Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

β-adrenoceptors in human tracheal smooth muscle: characteristics of binding and relaxation

Specific binding of [¹²⁵I]-(−)-cyanopindolol to human tracheal smooth muscle membranes was saturable, stereo-selective and of high affinity (K_d=5.3±0.9 pmol/l and R_T=78±7fmol/g tissue). The β₁-selective antagonists atenolol and LK 203-030 inhibited specific [¹²⁵I]-(−)-cyanopindolol binding according to a one binding site model with low affinity in nearly all subjects, pointing to a homogeneous β₂-adrenoceptor population. In one subject using LK 203-030 a small β-adrenoceptor subpopulation could be demonstrated. The beta-mimetics isoprenaline, fenoterol, salbutamol and terbutaline recognized high and low affinity agonist binding sites. Isoprenaline's pK_H- and pK_L- values for the high and low affinity sites were 8.0±0.2 and 5.9±0.3 respectively. In functional experiments isoprenaline relaxed tracheal smooth muscle strips having intrinsic tone with a pD₂-value of 6.63±0.19.     

Characterization of pancreatic somatostatin binding sites with a I-somatostatin 28 analog

Somatostatin binding to guinea pig pancreatic acinar cell plasma membranes was characterized with an iodinated stable analog of somatostatin 28 (S₂₈): ¹²⁵I-[Leu⁸, DTrp²²,Tyr²⁵] S₂₈. The binding was highly dependent on calcium ions. In 0.2 mM free Ca²⁺ medium, binding at 37°C was saturable, slowly reversible and exhibited a single class of high affinity binding sites (K_D=0.05±0.01 nM, B_max=157±33 fmol/mg protein). Dissociation of bound radioactivity occurred with biphasic kinetics. Rate of dissociation increased when dissociation was measured at a time before equilibrium binding was reached. In 30 nM free Ca²⁺ medium, binding affinity and maximal binding capacity were decreased by about 4-fold. Decreasing calcium concentrations increased the amount of rapidly dissociating form of the receptor. Somatostatin 14 antagonist, Des AA^1,2[AzaAla^4–5,DTrp⁸,Phe^12–13]-somatostatin was active at the membrane level in inhibiting the binding. We conclude that using ¹²⁵I-[Leu⁸,DTrp²²,Tyr²⁵]S₂₈ as radioligand allows us to characterize a population of specific somatostatin receptors which are not different from those we previously described with the radioligand ¹²⁵I-[Tyr¹¹]-somatostatin. Somatostatin receptors could exist in two interconvertible forms. Calcium ions are an essential component in the regulation of the conformational change of somatostatin receptors.     

Selective effect of castration on the anterior pituitary VIP receptor of male rats

We investigated the effect of surgical castration of male rats on the binding of [Tyr(¹²⁵I)¹⁰]VIP to receptors on the anterior pituitary gland, superior mesenteric artery, brain, liver, and prostate gland. In anterior pituitary membranes the maximum number of VIP binding sites was increased whereas binding affinity was decreased 24 hours following castration. In particular, the high affinity equilibrium dissociation constant (K_D) increased from 0.13±0.02 nM (mean±SEM) to 0.67±0.07 nM and the maximum number of high affinity binding sites (B_max) increased from 71±9 to 470±112 fmol/mg protein. No significant change was observed in the other tissues. Anesthesia or sham operation did not alter the anterior pituitary VIP receptor binding parameters. The changes in the VIP receptor 24 hours after castration were prevented by prior injection of testosterone. These findings demonstrate tissue-selective alterations to the anterior pituitary VIP receptor by castration that are likely mediated by withdrawal of testosterone.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Solubilization of binding sites for IAPP and CGRP from rat lung

Functional binding sites for [¹²⁵I]IAPP and [¹²⁵I]CGRP were solubilized from rat lung membranes with CHAPSO (10 mM). Rat IAPP had a higher affinity (K_i = 22.9 nM) for [¹²⁵I]IAPP binding and rat CGRP (K_i = 0.904 nM) had a higher affinity for [¹²⁵I]CGRP binding over related peptides. [¹²⁵I]IAPP binding was unaffected by GTPγS, but [¹²⁵I]CGRP binding was 50% inhibited, indicating solubilization of a G-protein-receptor complex for CGRP but not IAPP binding. Wheat germ agglutinin affinity columns gave a 25-fold purification of IAPP binding sites, but no CGRP binding sites were eluted from the column, indicating different patterns of glycosylation of the two sites.     

