HS-142-1, a novel polysaccharide of microbial origin, specifically recognizes guanylyl cyclase-linked ANP receptor in rat glomeruli.

HS-142-1, a novel polysaccharide, of microbial origin had been characterized as a specific antagonist of guanylyl cyclase-linked atrial natriuretic peptide (ANP) receptors (ANP-GC receptor) in bovine adrenal cortex. The effect of HS-142-1 on ANP receptors of rat glomeruli were examined. HS-142-1 blocked rat ANP (r-ANP)-stimulated cGMP production in a concentration-dependent manner, although it caused only slight inhibition in the specific binding of [125I]-rANP to the glomeruli where only a small portion of the binding sites are coupled to guanylyl cyclase. HS-142-1 recognized the 135K ANP receptor which is thought to be ANP-GC receptors but did not recognized 60K receptor, guanylyl cyclase-free type from affinity cross-linking studies with glomerular membranes. These results indicate that HS-142-1 is a specific antagonist for the ANP-GC receptor in rat glomeruli, and that it will be a powerful tool for understanding the physiological roles of ANP in renal responses.     

Mutagenicity of Isothiocyanates,Isocyanates and Thioureas on Salmonella typhimurium

During the screening program for atrial natriuretic peptide (ANP) receptor ligands of microbial origin, we isolated a novel nonpeptide ANP antagonist, HS-142-1, from a culture broth of Aureo-basidium pullulans var. melanigenum. Structural analysis showed that HS-142-1 was composed of 20–30 kinds of β-1,6-glucan esterified by caproyl groups; each component had an almost equal potency. HS-142-1 inhibited [¹²⁵I]-rANP binding to its receptor in rabbit kidney cortex membranes with an IC₅₀ of 0.3μg/ml and antagonized ANP-induced cGMP production by bovine lung membranes in a dose-dependent fashion. The discovery of this nonpeptide ANP antagonist, HS-142-1, will provide a useful tool to study the physiological significance of natriuretic peptide system.     

Evidence for vasoactive intestinal peptide receptors in apical membranes from tracheal epithelium

[125I]VIP (vasoactive intestinal peptide) bound to apical membranes isolated from the bovine tracheal epithelium with a half maximal inhibition by unlabeled VIP (IC50) of 0.6 x 10(-9)M and binding was reversible. Glucagon did not affect [125I]VIP binding to the membranes. [125I]VIP was covalently cross-linked to tracheal membrane proteins using disuccinimidyl suberate. SDS-polyacrylamide gel electrophoresis of labeled tracheal membranes revealed one major [125I]-receptor complex of Mr = 71,000 to which binding of [125I]VIP was inhibited by 10 microM unlabeled VIP. These results are consistent with the presence of a specific, high-affinity receptor for VIP, with a Mr = 71,000, in apical membrane vesicles isolated from the bovine tracheal epithelium.     

Stable expression of natriuretic peptide receptors: effects of HS-142-1, a non-peptide ANP antagonist.

We established clonal cell lines stably expressing each of two subtypes of membrane bound guanylate cyclases (GC-A and GC-B), which are known as natriuretic peptide receptors. Using these cell lines, we showed that GC-A is an ANP/BNP receptor, whereas GC-B is a specific receptor for CNP. Effects of HS-142-1, a novel non-peptide ANP antagonist, on GC-A and GC-B were examined by using these cells. In cells expressing either GC-A or GC-B, HS-142-1 inhibited cGMP production elicited by ANP or CNP with IC50 values of 1.8 micrograms/ml and 1.5 micrograms/ml, respectively, and also competitively blocked specific binding of the natriuretic peptides with IC50 values of 2.2 micrograms/ml and 3.3 micrograms/ml, respectively. These results indicate that HS-142-1 is a potent antagonist of CNP as well as ANP. We also showed that CNP suppressed the growth of cells expressing GC-B by 22% and that HS-142-1 blocked the antiproliferative action of CNP.     

HS-142-1, a Novel Non-peptide Antagonist for Atrial Natriuretic Peptide Receptor,Selectively Inhibits Particulate Guanylyl Cyclase and Lowers Cyclic GMP in LLC-PK1 Cells

