Reduced affinity of iodinated forms of Tyr0 C-type natriuretic peptide for rat natriuretic peptide receptor B

Tyr(O)CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr(O)CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr(O)CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always < 3% of the total binding observed in membranes from cells transfected with receptor expression plasmids. Receptor densities in transfected cells ranged from 500 to 2500 fmol/mg of protein. High performance liquid chromatography and ionspray mass spectrometry analyses revealed that the reagents used in the course of iodination (lactoperoxidase, chloramine T, or N-chloromorpholine altered the structure of Tyr(O)CNP, most likely by changing the thiol of the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr(O)CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability iodinated forms of Tyr(O)CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e. Tyr(O)Met(O)17CNP, 127I-Tyr(O)Met(O)17CNP, and 127I2-Tyr(O)Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr(O)CNP, ANP(99-126), BNP-32, and des[Gin18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr(O)CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr(O)CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr(O)CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.     

Mechanisms of natriuretic-peptide-induced growth inhibition of vascular smooth muscle cells

OBJECTIVE: While natriuretic peptides can inhibit growth of vascular muscle cells (VSMC), controversy exists as to whether this effect is mediated via the guanylate cyclase-coupled receptors, NPR-A and NPR-B, or the clearance receptor, NPR-C. The original aim of this study was to examine the mechanism by which the NPR-C receptor regulates growth. METHODS: Rat VSMC were characterized with regard to natriuretic peptide receptor expression by RT/PCR and radioligand binding studies. The effect on growth following addition of the peptides and the ligands for NPR-C was measured by [3H]thymidine incorporation. Cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay and mitogen activating protein kinase activity was based on the phosphorylation of myelin basic protein. RESULTS: In rat VSMC, passages 4-12, both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) dose-dependently inhibited serum and PDGF-induced VSMC growth. In contrast, NPR-C specific ligands alone had no effect on cell growth but enhanced growth inhibition when co-administered with ANP and CNP. ANP and CNP also decreased PDGF-BB-stimulated MAP kinase activity. Once again, NPR-C specific ligands alone had no effect but enhanced the effects of ANP. Furthermore, a cGMP specific phosphodiesterase inhibitor dose-dependently inhibited VSMC growth and markedly enhanced natriuretic-peptide-induced inhibition at low peptide concentrations. To examine a potential mechanism for the controversy concerning the NPR-C, we investigated the autocrine expression of ANP and CNP by VSMC and found that mRNA encoding both peptides could be detected by RT/PCR. CONCLUSION: Our findings indicate that the guanyl-cyclase-linked receptors mediate the antiproliferative actions of the natriuretic peptides on vascular smooth muscle cell growth. Moreover, we hypothesize that the apparent inhibition of growth by NPR-C specific ligands reported by others may be due to stabilization of natriuretic peptides produced by the cultured VSMC and subsequent action of these peptides at guanyl-cyclase-linked receptors.     

Distribution and characterization of natriuretic peptide receptors in the gills of the spiny dogfish, Squalus acanthias

The distribution and nature of natriuretic peptide receptors (NPR) in the gills of dogfish, Squalus acanthias, were examined by tissue section autoradiography, competition analysis, protein electrophoresis, guanylate cyclase (GC) assays, and molecular cloning. Specific NP binding occurred on the gill filaments, but not on the interbranchial septum or gill arch. The binding was densest on the efferent edge of the gills. Higher resolution light-microscopic examination of emulsion-coated sections showed that specific binding occurred mainly on the secondary lamellae and filament body and not on the arterial circulation. At least two types of NPR were revealed. One is linked to GC since NP binding stimulates the production of cGMP. The GC receptor may be similar to the NPR-B mammalian receptor since only pCNP stimulated cGMP production. The second receptor is not linked to GC and binds the specific ligand C-ANF [rat des(Gln18, Ser19, Gly20, Leu21, Gly22)]. The sequence of a cDNA generated using primers based on conserved regions of vertebrate NPR-C had considerable homology with mammalian and eel NPR-C and eel NPR-D. The presence of GC-linked NPR and NPR-C/ NPR-D suggests that the gills are an important target organ for NP action.     

