Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.     

Some properties of prostaglandin E2 receptors in rabbit alveolar bone cell membranes

[3H]prostaglandin E2 (PGE2) binding receptors exist in rabbit alveolar bone cell membranes. The presence of high (Kd = 3.9 X 10(-9) M) and low (Kd = 8.8 X 10(-8) M) affinity binding sites of [3H]PGE2 was demonstrated. The saturation values of [3H]PGE2 for high and low affinity binding sites were 0.13 pmol/mg protein and 1.22 pmol/mg protein, respectively. The digestion of the membranes with pronase, phospholipase C, D and neuraminidase led to a decrease of [3H]PGE2 binding but phospholipase A2 did not.     

Prostaglandin moieties that determine receptor binding specificity in the bovine corpus luteum.

This study provided a pharmacological evaluation of prostaglandin binding to bovine luteal plasma membrane. It was found that [3H]PGF2 alpha' [3H]PGE2' [3H]PGE1 and [3H]PGD2 all bound with high affinity to luteal plasma membrane but had different specificities. Binding of [3H]PGF2 alpha and [3H]PGD2 was inhibited by non-radioactive PGF2 alpha (IC50 values of 21 and 9 nmol l-1, respectively), PGD2 (35 and 21 nmol l-1), and PGE2 (223 and 81 nmol l-1), but not by PGE1 (> 10,000 and 5616 nmol l-1). In contrast, [3H]PGE1 was inhibited by non-radioactive PGE1 (14 nmol l-1) and PGE2 (7 nmol l-1), but minimally by PGD2 (2316 nmol l-1) and PGF2 alpha (595 nmol l-1). Binding of [3H]PGE2 was inhibited by all four prostaglandins, but slopes of the dissociation curves indicated two binding sites. Binding of [3H]PGE1 was inhibited, resulting in low IC50 values, by pharmacological agonists that are specific for EP3 receptor and possibly EP2 receptor. High affinity binding of [3H]PGF2 alpha required a C15 hydroxyl group and a C1 carboxylic acid that are present on all physiological prostaglandins. Specificity of binding for the FP receptor depended on the C9 hydroxyl group and the C5/C6 double bond. Alteration of the C11 position had little effect on affinity for the FP receptor. In conclusion, there is a luteal EP receptor with high affinity for PGE1' PGE2' agonists of EP3 receptors, and some agonists of EP2 receptors. The luteal FP receptor binds PGF2 alpha' PGD2 (high affinity), and PGE2 (moderate affinity) but not PGE1 due to affinity determination by the C9 and C5/C6 moieties, but not the C11 moiety.     

Effects of enzymes and protein modifying reagents on the binding of 3H-prostaglandin E2 to porcine oxyntic mucosa in vitro

In the present study we have investigated the macromolecular nature of porcine oxyntic mucosal PGE2 binding sites and the involvement of specific functional groups in the binding interaction. Incubation of oxyntic mucosal membranes with DNAse or RNAse did not influence binding. Phospholipase A2 was strongly inhibitory while phospholipases C and D exerted variable effects. Trypsinization of the membranes also reduced binding and this reduction was prevented by addition of soybean trypsin inhibitor. Neuraminidase and beta-galactosidase treatments resulted in variable increases in binding activity. The increase in binding was due to an increase in binding affinity and/or binding site concentration. Protein modifying reagents acetic anhydride, N-ethylmaleimide and mercaptoethanol all reduced binding. These results suggest the importance of protein, lipid and carbohydrate components of the membrane in the binding interaction between PGE2 and its binding site. The ability of mercaptoethanol and N-ethylmaleimide to reduce binding suggest the involvement of both sulphydryl and disulphide groups in the PGE2 binding reaction.     

Binding of (3H)prostaglandin E1 to putative receptors linked to adenylate cyclase of cultured cell clones.

