Number Of Specific Binding Sites Research Articles

Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells.

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.

Ontogeny of glucocorticoid receptors in the hyperstriatum-hippocampus-parahippocampal area and optic tectum of the embryonic chicken (Gallus domesticus) brain.

The importance of glucocorticoids and their perturbation during development is an active research area. Developmental insults, including direct and indirect consequences of exposure to drugs of abuse or withdrawal from them, may act upon or via the neuroendocrine axis of the pregnant experimental subject (e.g. rat) and/or directly upon the neuroendocrine axis of the embryo or fetus. The use of the domestic chicken embryo may constitute a good experimental subject for studying these effects in the absence of maternal influences. Thus, the pattern of brain glucocorticoid cytosolic receptors were characterized in an early developing brain region, the optic tectum (OT) and a later developing region with a different function, the hyperstriatum-hippocampus-parahippocampal (HHP) area, on embryonic days (E) 11, 15, 18 and on the day of hatching (HD). The influence of the glucocorticoid synthesis inhibitor metyrapone, injected into eggs on E14 and on E17, upon glucocorticoid receptors (on E15 and E18) was also studied to determine effects of a 'chemical adrenalectomy'. Receptors for this steroid are high on E11 and E15, decreasing as they approach the time of hatching, with the HHP generally showing greater numbers of specific binding sites for [3H]-corticosterone (CORT). Although metyrapone treatment did not alter the apparent number of receptors on E15, on E18 it unmasked receptors otherwise occupied by endogenous ligand(s) and/or induced their synthesis, resulting in significantly more receptors identified with [3H]-CORT. Nevertheless, the HHP continued to display more of these receptors than the OT on E15 and E18 after injection of metyrapone. These observations are consistent with the hypotheses that the HHP of embryos of this species contains a higher density of glucocorticoid receptors than does the OT; that glucocorticoid receptor quantification is related to steroid synthesis inhibition in late embryonic development; and that neuroendocrine feedback control of serum glucocorticoids may become functional between E15 and E18. The results also suggest the use of this experimental approach for assessing the effects of developmental insults with drugs, other than metyrapone, as a marker for altered neuroendocrine development and/or function.

Hyporesponsiveness of inflamed human gingival fibroblasts from patients with chronic periodontal diseases against cell surface components of Porphyromonas gingivalis

Inflamed human gingival fibroblasts (HGF) of patients with chronic periodontal diseases have less active interleukin-8 (IL-8) production compared with normal HGF of volunteers with healthy gingival tissues, after stimulation with Porphyromonas gingivalis surface components such as fimbriae, lipopolysaccharide (LPS) and its lipid A, but not LPS or lipid A from other bacterial species. A decrease in number of specific binding sites for P. gingivalis fimbrial molecules in inflamed HGF is also observed by Scatchard plot analysis. A short exposure (6 h) to P. gingivalis LPS resulted in significant potentiation of the LPS-dependent IL-8 production in normal HGF, whereas a long exposure (48 h) to the LPS significantly reduced IL-8 production. Tyrosine phosphorylation of proteins of 127 kDa and 186 kDa in inflamed HGF stimulated with P. gingivalis fimbriae or its LPS was observed by immunoblotting, and these two phosphoproteins were termed tolerance-induced protein, TIP. Protein bands of 45 kDa which bound to radioiodinated P. gingivalis fimbriae in the presence and absence of fetal bovine serum (FBS), and major 73-kDa and minor 30-kDa and 45-kDa bands which bound to radioiodinated P. gingivalis LPS in the presence of FBS in normal and inflamed HGF were observed by using photocrosslinking. These findings suggest that the hyporesponsiveness of HGF induced by a prolonged exposure to P. gingivalis may emerge because of HGF damage or result from host defense in chronic periodontal lesions.

Hyporesponsiveness of inflamed human gingival fibroblasts from patients with chronic periodontal diseases against cell surface components of Porphyromonas gingivalis.