Receptor-specific ligands distinguish natriuretic peptide receptors-A and -C in primate tissues

Systemic clearance of atrial natriuretic peptide (ANP) is in part due to neutral endopeptidase (NEP) proteolysis and natriuretic peptide receptor-C (NPR-C) mediated endocytosis. Biological responses to ANP are primarily mediated by the membrane guanylyl cyclase-A/natriuretic peptide receptor-A (NPR-A). Analogs of ANP selective for NPR-A and/or resistant to NEP may have increased activity in those tissues where NPR-C and NEP are coexpressed with NPR-A. The analog of ANP termed vANP; [(R3D, G9T, R11S, M12L, G16R)ANP] is selective for human NPR-A with at least 10,000 fold reduction in affinity for human NPR-C. We report that rat NPR-A is insensitive to 10 nM vANP, demonstrating the limitations of this species in evaluating human therapeutic candidates. As an alternative approach we tested the binding and potency of receptor-selective and NEP-resistant ANP analogs in rhesus monkey tissues. Competition binding studies with a simplified version of vANP, sANP [(G9T, R11S, G16R)rANP], in rhesus monkey kidney and lung membrane preparations shows displacement of 125I-ANP from only a fraction of the total ANP receptor population, 30 and 85%, respectively. The remaining ANP binding sites can be occupied with the NPR-C selective ligand cANP(4-23). These data strongly suggest that only two classes of ANP receptor are present in these membrane preparations, NPR-A and NPR-C. The NEP resistant sANP derivative called sANP(TAPR) was 8 fold more potent (ED50 = 0.6 nM) than rANP (ED50 = 5 nM) in stimulating cGMP production in the lung membrane preparation. Our results demonstrate that the rhesus monkey natriuretic peptide receptors reflect the pharmacology of the human receptors, and that this species may be suitable to determine the role of NPR-C and NEP in peptide clearance and attenuating functional responses.     

Production of an atrial natriuretic peptide variant that is specific for type A receptor

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.     

Cloning, properties and tissue distribution of natriuretic peptide receptor-A of euryhaline eel, Anguilla japonica

During the course of cloning and characterization of natriuretic peptide receptor-A (NPR-A) from the euryhaline fish eel, Anguilla japonica, we identified a splice variant with unique structural properties that affect ligand-inducible intrinsic guanylate cyclase activity. The variant, generated from a splice between a cryptic donor site and the normal acceptor site, lacked nine amino acid residues (VFTKTGYYK) in the kinase-like regulatory domain. This deletion of a very short segment resulted in the complete loss of the ligand inducibility of the cyclase activity. The nine-amino acid segment may therefore be useful as a target for studies aimed at clarifying the mechanism of activation of the guanylate cyclase domain. Characterization of the normal form of eel NPR-A also led to the following interesting findings. Although eel NPR-A had a domain structure very similar to that of mammalian counterparts, it lacked the third cysteine residue in the extracellular domain which is conserved among mammalian NPR-A molecules. The eel receptor bound both amidated and nonamidated eel atrial natriuretic peptide (eANP) with high affinity but, when assayed for ligand-inducible cGMP generation, it responded efficiently only to physiological concentrations of the amidated ligand, suggesting that the biologically active form is the amidated eANP, and the nonamidated form acts as a partial antagonist; similarly, nonhomologous rat ligands behaved like antagonists toward the eel receptor in the concentration range 0.1-10 nm. The receptor message was found to be relatively abundant in the osmoregulatory organs such as the gill, kidney, intestine and urinary bladder.     

Influence of respiratory afferents upon the proprioceptive reflex of skeletal muscles in healthy humans

Localization and gene expression of pituitary adenylate cyclase activating polypeptide receptor (PACAPR) in the rat retina were studied by immunocytochemistry and in situ hybridization, respectively. Antisera were raised against a synthetic peptide that corresponds to the carboxy-terminal cytoplasmic domain which is found in all subtypes of PACAPR. Strong PACAPR mRNA expression and PACAPR-like immunoreactivity (PACAPR-LI) were detected in ganglion cells, amacrine cells, and in the inner plexiform layer. PACAPR-LI appeared to be concentrated predominantly in the neuronal perikarya and processes. At the ultrastructural level, strong immunostaining for PACAPR was visible in the plasma membranes, rough endoplasmic reticulum and cytoplasmic matrix in neurons. This study provides the basis for a better understanding of the functions of PACAP in the rat retina.     

Pneumocystis carinii infection alters GTP-binding proteins in the lung

The GTP-binding regulatory proteins (G proteins) in the membranes of the lung parenchyma from normal, uninfected ferrets were compared to those from immunosuppressed animals with and without Pneumocystis carinii pneumonitis. In lung membranes, pertussis toxin (PT) catalyzed ADP ribosylation of a 41-kDa protein; treatment with cholera toxin (CT) led to ribosylation of a 44-kDa polypeptide. Compared to that in the normal ferrets, the level of the 44-kDa protein was dramatically suppressed in the P. carinii-infected animals. Western blot analysis with specific antibodies to alpha s (which recognized CT substrates), alpha common (which reacted to PT substrates), the alpha q/11 epitope, and the beta subunit demonstrated that these proteins were decreased in animals with P. carinii pneumonitis (PCP). Western blotting of PCP-free membranes with a pan-Ras antibody revealed a 21-kDa polypeptide (corresponding to small G proteins). The level of the 21-kDa protein in membranes from PCP-affected animals was only 30% of that in membranes from PCP-free animals. Finally, analogous studies performed with rat lung membranes revealed similar findings. These data suggest that, independent of its exacerbation of immunosuppression, PCP leads to extensive changes in the GTP-binding proteins in the lung.     

