The formylpeptide chemotactic receptor on rabbit peritoneal neutrophils. I. Evidence for two binding sites with different affinities

F Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe binding to rabbit peritoneal neutrophils and purified membranes were measured at 4 degrees C silicone oil centrifugation assays, and the results were analyzed by the LIGAND computer program, which permits analysis of ligand binding to multiple classes of binding sites. LIGAND analysis of peptide binding to intact neutrophil indicated that both f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe detected two population of binding sites. The apparent Kd values for f Met-Leu-[3H]Phe binding were 1.6 +/- 1.0 X 10(-9) M and 2.2 +/- 0.9 X 10(-8) M, respectively, and 3.1 +/- 0.2 X 10(-9) M and 1.2 +/- 0.6 X 10(-7) M for f Nle-Leu-[3H]Phe. Furthermore, the higher affinity sites detected on whole cells comprised approximately 15 to 30% of the total sites. Two populations of binding sites were also detected on purified neutrophil plasma membranes by both radiolabeled chemotactic peptides. LIGAND analysis of peptide binding to purified membranes yielded apparent Kd values of 5.0 +/- 2.5 X 10(-10) M and 4.8 +/- 0.6 X 10(-8) M for f Met-Leu-[3H]Phe binding, and 4.7 +/- 4.2 X 10(-10) M and 3.0 +/- 1.3 X 10(-8) M for f Nle-Leu-[3H]Phe. The percentage of higher affinity sites detected by f Met-Leu-[3H]Phe and f Nle-Leu-[3H]Phe on purified membranes were 1 to 5% of the total sites detected. These data are consistent either with the existence of two independent binding sites for formylpeptides on rabbit neutrophils or receptor negative cooperativity.     

Extracellular hydrolysis of formyl peptides and subsequent uptake of liberated amino acids by alveolar macrophages

The mechanism of accumulation of radioactive label from fNle-Leu-[3H]Phe by guinea pig alveolar macrophages was investigated. The binding of fNle-Leu-[3H]Phe to macrophages reached equilibrium within 5 min at 4 degrees C, but equilibrium could not be achieved at temperatures where fNle-Leu-Phe stimulated superoxide anion production is observed (e.g., 21-23 degrees C). At this temperature a rapid phase of initial binding of fNle-Leu-[3H]Phe to its receptor was followed by continued accumulation of cell-associated radioactivity which was linear and was dependent on the extracellular pH, i.e., the rate increased as the pH was lowered from pH 8 to pH 6. Examination for possible intracellular hydrolysis of fNle-Leu-[3H]Phe revealed the presence of extensive amounts of [3H]phenylalanine, both cell-associated and in the medium. The increases in cell-associated [3H]phenylalanine correlated in time and pH with cell-associated radioactivity that was accumulated after stimulation with fNle-Leu-[3H]Phe. The addition of 1 mM unlabelled phenylalanine blocked the long term accumulation of label from fNle-Leu-[3H]Phe by macrophages. 1 mM phenylalanine had no measureable effect on fNle-Leu-Phe stimulated O2- production, fNle-Leu-[3H]Phe hydrolysis or on fNle-Leu-[3H]Phe binding to its receptor. These results indicated that the long term accumulation of radioactivity by alveolar macrophages was due to extracellular hydrolysis of fNle-Leu-[3H]Phe followed by transport of liberated [3H]phenylalanine into the cells. A high affinity (Km = 3.56 X 10(-8) M) transport system for phenylalanine was measured in alveolar macrophages, which was not stimulated by the addition of fNle-Leu-Phe. The extracellular hydrolysis of fNle-Leu-[3H]Phe could not be attributed to release of macrophage enzymes into the medium. The responsible proteinase appears to be membrane bound and has a Km for the hydrolysis of fNle-Leu-[3H]Phe of 2.6 X 10(-7) M which is similar to the Kd (1.5 X 10(-7) M) for fNle-Leu-Phe binding. Taken together, these data suggest that for the alveolar macrophage: (1) formyl peptides are not internalized by a receptor-mediated process; (2) a surface proteinase can catalyze the hydrolysis of formyl peptides; and (3) [3H]phenylalanine formed by fNle-Leu-[3H]Phe hydrolysis is transported into the interior of the macrophage.     

