Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.     

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 1. Characterization of binding

A novel, specific, and potent biological action of leukotriene C4 (LTC4) was demonstrated in the Chinese hamster lung fibroblast cell line V79A03 (V79 cells), namely the confirment of protection against subsequent gamma-irradiation. Consequently, studies were conducted to determine whether LTC4-conferred radioprotection could be attributed to a receptor-mediated phenomenon. Specific binding sites for leukotriene C4 (LTC4) were identified and characterized using intact V79 cells incubated at 4 degrees C in the presence of serine-borate, during which time conversion of LTC4 to LTD4 or LTE4 was undetectable. Binding was maximal in a broad region between pH 6.2 and 8.8. Ca2+, Mg2+, and Na+ were not required for binding, and binding was not altered by GTP, ATP, or cAMP, by leukotrienes B4, D4, or E4, or by the leukotriene end point antagonists LY 171883, FPL 55712, or Revlon 5901-5. Scatchard analyses and kinetic experiments indicated the presence of high-affinity [Kd = 2.5 +/- 0.63 nM, approximately 9.9 x 10(5) sites/cell] and low-affinity [Kd = 350 +/- 211 nM, approximately 2.7 x 10(6) sites/cell] binding sites. The observed binding characteristics of LTC4 to V79 cells are consistent with a receptor-mediated phenomenon. In a companion communication which follows this report, we report the subcellular distribution of LTC4 binding to V79 cells and demonstrate that this binding is unlikely to be attributed principally to interaction with glutathione-S-transferase.     

Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells

The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation.     

Characterization of leukotriene C4 binding in anterior pituitary membrane preparations.

Pituitary cells produce leukotrienes (LTs) and respond to exogenous administration of LTs by releasing gonadotropins. Specific high affinity leukotriene C4 (LTC4) binding has been found in membrane preparations of bovine anterior pituitaries. Unlabelled LTC4 displaced specific [3H]LTC4 binding. Other leukotrienes (LTB4, LTD4, LTE4, LTF4) did not compete with [3H]LTC4 for binding sites when administered at increasing concentrations together with a constant amount of radioligand indicating that the binding is highly specific for LTC4. Scatchard analysis of binding data obtained from saturation studies revealed a single binding site for [3H]LTC4 with a Kd of 8.95 +/- 5.53 nM and a B max of 15.44 +/- 6.93 pmol per mg of membrane protein. Glutathione S-transferase, a possible LTC4 binding site, did not display activity in the membrane fraction although the two glutathione derivates S-octylglutathione and S-decylglutathione competed with LTC4 in binding experiments. As leukotrienes are potent stimulators of gonadotropin secretion and modulators of gonadotropin-releasing hormone (GnRH)-induced gonadotropin release it is concluded that leukotrienes may be involved in the signal transduction pathway of GnRH and that they may act via a specific and high affinity receptor.     

Specific endothelin binding sites in renal medullary collecting duct cells: lack of interaction with ANP binding and cGMP signalling.

The diverse biological actions of endothelins (ET) appear to be mediated by specific cell-surface receptors. Autoradiography and membrane binding studies have shown abundant ET binding sites in the kidney. However, their expression in specific types of renal cells is unclear. We studied the binding of 125I-labelled endothelin-1 in freshly isolated cell suspensions from canine inner medullary collecting duct. Competition binding experiments revealed the presence of specific high-affinity binding sites: unlabelled ET-1 and ET-2 compared with the radioligand with an IC50 of 135 and 83 pM, respectively, while the IC50 of ET-3 and big ET-1 were 2 and 4 orders of magnitude higher, indicating the presence of ETA-type receptor. Angiotensin II, vasopressin, and atrial natriuretic peptide (ANP) did not compete for ET binding even at a concentration of 10(-6) M. Saturation binding experiments showed a single class of binding sites of high density (Bmax = 56.7 +/- 10.3 fmol/10(6) cells) and high affinity (Kd = 69.8 +/- 10 pM). In contrast, ANP receptors in the same cell preparations appeared as two classes of binding sites with widely different affinity and density. The high-affinity ANP site (Kd = 311 +/- 48 pM) was compatible with ANP-B (guanylate cyclase-coupled) receptor. ET-1 did not compete for this receptor. ET-1 (10(-7) M) did not alter ANP-induced cGMP generation in these cells (3.8-fold increase at 10(-7) M ANP), nor basal levels of cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung

Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.     

