Affinity of binding of radiolabeled (125I) heparin and low molecular weight heparin fraction CY 222 to endothelium in culture

Binding of radiolabeled (125I) heparin and its low molecular weight fraction CY 222 (Choay) to human and porcine cultured endothelium was investigated. The binding was measured over a wide range of heparin or CY 222 concentration in culture medium, from less than 20-time up to more than 30-times of the therapeutic heparin level. A relatively small fraction (less than 1%) of tested products was bound to endothelium. The process of binding was temperature-independent. A comparable number of endothelial binding sites (approx. 10(12)/cm2) for both 125I-heparin and 125I-CY 222 was calculated. About 40% totally bound 125I-heparin and 30% of 125I-CY 222 was found in extracellular matrix of cultured endothelium. The endothelium exhibited a 2.4-times lower affinity for 125I-CY 222 (Kd = 5.59 +/- 1.77 microM) than that for 125I-heparin (Kd = 2.35 +/- 0.78 microM). A similar affinity of human (venous) and porcine (aortic) endothelium for 125I-heparin was demonstrated. Cultured endothelium exhibited the same affinity for unlabeled heparin as for 125I-heparin. The endothelium depleted of sialic acid residues bound 1.5-times more of 125I-CY 222 than the control endothelium in culture.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Islet amyloid polypeptide (IAPP) competes for two binding sites of CGRP.

Two distinct binding sites for [125I]human calcitonin gene-related peptide (hCGRP) were found in rat brain, skeletal muscle, and liver. Each tissue had a high affinity site with an average Kd of 46 pM and a low affinity site with an average Kd of 22 nM. Islet amyloid polypeptide (IAPP), which has N- and C-terminal sequence homology to CGRP and is produced by islet beta-cells, bound to both sites but had a potency closer to that of CGRP at the low affinity binding site. A C-terminal fragment of IAPP competed for [125I]hCGRP binding at the low affinity site with potency comparable to that of hIAPP. No specific binding to membrane preparations was found in experiments using [125I]rIAPP, which was iodinated at the C-terminal tyrosyl residue. These results suggest that some of the previously reported biological effects occurring at nM or microM concentrations of IAPP may be mediated by IAPP binding to low affinity CGRP receptors. This study further indicates that the C-terminal region of IAPP is important for binding to low affinity CGRP receptors, and suggests that C-terminal fragments of IAPP may be of biological importance.     

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.     

Multiple binding sites for phencyclidine on the nicotinic acetylcholine receptor from Torpedo ocellata electric organ

Binding of [3H]-phencyclidine ( [3H]-PCP) to acetylcholine-receptor enriched membrane from Torpedo ocellata electric organ was studied over a ligand concentration range of 1 to 200 microM. The results indicate that [3H]-PCP is bound to two classes of sites: high affinity (Kd = 6-9 microM) and low affinity (Kd = 85 microM) binding sites. In the absence of cholinergic drugs the ratio of high affinity [3H]-PCP binding sites to 125I-alpha-bungarotoxin (alpha-Bgt) binding sites is 0.37, and that of low affinity [3H]-PCP binding sites to 125I-alpha-Bgt is 1.06. Low affinity [3H]-PCP binding can be completely inhibited by alpha-bungarotoxin (alpha-Bgt), carbamylcholine and d-tubocurarine. This inhibition, together with the one to one stoichiometry with 125I-alpha-Bgt, suggests that the sites to which [3H]-PCP binds with low affinity are the acetylcholine (AcCho) binding sites. In the presence of 1 microM alpha-Bgt which blocks binding of [3H]-PCP to the AcCho binding sites, the ratio of high affinity [3H]-PCP sites to 125I-alpha-Bgt sites is 0.5, indicating the existence of one high affinity PCP site per receptor molecule, The toxin, however, decreases the apparent affinity of [3H]-PCP towards the AcCho receptor as well as the potency of tetracaine or dibucaine in inhibiting [3H]-PCP binding to that receptor. In the latter case the effect involves changes from a biphasic to a simple inhibition curve. The results suggest that non-competitive blockers to the AcCho receptors may affect their own sites as well, and that they do this also by binding to the AcCho binding sites. This is also inferred from the accelerated dissociation of [3H]-PCP from its high affinity binding sites by unlabeled PCP in the concentration range of 10(-3) to 10(-4) M, at which the drug occupies AcCho binding sites as well.     

