Enzymatic modifications of bovine adrenocortical angiotensin II receptors.

Several classes of proteolytic enzymes were used to gain an insight into the biochemical composition of the antiotensin II (ATII) receptor prepared from bovine adrenal cortices. Exposure of the receptor fractions to trypsin reduced their capacity to bind [3H]ATII. Phospholipases A2 and C similarly inhibited the [3H]ATII binding process, while phospholipase D had no effect. Binding was stimulated following addition of phosphatidylcholine but inhibited by lysophosphatidylcholine. Neuraminidase had no influence on [3H]ATII affinity for binding, while beta-galactosidase reduced binding of the radioligand. Concanavalin A did not displace [3H]ATII bound to receptor fractions. Very little aminopeptidase activity was detected in the receptor fraction, relative to the homogenate. The data suggest that the ATII recognition sites contain protein moieties, while phospholipids may play an essential role in ATII binding. Galactose units may form a part of the ATII receptor not directly associated with the binding site. The peptidase studies indicate that ATII probably cannot be hydrolyzed to its des-Asp1 metabolite at or near the site of binding.     

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)     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.     

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 the radiolabelled high-affinity anti-oestrogen [3H]H1285 with the cytoplasmic oestrogen receptor.

The high-affinity triarylethylene anti-oestrogen H1285 [4-(NN-diethylaminoethoxy)-beta-ethyl-alpha-(p-hydroxyphenyl) -4'-methoxystilbene] was tritiated to high specific radioactivity (35 Ci/mmol). Competition experiments between [3H]H1285 and H1285 or oestradiol demonstrated that both compounds would compete with [3H]H1285 for oestrogen-specific binding sites in rat uterine cytosol. [3H]H1285 had at least 10 times the affinity for the receptor compared with oestradiol at the 50% competition level. [3H]H1285 appeared to have at least twice the association rate for the oestrogen receptor compared with [3H]oestradiol. In addition, the dissociation half-life (t1/2) of specific binding of [3H]H1285 to oestrogen receptors at 0 degrees C was about 220 h compared with a value of 60 h for [3H]oestradiol. Because of the extremely slow dissociation of [3H]H1285 from the oestrogen receptor, we were able to compare the sedimentation profiles of [3H]H1285-receptor complexes with those of [3H]oestradiol-receptor complexes in the presence of 0.4 M-KCl on 5-20% sucrose density gradients. [3H]Oestradiol-receptor complexes had a major peak at 4.4 S with a smaller peak at 5.6 S, whereas with [3H]H1285-receptor complexes the 5.6 S peak was always higher than the 4.4 S peak. There was significant variation between the dissociation behaviour at 20 degrees C of [3H]H1285-receptor complexes and [3H]oestradiol-receptor complexes pre-activated at 25 degrees C for 30 min in the presence and in the absence of 10 mM-sodium molybdate. The dissociation t1/2 of [3H]oestradiol-receptor complexes at 20 degrees C decreased from 1.5 h to 0.5 h when molybdate was present during heat treatment whereas the dissociation t1/2 for [3H]H1285-receptor complexes was 5 h for both conditions. These observations indicate that there are fundamental differences in the initial interaction of H1285 and oestradiol with the oestrogen receptor.     

Glucocorticoids "receptors" in human gingiva.

Specific binding of [3H]-dexamethasone by the cytoplasmic fraction of normal human gingiva was carried out. Different concentrations of [3H]-dexamethasone were used with or without cold dexamethasone to determine the binding. Binding data by Scatchard plot yielded a straight line indicating a single class of specific receptors with equilibrium dissociation constant of 1.39 x 10(-9) M and B max of 80 fmol/mg protein. The proteins satisfied the high affinity and low capacity requirements of receptor.     

Dopamine Stimulates [3H]Phorbol 12,13-Dibutyrate Binding in Cultured Striatal Cells

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.     

Subcellular distribution of alpha 1-adrenergic receptors in rat liver

The distribution of alpha 1-adrenergic receptors in rat liver subcellular fractions was studied using the alpha 1-adrenergic receptor ligand [3H]prazosin. The highest number of [3H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane 'marker' enzymes in the same fractions a relative enrichment of [3H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with 125I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [3H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED50 values for displacement of [3H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10(-6), 10(-5) and 10(-4) mol/l, respectively. On the basis of lack of correlation between distribution of alpha 1-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of alpha 1-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [3H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that alpha 1-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

Electroconvulsive Shock (ECS) and the Adenosine Neuromodulatory System: Effect of Single and Repeated ECS on the Adenosine A1 and A2 Receptors, Adenylate Cyclase, and the Adenosine Uptake Site

The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.     

In vitro characterization of skeletal muscle beta-adrenergic receptors coupled to adenylate cyclase.

