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.     

Effect of topical application of estradiol-17 beta and PGE2 on PGE-binding sites in the porcine endometrium.

High- and low-affinity prostaglandin E2 (PGE2) binding sites were found on day 15 after estrus in the endometrium of cycling (Cy) and pregnant (Pr) gilts as well as gilts treated with intra-uterine Silastic beads containing estradiol-17 beta (E2) alone or in combination with PGE2 (E and PG gilts respectively) and inserted into the uterine lumen on day 10 of the cycle. The average apparent dissociation constants (Kd) and binding site concentrations (Bmax) for the high- and low-affinity sites were respectively (mean +/- SEM): 8.4 +/- 0.7 pM and 3.28 +/- 0.38 fmol/mg of protein and 5.3 +/- 0.8 nM and 71 +/- 9 fmol/mg of protein. Samples collected along the meso- and antimesometrial aspects did not differ (P greater than 0.05), although the low-affinity Bmax was higher on the antimesometrial aspect for Pr and Cy gilts only. No difference in Kd (P greater than 0.10) was found between treatments for high-affinity binding sites. For the low-affinity binding sites, Kd was higher for Pr compared to PG and E but not to Cy gilts (P less than 0.05). The high-affinity Bmax was higher (P less than 0.05) for PG, followed by E, Pr and Cy gilts (respectively: 5.50 +/- 0.26; 4.19 +/- 0.46; 1.78 +/- 0.40; 1.64 +/- 0.23 fmol/mg of protein), although Pr and Cy gilts were not different (P greater than 0.05). These results suggest that the localized presence of conceptuses in the uterus in early pregnancy does not markedly affect PGE binding sites but that intrauterine applications of Silastic beads containing E2 and PGE2 increase high-affinity Bmax and decrease low-affinity Kd.     

Existence of Three Subtypes of Bradykinin B2 Receptors in Guinea Pig

We describe the binding of [3H]bradykinin to homogenates of guinea pig brain, lung, and ileum. Analysis of [3H]bradykinin binding kinetics in guinea pig brain, lung, and ileum suggests the existence of two binding sites in each tissue. The finding of two binding sites for [3H]bradykinin in ileum, lung, and brain was further supported by Scatchard analysis of equilibrium binding in each tissue. [3H]Bradykinin binds to a high-affinity site in brain, lung, and ileum (KD = 70-200 pM), which constitutes approximately 20% of the bradykinin binding, and to a second, lower-affinity site (0.63-0.95 nM), which constitutes the remaining 80% of binding. Displacement studies with various bradykinin analogues led us to subdivide the high- and lower-affinity sites in each tissue and to suggest the existence of three subtypes of B2 receptors in the guinea pig, which we classify as B2a, B2b, and B2c. Binding of [3H]bradykinin is largely to a B2b receptor subtype, which constitutes the majority of binding in brain, lung, and ileum and represents the lower-affinity site in our binding studies. Receptor subtype B2c constitutes approximately 20% of binding sites in the brain and lung and is equivalent to the high-affinity site in brain and lung. We suggest that a third subtype of B2 receptor (high-affinity site in ileum), B2a, is found only in the ileum. All three subtypes of B2 receptors display a high affinity for bradykinin, whereas they show different affinities for various bradykinin analogues displaying agonist or antagonist activities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.     

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.     

Prostaglandin binding sites in human polymorphonuclear neutrophils

Prostaglandin (PG) E2 (greater than or equal to 1.6 nM) and PGD2 (greater than or equal to 16 nM) inhibited polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4 and platelet-activating factor (PAF) whereas PGF2 alpha was bioinactive. [3H]PGE2 and [3H]PGD2 bound to PMN and isolated, plasmalemma-enriched PMN membranes. Binding was time-dependent, specific, saturable, and reversible. Competitive studies indicated that the two PGs bound to distinctly different sites. PMN had high (Kd = 1 nM; Rt = 150/cell) and low (Kd = 100 nM; Rt = 5800/cell) affinity PGE2 binding sites. Only a single type of PGD2 binding site (Kd = 13 nM; Rt = 5100/cell) was detected. We conclude that PGE2 and PGD2 bind to their respective, plasmalemmal receptors to attenuate PMN function. The PGs may act as endogenous stop signals to limit the action of concurrently formed excitatory signals, eg., LTB4 and PAF.     