Specific substance P binding sites in rat thymus and spleen: in vitro autoradiographic study

Substance P binding sites were localized in rat thymus and spleen by incubation of tissue sections with [125I]Bolton-Hunter substance P, [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry and comparison with 125I standards. The tissue localization of the binding sites was determined with emulsion autoradiography. A single type of specific, saturable, high affinity binding sites was found associated with the vasculature in the medulla of the thymus and the marginal sinus of the spleen, with a Kd of 0.10 and 0.14 nM, respectively. Of all the unlabeled tachykinins tested (substance P, physalaemin, substance K, eledoisin, kassinin, and neuromedin K) substance P was the most potent inhibitor of [125I]Bolton-Hunter substance P binding, with an IC50 of approximately 0.5 nM, indicating the presence of substance P-P binding sites. Our results support the hypothesis of a role for substance P in the modulation of the immune system.     

Characterization of gingival epithelium epidermal growth factor receptor

The binding characteristics of gingival epithelium epidermal growth factor (EGF) receptor were investigated using epithelial cell membranes from bovine gingiva. The binding of [125I]EGF was found to be time and protein concentration dependent, reversible, and specific. Unlabeled EGF competed for [125I]EGF binding with IC50 of 0.25nM and maximum displacement of 93% at 0.81nM. Scatchard analysis of the binding data inferred the presence of two binding sites, one of high affinity (Kd = 3.3 nM and Bmax = 47.3fmol/mg protein) and the other of a low affinity (Kd = 1.6 microM and Bmax = 1.9pmol/mg protein). Crosslinking of [125I]EGF to gingival membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed a receptor protein of 170kDa.     

Interactions of thrombospondin with endothelial cells: receptor- mediated binding and degradation

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.     

Subcellular distribution of leukotriene C4 binding units in cultured bovine aortic endothelial cells

The subcellular distribution of specific binding sites for [3H]leukotriene C4 ([3H]LTC4) was analyzed after sedimentation of organelles from disrupted bovine aortic endothelial cells on sucrose density gradients and was shown to be in membrane fractions I (20% sucrose) and IV (35% sucrose). Saturation binding studies of [3H]LTC4 on endothelial cell monolayers at 4 degrees C demonstrated high-affinity binding sites with a dissociation constant (Kd) of 6.8 +/- 2.2 nM (mean +/- SD) and a density of 0.12 +/- 0.02 pmol/10(6) cells. At 4 degrees C, the specific binding of [3H]LTC4 by each of the subcellular fractions reached equilibrium at 30 min and remained stable for an additional 60 min. After 30 min of incubation with [3H]LTC4, the addition of excess unlabeled LTC4 to each subcellular fraction reversed more than 70% of [3H]LTC4 binding in 10 min. The [3H]LTC4 binding activities of subcellular fractions were enhanced approximately twofold to fourfold in the presence of Ca2+, Mg2+, and Mn2+, whereas Na+, K+, and Li+ were without effect. As measured by saturation experiments, the Kd and density of LTC4 binding sites in fraction I were 4.8 +/- 1.6 nM and 16.5 +/- 1.9 pmol/mg of protein, respectively, and in fraction IV were 4.7 +/- 1.5 nM and 81.4 +/- 19 pmol/mg of protein, respectively. Inhibition of [3H]LTC4 binding in membrane-enriched subcellular fractions I and IV by LTC4 occurred with molar inhibition constant (Ki) values of 4.5 +/- 0.1 nM and 4.7 +/- 1.2 nM, respectively, whereas Ki values for LTD4 were 570 +/- 330 nM and 62.5 +/- 32.8 nM, respectively, and for LTE4 were greater than 1000 nM for each fraction; LTB4 and reduced glutathione were even less active. FPL55712, a putative antagonist of the sulfidopeptide LT components of slow reacting substance of anaphylaxis, had Ki values of 1520 +/- 800 nM and 1180 +/- 720 nM for [3H]LTC4 binding sites on membrane-enriched subcellular fractions I and IV, respectively. Thus as defined by Kd, Ki, and specificity, the LTC4 binding units that are distributed to the plasma membrane and the binding units in the subcellular fraction of greater density were similar to each other. Pretreatment of the isolated subcellular membrane fractions with trypsin abolished [3H]LTC4 binding by fraction I, enriched for the plasma membrane marker 5' nucleotidase, and that by fraction IV, enriched for the mitochondrial membrane marker succinate-cytochrome C reductase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Properties and distribution of receptors for pituitary adenylate cyclase activating peptide (PACAP) in rat brain and spinal cord.