Interaction of beta-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes

Two H+/peptide cotransporters, PEPT1 and PEPT2, are expressed in the kidney, mediating the renal tubular reabsorption of oligopeptides and beta-lactam antibiotics. We examined the interactions of beta-lactam antibiotics with peptide transporters in rat renal brush-border membranes by evaluating the inhibitory potencies of the antibiotics against glycylsarcosine transport. Western blot analysis revealed that PEPT1 and PEPT2 were expressed in the renal brush-border membranes with the apparent molecular masses of 75 and 105 kDa, respectively. Using renal brush-border membrane vesicles, the uphill transport of glycylsarcosine was observed in the presence of an inward H+ gradient and an inside-negative membrane potential. Two transport systems with high affinity (Km of 50 microM) and low affinity (Km of 1.2 mM) appeared kinetically to mediate the glycylsarcosine uptake. The inhibition constants of the antibiotics for glycylsarcosine transport were more closely correlated with those in stable LLC-PK1 cells transfected with rat PEPT2 rather than PEPT1 cDNA. The beta-lactam antibiotics with an alpha-amino group showed trans-stimulation effects on the glycylsarcosine uptake, suggesting that these antibiotics and glycylsarcosine share a common peptide transporter. However, the antibiotics lacking an alpha-amino group failed to show the trans-stimulation effect. It is concluded that amino-beta-lactam antibiotics at therapeutic concentrations interact predominantly with PEPT2 localized in the brush-border membranes of rat kidney.     

Surgical pearl: Dermal advancement flaps for filling deep dorsal nasal defects under grafts

Endocytotic vesicles (EV) isolated from rat renal cortex were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. A monoclonal antibody against human type-1 porin (31 kDa) detected a strong band of 31 kDa. The same antibody has been used as the primary antibody in indirect immunocytochemistry. Light microscopy of cryostat sections of rat renal cortex showed a heavy staining of EV underneath the brush-border membrane. Electron microscopy was performed by "preembedding immunogold staining" of rat renal cortex, the sections of which showed an extensive labelling of EV with gold particles. These results demonstrate that the expression of type-1 porin is not restricted to outer mitochondrial membranes. The biological function of endosomal type-1 porin has as yet to be ascertained.     

Streptokinase contains two independent plasminogen-binding sites

Streptokinase (SK) exerts its thrombolytic effect by activating plasminogen (PG) indirectly, after the formation of an equimolar complex with either PG or plasmin (PN). The location and nature of the PG/PN-binding sites in SK have been explored using limited proteolysis with immobilized trypsin. Employing Western blotting with radiolabeled PG after SDS-PAGE of total tryptic digest, three fragments of MW 7 kD, 19 kD and 31 kD were found to possess PG-binding ability. Each of these fragments was then isolated by reverse phase HPLC and characterised with respect to its sequence, as well as its PG-binding properties by ELISA. These analyses revealed that in addition to a PG-binding site in the region 143-293 reported recently in the literature, there is another distinct, high-affinity and independent PG-binding site, located in the N-terminal region (residues 1-59) of SK. Using a synthetic peptide, the N-terminally located PG-binding-site has been further localised to the region 37-51 of SK. Further, we demonstrate that the PG-binding of this peptide is not mediated through the lysine-binding sites ("Kringles") of PG. This stretch contains a short sequence (LTSRPA) that is also present in the PG-binding domain of human fibronectin.     

Localization of ROMK channels in the rat kidney

Renal potassium secretion occurs in the distal segments of the nephron through apically located secretory potassium (SK) channels. SK may correspond to the ROMK channels cloned from rat kidney. In this study, the localization of ROMK at the cellular level in the rat kidney was examined using an affinity-purified polyclonal antibody raised against a C-terminal peptide of ROMK. The specificity of the antibody was demonstrated by immunoblots of membranes of Xenopus oocytes expressing ROMK2. Immunoblots of homogenates from rat renal outer medulla and cortex revealed predominant bands of 70 to 75 kD, which were ablated by preadsorption with an excess of peptide. These bands were specific for the rat kidney. Immunolocalization studies revealed that ROMK is expressed in specific nephron segments in both the cortex and medulla. In the cortex, ROMK was found in the apical domain of the thick ascending limb of Henle's loop, the connecting tubule, and in some, but not all, cells of cortical collecting tubules. In the medulla, expression in the apical membrane of the thick ascending limbs of Henle's loop was strong, whereas outer medullary collecting ducts were weakly stained. Expression in the thick ascending limb was also heterogeneous; some cells that expressed the Na-K-Cl cotransporter were weakly stained with the anti-ROMK antibody. No staining of glomeruli, proximal tubules, or inner medullary collecting ducts was found. The localization of ROMK agrees well with the findings of SK in patch-clamp studies and supports the view that ROMK is the SK channel of the distal segments of the nephron.     

