Atrial Natriuretic Peptide Binding Sites Research Articles

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 +/- 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4-23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.

Paradoxical antagonism of PACAP receptor signaling by VIP in Xenopus oocytes via the type-C natriuretic peptide receptor

Atrial natriuretic peptide (ANP) and the closely-related peptides BNP and CNP are highly conserved cardiovascular hormones. They bind to single transmembrane-spanning receptors, triggering receptor-intrinsic guanylyl cyclase activity. The “truncated” type-C natriuretic peptide receptor (NPR-C) has long been called a clearance receptor because it lacks the intracellular guanylyl cyclase domain, though data suggest it might negatively couple to adenylyl cyclase via G i. Here we report the molecular cloning and characterization of the Xenopus laevis type-C natriuretic peptide receptor ( XNPR-C). Analysis confirms the presence of a short intracellular C-terminus, as well as a high similarity to fish and mammalian NPR-C. Injection of XNPR-C mRNA into Xenopus oocytes resulted in expression of high affinity [ 125I]ANP binding sites that were competitively and completely displaced by natriuretic analogs and the unrelated neuropeptide vasoactive intestinal peptide (VIP). Measurement of cAMP levels in mRNA-injected oocytes revealed that XNPR-C is negatively coupled to adenylyl cyclase in a pertussis toxin-sensitive manner. When XNPR-C was co-expressed with PAC 1 receptors for pituitary adenylyl cyclase-activating polypeptide (PACAP), VIP and natriuretic peptides counteracted the cAMP induction by PACAP. These results suggest that VIP and natriuretic peptides can potentially modulate the action of PACAP in cells where these receptors are co-expressed.

Natriuretic Peptide Receptor A Activation Stabilizes a Membrane-distal Dimer Interface

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA(W74C) mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA(W74C). Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA(W74C). The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.

Effects of dehydration on endocrine regulation of the electrolyte and fluid balance and atrial natriuretic peptide-binding sites in perinatally malnourished adult male rats.

The first aim of this work was to investigate, under basal conditions in adult male rats, the long-term consequences of perinatal maternal food restriction on the plasma concentrations of vasopressin (VP), aldosterone and atrial natriuretic peptide (ANP) and on plasma renin activity (PRA). Furthermore, under these same conditions, the hypothalamic VP gene expression as well as the density (B(max)), affinity (K(d)) and gene expression of ANP receptors were determined in kidneys and adrenals. The second aim of this work was to examine the responsiveness to dehydration in perinatally malnourished rats. Thus, the latter parameters were studied in these rats after 72 h water deprivation. This study was conducted on 4-Month-old male rats from mothers exposed to 50% food restriction (FR50) during the last week of gestation and lactation and on age-matched control animals (C). At this stage, both C and FR50 rats were killed by decapitation between 0900 h and 1000 h in order to determine parameters under basal conditions or after 72 h water deprivation. Plasma VP, ANP and aldosterone levels and PRA were determined by radioimmunoassay. Hypothalamic VP gene expression was determined in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization. The B(max) and K(d) values of ANP receptors were evaluated from Scatchard plots. ANP receptor gene expression was determined by Northern blot analysis. Under basal conditions, perinatal malnutrition reduced body weight, absolute weight of kidneys and adrenals, and haematocrit. Compared with control rats, FR50 rats had significantly greater plasma VP and aldosterone levels but PRA, plasma ANP levels, plasma osmolality and hypothalamic VP gene expression were not significantly different. Perinatal malnutrition did not significantly affect glomerular ANP receptor density, but in adrenals it decreased both B(max) and K(d) values of ANP-B receptors (biological receptors) and increased B(max) of ANP-C receptors (clearance receptors). ANP-B(A) (receptor subtype A of ANP-B receptors) receptor gene expression was not significantly affected, whereas ANP-C receptor gene expression was enhanced in both adrenals and kidneys in FR50 rats. After 72 h dehydration, control rats showed a significant rise in haematocrit, plasma osmolality, PRA, circulating levels of VP and aldosterone and VP gene expression in both PVN and SON but showed a decrease in plasma ANP levels. B(max) of ANP-B receptors was decreased whereas B(max) of ANP-C receptors was enhanced in both adrenals and kidneys. ANP-B(A) receptor gene expression was not significantly affected in either kidneys or adrenals in dehydrated control rats. Similarly, ANP-C receptor gene expression was unaffected in kidneys whereas it was significantly enhanced in adrenals. In FR50 rats, the effects of water deprivation were qualitatively similar to those reported in controls concerning plasma parameters except for plasma VP levels which tended to rise (not significant) but this increase was only very slight compared with controls. Moreover, unlike controls, VP gene expression in both PVN and SON was not enhanced after dehydration in FR50 rats. In kidneys, dehydrated FR50 rats, like controls, upregulated ANP-C receptors, but they were unable to downregulate ANP-B receptors. In adrenals, unlike controls, FR50 rats enhanced ANP-B receptor density whereas they decreased both ANP-C receptor density and expression after 72 h dehydration. Similar to controls, the expression of ANP-B(A) receptors in both kidneys and adrenals as well as the expression of ANP-C receptors in kidneys, were unaffected in dehydrated FR50 rats. Perinatal malnutrition had long-lasting effects on regulation of the fluid and electrolyte balance under basal conditions. The main effects were a significant rise in circulating levels of VP and aldosterone, and changes in density of adrenal ANP-binding sites and ANP-C receptor gene expression in both adrenals and kidneys. Perinatal malnutrition also affects the responsiveness to water deprivation with alterations in both hypothalamic VP gene expression and regulation of ANP-binding sites.