[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify the adenylate cyclase-coupled beta-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 . 10(7) M-1 . min-1 and 3.21 . 10(-1) min-1, respectively, were obtained. The dissociation constant (Kd) of 15 mM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the Kd derived from the ratio of dissociation and association rate constants (K2/K1). Several beta-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol greater than epinephrine greater than norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (-)-isomers being more potent than (+)-isomers. Phenylephrine, an alpha-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known beta-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Ki values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the beta-adrenergic receptor.     

Intracellular transport of a newly synthesized asialoglycoprotein receptor in rat liver

Intracellular transport of a newly synthesized asialoglycoprotein receptor was studied biochemically using a monospecific antibody for the receptor. Pulse-labeling by intravenous injection of [3H]leucine and pulse-chasing after 10 min by cycloheximide injection resulted in the maximal labeling of the receptor in the rough microsomes at 15 min, in the smooth microsomes and the heavy Golgi subfraction (GF3) at 25 min and in the intermediate plus light Golgi subfraction (GF1+2) at 30 min. By 60 min, the labeling in GF1+2 had decreased and leveled off. In the plasma membrane fraction, the labeled receptor first appeared at 20 min, increased rapidly and also reached a constant level at 40-60 min. Intracellular movement of the newly synthesized receptor in the GF1+2 and plasma membrane fractions was also investigated by purifying the receptor protein from the GF1+2 and plasma membrane fractions by affinity chromatography. It was revealed that the specific radioactivities of the receptor in the two fractions become equilibrated after 60-120 min. The receptor of the various membrane fractions was also pulse-labeled in vivo for 20 min simultaneously with [3H]glucosamine and [14C]leucine, and pulse-chased for the following 40 min. After pulse-labeling for 20 min, the ratio of the radioactivity of [3H]glucosamine or [3H]sialic acid to [14C]leucine of the receptor from the rough and smooth microsomes, and GF3, GF2, and GF1 increased in that order. That of the receptor from the plasma membrane fraction was infinitely higher, because, while a significant amount of 3H-radioactivity was incorporated into the receptor in the Golgi apparatus, only a negligible amount of 14C-radioactivity was incorporated into the same receptor in the plasma membrane due to the delay in the arrival of [14C]leucine labeled receptor to the plasma membrane. After chasing for 40 min, however, the same radioactivity ratios of the GF1 and plasma membrane fractions approached each other. All these results strongly suggest that the distribution of the newly synthesized receptor becomes rapidly equilibrated between the trans-Golgi components and plasma membranes probably by repeated recycling of the receptor protein between the two membranes.     

Comparative study of nuclear binding sites for oestradiol in rat testicular and uterine tissue. Determination of low amounts of specific binding site by an [3H] oestradiol-exchange method.

1. An [3H]oestradiol-exchange method was developed for the determination of oestradiol-receptor complexes in the nuclear fraction of immature rat testicular tissue. This method permits the determination of nuclear oestradiol-receptor sites in the presence of a relatively large amount of non-specific oestradiol binding present in testicular nuclei. After incubation of nuclei for 60min at 20 degrees C in the presence of [3H]oestradiol with or without a 1000-fold excess of non-radioactive diethylstilboestrol, specific binding can be determined quantitatively in the KCl-extractabe fraction, which contains 40% of the total receptor population. 2. The amount of receptor-bound steroid present in the 0.4m-KCl extract of testicular neclei remained constant during incubation at 20 degrees C. For uterine nuclei incubated with [3H]oestradiol at 37 degrees C a shift of specifically bound [3H]oestradiol occurred from the KCl-soluble fraction to the KCl-insoluble fraction. 3. In intact rat testis, about 20% of the total receptor concentration was present in its nuclear form. Hypophysectomy 5 days before measurement resulted in a twofold decrease in the amount of receptor, which was present mainly in the cytosol. After injection of choriogonadotropin to intact animals, the total receptor concentration increased threefold. 4. This nuclear exchange method might be useful for determination of occupied specific receptor sites in tissues with relatively low contents of specific receptors.     

Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors

LTB4-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB4 to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [3H]-LTB4 binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20 degrees C, binding of [3H]-LTB4 to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [3H]-LTB4 bound to high affinity (dissociation constant, Kd = 1.5 nM), and low capacity (density, Bmax = 40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB4, LTB4-epimers, 20-OH-LTB4, 2-nor-LTB4, 6-trans-epi-LTB4 and 6-trans-LTB4, in decreasing order of affinity, bound to the [3H]-LTB4 receptors. The mean binding affinities (Ki) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB4 in human PMN plasma membrane.     

A Comparison of the Binding of [3H]Proprionyl-Neuropeptide Y to Rat and Human Frontal Cortical Membranes

The binding characteristics of [3H]proprionyl-neuropeptide Y ([3H]proprionyl-NPY) were studied in frontal cortical membranes prepared from rat and human postmortem tissue. The specific binding of NPY decreased as the magnesium concentration increased from 1.05 to 10 mM. The binding was also influenced by the concentration of GTP in the buffer medium, with a resulting 45% decrease in NPY binding in the presence of 10(-6) M GTP. Using equilibrium binding studies, [3H]proprionyl-NPY was found to bind in both tissues with high affinity to a single class of receptors with a similar KD (0.035 nM). However, kinetic experiments in both tissues provided evidence for two components of [3H]proprionyl-NPY binding which may be related to receptor states. Competition binding experiments showed that peptide YY (PYY) was equal to NPY in its ability to displace [3H]proprionyl-NPY, whereas rat and human pancreatic polypeptide were without effect up to a concentration of 10(-6) M. This suggests that, whereas PYY and NPY may compete for the same receptor(s), the pancreatic polypeptides probably act on a separate population of receptors.     

