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

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

Genetic analysis of the 6-thiobenzylpurine binding site of the nucleoside transporter in mouse lymphoblasts

From a mutagenized population of wild-type S49 T lymphoblasts, cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, and 30 microM p-nitrobenzyl-6-thioinosine (NBMPR), a potent inhibitor of nucleoside transport. Unlike wild-type parental cells, two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild-type cells, KAB1, and KAB5 cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that nucleoside incorporation was much less sensitive to inhibition by NBMPR in the mutant cells. Rapid transport studies indicated that the mutant cell lines, unlike the wild-type parent, had acquired an NBMPR-insensitive nucleoside transport component which was similar to the NBMPR-sensitive wild-type transporter with respect to affinities for nucleosides and sensitivities toward N-ethylmaleimide and dipyridamole. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild-type complement of NBMPR binding sites with wild-type binding characteristics. These data suggest that the NBMPR binding site in wild-type S49 cells is genetically distinguishable from the nucleoside carrier site and that the former may be a regulatory site.     

Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.     

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

An assay for inhibitors of nucleoside transport based upon the use of 5-[125I]iodo-2'-deoxyuridine as permeant

5-[125I]Iodo-2'-deoxyuridine (IdUrd) has been shown to serve as a permeant for the nucleoside transport system of human erythrocytes and to be matabolically inert in these cells. Linear initial velocities were obtained at 20 degrees C for 125IdUrd transport, yielding a Km of 73 +/- 18 microM (n = 6). Low-affinity inhibitors of 125IdUrd transport, such as adenosine (Ki = 32 +/- 2 microM, n = 2), could be characterized by Michaelis-Menten kinetics. However, high-affinity inhibitors, such as 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, caused nonlinear initial velocities when added to the cells simultaneously with 125IdUrd. Conditions were defined (viz., 20-min pretreatment of cells with test compound followed by 5.0-min incubation with 1.0 microM 125IdUrd, all at 20 degrees C) whereby high-affinity inhibitors of IdUrd transport can be identified and evaluated according to their 50% inhibitory concentrations. The use of 125IdUrd as permeant greatly expedites the testing of compounds as inhibitors of nucleoside transport by allowing the cell pellets generated in these assays to be monitored directly in a gamma spectrometer, thereby circumventing the solubilization and decolorization of cell pellets required by assays that use 3H- or 14C-labeled nucleoside permeants.     

Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and thiol reagents.

The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p-chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS.     

125I-DPDYN, monoiodo[D-Pro10]dynorphin(1-11): a highly radioactive and selective probe for the study of kappa opioid receptors

The mono- and diiodinated derivatives of the kappa-selective ligand [D-Pro10]dynorphin(1-11), DPDYN, were prepared. Their binding properties at the three opioid receptor types (mu, delta and kappa) were examined and compared to those of the parent peptide. The monoiodo derivative shows a general although moderate decrease in affinity and retains high kappa selectivity (KI mu/KI kappa = 48 and KI delta/KI kappa = 140). The binding properties of the diiodo derivative are found to be dramatically decreased. Radioiodination of DPDYN leads to the monoiodinated peptide with high specific activity (700-800 Ci/mmol). In guinea-pig cerebellum membranes, a kappa-specific tissue, [125I]-labelled monoiodo[D-Pro10]dynorphin(1-11), 125I-DPDYN, interacts specifically and reversibly with a single class of binding sites (Bmax = 118 fmol/mg protein) with a high affinity (KD = 0.12 nM from equilibrium experiments, 0.18 nM from kinetics studies). Therefore, because of its high specific radioactivity, high affinity and reasonably good selectivity, 125I-DPDYN designates itself as the probe of the k-opioid receptor type.     

Characterization of epidermal growth factor receptor in rat buccal mucosal cells

1. Receptor binding for epidermal growth factor (EGF) in rat buccal mucosa was characterized. Binding of [125I]EGF to rat buccal mucosa was time, temperature, cell number and [125I]EGF concentration dependent. 2. The [125I]EGF binding was reversible and specific. Unlabeled EGF competed for binding to buccal mucosal cells with an IC50 of 1.25 nM, whereas insulin failed to compete. 3. Scatchard analysis of the binding data revealed a curvilinear plot with dissociation constants of 3.39 nM and 2.14 microM, and binding capacities of 1.23 x 10(4) and 3.38 x 10(5) receptors per cell for high and low affinity sites, respectively. 4. Crosslinking of [125I]EGF to buccal mucosa followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one major protein with Mw 170,000 which shares similar molecular weight with other known EGF receptors from different tissues and species. 5. The study is the first report to provide biochemical parameters of the specific EGF receptors in rat buccal mucosa.     