Chronic hyperosmolality enhances ANP-dependent cGMP production via stimulation of transcription and protein synthesis in cultured rat IMCD cells.

Recently, we found that hyperosmolality acutely inhibited atrial natriuretic peptide (ANP) dependent cGMP production by reducing ANP binding sites in cultured rat inner medullary collecting duct (IMCD) cells. Therefore, the present study was undertaken to evaluate the chronic effect of hyperosmolality on ANP dependent cGMPproduction in IMCD cells. Cell culture was carried out either in an iso-osmotic or hyperosmotic solution consisting of equi-isomolar NaCl and/or urea. Incubations with ANP and/or other agents were performed under the same osmotic conditions. ANP or SNP stimulated cGMP production was enhanced in a chronically hyperosmotic medium. These changes occurred in an osmolality-dependent manner. Hyperosmolality with sodium alone or with sodium and urea, but not with urea alone, was effective for the enhancement of ANP action. There was no significant difference between 125I-ANP specific bindings under iso-osmotic and hyperosmotic conditions. Incubation with cytoskeleton modulators did not affect ANP-dependent cGMP production stimulated by hyperosmolality. On the other hand, both actinomycin D, an inhibitor of transcription, and cycloheximide, an inhibitor of protein synthesis, prevented the stimulatory effects of hyperosmolality. The results suggest that chronic hyperosmolality enhances ANP-dependent cGMP production via stimulation of transcription and protein synthesis in cultured rat IMCD cells.

Developmental modulation of specific receptor for atrial natriuretic peptide in the rat heart

Although cardiac distribution of specific receptors for atrial natriuretic peptide (ANP) was mainly observed in the ventricular endocardium, the modulation of ANP receptors in relation to cardiac development is not defined. The present study was undertaken to investigate ANP receptor modulation in rat during development. In the developmental stages examined (fetus, after postnatal 3‐days, 1‐, 2‐, 3‐, 4‐, and 8‐week‐old Sprague Dawley rats) specific ANP binding sites were localized in the right and left ventricular endocardia by quantitative in vitro receptor autoradiography using 125I‐rat ANP as labeled ligand. The specific bindings to endocardium were much higher in the right than the left ventricle. The binding affinities of ANP were much higher in the right than the left ventricular endocardium. The difference of these binding affinities among various developmental stages was not observed in the right ventricle, whereas the binding affinity in left ventricle was gradually increased with aging and reach...

Differential responses of newborn pulmonary arteries and veins to atrial and C-type natriuretic peptides.

Atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are important dilators of the pulmonary circulation during the perinatal period. We compared the responses of pulmonary arteries (PA) and veins (PV) of newborn lambs to these peptides. ANP caused a greater relaxation of PA than of PV, and CNP caused a greater relaxation of PV than of PA. RIA showed that ANP induced a greater increase in cGMP content of PA than CNP. In PV, ANP and CNP caused a similar moderate increase in cGMP content. Receptor binding study showed more specific binding sites for ANP than for CNP in PA and more for CNP than for ANP in PV. Relative quantitative RT-PCR for natriuretic peptide receptor A (NPR-A) and B (NPR-B) mRNAs show that, in PA, NPR-A mRNA is more prevalent than NPR-B mRNA, whereas, in PV, NPR-B mRNA is more prevalent than NPR-A mRNA. In conclusion, in the pulmonary circulation, arteries are the major site of action for ANP, and veins are the major site for CNP. Furthermore, the differences in receptor abundance and the involvement of a cGMP-independent mechanism may contribute to the heterogeneous effects of the natriuretic peptides in PA and PV of newborn lambs.

