The Effectiveness of Raloxifene-Loaded Liposomes and Cochleates in Breast Cancer Therapy.

Liposome (spherical vesicles) and cochleate (multilayer crystalline, spiral structure) formulations containing raloxifene have been developed having dimethyl-ß-cyclodextrin (DM-ß-CD) or sodium taurocholate (NaTC). Raloxifene was approved initially for the treatment of osteoporosis but it is also effective on breast tissue and endometrial cells. Raloxifene inhibits matrix metalloproteinase-2 (MMP-2) enzyme, which is known to be responsible for tumor invasion and the initiation of angiogenesis during the tumor growth. Therefore, raloxifene was selected as a model drug. A series of raloxifene-loaded liposome and cochleate formulations were prepared. In vitro release studies and in vivo tests were performed. Breast cancer cell lines (MCF-7) were also used to find the most effective formulation. Highest antitumor activity was observed, and MMP-2 enzyme was also found to be inhibited with raloxifene-loaded cochleates containing DM-ß-CD. These developed formulations can be helpful for further treatment alternatives and new strategies for cancer therapy.

[Enhancers on the transmembrane transport of chlorogenic acid].

To investigate the influence of the difference enhancers on the transport mechanism of chlorogenic acid (CGA) across Caco-2 cells model, a RP-HPLC method was adopted to detect the concentrations of CGA. At the concentrations of 20 to 80 microg x mL(-1), the difference of absorption rate constants (K(a)) was not statistically significant. At the concentrations of 40 and 20 microg x mL(-1), the ratios of apparent permeability coefficients (P(app)) of the apical to basolateral and the basolateral to apical were 1.14 and 1.18, respectively. With the effect of enhancers K(a) and P(app) increased, the absorption half-life (T1/2) decreased. CGA passed through the Caco-2 cell membrane mainly by passive transport. It showed that monocarboxylic acid transporter (MCT) could be involved in the across membrane transport process of CGA. Borneol had no effect on the cell membrane transport processes. The order of increasing absorption of CGA caused by the enhancers was sodium lauryl sulphate > sodium taurocholate > carbomer.

Enhancing effect of surfactants on fexofenadine.HCl transport across the human nasal epithelial cell monolayer.

The effect of various surfactants (sodium cholate, sodium taurocholate, Tween 80 and Poloxamer F68) on enhancing the transepithelial permeability of fexofenadine.HCl was evaluated in a human nasal epithelial cell monolayer model. The cytotoxicity of the surfactants on the human nasal epithelial cells was evaluated by the MTT assay. A dose-dependent reduction of cell viability was observed at higher than critical micelle concentration (CMC) of the surfactants, and the IC50 of non-ionic surfactants (Tween 80 and Poloxamer F68) was higher than that of ionic surfactants (sodium cholate and sodium taurocholate). The TEER values significantly decreased after 2 h incubation with the ionic surfactants, but were recovered after 24 h in the fresh culture media. Ionic surfactants significantly increased the transepithelial permeability (P(app)) of fexofenadine.HCl compared to the non-ionic surfactants. The reduction of TEER values upon exposing the cell monolayer to the surfactants for 2 h correlated well with the P(app) of fexofenadine.HCl, which suggests that the permeation-enhancing mechanism of the ionic surfactants is by altering the tight junction property of the paracellular pathway. F-actin staining showed that the effect of ionic surfactants on the tight junction is temporary and reversible, which is consistent with the TEER value recovery within 24 h. These results imply that ionic surfactants are potentially useful permeation enhancers for nasal delivery of hydrophilic compounds, such as fexofenadine.HCl. This study also indicated the usefulness of the human nasal epithelial cell monolayer model not only for evaluating the in vitro nasal drug transport but also for studying the mechanism and toxicity of enhancers.

The mechanism of liposomal damage by taurocholate.

