Radioligand Binding Analysis Research Articles

Autoradiographic labelling of P 2 purinoceptors in the guinea-pig cochlea

Two different radioligands were used to identify extracellular ATP binding sites specific to P 2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5′-(α-[ 35S]thio)triphosphate ([ 35S]dATPαS; 10 nM) provided a high activity probe for the P 2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP; 100 μM). [ 3H]α,β-methylene-ATP (10 nM), a high affinity probe for a P 2x purinoceptor subtype was selectively blocked by inclusion of the related compound β,γ-methylene-ATP (100 μM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P 2x purinoceptor probe also bound to lateral wall tissue below the spiral prominence and insertion point of the basilar membrane within the scala tympani compartment, a region which failed to show significant binding using [ 35S]dATPαS. Frozen sections of whole cochlea permitted analysis of radioligand binding to the cell body region (spiral ganglion in Rosenthal's canal) of the primary auditory afferents and the auditory nerve itself, which lies within the central region of the modiolus of the cochlea. Both these regions exhibited 2MeSATP blockable [ 35S]dATPαS binding whereas specific [ 3H]α,β-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P 2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites.

Characterisation of melanocortin receptor subtypes by radioligand binding analysis

The DNAs encoding three melanocortin receptor subtypes (melanocortin MC 1 receptor, melanocortin MC 3 receptor and melanocortin MC 5 receptor) were expressed individually in COS ( C V-1 O rigin, SV40) cells to characterise their ligand binding properties. The results indicated that [ 125I][Nle 4, D-Phe 7]α-MSH (melanocyte stimulating hormone) bound to a single saturable site with K d values of 85.1 ± 8.0 pmol/l (mean ± S.E.M.), 396 ± 65 pmol/l and 5.05 ± 1.00 nmol/l for melanocortin MC 1 receptor, melanocortin MC 3 receptor and melanocortin MC 3 receptor, respectively. The melanocortin MC 1 receptor and the melanocortin MC 5 receptor showed a similar potency order to the melanocortic peptides examined which was markedly different from the potency order of the melanocortin MC 3 receptor. The melanocortin MC 1 receptor and melanocortin MC 5 receptor had a relatively higher affinity for α-MSH than γ-MSH and β-MSH, whereas the melanocortin MC 3 receptor had higher affinity for desacetyl-α-MSH, γ-MSH and β-MSH compared to α-MSH. The inclusion of the endopeptidase inhibitor phosphoramidon to prevent the breakdown of ACTH-(1-39) (adrenocorticotrophic hormone) to α-MSH, decreased ACTH-(1-39) binding affinity showing that ACTH-(1-39) had a much lower affinity for melanocortin MC 1 receptor than reported earlier.

Lipopolysaccharide-dependent induction of IL-10 receptor expression on murine fibroblasts.

Although various biologic activities of IL-10 have been identified, little is known about IL-10's molecular mechanism of action. Herein we report the characterization of IL-10R on different murine cell lines and demonstrate the LPS inducibility of this protein. With the use of purified recombinant murine IL-10 and labeled IL-10-specific mAb, IL-10Rs were detected by flow cytometry. Radioligand binding analyses showed that RAW264.7 cells expressed 238 +/- 87 receptors per cell and bound ligand with a single affinity of 8.3 +/- 2.4 x 10(9) M-1. Similar studies documented IL-10R expression on murine B cells (CH27) and CD4+ Th1 cells but not murine Th2 cells, L929 or WA-17 fibroblasts, or fibrosarcoma cells. Exposure of fibroblasts to LPS-induced cellular IL-10 binding activity in a dose- and time-dependent manner. Radioligand binding analyses performed on LPS-treated fibroblasts showed cellular expression of 325 +/- 59 IL-10 binding sites and a binding affinity of Ka = 7.5 +/- 2.5 x 10(8) M-1. RT-PCR analysis confirmed the induction of the IL-10R. Functional analyses of IL-10R-expressing cells revealed that IL-10 activated a cellular transcription factor in RAW264.7 cells that bound a DNA probe containing the IFN-gamma response region from the Fc gamma RI gene. In contrast, IL-10 did not induce gamma response region binding activity in L929 fibroblasts either treated with LPS or transfected with the murine IL-10R cDNA. These results thus indicate that cellular responses to IL-10 may be influenced by both the external environment and the presence of additional signaling components within the cell.