Inflamed human gingival fibroblasts (HGF) of patients with chronic periodontal diseases have less active interleukin-8 (IL-8) production compared with normal HGF of volunteers with healthy gingival tissues, after stimulation with Porphyromonas gingivalis surface components such as fimbriae, lipopolysaccharide (LPS) and its lipid A, but not LPS or lipid A from other bacterial species. A decrease in number of specific binding sites for P. gingivalis fimbrial molecules in inflamed HGF is also observed by Scatchard plot analysis. A short exposure (6 h) to P. gingivalis LPS resulted in significant potentiation of the LPS-dependent IL-8 production in normal HGF, whereas a long exposure (48 h) to the LPS significantly reduced IL-8 production. Tyrosine phosphorylation of proteins of 127 kDa and 186 kDa in inflamed HGF stimulated with P. gingivalis fimbriae or its LPS was observed by immunoblotting, and these two phosphoproteins were termed tolerance-induced protein, TIP. Protein bands of 45 kDa which bound to radioiodinated P. gingivalis fimbriae in the presence and absence of fetal bovine serum (FBS), and major 73-kDa and minor 30-kDa and 45-kDa bands which bound to radioiodinated P. gingivalis LPS in the presence of FBS in normal and inflamed HGF were observed by using photocrosslinking. These findings suggest that the hyporesponsiveness of HGF induced by a prolonged exposure to P. gingivalis may emerge because of HGF damage or result from host defense in chronic periodontal lesions.

Affinity labelling of frog brain opioid receptors by dynorphin(1-10) chloromethyl ketone.

It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphin(1-10)-Gly11 chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog (Rana esculenta) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the kappa-opioid binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta sites ([3H]D-Ala2-Leu5]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.

Influence of oestrogens on muscarinic receptor density and contractile response in the guinea-pig uterus.

The objective of this study was to investigate the contractile response in vitro of uterine and myometrial strips from control and oestrogen-treated guinea-pigs to carbachol. In addition, the muscarinic receptor parameters in subcellular preparations from the myometrium of the same animals were evaluated. Strips of guinea-pig uterus contract in response to carbachol in a concentration dependent manner and the effect of carbachol is independent of the presence of an intact endometrium. However, treatment with oestrogen reduces the response to carbachol, when expressed with respect to the effect of KCl. Measurement of the specific binding of [3H]quinuclidinyl benzylate ([3H]QNB) to myometrial membranes from control and oestradiol-treated immature guinea-pigs showed that oestradiol significantly decreased the number of specific binding sites of [3H]QNB, but did not change the apparent affinity of specific [3H]QNB binding. Moreover, the apparent affinity constants for the different antagonists showed a close similarity to those obtained at muscarinic receptors present in the myometrium from control and oestradiol-treated guinea-pigs.

The Agonist Binding Site on the Bovine Bradykinin B2 Receptor Is Adjacent to a Sulfhydryl and Is Differentiated from the Antagonist Binding Site by Chemical Cross-linking

Chemical cross-linking was used to analyze the binding sites for the agonist bradykinin (BK) and the antagonists NPC17731 and HOE140 on the bovine B2 bradykinin receptor. [3H]BK and [3H]NPC17731 bound with high affinity to the same B2 receptor in bovine myometrial membranes as determined by the total number of specific binding sites and pharmacological specificity of the binding of these two radioligands. Cross-linking experiments were done using a series of bifunctional reagents reactive either primarily to amines (homobifunctional) or reactive to amines in one end and to sulfhydryls in the opposite end (heterobifunctional). All the heterobifunctional reagents plus the homobifunctional arylhalide 1,5-difluoro-2,4-dinitrobenzene were effective in cross-linking the [3H]BK N terminus specifically to a sulfhydryl in the receptor, and this cross-linking occurred at 5-100 microM reagent. In contrast, the homobifunctional N-hydroxysuccinimide ester reagents, at < or = 1 mM, were only able to cross-link [3H]BK to membrane proteins nonspecifically. The sulfhydryl reagents N-ethylmaleimide, iodoacetamide, and phenylarsine oxide blocked cross-linking, whereas these reagents did not inhibit reversible specific [3H]BK binding. Immunoblotting with anti-BK antiserum revealed that low concentrations of BK (5-50 nM) were cross-linked to a receptor-specific species of 65 kDa. All cross-linking of [3H]NPC17731 was nonspecific with both homobifunctional and heterobifunctional reagents. The 65-kDa receptor-specific species was observed on anti-HOE140 immunoblots, but only when proteins were cross-linked with very high concentrations of HOE140 (> or = 500 nM). Our results provide direct biochemical evidence that the binding site for the agonist BK in the bovine B2 receptor is adjacent to a cysteine and is differentiated from the binding site(s) for the antagonists NPC17731 and HOE140.