The stability of small unilamellar vesicles formed by egg-yolk phosphatidylcholine (PC) has been examined in the presence of sodium taurocholate. The permeability of the vesicular membrane changes as the total taurocholate concentration increases, until a transformation from mixed bile salt/PC vesicles to mixed micelles occurs. Based on experiments in which the bile salt-induced release of either hydrophilic (carboxyfluorescein) or hydrophobic (Bromothymol blue) probes was studied, and on fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene and turbidity measurements, a two-step process for the initial stage of liposomal damage by taurocholate is postulated.

The effect of the interdependence of protein and bile salt concentrations on the measurement of cerebroside sulphate sulphatase activity in human leucocyte and fibroblast extracts.

The previously observed differences in properties of human leucocyte and fibroblast cerebroside sulphate sulphatase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) measured in vitro have been found to be due to subtle differences in incubation conditions. Maximum enzyme activity was observed with either crude sodium taurocholate or with pure sodium taurodeoxycholate. The optimum bile salt concentration of the enzyme in leucocyte or fibroblast extracts, but not the pure ox liver enzyme, was critically dependent on protein concentration. At low concentrations of the latter (less than 0.1 mg/ml), maximum activity was observed at taurocholate concentrations less than 0.5 mg/ml; at protein concentrations greater than 0.20 mg/ml substantially more bile acid (more than 1.3 mg/ml) was required to stimulate maximum activity. Addition of Triton X-100 or bovine serum albumin to the incubation mixtures increased the optimum taurocholate concentration. The dependence of the bile salt optimum on protein concentration appears to be related to the binding of the lipid substrate to membranous protein present in the tissue extracts. Release of the bound lipid is effected either by increasing the bile salt concentration or by adding Triton X-100. In the presence of excess bile salt human leucocyte, fibroblast and liver cerebroside sulphate sulphatase activity is stimulated by Triton at low protein concentrations; under identical conditions the pure or crude ox-liver enzyme is substatially inhibited. Our data also show that cerebroside sulphate sulphatase activity measured in extracts from leucocytes and fibroblasts, the tissues normally used to effect a diagnosis of metachromatic leucodystrophy, is the result of a complex interaction of bile salt, protein, Triton X-100 and probably the substrate itself. Any slight alteration in any of those factors, without a corresponding change in any or all of the others, can have a marked effect on the measured enzyme activity, and may lead to errors in the diagnosis of metachromatic leucodystrophy.

Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells.

Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.

Cholesterol ester hydrolase in human red blood cells.

1. Cholesterol ester hydrolytic activity (sterol-ester hydrolase EC 3.1.1.13) was detected in human red blood cells. Enzyme activity appeared confined to the cell membrane and was most marked in washed preparations of red cell ghosts. 2. Hydrolytic activity was stimulated by the anti-oxidants D-alpha-tocopherol and butylated hydroxytoluene. Marked inhibition was produced by erythrocyte hemolysate, sodium taurocholate, and Triton X-100. 3. Optimal pH for the reaction was 5.4--5.7. 4. Because red cell cholesterol is all unesterified, it is speculated that the hydrolase serves to maintain the erythrocyte membrane free of esterified cholesterol.

Effects of detergents and choline-containing phospholipids on human spleen glucocerebrosidase.

1. Glucocerebrosidase, extracted from human spleen lysosomal membrane by sodium cholate and recovered in a high speed centrifugation supernatant, aggregated following removal of the detergent. 2. Re-solubilization of the enzymatic activity from the aggregate was achieved by treatment with the non-ionic detergents Triton X-100 and Tween 20. The anionic detergents sodium cholate and sodium taurocholate and the cationic detergents cetyltrimethylammonium bromide and cetylpyridinium chloride were also effective. The solubilizing capacity of the anionic detergents was smaller than that of the nonionic detergents. Quantitative evaluation of the solubilizing capacity of the cationic detergents was not feasible because of their being potent inhibitors of glucocerebrosidase activity. 3. Treatment of the enzyme aggregate with acetone rendered it buffer-soluble. 4. In addition to the above cationic detergents some choline-containing and highly hydrophobic phospholipids were found to inhibit the glucocerebrosidase activity.