Immunoreactive atrial natriuretic peptide and autoradiographic distribution of atrial natriuretic peptide binding sites in the brain of the Antarctic fish, Chionodraco hamatus

The anatomical distribution of atrial natriuretic peptide (ANP)-immunoreactive structures and the autoradiographic localization of ANP binding sites were studied in the brain of the Antarctic fish, Chionodraco hamatus. ANP-containing elements were colocated with ANP binding sites in the dorsal medial and lateral subdivisions of the telencephalon, prethalamic nuclear complex, and in the nucleus of the medial longitudinal fasciculus of the mesencephalon. However, mismatching was observed in other brain regions, particularly at mesencephalic and metencephalic levels. In the pituitary, ANP immunoreactivity occurred only in the pars distalis, whereas ANP binding sites were localized in the whole pituitary. In this paper we describe the occurrence of ANP immunoreactivity and ANP binding sites in the brain and pituitary of an Antarctic fish. In particular, in the cerebellum and pituitary of C. hamatus, ANP binding sites are distributed in corresponding brain regions of dipnoans, amphibians and mammals. The immunocytochemical and histoautoradiographic data suggest that ANP acts as neuromodulator in the brain of C. hamatus. Moreover, the presence of ANP-like substances in tanycytes lining the diencephalic ventricle suggests a chemosensorial role for such liquor-contacting cells and a possible modulatory effect of ANP on the osmoregulation of the cerebrospinal fluid.

Natriuretic peptides: a new lipolytic pathway in human adipocytes

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.

Atrial natriuretic peptide and aldosterone secretions, and atrial natriuretic peptide-binding sites in kidneys and adrenal glands of pregnant and fetal rats in late gestation in response to a high-salt diet.

This study aimed at determining, in the term pregnant rat, whether maternal and fetal plasma atrial natriuretic peptide (ANP) concentrations were modified in response to an oral sodium load, and to investigate whether any changes in plasma concentrations were able to modify the density and affinity of the different ANP-binding site subtypes in maternal and fetal kidneys and adrenal glands. Pregnant rats kept in metabolic cages were divided into two groups. The normal sodium diet group had free access to rat chow and tap water whereas the high sodium diet group received 1% NaCl as drinking water for 10 consecutive days from day 11 to day 21 of gestation with free access to standard rat chow. Pregnant rats from both groups were killed by decapitation on day 21 of gestation. The plasma ANP and aldosterone concentrations were determined by RIA. The density and affinity of ANP receptors were determined in the maternal and fetal adrenal glands and kidneys. In the pregnant rats on the high-salt diet, the sodium and water intakes, as well as the urine volume and sodium excretion, were significantly higher than in the control group. After 10 days of high-salt intake, water and sodium retentions were not significantly different in the two groups, indicating that the pregnant rats were able to excrete excess salt. The high sodium intake did not change the body weight of the pregnant rats but did increase the body weight of the fetal rats. Maternal and fetal hematocrits remained unchanged in both groups, the high sodium intake did not modify plasma sodium concentration in the maternal rats but increased that of the fetuses, indicating an accumulation of sodium in the fetal rats. The dietary sodium intake did not change the plasma ANP concentrations but significantly decreased the plasma aldosterone concentrations in both the maternal and fetal rats. In response to the high-salt diet, the density and affinity of total ANP, ANPb and ANPc receptors were not altered in the maternal isolated renal glomeruli or the adrenal zona glomerulosa membranes or the fetal adrenal gland and kidney membrane preparations. These results suggest that ANP is not involved in the regulation of water and electrolyte balance in maternal and fetal rats during salt-loaded intake.

