Comparison between endothelin binding sites and sarafotoxin S6B binding sites in rat tissues: studies by quantitative in vitro receptor autoradiography]

Sarafotoxin S6B(SRT), a peptide in snake venom, has a high degree of sequence homology with endothelin (ET) and both are potent vasoconstrictors. In order to determine whether SRT acts via the ET receptor, we performed autoradiographic binding studies on rat tissues using the radioligands, 125I-ET-1 and 125I-SRT and computerized in vitro autoradiography. In the heart, a high density of ET binding was found in the atria, and moderate density was found in the ventricles. A high density of ET-1 was found in the heart. In the kidney, ET-1 binding occurred in association with glomeruli, outer cortex, and inner stripe and inner medulla. In the adrenal, a high density of ET-1 binding occurred in the medulla as well as the zona glomerulosa. The binding affinity constant (KA) for ET-1 binding in these sites ranged from 1-10 x 10(9) M-1. Although SRT was 5-100 fold weaker than ET-1 in displacing 125I-ET-1 from these sites, 1 microM unlabelled SRT completely abolished 125I-ET-1 binding in all sites. Other venom peptides or unrelated peptides did not affect 125I-ET-1 binding. Moreover, the pattern of 125I-SRT binding in rat tissues by in vitro autoradiography was identical with that of 125I-ET-1 binding, and both unlabelled SRT and unlabelled ET-1 fully competed with 125I-SRT for binding. These results provide evidence that SRT binds to the ET binding sites in a range of rat tissues.     

Characterization and solubilization of vasoactive intestinal peptide receptors from rat lung membranes

Receptors for vasoactive intestinal peptide (VIP) were characterized in rat lung membranes by binding and covalent cross-linking of [125I]VIP using ethylene glycolbis-(succinimidylsuccinate). Binding studies indicated the presence of two classes of binding sites for VIP in rat lung membranes: 0.28 +/- 0.11 pmol/mg protein high affinity receptors (Kd = 79.2 +/- 26.4 pM) and 3.3 +/- 0.9 pmol/mg protein lower affinity receptors (Kd = 4.8 +/- 2.1 nM). Furthermore, binding of [125I]VIP to rat lung receptors was inhibited by micromolar concentrations of GTP analogs, guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), and guanylylimidodiphosphate, suggesting that VIP receptors in rat lung membranes were tightly coupled to the guanine nucleotide regulatory protein (Ns). Scatchard analysis of VIP binding in the presence of GTP gamma S revealed selective inhibition of binding to high affinity sites. A 58K band was specifically labeled when membranes covalently labeled with [125I]VIP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The apparent size of this species was not altered when sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was carried out in the absence of reducing agent. Unlabeled VIP inhibited the labeling, with an IC50 of about 1 nM. A related peptide, GH-releasing factor-(1-40)OH, exhibited a much lower binding affinity, and two unrelated peptides, insulin and atrial natriuretic factor, did not inhibit labeling of the 58K species, even at micromolar concentrations. Labeling of the 58K species was inhibited in a GTP gamma S-dependent manner, suggesting the involvement of this species in the coupling to Ns. These data collectively indicated that the 58K species was a VIP-binding unit of VIP receptors in rat lung membranes. Several nondenaturing detergents were tested for extraction of labeled receptors from the membrane; the best extraction was obtained using 1% n-octyl-beta-D-glucopyranoside.     

Identification of a single binding protein for endothelin-1 and endothelin-3 in bovine cerebellum membranes.