Leukotriene C4 binding to rat lung membranes

A high affinity binding site for leukotriene C4 (LTC4), one component of slow reacting substance of anaphylaxis, has been identified in a membrane preparation from rat lung. As measured by a filtration technique, [3H]LTC4 binding was saturable, specific, reversible, and heat-sensitive. In the presence of 20 mM CaCl2, the dissociation constant (KD) was 41 +/- 9 nM and the maximum number of binding sites (Bmax) was 31 +/- 10 pmol/mg of protein. Specificity was demonstrated by competition studies in which LTC4 had a Ki of 40 nM against specifically bound [3H]LTC4, whereas leukotriene D4 (LTD4) had a Ki of 4 microM. The stereoisomers (5R, 6R) LTC4, (5S, 6S) LTC4, and (5R, 6S) LTC4 had Ki values 3-, 15-, and 25-fold higher than that of natural (5S, 6R) LTC4. Leukotrienes E4 and B4, several prostaglandins and fatty acids, glutathione, and platelet activating factor were even less effective with Ki values above 10 microM. A slow reacting substance of anaphylaxis antagonist, FPL 55712, which, in some systems, distinguishes LTC4- from LTD4-induced contractions, was a weak competitor with a Ki of 16 microM. Serine-borate complex which inhibits gamma-glutamyl transpeptidase, an enzyme responsible for LTC4 metabolism, did not alter binding. In addition, 100 microM FPL 55712 did not reduce metabolism. These observations suggest that the binding observed for LTC4 may represent association with a physiological receptor for this molecule which has a relatively low affinity for LTD4.     

Identification of leukotriene D4 receptor binding sites in guinea pig lung homogenates using [3H]leukotriene D4

We have characterized [3H]leukotriene D4 binding to guinea pig lung homogenates. Both biphasic dissociation kinetics and curvilinear Scatchard plots indicated the presence of [3H]leukotriene high and low affinity states of the binding sites. The rank order of potency for the competition study was leukotriene C4 = leukotriene D4 greater than leukotriene E4 much greater than arachidonic acid, and for their contractile effect on lung strips was leukotriene C4 = leukotriene D4 = leukotriene E4 much greater than arachidonic acid. FPL-55712 was the only other agent tested that inhibited binding. These results suggest that binding of [3H]leukotriene D4 to the homogenate is consistent with its binding to specific leukotriene D4 receptor sites.     

Evidence for specific binding of 15 hydroxyeicosatetraenoic acid to pituitary rat cells

Specific receptors for [3H]-15 HETE have been identified on GH3 cells, a cloned strain of rat pituitary cells. With incremental inputs of radioligand and a constant cell number, specific [3H]-15 HETE binding reached a plateau indicative of saturable binding sites. Ligand analysis of the Scatchard plot demonstrated a single class of high affinity binding sites with a dissociation constant (Kd) of 0.75 nM. 12 HETE competed with radiolabeled 15 HETE (IC50 = 1 x 10(-6) +/- 0.8 M). In contrast, arachidonic acid, leukotriene B4, prostaglandins E2 and F2 alpha did not compete with [3H]-15 HETE.     