Effects of tumor necrosis factor-alpha on secretion of prostaglandins E2 and F2alpha in bovine endometrium throughout the estrous cycle

To determine the physiological significance of tumor necrosis factor-alpha (TNFalpha) in the regulation of endometrial prostaglandin (PG) release in cattle, we investigated the effects of TNFalpha on the secretion of PGE2 and PGF2alpha by bovine endometrium during the estrous cycle. Bovine uteri were classified into six stages (estrus: Day 0, early luteal 1: Days 2 to 3, early luteal 11: Days 5 to 6, mid-luteal: Days 8 to 12, late luteal: Days 15 to 17 and follicular: Days 19 to 21). After 1 h of pre-incubation, endometrial tissues (20 to 30 mg) were exposed to 0 or 0.6 nM TNFalpha for 4 h. The PGE2 concentrations in the medium were higher in the luteal stages than in the follicular stage and in estrus. In contrast, PGF2alpha concentrations were higher in the follicular stage and in estrus than in the luteal stages. The ratio of the basal concentrations of PGE2 and PGF2alpha (PGE2/PGF2alpha ratio) was higher in the luteal stages than in the follicular stage and in estrus. Although TNFalpha stimulated both PGE2 and PGF2alpha secretion during the entire period of the estrous cycle, the level of stimulation of TNFalpha on PGE2 output by the bovine endometrium does not show the same cyclical changes as that shown on PGF2alpha output. The stimulation of TNFalpha resulted in a decrease in the PGE2/PGF2alpha ratio only in the late luteal stage. Furthermore, TNFalpha stimulated PGE2 secretion in stromal, but not epithelial cells. The overall results suggest that TNFalpha is a potent regulator of endometrial PGE2 secretion as well as PGF2alpha secretion during the entire period of estrous cycle, and that TNFalpha plays different roles in the regulation of secretory function of bovine endometrium at different phases of the estrous cycle.     

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.     

The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain.

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.     

Constitutive expression of uterine receptors for insulin-like growth factor-I during the peri-implantation period in the pig.

Insulin-like growth factor-I (IGF-I), synthesized by the uterine endometrium of cyclic and early pregnant gilts, accumulates in the uterine luminal fluid, where it comes in contact with the developing conceptus and the rapidly growing uterus. The uterus and the conceptus thus represent potential target sites for the biological effects of IGF-I, provided high-affinity Type I receptors are present. This study was undertaken to evaluate the expression of functional IGF-I receptors in the endometrium and myometrium of pregnant (Day 10, 12, and 15) gilts and in the endometrium of cyclic (Day 15) and pseudopregnant (Day 15) gilts and to correlate levels of these receptors with temporally regulated uterine production of IGF-I. Specific binding of 125I-IGF-I to endometrial membranes pretreated with MgCl2 (4 M) at 4 degrees C for 16 h, was saturable and membrane concentration-dependent. Competition of 125I-IGF-I binding to endometrial membranes was highest with unlabeled IGF-I greater than IGF-II much greater than insulin, whereas porcine relaxin was noncompetitive. Affinity cross-linking of endometrial membranes with 125I-IGF-I followed by SDS-PAGE and autoradiography revealed two labeled bands of Mr greater than 200,000 and Mr 135,000, with the major band being the Mr 135,000 species. Scatchard analysis of 125I-IGF-I binding to endometrial membranes from Day 12 pregnant gilts revealed a single class of binding sites with a dissociation constant (Kd) = 4.08 +/- 0.09 nM. Membranes prepared from endometrium of Day 10, 12, and 15 pregnant gilts exhibited comparable 125I-IGF-I binding (p greater than 0.05) that was higher (p less than 0.001) than that for the corresponding myometrial membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of modifiers of a radiation lesion on the binding of prostaglandin E2 with liver membranes in F1 CBA X C57BL strain mice

Prostaglandin E2 (PGE2) was specifically bound by the membrane fraction prepared from the mouse liver. The binding constants indicate the presence of high-affinity PGE2 binding sites with an apparent dissociation constant (Kd) of 0.82 X 10(-9) M and a capacity of 0.36 X 10(9) M/mg protein and a lower affinity PGE2 binding site with Kd = 15.73 X 10(-9) M and a capacity of 5.31 X 10(9) M/mg protein. The radioprotectors, MEA and APAETP inhibit PGE2 binding and alter its kinetics. Apparently the mechanism of PGE2 binding by membranes is related to interaction of prostaglandins with thiols and sufhydryl groups of membrane lipoproteins, while the radioprotectors modify the functional groups participating in receptor PGE2 binding.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

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) μM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9±0.9 (2.0±0.8) μM. The Scatchard analysis on [3H]PGE1- as well as on [3H]ilioprost-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 (15.3±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 stable 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.     