Interaction between brain natriuretic peptide and atrial natriuretic peptide in caecal circular smooth muscle cells

Guinea pig caecal circular smooth muscle cells were used to determine whether brain natriuretic peptide (BNP) can inhibit the contractile response produced by cholecystokinin-octapeptide (CCK-8). In addition, we examined the effect of an inhibitor of cAMP-dependent protein kinase, an inhibitor of particulate or soluble guanylate cyclase, an atrial natriuretic peptide (ANP) antagonist (ANP 1-11), and selective receptor protection on the BNP-induced relaxation of these muscle cells. The effect of BNP on cAMP formation was also examined. BNP inhibited the contractile response produced by CCK-8 in a dose–response manner, with an IC 50 value of 8.5 nM, and stimulated the production of cAMP. The inhibitor of cAMP-dependent protein kinase and the inhibitor of soluble guanylate cyclase significantly inhibited the relaxation produced by BNP. In contrast, the inhibitor of particulate guanylate cyclase did not have any significant effect on the relaxation produced by BNP. ANP 1-11 significantly but partially inhibited the relaxation produced by BNP. The muscle cells where CCK-8 and ANP binding sites were protected completely preserved the inhibitory response to ANP, but partially preserved the inhibitory response to BNP. The muscle cells where CCK-8 and BNP binding sites were protected completely preserved the inhibitory response to both ANP and BNP. This study demonstrates that BNP induces relaxation of these muscle cells via both ANP binding sites coupled to soluble guanylate cyclase and distinct BNP binding sites coupled to adenylate cyclase.

Natriuretic peptides receptors in human aldosterone-secreting adenomas.

Atrial natriuretic peptide (ANP) and B-type or brain natriuretic peptide (BNP) inhibit aldosterone secretion in humans both in vitro and in vivo. Unresponsiveness of aldosterone-secreting adenomas (aldosteronomas) to ANP in vitro and in vivo, might be due to reduced expression of the biologically-active natriuretic peptide receptor type A (NPr-A) and/or increased expression of the clearance receptor for natriuretic peptides (NPr-C). Therefore, we have analyzed NPr gene expression and ANP binding sites in human adrenals and aldosteronomas. Using reverse transcription and polymerase chain reaction, we cloned and characterized cDNAs for NPr-A, NPr-C, and the receptor (NPr-B) for the C-type natriuretic peptide (CNP). Total RNA from three normal human adrenals (obtained at surgery from patients with renal cancer) and five aldosteronomas were used for Northern analysis. NPr-A mRNA (approximately 4 kb) and NPr-B mRNA (approximately 4 kb) were expressed without significant differences in adrenals and in aldosteronomas except in an aldosteronomas that contained only very low amounts of NPr mRNAs. The gene expression of NPr-C was barely detectable both in adrenals and in aldosteronomas. ANP binding sites were analyzed by autoradiography with 125I-labeled ligand in other six aldosteronomas. Only one of the adenomas analyzed showed ANP binding sites with density of granules similar to nonadenomatous glomerulosa, whereas the others had significantly reduced densities. In summary, aldosteronomas express the genes encoding for NPr but mainly NPr-A, similarly to control adrenals. On the contrary, the binding sites for ANP are greatly reduced in most aldosteronomas. A somatic mutation or a post-transcriptional defect that reduces ANP binding sites might be present in some aldosteronomas.

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.

Effects of severe hemorrhage on plasma ANP and glomerular ANP receptors.

Atrial natriuretic peptide (ANP) plays an important role in blood volume and electrolyte homeostasis in normovolemia and in hypervolemic states. The currently available information on the effects of hypovolemia on plasma ANP is contradictory. Moreover, possible regulation of ANP receptors during severe hemorrhagic hypovolemia has not been investigated. This study evaluated the effects of severe hemorrhage on plasma ANP and on the regulation of glomerular ANP receptor subtypes in anesthetized rats. Constant rate bleeding of 50% of total blood volume within 2 h induced a reproducible shock state characterized by marked decreases in blood pressure, heart rate, and hematocrit and an increase in plasma renin activity and aldosterone. Hemorrhaged rats exhibited a gradual significant increase in plasma ANP from 39.3 +/- 2.9 to 114.7 +/- 20.0 pmol/l 1 h after the bleeding (P < 0.001 from the initial value and P < 0.02 from the final value of sham-shock rats). Hemorrhage induced a significant decrease in total glomerular ANP binding sites (172 +/- 25 vs. 363 +/- 39 fmol/mg protein in hemorrhaged and sham-shock rats, respectively, P < 0.05). This decrease was mainly due to a significant decrease in ANPC receptors (132 +/- 22 vs. 312 +/- 40 fmol/mg protein in hemorrhaged and sham-shock rats, respectively, P < 0.05). Hemorrhage did not change glomerular ANPA receptor density. No significant differences in the affinity of the glomerular receptor subtypes for ANP were detected. Our data indicate that plasma ANP increases after prolonged severe hemorrhage. It is suggested that downregulation of renal ANPC receptors leads to reduced clearance of ANP and contributes to elevation of its plasma level after severe hemorrhage.

