Binding of prostaglandin E1 to human erythrocyte membrane

Prostaglandin E1 is known to alter the structural and functional characteristics of red blood cells, yet, little is understood about the membrane receptors mediating this process. We therefore studied the binding of tritium-labeled prostaglandin E1 to the intact human erythrocyte membrane and demonstrated that the interaction is highly specific, rapid, saturable and reversible. Scatchard analysis of prostaglandin E1 binding to the membrane preparations showed the presence of two independent classes of prostaglandin E1 binding sites which differed in their affinity for the autacoid. The high-affinity class had Kd = 3.6 X 10(-9) M and the low-affinity class had Kd = 5.6 X 10(-5) M. The optimum pH for the binding of [3H]prostaglandin E1 to the erythrocyte membrane was found to be around 7.5 and maximum specific binding occurred at a concentration of 5 mM Mg2+ in the incubation mixture. [3H]Prostaglandin E1 bound to the membrane preparation could not be displaced by GTP or by its stable derivative Gpp[NH]p. However, prostaglandin E1 bound to the erythrocyte membrane preparation could be rapidly displaced by cyclic AMP. The IC50 (concentration of the nucleotide displacing 50% bound [3H]prostaglandin E1 from the membrane) was 75 nM. Other adenine nucleotides or cyclic GMP could not substitute for cyclic AMP. Unlike the right-side-out erythrocyte membrane, the inside-out membrane preparations do not bind [3H]prostaglandin E1. Treatment of right-side-out erythrocyte membrane preparation with neuraminidase markedly decreases the binding of prostaglandin E1. Incubation of the erythrocyte membrane preparation with trypsin resulted in total loss of the binding activity. These results indicate that the prostaglandin E1 binding sites located on the cell surface and sialic acid residues are required for prostaglandin E1 binding to the human erythrocytes. These results also indicated that the binding sites are glycoprotein in nature.     

Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it.

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.     

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

Localization and characterization of prostaglandin E1 receptors in rat small intestine

While prostaglandins of the E series are known to affect several small intestinal functions, their cellular mechanisms are poorly understood. The purposes of our study were to determine whether receptors for PGE are present in rat small intestine and to locate and characterize the receptor binding in the subcellular fractions. Small intestinal binding of prostaglandin E1 was significantly higher than that of prostaglandin E2. Highest receptor binding for prostaglandin E1 was found in the plasma membrane fraction of isolated small intestinal enterocytes. Curvilinearity of prostaglandin E1 binding in plasma membranes upon Scatchard analysis indicated two receptor binding sites in rat small intestine. Competitive binding studies demonstrated that receptor binding was highest for prostaglandins of the E series. These studies are the first to demonstrate specific prostaglandin E1 receptors in different subcellular fractions of rat small intestine. We suggest that receptor binding of prostaglandin E may be an important initial step in the mechanism of prostaglandin-E-induced responses in the small intestine.     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Development of affinity labeling agents based on nonsteroidal anti-inflammatory drugs: labeling of the nonsteroidal anti-inflammatory drug binding site of 3 alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs (NSAIDs) exert their effect by inhibiting the target enzyme cyclooxygenase (prostaglandin H2 synthase); however, little is known about the peptides comprising its NSAID binding site. Hydroxyprostaglandin dehydrogenases also bind NSAIDs, but their NSAID binding sites have not been well characterized. Using existing synthetic strategies, we have incorporated the bromoacetoxy affinity labeling moiety around the perimeter of two potent NSAIDs, indomethacin and mefenamate, a N-phenylanthranilate. The compounds synthesized were 1-(4-(bromoacetamido)benzyl)-5-methoxy-2-methylindole-3-acetic acid (1), 3-(2-(2-bromoacetoxy)ethyl)-1-(4-chlorobenzyl)-5-methoxy-2-methylindole (2), 4-(bromoacetamido)-N-(2,3-dimethylphenyl)anthranilic acid (3), N-(3-(bromoacetamido)phenyl)-anthranilic acid (4), and N-(4-(bromoacetamido)phenyl)anthranilic acid (5). To access whether these compounds have general utility in labeling NSAID binding sites, the compounds were evaluated as affinity labeling agents for 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from rat liver cytosol. This enzyme displays 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase activity, is inhibited potently by NSAIDs, and is homologous to bovine lung prostaglandin F synthase. Compounds 1-5 were shown to affinity label the NSAID binding site of 3 alpha-HSD. They inactivated 3 alpha-HSD through an E.I complex in a time- and concentration-dependent manner with t1/2 values ranging from seconds to hours. Ligands that compete for the active site of 3 alpha-HSD (NAD+ and indomethacin) afforded protection against inactivation, and the inactivators could demonstrate competitive kinetics against 3 alpha-hydroxysteroid substrates by forming an E.NAD+.I complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone action at the membrane level. IV. Epinephrine binding to rat liver plasma membranes and rat epididymal fat cells.

