory effects in whole-cell competition binding studies (at 37 degrees C) and in TAT induction studies were analyzed by Dixon plots and Schild plots, respectively. We show that: In both cases, there is an actual competition of each antiglucocorticoid with the agonist dexamethasone for the same binding site; the two Kd values derived from the two plots are almost identical for each antiglucocorticoid; RU486 can be distinguished from the three other antiglucocorticoids by its high biological efficacy and its high affinity for the glucocorticoid receptor in whole cells at 37 degrees C (identical to its affinity in cytosol at 0 degree C). These results imply that: There is a linear correlation between the antagonist efficacies of antiglucocorticoids and their affinities for the glucocorticoid receptor in whole cells at 37 degrees C; the antagonistic action is solely mediated by competition with the agonist for the receptor binding site; this is verified by the fact that in all cases, in the presence or absence of antiglucocorticoids, a specific TAT induction level was always related to the same level of receptor saturation by the agonist in whole cells; the phenomena responsible for the high antagonist efficacy of RU486 are also responsible for its high affinity in whole cells at 37 degrees C.

Animals↗
PubMed1986

Identification of two forms of progesterone receptor from chick oviduct cytosol using non-denaturing gel electrophoresis.

Non-denaturing polyacrylamide gel electrophoresis and non-denaturing agarose gel electrophoresis have been used to resolve [3H]R5020-binding components from chick oviduct cytosol. From both gel systems 2 peaks of bound radioactivity are resolved which display these properties of authentic progesterone receptor: binding of R5020: steroid specificity, saturability, and restriction to target tissues. The two peaks are approximately equal in magnitude, and there is no evidence for interconversion of the 2 peaks. The presence or absence of 10-20 mM sodium molybdate during cytosol preparation had no effect on the magnitude or mobility of either peak. Neither peak contains salt-dissociable components which affect its electrophoretic properties, suggesting a possible alteration of native receptor forms during electrophoresis.

Animals↗
PubMed1986

The effect of dithiothreitol on the kinetics of dissociation of dexamethasone from the non-transformed mammary cytosolic glucocorticoid receptor.

Sulfhydryl reducing agents such as dithiothreitol are required for maximum binding of dexamethasone to the mammary cytosolic glucocorticoid receptor, but little is known concerning the effects of dithiothreitol on the kinetics of the binding reaction. In this report we have examined the influence of dithiothreitol on the dissociation kinetics of dexamethasone from the non-transformed glucocorticoid-receptor complex at 0-4 degrees C under various experimental conditions. Without dithiothreitol, the rate of dissociation of dexamethasone remains essentially the same (t1/2 approximately 17 h) regardless of the method chosen to monitor dissociation. With dithiothreitol, however, there is a marked acceleration in the rate of dissociation of receptor-bound dexamethasone when an excess of unlabeled dexamethasone is used to study dissociation (t1/2 approximately 5 h) but not when dissociation is investigated by removal of free labeled dexamethasone by charcoal adsorption (t1/2 approximately 21 h); dithiothreitol also accelerates the observed rate of dissociation when a combination of these methods is used. An acceleration in the rate of receptor-bound dexamethasone is also observed when an excess of the synthetic progestin, R5020, is used in the dissociation assay. The possible reasons and importance underlying these findings have been discussed.

Animals↗
PubMed1987

Occupied and unoccupied type II estrogen binding sites in human breast cancer.

Using a saturation analysis over a wide range of [3H]estradiol at two temperatures 4 and 22 degrees C we have determined unoccupied (4 degrees C) and total (22 degrees C) type II estrogen binding site (EBS) levels in individual cytosols of 100 patients with breast cancer (50 post and 50 premenopausal). Exchange was found to be complete after 18 h at 22 degrees C and receptor degradation was negligible during this treatment. Steroid specificity and affinity determined by Scatchard and Rosenthal plot analysis were not altered at 22 degrees C. Carcinomas presented a higher total type II REBS level as compared unfilled type II binding sites or the classical ER, independently of menopausal status, phase of the menstrual cycle or positivity of ER. On the other hand, unoccupied type II EBS level was strongly correlated to the concentration of type I ER, being higher on the post-menopausal group and older patients.