HS-142-1, a novel atrial natriuretic peptide (ANP) antagonist isolated from the culture broth of Aureobasidium sp., selectively inhibits ANP-induced cyclic GMP accumulation in porcine kidney epithelial LLC-PK₁ cells. At concentrations from 0.1 to 100 μg/ml (= 2.5 × 10^–8 – 2.5 × 10^–5 M, given the mean molecular weight is 4, 000), HS-142-1 prevents intracellular cyclic GMP accumulation initiated by 10^–8 M rat ANP in a dose-dependent manner, but not cyclic GMP accumulation produced by 10^–5 M sodium nitroprusside. HS–142–1 alone has no effects on the basal level of cyclic GMP seen in the absence of ANP. No change of intracellular cyclic AMP was observed upon the treatment of the cells with HS-142-1. Further, the selectivity of HS-142-1 for the guanylyl cyclase-linked receptor was confirmed by affinity labeling studies with bovine adrenocortical membranes. HS-142-1 specifically abolished the labeling of the guanylyl cyclase-linked 135-kDa band in a dose-dependent manner, but not the labeling of the 60-kDa band not coupled to the guanylyl cyclase. These results show that HS-142-1 selectively inhibits ANP-mediated accumulation of cyclic GMP in LLC-PK₁ cells through interacting with guanylyl cyclase-linked receptors.     

Opioid and non-opioid binding of beta-endorphin to bovine adrenal medullary membranes

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.     

Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

We have recently reported that the highly potent beta-adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM-CYP) irreversibly blocks the turkey erythrocyte beta-adrenoceptor binding site by combining with a receptor-associated non-protein component. In this communication, we report: lipid labelling is inhibited by beta 1-adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1-adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM-CYP-blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM-CYP-glycolipid with restoration of the binding capacity of the beta 1-adrenoceptor protein; the tightly associated [125I]BAM-CYP-labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor-mediated signal transduction is discussed in view of these findings and in view of data from the literature.     

Atrial and brain natriuretic peptide in adrenal steroidogenesis.

We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cyclic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic alpha-human ANP (alpha-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. alpha-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinity labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma alpha-hANP level was 25 +/- 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of alpha-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.     

Characterisation of atrial natriuretic peptide receptors in bovine ventricular sarcolemma

Analysis of [125I]-ANP binding data in an isolated bovine ventricular sarcolemmal membrane fraction revealed a single high affinity binding site (Kd approximately 5 x 10(-11) M). The ring deleted ANP analogue des [QSGLG]-ANP (4-23)-NH2 bound with a 1000-fold lower affinity indicating the absence of C-type receptors in this preparation. ANP stimulated guanylate cyclase activity by up to 2-fold with half-maximal activation at approximately 10(-9) M. Crosslinking [125I]-ANP to its receptor with disuccinimidyl suberate (DSS) revealed two radiolabelled bands of 120 kDa and 65 kDa on non-denaturing SDS-PAGE. Radioactive signals from both bands were lost by reducing the sample with beta-mercaptoethanol prior to electrophoresis, in which case a radioactive fragment of less than 5 kDa migrated with the dye front. These results suggest that the binding of ANP to both high and low molecular weight "receptor" proteins may be associated with the hydrolysis of the peptide.     

Neuropeptide Y receptor in bovine hippocampus is a Y2 receptor.

[125I]NPY bound to a single class of saturable binding sites on bovine hippocampus membranes with a KD of 0.1 mM and Bmax of 165 fmol/mg of protein. The rank order of potency of NPY fragments and other structurally related peptides to inhibit [125I]NPY binding was: PYY greater than or equal to NPY much greater than BPP greater than or equal to APP and NPY greater than NPY-(13-36) greater than NPY-(18-36) greater than or equal to NPY-(20-36) much greater than NPY-(26-36) greater than NPY-(free acid). The identity of the NPY binding site was investigated by affinity labeling. Gel electrophoresis followed by autoradiography revealed a band with a mol mass of 50 kDa. Unlabeled NPY or PYY, but not BPP, HPP and APP, inhibited labeling of [125I]NPY to the 50 kDa protein band. Moreover, labeling was inhibited by NPY greater than NPY-(18-36) greater than or equal to NPY-(13-36) greater than or equal to NPY-(20-36) greater than NPY-(26-36) greater than NPY-(free acid). The binding of [125I]NPY and the intensity of the cross-linked band were reduced in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.7 nM and 0.6 mM, respectively). These studies demonstrate that bovine hippocampal membranes contain a 50 kDa [125I]NPY binding site that has the ligand specificity characteristic of the Y2 receptor subtype.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Identification and characterization of endothelin binding sites in rat renal papillary and glomerular membranes