The protective effects of stress control may be mediated by increased brain levels of benzodiazepine receptor agonists

Control over stress protects against many of the deleterious effects of stress exposure, but the endogenous mediators responsible for these prophylactic effects have remained elusive. Using behavioral pharmacology, in vitro radioligand binding and neurochemical analyses, we demonstrate that exposure to escapable stress results in brain and behavior changes reminiscent of benzodiazepine administration. The stress control group shows significant protection against picrotoxinin-induced seizures, reductions in [ 35 S]t- butylbicyclophosphorothionate (TBPS) binding and a 3-fold increase of benzodiazepine-like substances in brain in comparison to both yoked-inescapable shock and non-shock controls. These observations suggest that coping behavior leads to the release of endogenous benzodiazepine-like compounds in brain which protect the organism from stress pathology.

Corticotropin-releasing factor receptors in human small cell lung carcinoma cells: radioligand binding, second messenger, and northern blot analysis data

Corticotropin-releasing factor receptors in human small cell lung carcinoma cells: radioligand binding, second messenger, and northern blot analysis data

Immunohistochemical mapping of angiotensin type 2 (AT 2) receptors in rat brain

Recently developed antisera selective for angiotensin Type 2 (AT 2) receptors were use to localize AT 2 receptors in rat brain by immunohistochemistry. While the results from these experiments were largely consistent with previous autoradiographic and radioligand binding analyses of AT 2 receptor populations in brain, there were also some notable differences in the distribution of immunoreactivity. More specifically, in agreement with previous studies, AT 2 antisera detected apparent receptor populations in the locus coeruleus and the bed nucleus of the accessory olfactory tract, whereas AT 2 receptor-immunoreactivity in the cerebellum was primarily associated with the Purkinje cell layer and the deep cerebellar nuclei rather than the molecular layer as has been previously reported in autoradiographic studies. Other regions with prominent immune-staining included all subfields of the hippocampus, which had been previously reported to contain exclusively AT 1 receptors. Limbic structures such as the amygdala, thalamic areas such as the rhomboid thalamic nucleus, the paraventricular thalamic nucleus, hypothalamic areas such as the paraventricular hypothalamic nucleus, and the supraoptic nucleus also exhibited prominent AT 2-immunoreactivity. In the paraventricular hypothalamic nucleus, AT 2 receptor staining appeared to be associated primarily with the magnocellular neurons. In all regions examined, AT 2 receptor immunoreactivity was associated with the cytoplasm and cell membrane and was not localized within the nucleus. Collectively, these results confirm and extend the neuroanatomical resolution of previous autoradiographic studies as well as identify new AT 2 receptor populations in rat brain.

Developmental appearance of peptide-binding sites in the small intestine of the neonatal piglet

The ontogeny of the expression of specific binding sites for bombesin (BBS), vasoactive intestinal polypeptide (VIP), and gastrin in the neonatal pig was examined. Piglets were sacrificed at birth, 1 d, 1 wk, 3 wk and 4 wk, and samples of duodenal, jejunal and ileal tissue were harvested for autoradiographic analysis of saturable radioligand binding. Monoiodinated, biologically active 125I-Tyr4-BBS, VIP or gastrin (100 pM) was applied to slide-mounted sections of piglet intestinal tissue and localized using autoradiographic analysis of radioligand binding. Saturable binding sites for 125I-BBS first appeared in duodenal tissue of 7-d-old piglets, with evidence for continued presence in this tissue through 21 d. BBS binding sites were localized in duodenal mucosa, with no detectable binding in any other tissue layers. No evidence for saturable binding of 125I-Tyr4-BBS was found in intestinal sections from either newborn, 1-d-old or 4-wk-old piglets. Saturable binding sites for 125I-VIP were present in duodenal, jejunal and ileal tissues of piglets at all ages studied and were present in both the mucosa and the muscularis externa. No saturable binding sites for 125I-gastrin were observed in intestinal sections from piglets at any of the ages studied. These results suggest potential sites of direct action of VIP in newborn, suckling and newly weaned piglets. Bombesin, however, may have direct actions only on the duodenal mucosa at 7–21 d of age. Key words: Bombesin, vasoactive intestinal polypeptide, gastrin, piglet, small intestine, newborn

