Natriuretic Peptide Binding Sites Research Articles

Natriuretic peptide binding sites in the gills of the pouched lamprey Geotria australis

Iodinated atrial natriuretic peptide (ANP) binding sites were examined in the gills and ventral aorta of the adult upstream-migrating lamprey Geotria australis using tissue section autoradiography, in vitro competition analysis and affinity cross-linking, while guanylate cyclase assays were performed on gill membranes of both adult and juvenile lampreys. A partial natriuretic peptide (NP) receptor sequence was amplified using reverse transcription/polymerase chain reaction (RT-PCR). The results indicated that there was specific NP binding to the aortic endothelium and to pillar cell regions in the axial plate and secondary lamellae. In competition studies, 50 % of NP binding was abolished by 4 nmol l-1 rat ANP, 35 nmol l-1 porcine C-type NP (CNP) and 45 nmol l-1 C-ANF (a truncated ANP). Affinity cross-linking followed by SDS-PAGE demonstrated two binding sites at 205 and 65 kDa under non-reducing conditions and at 85 and 65 kDa under reducing conditions. Guanylate cyclase assays demonstrated that, while no NP-stimulated GC activity occurred in adult lampreys, NP-stimulated enhancement of cyclic GMP accumulation was found in juveniles in fresh water and more particularly in salt water. RT-PCR amplified a 471 base pair fragment with 68 % amino acid sequence homology to the eel natriuretic peptide receptor D (NPR-D). This study suggests that NP binding sites in the adult gill and aorta are of an NPR-C/D type, whereas an additional GC-coupled site exists in juveniles.

Functional Analysis of Natriuretic Peptide Receptors in the Bladder of the Toad, Bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific 125I-rat atrial natriuretic peptide (125I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with 125I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to 125I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Natriuretic peptide binding sites in the gills of the pouched lamprey Geotria australis.

Iodinated atrial natriuretic peptide (ANP) binding sites were examined in the gills and ventral aorta of the adult upstream-migrating lamprey Geotria australis using tissue section autoradiography, in vitro competition analysis and affinity cross-linking, while guanylate cyclase assays were performed on gill membranes of both adult and juvenile lampreys. A partial natriuretic peptide (NP) receptor sequence was amplified using reverse transcription/polymerase chain reaction (RT-PCR). The results indicated that there was specific NP binding to the aortic endothelium and to pillar cell regions in the axial plate and secondary lamellae. In competition studies, 50 % of NP binding was abolished by 4 nmol l-1 rat ANP, 35 nmol l-1 porcine C-type NP (CNP) and 45 nmol l-1 C-ANF (a truncated ANP). Affinity cross-linking followed by SDS-PAGE demonstrated two binding sites at 205 and 65 kDa under non-reducing conditions and at 85 and 65 kDa under reducing conditions. Guanylate cyclase assays demonstrated that, while no NP-stimulated GC activity occurred in adult lampreys, NP-stimulated enhancement of cyclic GMP accumulation was found in juveniles in fresh water and more particularly in salt water. RT-PCR amplified a 471 base pair fragment with 68 % amino acid sequence homology to the eel natriuretic peptide receptor D (NPR-D). This study suggests that NP binding sites in the adult gill and aorta are of an NPR-C/D type, whereas an additional GC-coupled site exists in juveniles.

Different binding activities of A- and B-type natriuretic hormones in the heart of two Antarctic teleosts, the red-blooded Trematomus bernacchii and the hemoglobinless Chionodraco hamatus

Different binding activities of 125I-rat atrial natriuretic peptide were evaluated using in vitro quantitative autoradiography in the heart of two antarctic notothenioid teleosts, the red-blooded Trematomus bernacchii and hemoglobinless Chionodraco hamatus. Saturable and specific binding sites for rat atrial natriuretic peptide were found in the atrium, ventricular myocardium, ventricular endocardium and inner and outer layers of the bulbus arteriosus of both fishes. Scatchard analysis of the saturation data showed that the atrium, ventricular endocardium and outer bulbar layer of T. bernacchii were characterized by a single class of high affinity natriuretic peptide binding sites ( K d = 14 ± 3.2, 9.7 ± 2.3 and 6.2 ± 1.3 pM, respectively), whereas the ventricular myocardium and the inner bulbar layer contained elevated numbers of two classes of high and low affinity natriuretic peptide binding sites (1.8 ± 0.6 < K d < 209 ± 66 pM). In contrast, in C. hamatus, both high and low affinity binding sites were detected in all cardiac regions (2.1 ± 0.7 < K d < 262 ± 90 pM). In both fishes, competition experiments in the presence of either unlabeled rat atrial natriuretic peptide or porcine brain natriuretic peptide indicated different displacement capacities. Porcine brain natriuretic peptide, able to bind to natriuretic peptides sites in all the heart regions of both notothenioids, provided a higher displacement capacity with respect to that of rat atrial natriuretic peptide in the atrium of T. bernacchii.

Localization and analysis of natriuretic peptide receptors in the gills of the toadfish, Opsanus beta (teleostei).