A method for assessing the binding of 3H-labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1-binding activity with the presence or absence of PGE1-sensitive adenylate cyclase in several clones. In clone B82, a murine L-cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 X 10(8) M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (KD =30 nM) and kinetic analysis of [3H]PGE1 binding (k1 = 4 X 10(6) liters/mol/min, k-1 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clone N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1-responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.     

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein.

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 microM 5'-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 +/- 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 +/- 0.7 nM) to one site of intermediate affinity (KD = 0.52 +/- 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-affinity prostaglandin E receptors attenuate adenylyl cyclase activity in isolated bovine myometrial membrane

Purified bovine myometrial plasma membranes were used to characterize prostaglandin (PG) E2 binding. Two binding sites were found: a high-affinity site with a dissociation constant (KD) of 0.27 +/- 0.08 nM and maximum binding (Bmax) of 102.46 +/- 8.6 fmol/mg membrane protein, and a lower affinity site with a KD = 6.13 +/- 0.50 nM and Bmax = 467.93 +/- 51.63 fmol/mg membrane protein. Membrane characterization demonstrated that [3H]PGE2 binding was localized in the plasma membrane. In binding competition experiments, unlabelled PGE1 displaced [3H]PGE2 from its receptor at the same concentrations as did PGE2. Neither PGF2 alpha nor PGD2 effectively competed for [3H]PGE2 binding. Adenylyl cyclase activity was inhibited at concentrations of PGE2 that occupy the high-affinity receptor. These data demonstrate that two receptor sites, or states of binding within a single receptor, are present for PGE2 in purified myometrial membranes. PGE2 inhibition of adenylyl cyclase activity support the view that cAMP has a physiological role in the regulation of myometrial contractility by PGE2.     

Chronic morphine treatment increases the number, but decreases the affinity of [3H]-U69,593 binding sites in the rat heart.

In the present study, the effects of chronic morphine treatment on the binding properties of tritiated U69,593, a specific kappa-ligand, in the rat heart were determined. Adult rats were given morphine through osmotic pumps at a rate of 80 micrograms/hour supplemented by daily injection of morphine at increasing doses for 9 days. The increase in colonic temperature to morphine, used as an indicator of development of tolerance in the rat, was measured daily. It was shown that, on day 7 following morphine treatment, the rats developed tolerance to morphine as indicated by an attenuated hyperthermic response to morphine. The [3H]-U69,593 specific binding properties were determined by direct receptor binding assay. The binding sites increased gradually and reached a significantly higher level at day 10. Scatchard analysis showed that both Bmax and Kd increased at day 10 following morphine treatment, indicating an increase in number of sites and a reduction in affinity to the kappa-ligand. Acute morphine injection at a dose of 10 mg/kg did not cause any significant alteration of [3H]-U69,593 binding sites. Two days after withdrawal of morphine, the [3H]-U69,593 binding sites returned to the original level. The finding of the present study indicates that there is an up-regulation of number, but a reduction in affinity of kappa-binding sites following chronic morphine treatment.     

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.     

3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes.

We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.     

PGE receptor characteristics on porcine luteal cells during the estrous cycle and early pregnancy.

This study examined the affinities and concentrations of prostaglandin E (PGE) receptors on porcine luteal cells during the estrous cycle and early pregnancy. Corpora lutea (CL) were obtained from nonpregnant gilts at days 9 (n = 4), 12 (n = 3), and 14 (n = 6); three gilts possessed red, vascular CL and three gilts had white nonvascular CL) of the estrous cycle, and days 9 (n = 4), 12 (n = 3), 14 (n = 5), and 30 (n = 5) of pregnancy. The CL were dissociated enzymatically to disperse single cells and the red blood cells were removed by elutriation. The luteal cells were assayed for specific PGE binding by displacement analysis with use of [3H] PGE2 and varying concentrations of unlabeled PGE2. The specific binding of [3H] PGE2 to luteal cells decreased (p < 0.05) from days 9 to 14 of the estrous cycle, but only decreased (p < 0.05) from days 9 to 12 of pregnancy. Specific binding was higher (p < 0.05) on day 14 of pregnancy than the comparable stage of the estrous cycle. The affinities of PGE receptors decreased (p < 0.05) only on the luteal cells dissociated from red, vascular CL of day 14 nonpregnant gilts compared with those of other days of the estrous cycle and pregnancy. The number of PGE receptors on porcine luteal cells was similar (p > 0.05) in pregnant and nonpregnant gilts, but decreased (p < 0.05) on days 12-14 postestrus. During early pregnancy, it was evident that high affinity PGE receptors are sustained on porcine luteal cells; however, the role of the PGE receptors in maternal recognition of pregnancy remains speculative.     