Intestinal Mucosal Lymphocytes Have H 1 Receptors: H 1 Antagonists Reduce Their Proliferation and Cytotoxicity

Histamine and H 1 antagonists inhibit the proliferation and natural killer activity of peripheral blood mononuclear cells, which express large numbers of H 1 receptors. This study examined the presence of H 1 receptors and the effects of histamine, H 1 antagonists (pyrilamine and diphenhydramine), and H 2 antagonists (cimetidine and ranitidine) on human intestinal lymphocyte proliferation and cytotoxicity. Intestinal lymphocytes were obtained by chemical and enzymatic treatment of surgical specimens and purified by Percoll density gradient centrifugation. Proliferation was measured by [ 3H]thymidine incorporation of mitogen-stimulated lymphocytes; cytotoxicity was measured by the standard 51 Cr-release assay using HT-29 adenocarcinoma target cells. Scatchard analysis of radioligand binding using [ 3H]pyrilamine demonstrated H 1 receptors. The mitogen-induced proliferative responses of intraepithelial lymphocytes and lamina propria lymphocytes were inhibited by histamine and the H 1 antagonists but not the H 2 antagonists. Cytotoxic activities of fresh or IL-2-stimulated mucosal lymphocytes (spontaneous and lymphokine-activated killing, respectively) were also reduced by the H 1 antagonists. A large number of H 1 receptors were found on intraepithelial lymphocytes and peripheral blood mononuclear cells and still more on lamina propria lymphocytes. Intestinal lymphocytes bear H 1 receptors; histamine and H 1 antagonists have immunomodulatory effects on these cells.

Mutational analysis of human endothelin receptors ETA and ETB identification of regions involved in the selectivity for endothelin 3 or cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu).

Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET-1-selective profile and ETB a non-selective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-125I]iodotyrosyl ET-1 (125I-ET-1) binding, with affinities comparable to those of the wild-type receptors (apparent Ki approximately 1-6 x 10(-9) M). An average of 10(5)-10(6) binding sites/cell was calculated for the wild-type and mutant forms. In competition experiments using 125I-ET-1 and unlabeled ET-3, an ETB-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ETB. The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ETB chimeras where the symmetrical exchange had been operated. Using 125I-ET-1 and the ETA-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the N-terminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.

Pharmacological characterization and anatomical localization of prejunctional beta-adrenoceptors in the rat kidney.

1. The subtype and anatomical localization of beta-adrenoceptors mediating facilitation of stimulus-induced overflow of noradrenaline ('prejunctional beta-adrenoceptors') are not conclusively known to date. The present study was undertaken to characterize these receptors by use of pharmacological methods as well as to define their localization (prejunctional or postjunctional) with radio-ligand binding and autoradiography techniques combined with surgical denervation of the sympathetic innervation to the rat kidney. 2. Exposure of the kidney to (-)-isoprenaline, the nonselective beta-adrenoceptor agonist, resulted in a dose-dependent facilitation of stimulus-induced neurotransmitter overflow. This response was inhibited by propranolol, the beta 1- and beta 2-adrenoceptor antagonist, with a pA2 of 9.20 suggesting that the prejunctional beta-adrenoceptors are not of the beta 3-subtype. 3. The rank order of potency and potency ratios of beta-adrenoceptor agonists at renal prejunctional beta-adrenoceptors (EC50 for agonist/EC50 for (-)-isoprenaline) were: (-)-isoprenaline (1) > procaterol (2) > salbutamol (3) > adrenaline (10) > (+)-isoprenaline (25). However, dobutamine, the beta 1-adrenoceptor agonist, failed to enhance stimulus-induced overflow of noradrenaline. These results are indicative of the presence of beta 2-adrenoceptors as prejunctional beta-adrenoceptors. 4. Facilitation elicited by (-)-isoprenaline and procaterol, the selective beta 2-adrenoceptor agonist, was inhibited by ICI 118,551, the selective beta 2-adrenoceptor antagonist, with pKb values of 9.20 and 9.35, respectively at renal prejunctional beta-adrenoceptors. Similarly, the pKb values of metoprolol, the selective beta 1-adrenoceptor antagonist, at renal prejunctional beta-adrenoceptors were determined to be 6.25 and 6.18 against (-)-isoprenaline and procaterol, respectively. These results suggest the presence of a homogeneous population of beta 2-adrenoceptors as prejunctional beta-adrenoceptors. 5. Radio-ligand binding analysis of renal beta-adrenoceptors revealed the prevalence of the beta 1-subtype as compared to the beta 2-subtype (63% vs 37%). However, surgical denervation of the rat kidney, resulting in more than 90% reduction in renal noradrenaline content, selectively reduced the beta 2-adrenoceptor population by 80%, implying the presence of beta 2-adrenoceptors on renal sympathetic nerve terminals. 6. Autoradiographic analysis demonstrated the presence of beta 1-adrenoceptors on cortical structures such as glomeruli and tubules. beta-Adrenoceptors were found to be present on tubules (minor population), collecting tubules in outer medulla and the adventitia and adventitial-medial border of intraparenchymal branches of the renal artery. Surgical denervation of the rat kidney resulted in the disappearance of Beta2-adrenoceptors associated with the intraparenchymal branches, without affecting the Beta-adrenoceptor populations at other sites. These results support the notion that the Beta2-subtype is present on renal sympathetic nerve terminals and demonstrate that these prejunctional Beta2-adrenoceptors are associated with the renal vasculature and not with renal tubules.7. The results of the present investigation demonstrate that renal prejunctional Beta-adrenoceptors are of the Beta2-subtype in nature. These receptors are present on sympathetic nerve terminals which are associated with the renal vasculature.

