Autoradiographic localization of binding sites for atrial natriuretic factor

The distribution of atrial natriuretic factor (ANF) binding sites in Wistar rat tissues, as well as tissues from other species was studied. Using autoradiography of slide mounted tissue sections incubated with 125I-labelled ANF, high densities of binding sites were found in the renal glomeruli and papilla, aortic smooth muscle, iliac vein, choroid plexus, anterior pituitary, lung, and adrenal zona glomerulosa. Results from renal, aortic, adrenal, and lung tissues from spontaneously hypertensive rats did not differ from those of Wistar rats. Binding sites were also observed in guinea pig glomeruli, renal medulla, and aorta as well as in the rabbit aorta.     

Nature of atrial natriuretic polypeptide in rat brain

Using reverse phase high performance liquid chromatography (RP-HPLC) coupled with two radioimmunoassays for atrial natriuretic polypeptide (ANP) with different specificities, we investigated the nature of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) with a low molecular weight in the rat brain. Two major peaks with alpha-rANP-LI in the extract from rat whole brains were eluted in the vicinity of the elution position of synthetic alpha-rANP, a 28-amino acid polypeptide. These two components co-migrated with synthetic alpha-rANP (4-28) and alpha-rANP (5-28), respectively. The peak corresponding to alpha-rANP (4-28) was the highest, and only a little alpha-rANP-LI was detected at the elution position of alpha-rANP. An identical profile in RP-HPLC was also observed in the extract from the rat hypothalamus. These results indicate that the major components of alpha-rANP-LI with a low molecular weight in the rat brain are alpha-rANP (4-28) and alpha-rANP (5-28).     

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

Summary 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. Alphaatrial natriuretic peptide (¹²⁵I-ANP_1-128) binding 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 ANP_1-28 binding sites was substantially greater in guinea-pig cultures than in rat cultures and was least in rat ventricular cultures. ANP_1-28-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 ANP_1-28 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 ANP_1-28 binding sites.     

Autoradiography of atrial natriuretic peptide (ANP) receptors in the rat brain.

Quantitative autoradiography was used to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat brain and to study their regulation. Peptide receptors are selectively located to circumventricular organs outside the blood brain barrier, such as the subfornical organ, and to brain areas involved in fluid and cardiovascular regulation. Dehydration, either by water deprivation of normal rats, or chronic dehydration present in homozygous Brattleboro rats lacking vasopressin, results in large increases in ANP binding in receptor number in the subfornical organ. In the deoxycorticosterone acetate (DOCA)-salt hypertensive model, only salt treatment, but not DOCA alone or the combination of DOCA-salt, increased the ANP receptor number in the subfornical organ and the choroid plexus. Both young and adult genetically hypertensive rats have a greatly decreased ANP receptor number in the subfornical organ and the choroid plexus. Selective displacement with an inactive analog lacking the disulfide bond (ANP 111-126) suggests that genetically hypertensive rats may lack C (clearance) atrial natriuretic peptide receptors. Our results implicate brain atrial natriuretic peptide receptors in the central response to alterations in fluid regulation and blood pressure.     

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. Accepted: 3 April 2000     

Characterization, distribution and plasticity of atrial natriuretic factor binding sites in brain

The [125I]iodotyrosyl derivative of atrial natriuretic factor [( 125I])ANF) apparently binds to a single class of high affinity sites in guinea pig brain membrane preparations. Ligand selectivity pattern reveals that the structural requirements of brain [125I]ANF binding sites are similar to those reported in most peripheral tissues. In vitro receptor autoradiographic studies demonstrate that the brain distribution of [125I]ANF binding sites is species dependent. In rat, high levels of binding are found in olfactory bulb, subfornical organ, area postrema, choroid plexus, and ependyma. In guinea pig, these regions are also enriched with [125I]ANF binding in addition to various thalamic nucleic, amygdala, hippocampus, and cerebellum. In monkey, high densities of sites are seen in the cerebellar cortex. This suggests that brain ANF receptor sites could mediate ANF effects related to the central integration of cardiovascular parameters, as well as other actions not associated with these systems. As in the periphery, it appears that brain [125I]ANF binding sites are associated with guanylate cyclase. Moreover, the density of [125I]ANF receptor binding sites is altered in certain brain regions in spontaneously hypertensive rats and in cardiomyopathic hamsters, demonstrating the plasticity of brain ANF receptors. Thus, ANF and ANF receptors are complementary facets of a new neurotransmitter-neuromodulator system present in mammalian brain.     