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [³H](−)quinuclidinyl benzilate ([³H](−)QNB) and [³H]pirenzepine ([³H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K_d) of 47 ± 6.3 pM and a maximum number of binding sites (B_max) of 627 ± 65 fmol/mg protein were obtained for [³H](−)QNB, with a K_d value of 3.85 ± 0.67 nM and a B_max value of 95.3 ± 6.25 fmol/mg protein for [³H]PZ binding. The [³H]PZ binding amounted to only 15% of the [³H](−)QNB-labeled sites, as estimated from the ratio of the B_max values of [³H](−)QNB and [³H]PZ, suggesting a low density of M₁ subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n_H) close to unity. The n_H value of AF-DX 116 was close to 1 against [³H](−)QNB binding, while it was 0.75 against [³H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [³H](−)QNB binding (K_i nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [³H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M₂ receptors in the carp brain.     

Characterization of the oligosaccharide moiety of VIP receptor from the human pancreatic cell line BxPC-3

The human pancreatic cell line BxPC-3 displays two classes of binding sites with high and low affinity for VIP. The order of potency of VIP-related peptides in inhibiting either [¹²⁵I]VIP or [¹²⁵I]N-AcPACAP27 binding and in stimulating cAMP production was typical of the human VIP receptor. By combining affinity labeling with glycosidase treatments, we have characterized the VIP receptor as a M_r = 68,200 glycoprotein, consisting of a M_r = 39,300 polypeptide core with at least three N-linked oligosaccharide chains. In addition, our results revealed the presence of a low amount of sialic acid residues in the carbohydrate moiety of receptor.     

Influence of lipid peroxidation on [H]ketanserin binding to 5-HT2 prefrontal cortex receptors

The influence of lipid peroxidation on 5-HT₂ receptor binding was examined in prefrontal cortex membranes from sheep brain. Lipid peroxidation was induced with ascorbic acid and ferrous sulphate and measured by the thiobarbituric acid method. In lipid-peroxidized membranes, [³H]ketanserin specific binding was inhibited. The B_max values decreased by 80%, from 50.1±3.5 fmol/mg protein in control membranes to 10.1±2.0 fmol/mg protein in peroxidized membranes, indicating a decrease in the number of 5-HT₂ binding sites. However, the K_D values for the [³H]ketanserin specific binding did not significantly change. In order to further characterize [³H]ketanserin binding, the inhibition potency (IC₅₀ values) of antagonists or agonists of serotonin and dopamine receptors for [³H]ketanserin specific binding was determined. In control membranes, the order of the inhibition potency of the drugs tested was the following: ketanserin (−log [IC₅₀] = 8.56±0.70) ritanserin (−log [IC₅₀] = 8.13±0.30) methysergide (−log [IC₅₀] = 7.42±0.50) spiperone (−log [IC₅₀] = 7.23±0.18) serotonin (−log [IC₅₀] = 6.99±0.65) haloperidol (−log [IC₅₀] = 6.95±0.65) dopamine (−log [IC₅₀] = 5.82±0.76). After membrane lipid peroxidation, the IC₅₀ value for ritanserin was significantly increased, suggesting a decreased capacity for displacing [³H]ketanserin specific binding. Other antagonists of 5-HT₂ receptors showed apparent increases in IC₅₀ values upon peroxidation, whereas spiperone was shown to be the most potent drug (−log [IC₅₀] = 7.19±1.06) in inhibiting [³H]ketanserin specific binding. A decrease in polyunsaturated fatty acids, namely docosahexaenoic acid (22:6) was also observed in peroxidized membranes. These results indicate a modulating role of the surrounding lipids and of the physical properties of the membranes on the binding activity of 5-HT₂ receptors upon the lipid peroxidation process, which can be involved in the tissue impairment that occurs during the aging process and in post-ischemic situations.     