Competition binding experiments of [125I]endothelin-3 ([125I]ET-3) to bovine cerebellum membrane preparations in the presence of either ET-3 or ET-1 have indicated the presence of a single class of high affinity binding sites for these two peptides in the brain. Cross-linking of [125I]ET-3 to cerebellum membrane preparations with dissuccinimidyl suberate (DSS) resulted in the labeling of two bands with apparent mol wt of 52 and 30 kDa. Under these conditions the labeling intensities of these two bands were similar. However, addition of 5 mM EDTA to the protease inhibitor mixture during membrane preparations as well as the binding and cross-linking reaction increased the labeling of the 52-kDa protein while reducing the labeling of the 30-kDa protein. Peptide map comparisons of the 52- and 30-kDa protein bands using Staphylococcus aureus V8 protease and papain revealed that the 30-kDa band is a proteolytic degradation product of the 52-kDa protein. These results suggest that the 52-kDa protein represents the specific binding protein of ET-3, and thus, the apparent mol wt of the ET receptor is 50 kDa, subtracting the mol wt of the iodinated ET. Since cross-linking of [125I]ET-1 to cerebellum membrane preparations revealed the same two bands of 52 and 30 kDa, peptide mapping of the 52-kDa proteins, cross-linked with either [125I]ET-1 or ET-3, was conducted. Under these experimental conditions, identical peptide fragments were generated by both Staphylococcus aureus V8 protease and papain. These results suggest that ET-1 and ET-3 bind to a common brain binding protein with an apparent mol wt of 50 kDa.     

Direct identification of beta-adrenergic receptors on isolated bovine parathyroid cells.

The radioiodinated beta-blocker iodohydroxybenzylpindolol ([125I]HYP) has been used to identify directly and characterize beta-adrenergic receptors in isolated bovine parathyroid cells. [125I]HYP was bound rapidly and reversibly to isolated bovine parathyroid cell membranes. Scatchard analysis revealed a single class of binding sites with high affinity (4 X 10(10M-1) and low capacity (0.7 pmol/mg). Saturation analysis of [125I]HYP binding to intact bovine parathyroid cells suggested a site with similar affinity on whole cells and with a binding capacity of 5000-10,000 sites/cell. True dissociation constants (Kd's) for beta-adrenergic agonists and antagonists were in good agreement with activation constants (KA'S) and inhibition constants (KI'S) for effects on adenylate cyclase in membrane preparations. These constants also were in reasonable agreement with KA'S and KI'S previously shown for effects of agonists and antagonists on cAMP accumulation and PTH release in whole cells. This study shows by direct analysis that beta-adrenergic receptors exist on isolated bovine parathyroid cells, and that there is close coupling between receptor binding, effects on cAMP and hormonal release. This represents still another system in which [125I]HYP has been successfully used to study beta adrenergic receptors in membrane as well as intact cell preparations.     

Covalent cross-linking of the Phytophthora megasperma oligopeptide elicitor to its receptor in parsley membranes.

An oligopeptide elicitor from Phytophthora megasperma f.sp. glycinea (Pep-13) that induces phytoalexin accumulation in cultured parsley cells was radioiodinated and chemically cross-linked to its binding site in microsomal and plasma membrane preparations with each of three homobifunctional reagents. Analysis by SDS/PAGE and autoradiography of solubilized membrane proteins demonstrated labeling of a 91-kDa protein, regardless of which reagent was used. Cross-linking of this protein was prevented by addition of excess unlabeled Pep-13. The competitor concentration found to half-maximally reduce the intensity of the cross-linked band was 6 nM, which is in good agreement with the IC50 value of 4.7 nM, obtained from ligand binding assays. No crosslinking of 125I-labeled Pep-13 was observed by using microsomal membranes from three other plant species, indicating species-specific occurrence of the binding site. Coupling of 125I-Pep-13 to the parsley 91-kDa protein required the same structural elements within the ligand as was recently reported for binding of 125I-Pep-13 to parsley microsomes, elicitor-induced stimulation of ion fluxes across the plasma membrane, the oxidative burst, the expression of defense-related genes, and phytoalexin production. These findings suggest that the 91-kDa protein identified in parsley membranes is the oligopeptide elicitor receptor mediating activation of a multicomponent defense response.     