Degradation of atrial natriuretic factor in the rat.

The biologically active circulating form of atrial natriuretic factor (ANF) in the rat is the 28-amino-acid peptide ANF-(Ser-99-Tyr-126). Degradation of this peptide in vivo as well as in vitro, in whole blood, in plasma and by the isolated mesenteric artery was investigated. Studies in vivo in the rat demonstrated that the elimination and degradation of ANF was extremely fast: within 3 min more than 95% of the injected immunoreactive material was eliminated from circulation. The production of a short C-terminal peptide was detected on injection of 125I-ANF-(Ser-99-Tyr-126) into the rat. This peptide increased proportionately with incubation time. Experiments in vitro in the presence of whole blood or plasma did not cause any major destruction of ANF even after incubation for 60 min. After this prolonged incubation in plasma, ANF-(Ser-99-Tyr-126) was partially converted into ANF-(Ser-103-Tyr-126), a less potent peptide. Isolated mesenteric-artery preparation appeared to degrade ANF in a manner very similar to the system in vivo. These results suggest that degradation of ANF may occur either after internalization in the vascular cells or by a membrane-bound enzyme in the vasculature.     

Autoradiographic localization of 125I-galanin binding sites in the blowfly brain.

The localization of porcine galanin (pGAL) binding sites in the brain of the blowfly Phormia terraenovae was investigated by autoradiography using the following radioiodinated ligands: pGAL 1-29 (two isoforms), pGAL 15-29 and rat (r) GAL 1-29. The different porcine radioligands bound specifically with the following intensity: 125I-[Tyr26]-pGAL15-29 > > 125I-[Tyr26]-pGAL1-29 > > 125I-[Tyr9]-pGAL1-29. With rat galanin 125I-[Tyr9]-rGAL1-29 no specific binding could be shown. In addition, displacement of 125I-[Tyr26]-pGAL1-29 was tested with pGAL 1-29, pGAL 1-22 and pGAL 15-29 (at 0.1 nM-1 microM). A gradual displacement was achieved with increasing concentrations of pGAL 1-29 and pGAL15-29, whereas no displacement with pGAL 1-22 was detected. The results indicate that the C-terminal portion of pGAL is important for binding in the blowfly. The pGAL binding sites were localized in synaptic neuropils of the central body, the antennal lobes, the optic lobes, the pars intercerebralis and the subesophageal ganglion, all of which contain GAL-like immunoreactive neural processes.     

Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland

With in vitro autoradiography, specific receptors for atrial natriuretic peptide (ANP) were localized in fetal rat kidney and adrenal glands. Receptors were present over renal vesicles, in the primitive renal medulla and throughout the adrenal gland as early as 16 days gestation. By 20 days gestation, several layers of developing renal corpuscles were present and ANP receptors were localized over developing glomeruli in each layer. Larger accumulations occurred over the juxtamedullary glomeruli. In the medulla, the receptors were localized in a reticular pattern near the pelvis. With emulsion coated sections, ANP receptors in developing renal corpuscles were seen primarily over the lower curve of S-shaped vesicles and around the periphery of the more mature corpuscles. In the renal medulla, receptors were localized over the interstitial cells. In the 16-day-old adrenal gland, ANP receptors were present throughout the cortical area but at 20 days gestation and 1 day postpartum receptors appeared more numerous in the peripheral region. These data suggest that ANP has important developmental effects in the kidney and adrenal gland and may be involved in regulation of body fluid homeostasis in the late gestation rat fetus.     

Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland

Summary With in vitro autoradiography, specific receptors for atrial natriuretic peptide (ANP) were localized in fetal rat kidney and adrenal glands. Receptors were present over renal vesicles, in the primitive renal medulla and throughout the adrenal gland as early as 16 days gestation. By 20 days gestation, several layers of developing renal corpuscles were present and ANP receptors were localized over developing glomeruli in each layer. Larger accumulations occurred over the juxtamedullary glomeruli. In the medulla, the receptors were localized in a reticular pattern near the pelivis. With emulsion coated sections, ANP receptors in developing renal corpuscles were seen primarily over the lower curve of S-shaped vesicles and around the periphery of the more mature corpuscles. In the renal medulla, receptors were localized over the interstitial cells. In the 16-day-old adrenal gland, ANP receptors were present throughout the cortical area but at 20 days gestation and 1 day postpartum receptors appeared more numerous in the peripheral region. these data suggest that ANP has important developmental effects in the kidney and adrenal gland and may be involved in regulation of body fluid homeostasis in the late gestation rat fetus.     