A high density (in the pmol/mg protein range) of specific functional receptors for PACAP (pituitary adenylate cyclase activating polypeptide) was observed in membranes from rat brain cortex, olfactory bulb, hypothalamus, hippocampus, striatum, cerebellum, pons and cervico-dorsal spinal cord, using [125I]PACAP-27 (PACAP 1-27). The tracer bound rapidly, specifically and reversibly. Competition binding curves were compatible with the coexistence, in the eight central nervous areas explored, of high and low affinity binding sites for PACAP-27 (Kd of 0.2 nM and 3.0 nM, respectively), and of only one class of binding sites for PACAP-38 (PACAP (1-38), Kd 0.2-0.9 nM). VIP inhibited only partially the binding of [125I]PACAP-27, and PHI, GRF(1-29)NH2 and secretin were ineffective at 1 microM. Chemical [125I]PACAP-27 cross-linking revealed a single specific 64 kDa protein species. In rat brain cortical membranes, saturation and competition experiments, using [125I]PACAP-38 as radioligand, indicated the presence of both high (Kd 0.13 nM) and low (Kd 8-10 nM) affinity binding sites for PACAP-38 and of low affinity (Kd 30 nM) binding sites for PACAP-27. These data taken collectively suggest the coexistence of PACAP-A receptors with a slight preference for PACAP-27 over PACAP-38 and of PACAP-B receptors that recognize PACAP-38 with a high affinity and PACAP-27 with low affinity. Both PACAP-27 and PACAP-38 stimulated adenylate cyclase with similar potency and efficacy. VIP was markedly less potent in this respect and also less efficient, except on cerebellar membranes.     

Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes

The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega-tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA-OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor.     

Galanin binding sites in rat gastric and jejunal smooth muscle membrane preparations.

Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.     

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro.

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 microM) increased specific [125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 microM) increased specific [125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in Bmax to 119% by cortisol (0.5 microM, 48 h) and to 194% by dexamethasone (0.5 microM, 48 h), and no change in Kd for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.     

Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thioglycolate-activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET-1 or [125I]ET-3 revealed the existence of a single class of binding sites. The binding parameters (Kd and Bmax) for [125I]ET-1 were almost identical to those for [125I]ET-3. In addition, unlabeled 3 ET isopeptides (ET-1, ET-2 and ET-3) inhibited the specific binding of both ET-1 and ET-3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose-dependent manner (0.01-100 nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET-induced intracellular Ca2+ mobilization in murine peritoneal macrophages.     

Specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat cultured astrocytes: molecular identification and interaction with vasoactive intestinal peptide (VIP)

Molecular identification of the binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and the effect of vasoactive intestinal peptide (VIP) on the specific binding sites for PACAP in rat cultured astrocyte membrane preparations were investigated. Affinity cross-linking of astrocyte membrane preparations with [125I]PACAP27 showed the presence of a 60 kDa radiolabeled ligand-receptor complex. The labeling of this band was completely abolished in the presence of 10(-8) M or higher concentrations of unlabeled PACAP27. The molecular weight of this binding protein was estimated to be 57 kDa assuming an equimolar interaction of ligand and receptor in the 60 kDa complex. The labeling of [125I]PACAP27 binding to this binding protein was partly reduced by the addition of 10(-6) M VIP, but not by 10(-8) M. In the binding assay, VIP displaced the specific binding of [125I]PACAP27 at 10(-7) M or a greater concentration. Displacement of [125I]PACAP27 binding by unlabeled PACAP27 was analyzed in the presence or absence of 10(-6) M VIP. VIP at 10(-6) M reduced the maximal binding capacity (Bmax) of the high affinity binding site for PACAP27 by about 50% but did not alter the Bmax of the low affinity binding site. The dissociation constants (Kd) for both the high and low affinity binding sites were unaltered. These results indicate that PACAP binds to a 57 kDa membrane protein with high affinity and that VIP, at much higher concentrations, binds to this same binding site, suggesting that VIP mimics the biological action of PACAP in astrocytes at high concentrations.