Deactivation of human neutrophil chemotaxis by chemoattractants: effect on receptors for the chemotactic factor f-Met-Leu-Phe

Normal human peripheral blood PMN were exposed to varying concentrations of partially purified chemotactic complement fragments (C5fr) and a chemotactic peptide N-formyl methionylleucylphenylalanine (f-Met-Leu-Phe). This exposure resulted in a decreased chemotactic response termed deactivation of chemotaxis. Deactivation was found to be nonpreferential for the deactivating stimulus when high concentrations of either f-Met-Leu-Phe (10(-6) M) or C5fr (20 micrograms/ml) were used. When PMN were incubated with lower concentrations of C5fr (10 micrograms/ml), there was preferential deactivation towards C5fr. Similarly, preferential deactivation of chemotaxis was observed when PMN were incubated with 10(-6) M f-Met-Leu-Phe, but this was transient and cells were nonpreferentially deactivated 60 min after the initial exposure to f-Met-Leu-Phe. The availability of receptors for tritiated f-Met-Leu-Phe was examined by Scatchard analyses and measurement of reversible f-Met-Leu-[3H]Phe binding to C5fr and f-Met-Leu-Phe-deactivated PMN. When PMN f-Met-Leu-Phe receptors were studied immediately after exposure to concentrations of C5fr causing either preferential or nonpreferential deactivation, there was increased receptor availability compared with control PMN. In contrast, PMN deactivated with high concentrations of f-Met-Leu-Phe 10(-6) M) had a transient decrease in the number of receptors followed 1 hr later by an increase in the number of receptors. This was similar to the functional correlate of preferential deactivation of chemotaxis immediately after incubation with f-Met-Leu-Phe followed by nonpreferential deactivation in these same PMN. The data indicate that preferential deactivation of chemotaxis may be associated with a preferential decrease (down-regulation) of chemoattractant receptors and that nonpreferential deactivation is associated with an increase in chemoattractant receptors.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.     

Detergent solubilisation of the rabbit neutrophil receptor for chemotactic formyl peptides

Digitonin was found to be the only detergent (out of 24 tested) capable of solubilising the chemotactic formyl peptide receptor from rabbit neutrophil membranes in a form which retained its [3H]fMet-Leu-Phe binding activity. The solubilised material retained many of the characteristics of the membrane-bound receptor. [3H]fMet-Leu-Phe binding to the digitonin extract was measured at 4 degrees C using an equilibrium dialysis assay. Binding was saturable and of high affinity (Kd = 3.5 +/- 0.7 nM). The potencies of a series of synthetic peptides as inhibitors of [3H]fMet-Leu-Phe binding to the soluble receptor showed the same rank order as for inhibition of the membrane-bound receptor. In addition, binding to both preparations was sulphydryl dependent showing a parallel inhibition by p-chloromercuribenzene sulphonate which could be partially reversed by subsequent incubation with dithiothreitol.     

Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor

The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.     

Formyl peptide chemotaxis receptors on the rat neutrophil: experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[3H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analysis of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P less than .010). In addition, Hill coefficients of 0.80 +/- 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[3H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[3H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone. In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[3H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[3H]Phe receptor/f Met-Leu-[3H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.     

Substance P binds to the formylpeptide chemotaxis receptor on the rabbit neutrophil

Substance P, a potent vasodilatory and smooth muscle contracting agent, binds specificially to the formyl peptide receptor on the rabbit neutrophil. Substance P stimulates chemotaxis and induces lysosomal enzyme secretion in concentrations which similarily inhibit f Met-Leu-(³H)Phe receptor binding. Competitive antagonists of the formyl peptide receptor also inhibit the activity of Substance P. The finding of a naturally occurring eukaryotic peptide interacting with the neutrophil formyl peptide receptor is of importance.     

Effect of membrane fluidizers on the number and affinity of chemotactic factor receptors on human polymorphonuclear leukocytes

Chemotaxis by leukocytes appears to be initiated by the binding of chemo-attractants to specific cell surface receptors. In other biological systems, the affinity and functional activity of membrane receptors are regulated by the local microviscosity. The present studies were undertaken to determine if the number and/or affinity of chemotactic factor receptors expressed on human polymorphonuclear leukocytes were similarly affected. Aliphatic alcohols and cis-vaccenic acid, agents known to decrease membrane microviscosity, were studied for their effects on the binding of the radiolabeled chemoattractant f-Met-Leu-[3H]Phe to human polymorphonuclear leukocytes. Butanol and propanol increased the number of f-Met-Leu-[3H]Phe binding sites approximately 1.5 fold. More dramatically, these same agents enhanced the affinity of the receptor by ten-fold, without affecting the specificity of the receptor. Similarly, cis-vaccenic acid enhanced both the number and affinity of this chemotactic factor receptor on human polymorphonuclear leukocytes contain cryptic receptors for the N-formylated peptide chemotactic factors, but more importantly that the affinity of these receptors can exist in more than one state and can be modulated by membrane microviscosity. Alterations of membrane fluidity in leukocytes during chemotaxis may be an important mechanism for regulating their sensitivity to chemoattractants.     