Integrin-mediated collagen gel contraction by cardiac fibroblasts. Effects of angiotensin II.

Angiotensin II (Ang II), a vasoactive octapeptide, has been implicated in cardiac growth and the development of hypertrophy and fibrosis secondary in hypertensive disease. These consequences of Ang II imply an effect on the function and morphology of cardiac interstitial cells (fibroblasts). The present investigation was designed to (1) determine whether neonatal heart fibroblasts (NHFs) possess functional Ang II receptors on their plasma membrane and (2) examine the effects of Ang II on NHFs in vitro using three- and two-dimensional (3D and 2D, respectively) cultures. Several analytic techniques were used to test the specific questions of the present study. Since cardiac fibroblast phenotype can be influenced by culture conditions, both 2D and 3D cultures were used in the present investigations. Reverse-transcriptase polymerase chain reaction and radioligand binding analysis were used to test for the presence of Ang II receptors on NHFs. Both revealed that NHFs in 2D culture possess Ang II receptor mRNA and Ang II receptors. When isolated NHFs were cultured in 3D collagen gels and treated with Ang II, gel contraction was stimulated by NHFs. This effect was attenuated by the specific Ang II receptor antagonist [Sar1,Ala8]Ang II. Ang II-stimulated gel contraction was completely inhibited by extracellular matrix receptor (beta 1-integrin) antibodies (P < .05), supporting previous studies indicating that collagen gel contraction is mediated via the integrins. Immunofluorescent staining was used to test the localization of cell-surface integrins. A more intense staining pattern for beta 1-integrin in Ang II-treated versus control cells was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the receptor subtype responsible for endothelin-mediated protein kinase C activation and atrial natriuretic factor secretion from atrial myocytes.

Endothelin-1 (ET-1) is a potent stimulator of atrial natriuretic factor (ANF) secretion from myocardial cells. In heart tissue there are two ET receptor subtypes (ETA-R and ETB-R), which can be pharmacologically distinguished by the ET isopeptides ET-1 and ET-3. However, the identification of the ET-R subtype responsible for the rapid enhancement of ANF release, which occurs within minutes of exposing cardiac myocytes to ET, has not been investigated. In the present study ET-1 was about 100-fold more potent than ET-3 at stimulating membrane phosphoinositide hydrolysis, protein kinase C activation, and ANF release from purified primary atrial myocytes. These responses were completely abolished by BQ123, an ETA-R antagonist. Radioligand binding analyses showed that competitor peptides displaced 125I-ET-1 binding to atrial myocyte ET-Rs with a rank order of potency of ET-1 >> BQ123 > ET-3, a characteristic ETA-R pharmacological profile. While neither ET-1 or ET-3 altered forskolin-stimulated cAMP levels, suggesting the absence of the ETB-R, basal cAMP levels were also unaffected by the ETs. Northern analysis using ET-R subtype-specific probes demonstrated that the ETA-R transcript was present in the cultures at levels at least 50-fold greater than the ETB-R transcript. These findings demonstrate that the stimulation of the phosphatidylinositol/protein kinase C pathway, which is required for maximal ET-stimulated ANF release from primary atrial myocytes, is associated with the activation of only the ETA-R, thus defining a specific function for an endogenous ET-R in myocardial cells.

