Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells

In a previous study, we found that atriopeptin I was much weaker (EC50 greater than 500 nM) than atrial natriuretic factor (ANF-(8-33)) (EC50 = 0.3 nM) at increasing cyclic GMP in cultured endothelial cells. In this study, we used the cross-linking reagent disuccinimidyl suberate to investigate whether the differences in activity were due to the presence of multiple ANF receptors. When 98% of the ANF-binding sites on endothelial cells were occupied by tyrosine-atriopeptin I after cross-linking, there was no difference in the concentration-response curve to ANF-(8-33) with regard to cyclic GMP accumulation. In contrast, when 96% of the binding sites were occupied by cross-linked ANF-(8-33), a 60% decrease in the maximal cyclic GMP response was observed after the readdition of ANF-(8-33). These results suggest that ANF-(8-33) is binding to an additional site that atriopeptin I does not effectively bind. Affinity cross-linking of 125I-ANF to intact endothelial cells resulted in the labeling of two sites of Mr approximately 66,000 and approximately 130,000. Approximately 94% of the 125I-ANF binding sites had an Mr approximately 66,000. Labeling of this site was inhibited by both tyrosine-atriopeptin I (KI = 0.9 nM) and ANF-(8-33) (KI = 0.09 nM). Although 0.1 microM tyrosine-atriopeptin (AP I) inhibited labeling of the 66,000-dalton site to nearly the same degree as ANF-(8-33), it produced only a 4-fold increase in cyclic GMP compared to a 400-fold increase with ANF-(8-33). These results suggest that the 66,000-dalton site is not coupled to guanylate cyclase and cyclic GMP formation. Tyrosine-AP I (KI greater than 10 nM) was much weaker at competing for the 130,000-dalton site than ANF-(8-33) (KI = 0.075 nM). Because the EC50 for cyclic GMP stimulation for tyrosine-AP I (greater than 100 nM) and ANF-(8-33) (0.4 nM) is closer to the KI values for the 130,000-dalton protein, this site probably mediates the marked stimulation of cyclic GMP. Our results demonstrate that endothelial cells contain two binding sites for ANF-(8-33) and suggest that only the less abundant site (Mr approximately 130,000) is the receptor coupled to the activation of guanylate cyclase.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Photoaffinity labeling of atrial natriuretic factor receptor in bovine and rat adrenal cortical membranes

Radioiodinated synthetic atrial natriuretic factor (ANF) bound to a single class of high affinity binding sites in the plasma membrane from bovine adrenal cortex with a KD of 7.4 X 10(-10) M. The binding affinities of related peptides showed close parallelism to their potencies in natriuretic and vasorelaxant activities. Incubation of adrenal membranes with radioiodinated 4-azidobenzoyl ANF or a similar derivative of its analogue followed by photolysis resulted in specific radiolabeling of a protein band in SDS gel electrophoresis with an apparent Mr of 124,000 in bovine or Mr of 126,000 in rat, which was abolished by inclusion of unmodified ANF in the incubation. Prevention of the labeling was dependent on the concentration of ANF and was not observed with atriopeptin I or with unrelated peptides, angiotensin II, ACTH or [Arg8] vasopressin. These results indicate specific covalent labeling of ANF-receptor or its subunit by the photoaffinity ligands.     

Association of the atrial natriuretic factor receptor with guanylate cyclase in solubilized rat glomerular membranes

The elution profile of solubilized rat glomerular membranes from a gel filtration column showed two peaks of 125I-ANF (atrial natriuretic factor) binding (367 +/- 21, 156 +/- 12 KDa). Over 85% of the total binding for the extract was in the 367 KDa peak. Guanylate cyclase activity was correlated with 125I-ANF specific binding. ANF activation of guanylate cyclase was also observed. As observed previously with particulate membrane, Scatchard-analysis of ANF binding data with the solubilized extract was consistent with a two-site model. Both affinities (Kd's), 4 pM and 1 nM, are within the range of blood concentrations reported for ANF. These observations suggest that most rat glomerular ANF receptors are large molecular complexes coupled with guanylate cyclase in the 300-350 KDa size range.     