The present study was designed to identify and characterize specific endothelin binding sites in membranes of rat renal papillae and glomeruli which appear to be target tissues for this new peptide hormone. Saturation binding studies indicate that the sites have a high and uniform affinity. The dissociation constants averaged 662 +/- 151 and 1309 +/- 123 pM and the receptor densities 7666 +/- 920 and 5831 +/- 348 fmol/mg protein for papillary and glomerular membranes, respectively. Endothelin 1, endothelin 3 and sarafotoxin all inhibited [125I]-endothelin binding with IC50's in the 100-300 pM range, whereas unrelated peptides, namely angiotensin II, atrial natriuretic peptide, and platelet-derived growth factor failed to compete for [125I]-endothelin binding. Deletion of the carboxyterminal tryptophan in endothelin 1 reduced its affinity for glomerular binding sites by 2 orders of magnitude. Specific endothelin binding to these membranes was maximal at pH 4 and was markedly inhibited as the pH was raised above 8. When [125I]-endothelin is covalently linked to glomerular membrane binding sites, SDS-PAGE of these solubilized membranes followed by autoradiography reveals a predominant specifically labeled band of 45 kDa. Whether this band represents a subunit of the endothelin receptor(s), the receptor proper, or an intracellular endothelin binding protein remains to be determined.     

Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.     

Photoaffinity labeling of dopamine D1 receptors

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.     

Internalization of atrial natriuretic peptide by adrenal glomerulosa cells

Internalization of 125I-labelled atrial natriuretic peptide ([ 125I]ANP) by rat adrenal glomerulosa cells in vivo was investigated by means of an ultrastructural autoradiographic approach. One to 30 min after IV injection of [125I]ANP, silver grains were found, at the light microscope level, over all glomerulosa cells; coinjection of 20 micrograms of unlabelled ANP inhibited this binding by 64%. At the electron microscope level, the time-course study indicated maximal silver grain densities in plasma membranes 1 min after IV injection; grains were detected in mitochondria (external membranes and matrix) 2 min after injection, with maximal labelling at 15 min. The cytoplasmic matrix was labelled only 30 min after injection. During the time-course, labelling of nuclei, Golgi apparatus, and lysosomes was minimal. The data suggest that after binding to plasma membranes ANP is rapidly internalized and distributed within glomerulosa cells. The association of radioactivity with mitochondria suggests that ANP may have intracellular sites of action complementary to those on plasma membranes.     

beta-Adrenergic receptors in rat brain.

125I-Iodohydroxybenzylpindolol ([125I] IHYP), a potent beta-adrenergic receptor antagonist, has been used to study beta-adrenergic receptors in rat brain. Binding of [125I] IHYP (30 pM) to a membrane fraction min and dissociation took place with a half time of about 16 min. Phentolamine (10(-4) M) decreased non-receptor binding but it had no effect on the binding of [125I] IHYP to beta-adrenergic receptors in cortex, cerebellum or caudate. In the presence of phentolamine specific binding (defined as binding which was blocked by 0.3 muM dl-propranolol) represented 70-85% of total binding. The binding of [125I] IHYP was inhibited by beta-adrenergic agonists and antagonists. d-Stereoisomers were 2-3 orders of magnitude less potent than the corresponding 1-isomers. The denstiy of [125I] IHYP binding sites was studied in membrane fractions from cerebral cortex, cerebellum, and caudate nucleus by means of Scatchard analysis. The K(D) of [125I] IHYP was similar in the three regions studied, and the density of [125I] IHYP binding sites was approximately 50% greater in the cortex and caudate than in the cerebellum. The Hill coefficient for the binding of [125I] IHYP to membranes from cerebral cortex was 1.02. The properties of the binding of [125I] IHYP are similar to those which would be expected of binding to beta-adrenergic receptors in vitro.     

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.     

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 an NPY-Y1 receptor subtype in bovine chromaffin cells

Bovine chromaffin cells have been used in a variety of studies designed to reveal different aspects of neuropeptide Y (NPY) action. Pharmacological data have defined five NPY receptor subtypes, only one of which (Y3) has not been cloned. Some studies with bovine chromaffin cells have concluded that the effects of NPY on this cell type are mediated by the Y3 subtype. Previous work from our laboratory demonstrates that a Y1 subtype mediates the effect of NPY in this tissue. In the current studies we provide further evidence for the existence of the Y1 subtype in bovine chromaffin cells. BIBP3226, the selective Y1 antagonist, potently displaces [125I]NPY from its binding site IC50 = 1.91 x 10(-9) M. Moreover, [125I]BIBP3226 binds to bovine chromaffin cell membranes with high affinity (IC50 = 5.9 x 10(-8) M). Examination of BIBP3226 antagonism of NPY inhibition of forskolin stimulated cyclic AMP accumulation reveals that it is a competitive antagonist with a K(B) similar to the IC50 for [125I]BIBP3226 binding. Northern blot analysis using a porcine cDNA clone for the Y1 subtype demonstrates a 3.5-kb mRNA species in chromaffin cells. These data identify the bovine chromaffin cell NPY receptor as a Y1 subtype.