Uterine progesterone metabolism during early pseudopregnancy in the rat

We measured uptake and metabolism of progesterone (P4) during the estrous cycle and Days 2-6 of pseudopregnancy (PSP) to determine uterine P4 dynamics during preimplantation. Rats were infused with [3H]P4 for 60 min, blood was obtained, the uterus was removed, and endometrium and myometrium were isolated. Tissue radiolabeled P4 and P4 metabolites (5 alpha-pregnane-3,20-dione, DHP; 20 alpha-hydroxy P4; 17 alpha-hydroxy P4, and hydroxylated DHP derivatives) were extracted and separated by thin-layer chromatography (TLC). Serum P4 was measured by radioimmunoassay (RIA) in another group of rats. Endometrial and myometrial concentrations of [3H]P4 were greater (p less than 0.05) than plasma values. In contrast, [3H]DHP levels in the endometrium were higher (p less than 0.01) than values in myometrium or plasma. Compared to values in the estrous cycle, endometrial ratios of [3H]DHP/[3H]P4 and [3H]metabolites/[3H]P4 decreased (p less than 0.02) on Days 3-5 of PSP. Serum P4 levels during the estrous cycle (13-25 ng/ml) increased (p less than 0.01) to 120 ng/ml on Days 3-5 of PSP. Estimated concentration of P4 in the endometrium during the estrous cycle (90 ng/g) increased (p less than 0.05) to 580 ng/g by Day 5 of PSP. Similar observations were noted for the estimated endometrial concentrations of DHP and all P4 metabolites. We suggest that both endometrium and myometrium take up and metabolize P4 during the estrous cycle and early PSP. However, endometrial P4 metabolism during PSP is greater than during the estrous cycle, in part because of increased ovarian secretion and endometrial concentration of P4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxytocin-stimulated inositol phosphate turnover in endometrium of ewes is influenced by stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins

Three experiments (Exp) assessed the influence of stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins (oCSP) on turnover of inositol trisphosphate (the putative second-messenger for oxytocin-stimulated secretion of prostaglandin F2 alpha) in ovine endometrium during luteolysis and maternal recognition of pregnancy. In Exp 1, endometrium was collected from 5 cyclic (Cy) and 6 pregnant (P) ewes on Day 16 after onset of estrus. In Exp 2, endometrium was collected from Day 12 Cy (n = 5), Day 12 P (n = 3), Day 16 Cy (n = 4), and Day 16 P (n = 3) ewes. In Exp 3, 12 Cy ewes were allotted randomly, in a 2 x 2 factorial arrangement, to receive serum protein (SP), or oCSP and estradiol-17 beta (E2), or vehicle treatments. Ewes were injected i.v. with 0.5 mg E2 or vehicle on Day 12 and received twice-daily infusions of 1.5 mg SP or oCSP (containing 25 micrograms ovine trophoblast protein-1 by radioimmunoassay [RIA]) + SP (1.5 mg total protein) into each uterine horn on Days 12, 13, and 14. Blood samples for RIA of plasma progesterone were collected on Days 10-15 (before treatment on each day) and endometrium was collected on Day 15. For each Exp, 100 mg endometrium was incubated, in duplicate, for 2 h with 10 microCi [3H] inositol and treated with 0 or 100 nM oxytocin (OT) for 20 min, then [3H]inositol mono-, bis-, and trisphosphates (IP1, IP2, and IP3, respectively) were quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high'-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 microM. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 microM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4 degrees C to one site with a Kd value of 3 microM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 microM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 microM to 0.17 microM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 microM to 40 microM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1.     

Differentiation of the drug-binding sites of calmodulin

Calmodulin contains several binding sites for hydrophobic compounds. The apparent specificity of various 'calmodulin antagonists' for these sites was investigated. The Ki values for the inhibition of calmodulin-activated cyclic-nucleotide phosphodiesterase and myosin light-chain kinase was determined. In addition, the Kd values of the same compounds for binding to calmodulin were measured. The compounds could be separated into four groups. Group I and II compounds inhibited competitively the activation of the phosphodiesterase and myosin light-chain kinase by calmodulin. Group I compounds inhibited the activation of the phosphodiesterase and myosin light-chain kinase at identical concentrations. In contrast, group II compounds inhibited the activation of the phosphodiesterase at 5-10-fold lower concentrations than that of myosin light-chain kinase. Group III compounds inhibited the activation of these enzymes by an uncompetitive mechanism. Group IV compounds inhibited the activation of the phosphodiesterase with Ki values above 10 microM and did not affect the activation of myosin light-chain kinase. Binding of [3H]bepridil to calmodulin under equilibrium conditions yielded one high-affinity site (apparent Kd 0.4 microM) and four low affinity sites (apparent Kd 44 microM). Group I compounds interfered with the binding of bepridil to the high and low-affinity sites in a competitive manner. Group II compounds interfered in a non-competitive manner with the high-affinity site and apparently competed only with one of the low-affinity sites. Group III compounds did not compete with any of the bepridil-binding sites. Nimodipine, a group III compound, bound to one site on calmodulin with a Kd value of 1.1 microM. Other dihydropyridines competed with [3H]nimodipine for this site. The group I and II compounds, trifluoperazine and prenylamine, did not affect the binding of [3H]nimodipine. These data show that 'calmodulin antagonists' can be differentiated into at least three distinct groups. Kinetic and binding data suggest that the three groups bind to at least three different sites on calmodulin. Selective occupation of these sites may inhibit specifically the activation of distinct enzymes.