Vascular atrial natriuretic factor receptor subtypes are not independently regulated by atrial peptides

The regulation of the atrial natriuretic factor (ANF) receptor system in cultured rat vascular smooth muscle cells (RVSMC) was examined following long term pretreatment of these cells with rANF99-126 or with any one of a series of truncated and ring-deleted analogs. The latter analogs are reported to bind selectively the ANF-C or clearance receptor. Initial competition binding studies revealed that all analogs examined showed comparable apparent receptor binding affinities (Ki values did not differ by more than 10-fold). In contrast, the extent of interaction of the ANF analogs with the receptor pool coupled to particulate guanylate cyclase (the ANF-B receptor) was much more variable, with some ligands failing to stimulate cGMP production or particulate guanylate cyclase over the concentrations tested. Pretreatment of cells for 24 h with rANF99-126 or any of the truncated analogs that interact with the ANF-B receptor caused a dose- and time-dependent decrease in the number of ANF binding sites (99% of which are uncoupled in RVSMC) without any change in affinity. Examination of the binding activity following pretreatment of the cells with ANF suggested that the observed reduction in 125I-rANF99-126 binding capacity was not because of the retention of the peptide on its receptor. Furthermore, this down-regulation was associated with desensitization of particulate guanylate cyclase resulting in a decreased responsiveness of intracellular cGMP accumulation to ANF. In contrast, however, analogs selective for the ANF-C receptor pool failed to cause down-regulation or desensitization. These findings suggest that ANF-C receptors in RVSMC are not independently down-regulated by selective ligands but that nonselective analogs that down-regulate and desensitize the ANF-B receptor system can by some cooperative mechanism reduce the size of the predominant ANF-C receptor pool in these cells.     

Identification of a receptor for peptides of the bombesin family in Swiss 3T3 cells by affinity cross-linking

The cross-linking agent ethylene glycol-bis(succinimidyl succinate) was used to covalently link 125I-labeled gastrin releasing peptide (125I-GRP) to an Mr 75,000-85,000 surface protein in Swiss 3T3 cells that displays many characteristics of a specific receptor for peptides of the bombesin family. This protein was not present in other cell lines which do not exhibit receptors for bombesin-like peptides. Unlabeled GRP competed for affinity labeling of the Mr 75,000-85,000 protein in a concentration-dependent manner, and other bombesin-related peptides also inhibited the cross-linking of 125I-GRP to this component. In contrast, high concentrations of a variety of other peptide hormones and mitogens had no effect. Affinity labeling of the Mr 75,000-85,000 protein was dependent on the concentration of 125I-GRP and exhibited saturability. 125I-GRP affinity labeling of this protein was also demonstrated by two-dimensional gel electrophoresis. These studies suggest that an Mr 75,000-85,000 surface protein with an isoelectric point of 6.0 to 6.5 is a major component of the receptor for peptides of the bombesin family in Swiss 3T3 cells.     

Characterization of high affinity receptor sites for atrial natriuretic factor in anterior pituitary gland: evidence for the existence of two receptor forms

The present study shows that in rat anterior pituitary tissue atrial natriuretic factor (ANF) binds to two distinct receptor forms, with apparent molecular weights of about 166K and 58K. Binding assays carried out with (125I)-ANF revealed specific and high affinity non-interacting binding sites, with Ko values of 1-1.7 nM and a density of 10-15,000 sites/cell. rANF fragments (5-25), (5-27) and (5-28) exhibited apparent equipotency in displacing tracer binding, while fragment (13-28) and various other peptides were ineffective. ANF (5-25) was about 100-times less potent than ANF (8-33) in stimulating half-maximum pituitary cGMP production. These data indicate the presence of multiple binding sites for ANF in the pituitary gland and suggest that only part of these sites may be coupled to activation of guanylate cyclase.     

Regional vasorelaxant selectivity of atrial natriuretic factor in isolated rabbit vessels

Synthetic atrial natriuretic factor (ANF) exhibited a marked selectivity in its ability to relax isolated rabbit arteries and veins. The aorta, renal and mesenteric arteries and the facial vein were the most sensitive vessels with the more distal arteries and most veins being relatively unresponsive to ANF. All preparations were effectively relaxed by sodium nitroprusside. ANF (up to 10(-7) M) failed to elicit any effect on isolated rabbit right atria or papillary muscles. The profound regional vasorelaxant selectivity of ANF may help to explain the hemodynamic effects of this substance in vivo.     

Identification of the V1 vasopressin receptor by chemical cross-linking and ligand affinity blotting

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased glomerular atrial natriuretic peptide receptor affinity in experimental nephrotic syndrome.

Atrial natriuretic peptide (ANP) is a cardiac hormone with natriuretic and diuretic effects. To better define the ANP hormonal system in the nephrotic syndrome, a condition associated with renal sodium retention, we undertook a study of glomerular ANP receptors in rats with puromycin aminonucleoside-induced nephrotic syndrome and in pair-fed controls. Nephrotic rats had significantly decreased serum albumin and total protein and significantly increased serum cholesterol, triglycerides and 24 hour urinary protein excretion. Plasma level of atrial natriuretic peptide was similar in both groups of rats. Competition binding inhibition studies in isolated glomeruli demonstrated one binding site in both groups of rats. The density of ANP binding sites in isolated glomeruli was similar in nephrotic and pair-fed rats while the binding affinity was increased significantly in the nephrotic rats. This is the first study to demonstrate alterations in renal ANP receptors in the nephrotic syndrome. Further studies will be necessary to determine whether alterations in glomerular ANP receptors contribute to renal sodium retention in the nephrotic syndrome.     