Breast Neoplasms↗
PubMed1987

A progesterone receptor affinity chromatography reagent: 17 alpha-hexynyl nortestosterone sepharose.

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17 alpha-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17 alpha-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17 alpha-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred microliter of these beads adsorbed 71% of the PgR present in 1 ml of cytosol from T47D cells. This adsorption was inhibited by 10 microM progesterone but not cortisol, indicating the specificity of the binding. Comparisons with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.

Animals↗
PubMed1987

Modulation of brain progestin and glucocorticoid receptors by unsaturated fatty acid and phospholipid.

In an attempt to learn how nonsteroidal factors modulate brain progestin and glucocorticoid receptors, the effects of saturated and unsaturated fatty acids, and phosphatidylinositol on the binding of [3H]R5020 or [3H]dexamethasone, determined by sucrose density gradient and gel filtration on LH20, were examined in the cerebral cortical cytosol from 10-day-old female rats which contain a considerable amount of progestin and glucocorticoid receptors. Unsaturated fatty acids such as oleic (C18:1), arachidonic (C20:4) and docosahexaenoic acid (C22:4) depressed the [3H]R5020 or [3H]dexamethasone binding in increasing order, but saturated fatty acids had no effect. Arachidonic and docosahexaenoic acids, which were strong inhibitors, lowered the binding dose dependently. The fatty acid inhibition on brain progestin and glucocorticoid receptors was thus a function of acid dose and degree of acid unsaturation. Interestingly, prostaglandin D2 did not show any effect. Among phospholipids tested the inhibitory effect of phosphatidylinositol on the [3H]R5020 binding was evident, but no significant effect was found with phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine or sphingomyelin. The phosphatidylinositol inhibition was dose dependent. Analysis on kinetics and Scatchard plot have revealed the noncompetitive type of inhibition by arachidonic acid and phosphatidylinositol. From these results it is suggested that the unsaturated nonestrified fatty acid, arachidonic acid, and phosphoinositides modulate the brain progestin and, possibly, glucocorticoid receptors through their binding at sites different from steroid binding sites on the respective receptor molecules.

Animals↗
PubMed1987

Antiprogesterone and antiglucocorticoid actions of RU 486 on rabbit mammary gland explant cultures. Evidence for a persistent inhibitory action of residual progesterone upon the mammary tissue.

The antiprogesterone and antiglucocorticoid compound RU 486 added to pregnant rabbit mammary gland explant cultures had no effect alone but significantly stimulated casein production in the presence of ovine prolactin (PRL) in a dose dependent manner. This stimulation was inhibited by progesterone (Pg) and the Pg agonist R5020. When the explants were cultured for 5 days with two changes of medium, to eliminate all steroids, and hormones added afterwards, the effect of PRL was potentiated, Pg was no longer inhibitory and RU 486 had no effect, RU 486 also could inhibit the stimulatory action of glucocorticoids added to the cultures along with PRL. The compound was able to displace [3H]dexamethasone and [3H]R 5020 from mammary gland glucocorticoid and Pg receptors respectively and proved to have a high relative binding affinity (RBA) for both receptors when compared with typical ligands for each receptor. The RBAs of RU 486 and the steroids used in this study to mammary gland glucocorticoid and Pg receptors correlated well with the ability of RU 486 to block their biological activities. These results demonstrate that RU 486 has both antiglucocorticoid and antiprogesterone activities in pregnant rabbit mammary glands as well as the existence of a strong inhibitory residual action of Pg in the gland that persists during the first 48 h of culture and that can be eliminated by RU 486 or after several days of culture with no hormones.

Animals↗
PubMed1988

RU486, a progestin antagonist, binds to progesterone receptors in a human endometrial cancer cell line and reverses the growth inhibition by progestins.

