Atrial natriuretic peptide and endothelin-3 target renal sodium-glucose cotransporter

Atrial natriuretic peptide (ANP) and endothelin (ET) are endogenous vasoactive factors that exert potent diuretic and natriuretic actions. We have previously shown that ANP and ET-3 act through an NO pathway to inhibit the sodium-glucose cotransporter (SGLT) in the intestine [Gonzalez Bosc LV, Elustondo PA, Ortiz MC, Vidal NA. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 1997; 18: 1491-5; Gonzalez Bosc LV, Majowicz MP, Ortiz MC, Vidal NA. Effects of endothelin-3 on intestinal ion transport. Peptides 2001; 22: 2069-75.]. Here we address the role of ANP and ET-3 on SGLT activity in renal proximal tubules. In rat renal cortical brush border membranes (BBV), fluorescein isothiocianate (FITC) labeling revealed a specific 72-kD peptide that exhibits increased FITC labeling in the presence of Na+ and D-glucose. Using alpha-14C-methylglucose active uptake, rat BBV were shown to possess SGLT activity with an affinity constant (K(0.5) approximately 2.4 mM) that is consistent with the expression of the low-affinity, high-capacity SGLT2 isoform. SGLT2 activity in these preparations is dramatically inhibited by ANP and ET-3. This inhibition is independent of changes in membrane lipids and is mimicked by the cGMP analogue, 8-Br-cGMP, suggesting the involvement of cGMP/PKG pathways. These results are the first demonstration that both ANP and ET-3 inhibit rat cortical renal SGLT2 activity, and suggest a novel mechanism by which these vasoactive substances modulate hydro-saline balance at the proximal tubular nephron level.     

Renal effects of administered atrial natriuretic peptide in the conscious, aging rat

Studies were performed in conscious, chronically catheterized male Sprague-Dawley rats to investigate the effect of administered atrial natriuretic peptide (ANP) on blood pressure, renal hemodynamics and urinary electrolyte excretion. Studies were performed on young adult (3-4 month old) rats and on aging rats (18-24 months of age). Low dose ANP (80 ng/kg/min for 60 min) had no effects on renal hemodynamics in either young or old rats and produced only a slight blood pressure reduction in young animals. No effect on urinary electrolyte excretion was evident in young rats whereas in the old animals, low dose ANP produced large rises in the rate of sodium excretion, fractional excretion of sodium and urine flow rate. A four fold higher dose of ANP evoked a moderate natriuretic and a marked antihypertensive response in young rats. Time control studies indicated that time alone had no influence on urinary sodium excretion rate, the fractional excretion of sodium or urine flow rate. These studies indicate a much enhanced sensitivity to the natriuretic effects of administered ANP by the kidneys of old rats.     

Microvascular effects of atrial natriuretic peptide in rat cremaster

Experiments utilized the open cremaster preparation to test the hypothesis that atrial natriuretic peptide (ANP)-induced volume changes result from microvascular resistance alterations. Atrial natriuretic peptide (25, 100, and 500 ng/kg/min, IV) or vehicle was infused into anesthetized rats. At the two highest ANP infusion rates, mean arterial pressure was significantly reduced from 104 ± 3 (control) to 87 ± 2 and 77 ± 2 mmHg, respectively. Hematocrit was 41.0 ± 0.8 and 45.6 ± 0.9% (p < 0.05) at the end of vehicle and ANP infusions, respectively. Despite these effects of ANP, there were no significant arteriolar or venular diameter alterations. Thirty μM nitroprusside significantly dilated all vessel segments except large venules. These observations suggest that resistance alterations in the skeletal muscle microvasculature are not the cause of ANP-induced fluid movement.     

Cardiac and intestinal natriuretic peptides: insights from genetically modified mice

Since the original discovery of atrial natriuretic peptide (ANP) more than two decades ago, the application of gene targeting technology in mice has provided new insights into the diverse physiological functions of natriuretic peptides and their membrane guanylyl cyclase (GC) receptors. Disruption of the genes for ANP or its receptor, GC-A, demonstrated that this system is not only essential for the maintenance of normal blood pressure and volume, but in addition exerts local antihypertrophic effects in the heart. Disruption of the genes encoding B-type (BNP) or C-type natriuretic peptides (CNP) or the CNP-receptor, GC-B, demonstrated that these "natriuretic" peptides are in fact unlikely to physiologically regulate renal sodium excretion but instead exert important autocrine/paracrine cGMP-mediated effects on cellular proliferation and differentiation in various tissues. Notably, the intestinal peptide uroguanylin, which activates a third guanylyl cyclase receptor (GC-C), exerts diuretic/natriuretic activity and links the intestine and kidney in an endocrine way to modulate renal function in response to oral salt load. Reviewed here is the physiology of cardiac and intestinal natriuretic peptides and their guanylyl cyclase receptors, with special focus on the information gained to date from genetically modified mice.     

Dendroaspis natriuretic peptide is the most potent natriuretic peptide to cause relaxation of lower esophageal sphincter

Atrial natriuretic peptide (ANP) causes relaxation in the opossum lower esophageal sphincter. The effects of dendroaspis natriuretic peptide (DNP) and other natriuretic peptides in the lower esophageal sphincter were not known. We measured the relaxation of transverse strips from the guinea pig lower esophageal sphincter caused by DNP, ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and a natriuretic peptide receptor-C agonist des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANF(4-23)) in vitro. In resting strips of the guinea pig lower esophageal sphincter DNP and BNP caused marked relaxations. Furthermore, in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips, DNP caused marked and BNP caused moderate, concentration-dependent relaxations. ANP as well as CNP caused mild relaxations. In contrast, cANF(4-23) did not cause relaxation. The relative potencies for natriuretic peptides to cause relaxation were DNP>BNP>ANP>=CNP in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips. The DNP and BNP-induced relaxations were not affected by tetrodotoxin or atropine, suggesting that the natriuretic peptide-induced response was not neutrally mediated. In conclusion, these results demonstrate that natriuretic peptides cause the relaxation of the guinea pig lower esophageal sphincter. DNP is the most potent natriuretic peptide to cause lower esophageal sphincter relaxation, which might be mediated by natriuretic peptide receptor-A or a novel DNP-selective natriuretic peptide receptor.     