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line

R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [¹²⁵I]-PACAP or [¹²⁵I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC₅₀ of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC₅₀ in agreement with the pharmacological profile of the VPAC₁ receptor subtype: PACAP = VIP > [K¹⁵, R^16, L²⁷]VIP(1–7)/GRF(8–27) = [R¹⁶]ChSn (two VPAC₁ agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC₂ receptor was inactive at 1 μM. Dose-response curves of VPAC₁ agonist molecules (PACAP, VIP, [K¹⁵, R¹⁶, L²⁷]VIP(1–7)/GRF(8–27), [R¹⁶]ChSn) were shifted to the right by the VPAC₁ receptor antagonist [AcHis¹, D-Phe², Lys¹⁵, Leu¹⁷]VIP(3–7)/GRF(8–27), with a K_i of 3 ± 1 nM (n = 3). The presence of VPAC₁ receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC₁ receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC₁ receptors undergoing rapid desensitization in HEL cells.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.     

Binding of sex steroid binding protein to plasma membranes of human testis

The existence of a specific binding site for sex steroid binding protein (SBP or SHBG) was detected on plasma membranes prepared from the testis of a patient affected by a variant form of testicular feminization. A binding technique using [¹²⁵I]SBP as a tracer allowed us to identify a single set of binding sites, characterized by a K_d of 1.917 × 10⁻¹¹ M. The maximum number of binding sites was 5.2 fmol/mg membrane protein. Membranes were also prepared from a sample of genital skin from the same patient, but no binding for [¹²⁵I]SBP was detectable. The evidence of the SBP membrane receptor in the testis of a patient affected by Morris syndrome extends our knowledge about the tissue distribution of the SBP receptor and suggests the possible implication of SBP and its recognition system in a disorder related to peripheral androgen insensitivity.     

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: Characterization by radioligand binding and photoaffinity labelling

A₁ adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) gave a K_d value of 0.7 nM and a B_max value of 82 ± 13 fmol/mg protein. For the highly A₁-selective agonist 2-chloro-N⁶-[³H]cyclopentyladenosine ([³H]CCPA) a K_d value of 1.7 nM and a B_max value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [³H]DPCPX binding gave a pharmacological profile with R-N⁶-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5′-N-ethylcarboxamido-adenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine ([¹²⁵I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A₁ receptors labelled in brain membranes. Therefore, coated vesicles contain A₁ adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A₁ receptors in these vesicles are not a processed receptor form. These results confirm that A₁ receptors in coated vesicles are coupled to a G-protein, and it appears that the A₁ receptor systems in coated vesicles and in plasma membranes are identical.     

Fate of intraduodenally administered somatostatin in rats in vivo

Intraduodenally administered somatostatin-14 (a milk-borne peptide) has been shown to influence the pancreatic secretions in rats and dogs. To delineate the mechanism involved in the intraduodenal somatostatin-14, the fate of intraduodenal somatostatin-14 was investigated by administering [¹²⁵I][Tyr¹¹]somatostatin-14 ([¹²⁵I][Tyr¹¹]SS14) into the lumen of isolated duodenum in vivo of suckling and adult rats. At 2, 5, 10, and 30 min after administration, the radioactivity in the duodenal contents, duodenal wall, blood, liver, and kidney was measured, and the extracted radioactivity was analyzed for intact [¹²⁵I][Tyr¹¹]SS14 by reversed-phase high performance liquid chromatography. Radioactivity disappeared rapidly from the duodenal lumen with 50% of loss occurring at 2 min. No trace of intact [¹²⁵I][Tyr¹¹]SS14 was detected in any of the samples, except duodenal contents of suckling rats at 2 min, suggesting a rapid metabolism of SS14 in the duodenal lumen and a lack of duodenal absorption of intact SS14 in the rat.     