Cholecystokinin-A Specific Antagonism of KSG-504 to Cholecystokinin Receptor Binding and Pancreatic Secretion in Mammals

The effects of KSG-504 ((S)-arginium (R)-4-[-N-(3-methoxypropyl)-Ar-pentylcarbamoyl]-5-(2-naphthylsulfonyl) pentanoate monohydrate), a new cholecystokinin (CCK)-receptor antagonist, onl25I-CCK-8 binding to rat pancreatic, canine gallbladder and guinea pig cerebrocortical membranes and the pancreatic amylase release from isolated rat acini stimulated by several kinds of secretagogues, including CCK, were investigated. The125I-CCK-8 saturation experiment showed that pancreatic, gallbladder and cerebrocortical CCK receptors had a single high affinity binding component with dissociation constants (Kd) of 0.18, 0.31 and 0.88 nM, respectively. The maximum numbers of specific binding sites (Bmax) in these membranes were 1012, 52 and 20 fmol/mg protein, respectively. KSG-504 and CCK-8 displaced specific125I-CCK-8 binding to CCK receptors in all membrane preparations in a competitive manner. The affinity of KSG-504 for pancreatic (Ki= 173 nM) and gallbladder (Ki = 283 nM) CCK receptors were >3 orders of magnitude higher than its affinity for cerebrocortical CCK receptors. KSG-504 also inhibited125I-gastrin-I binding to guinea pig gastric glands, but the IC50 value (18.2 μΜ) was apparently much higher. CCK-8-stimulated amylase release from isolated pancreatic acini of rats was antagonized by KSG-504 in a concentration-dependent manner. KSG-504 did not affect amylase release stimulated by secretagogues such as gastrin-releasing peptide, carbachol, vasoactive intestinal peptide and A23187. These results indicate that KSG-504 acts as a CCK-A-receptor-specific antagonist in the pancreas and gallbladder.

Localization of calcitonin gene-related peptide binding sites in the eye of different species.

The localization of calcitonin gene-related peptide (CGRP) binding sites in the eye of monkey, pig, cat and guinea pig was studied by autoradiography. Specific binding of CGRP was found in ciliary muscle, ciliary processes and limbal conjunctiva in all tested species. Furthermore, specific binding sites of CGRP was found in the choroidea of monkey, pig and guinea pig, in the iris of pig, cat and guinea pig, in the retina of pig and in the anterior chamber angle of cat. The number of specific binding sites varied depending on the tissue and species. The present study shows that there are specific binding sites of CGRP in the eye of monkey, pig, cat and guinea pig. CGRP binding sites found in vascular system of ciliary body, choroidea and iris further demonstrates the role of CGRP as a vasoregulatory peptide. Binding sites in the ciliary muscle, in the limbal conjunctiva and in the chamber angle area may indicate a role in the regulation of ciliary muscle tone, epithelial cell regeneration and aqueous humour outflow.

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to opioid receptors in frog brain membrane preparations.

A tritiated heptapeptide, [3H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([3H]Met-enkephalin-Arg6-Phe7), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent KD value of the radioligand calculated from homologous displacement experiments was 3.4 nM, and the maximal number of specific binding sites was 630 fmol/mg of protein. The KD determined from equilibrium saturation binding studies was found to be 3.6 nM. However, the Hill coefficient was far below unity (nH = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3H]Met-enkephalin-Arg6-Phe7 binding reflects a kappa 2- and/or delta-subtype specificity of the heptapeptide. Binding to a kappa 1 and/or mu site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg6-Phe7 in frogs cannot be ruled out. The [3H]-Met-enkephalin-Arg6-Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.

Physicochemical Parameters Affecting the Charcoal Adsorption Assay for Quantitative Retinoid-Binding Measurement