Conditions affecting the activity of glucocerebrosidase purified from spleens of control subjects and patients with type 1 Gaucher disease.

Glucocerebrosidase was purified to homogeneity from spleens of control subjects and Type 1 Gaucher disease patients by immunoaffinity chromatography. Activation of the enzyme by taurocholate, phosphatidylserine and sphingolipid activator protein 2 (saposin C; SAP-2) was investigated by titration of combinations of various effectors in the absence and presence of Triton X-100. The specific activity of Type 1 Gaucher glucocerebrosidase was found to be less than 20% of the corresponding control value under most conditions. However, in the presence of optimal amounts of activator protein SAP-2 and phosphatidylserine (and in the absence of Triton X-100 and/or taurocholate), the specific activity of mutant enzyme towards artificial and natural lipid substrates was close to normal when measured at pH 5.0-5.5. At pH values below 5.0, the specific activity of mutant enzyme decreased more rapidly compared to that of control enzyme. The activity of Type 1 Gaucher glucocerebrosidase in the intact cell might, in a comparable manner, be highly dependent on the extent of activation by endogenous activators and on the intralysosomal pH. Values for residual glucocerebrosidase activity, as measured in vitro in extracts of cells and tissues from Type 1 Gaucher disease patients, are indeed highly dependent on the assay conditions employed. Consequently such measurements are of little value in the assessment of the actual capacity for glucosylceramide hydrolysis and for prediction of the clinical severity of the disease.

Inhibition of rat liver cholesterol esterase by local anaesthetics.

1. A number of local anaesthetics was shown to inhibit rat liver cholesterol esterase activity towards radioactively labelled cholesterol oleate. The anaesthetics inhibited in the order dibucaine greater than chlorpromazine greater than tetracaine greater than benzocaine greater than procaine greater than lidocaine greater than cocaine. 2. The mode of inhibition was seen to be non-competitive with respect to the substrate and is probably independent of any involvement of Ca2+. 3. The inhibition by tetracaine is partially reversed by sodium deoxycholate. However, all ionic and non-ionic detergents studied, sodium deoxycholate, sodium taurocholate, Triton X-100, and cetyltrimethylammonium bromide are capable of inhibiting the rat liver cholesterol esterase in a concentration dependent manner. Only sodium taurocholate stimulates enzymic activity.

Activity of human hepatic beta-galactosidase toward natural glycosphingolipid substrates.

1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactoside, galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide (GM1-Ganglioside) and galactosyl-Cacetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. Teh apparent Km values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimualting hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude tauroxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 Were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.

Studies on the mechanism of cholesterol uptake and on the effects of bile salts on this uptake by brush-border membranes isolated from rabbit small intestine.

The effect of bile salts and other surfactants on the rate of incorporation of cholesterol into isolated brush-border membranes was tested. At constant cholesterol concentration, a stimulatory effect of taurocholate was noticed which increased as the bile salt concentration was raised to 20 mM. Taurodeoxycholate was as effective as taurocholate at concentrations of up to 5 mM and inhibited at higher concentrations. Glycocholate was only moderately stimulatory whereas cholate was nearly as effective as taurocholate at concentrations above 5 mM. Other surfactants such as sodium lauryl sulfate and Triton X-100 were very inhibitory at all concentrations tried whereas cetyltrimethyl ammonium chloride was stimulatory only at a very low range of concentrations. These micellizing agents all caused some disruption of the membranes and the greater effectiveness of taurocholate in stimulating sterol uptake was partly relatable to the weaker membrane solubilizing action of this bile salt. Preincubation of membranes with 20 mM taurocholate followed by washing and exposure to cholesterol-containing lipid suspensions lacking bile salt, did not enhance the incorporation of the sterol. In the absence of bile salt the incorporation of cholesterol was unaffected by stirring of the incubation mixtures. Increasing the cholesterol concentration in the mixed micelle while keeping the concentration of bile salt constant caused an increase in rate of sterol incorporation. This increased rate was seen whether the cholesterol suspension was turbid, i.e., contained non-micellized cholesterol, or whether it was optically-clear and contained only monomers and micelles. When the concentration of taurocholate and cholesterol were increased simultaneously such that the concentration ratio of these two components was kept constant, there resulted a corresponding increase in rate of cholesterol uptake. The initial rates of cholesterol incorporation from suspensions containing micellar and monomer forms of cholesterol were much larger than from solutions containing only monomers of the same concentration. The rates of incorporation of cholesterol and phosphatidylethanolamine from mixed micelles containing these lipids in equimolar concentrations were very different. The results as a whole suggest at least for those experimental conditions specified in this study, that uptake of cholesterol by isolated brush-border membranes involves both the monomer and micellar phases of the bulk lipid and that the interaction of the micelles with membrane does not likely involve a fusion process.

Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation-dephosphorylation.

The characteristics of neutral cholesteryl ester hydrolase activities found in the microsomal and cytosolic subcellular fractions of rat lactating mammary tissue were investigated. The enzymes were assayed using cholesteryl oleate dispersed as a mixed micelle with phosphatidylcholine and sodium taurocholate (molar ratio 1:4:2) as substrate. This method gave activities approx. 20-fold higher than those seen when cholesteryl oleate was added in ethanol. Addition of phosphatidylcholine and sodium taurocholate to the assays using the ethanol-dissolved substrate did not increase the activities observed. When the cholesteryl oleate was dispersed with phosphatidylcholine only (molar ratio, 1:4) the activity of the two neutral cholesteryl ester hydrolases was also decreased considerably compared to that found with mixed micelles. In this case, however, approx. 60% of the cytosolic, but only 10% of the microsomal activity, was restored by separate addition of sodium taurocholate. The activities of both the microsomal and the cytosolic neutral cholesteryl ester hydrolases were inhibited by MgCl2, and this inhibition was almost completely reversed by the addition of an equimolar concentration of ATP. At a fixed concentration of MgCl2 increasing concentrations of ATP increased the enzyme activities in a dose-dependent way. The activity of the microsomal, but not the cytosolic enzyme was enhanced by a cyclic AMP-dependent protein kinase and both activities were inhibited by alkaline phosphatase (bovine milk). These results provide evidence for the regulation of neutral cholesteryl ester hydrolases in the rat lactating mammary gland by mechanisms involving phosphorylation-dephosphorylation and therefore suggest that these enzymes may be under hormonal control.

Degradation of lysophosphatidylcholine by lysosomes. Stimulation of lysophospholipase C by taurocholate and deficiency in Niemann-Pick fibroblasts.

Hydrolysis of 2-[1-14C]oleoyl phosphatidylcholine and of 1-[1-14C]oleoyl lysophosphatidylcholine by lysosomes prepared from rat liver using Triton WR-1339 has been studied. At pH 5.0 sodium taurocholate stimulated the release by the soluble lysosomal fraction of labelled lysophosphatidylcholine, diacyl- and monoacylglycerol and fatty acids from [14C]phosphatidylcholine. The time course of appearance of labelled products suggested that monoacylglycerol could be released as a result of the action of phospholipase A1 followed by lysophospholipase C or by the initial action of phospholipase C followed by monoacylglycerol lipase. The hydrolysis of 1-[14C]acyl lysophosphatidylcholine was also stimulated by sodium taurocholate under similar conditions; however, only release of monoacylglycerol was increased, whereas release of fatty acid was inhibited. Mg2+ inhibited the release of labelled monoacylglycerol and of fatty acid from lysophosphatidylcholine. The detergents deoxycholate and Triton X-100 and phospholipids were strongly inhibitory. 5'-AMP almost completely suppressed release of monoacylglycerol but increased release of fatty acid. Chloroquine strongly suppressed release of monoacylglycerol and only at high concentration (1.25 mM) diminished fatty acid release. In the presence of sodium taurocholate the predominant mechanism for degradation of phosphatidylcholine by the soluble fraction of lysosomes involves phospholipase A followed by phospholipase C. Assay of release of monoacylglycerol from [14C]lysophosphatidylcholine catalyzed by extracts of fibroblasts from patients with Niemann-Pick disease and controls in the presence of taurocholate revealed that lysophospholipase C activity was lacking in those cell lines that were deficient in sphingomyelinase. This suggests that lysophospholipase C and sphingomyelinase activities may be catalyzed by one enzyme.