[3-H]Epinephrine binding to isolated purified rat liver plasma membranes is a reversible process. An initial peak in binding occurs at about 15 min and a plateau occurs by 50 min. Optimal binding occurred at a membrane protein concentration of 125mug. Rat liver plasma membranes stored at-70 degrees C up to 4 weeks showed no difference in epinephrine binding capacity as compared to control fresh membranes. Epinephrine binding to liver plasma membranes was decreased by 79% by phospholipase A2 (phosphatide acylhydrolase EC 3.1.1.4), 81% by phospholipase C (phosphatidylcholine choline phosphohydrolase EC 3.1.4.3) and 59% by phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4). Trypsin and pronase digestion of the membrane decreased epinephrine binding by 97 and 47% respectively. In the presence of 10-3M Mg-2+ ions, increasing concentrations of QTP decreased epinephrine binding to liver plasma membranes. A maximal effect was demonstrated with 10-5M GTP, representing an inhibition of 52% of the control. In a Mg-2+ -free system, epinephrine binding was unaffected by GTP. However, in a Mg-2+ -free system, increasing concentrations of ATP cause increasing inhibition of hormone binding. ATP at 10-3 M reduced epinephrine binding to 28% of the control. GRP (10-5 M) was shown to inhibit epinephrine uptake rather than epinephrine release from the membrane. [3-H]Epinephrine binding to isolated rat epididymal fat cells shows an initial peak within 5 min followed by a gradual rise which plateaus after 60 min. Epinephrine binding increased nearly linearly with increasing fat cell protein concentration (40-200 mug protein). GTP (10-5 M) and ATP (10-4 M) decreased epinephrine binding to rat epididymal fat cells by 41%. Nearly complete inhibition of binding was demonstrated with 10-2-10-3M ATP. Epinephrine analogs that contain two hydroxyl groups in the 3 and 4 position on the benzene ring act as inhibitors of [3-H]epinephrine binding to rat adipocytes. Alteration of the epinephrine side chain has relatively little influence on binding. Analogs in which one of the ring hydroxyl groups is missing or methylated are poor inhibitors of [3-H]epinephrine binding. Alpha-(phentolamine and phenoxybenzamine) and beta-(propranolol and dichorisoproterenol) adrenergic blocking agents were tested with respect to their ability to influence [3-H]epinephrine binding and their influence on epinephrine-stimulated lipolysis. Only dichloroisoproterenol significantly inhibited epinephrine binding (by 25%). The two beta-adrenergic blocking agents caused an inhibition of epinephrine-stimulated glycerol release, with propranolol being most effective. Phentolamine and phenoxybenzamine had no significant effect on the epinephrine stimulation of glycerol release by fat cells.     

Determination of the structural domain of ApoAI recognized by high density lipoprotein receptors.

There is good evidence that high density lipoprotein (HDL) interacts with high affinity sites present on hepatocytes. The precise nature of the ligand recognized by putative HDL receptors remains controversial, although there is a consensus that apolipoprotein AI (apoAI) is involved. This suggestion would be strengthened if a biologically active site demonstrating a high affinity for the receptor could be isolated. Cyanogen bromide fragments (CF) of apoAI (CF1-CF4) were complexed with phospholipid, and their ability to associate with the receptor was compared in various binding studies. Careful analysis of the concentration-dependent association of 125I-labeled dimyristoyl phosphatidylcholine (DMPC) recombinants to rat liver plasma membranes revealed high and low affinity binding components. As all DMPC recombinants displayed the low affinity binding component, it was postulated that this interaction was independent of the protein present in the particle and may well represent a lipid-lipid or lipid-protein association with the membranes. Only 125I-labeled CF4.DMPC displayed a high affinity binding component with similar Kd and Bmax (8 x 10(-9) M, 1.6 x 10(-12) mol/mg plasma membrane protein) to that of 125I-labeled AI.DMPC (7 x 10(-9), 1.4 x 10(-12) mol/mg plasma membrane protein). Similarly, egg yolk phosphatidylcholine complexes containing CF4 (CF4.egg PC) showed higher affinity binding than CF1-egg yolk phosphatidylcholine complexes confirming the results obtained with DMPC complexes. Furthermore, ligand blotting studies showed that only 125I-labeled CF4.DMPC associated specifically with HB1 and HB2, two HDL binding proteins recently identified in rat liver plasma membranes. We conclude that a region within the carboxyl-terminus of apoAI is responsible for the interaction with putative HDL receptors present in rat liver plasma membranes.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