Multiple forms of atrial natriuretic factor receptor in human placenta

Multiple forms of atrial natriuretic factor receptor have been identified in human placental membranes. Atrial natriuretic factor binds specifically to placental membranes and the binding activity could be solubilized using non ionic detergent, Triton X-100. Binding to the detergent solubilized preparation was inhibited 80% by the addition of 0.5 M sodium chloride. Affinity cross-linking analysis indicated that this binding was associated with a single protein band of molecular weight 170-kDa. On the other hand, if sodium chloride was added together with a chelator, o-phenanthroline, ANF binding to this preparation was stimulated 300%. Binding under these conditions was not to the 170-kDa protein but was associated with a broad band in the region of 100/110-kDa and a minor band at 200-kDa. These observations clearly indicated that in human placental membranes, atrial natriuretic factor binds to distinctly different molecular species depending on the presence or absence of certain ions and chelators. The two types of binding could be conveniently assayed in the presence of each other by elimination or inclusion of sodium chloride and o-phenanthroline in the assay system.     

Functional heterogeneity of atrial natriuretic factor receptor in bovine adrenal zona glomerulosa is explained by an amiloride-sensitive high affinity molecular complex

The effects of amiloride on the molecular characteristics of the atrial natriuretic factor (ANF) receptor from bovine adrenal zona glomerulosa were studied by computer modeling of competitive binding data, by affinity labeling experiments, and by steric exclusion high performance liquid chromatography of solubilized receptor. The order of potency of a series of truncated ANF analogs in competing for 125I-ANF binding to bovine adrenal zona glomerulosa membranes was the same as that obtained for inhibition of aldosterone secretion. Deletion of amino acids at the COOH-terminal end drastically reduced the affinities of the peptides. Computer analysis of competition curves revealed that all ANF analogs tested show similar binding characteristics: shallow competition curves, discrimination of varying proportions of high and low affinity binding states, and sensitivity to amiloride which increases the proportion of the high affinity binding component. These results from binding studies are suggestive of potential heterogeneity of ANF binding sites. In contrast, results from affinity cross-linking experiments are consistent with the notion of a single receptor protein. Incubation of membranes with increasing concentrations of 125I-ANF-(99-126) up to 3 nM resulted in the labeling of a single band of Mr 130,000. The ability of ANF analogs to compete for the labeling of the Mr 130,000 band by 125I-ANF-(99-126) agreed well with their potency as inhibitors of 125I-ANF binding to intact membranes. Addition of amiloride caused a dose-dependent increase in the labeling of the Mr 130,000 band. A single Mr 130,000 band was also labeled in bovine aorta and LLC-PK1 cell membranes. In order to further investigate the molecular basis for the apparent heterogeneity of ANF binding we have prelabeled the membrane receptor with 125I-ANF-(99-126) prior to solubilization with octyl-beta-D-glucoside and chromatography on a Superose 6 steric exclusion column. The elution profile of the prelabeled receptor consistently showed two peaks of radioactivity with mean Stokes radii of 70 and 50 A. When amiloride was added to the incubation medium, the elution profile consisted almost exclusively of the 70-A peak. Quantitative analysis of the chromatographic profiles revealed that amiloride increases by 2-3 times the area of the 70-A peak. We conclude that the 70-A form represents a ternary complex of the receptor with an amiloride-sensitive effector protein.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Identification of an atrial natriuretic peptide specific receptor in human kidney

A specific receptor for human atrial natriuretic peptide (h-ANP) was identified in the human kidney using the radioligand binding assay. Samples were prepared from non-malignant renal tissues obtained at nephrectomy of patients with renal carcinoma. Binding studies using [125I]hANP were performed at 0 degree C for 20 minutes and terminated by a rapid filtration technique. Scatchard plot analysis revealed [125I]hANP bound to a single class of binding site (Kd = 0.4 +/- 0.2 nM) with a density of 16 +/- 4 fmol/mg protein in the renal cortex (n = 7). The binding was rapid and maximal binding was obtained within 20 minutes after the start of incubation. Radioligand displacement was observed in a dose dependent fashion when cold hANP was entered into the reaction mixture. However, unrelated agents, such as angiotensin II or 1-epinephrine, did not affect the binding. This is the first time characterization of the hANP receptor in the human kidney has been conducted using a Scatchard plot analysis.     