Endothelin receptors and endothelin-1 immunoreactivity in the human lung]

We investigated in human lung preparations the characteristics of endothelin-1 (ET-1) binding and the amount of ET-1-like immunoreactivity. Saturation experiments revealed the presence of a large number of high affinity specific ET-1 binding sites with a dissociation constant (Kd) of 1.35 nM and a binding capacity (Bmax) of 9.74 pmol/mg of protein. The binding was time- and temperature-dependent and dissociated by only 10% by the addition of 1 microM unlabeled ET-1. In competition experiments, [125I]ET-1 binding was totally inhibited by unlabeled ET-1 and ET-2 with inhibition constant (Ki) values of 0.20 and 0.21 nM respectively, and 80% inhibited by ET-3 with Ki value of 0.50 nM. The binding was not affected by 1 microM structurally unrelated compounds. Moreover a high level of ET-1-like immunoreactivity (2.3 pg/mg wet weight) was found in human lung by using a specific radioimmunoassay of ET-1 after extraction. HPLC analysis revealed the presence of both ET-1 and Big-ET. These results suggest that the lung may be an important target organ for ET-1 action and/or metabolism in human.     

Expression of hepatic transforming growth factor receptors during late gestation in the fetal rat

Transforming growth factor-alpha (TGF alpha) promotes DNA synthesis in adult rat hepatocytes, an effect opposed by picomolar concentrations of TGF beta. Recently, the presence of these growth factors in fetal rat liver has been demonstrated. Since a regulatory role for TGF alpha and TGF beta in fetal hepatic growth requires the presence of high affinity receptors, the receptors for these hormones were studied in membranes from normal fetuses at 17-21 days gestational age and growth-retarded fetuses of mothers fasted for 48 h. Fetal liver membranes bound [125I]epidermal growth factor ([125I]EGF) with high affinity (Kd = 1-2 nM). TGF alpha could compete with EGF for the same binding site, albeit at 4-fold lower affinity. EGF receptor number increased from nearly undetectable levels at 17 days to adult levels (0.15-0.3 nmol/mg membrane protein) by 21 days. Affinity labeling of fetal liver membranes with [125I]TGF alpha identified the 170,000 mol wt (Mr) EGF receptor. The intensity of labeling correlated with EGF receptor number based on binding analyses. TGF beta bound to fetal liver membranes with high affinity (Kd = 30 pM) and at a level (20-30 pmol/mg throughout late gestation) that was 3-fold higher than in adult liver. Affinity labeling of fetal hepatic membranes with [125I]TGF demonstrated high affinity 85,000 Mr TGF beta receptors and lower affinity 66,000 and 130,000 Mr receptors. Although TGF beta binding did not change with advancing gestation, affinity labeling of the 85,000 Mr protein doubled from day 18 to day 21 and was decreased by 50% in fetuses from fasted mothers. These data, demonstrating the presence and regulation of the receptors for TGF alpha and TGF beta, support roles for these hormones in the regulation of fetal hepatic growth.     

Galanin receptors in a hamster pancreatic beta-cell tumor: identification and molecular characterization

High affinity binding sites for galanin are identified and characterized in membranes from a hamster pancreatic beta-cell tumor. Using the radioiodinated peptide [125I] galanin, interaction of the peptide with pancreatic membranes is shown to be saturable, reversible, and time, temperature, membrane protein concentration, pH, and ionic strength dependent. In optimized equilibrium conditions of binding (90 min at 10 C), native galanin competitively inhibits the binding of [125I]galanin in a dose-dependent manner (from 10(-11)-10(-8) M); half-maximal inhibition is induced by 1 nM peptide. Scatchard analysis indicates the existence of a single population of sites of high affinity (Kd = 1.5 nM) and low capacity (44 fmol/mg protein). The monophasic dissociation process confirms the homogeneity of galanin-binding sites. Galanin-binding sites are highly specific, since apart from native galanin, none of the numerous biologically active peptides tested competes with [125I] galanin for binding to pancreatic membranes. The cross-linking of [125I]galanin to beta-cell membranes is performed using the chemical bifunctional reagent ethylene glycol bis-(succinimidyl succinate). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the presence or absence of dithiothreitol, one single band of 57,000 mol wt is observed, which may be corresponding to the [125I]galanin-receptor complex. Indeed, labeling of this 57,000 mol wt component is abolished only by native galanin but is unaffected by various other digestive peptides. Assuming one molecule of [125I]galanin is bound per molecule of protein, a 54,000 mol wt protein is identified as the pancreatic galanin receptor. In conclusion, our results indicate for the first time the identification of galanin receptors. Their presence in pancreatic beta-cells suggests a direct role of galanin in regulating endocrine beta-cell function.     