Differential regulation of tumour necrosis factor receptors (TNF-R) by IL-4; upregulation of P55 and P75 TNF-R on synovial joint mononuclear cells

Interleukin 4 (IL-4) has previously been shown to downregulate the production of pro-inflammatory cytokines such as TNF-α, and hence has been considered to be a potential anti-inflammatory agent. In this study we have investigated the effects of IL-4 on the expression of both p55 and p75 TNF receptors (TNF-R) by flow cytometry and radioligand binding analyses and demonstrate that IL-4 downregulates both p55 and p75 TNF-R on HeLa and Jijoye cell lines in a dose dependent manner. IL-4 reduced the number of p55 TNF-R on HeLa cells from 6400 ( K d 5.1 nM) to 3900 ( K d 3.7 nM), and p75 TNF-R on Jijoye cells from 4800 ( K d 1.6 nM) to 3250 ( K d 1.5 nM). However, different effects were observed on peripheral blood mononuclear cells (PBMC). IL-4 inhibited the increase in p55 and p75 TNF-R on PBMC following adherence, whereas IL-4 upregulated p75 TNF-R expressed on PHA induced T cell blasts. To assess further the possible anti-inflammatory properties of IL-4, we studied its effects on synovial joint mononuclear cell cultures from 15 patients with inflammatory synovitis. In contrast to the differential effects of IL-4 on monocytes and T cells, IL-4 upregulated both p55 ( P < 0.05) and p75 TNF-R ( P < 0.005) on synovial joint cells in culture. IL-4 treatment caused a small decrease in levels of bioactive TNF-α in RA synovial culture supernatants, together with an increase in soluble p75 TNF-R levels although differences were not significant. These data demonstrate that IL-4 can modulate both TNF-α and TNF-R expression, and provides further evidence for the role of soluble TNF-R as endogenous regulators of TNF-α function.

Distinct subdomains of human endothelin receptors determine their selectivity to endothelinA-selective antagonist and endothelinB-selective agonists.

The endothelin (ET) family of peptides acts via two subtypes of G-protein-coupled heptahelical receptors termed ETA and ETB, which have distinct rank orders of affinity to endothelin receptor agonists and antagonists. To delineate which portions of the receptor molecules determine ligand selectivity, we have constructed a series of chimeras between human ETA and ETB receptors and characterized the chimeric receptors expressed in heterologous cell lines by competitive radioligand binding analysis and by measuring agonist-induced transients of intracellular Ca2+. We demonstrate that the binding determinant for the ETB-selective agonists ET-3, BQ3020, and IRL1620 residues within the region spanning the putative transmembrane helices IV-VI and the adjacent loop regions. In contrast, the transmembrane helices I, II, III, and VII plus the intervening loop regions specify the selectivity for BQ123, an ETA-selective antagonist. BQ123 exhibited no detectable agonistic activity in all wild-type and chimeric receptors tested. A chimeric receptor that has the transmembrane helices IV-VI (and adjacent loops) from the ETB receptor inserted into the remaining regions from the ETA receptor binds both the ETA- and ETB-selective ligands with high affinities. Moreover, BQ123 competitively inhibits the binding of the amino-terminally truncated ETB agonists, 125I-BQ3020 and 125I-IRL1620, to this chimeric receptor, suggesting that BQ123 is a mimic of the carboxyl-terminal linear portion of endothelins. These findings indicate that there are at least two separable ligand interaction subdomains within the endothelin receptors.

Both tumor necrosis factor receptor types mediate proliferative signals in human mononuclear cell activation.