Whole body autoradiography and microautoradiography in eels after intra-arterial administration of 125I-labeled eel ANP.

125I-labeled eel atrial natriuretic peptide (ANP) was administered into the ventral or dorsal aorta of freshwater (FW) and seawater (SW) eels, Anguilla japonica, and the major target organs were explored by whole body autoradiography. Localization of the ANP binding in the target organs was also examined at tissue and cell levels by microautoradiography using tissue sections. Whole body autoradiography revealed that the specific label was accumulated predominantly in the gill, with lesser amounts in the atrium, kidney, liver, and urinary bladder. Autoradiographic grains were most dense in the secondary lamellae of the gill, particularly on the side of the efferent filamental artery. Other binding sites in target tissues were the glomerulus of the kidney, epicardium and endocardium of the atrium, bile duct/blood vessels of the liver, and interrenal cells of the head kidney. There was no difference in the distribution and density of grains between injections into the ventral aorta and dorsal aorta, although, in the former, injected 125I-labeled eel ANP passes through the gill before reaching peripheral target tissues. There was a tendency for downregulation of ANP binding sites in SW eels, especially in the gill. These results show that specific ANP binding sites are present in organs that are implicated in osmoregulation and cardiovascular regulation in eels and further suggest that the number of ANP binding sites varies according to changes in the environmental salinity.

The Guanylyl Cyclase-A Receptor Transduces an Atrial Natriuretic Peptide/ATP Activation Signal in the Absence of Other Proteins

Attempts to activate partially purified preparations of the guanylyl cyclase-A (GC-A) receptor with atrial natriuretic peptide (ANP) have previously failed, leading to speculation that essential cofactors are lost during purification procedures. The receptor was modified to contain the FLAG epitope (DYKDDDDK), expressed in Sf9 cells, and purified to apparent homogeneity (4.3 mumol cyclic GMP formed/min/mg protein; 5.8 mmol 125I-ANP binding site/mg protein) by a combination of immunoaffinity, Q-Sepharose FF, and wheat germ agglutinin batch chromatography. High initial protein/detergent ratios, the presence of glycerol (40%), and the inclusion of protein phosphatase inhibitors in all buffers resulted in the purification of a receptor that continued to transduce the ANP/ATP activation signal. Both native and purified GC-A contained a single class of high affinity ANP binding sites (Kd = 60 pM) and an equivalent EC50 for ATP (0.3 mM). Positive cooperativity as a function of MnGTP was retained during purification. Thus, GC-A is capable of transducing a ligand binding signal in the absence of other proteins.

Endogenous ANP in postischemic acute renal allograft failure.

Circulating atrial natriuretic peptide (ANP) levels and glomerular binding sites for ANP were examined in 23 subjects undergoing renal transplantation. Subjects were divided into two groups, group 1 (n = 12) with prompt and group 2 (n = 11) with delayed allograft function. Sixty to 180 min after graft reperfusion, renovascular resistance was threefold higher and glomerular filtration rate (GFR) depressed by 79% in group 2 vs. group 1. Corresponding median plasma ANP (114 vs. 140 pg/ml) and guanosine 3',5'-cyclic monophosphate (cGMP) levels (22 vs. 28 pmol/ml) were similarly elevated in the two groups [P = not significant (NS)]. Autoradiographic analysis of glomeruli in an allograft biopsy revealed the median density of total receptors (24 vs. 28 fmol/mm3), A receptors (15 vs. 19 fmol/mm3), and C receptors (6 vs. 9 fmol/mm3) for ANP to also be similar in group 2 vs. group 1, respectively (P = NS). By postoperative day 3, allograft GFR averaged only 6 +/- 2 in group 2 vs. 59 +/- 4 ml/min in group 1. Median plasma ANP levels doubled in each group to 262 and 251 pg/ml, respectively (P = NS). However, median values for plasma levels (38 vs. 17 pmol/ml) and the fractional clearance of cGMP (1.9 vs. 1.2) were significantly higher in group 2 than group 1. We conclude that, despite an adequate density of glomerular ANP receptors and enhanced cGMP generation, neither renal vasoconstriction nor hypofiltration is alleviated by a progressive elevation of plasma ANP levels in renal transplant recipients with sustained postischemic injury. We infer that constricted afferent arterioles are unresponsive to the vasorelaxant action of endogenous ANP in this form of postischemic, acute renal failure.