Heterogeneity of calcium channel agonist binding sites in the coronary artery

The binding properties (3H) BAY k 8644 a 1,4-dihydropyridine calcium channel agonist were studied in the subcellular membrane fraction isolated from the coronary artery by differential centrifugation. The specific binding of (3H) BAY k 8644 to microsomal membranes of the coronary smooth muscle was rapid, saturable, reversible and of both high and low affinity. The dissociation constants obtained from Scatchard analysis with (3H) BAY k 8644 and nitrendipine were 0.60 +/- 0.02 nmol.l-1 and 9.1 +/- 0.1 nmol.l-1 for the high and low affinity binding site respectively and the estimated maximal numbers of binding sites in the plasma membrane fraction were 0.76 +/- 0.02 and 3.15 +/- 0.18 pmol.mg-1 of protein respectively. The substituted dihydropyridine calcium channel antagonists nitrendipine and nifedipine competitively inhibited specific (3H)BAY k 8644 binding suggesting a common high affinity 1,4-dihydropyridine binding site in the coronary microsomal fraction for calcium channel activator and antagonists. The low affinity agonist binding sites were significantly inhibited by adding nucleoside carrier inhibitors, 2-deoxyadenosine and dipyridamole, and by -SH alkylating agent N-ethylmaleimide. The results suggests that the coronary artery contains both high and low affinity calcium channel binding sites (in a 1:5 ratio) with the low affinity calcium channel agonist binding sites being associated with nucleoside carrier and/or with-SH groups.     

Platelet-activating factor induces leukotriene C4 synthesis by purified human eosinophils

Platelet-activating factor, at a concentration of 10 microM, was capable of inducing leukotriene C4 synthesis by eosinophils of healthy donors, i.e. (3.1 +/- 0.3) x 10(6) molecules leukotriene C4/cell (n = 31, mean +/- SEM, cell purity 87 +/- 2%). Reversed-phase high performance liquid chromatography analysis demonstrated the exclusive synthesis of leukotriene C4. At a concentration of 1 microM, platelet-activating factor was capable of significantly enhancing the calcium ionophore A23187, the opsonized zymosan or the arachidonic acid induced leukotriene C4 synthesis by eosinophils. These results show that PAF is capable of inducing and enhancing the leukotriene C4 formation by human eosinophils.     

Calcium-induced membrane metabolic alterations modify the sex steroids binding into dog brain synaptosomal plasma membranes

The binding of 45Ca2+ into synaptosomal plasma membranes (SPM) of dog brain follows a sigmoid path. In graphical analysis of this binding the mean Hill coefficient (h) was 1.64 +/- 0.09 (r2 = 0.96 +/- 0.02). Binding of Ca2+ into SPM was saturable, with an apparent binding constant of 1.2 +/- 0.1 microM. At saturation, such calcium specific binding sites corresponded to 11.2 +/- 0.9 nmol/mg SPM protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of calcium into SPM of at least one class of high affinity specific binding sites. [14C]estradiol, [14C]estrone and [14C]progesterone, when incubated with SPM up to a concentration of 10 microM for 2 hr at 37 degrees C, bind into SPM at nmolar concentrations. Ca2+ ions up to 5 mM considerably increase steroids binding into SPM. This effect of calcium was concentration-dependent, reached saturation at approx 4-5 mM. Once calcium has promoted steroids binding, the subsequent addition of 25 mM EGTA failed to displace bound steroids. Molecular interactions between calcium and SPM was assessed by measuring the steady-state fluorescence polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the production of malondialdehyde (MDA) during 2 hr incubation of Ca2+ (5 mM) with SPM at 37 degrees C. The effect of Ca2+ on the SPM structure was to increase both the rigidity of the membrane and the MDA production. Chelation of Ca2+ (5 mM) with EGTA (25 mM) did not reverse the increase in the rigidity owing to metabolic alterations of SPM lipids (e.g. production of MDA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.     