The different parameters affecting the accuracy and reliability of the dextran-coated charcoal adsorption assay for characterization of retinoic acid receptors ligand binding activity were investigated. Using dextran-coated charcoal (DCC) at a final 10 mg/ml concentration, an efficient adsorption of free [3H]retinoic acid was observed with a yield in the range 99.2 to 99.8% for ligand concentrations varying from 10−9 to 10−4 M. Nonspecific adsorption of retinoic acid reached 50% to polystyrene and silanized glass and 70% to uncoated glass. Results obtained by the DCC method and by gel-filtration assay were correlated; however, the DCC assay appeared easier to perform and gave more reproducible results. When a careful measurement of free retinoid concentration was performed, the apparent equilibrium dissociation constant (KD) of retinoic acid was 3.1 ± 0.4 nM and the KD of CD367, a synthetic retinoid, was 1.8 ± 0.3 nM. Optimal pH for the binding of [3H]retinoic acid or [3H]CD367 was in the range 7.5 to 8.5. Under the conditions described for the adsorption assay, bound retinoid measurement was linearly related to the protein concentration between 0.05 and 0.25 mg/ml. At a lower protein concentration, addition of bovine serum albumin exerted a stabilizing effect on retinoid binding, allowing an accurate measurement of the number of specific binding sites. Using retinoic acid as ligand, bacterial extracts often resulted in a level of nonspecific binding in the range 10-25%. It could be lowered (4-10%) when resorting to [3H]CD367. The extent of nonspecific binding could be significantly reduced when organic solvents (dimethylformamide or dimethyl sulfoxide) were added at a 3-6% final concentration during the adsorption step with charcoal, whereas gelatin was unefficient. The DCC adsorption assay used under optimized conditions provides a rapid and useful method for an accurate and quantitative measurement of retinoic acid receptors.

Characteristics of a novel low affinity complement C3dg-binding protein of human platelets.

The breakdown products of the complement protein C3 function in receptor-mediated immune clearance. The catabolism of the C3 molecule, mediated by factors I and H, results in the generation of the fragments C3c and C3dg. C3dg binds to human platelets in a specific and saturable manner. The direct interaction of platelets with soluble C3dg may contribute to immune-mediated platelet destruction. More importantly, platelets may interact with opsonized pathogens or complement-activating immune complexes via C3dg. In this report, we investigated the interaction of C3dg with platelets and calculated the Ka to be 3.2 x 10(6) M-1 with 1100 to 2200 specific binding sites/platelet. In the presence of 5 mM calcium, both the Ka and the number of specific binding sites were modestly decreased to Ka = 2.8 x 10(6) M-1 with 1400 to 2400 specific binding sites/platelet. The Scatchard plots demonstrated a curvilinear character. On labeling C3dg with peroxidase and visualizing platelet-bound C3dg by electron microscopy, it was shown that binding sites for C3dg were restricted to the platelet plasma membrane. Using a cell attachment assay, platelet adhesion to C3dg was readily detectable; attachment to C3dg-coated plates was not blocked by fibrinogen or fibronectin. We have characterized the C3dg-binding protein of platelets using the chemical cross-linkers, SASD and DSS, to cross-link C3dg to thrombin-stimulated platelets. Gel filtration of the 125I-labeled C3dg-platelet complex revealed the presence of a large protein complex that was absent when 125I-labeled C3dg alone was analyzed. SDS-PAGE of the radiolabeled cross-linked protein, followed by autoradiography, identified a 95-kDa membrane protein. The relationship of this C3dg-binding protein to other platelet membrane proteins has yet to be determined.

Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon.

Aldosterone is a major regulator of Na(+)-absorptive and K(+)-secretory processes in the distal segment of mammalian colon. In this study, the distribution of aldosterone-sensitive cell types in isolated rat distal colon was determined using site-directed intracellular microelectrodes, specific Na(+)- and K(+)-channel blockers, and aldosterone-receptor binding techniques. Electrophysiological data indicated that aldosterone induced parallel apical membrane Na+ and K+ conductances, mainly in surface cells and to a significantly lesser degree in crypt cells. Scatchard analyses of aldosterone-receptor binding in cytosolic fractions revealed the maximum number of specific binding sites in whole mucosal homogenate and in the upper one-third and lower two-thirds of isolated crypt units to be 74.9 +/- 2.0, 59.8 +/- 2.4, and 59.3 +/- 3.2 fmol/mg protein, respectively, indicating the presence of aldosterone receptors in the crypt cell population. We conclude that in rat distal colon aldosterone-induced Na+ and K+ conductances (and by inference, electrogenic Na(+)-absorptive and K(+)-secretory processes) are located predominantly in the surface cell population and to a lesser extent in crypt cells, which also contain aldosterone receptors. This spectrum of aldosterone-induced Na+ and K+ conductances may reflect varying stages of differentiation along the surface cell-crypt cell axis.