Multiple types of binding sites for atrial natriuretic peptide in rat olfactory bulb membranes and synaptosomes

The binding of atrial natriuretic peptide (ANP) to rat olfactory bulb membranes and synaptosomes was examined. [ 125I]ANP (rat, 99–126) bound specifically to a single class of binding site on olfactory bulb membrane preparation with dissociation constant ( K d) of 106 pM and maximum binding capacity ( B max) of 13.6 fmol/mg protein. Comparable results were obtained when the binding was characterized using displacement and kinetic experiments. The ring deleted analog of ANP, C-ANP (rat, 4–23) displaced [ 125I]ANP only minimally from its binding site in the membrane preparation. Saturation, displacement and blocking experiments on [ 125I]ANP binding to rat olfactory bulb synaptosomes revealed the presence of two distinct binding site. Simultaneous analysis of homogeneous and heterogeneous displacement curves and blocking experiments revealed the quantitative characteristics of these receptors to be: K d1 = 44 pM, B max1 = 42 fmol/mg protein and K d2 = 1050 pM, B max2 = 173 fmol/mg protein, for the high and low affinity binding sites, respectively. Kinetic experiments further confirmed the differences between the receptors present in the membranes and the synaptosomes preparations. The demonstration of multiple ANP binding sites in olfactory bulb synaptosomes but not membrane preparations raises the possibility of a particular function of ANP in nerve terminals.

Hyperosmolality rapidly reduces atrial-natriuretic-peptide-dependent cyclic guanosine monophosphate production in cultured rat inner medullary collecting duct cells

The present study was undertaken to explore the acute effect of hyperosmolality on the response of cultured rat inner medullary collecting duct (IMCD) cells to atrial natriuretic peptide (ANP). In contrast to the stimulatory effect of chronic incubation (12 h) in hypertonic medium, it was found that short-term incubation (< 2 h) reversibly suppressed the ANP-dependent cyclic guanosine monophosphate (cGMP) production. Urea, NaCl and mannitol were equi-potent as the osmolyte in suppressing the ANP-dependent cGMP production. Receptor binding assay revealed that hyperosmolality induced a rapid and marked reduction of the maximum binding (Bmax) of ANP without a significant change of the dissociation constant (Kd). Pretreatment with protein kinase C inhibitors (calphostin-C, staurosporin) or with cytoskeleton modulators (cytochalasin-B, colchicine) did not affect the inhibitory effect of hyperosmolality. In conclusion, acute hypertonicity inhibited the ANP-induced cGMP production in contrast to chronic hypertonicity, and reduction of the number of ANP binding sites was considered to be a mechanism responsible for the inhibitory effect of hypertonicity.

Role of the atrial natriuretic peptide for adrenal regulation in the teleost fish Cyprinus carpio.

The biological role of atrial natriuretic peptide (ANP) for the adrenal tissue of the teleost fish Cyprinus carpio, the carp, was investigated using in vitro autoradiography to study ANP binding sites, immunohistochemistry to localize ANP-like peptides, and in vitro perifusion to measure adrenal cortisol and catecholamine release. 125I-labeled rat ANP-(99-126) [125I-rANP-(99-126)] binding was present in adrenal tissue exhibiting positive cooperativity with a half-maximal binding concentration (EC50) of 87 +/- 15 pM and a maximal binding capacity (Bmax) of 56 +/- 11 amol/mm2 (n = 3). 125I-rANP-(99-126) binding was competitively displaced by unlabeled ANP analogues with an intact disulfide bridge showing a lower affinity than the iodinated ligand. By the use of immunohistochemistry, ANP immunoreactivity was found in the majority of the phenylethanolamine N-methyl-transferase-immunoreactive, i.e., epinephrine-synthesizing cells. In vitro, ANP elevated both basal and acetylcholine-stimulated cortisol release by carp adrenal tissue, whereas adrenal catecholamines were significantly affected only in acetylcholine-stimulated epinephrine secretion. Thus ANP derived from the ANP-immunoreactive chromaffin cells of the carp adrenal seems to act in an autocrine and/or paracrine manner on steroidogenic interrenal and catecholaminergic chromaffin cells. Furthermore, the study indicates that the ANP effects on fish catecholamine and steroid release are reversed in mammals.