Aortic cholesteryl esterase. Influence of bile salts.

The in vitro activity of cholesteryl ester hydrolase preparations of rat and rabbit aortas was assayed in the presence of the taurine and glycine conjugates of cholic, chenodeoxycholic, deoxycholic and lithocholic acids or in the presence of Triton X-100 and Tween-20. Maximum activity was obtained with tauro- or glycocholic acids. As in the case of pancreatic cholesteryl esterase, trihydroxycholanoic acid derivatives may serve an obligatory function.

Characterization of bile salt uptake by Giardia lamblia.

We have shown previously that Giardia lamblia takes up conjugated bile salt in vitro, and have now investigated the mechanism by which this occurs. Uptake of sodium taurocholate (TC) and glycocholate (GC) with respect to time had an initial exponential component followed by a linear component, consistent with a combination of both active and passive transport processes. The presence of an active transport process was further supported by experiments which showed that bile salt uptake: (i) was concentration dependent (apparent Km's for TC and GC were 0.21 and 0.63 mM, respectively); (ii) was competitively inhibitable; (iii) was reduced by the metabolic inhibitor sodium fluoride (50 mM) and low temperature (4 degrees C). Bile salt was not taken up by glutaraldehyde-fixed parasites, indicating that bile salt was not merely being adsorbed on to the parasite surface. Differential centrifugation of lyzed parasites following exposure to radiolabelled GC, showed that the majority of bile salt was located in the cytosol fraction (76%) with a relatively minor component associated with cell membrane, indicating that bile salt had been internalized. Bile salt analysis of extracts of parasites and culture medium indicated that GC had not been metabolized by Giardia. Thus, like the mammalian ileum, Giardia appears to take up conjugated bile salts by active and passive transport processes. Conjugated bile salts are known to promote encystation and thus these uptake mechanisms may constitute an important survival mechanism for the parasite enabling it to complete its life cycle.

Human macrophage-mediated biodegradation of polyurethanes: assessment of candidate enzyme activities.

A predominant cell type associated with explanted failed devices is the monocyte-derived macrophage (MDM). However, there is still very little known about the specific cellular enzyme activities involved in interactions with these devices. The current study investigates the nature of candidate enzymes that may be involved in the degradation of polymeric biomaterials through the use of specific enzyme inhibitor agents. When MDM were incubated with a polycarbonate-based polyurethane (PCNU) synthesized with 14C-labeled hexane diisocyanate (HDI), polycarbonate diol and butanediol (BD) (referred to as 14C-HDI431), the radiolabel release (RR) measured was inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate (serine protease/esterase inhibitors), and sodium fluoride (NaF) (a carboxyl esterase (CXE) inhibitor). Sodium taurocholate (NaT) (a cholesterol esterase (CE) stimulator) had little effect on RR. The two candidate enzymes proposed were CE and CXE, based on the fact that both were identified by immunoblotting in the releasate of MDM following 48 h incubation with 14C-HDI431. The effect of the above reagents on the RR caused by purified CE and CXE, was measured and compared to changes in their activity with p-nitrophenylbutyrate (PNB). The effect of NaF on MDM was similar to that of purified CXE (inhibitory on both RR and lysate esterase activity), suggesting the involvement of CXE. However, NaT inhibited the PNB activity of purified CXE, but had no effect on MDM-mediated RR or PNB activity, implicating another esterase in the biomaterial degradation. Since NaT stimulated CE-mediated RR and PNB activity, it may also be involved in MDM-mediated biodegradation of PCNUs. The results of these studies point to both esterases as being candidates. However, the current methods were unable to determine the relative contribution of each one to the observed biodegradation.

