Occurrence of a novel cardiac natriuretic peptide in rats

We established a specific radioimmunoassay for the ring structure of "iso-ANP" and detected iso-ANP[23-46]-like immunoreactivity (-LI) in the rat atrium (2.76 +/- 0.5 micrograms/g) and ventricle (13.9 +/- 5.7 ng/g). High performance-gel permeation chromatography revealed that iso-ANP[23-46]-LI in the rat heart was composed of two components with molecular weights of 10K and 5K. In reverse phase-high performance liquid chromatography, the retention times of these components were clearly different from that of synthetic iso-ANP. The 5K peptide was demonstrated to be present in the perfusate from isolated rat hearts and possessed binding ability to ANP receptors. This natriuretic peptide was, however, not detectable in other tissues including the brain. We conclude that the novel cardiac natriuretic peptide distinct from iso-ANP and ANP occurs in the rat heart and is secreted from the heart.     

Differential changes in atrial natriuretic peptide and vasopressin receptor bindings in kidney of spontaneously hypertensive rat

To elucidate the role of atrial natriuretic peptide (ANP) and vasopressin (VP) in a hypertensive state, ANP and VP receptor bindings in spontaneously hypertensive rat (SHR) kidney were analyzed using the radiolabeled receptor assay (RRA) technique. Systolic blood pressure of SHR aged 12 weeks was statistically higher than that of age-matched Wistar Kyoto (WKY) rats. Maximum binding capacity (Bmax) of [125I]-ANP binding to the SHR kidney membrane preparations was statistically lower than that of WKY rats, but dissociation constant (Kd) was not significantly different. On the other hand, Bmax of [3H]-VP binding to the SHR kidney membrane preparations was statistically higher than that of WKY rats, but Kd were similar. Since the physiological action of ANP is natriuresis and VP is the most important antidiuretic hormone in mammalia, these opposite changes of ANP and VP receptor bindings in SHR kidney suggested that these peptides may play an important role in the pathophysiology of the hypertensive state, although it has not been confirmed as yet.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Organization of the gene for iso-rANP, a rat B-type natriuretic peptide

Using the polymerase chain reaction and oligonucleotide primers constructed from knowledge of the cDNA sequence we have sequenced the gene for iso-rANP, a peptide of the B-type of atrial natriuretic peptides. The overall organization of the rat iso-ANP gene is the same as that of ANP and BNP consisting of three exons and two introns at relatively similar positions. Iso-rANP and it's gene are more closely related to BNPs than ANP and yet there are significant differences at both the protein and DNA levels. Our results suggest that iso-rANP and BNP are distinct members of the same sub-family (B-type natriuretic peptides) within the family of natriuretic peptide genes.     

Atrial natriuretic peptide elevates cGMP contents in glomeruli and in distal tubules of rat kidney

Effect of synthetic rat atrial natriuretic peptide (1-28) (ANP) on the cGMP content was studied using defined nephron segments of rat kidney. ANP elevates cGMP contents in glomeruli in a concentration and time-dependent manner. The increase of cGMP was observed in glomeruli, distal convoluted tubule (DCT) and cortical collecting tubule (CCT) (delta %; 279 +/- 35, 148 +/- 10 and 152 +/- 18, respectively), and no effect was observed in proximal convoluted (PCT) and straight tubule (PST). These results suggest that ANP may act directly on the tubular cells as well as glomeruli. In glomeruli, effects of ANP and carbamylcholine on cGMP contents were additive suggesting that these two agents may act on different receptors. Angiotensin II and norepinephrine failed to affect the ANP-induced cGMP production in the glomeruli.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Atrial natriuretic peptide, ANP(99-126), receptors in rat thymocytes and spleen cells

A single class of saturable, specific binding sites for the circulating form of atrial natriuretic peptides, ANP(99-126), was identified in rat thymus and spleen and in isolated thymocytes and spleen cells using quantitative autoradiographic techniques. In the thymus, the relative potency of ANP analogs to inhibit [125I] ANP(99-126) binding was ANP(99-126) = ANP(103-126) greater than ANP(111-126) greater than ANP(103-125). ANP(103-123) could not displace [125I]ANP(99-126) binding. Addition of ANP(99-126) stimulated the formation of cyclic GMP in isolated thymocytes and spleen cells in a dose-dependent manner. Our results indicate that immune cells have specific ANP receptors which could be coupled to guanylate cyclase activation and may play a role in the regulation of the immune response.     

Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques 1) to identify possible sites of synthesis; 2) to compare the localization of ANP to known physiological effects; 3) to determine species differences, if any, in ANP localization; and 4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP, IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.     

Apparent B-type natriuretic peptide selectivity in the kidney due to differential processing

Two natriuretic peptides, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), are found principally in the heart. In preliminary experiments with mouse kidney cells or slices, we found mouse BNP1-45 much more potent than ANP1-28 in causing elevations of cGMP (>50-fold). The guanylyl cyclase-A (GC-A) receptor has been suggested to represent the primary means by which both peptides signal. In cultured cells overexpressing GC-A, BNP and ANP were almost equivalent in potency, suggesting that a receptor unique for BNP exists in the kidney. However, in mice lacking the GC-A gene, neither BNP nor ANP significantly elevated cGMP in kidney slices. Phosphoramidon, a neutral endopeptidase inhibitor, shifted the apparent potency of ANP to values equivalent to that of BNP, suggesting these kidney cell/slices rapidly degrade ANP but not BNP. Mass spectroscopic analysis confirmed that ANP is rapidly cleaved at the first cysteine of the disulfide ring, whereas BNP is particularly stable to such cleavage. Other tissues (heart, aorta) failed to significantly degrade ANP or BNP, and therefore the kidney-specific degradation of ANP provides a mechanism for preferential regulation of kidney function by BNP independent of peripheral ANP concentration.     

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, ?B and ?C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.     

Atrial natriuretic peptide in kidney of renal disease patients and healthy persons

Regulation of renal excretion of atrial natriuretic peptide (ANP) was studied in kidney disease patients and healthy kidney donors. The measured ANP concentration in the patient's plasma did not correlate with their creatinine clearance (Ccr), while the fractional excretion of ANP (FEANP) significantly correlated with Ccr. FEANP in healthy persons is less than 1%. In the healthy donors of kidneys for transplantation, approximately 80% of the plasma ANP from the renal artery appeared in the renal vein. From these results, this high recovery of ANP in the veins does not appear to be adequately explained by its degradation in the renal arterioles and nephrons. The FEANP from kidney disease patients significantly correlated with FENa, FEK and FEP, but not with FECa and FEMg. The manner of ANP handling in the nephron may possibly differ from that of Ca or Mg.     

Vascular receptors for angiotensin, vasopressin, and atrial natriuretic peptide in experimental hypertension

Angiotensin II (ANG II) and vasopressin (AVP) are two powerful vasoconstrictors, and atrial natriuretic peptide (ANP) is a potent vasorelaxant. The changes in the density or affinity of binding sites for these agents that may alter target organ responsiveness in hypertension are reviewed. ANG II binding in mesenteric arteries was unaltered in one-kidney, one-clip (1-K, 1-C) and in 2-K, 1-C hypertensive rats, while in deoxycorticosterone acetate (DOCA)-salt hypertensive rats ANG II binding to blood vessels was significantly increased. A role of mineralocorticoids to increase the number of vascular ANG II sites in some hypertensive models is suggested. In spontaneously hypertensive rats (SHR) ANG II receptors were increased in young rats in the prehypertensive stage with respect to Wistar-Kyoto (WKY) control rats, but normal in older rats. AVP binding in the vasculature of hypertensive rats was uniformly decreased in inverse correlation to plasma AVP levels, but vascular responsiveness to AVP was exaggerated. Inositol trisphosphate production by blood vessels of SHR in response to AVP showed that increased AVP receptor-coupled phospholipase C activity may mediate in part the exaggerated pressor response in spite of reduced or normal density of receptors for vasoconstrictor peptides. Vascular ANP sites in 2-K, 1-C, 1-K,1-C, and DOCA-salt hypertensive rats varied inversely with plasma concentrations of ANP. Normal densities of ANP receptors in saralasin-sensitive 2-K, 1-C hypertensive rats correlated with ANP sensitivity, while saralasin-insensitive 2-K, 1-C hypertensive rats, which did not respond to ANP, had significantly decreased density of ANP vascular receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different patterns of expression of five neuropeptides in the adrenal gland and kidney of two species of frog