Characterization of the epidermal growth factor receptor in the gastric mucosa

The effects of epidermal growth factor (EGF), a potent mitogen involved in mucosal protection, are mediated by specific cellular receptors. Here, we present the characteristics and binding properties of EGF receptors in the gastric mucosa. The studies were conducted using cell membranes isolated by subcellular fractionation of rat stomach mucosal scrapings. Specific binding of [125I]-EGF to the membrane preparations was assessed at room temperature for various periods of time and at different pHs. The results showed that the binding was proportional to the incubation time up to 1 h and was not affected by a pH change between 4.0 and 7.0. Scatchard analysis of the binding data infer the presence of 2 binding sites, one of high affinity (Kd = 1.34 nM, Bmax = 34 fmol/mg protein) and the other of low affinity (Kd = 484 nM, Bmax = 2.29 pmol/mg protein). Cross-linking experiments using disuccinimidyl suberate to link the [125I]-EGF to gastric membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed that the major receptor for EGF was a protein of 170 kilodaltons. When the solubilized membranes were subjected to wheat germ agglutinin affinity chromatography, the purified material was found to act as substrate for EGF-stimulated phosphorylation. The major component which was labeled by the [gamma-32P]-ATP was also found to be a 170-kilodalton protein. The data are the first to provide evidence that the gastric mucosa possesses a functional EGF receptor and describe its binding characteristics.     

LHRH-PITUITARY PLASMA MEMBRANE BINDING: THE PRESENCE OF SPECIFIC BINDING SITES IN OTHER TISSUES

Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.     

The insulin receptor of embryonic chicken cartilage.

Highly purified plasma membranes have been obtained from embryonic chicken cartilage by physical means rather than enzymatic digestion. Rapid and reversible binding of [125I]iodoinsulin to these membranes is demonstrated. Similar to the insulin-binding properties of rat liver and adipocytes and human mononuclear cells, optimal specific binding of insulin to chondrocyte plasma membranes has a sharp pH optimum at 8.0, and maximal binding occurs at 2--4 C. Analysis of equilibrium binding reveals a curvilinear Scatchard plot, whose high affinity segment generates a maximum affinity of 1.0 X 10(9) M-1, and a receptor concentration of 0.4 pmol/mg membrane protein. This affinity constant is similar to those generated for insulin binding to membranes prepared from embryonic chicken liver (2.5 X 10(9) M-1), rat liver (1.4 X 10(9) M-1), and mouse liver (0.6 X 10(9) M-1), whereas the receptor concentration is less than that of embryonic chicken liver membranes (1.1 pmol/mg), which in turn was less than those of rat liver membranes (2.8 pmol/mg) and mouse liver membranes (3.5 pmol/mg). Kinetic studies show augmentation of insulin-receptor dissociation by excess insulin when initial receptor occupancy, is low, suggesting that negative cooperativity is present. There is little or no interaction of other hormones with the chondrocyte insulin receptor, with the exception of proinsulin and the insulin-like growth factors. Porcine proinsulin, bovine proinsulin, somatomedin C, and nonsuppressible insulin-like protein prevent [125I]iodoinsulin binding to chondrocyte plasma membranes with dose-response curves which are parallel to that of unlabeled porcine insulin itself, but with molar potencies relative to porcine insulin of 15%, 9%, 2.5%, and 1.4%, respectively. Porcine insulin and proinsulin both prevent binding of [125I]iodosomatomedin C to chondrocyte plasma membranes but with molar potencies less than 1% that of unlabeled somatomedin C. These observations are consistent with the presence of a specific independent insulin receptor in embryonic chicken cartilage which is similar in its characteristics to the insulin receptor in previously described tissues. Insulin has a weak interaction with the chondrocyte receptor for somatomedin C. Interaction with the somatomedin receptor may be the mechanism by which insulin exerts anabolic effects on cartilage when used in pharmacological amounts.     