Receptor sites for atrial natriuretic factors in brain and associated structures: an overview

1. Recent data have clearly shown the existence of specific receptor binding sites for atrial natriuretic factors (ANF) or polypeptides in mammalian brain tissues. 2. Ligand selectivity pattern and coupling to cGMP production suggest that brain ANF sites are similar to high-affinity/low-capacity sites found in various peripheral tissues (kidney, adrenal gland, blood vessels). These brain ANF sites possibly are of the B-ANP subtype. 3. High densities of ANF binding sites are found especially in areas of the central nervous system associated with the control of various cardiovascular parameters (such as the subfornical organ and area postrema). However, high densities of sites are also present in other regions such as the hippocampus, cerebellum, and thalamus in the brain of certain mammalian species, suggesting that brain ANF could act as a neuromodulator of noncardiovascular functions. 4. The density of brain ANF binding sites is modified in certain animal models of cardiovascular disorders and during postnatal ontogeny, demonstrating the plasticity of these sites in the central nervous system (CNS). 5. Specific ANF binding sites are also found in various other CNS-associated tissues such as the eye, pituitary gland, and adrenal medulla. In these tissues ANF appears to act as a modulator of fluid production and hormone release. 6. Thus, ANF-like peptides and ANF receptor sites are present in brain and various peripheral tissues, demonstrating the existence of a family of brain/heart peptides.     

Existence of atrial natriuretic polypeptide in kidney

Using a specific radioimmunoassay (RIA) for alpha-atrial natriuretic polypeptide (alpha-ANP), we have demonstrated the presence of alpha-rat ANP-like immunoreactivity (alpha-rANP-LI) in the rat kidney which is considered to be a target organ for atrial natriuretic polypeptides released from the heart. Most of alpha-rANP-LI was localized in the cortex. High performance gel permeation chromatography coupled with the RIA revealed that renal alpha-rANP-LI was eluted at the position of a low molecular weight form corresponding to alpha-rANP without detectable amounts of high molecular weight forms. This is in contrast to the observation that gamma-rANP, a high molecular weight form of 13k daltons, is the dominant form of alpha-rANP-LI in the rat atrium. In water-deprived rats, the concentration and content of alpha-rANP-LI in the kidney showed a significant decrease compared with control rats. In addition, the alpha-rANP-LI concentration and content in this organ revealed a substantial decrease after perfusion with physiological saline. These results indicate the existence of atrial natriuretic polypeptide (ANP) in the kidney and suggest that part of renal ANP may originate from the heart.     

Binding sites for atrial natriuretic peptide in the kidney and aorta of the hagfish (Myxine glutinosa)

1. In the atlantic hagfish (Myxine glutinosa) binding sites for atrial natriuretic peptide (ANP) were visualized by autoradiography in contractile structures of the renal system (glomeruli, neck segment, and archinephric duct) and in the aorta. 2. Since the location of binding sites is comparable to that in higher vertebrates including man, these results suggest that ANP may act as a hormone already in cylcostomata.     

Computerized approach using autoradiography to quantify atrial natriuretic peptide receptors in the DOCA-salt hypertensive rat kidney.

The alteration of atrial natriuretic peptide (ANP) receptors was investigated in the kidney of deoxycorticosterone acetate (DOCA)-salt treated hypertensive rats. The absolute amount of renal ANP receptors was determined in a membrane homogenate binding study of rat whole kidneys. Administration of DOCA-salt led to a decrease in renal ANP receptors after 3 weeks (prehypertensive state) and 6 weeks (established hypertensive state) of treatment. In vitro macro-autoradiography (ARG) was then performed with [125I]ANP to localize and to quantitate specific renal ANP receptors. ARG revealed that specific ANP binding was distributed mainly over the renal cortex with the inner medulla next in frequency. Renal ANP receptors were therefore quantified over the cortex and the inner medulla using the computerized microdensitometry of ARG. A significant reduction in renal ANP receptors was observed in the DOCA-salt treated rats after 3 and 6 weeks of treatment with decrements observed in both the cortex and inner medulla. These alterations may be related to the pathophysiology of hypertension.     

Effects of atrial natriuretic factor, nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney.

In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 microM) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 microM) and ANF (0.01 microM) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 microM) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.