The human endometrial cancer cell line, IK-90 cells, contains estrogen-independent progesterone receptors (PR) and is progestin sensitive. Accumulation of glycogen in the cytoplasm of IK-90 cells as well as growth inhibition of the cells in response to progestins are observed. In the present study, the effects of RU486, a progestin antagonist, on IK-90 cells were investigated in a serum-supplemented medium. Scatchard plot analysis of cytoplasmic binding data in the cells revealed a high affinity binding site for RU486 (Kd, 2.6 nM) with maximum binding sites of 169 fmol/mg protein. However, the binding ability to DNA-cellulose of heat activated [3H]RU486-PR complexes was lower when compared with that of the progestin agonist [3H]R5020-PR complexes, suggesting a decrease in progestin activity of RU486 in IK-90 cells. The addition of 1 microM RU486 to culture medium produced periodic acid-Schiff-positive granules in the cytoplasm of the cells. On the other hand, RU486 (1 nM-1 microM) did not significantly inhibit the growth of cells. However, RU486 (0.1-1 microM) totally prevented the growth-inhibitory effect of R5020 (0.1-1 microM) on IK-90 cells. In conclusion, RU486, an antiprogestin, had a dual activity both a progestin antagonist and weak agonist in human endometrial cancer cells, which was not mediated through the estrogen receptor system.

Binding, Competitive↗
PubMed1988

Patterns of estrogen and progesterone receptors in rhesus monkey endometrium during secretory phase of normal menstrual cycle and preimplantation stages of gestation.

Using validated methods, estradiol receptor (ER) and progesterone receptor (PgR) levels have been estimated in endometria collected in secretory phase of normal menstrual cycle and preimplantation stages of gestation from rhesus monkeys (Macaca mulatta). Endometrial PgR in both cytoplasmic and nuclear compartments decreased significantly (P less than 0.001) from day 2 to day 6 post-ovulation in both groups, but in fertile cycle, absolute levels of nuclear PgR remained significantly higher (P less than 0.05) on days 4, 5 and 6 of gestation, ER concentrations, both total (P less than 0.02), as well as cytoplasmic (P less than 0.01) declined significantly in secretory phase of normal menstrual cycle while nuclear ER levels remained unchanged. In the preimplantation period, ER patterns remained unvarying on days 2-6 of gestation in both cytoplasmic and nuclear compartments; their levels in nuclear fraction were significantly higher from day 3 onwards while, total cytoplasmic ER concentrations were higher from day 4 of gestation compared with the values obtained for secretory phase tissues from normal ovulatory cycles. No changes were, however, detected in apparent equilibrium dissociation constants (Kd) for the sex steroid receptors in endometria obtained from fertile and non-fertile cycles. It has been suggested that in prenidatory stage rhesus monkey endometrium elevated concentrations of nuclear ER and PgR possibly indicate higher degree of nuclear occupancy required for endometrial differentiation permitting blastocyst implantation.

Animals↗
PubMed1988

Molybdate stabilized rat uterine progesterone receptors: evidence for two mechanisms.

Molybdate (Mo), EGTA, or protease inhibitors substantially increase detectable rat uterine progesterone (Pg) receptors. Rehomogenization experiments demonstrated that receptor levels decreased in the absence of Mo or protease inhibitors and were not regenerated. Thus Mo prevents an EGTA and protease-inhibitor-sensitive loss of uterine Pg receptors during homogenization. This effect was compared with receptor stabilization at elevated temperature. In contrast to the stability of receptors in the presence of Mo, receptors decreased rapidly to minimal levels by 30 min at 30 degrees C in TESHG (10 mM Tris, 1.5 mM EGTA, 12 mM thioglycerol, 10% glycerol) or TG buffers. The ability of EGTA to mimic receptor stabilization by Mo during homogenization, compared with its ineffectiveness at 30 degrees C, suggested fundamentally different mechanisms for these two phenomena. Similarly, 0.3 M KCl prevented Mo stabilization of the receptors at 30 degrees C, but did not change their recovery after homogenization. Results with protease inhibitors were also consistent: addition of 2-5 mM leupeptin and 500 microM PMSF to TG during homogenization resulted in substantially increased (P less than 0.01) receptor recovery, but leupeptin (+/- the temperature-labile PMSF) did not prevent the Pg receptor losses at 30 degrees C. The transformation state of the receptors may be important, since receptors were untransformed in the presence of either EGTA or Mo. Moreover, KCl transformed the receptors in parallel to their instability at 30 degrees C. In conclusion, Mo stabilizes Pg receptors during temperature elevation by a different mechanism from that involved during homogenization. Although the parallel effects by EGTA, molybdate, and the protease-inhibitors during homogenization is consistent with inhibition of Ca2+-dependent proteolysis, other possible mechanisms must be considered in future studies.

Animals↗