Evaluation of prostaglandin-receptors in human and rat liver: interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50:2.5 +/- 1.7, (6.1 +/- 2.1) microM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9 +/- 0.9 (2.0 +/- 0.8) microM. The Scatchard analysis on [3H]PGE1- as well as on [3H]iloprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3 +/- 5.2 (21.3 +/- 4.3) fmol/mg protein and a Kd of 2.1 +/- 1.8 (1.9 +/- 0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4 +/- 18.2 (86.1 +/- 13.2) fmol/mg protein and a Kd of 10.5 +/- 2.9 (15.1 +/- 3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4 +/- 13.9 (35.9 +/- 8.2) fmol/mg protein and a Kd of 4.1 +/- 1.2 (1.7 +/- 1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3 +/- 42.1 (142.9 +/- 17.8) fmol/mg protein and a Kd of 16.3 +/- 4.9 (9.2 +/- 7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4 +/- 51.9 (38.8 +/- 7.4) fmol/mg protein and a Kd of 16.2 +/- 3.2 (2.5 +/- 1.2) nM. It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are able to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Failure to detect specific binding sites for prostaglandin F-2 alpha in membrane preparations from rat endometrium

Membrane preparations from endometria of rats in different physiological states (e.g. pseudopregnancy, ovariectomized animals receiving progesterone + oestradiol or oestradiol alone) were studied for [3H]PGF-2 alpha binding by methods which detected PGF-2 alpha binding in ovary preparations and PGE binding in the same endometrial preparations. There was no evidence of high-affinity binding sites for [3H]PGF-2 alpha. Saturable [3H]PGF-2 alpha binding that increased with the onset of uterine sensitivity was detected but this binding does not fulfil all the criteria required for a PGF-2 alpha receptor and is probably due to binding to PG metabolizing enzymes in our preparations, or to binding of [3H]PGF-2 alpha to PGE binding sites. The failure to detect specific PGF-2 alpha binding sites seems to reflect a true absence of these sites in the rat endometrium.     

Decrease of prostaglandin E2 receptor binding is accompanied by reduced antilipolytic effects of prostaglandin E2 in isolated rat adipocytes

The effect of treatment of isolated rat adipocytes with prostaglandin E2 (PGE2) on subsequent [3H]PGE2 binding was studied. In addition, the antilipolytic effects of was studied. In addition, the antilipolytic effects of PGE2, adenosine, and insulin were studied in control and PGE2-treated adipocytes. Treatment of adipocytes with PGE2 (1 microM) decreased the binding of [3H]PGE2 by 61% (from 11.0 to 4.6 fmol/10(6) cells, P less than 0.005). Scatchard analysis of the binding data demonstrated that the decrease of PGE2 receptor binding was due to a decrease in the apparent number of PGE2 receptors while the apparent receptor affinity was unaltered. Reduction of the PGE2 receptor binding was specifically regulated inasmuch as structurally related compounds such as PGF2 alpha and arachidonic acid had only minor effects on subsequent [3H]PGE2 receptor binding. Reduction of the available receptor number was associated with a significant decrease in the antilipolytic effect of PGE2 on the isoproterenol-stimulated lipolysis (P less than 0.05). The maximal antilipolytic effect of PGE2 was decreased by 45%. Desensitization of the biological effect of PGE2 (antilipolysis) was only partially specifically regulated inasmuch as the antilipolytic compound phenylisopropyladenosine also had reduced antilipolytic effect in PGE2-treated cells. However, the antilipolytic effect of insulin was similar in control and PGE2-treated cells. It was found that the PGE2-induced decrease of [3H]PGE2 receptor binding may be due to a very tight coupling between the PGE2 molecule and its specific receptor. This tight coupling may then represent an occupancy of the receptor rather than a true loss of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of trypsin on binding of [3H]epinephrine and [3H]-dihydroergocryptine to rat liver plasma membranes. Evidence for interconversion of binding sites