Purification and immunological characterization of the rat liver insulin-like growth factor-II receptor

Rat liver microsomal insulin-like growth factor-II (IGF-II) receptor has been purified to homogeneity using a single step affinity chromatographic procedure on agarose-IGF-II with elution at pH 4. Determined by either IGF-II binding or a direct RIA for receptor, purification of 2000-fold was obtained. The mean recovery was 28% for five such preparations. Sodium dodecyl sulfate-electrophoresis and autoradiography of purified receptor, radioiodinated receptor, and affinity-labeled receptor all indicated a molecular mass of approximately 250K. Scatchard analysis of IGF-II binding to purified receptor, solubilized microsomal membranes, or plasma membranes showed a single class of binding site with an affinity constant of 6 X 10(10) liter/mol in all cases. Potent antibodies to the purified receptor were raised in rabbits, capable of inhibiting 50% of IGF-II binding at dilutions of 1:170,000 and also of fully precipitating IGF-II-prelabeled receptor at 1:50,000. Both types of antibodies reacted with IGF-II receptors in rat adipose tissue, brain, heart, kidney, lung, and spleen. However, little cross-reactivity was seen with other species. Comparison of the ability of receptor antibodies to inhibit IGF-II binding to microsomal and plasma membranes indicated a specific immunological difference between the IGF-II receptors in the two membrane preparations.     

Uterine insulin-like growth factor-I receptors: regulation by estrogen and variation throughout the estrous cycle

This study was undertaken to identify uterine insulin-like growth factor-I (IGF-I) receptors and examine the regulation of these receptors throughout the estrous cycle and after 17 beta-estradiol (E2) administration to immature rats. We have demonstrated type I IGF receptors in crude uterine membranes by binding and cross-linking experiments. The characteristics of the uterine IGF-I receptor are similar to those reported for other tissues, with a high affinity component (Kd = 0.12 nM; binding capacity = 0.028 pmol/mg protein) and a low affinity component (Kd = 0.98 nM; binding capacity = 0.041 pmol/mg protein). Autoradiographic visualization of [125I]IGF-I binding to uterine sections localized the IGF-I receptors to the smooth muscle cells of the myometrial layer of the uterus, with a higher density of IGF-I receptors in the outer longitudinal layer than in the inner circular layer. In immature rats administration of E2 significantly increased total [125I]IGF-I binding per uterus as early as 6 h after E2 injection. Although [125I]IGF-I binding was significantly increased per mg DNA, because of a more marked increase in membrane protein after E2, [125I]IGF-I binding, when expressed per mg membrane protein, decreased. This change in [125I]IGF-I binding resulted from a change in receptor number with no change in receptor affinity. In mature cycling rats, the proestrous uteri showed the lowest level of [125I]IGF-I binding per mg membrane protein, although because of the greater yield of protein from proestrous uteri, the total [125I]IGF-I-binding capacity of these uteri were greater than that of uteri from other stages of the estrous cycle. The lowest [125I]IGF-I binding was seen in the diestrous uteri. These studies demonstrate the presence of authentic type I IGF-I receptors in the rat uterus localized predominantly to the myometrial smooth muscle cells. In addition, E2 appears to regulate the uterine IGF-I receptor in the immature rat, and the cyclical changes in the mature rat are consistent with a role of E2 in regulation of this receptor in vivo.     

Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.