The distribution and nature of natriuretic peptide binding sites was determined in the gills of the toadfish, Opsanus beta. Specific 125I-labeled rat atrial natriuretic peptide (rANP) and 125I-labeled porcine C-type natriuretic peptide (pCNP) binding sites were observed on the afferent and efferent filamental arteries and lamellar arterioles, and on the marginal channels of the secondary lamellae. In both section autoradiography and competition assays, the binding of both ligands was completely displaced by 1 microM rANP and 1 microM pCNP, but residual binding was observed with 1 microM of the type C natriuretic peptide receptor (NPR-C)-specific ligand C-ANF. Electrophoresis of gill membranes cross-linked with 125I-rANP showed a major band at 75 kDa and a fainter band at 140 kDa. Both rANP and pCNP significantly stimulated the production of cGMP above basal levels; C-ANF had no stimulatory effect. These data show that the intrafilamental gill vasculature of toadfish contains a major population of natriuretic peptide receptors very similar to mammalian clearance receptors and a smaller population of receptors that are linked to a membrane-bound guanylate cyclase.

Autoradiographic discrimination of brain and atrial natriuretic peptide-binding sites in the rat kidney.

Brain (BNP) and atrial natriuretic peptides (ANP) have been identified which may represent endogenous agonists of kidney receptor subtypes. Quantitative in vitro autoradiography was used to investigate the regional distribution of receptor subpopulations and the competitive inhibition of 125I porcine BNP1-26 (pBNP1-26) and 125I rat alpha-ANP1-28 (rANP1-28) renal binding sites. Specific, high affinity binding (Kd 0.2-1.37 nM range) was localized to glomeruli, inner medulla, interlobar and arcuate arteries, vasa recta bundles, and smooth muscle in the renal pelvis. pBNP1-26 competed for the same sites as rANP1-28 but displayed a lower potency and was less selective for nonclearance sites. Clearance binding sites were discriminated by competitive inhibition with C-ANP4-23 and comprised some 65% of glomerular sites as well as the vast majority of sites in the renal pelvis. Nonclearance sites predominated in the inner medulla and intrarenal arteries. C-terminal changes in amino acid sequence induced a significant loss of inhibitory potency. Immunohistochemical studies identified a distinct population of BNP-like immunoreactive renal nerve fibers, associated with intra-renal arteries. Circulating natriuretic peptides and BNP sequences derived from renal nerves may influence renal function by interacting with specific receptor subpopulations in the kidney.

Autoradiographic localization of specific atrial natriuretic peptide binding sites on immunocytochemically identified cells in cultures from rat and guinea-pig hearts

Dissociated cell culture preparations from rat and guinea-pig atria and interatrial septum, and from rat ventricles were studied using a combined autoradiographic and immunocytochemical approach. Alpha-atrial natriuretic peptide (125I-ANP1-128) bindings sites were confined to subpopulations of identified non-neuronal cells in each type of culture preparation, and had distinct patterns of labelling. The density of ANP1-28 binding sites was substantially greater in guinea-pig cultures than in rat cultures and was least in rat ventricular cultures. ANP1-28-labelled subpopulations of S-100-like immunoreactive glial cells were only seen in guinea-pig cultures. Von Willebrand factor (vWF)-like immunoreactive endothelial cells and vWF-negative endothelioid cells expressed ANP1-28 binding sites in both the guinea-pig and rat atrial cultures, but were unlabelled in rat ventricular cultures. In contrast, labelled subpopulations of fibronectin-like immunoreactive fibroblasts were present in all of the three types of culture preparation studied. ANP-like immunoreactive myocytes were present in both atrial and ventricular cultures. These cells did not, however, express ANP1-28 binding sites.

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat 125I-atrial natriuretic peptide(1-28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.

Localization of the Atrial Natriuretic Peptide Binding Sites in the Inner Ear Tissue—Possibly an Additional Regulating System

Specific high affinity atrial natriuretic peptide (ANP) binding sites were identified and localized by autoradiography (125J-ANP 1-28) in the inner ear of guinea pigs. ANP is a cardiac hormone known to mediate increased capillary permeability, vasodilation, and natriuresis. Since specific receptors for ANP exist in both eye and brain, possibly playing a role in regulation of fluid and pressure (14, 15), we hypothetized that ANP might be involved also in inner ear fluid dynamics via interaction with other volume/electrolyte balance regulating hormones including vasopressin, renin, and aldosteron. The distribution of ANP receptor in the inner ear tissue appears unequal, which may play a significant role in view of some functional disturbances such as Meniere's disease.

Increased atrial natriuretic peptide binding sites in the rat subfornical organ after water deprivation.

Binding sites for rat atrial natriuretic peptide (99-126) were localized and quantified in the subfornical organ and choroid plexus of control and water-deprived rats. Brain sections were analyzed after incubation with 125I-rat atrial natriuretic peptide (99-126), autoradiography, computerized microdensitometry and comparison to 125I-standards. Higher numbers of atrial natriuretic peptide binding sites were present in both the subfornical organ and the choroid plexus after 4 days of water deprivation. Our results indicate a central role for atrial natriuretic peptide in the regulation of water balance.