The aim of this study was to demonstrate in the adrenocortical and renal tissues of two species of frog, Rana italica and Rana esculenta, the presence and distribution of five neuropeptides: atrial natriuretic peptide (ANP), Leu-enkephalin (Leu-ENK), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP).In anurans, the adrenal medulla is the site for the synthesis, storage and secretion of not only catecholamines but also various peptides. These peptides should not be regarded only as neurotransmitters or modulators for the secretion of catecholamines, but also as hormonal substances that induce systemic effects.All the peptides studied (ANP, Leu-ENK, NPY, SP and VIP) are present in both organs. However, different patterns of expression were observed for some of the peptides in two frogs.Immunopositivity to ANP was found in small clusters of chromaffin cells in both frogs whereas a clear strong positivity was present only in Rana esculenta kidney. Large clusters of chromaffin cells were immunoreactive to Leu-ENK in Rana italica but there were approximately 25% fewer compared to the positive cells present in Rana esculenta. Epithelial cells of renal tubules showed strong immunopositivity to Leu-ENK in Rana esculenta but not in Rana italica. A large number of adrenal cells (70–80%) were immunoreactive to NPY in Rana italica, while in Rana esculenta this peptide was localized in small clusters of chromaffin cells. Both frogs showed many NPY-positive cells in kidney. Many chromaffin cells were found positive to SP and VIP. A strong positivity was also observed in kidney in both frogs. These observations suggest a possible role of these peptides in the control of the physiological functions of adrenal glands and kidney of the two species of frogs studied.     

Presence of dendroaspis natriuretic peptide and its binding to NPR-A receptor in rabbit kidney

Natriuretic peptides help to maintain sodium and fluid volume homeostasis in a healthy cardio-renal environment. Since the identification of Dendroaspis natriuretic peptide (DNP) as a new member of the natriuretic peptide family, DNP has been considered as an important regulator of natriuresis and dieresis. The present study was undertaken to investigate the presence of immunoreactive Dendroaspis natriuretic peptide (DNP) and its specific receptor in rabbit. DNP was detected in heart, kidney, liver, brain, and plasma by radioimmunoassay (RIA). DNP contents of cardiac atrium and ventricle, renal cortex and medulla, liver, and brain were 1.42 ± 0.15, 1.0 6 ± 0.08, 2.55 ± 0.21, 1.81 ± 0.16, 1.36 ± 0.22, and 0.69 ± 0.15 pg/mg of wet weight, respectively. The concentration of DNP in plasma was 235.44 ± 15.44 pg/ml. By quantitative in vitro receptor autoradiography, specific 12?I-DNP binding sites were revealed in glomeruli, interlobular artery, acuate artery, vasa recta bundle, and inner medulla of the kidney with an apparent dissociation constant (K(d)) of 0.29 ± 0.05, 0.36 ± 0.03, 0.84 ± 0.19, 1.18 ± 0.23, and 10.91 ± 1.59 nM, respectively. Basal rate of 3', 5'-cyclic guanosine monophosphate (cGMP) production by particulate guanylyl cyclase (GC) activation of glomerular membranes was basally 13.40 ± 1.70 pmol/mg protein/min. DNP caused an increment of cGMP production in similar magnitude to that caused by ANP, BNP, and urodilatin, while the production of cGMP by CNP was significantly lower than that by DNP. Our results show that plasma levels of DNP were higher when compared to other tissues. DNP produces cGMP via the NPR-A receptor subtype in the kidney, similarly to ANP and BNP, suggesting that plasma DNP could have similar functions as ANP and BNP.     

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma: preferential increase of free ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP)

Free and bound forms of atrial natriuretic peptide (ANP) in rat plasma were analysed by gel permeation chromatography combined with a radioimmunoassay (RIA) for rat ANP (rANP). Gel permeation chromatography showed two immunoreactive peaks in rat plasma, one corresponding to alpha-rANP, rANP(99-126), and the other eluted at a high molecular weight, clearly different from gamma-rANP, rANP(1-126). The chromatographic profile of rat plasma after incubation with synthetic alpha-rANP demonstrated that the high molecular immunoreactivity had ANP-binding capacity. This bound form of ANP was almost totally excluded following extraction procedure, therefore, the immunoreactive ANP (ir-ANP) measured with the extraction assay was mainly free ANP. On the other hand, direct RIA may detect not only the free but also the bound form of ANP. Using both direct RIA and the extraction method, bound forms of plasma ANP in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) were compared to normotensive Wistar Kyoto rats (WKY). Bound forms of plasma ANP in 20-week-old SHR and SHRSP were significantly higher than that in age-matched WKY. The ratio of free/bound form of plasma ANP in SHR and SHRSP also significantly increased compared to WKY, indicating a preferential increase in free ANP in the plasma of these hypertensive rats. These findings suggest that a bound form of ANP may be present in rat plasma and that it may play some pathophysiological role in the hypertension of SHR and SHRSP. Increased free ANP in plasma may indicate a compensatory increase in ANP release in these hypertensive rats.     