A receptor that binds the phosphomannosyl recognition marker of bovine testicular beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was isolated from bovine liver membranes. The receptor was extracted from crude plasma membrane preparations with Triton X-100 and immunoprecipitated as a receptor--beta-galactosidase complex with anti-beta-galactosidase. The receptor was dissociated from the precipitate with mannose 6-phosphate, labeled with 125I, and purified on a beta-galactosidase-Sepharose 4B affinity matrix. A quantitative binding assay employing anti-beta-galactosidase and IgGsorb (formalin-fixed Staphylococcus aureus) was devised to study the binding of 125I-labeled receptor to beta-galactosidase. Maximal binding of receptor to enzyme occurred at pH values between 5.7 and 6.5. Divalent cations were not required for binding. The values of the dissociation constant obtained for beta-galactosidase varied between 200 nM observed with "lower uptake" forms and 20 nM for "higher uptake" forms of the enzyme. A number of phosphorylated monosaccharides were tested as inhibitors of binding of enzyme to receptor; mannose 6-phosphate and fructose 1-phosphate served as inhibitors and exhibited Ki values of 0.064 mM and 0.24 mM, respectively. The receptor has a subunit molecular weight of 215,000. Similar receptors were also demonstrated in Triton X-100 extracts of human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. These cell types are known to assimilate lysosomal enzymes containing covalently bound mannose 6-phosphate residues.     

A specific, high affinity, saturable binding site for the 16-kilodalton fragment of prolactin on capillary endothelial cells.

A 16-kilodalton N-terminal fragment of PRL (16K PRL) is formed by enzymatic cleavage of intact 23-kilodalton PRL (23K PRL) in the pituitary gland and in target tissues for PRL. 16K PRL inhibits the growth of capillary endothelial cells, while intact PRL was inactive suggesting that 16K PRL acted via a receptor other than the PRL receptor. To analyze whether this inhibitory effect could be mediated through an specific 16K PRL receptor, we characterized the binding of 16K PRL to membrane preparations of bovine brain capillary endothelial (BBE) cells. 16K PRL was generated by the proteolysis of rat 23K PRL with a particulate fraction from rat mammary gland homogenates and purified by gel filtration. The specific binding of [125I]16K PRL to BBE cell membranes was high affinity (Kd = 9.9 nM), saturable (Bmax = 4.8 pmol/mg protein), and reversible. In competition studies for [125I]16K PRL binding, 16K PRL was most potent, while little displacement was observed with high concentrations of 23K PRLs, growth hormones, and basic fibroblast growth factor. Blockade of reformation of disulfide bonds by carbamidomethylation of 16K PRL, a procedure which increases the biological activity of the molecule, increased its binding affinity (Kd = 0.9 nM). Cross-linking experiments identified a 52,000 and a 32,000 mol wt protein as the major 16K PRL binding species. These data demonstrate the presence of specific, high affinity, saturable binding sites for 16K PRL on BBE cell membranes and support biological findings that 16K PRL inhibits capillary endothelial cell proliferation, through a novel, high affinity receptor.     

Monoclonal antibodies to the thyrotropin receptor: implications for receptor structure and the action of autoantibodies in Graves disease.

Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.     

(125I)iodoazidococaine, a photoaffinity label for the haloperidol-sensitive sigma receptor.

A carrier-free radioiodinated cocaine photo-affinity label, (-)-3-(125I)iodo-4-azidococaine [(125I)IACoc], has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM (125I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. Covalent labeling of the 26-kDa polypeptide was inhibited by 1 microM haloperidol, di(2-tolyl)guanidine (DTG), 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP), dextromethorphan, and carbetapentane. Stereoselective protection of (125I)IACoc photolabeling by 3-PPP [(+)-3-PPP more potent than (-)-3-PPP] was observed. (125I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 microM imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the (125I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-[3H]DTG. Kinetic analysis of (125I)IACoc binding to rat liver microsomes revealed two sites with Kd values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, (125I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.     