Partial purification and properties of porcine thymus lactosylceramide beta-galactosidase.

Porcine thymus lactosylceramide beta-galactosidase was purified by a simple procedure. In the final step of isoelectric focusing the enzyme was separated into two peaks of pI 6.3 (peak I) and 7.0 (peak II), which showed 3,600- and 4,000-fold enhancement of lactosylceramide-hydrolysing activity, respectively. The two peaks had identical mobility on polyacrylamide gel electrophoresis. The apparent molecular weight was 34,000. Neither monosialoganglioside (GM1) nor galactosylceramide was hydrolysed by the purified enzyme fractions. The optimal pH was at 4.6, and sodium taurocholate was essential for the reaction. The apparent Km was 2.3 x 10-5 M. The reaction was stimulated by sodium chloride and linoleic acid, while it was strongly inhibited by Triton X-100 and bovine serum albumin. Galactosylceramide, p-nitrophenyl beta-galactoside, and p-nitrophenol were weak inhibitors. No effects of GM1 and galactose were observed on the hydrolysis of lactosylceramide.

Purification and properties of sterol-ester hydrolase from Saccharomyces cerevisiae.

Sterol-ester hydrolase [EC 3.1.1.13] from Saccharomyces cerevisiae grown aerobically was solubilized with 1% Tween 20 and purified about 700-fold by the protamine sulfate treatment, DEAE-cellulose-, Sepharose 6B- and DEAE-cellulose column chromatographies. The molecular weight of the enzyme was estimated to be 70,000 by Sepharose 6B gel filtration. The enzyme activity showed two peaks of pH optimum at 4.4 and 6.8. Triton X-100 stimulated the activity as its low concentrations at both pH regions, but decreased the activity at its high concentrations at pH 6.8. The presence of Tween 20 or Tween 80 also stimulated the activity. These results were different from those in the previous report showing no stimulation of the crude enzyme by these detergents. The stimulation of the activity by phosphatidylcholine or low concentrations of lysophosphatidylcholine was similar to that by Triton X-100, and taurocholate was less effective than Triton X-100. The enzyme activity was inhibited by divalent cations such as Hg2+ and Cu2+.

Myometrial estrogen and progesterone receptor binding in pregnancy: inhibition by the detergent action of phospholipids.

We characterized the phospholipid inhibition of estradiol and progesterone binding to guinea-pig and human myometrial receptors. Of twelve compounds studied, phosphatidylinositol (PI), lysophosphatidic acid and lysophosphatidylcholine (lyso-PC) were the most active inhibitors (50% inhibition at 10(-5) M). Lyso-PC with fatty acid chain length C14:0 inhibited ligand binding both to estrogen receptor (ER) and progesterone receptor (PR), C16:0 only to PR and C18:0 neither to ER nor to PR. The lyso-derivates were more inhibitory than the parent compounds. The ionic detergent (sodium taurocholate) inhibited both ER and PR binding, but the non-ionic detergent (Triton X-100) only PR. Triton X-100 enhanced the PI-induced inhibition of ER binding by a factor of 10. PR was more sensitive to inhibition than ER in all cases. The type of inhibition was non-competitive. At term pregnancy, ligand binding to myometrial ER or PR was low or absent in humans, but moderate in the guinea-pig. Phospholipid extracts of human decidua and fetal membranes contained PI and phosphatidylserine rather than lyso-PC. The extract was a potent inhibitor of ligand binding to PR (50% inhibition at 10(-6) M phospholipid phosphorus), but not to ER. The physicochemical environment, modulated by phospholipids acting as detergents, may regulate sex steroid function also in vivo. This might have special significance for pregnancy maintenance.