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.     

Different responses of atrial natriuretic peptide secretion and its receptor density to salt intake in rats

This study investigated whether high-salt intake influences atrial natriuretic peptide (ANP) system, atrial content, and release rate of ANP as well as receptor density in the kidney were measured in salt intake rats. Male Sprague-Dawley rats received either 0.9% or 2% salt in their drinking water for 10 days. The stretch-induced ANP secretion from isolated perfused non-beating left atria was accentuated, and the production of cGMP by ANP in renal cortical tissue membranes were pronounced in rats exposed to 0.9% salt for 10 days but not in rats exposed to 2% salt. The levels of ANP receptor density and expression in renal cortex were decreased in 2% salt intake rats but not in 0.9% salt intake rats. No significant differences in atrial and plasma concentrations of ANP and water balance were observed in both salt intakes. Therefore, these results suggest that atrial ANP secretion and its binding sites in the kidney may respond differently to ingested salt concentrations in rats.     

Vasorelaxant effects of and receptors for atrial natriuretic peptides in the mesenteric artery and aorta of the rat

Vasorelaxant effects of different atrial natriuretic peptides (ANP) were measured on rat aortic strips and mesenteric artery rings. These results were compared with the potency of the same peptides to displace 125I-labelled ANP (101-126) on membrane preparations of aorta and of mesenteric vascular bed. In aortic strips and mesenteric artery rings precontracted with phenylephrine (3 X 10(-8) and 10(-6) M, respectively), the order of potency of ANP was as follows: ANP (99-126) greater than ANP (101-126) greater than ANP (103-126) = ANP (103-125) much greater than ANP (103-123). In the displacement binding assays, the order of potency of ANP peptides was similar to that of the relaxation experiments: ANP (99-126) = ANP (101-126) greater than ANP (103-126) = ANP (103-125) much greater than ANP (103-123). When the vessels were precontracted by a smaller concentration of phenylephrine (10(-7) M in mesenteric artery and 10(-8) M in aorta), the IC50 of ANP (101-126) was significantly lower than when the higher concentration of phenylephrine was used. These results show that ANP receptors in the mesenteric artery and in the aorta have similar structural requirements, according to the order of potency of different length ANP, both for binding and myotropic responses.     

A comparison of the uptake of [75Se]selenite, [75Se]selenomethionine and [35S]methionine by tissues of ewes and lambs.

The fate of selenium, given as Na2(75)SeO3, or [75Se]selenomethionine, and of [35S]methionine administered intravenously to ewes and lambs, has been examined. The main intention was to follow the incorporation of selenium into protein in a number of tissues, including liver and kidney, and to measure the extent of that incorporation of selenoamino acid, particularly with respect to the administration of selenite. The ewes chosen were lactating ewes with lambs at foot, and the lambs were animals which had been weaned on to fodder low in selenium and were recovering from white muscle disease with selenium therapy. These two experimental situations were chosen as they offered conditions under which selenium incorporation might be considered to be maximal. Entry of isotope into milk was rapid and was greater when 75Se was given as the selenoamino acid than as selenite. In both ewes and lambs greater amounts of activity, derived from selenite, were bound to plasma proteins than to the proteins of milk. This was particularly evident in samples taken some hours after administration. This ability of the plasma to bind selenium was demonstrated by alkaline dialysis. Small, though significant amounts of selenium, derived from Na2(75)SeO3, were incorporated as selenoamino acids into the proteins of liver, kidney and pancreas, as well as into the proteins of milk and plasma. In ewes, both selenomethionine and selenocystine were identified chromatographically in enzyme digests of defatted liver and kidney. Some differences occurred in the distribution of labelled compounds in organs from lactating ewes and recovering lambs. The incorporation of selenium into protein is discussed briefly in relation to the recent findings of an association between selenium and the enzyme glutathione peroxidase.     

Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites

The present study demonstrates a differential effect of various detergent treatments on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 (0.0125%) caused a decrease of about 70% in [3H]Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on [3H]PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in [3H]Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in [3H]Ro 5-4864 and [3H]PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in [3H]Ro 5-4864 binding, while [3H]PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the peripheral-type benzodiazepine binding site molecule.