Identification and solubilization of atrial natriuretic factor receptors in human placenta

Specific high affinity 125I-atrial natriuretic factor binding sites have been identified in human placental membranes. Using the nonionic detergent, Triton X-100, these binding sites were quantitatively solubilized and retained binding activity. In the solubilized preparation, the macromolecular component that binds atrial natriuretic factor is a 160,000 dalton protein as shown by covalently cross-linking it to 125I-atrial natriuretic factor with the bifunctional chemical crosslinker, disuccinimidyl suberate, followed by gel electrophoresis and autoradiography. On Sephadex G-200 gel filtration in the presence of detergent, the hormone-receptor complex elutes in the molecular weight range of 140,000. These observations suggest strongly that a 140- 160,000 dalton protein present in human placental membranes is the receptor for specific recognition of atrial natriuretic factor.     

Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling

Two homobifunctional cross-linking reagents have been used to cross-link 125I-platelet-derived growth factor (PDGF) to a cell surface component with an approximate Mr = 164,000 that has many characteristics of a specific PDGF receptor. Excess unlabeled PDGF competed for labeling of this component, while high concentrations of fibroblast growth factor, insulin, epidermal growth factor, low density lipoprotein or acetylated low density lipoprotein had no effect. Preincubation of cells with 125I-PDGF at 37 degrees C reduced specific 125I-PDGF binding (down regulation) and produced a parallel decrease in the amount of the 164,000-dalton receptor available for labeling. The 164,000-dalton component contains at least some protein that is accessible to trypsin in the extracellular medium. A complex of comparable Mr is seen on all PDGF-responsive cell types examined, but not on a nonresponsive cell type. 125I-PDGF does not become covalently cross-linked to this component in the absence of a cross-linking reagent.     

Stimulation of protein phosphorylation by atrial natriuretic factor in plasma membranes of bovine adrenal cortical cells

Atrial natriuretic factor (ANF) rapidly enhanced phosphorylation of plasma membrane proteins of bovine adrenal cortical cells. Pretreatment of the membranes with ANF (1 x 10(-8)M to 1 x 10(-7)M) resulted two- to four-fold in an incorporation of 32p-radioactivity from [gamma -32p]ATP as compared to the controls. The guanosine 3', 5' monophosphate (cGMP) which has been considered a second messenger of ANF also enhanced the phosphorylation of several proteins which were stimulated by ANF. However, the phosphorylation of certain proteins was stimulated differentially only by either ANF or cGMP. These results suggest that ANF-induced protein phosphorylation may play a role in transmembrane signalling pathway involving other second messenger(s) in addition to cGMP during the biological action of ANF.     

Radioreceptor assay for atrial natriuretic factor

Interest in accurate measurement of atrial natriuretic factor (ANF) in biological fluids and various tissues has been stimulated by recent data indicating the possible role of ANF in the homeostasis of salt and water. The presence of high-affinity binding sites for ANF in rat glomeruli has allowed us to develop a rapid, sensitive, and simple radioreceptor assay (RRA). A saturable high-affinity binding site on the membranes of rat glomeruli has been characterized by a dissociation constant of 33 pM and binding capacity of 396 fmol/mg protein. Rat plasma extracts or atrial homogenates or standards were incubated with radioiodinated ANF and a preparation of rat glomerular membranes. The receptor-bound and free radioactivity were separated by filtration on Whatman GF/C paper after 1 h incubation at room temperature. The sensitivity of the RRA was 2.08 fmol. The effective concentration of standard ANF that displaced 50% of labeled receptor-bound ANF (EC50) was 43.3 +/- 2.6 fmol/ml (n = 7). Both intra- and interassay coefficients of variation were smaller than 11%. This RRA assay has been compared with radioimmunoassay (RIA). High correlations for 19 plasma extracts and 34 atrial homogenates (r = 0.973 and r = 0.954, respectively) tested by RRA and RIA were obtained. This good correlation between the two methods suggests that the immunoreactive material found in rat plasma and atrial homogenates also displays biological activity.     

Identification by cross-linking of a beta-bungarotoxin binding polypeptide in chick brain membranes.

beta-Bungarotoxin (beta-BTX) is a snake venom neurotoxin which inhibits neurotransmitter release from different types of nerve terminals. To identify presynaptic membrane components potentially important in neurosecretion, 125I-labeled beta-BTX (mol. wt. 21 000) was cross-linked to a high-affinity binding site in synaptic membrane fractions of chick brain using the photoactivable cross-linker N-succinimidyl-6(4'-azido-2'-nitrophenylamino)-hexanoate. Electrophoretic analysis of the cross-linked membrane proteins under both reducing and non-reducing conditions revealed a single [125I]beta-BTX-polypeptide adduct of apparent mol. wt. 116 000 (+/- 2000). The labeling of this band was prevented under conditions previously shown to inhibit the binding of [125I]beta-BTX to its high-affinity binding site. It is concluded that the cross-linking procedure identified a polypeptide of the presynaptic binding site for beta-BTX, and that this polypeptide has a mol. wt. of 95 000.