Testing the quality of electron microscope mapping data for DNA molecules with sequence-specific ligands

A procedure for the testing of Electron Microscope (EM) mapping data for DNA molecules with site-specific bound ligands is suggested. The difficulty of distinguishing DNA molecule ends on electron micrographs indicates that their true orientations are not known. This in turn presents problems in obtaining correct maps relating to their alignment, and complicates checking the maps' validity. For these reasons a computer simulation of the EM study of double-stranded DNA molecules with site-specific bound ligands was carried out. The knowledge of the true orientations of the simulated DNA molecules allowed us to examine their final orientations after alignment. We used the number of improper-oriented molecules as the quantitative measure of the map quality. Detailed investigation based on this parameter permitted us to invent the criterion for the map validity, and to suggest the procedure for the testing of alignment of real DNA molecules. This procedure implies multiple randomization of initial orientations of the DNA molecules and minute analysis of the final maps. Most of the molecular, statistical and experimental parameters inherent to EM investigation of site-specific binding, such as the number of specific binding sites ( N), the mean number of bound ligands ( A), the length of the DNA molecules ( L), the specific/non-specific ratio of binding ( K), together with the standard deviation of DNA molecule lengths ( H L were tested for their influence upon the quality of EM mapping data. An empirical equation for the ultimate values of these parameters has been found, allowing us to predict the success of EM mapping.

Quantitative receptor autoradiographic analysis for angiotensin II receptors in bovine retinal microvessels: quantitation with radioluminography.

1. Specific 125I-Sar1, Ile8-Angiotensin II (125I-Sar1, Ile8-AII) binding sites in bovine retinal microvessels were investigated using the quantitative receptor autoradiographic method with pellet sections. 2. A quantitation was made with the computerized radioluminographic imaging plate system, a newly developed and highly sensitive method. Binding characteristics of the retinal microvessels were compared with those of the cerebral microvessels and the retinal macrovessels. 3. We isolated microvessels from the bovine retina and bovine cerebral cortex using the method composed of two-size sievings and high-speed homogenization with a Polytron. The isolated microvessels were composed of capillaries, and the retinal macrovessels contained vessels with smooth muscle. 4. There were specific binding sites for 125I-Sar1, Ile8-AII which were single and of a high affinity, in both the cerebral and the retinal microvessels and the retinal macrovessels. There were no differences in affinity between the vessels, but the retinal microvessels did have a higher density of binding sites than the cerebral microvessels. 5. The method we used is simple and sensitive for detecting and characterizing 125I-Sar1, Ile8-AII binding sites in retinal capillaries. Knowledge of the existence of large numbers of specific binding sites, candidates of physiologically active angiotensin II receptors, aids with understanding the regulatory roles of angiotensin II in the blood-retinal barrier.

Measurement of electrostatic and site‐specific associations of alkali metal cations with humic acid

SUMMARYA discontinuous acidimetric titration method incorporating ultrafiltration was developed to measure the association of a soil humic acid with Li+ t, Na+ and K+ (pH 3 to 8). In addition, possible site‐specific binding of these alkali metal cations was investigated using desorption experiments at pH 1. Li, Na and K cations behaved equivalently in the titrations and the amounts of these cations associated with the humic acid was measurable at all pH values between 3 and 8. Up to 90% of the total alkali metal cation was humate‐associated at pH 8. The absolute amount of humic‐associated cation did not depend on the alkali metal cation concentration, but rather on the solution alkalinity. In addition, the net charge of the humate polyanion made a negligible contribution to the electroneutrality of the bulk solution under all conditions. These results are consistent with a diffuse layer model of hydrated humic acid in which the alkali metal cations neutralize the humic charge. The association of Na+ andK+ with humic acid at pH 1 was successfully described by a Langmuir adsorption model. The number of sites per g of humic acid was very small, and greater for K+ than for Nat. Lithium cations exhibited no detectable humic association at pH 1. These differences suggest that humic acids may have a small number of specific binding sites for which the size of the hydrated cation is important.