Quantitative and qualitative differences in guanine nucleotide binding protein activation by formyl peptide and leukotriene B4 receptors.

Formyl peptides and leukotriene B4 (LTB4) stimulate disparate neutrophil functional responses and second messenger generation. The hypothesis that differences in receptor-guanine nucleotide-binding proteins (G protein) interaction account for the disparate responses was examined using HL-60 granulocyte plasma membranes. The quantity of receptor-coupled G proteins was determined by guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) equilibrium binding in the presence or absence of f-Met-Leu-Phe and/or LTB4. About one-third of the total GTP gamma S binding sites were coupled to f-Met-Leu-Phe receptors, to LTB4 receptors, and to receptors when both ligands were added simultaneously. The dissociation constant of GTP gamma S-binding sites in the presence of LTB4 was significantly greater than that in the presence of f-Met-Leu-Phe. f-Met-Leu-Phe shifted the GDP dose-inhibition curve for GTP gamma S binding further to the right than did LTB4. The apparent initial rate of GTP hydrolysis and GTP gamma S binding stimulated by f-Met-Leu-Phe was significantly greater than that stimulated by LTB4. There were significantly more formyl peptide receptors than LTB4 receptors, however, formyl peptide and LTB4 receptor density did not differ under GTP gamma S binding assay conditions. The rate of GTP hydrolysis stimulated by LTB4 was not increased in membranes containing twice the LTB4 receptor density. We conclude that formyl peptide receptors stimulate more rapid activation of a common pool of G proteins than LTB4 receptors because of a significantly reduced affinity of formyl peptide receptor-activated G proteins for GDP.     

Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes

The mobilization of internally sequestered stores of Ca2+ and activation of protein kinase C appear to be involved in neutrophil activation. We have examined the inter-relationship of these two pathways by investigating the effects of modulating Ca2+ activity on the binding of [3H]phorbol 12,13-dibutyrate (PDBU) to protein kinase C in intact phagocytes. Differentiated HL-60 cells were equilibrated with [3H]PDBU prior to stimulation with various agents known to alter Ca2+ homeostasis in cells. Agents that elevated cytosolic Ca2+, such as f-Met-Leu-Phe and A23187, up-regulated radioligand binding by increasing the affinity of the PDBU/protein kinase C interaction. These effects were time- and agonist concentration-dependent and temperature-sensitive. The kinetics of the up-regulation of binding by f-Met-Leu-Phe coincided with the kinetics of Ca2+ mobilization (by quin2 fluorescence measurements). The putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate alone down-regulated [3H]PDBU binding and inhibited the up-regulation of ligand binding by f-Met-Leu-Phe and A23187. Low concentrations of La3+ (0.1-10 microM) also inhibited up-regulation of radioligand binding to f-Met-Leu-Phe and A23187, whereas higher concentrations (0.1-1 mM) alone increased [3H] PDBU binding and supported further up-regulation of ligand binding by the Ca2+-mobilizing agents. These data suggest a role for Ca2+ in the regulation of phorbol diester binding to protein kinase C in intact cells.     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.     

Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega-oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.     

The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.     

ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells

To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.     

Endocytosis and degradation of chondroitin sulphate by liver endothelial cells.

Intravenously administered chondroitin sulphate, chemically labelled by [3H]acetylation of partially deacetylated polysaccharide, was taken up and degraded by the non-parenchymal cells of the liver. Studies using primary monolayer cultures of pure Kupffer cells, liver endothelial cells and parenchymal cells revealed that [3H]chondroitin sulphate was taken up and degraded by the liver endothelial cells only. Binding studies at 4 degrees C with [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan indicated that the glycosaminoglycan and the proteoglycan are recognized by the same binding sites on the liver endothelial cells. The ability of hyaluronic acid to compete with the labelled ligands for binding suggested that the binding site is identical with the recently described hyaluronate receptor on the liver endothelial cells [Smedsrød, Pertoft, Eriksson, Fraser & Laurent (1984) Biochem. J. 223, 617-626]. Fluorescein-labelled chondroitin sulphate proteoglycan accumulated in perinuclear vesicles of the liver endothelial cells, indicating that the proteoglycan is internalized and transported to the lysosomes. The finding that [3H]chondroitin sulphate and 125I-chondroitin sulphate proteoglycan were degraded by the liver endothelial cells to low-molecular-mass radioactive products suggested that both the polysaccharide chain and the core protein were catabolized by the cells.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between the binding of N-formylmethionylleucylphenylalanine and the respiratory responses in human neutrophils