Treatment of liver plasma membranes with trypsin at low concentrations (1 to 2 microgram/mg of protein) caused at 3- to 4-fold increase in alpha-specific [3H]epinephrine binding. The change was due to an increase in the number of high affinity binding sites, with no change in the dissociation constant. With increasing trypsin concentrations, the dissociation constant was decreased and there was a progressive loss of binding. Elastase, papain, and thermolysin caused similar effects, whereas the thrombin, leucine aminopeptidase, phospholipase A2, phospholipase C, phospholipase D, and detergents did not cause an increase in [EH]epinephrine binding. The increase in epinephrine high affinity binding sites was correlated with a loss of high affinity [3H]-dihydroergocryptine binding sites which also bind [3H]epinephrine with low affinity (El-Refai, M. F., Blackmore, P. F., and Exton, J. H. (1979) J. Biol. Chem. 254, 4375-4386). Incubation of membranes with the alpha blockers dihydroergocryptine (50 nM) and phenoxybenzamine (20 nM) prior to protease treatment diminished the increase in [3H]epinephrine binding induced by trypsin (1.5 microgram/mg). The concentration dependence and time course of trypsin actions on 70 nM [3H]epinephrine binding and 10 nM [3H]dihydroergocryptine binding are consistent with a trypsin-mediated conversion of low affinity epinephrine binding sites to high affinity epinephrine binding sites.     

Characterization of dog platelet alpha-adrenergic receptor: lack of in vivo down regulation by adrenergic agonist treatments

The dog platelet alpha-adrenergic receptor was characterized using [3H]clonidine and [3H]yohimbine. The binding of both radioligands was rapid and reversible at 25 degrees C; saturation and kinetic experiments revealed a single population of binding sites. The number of [3H]yohimbine sites was 2-3-fold higher than the number of [3H]clonidine sites as reported in other tissues containing alpha2-adrenoceptors. The various alpha-agonists and antagonists displaced [3H]clonidine and [3H]yohimbine with an order of potency indicating alpha2-adrenoceptor specificity. Neither (-)adrenaline nor clonidine infusions (0.5 micrograms/min/kg during 3 hr) modified the number of [3H]yohimbine and [3H]clonidine sites or the affinity of the ligands for the alpha2-sites of the dog platelet. Oral administration of clonidine (3 X 150 micrograms/day) did not alter the binding parameters of either ligand.     

Dopaminergic and cholinergic muscarinic receptor effects of L-alphacetylmethadol (LAAM) and its metabolites

In isolated rat hearts L-alphacetylmethadol (LAAM) produced a concentration-dependent decrease in the spontaneous beating rate. This effect was completely prevented by 1.0 microM atropine. Chronic treatment of rats with LAAM increased the number of striatal dopamine receptors measured by [3H]spiroperidol binding. The affinity of these binding sites for [3H]spiroperidol was unchanged by LAAM treatment. There were no significant changes in the number or affinity of binding sites for the labeled muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) with chronic LAAM treatment. The ability of LAAM, nor-LAAM, or dinor-LAAM to antagonize the binding of [3H]spiroperidol (40 pM) or [3H]QNB (125 pM) to striatal membrane fragments was tested. The measured affinity constants for LAAM and metabolites were 100-3000 times higher than the affinity constants of unlabeled spiroperidol at [3H]spiroperidol binding sites. The affinity constants of LAAM and metabolites at muscarinic binding sites were 10-20 times higher than pilocarpine and 5000-8000 times higher than atropine. These results suggest that LAAM can produce some of its effects by acting as a weak agonist at muscarinic receptor sites.     