Olfactory receptor proteins. Expression, characterization and partial purification

A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation was a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in vitro, followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.     

Intracellular third loop domain of angiotensin II type-2 receptor. Role in mediating signal transduction and cellular function

The present study tests the hypothesis that the unique intracellular third loop domain of angiotensin II type-2 (AT2) receptor is essential for the subsequent intracellular signaling and plays an important role in mediating receptor function. Synthetic intracellular third loop peptide of the AT2 receptor (AT2-3LP, 22 amino acids) and control peptide consisting of the same amino acid composition in random sequence were delivered into adult rat aortic vascular smooth muscle cells by cationic liposome-mediated transfection. Successful intracellular peptide delivery was confirmed by microscopic localization of the fluorescein-labeled AT2-3LP within the cells and also by co-immunoprecipitation of the 125I-labeled 3LP complexed with Gi protein using anti-Gialpha antibody. The AT2-3LP-transfected cells showed reduction of serum-stimulated DNA synthesis and cell proliferation as well as a decrease in mitogen-activated protein kinase activity, simulating the effects of AT2 receptor stimulation. The antagonistic effect of the AT2-3LP on mitogen-activated protein kinase activity and DNA synthesis were reversed by pertussis toxin and sodium orthovanadate. Thus, our data suggest that the intracellular third loop domain of the AT2 receptor is closely linked with the cellular signaling pathways of vascular smooth muscle cells in which Gi and protein-phosphotyrosine phosphatase are involved, resulting in the alteration of mitogen-activated protein kinase activity and in growth inhibition.     

Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.     

A synthetic peptide corresponding to the third cytoplasmic loop (residues 533 to 555) of the testicular follicle-stimulating hormone receptor affects signal transduction in rat testis membranes and in intact cultured rat Sertoli cells

Involvement of the third cytoplasmic (3i) loop (residues 533 to 555) of the rat testicular FSH receptor in the mechanism of FSH signal transduction was examined using light membranes prepared from immature rat testes, monolayer cultures of rat Sertoli cells, and a synthetic peptide strategy. This region of the FSH receptor is structurally related to G protein-activator regions identified in other G protein-coupled receptors. FSHR-(533-555) peptide amide stimulated guanine nucleotide exchange in rat testis light membranes, presumably via its interaction with membrane-associated G protein. The peptide failed to inhibit FSH binding to testis membrane receptors, indicating that the nucleotide exchange effect was not a result of peptide interaction with receptor. When incubated with cultured Sertoli cells from immature rat testes, FSHR-(533-555) peptide amide consistently and significantly inhibited FSH stimulation of cAMP and estradiol biosynthesis, but failed to inhibit forskolin stimulation of each. The peptide effect, therefore, was not due to a direct interaction with adenylyl cyclase. Since FSHR-(533-555) peptide amide did not inhibit FSH binding to membrane receptor, these results imply entry of the peptide into the Sertoli cell, possibly by vesicular internalization or diffusion. Indeed, the inhibitory effects of FSHR-(533-555) peptide amide on FSH-stimulated estradiol biosynthesis were prevented by pretreating Sertoli cells with phenylarsine oxide, an inhibitor of FSH receptor internalization. FSHR-(533-555) was without effect on basal levels of cAMP and estradiol biosynthesis, indicating absence of toxicity at the concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid pIP501 encoded transcriptional repressor CopR binds to its target DNA as a dimer

The Y1 receptor, which belongs to the family of rhodopsin-like GTP-binding protein-coupled, seven-transmembrane helix-spanning receptors, binds the 36-mer neuromodulator neuropeptide Y (NPY) with nanomolar affinity. Synthetic fragments of the N-terminus, extracellular loops and C-terminus of the Y1 receptor were used to generate 18 anti-receptor antibodies; ten of them recognize the receptor expressed on intact cells as well as on membranes that have been prepared (with the exception of one antibody raised against the intracellular C-terminus) as investigated by ELISA. SDS/PAGE of solubilized membranes, subsequent Western blotting and staining with the antibodies revealed two proteins of 73 kDa and 51 kDa for both, the rat and the human receptor. Competition with neuropeptide Y showed that the binding of seven antibodies is strongly inhibited in the presence of the native ligand. Using photoactivatible analogues, it could be demonstrated that the competition efficiency strongly depends on the position of the crosslinker within the ligand. Based on these studies, a model for the ligand-receptor interaction is suggested. These antibodies represent novel tools for the structural characterization of the Y1 receptor and its interaction with NPY and antagonists as well as for localization studies.