The results presented in this paper demonstrate that the chemotactic peptide N-formylmethionylleucylphenylalanine (-Met-Leu-Phe) is rapidly inactivated by the products of the respiration of human neutrophils stimulated by the peptide itself. The process of inactivation is impeded by the addition of inhibitors of myeloperoxidase (KCN, NaN₃), of catalase, of methionine but not by the addition of superoxide dismutase, indicating that the mechanism of inactivation is the oxidation of methionine residue by myeloperoxidase-H₂O₂-halide system. The oxidation of the peptide causes the rapid cessation of the respiratory burst, since the sulfoxide derivative loses its ability to bind the specific receptors of neutrophil surface and, hence, its biological activity. The comparison between the time course of the binding of f-Met-Leu-[³H]Phe to the specific receptors and the rate of the respiratory response of neutrophils in the presence and in the absence of the process of peptide oxidation was used to investigate the mechanism of the activation of the respiratory burst by the peptide-receptor complexes. In conditions where the inactivation of the stimulatory agent takes place the stimulated respiration slows down and resumes the resting state shortly after the cessation of the binding, although a substantial amount of the peptide remains bound to the specific receptors. In conditions where the degradation of the peptide does not occur the binding of the peptide and the respiratory burst continue for a longer period of time, but the rate of the respiration, calculated in terms of the instantaneous velocity (V_ist), is not correlated to the amount of the ligand bound to the membrane receptors measured at various times, indicating that a summation of the effects ofthe ligand-receptor complexes does not occur as they form. These findings demonstrate, as far as the respiratory response is concerned, that the bioogical activity of the peptide-receptor complexes is short-lived and that continuous de-novo receptor occupancy is necessary for the maintenance of the activated respiration.     

Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation.

Cyclosporin (Cs)A but not CsH inhibits activation of human lymphocytes. We studied the effects of CsA, CsD, and CsH on human neutrophil activation induced by chemoattractants and by various substances that circumvent receptor stimulation. CsH inhibited superoxide (O2-) formation induced by the chemotactic peptide, FMLP (30 nM), with a half-maximal effect at 40 nM. O2- formation was abolished by CsH at 1 microM. CsH increased the concentration of FMLP causing half-maximal activation of O2- formation from 30 nM to 0.8 microM and substantially reduced the stimulatory effect of FMLP at supra-maximally effective concentrations. The inhibitory effect of CsH on O2- formation was evident immediately after addition to neutrophils. CsH also markedly inhibited the increase in cytosolic Ca2+ ([Ca2+]i), beta-glucuronidase, and lysozyme release and aggregation stimulated by FMLP. CsA and CsD were considerably less effective than CsH to inhibit FMLP-induced O2- formation. CsA and CsD were without effect on exocytosis, rises in [Ca2+]i, and aggregation induced by the chemotactic peptide. Cyclosporines inhibited FMLP-induced O2- formation in an additive manner, indicating that they acted through a mechanism they had in common. Cyclosporines only slightly inhibited O2- formation and lysozyme release induced by C5a. Aggregation and rises in [Ca2+]i stimulated by C5a were not affected by cyclosporines, and they did not inhibit O2- formation and exocytosis induced by platelet-activating factor and leukotriene B4. Cyclosporines partially inhibited O2- formations induced by NaF and gamma-hexachlorocyclohexane. CsA marginally inhibited PMA-induced O2- formation and lysozyme release. CsA, CsD, and CsH did not inhibit arachidonic acid-induced O2- formation and its potentiation by NaF or stable guanine nucleotides in a cell-free system from DMSO-differentiated HL-60 cells. CsH partially inhibited binding of FML [3H]P to formyl peptide receptors in membranes from DMSO- or dibutyryl cAMP-differentiated HL-60 cells. Our data show that: 1) cyclosporines differentially inhibit activation of human neutrophils; and 2) CsH is, indeed, not immunologically inactive but is a potent and effective inhibitor of FMLP-induced O2- formation. 3) CsH interferes with agonist binding to formyl peptide receptors and in addition, cyclosporines may also act at sites distal to chemoattractant receptors.