Endometrial prostaglandin E2 binding: characterization in rats sensitized for the decidual cell reaction and changes during pseudopregnancy

As an initial step in testing the hypothesis that uterine receptivity for blastocyst implantation and sensitivity for decidualization are controlled in part by the presence of functional receptors for prostaglandin E2 (PGE2) in the endometrium, we have characterized the high-affinity binding of [3H]PGE2 to an endometrial membrane preparation from ovariectomized rats treated with progesterone and estradiol so that their uteri were sensitized for the decidual cell reaction. As determined by Scatchard analysis, a single class of [3H]PGE2 binding sites with an apparent Kd ranging from 2 to 6 nM and a capacity of approximately 100 fmol/mg protein was found. Prostaglandins E1 and E2 competed equally for binding while relative cross-reactivity of other prostanoids and compounds tested was less than 3%. Binding was temperature-dependent and reversible. Under the assay conditions used, no metabolism of [3H]PGE2 was detectable. Pretreatment of the membrane preparation with proteolytic enzymes, or by heating, reduced subsequent specific [3H]PGE2 binding. These data are consistent with the presence of endometrial PGE receptors in the sensitized endometrium. The binding of [3H]PGE2 to endometrial membrane preparations from rats on Days 2 to 7 pseudopregnancy was determined. No specific binding could be detected on Day 2. A low binding capacity was found on Days 3 and 4; this increased markedly on Day 5 and reached a maximum on Day 6. These data indicate that the onset of uterine receptivity/sensitivity is temporally correlated with the appearance of endometrial PGE binding sites.     

Prostaglandin-sensitive adenylate cyclase of a murine macrophage-like cell line (P388D1): II. Isolation and partial characterization of PGE2-binding proteins

Properties of prostaglandin (PG) E2 binding sites of a murine macrophage cell line (P388D1) were investigated. The specific binding of [3H]-PGE2 to intact P388D1 cells at 4 degrees C in the presence of cytochalasin D (10 micrograms/ml) approached saturation at concentration greater than 7.5 X 10(-9) M, and could be displaced most effectively by unlabeled PGE2 and less effectively by unlabeled PGI2. The Scatchard analysis of the binding data clearly indicated the heterogeneity with respect to the PGE2 binding affinity and showed the presence of about 3.9 fmol/10(8) cells of the high affinity sites (KD = 1.1 X 10(-9) M) and about 24 fmol/10(8) cells of the low affinity sites (KD = 2 X 10(-8) M). PGE2-binding proteins were isolated from the detergent lysate of the radiolabeled P388D1 cells by affinity chromatography on Sepharose 4B coupled to PGE2. The affinity-isolated materials were further purified by successive use of Sephadex G-100 gel filtration and isoelectric focusing in the presence of dithiothreitol (1 mM) and Triton X-100 (0.5%). The final step yielded about 0.25% of the original radioactivity, which sharply focused as a single peak at pH near 6.5. The electrofocused PGE2-binding proteins migrated as a single band with a m.w. of 95,000 during SDS-PAGE. The electrofocused PGE2-binding proteins bound specifically to [3H]-PGE2 but showed again the heterogeneity with respect to their affinity.     

Effects of thyroid status on the characteristics of alpha 1-, alpha 2-, beta, imipramine and GABA receptors in the rat brain.

The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alpha 1, alpha 2, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [3H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [3H]prazosin, [3H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [3H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status.