TNF is a highly pleiotropic cytokine. The recent identification of two distinct cellular receptors for TNF may provide explanations for the many different TNF activities. We have investigated the expression of the two receptor types, TNFR alpha (75 kDa) and TNFR beta (55 kDa), in human PBMC. Both receptors were found simultaneously expressed by cytofluorimetric, radioligand binding and Northern analysis of naive as well as PHA-activated PBMC. The expression levels in the CD14+ and CD14- subsets were different. Both receptors were strongly expressed in the CD14+ subset. The expression of the receptors in the CD14-, CD3+, CD4+, and CD8+ subsets was lower and similar among these subsets, but TNFR alpha was expressed at higher level than TNFR beta. To dissect the functional roles of the two receptors, we studied the growth factor activity of TNF in the late proliferative responses of PBMC to PHA. In the first approach, the activity of either receptor was blocked by neutralizing, receptor type specific antibodies. In a second approach, the ligand, TNF, was inhibited by a neutralizing antiserum, and the cells were restimulated using type-specific anti-TNFR antibodies with agonistic activity. It was found that both receptor types mediated signals required for proliferative responses of PBMC to PHA from day 4 to day 8 in culture. The cell responses to the activation of either receptor type appeared to be independent, because one receptor could not compensate for the reduction in cell activation caused by blocking the other receptor type.

Human lymphocytes lack substance P receptors

Substance P (SP), a neuropeptide found in high concentrations in the gut, is reported to have many potent immunomodulatory actions. This study evaluated some effects of SP on human peripheral blood lymphocytes (PBL) and jejunal intraepithelial lymphocytes (IEL) and the expression of SP receptors on these and other lymphocyte types. In contrast to previous studies, SP (10 −8 or 10 −12 M) did not affect the proliferation (spontaneous or mitogen-induced) nor spontaneous cytotoxicity by PBL or IEL. To determine whether this unresponsiveness was due to an absence of SP receptors, the SP binding potential of these and other human lymphocyte types was determined by Scatchard analysis of radioligand binding. The IM-9 B lymphoblastoid cell line, used as a positive control, demonstrated 4838 ± 603 or 3131 ± 832 receptors per cell, with a K d of 0.21± 0.01 or 0.18 ± 0.09 M, using [ 3H]SP or 125I-SP, respectively. No receptors were found on PBL, polymorphonuclear leukocytes, splenocytes, IEL, or jejunal lamina propria lymphocytes using either radioligand. These findings dispute the presence of large numbers of SP receptors on lymphocytes in peripheral blood, spleen, or intestinal mucosa, and argue against any major effect of SP on T cell proliferation or spontaneous cytotoxicity.

Interferon-gamma receptor: mRNA half-life, receptor mass, and abundance on A431 human epidermoid carcinoma cells.

The human interferon-gamma (IFN-gamma) receptor protein and mRNA were identified in the epidermoid carcinoma cell line A431 by radioligand binding and Northern hybridization, respectively. Receptor affinity and receptor number per cell were calculated by Scatchard analysis of saturation binding data. The half-life of the receptor mRNA was determined by actinomycin D inhibition of de novo transcription. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of covalently cross-linked 32P-labeled IFN-gamma and its receptor is consistent with a molecular mass of approximately 99 kD. Radioligand binding analysis revealed 27,000 high affinity (KD = 7.1 x 10(-10) M) receptors per cell. Northern hybridization using an IFN-gamma receptor cDNA probe revealed a single mRNA of 2.2 kb. The receptor mRNA half-life in actinomycin D-treated cells was 4.7 h.

Dual Transmembrane Signalling Mechanisms in Eosinophils: Evidence for Two Functionally Distinct Receptors for Platelet-Activating Factor.

The effect of PAF on eosinophil activation was investigated. TxB2 release required a lower concentration of PAF (ED50 = 17.2 nM) whereas for superoxide anion (O2) production doses in excess of 1 μM (ED50 = 31.7 μM) were needed. The PAF-induced O2 release occurred in the absence of increased [Ca2+]i whereas the production of TxB2 paralleled the magnitude of the [Ca2+]i increase. Pretreatment of the eosinophils with pertussis toxin (PTX) reduced both the PAF-induced release of TxB2 and the PAF-induced rise in [Ca2+]i. However, PTX failed to inhibit PAF-induced O2 generation. Experiments with the microsomal (100,000 g) fraction from these cells demonstrated that PTX pretreatment had ADP-ribosylated a 41-kD protein in the membrane, confirming that GTP binding proteins are present in eosinophil membranes. Scatchard plot analysis of radioligand binding to eosinophil membranes indicated the presence or two binding sites with an apparent Kd of 0.33 and 11.5 nM, respectively. The results suggest that two distinct forms of the PAF receptor can be identified in eosinophil membranes.