CRF receptors in the rodent and human cardiovascular systems: species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with ¹²⁵I-[Tyr⁰, Glu¹, Nle¹⁷]-sauvagine or [¹²⁵I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF₂ receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF₂ receptors were detected with ¹²⁵I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with ¹²⁵I-[Tyr⁰, Glu¹, Nle¹⁷]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF₂ receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.     

Atrial natriuretic factor (ANF) binding sites in brain and related structures

Visualization of [¹²⁵I]ANF binding sites in rat brain by an autoradiographic technique demonstrated that these sites are highly localized in areas such as the olfactory bulb, subfornical organ, area postrema and nucleus tractus solitarius. This distribution suggests that certain cardiovascular effects of ANF could be centrally mediated and that the existence of brain ANF-related peptides should be considered. Finally, moderate densities of [¹²⁵I]ANF binding sites are found in the rat and guinea pig eye while low densities are seen in pituitary and pineal gland.     

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [¹²⁵I]rAM revealed the presence of a single class of high-affinity (K_d1.3 × 10⁻⁸ M) binding sites for rAM in VSMC. The apparent K_i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10⁻⁷ M. Affinity labeling of VSMC membranes with [¹²⁵I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC₅₀ of 10⁻⁸ M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.     

BW1023U90: A new GRP receptor antagonist for small-cell lung cancer cells

Gastrin-releasing peptide (GRP) receptor antagonists were synthesized and their ability to interact with small-cell lung cancer (SCLC) cells determined. [¹²⁵I]BW1023U90, bound with high affinity (K_d = 2 nM) to a single class of sites (Bmax = 55 fmol/mg protein) using SCLC cell line NCI-H345. [¹²⁵I]BW1023U90 binding was time dependent and reversible even at 37°C as the ligand was minimally internalized. Specific [¹²⁵I]BW1023U90 binding was inhibited with high affinity by GRP as well as bombesin (BB) but not neuromedin B (NMB). BW1023U90 inhibited the ability of BB to elevate cytosolic Ca²⁺ and increase the growth of SCLC cells. A BW1023U90 analogue, BW2258U89 (10 μg/day, SC) slowed SCLC xenograft formation in nude mice and [¹²⁵I]BW1023U90 localized to SCLC tumors 1 h after injection into nude mice. BW2258U89 (4% by weight) was placed in microspheres and slowly released over a 3-week period in nude mice bearing SCLC xenografts. The microspheres containing BW2258U89 strongly inhibited SCLC growth in vivo. A radioimmunoassay was developed for the GRP receptor antagonists and the rabbit antiserum cross-reacted totally with BW2258U89 or BW1023U90. BW2258U89 immunoreactivity (5 nM) was detected in the plasma of nude mice containing the microspheres after 1 week. These data suggest that GRP receptor antagonists bind to receptors on SCLC tumors.     

Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10⁻¹⁰−10⁻⁶M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [¹²⁵I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500–10,000-fold). A specific, time-dependent binding of [¹²⁵I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.     

Identification of [I]endothelin binding sites in human coronary tissue

In vitro receptor autoradiography has been used to study the distribution of [¹²⁵I]endothelin binding sites in human coronary tissue from patients undergoing cardiac transplantation. Dense binding of [¹²⁵I]endothelin was associated with the smooth muscle of epicardial coronary arteries as well as to perivascular regions of these vessels. Binding was also associated with the ventricular myocardium. There was an increased binding of [¹²⁵I]endothelin to atheromatous tissue, both coronary arteries and vein graft.

The [¹²⁵I]endothelin binding sites identified using in vitro autoradiography are likely to be functionally relevant since endothelin causes a concentration-dependent contraction of segments of human epicardial coronary arteries in vitro and also has positive inotropic activity on isolated human cardiomyocytes.

The presence of specific binding sites for [¹²⁵I]endothelin on coronary tissue and the increased binding in atheromatous tissue suggest that endothelin is a peptide which may play a role in the maintenance of vascular tone and/or the pathogenesis of ischaemic heart disease.