Somatostatin receptors in the rat adrenal cortex: characterization and comparison with brain and pituitary receptors

Specific receptors for tetradecapeptide somatostatin (S-14) in rat adrenal cortical membranes were quantitated by direct binding studies using [125I-Tyr11]S-14. Competitive inhibition of this radioligand by S-14 showed that these receptors constitute a single class of high affinity binding sites [dissociation constant (Kd) = 1.08 nM and maximum binding capacity (Bmax) = 0.35 pmol/mg membrane protein]. Structural analogs of S-14 with halogenated Trp8 moiety exhibited 4- to 46-fold greater binding affinity than S-14, [D-F5-Trp8]S-14 being the most potent. [Tyr11]S-14 and [des-Ala1]S-14 bound to these receptors with reduced affinity whereas [Phe4]S-14 exhibited 1.5-fold greater affinity than S-14. Somatostatin-28 (S-28) and S-14 were equipotent, whereas the N-terminal fragments of S-28 [S-28(1-14) and S-28(1-12)] were inactive. High affinity binding sites were also quantitated using a radioligand prepared from the tyrosinated S-28 analog, [Leu8, D-Trp22, Tyr25]S-28 (Kd = 1.2 nM; Bmax = 0.21 pmol/mg membrane protein). Both S-14 and S-28 exhibited comparable relative potencies for inhibiting the specific binding of this radioligand and [125I-Tyr11]S-14. Extracts of whole adrenal or the adrenal medulla and cortex contained very low levels of S-14-like immunoreactivity (2.4 pg/mg protein). These studies confirm the presence of specific receptors for S-14 in the adrenal cortex and suggest that 1) with respect to S-14 biological activity, Trp8-modified S-14 analogs should be more potent than S-14, S-28 equipotent with S-14, and N-terminal fragments of S-28 inactive in this tissue. 2) Direct binding studies using radioiodinated [Tyr11]S-14 and [Leu8,D-Trp22, Tyr25]S-28 appear to quantitate the same receptor sites in adrenocortical tissue. 3) The ligand specificity of the adrenocortical S-14 receptor differs from that previously reported for the pituitary and brain providing further evidence for the heterogeneity of the S-14 receptor. 4) In view of the very low concentrations of endogenous S-14-like immunoreactivity, the adrenal actions of S-14 and S-28 are probably mediated through an endocrine mechanism.     

Specific 125I-Endothelin-1 Binding Sites in the Central Nervous System

The quantitative receptor autoradiographic method we used revealed that specific ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites are highly concentrated in the choroid plexus (ChP), subfornical organ (SFO), lacunosum molecular layer of the hippocampus (LMol) and granular layer of the cerebellum (GC) of the rat brain. High densities of the binding sites were also observed in the laminae I-III and intermediolateral nucleus of the porcine spinal cord. ¹²⁵I-ET-1 bound to the rat ChP and LMol with a high-affinity and to the SFO and GC with a low-affinity. The possibility that ET-1 acts as a neuropeptide within the central nervous system by interacting with specific receptors would have to be considered.     

Serotonin receptors in rat lung

Mammalian lungs have been shown to store and to inactivate serotonin by an active process involving uptake and metabolism. Serotonin has direct action on lung including constrictor effects of pulmonary vascular and tracheobronchial smooth muscle suggesting the presence of serotonin receptors in lung. We have identified several serotonin binding receptors in rat lung. Two separate binding sites are present in a purified mitochondrial fraction. Saturation analysis of (3H)-serotonin binding to outer mitochondrial membranes exhibits temperature-dependent association kinetics and demonstrates a single, high affinity, high capacity binding (dissociation constant = 8.3 +/- 1.2 nM, maximum binding capacity = 0.819 +/- 0.046 pmol/mg protein). The dissociation constant of inner mitochondrial membrane demonstrates a low affinity, low capacity site (dissociation constant = 25.2 +/- 2.2 nM, maximum binding capacity = 0.453 +/- 0.037 pmol/mg protein). The purified microsomal fraction of lung exhibits a moderate affinity, high capacity binding site for (3H)-serotonin (dissociation constant = 14.8 +/- 1.6 nM, maximum binding capacity = 0.760 +/- 0.03 pmol/mg protein). In addition to the lung being the major site for its inactivation, the presence of several specific serotonin receptors may be related to some of the known serotonin actions in lung and may suggest other unknown actions of this amine.