Characterization of gingival epithelium epidermal growth factor receptor

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

Identification of the nucleoside transporter in cultured mouse lymphoma cells. Photoaffinity labeling of plasma membrane-enriched fractions from nucleoside transport-competent (S49) and nucleoside transport-deficient (AE1) cells with [3H]nitrobenzylthioinosine

Plasma membrane-enriched fractions from disrupted S49 lymphoma cells contained high affinity sites for [3H]nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport. These sites were absent from similar preparations from AE1 cells, a nucleoside-transport deficient clone derived from the S49 cell line. Reversible binding of [3H]nitrobenzylthioinosine to the S49 membrane preparations was inhibited by adenosine, nitrobenzylthioguanosine, and dipyridamole. Exposure of S49 membrane preparations to UV light in the presence of [3H]nitrobenzylthioinosine resulted in the covalent radiolabeling of a membrane protein(s) which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of 45,000 to 66,000. Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine and markedly reduced in the presence of adenosine and dipyridamole. AE1 membrane proteins were not covalently labeled under these conditions.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of binding sites for the transport inhibitor nitrobenzylthioinosine on nucleoside transport-deficient mouse lymphoma cells

Cells of an adenosine-resistant clone (AE₁) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthionisine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2′-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE₁ cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 · 10⁴ sites/cell, K_d  0.2 nM), whereas site-specific binding of NBMPR to AE₁ cells was not demonstrable, indicating that loss of nucleoside transport activity in AE₁ cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE₁ cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine.     

Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.

The interaction of (E)-N-(3-iodoprop-2-enyl)-2beta-Carbomethoxy-3beta-(4'-methylphenyl) nortropane (PE2I) with the rat neuronal dopamine transporter (DAT) was studied in transfected COS cells by measuring its ability to inhibit DA uptake and by measuring its affinity in radioligand binding experiments. Saturable [3H]DA uptake was measured in COS cells transiently transfected with the cDNA sequence encoding the rat DAT. Pharmacological characterisation of this uptake revealed functional properties with a V(max) value of 45.05+/-2.62 pmol/mg protein per min and a K(m) value of 2.86+/-0.28 microM. The specific [3H]DA uptake was fully inhibited by 1 microM PE2I. Concentration response curves revealed the high potency of PE2I in inhibiting DA uptake (pEC(50) value of 8.70+/-0.33), 25 times higher than that observed for the reference DAT inhibitor, GBR 12935. On crude homogenates from transfected COS cells, PE2I displaced the specific binding of [3H]GBR 12935 with a pK(i) value of 7.73+/-0.13. Accordingly, [125I]PE2I was found to specifically recognise a single binding site population which is almost completely displaced by GBR 12935 and nomifensine. Saturation experiments revealed the high affinity of [125I]PE2I (K(D) value of 3.8+/-0.63 nM) that correlates with the high potency of PE2I in inhibiting the [3H]DA uptake. This contrasts with the results obtained with GBR 12935 for which a discrepancy was found between its high affinity in binding assays (K(D) value of 0.43+/-0.04 nM) and its rather low potency in functional assays (pEC(50) value of 7.30+/-0.05). A relatively high level of [3H]GBR 12935 binding was detected in non transfected COS cells. Such nomifensine resistant binding is attributed to the interaction of GBR 12935 with cytochrome P-450 as it was displaced by cis-(Z)-flupentixol (an inhibitor of cytochrome P-450). Such interaction was not observed using PE2I. Taken together, these data demonstrate that PE2I was a highly potent inhibitor of cloned DAT compared with GBR 12935 and provided a useful tool for further investigations in cells transfected with cDNA encoding the DAT.     

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

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

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Bombesin receptor from Swiss 3T3 cells. Affinity chromatography and reconstitution into phospholipid vesicles

Bombesin and its mammalian counterpart gastrin releasing peptide (GRP) are potent mitogens for Swiss 3T3 cells in which distinct high affinity receptors have been identified. We developed here a probe for specific ligand affinity chromatography by coupling biotin to [lys3]bombesin. The resulting biotinylated [lys3]bombesin (BLB) retained biological activity as judged by inhibition of [125I]GRP binding to intact cells and membrane preparations and stimulation of rapid Ca2+ mobilization and DNA synthesis in intact cells. Using this ligand and magnetised beads coated with streptavidin, we extracted differentially a single protein from detergent-solubilized Swiss 3T3 membranes in a BLB-dependent manner. Visualization was achieved either after autoradiograph of metabolically labelled proteins with [35S]methionine or by silver staining of larger preparations. In other experiments, elution of BLB-receptor complexes bound to streptavidin beads was carried out at neutral pH and the eluted fraction was reconstituted into phospholipid vesicles. This procedure revealed [125I]GRP binding activity that exhibited saturability, specificity and a 1946-fold increase in specific activity.     