Solubilization and Characterization of Prostaglandin E2 Binding Protein from Porcine Cerebral Cortex

The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 4 degree C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time-dependent and the equilibrium was reached at around 60 min at 37 degrees C. By dilution of the reaction mixture, the binding site-[3H]PGE2 complex formed after 5-min incubation slowly dissociated, whereas that formed after 60-min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 greater than PGE1 much greater than PGF2 alpha greater than PGE2 methyl ester greater than PGA2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high- and low-affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion-exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.     

Binding and subcellular distribution of cyclosporine in human fibroblasts

The uptake, binding, and subcellular sites of accumulation of [³H]-cyclosporine (CS) in two human gingival fibroblast strains, GN 23 and GN 54, have been examined. GN 23 responds to CS treatment with a decrease in collagenolysis, while GN 54 does not. Binding of the drug was determined using [³H]-CS concentrations ranging from 10^?5 to 10^?8 M in the absence or presence of excess unlabeled CS (1 mM). The binding of the drug to both strains was specific and reached a plateau within 10 min, remaining at that level for up to 1 h. Scatchard analysis of the specific binding of [³H]-CS to the responsive GN 23 strain revealed two dissociation constants: K_D = 5 × 10^?8 M (1.2 × 10⁷ sites/cell) and K_D = 1.4 × 10^?6 M (2.2 × 10⁸ sites/cell). GN 54, on the other hand, had only one class of low affinity binding site (K_D = 0.47 × 10^?6 M [1.2 × 10⁸ sites/cell]). Unlabeled CS (0.01–1 mM) inhibited the binding of [³H]-CS in a dose-dependent manner to both strains, as did the calmodulin antagonist W-7, to a lesser extent. However, W-7 inhibited CS binding much more efficiently in GN 54 than in GN 23, suggesting that calmodulin may be the predominant CS receptor in GN 54. In both strains, 70% of the drug accumulated in the crude nuclear fraction after a 1 min incubation, with very little (? 4%) being membrane associated, and the remainder was in the cytosol. In GN 23, CS levels in the crude nuclear fraction reached 80% by 20 min, and remained at this level for up to 1 h. In contrast, in GN 54, at incubation times of more than 1 min, the drug did not selectively accumulate in the crude nuclear fraction, but appeared to be in equilibrium between the nuclear and cytosolic fractions. These data show that the CS resistance of human gingival fibroblasts was not due to their inability to take up and bind CS. Rather, the different effects of CS on the collagenolysis of the responder and non-responder fibroblast strains may be related to the types of CS receptors they possess and differences in the cellular metabolism of CS occurring after binding, including the subcellular sites of drug accumulation. © 1993 Wiley-Liss, Inc.     

Identification of thromboxane A2 receptor in cultured vascular endothelial cells of rat aorta

The binding site for [3H]SQ29,548, a potent and selective thromboxane A2 (TXA2) receptor antagonist, was studied in cultured vascular endothelial cells (VEC) of the rat aorta. Specific binding of [3H]SQ29,548 to rat VEC at 24 degrees C was saturable, displaceable and of high affinity. Scatchard analysis of equilibrium binding studies indicated that rat VEC contain a single class of binding sites with a Kd of 2.7 nM. The number of maximum binding sites (25.8 fmol/10(6) cells) for [3H]SQ29,548 on rat VEC was respectively 23 and 3.2 times more than that on rat platelets and rat vascular smooth muscle cells. Four TXA2 receptor antagonists and U46619 completely suppressed [3H]SQ29,548 binding to rat VEC, whereas other prostanoids, such as PGD2, PGF2 alpha, PGE1 and Iloprost, displaced the ligand binding only at considerably higher concentrations. These results suggest that the specific receptor for TXA2 is present in rat vascular endothelial cells.     

Binding of a thromboxane A2/prostaglandin H2 agonist [3H]U46619 to washed human platelets

The binding characteristics of [3H]U46619 to washed human platelets were studied. [3H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a Kd of 11 +/- 4 nM (n = 4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a Kd of 20 +/- 7 nM and a Bmax of 9.1 +/- 2.3 fmole/10(7) platelets (550 +/- 141 binding sites per platelet) (n = 4). A number of compounds that act as either agonists or antagonists of the TXA2/PGH2 receptor were tested for their ability to inhibit the binding of [3H]U46619 to washed human platelets. The Kds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [3H]U46619 binds to the putative human platelet TXA2/PGH2 receptor.