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

3H]nitrendipine binding to adrenal capsular membranes

The physiologic regulation of aldosterone secretion is dependent on extracellular calcium and appears to be mediated by increases in cytosolic free calcium concentration in the zona glomerulosa cell. A specific role for voltage-dependent calcium channels was suggested by previous studies with the calcium channel antagonist verapamil. We therefore studied the [3H]nitrendipine calcium channel binding site in adrenal capsules. These studies revealed a single class of saturable, high affinity sites with KD = .26 +/- .04 nM and Bmax = 105 +/- 5.7 fmol/mg protein. Specific binding of [3H]nitrendipine was inhibited by calcium channel antagonists with potencies nitrendipine = nifedipine much greater than verapamil, while diltiazem had no inhibitory effect. In the rat, binding sites for [3H]nitrendipine were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Physiologic studies with collagenase-dispersed adrenal glomerulosa cells demonstrated that nifedipine selectively inhibited angiotensin-II and potassium-stimulated steroidogenesis. These observations suggest both a pharmacologic and physiologic role for the nitrendipine binding site in aldosterone production.     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Identification and characterization of leukotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

We studied the characteristics of the leukotriene (LT) C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [3H]LTC4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant (Kd) of 14.3 +/- 2.0 nM (n = 9). The association and dissociation of [3H]LTC4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [3H]LTC4 binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC4 to compete for the specific binding of [3H]LTC4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The rank order of potency of the unlabeled competitors for competing specific [3H]LTC4 binding was LTC4 much greater than LTD4 greater than LTE4 greater than FPL-55712. The maximum number of binding sites (Bmax) of [3H]LTD4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [3H]LTC4. The calculated values of Kd and Bmax of [3H]LTD4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg protein, respectively (n = 3). The rank order of potency or the unlabeled competitors for competing specific [3H]LTD4 binding was LTD4 = LTE4 greater than FPL-55712 much greater than LTC4. These findings demonstrate that BC3H-1 cell line possess both LTC4 and LTD4 receptors with a predominance of LTC4 receptors. Thus BC3H-1 cell line is a good model to study the regulation of LTC4 and LTD4 receptors.     

Identification of functional platelet-activating factor receptors in Raji lymphoblasts

The binding and metabolism of platelet-activating factor (PAF) were characterized in Raji, a human Burkitt's lymphoma-derived cell line. Raji lymphoblasts readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies conducted at 4 degrees C demonstrated specific binding that reached saturation within 80 min. This binding was only partially reversible. Scatchard analysis of PAF binding data revealed a single class of PAF binding sites (17,800 +/- 3,600/cell) with a K of 2.3 +/- 0.3 nM. These high-affinity PAF binding sites were shown to be functional receptors, as 100 pM to 1 microM PAF increased free intracellular calcium in a dose-dependent manner. The dose of PAF necessary to achieve half maximal calcium mobilization response was 6.3 nM, which was in the range of the K for the receptor calculated from the binding studies. The structurally dissimilar PAF receptor antagonists CV-3988 and BN52021 inhibited the PAF-induced calcium changes at doses that competed with PAF binding. These studies provide the first evidence for a functional PAF receptor expressed on a lymphocyte cell line.     

Endothelin binding sites in porcine aortic and rat lung membranes

High-affinity binding sites for endothelin were identified on porcine aortic and rat lung membranes. Interaction of 125I-labelled endothelin with its binding site was specific, saturable, time- and temperature-dependent but dissociation of receptor-bound ligand was minimal. Maximal binding was observed at pH 7.0 in porcine aorta and at pH 3.1 in the rat lung. Treatment of membranes with trypsin destroyed the binding site in both tissues. Porcine endothelin showed a higher affinity for receptors in both tissues compared to rat endothelin. Vasoactive peptides and Ca2+ channel antagonists did not interact with this site suggesting high specificity of binding. Analysis of saturation binding showed that the number of binding sites was 1250 +/- 104 and 1650 +/- 170 fmol/mg protein and the affinity of binding sites was 0.47 +/- 0.15 and 0.16 +/- 0.07 nM in the aorta and the lungs respectively (n = 5). Presence of protease inhibitors did not alter binding suggesting that the label was stable under the incubation conditions. This was further confirmed by HPLC. Removal of the endothelium from the aorta did not change the binding characteristics of this tissue. Ca2+ and Mg2+ ions caused an increase in binding by increasing the affinity. Binding was completely abolished in the presence of Triton and dithiothreitol. The binding sites identified in this study may be responsible for the actions of endothelin in the aorta and the lung.