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.     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

Regulation of insulin receptor activity of human erythrocyte membrane by prostaglandin E1

Incubation of human erythrocyte membrane with low concentration of prostaglandin E1 or prostacyclin increased the binding of 125I-labeled insulin to the membrane. The binding of the radioiodinated hormone was maximally stimulated at 3 nM prostaglandin E1 and the use of higher concentrations (above 8 nM) of the autacoid tended to reverse its own effect at lower concentrations. While prostaglandins A1, A2, B1, B2, D2, F1 alpha, F2 alpha or 6-keto-prostaglandin F1 alpha had no effect on the binding of insulin to the erythrocyte membrane, prostaglandin E2 at similar concentrations decreased the binding of the hormone. The effect of prostaglandin E1 on the increased binding of the insulin was found to be reversible and depended on the occupancy of the autacoid molecules on the membrane and showed positive cooperativity. Scatchard analysis of the binding of 125I-labeled insulin to the erythrocyte ghosts indicated that in the presence of the autacoid, the binding capacity of the insulin receptor increased 2-fold (from 207 to 424 fmol/mg protein) without any change in the ghosts affinity for the ligand (Kd 2.4 X 10(-9) versus 2.49 X 10(-9) M). As a consequence of increased binding of insulin to the erythrocyte membrane in the presence of prostaglandin E1 (3.0 nM), the optimal concentration of the peptide hormone for the maximal reduction of the membrane microviscosity decreased from approx. 1.6 to approx. 0.4 nM. Addition of prostaglandin E1 alone at the above concentration to the assay mixture had no effect on the membrane microviscosity.     

Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator.

Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues.

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 microgram/ml) when compared to other naturally occurring glycosamin oglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin E1-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Ouabain binding sites and (Na+,K+)-ATPase activity in rat cardiac hypertrophy. Expression of the neonatal forms

The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involves several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of [3H]ouabain and ouabain-induced inhibition of (Na+,K+)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (Kd) of 1.8-3.2 X 10(-8) M and 1-8 X 10(-6) M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na+,K+)-ATPase (IC50 = 1-3 X 10(-8) M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that we observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms (Schwartz, K., Lompre, A.M., Bouveret, P., Wisnewsky, C., and Whalen, R.G. (1982) J. Biol. Chem. 23, 14412-14418), our data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor.     

Multiple sites of interaction between prostaglandins and non-steroidal anti-inflammatory agents.

Several non-steroidal anti-inflammatory agents (NSAIA) are shown to inhibit the net production of prostaglandin (PG)- like activity from arachidonic acid by a cell-free preparation of guinea-pig lung. Moreover, these agents also antagonize PGE-1-induced contractions of the isolated gerbil colon. The anti-spasmogenic effects are reversible and specific. At high concentrations, indomethacin and mefenamic acid interfere with the binding of PGE-1 to a broken cell preparation of rat epididymal adipocytes. Taken together the data indicate that NSAIA interact with prostaglandins at multiple sites and are consistent with the suggestions reported previously that NSAIA may have multiple in vivo actions.     

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans.

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.     

Inhibition of prostaglandin E1-induced activation of adenylate cyclase in human blood platelet membrane

Activation of human blood platelet adenylate cyclase is initiated through the binding of prostaglandin E1 to the membrane receptors. Incubation of platelet membrane with [3H]prostaglandin E1 at pH 7.5 in the presence of 5 mM MgCl2 showed that the binding of the autacoid was rapid, reversible and highly specific. The binding was linearly proportional to the activation of adenylate cyclase. Although the membrane-bound radioligand could not be removed either by GTP or its stable analogue 5'-guanylylimido diphosphate, 150 nM cyclic AMP displaced about 40% of the bound agonist from the membrane. Scatchard analyses of the binding of the prostanoid to the membrane in the presence or absence of cyclic AMP showed that the nucleotide specifically inhibited the high-affinity binding sites without affecting the low-affinity binding sites. Incubation of the membrane with 150 mM cyclic AMP and varying amounts of prostaglandin E1 (25 nM to 1.0 microM) showed that the percent removal of the membrane-bound autacoid was similar to the percent inhibition of adenylate cyclase at each concentration of the agonist. At a concentration of 25 nM prostaglandin E1, both the binding of the agonist and the activity of adenylate cyclase were maximally inhibited by 40%. With the increase of the agonist concentration in the assay mixture, the inhibitory effects of the nucleotide gradually decreased and at a concentration of 1.0 microM prostaglandin E1 the effect of the nucleotide became negligible. These results show that cyclic AMP inhibits the activation of adenylate cyclase by low concentrations of prostaglandin E1 through the inhibition of the binding of the agonist to high-affinity binding sites.     

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.