Involvement of urokinase and its receptor in the invasiveness of human prostatic carcinoma cell lines.

We have investigated the role of urokinase (UK) and its cell-surface receptor in determining the invasiveness of prostate cancer cells. Three human cell lines, DU-145, PC-3 and LNCaP, that differ in androgen-responsiveness and growth characteristics, were tested. Analysis of the conditioned medium by an enzyme-linked immunosorbent assay showed secretion of UK by DU-145 (63 ng/mL/10(6) cells/48 hr) and PC-3 (682 ng/mL/10(6) cells/48 hr), but absence of secretion by LNCaP cells. Western blot analysis and enzyme activity assay of the conditioned medium confirmed these results. Scatchard analysis of radioligand binding with acid pretreated cells showed the presence of a single population of high affinity UK receptors on DU-145 cells (93,000 sites/cell, Kd = 0.9 nM) and PC-3 cells (25,000 sites/cell, Kd = 1.0 nM) but not on LNCaP cells. DU-145 and PC-3 cells were found to be highly invasive in in vitro invasion assays: 4.5 +/- 0.5% and 6.5 +/- 0.5%, respectively, of total tumor cells (approximately 2 x 10(5)) had penetrated reconstituted basement membrane (Matrigel) in a 72 hr incubation in serum-free growth medium. Under similar conditions, less than 0.25% LNCaP cells invaded Matrigel. The data indicate that androgen unresponsive, aggressive prostate tumor cells of high metastatic potential, DU-145 and PC-3, secrete UK and display cell-surface UK receptors, fully charged with the protease. Conversely, relatively indolent LNCaP cells of low metastatic potential do not secrete UK nor do they possess its binding sites. UK receptor antagonists, UK 12-32 and UK 6-135, which compete with labeled UK for binding to prostatic cells but do not inhibit cellular proliferation or UK secretion, markedly reduced DU-145 and PC-3 cell invasion (80-85% inhibition), thereby suggesting an important role of receptor-bound UK in prostate tumor cell invasion.

Graphical Analysis of Reversible Radioligand Binding from Time—Activity Measurements Applied to [N-11C-Methyl]-(−)-Cocaine PET Studies in Human Subjects

A graphical method of analysis applicable to ligands that bind reversibly to receptors or enzymes requiring the simultaneous measurement of plasma and tissue radioactivities for multiple times after the injection of a radiolabeled tracer is presented. It is shown that there is a time t after which a plot of integral of t0ROI(t')dt'/ROI(t) versus integral of t0Cp(t')dt'/ROI(t) (where ROI and Cp are functions of time describing the variation of tissue radioactivity and plasma radioactivity, respectively) is linear with a slope that corresponds to the steady-state space of the ligand plus the plasma volume,.Vp. For a two-compartment model, the slope is given by lambda + Vp, where lambda is the partition coefficient and the intercept is -1/[kappa 2(1 + Vp/lambda)]. For a three-compartment model, the slope is lambda(1 + Bmax/Kd) + Vp and the intercept is -[1 + Bmax/Kd)/k2 + [koff(1 + Kd/Bmax)]-1) [1 + Vp/lambda(1 + Bmax/Kd)]-1 (where Bmax represents the concentration of ligand binding sites and Kd the equilibrium dissociation constant of the ligand-binding site complex, koff (k4) the ligand-binding site dissociation constant, and k2 is the transfer constant from tissue to plasma). This graphical method provides the ratio Bmax/Kd from the slope for comparison with in vitro measures of the same parameter. It also provides an easy, rapid method for comparison of the reproducibility of repeated measures in a single subject, for longitudinal or drug intervention protocols, or for comparing experimental results between subjects. Although the linearity of this plot holds when ROI/Cp is constant, it can be shown that, for many systems, linearity is effectively reached some time before this. This analysis has been applied to data from [N-methyl-11C]-(-)-cocaine ([11C]cocaine) studies in normal human volunteers and the results are compared to the standard nonlinear least-squares analysis. The calculated value of Bmax/Kd for the high-affinity binding site for cocaine is 0.62 +/- 0.20, in agreement with literature values.