Interaction of the catecholamine release-inhibitory peptide catestatin (human chromogranin A(352-372)) with the chromaffin cell surface and Torpedo electroplax: implications for nicotinic cholinergic antagonism

The catecholamine release-inhibitory chromogranin A fragment catestatin (chromogranin A(344-364)) exhibits non-competitive antagonism of nicotinic cholinergic signaling in chromaffin cells. A previous homology model of catestatin's likely structure suggested a mode of interaction of the peptide with the nicotinic receptor, but direct evidence has been lacking. Here we found that [125I]-catestatin binds to the surface of intact PC12 and bovine chromaffin cells with high affinity (K(D)=15.2+/-1.53 nM) and specificity (lack of displacement by another [N-terminal] fragment of chromogranin A). Nicotinic agonist (carbamylcholine) did not displace [125I]-catestatin from chromaffin cells, nor did catestatin displace the nicotinic agonist [3H]-epibatidine; these observations indicate a catestatin binding site separate from the agonist binding pocket on the nicotinic receptor, a finding consistent with catestatin's non-competitive nicotinic mechanism. [125I]-catestatin could be displaced from chromaffin cells by substance P (IC(50) approximately 5 microM), though at far lower potency than displacement by catestatin itself (IC(50) approximately 350-380 nM), suggesting that catestatin and substance P occupy an identical or overlapping non-competitive site on the nicotinic receptor, at different affinities (catestatin > substance P). Small, non-peptide non-competitive nicotinic antagonists (hexamethonium or clonidine) did not diminish [125I]-catestatin binding, suggesting distinct non-competitive binding sites on the nicotinic receptor for peptide and non-peptide antagonists. Similar binding and inhibitory profiles for [125I]-catestatin were observed on chromaffin cells as well as nicotinic receptor-enriched Torpedo membranes. Covalent cross-linking of [125I]-catestatin to Torpedo membranes suggested specific contacts of [125I]-catestatin with the delta, gamma, and beta subunits of the nicotinic receptor, a finding consistent with prior homology modeling of the interaction of catestatin with the extracellular face of the nicotinic heteropentamer. We conclude that catestatin occludes the nicotinic cation pore by interacting with multiple nicotinic subunits at the pore vestibule. Such binding provides a physical explanation for non-competitive antagonism of the peptide at the nicotinic receptor.     

4-Iodotomoxetine: a novel ligand for serotonin uptake sites.

The tomoxetine analog, R-4-iodotomoxetine, binds in vitro to a single site of rat cortical membranes with high affinity (Kd = 0.03 +/- 0.01 nM, n = 4) and can be blocked by a selective serotonin reuptake site inhibitor, paroxetine. The [125I]R-4-iodotomoxetine binding at equilibrium is saturable and is temperature- and Na(+)-dependent. The number of specific [125I]R-4-iodotomoxetine binding sites (Bmax = 356 +/- 20 fmol/mg protein) is similar to that of [3H]citalopram (329 +/- 30 fmol/mg protein), a known serotonin uptake inhibitor. The binding of [125I]R-4-iodotomoxetine is selectively inhibited by several serotonin uptake blockers, and a good correlation is demonstrated between the potency of various drugs to inhibit in vitro binding of [125I]R-4-iodotomoxetine and [3H]citalopram. In addition, lesions performed with the neurotoxin p-chloroamphetamine, which destroys monoamine neurons, including serotonergic neuronal system, result in a 90% reduction of [125I]R-4-iodotomoxetine binding when compared to sham controls. These results indicate that the binding sites labeled by [125I]R-4-iodotomoxetine are associated with the neuronal serotonin uptake sites. However, the in vivo and ex vivo results do not show regional localization corresponding to the distribution of serotonin uptake sites. The nonspecific uptake may be related to this compound's high lipophilicity (octanol-buffer partition coefficient = 1100 - 1400 at pH 7). Although the in vivo properties of [125I]R-4-iodotomoxetine make it an unlikely candidate for mapping serotonin uptake sites with SPECT, the high affinity and selectivity should make it a useful tool for in vitro studies of the serotonin uptake sites.