Effect of VLDL on the inhibition of arachidonic acid transformation by dexamethasone in cultured smooth muscle cells

Very-low-density lipoproteins (VLDL) induce a dose-dependent reduction (up to 55%) in the number of specific binding sites and about a 2-fold increase in binding affinity for [ 3H]dexamethasone in human and rat smooth muscle cells (SMC). Maximal effect of VLDL was achieved within 3–5 h at a lipoprotein concentration 60 μg protein/ml. Lipoprotein-mediated reduction in the number of [ 3H]dexamethasone binding sites resulted in partial loss of cellular sensitivity to hormone action: dexamethasone (1·10 −6 M) inhibited the transformation of [ 14C]arachidonic acid (AA) into metabolites to a lesser extent in SMC preincubated with VLDL (11.5%) than in untreated cells (29.0%). In particular, under these conditions the inhibitory effect of dexamethasone on prostaglandin I 2 (PGI 2) formation in VLDL-treated SMC was lower than in untreated cells (42.1% vs. 60%). We propose that VLDL is able to counteract the inhibitory effect of glucocorticoids on AA release and PGI 2 formation in vascular SMC by reduction of cellular specific glucocorticoid binding sites.

Eicosapentaenoic acid inhibits cholesteryl ester accumulation in rat peritoneal macrophages by decreasing the number of specific binding sites of acetyl LDL

We found that rat peritoneal macrophages bind acetyl low density lipoprotein (AcLDL) by a saturable and specific manner and accumulate a substantial amount of cholesteryl ester (CE) when incubated with AcLDL. In macrophages enriched with eicosapentaenoic acid (EPA) by the ingestion of highly purified eicosapentaenoic acid-ethyl ester (EPA-E), the accumulation of CE was significantly decreased in a dose-dependent manner. In addition, the contents of EPA and docosapentaenoic acid (DPA) in macrophage phospholipids were dose dependently and significantly increased by EPA-E feeding. In contrast, the contents of arachidonic acid (AA) and docosahexaenoic acid (DHA) were unchanged. Furthermore EPA-E ingestion significantly decreased the Bmax of the AcLDL receptor without affecting the Kd in rat peritoneal macrophages. In addition, specific proteolytic degradation of AcLDL was also dose dependently inhibited by EPA-E feeding, indicating that the number of AcLDL receptor was significantly decreased after EPA-E ingestion. These findings indicate that EPA-E feeding inhibited CE accumulation mainly by decreasing the AcLDL receptors in macrophages. We speculate that EPA inhibits foam cell formation and this inhibitory effect may partly account for its anti-atherogenic action.

Binding characteristics of the new thromboxane A 2/prostaglandin H 2 receptor antagonist [ 3H]BAY U 3405 to washed human platelets and platelet membranes

The new thromboxane A 2 antagonist [ 3H]BAY U 3405 was characterized for its binding to washed human platelets and platelet membranes. In washed platelets the specific binding was reversible, selective and stereospecific, but not saturable. The dissociation constant ( K d) was 6 ± 2.5 nM, the number of specific binding sites 1177 ± 306 per platelet. Three structurally different thromboxane A 2 (TXA 2)/prostaglandin H 2 (prostaglandin endoperoxide) (PGH 2) receptor ligands completely inhibited the specific binding of [ 3H]BAY U 3405 in a concentration-dependent manner, indicating that the observed high affinity binding site is the TXA 2/PGH 2 receptor. In platelet membranes, however, specific [ 3H]BAY U 3405 binding showed saturability in addition to reversibility, selectivity, and stereospecifity. The K d of the binding was 9.6 ± 2.3 nM in kinetic studies and 8.7 ± 3.7 nM in saturation studies, the inhibition constant ( K i) was 10 ± 1.1 nM in displacement studies. The TXA 2/PGH 2 receptor agonists U 46619 and CTA 2, and the antagonists Daltroban (BM 13505), I-PTA-OH and SQ 29548 all completely inhibited the specific binding of [ 3H]BAY U 3405 thus defining the observed binding site as the TXA 2/ PGH 2 receptor. In conclusion, the data suggest that the previously reported TXA 2 antagonism of BAY U 3405 is mediated by binding to a specific high affinity binding site of human platelets and platelet membranes that represents the TXA 2/PGH 2 receptor.

The human platelet as a reproducible and sensitive cell for the detection and assay of platelet-activating factor

A new procedure for the preparation of human platelets consistently sensitive to platelet-activating factor (PAF) in the low nanomolar range has been developed. Key to the success of this approach was the addition of adenosine during the isolation phase, providing an excellent recovery of stable cells, and the inclusion of ADP in the aggregation assay, providing increased sensitivity to PAF. Examination of the binding profile of tritium-labeled PAF to these platelets in the presence or absence of ADP revealed significant difference in the K d values but not in the number of specific binding sites. Other reagents having an influence on the reactivity and stability of the human platelets, as regards its interaction with PAF, are described.