Potentiation of platelet aggregation by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) has binding sites on a variety of tissues, including human platelets. We have used a new, quenched-flow approach coupled to single-particle counting to investigate the effects of ANP (rat, 1-28) on the initial events (within the first several seconds) following human platelet activation. While ANP alone (1 pM-100 nM) had no effect, ANP significantly potentiated thrombin (0.4 units/ml)-, epinephrine (15 microM)- and ADP (2 or 10 microM)-induced aggregation. Maximum stimulation occurred between 10 to 100 pM. ANP had no influence on the thrombin or ADP-induced increase in platelet volume associated with the "shape change." Since ANP receptors are coupled to a particulate guanylate cyclase and some ANP-induced effects may be mediated through cyclic GMP, we studied how another activator of platelet guanylate cyclase, sodium nitroprusside, affected platelet activation and cyclic nucleotide levels. Sodium nitroprusside (1 microM) inhibited ADP, but not thrombin or epinephrine-induced aggregation. Both sodium nitroprusside (1 microM) and ANP (10 nM) increased cyclic GMP levels by 80% and 37%, respectively, within 60 sec in washed platelets. ANP had no effect on platelet cyclic AMP, while sodium nitroprusside induced a 77% increase. These data suggest that the platelet ANP receptor may be coupled to guanylate cyclase and the rise in cyclic GMP may potentiate platelet function.     

Natriuretic peptides differentially attenuate thrombin-induced barrier dysfunction in pulmonary microvascular endothelial cells

Previous studies have described a protective effect of atrial natriuretic peptide (ANP) against agonist-induced permeability in endothelial cells derived from various vascular beds. In the current study, we assessed the effects of the three natriuretic peptides on thrombin-induced barrier dysfunction in rat lung microvascular endothelial cells (LMVEC). Both ANP and brain natriuretic peptide (BNP) attenuated the effect of thrombin on increased endothelial monolayer permeability and significantly enhanced the rate of barrier restoration. C-type natriuretic peptide (CNP) had no effect on the degree of thrombin-induced monolayer permeability, but did enhance the restoration of the endothelial barrier, similar to ANP and BNP. In contrast, the non-guanylyl cyclase-linked natriuretic peptide receptor specific ligand, cyclic-atrial natriuretic factor (c-ANF), delayed the rate of barrier restoration following exposure to thrombin. All three natriuretic peptides promoted cGMP production in the endothelial cells; however, 8-bromo-cGMP alone did not significantly affect thrombin modulation of endothelial barrier function. ANP and BNP, but not CNP or c-ANF, blunted thrombin-induced RhoA GTPase activation. We conclude that ANP and BNP protect against thrombin-induced barrier dysfunction in the pulmonary microcirculation by a cGMP-independent mechanism, possibly by attenuation of RhoA activation.     

Natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with natriuretic peptide receptor-B

Atrial natriuretic peptide (ANP) binding sites have been demonstrated in the guinea-pig gallbladder muscle with unclear function. To investigate effects of natriuretic peptides in the gallbladder, we measured relaxation of isolated human and guinea-pig gallbladder strips caused by natriuretic peptides, including C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP) and ANP, as well as des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. Results in the human gallbladder were similar to those in the guinea-pig gallbladder. CNP, BNP, ANP and cANP(4-23) alone did not cause contraction or relaxation in resting gallbladder strips. However, in carbachol or endothelin-1-contracted strips, CNP caused moderate, sustained and concentration-dependent relaxation. The relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted gallbladder strips and not by tetrodotoxin in carbachol-contracted strips. These indicate a direct effect of CNP on the gallbladder muscle. The relative potencies for natriuretic peptides to cause relaxation were CNP>BNP> or = ANP. cANP(4-23) did not cause relaxation. These indicate the existence of the natriuretic peptide receptor-B (NPR-B) mediating the relaxation. Taken together, these results demonstrate that natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with the natriuretic peptide receptor-B.     

Modification of the renal response to endopeptidase inhibition and atrial natriuretic peptide infusion in normal dogs.

Inhibition of intrarenal neutral endopeptidase 24:11 (NEP) increases the natriuretic response to infused atrial natriuretic peptide (ANP). In various models of canine heart failure, angiotensin and kinins have been shown to modulate ANP and (or) NEP activity. In the present study, we examined possible modulators of NEP activity in normal dogs by infusing various agents into the left renal artery (or by denervating the left kidney) and comparing the response of this kidney with that of the contralateral one following the combined intravenous infusion of Squibb 28603 (a potent NEP inhibitor) and ANP (75 ng.kg-1.min-1). Four dogs received angiotensin (1.5 ng.kg-1.min-1) into the left renal artery, 8 dogs received saralasin (5 micrograms/min), 5 dogs received noradrenaline (2 micrograms/min), and 6 dogs received bradykinin (3 micrograms/min). Five dogs underwent left renal denervation. Angiotensin inhibited sodium excretion following the NEP inhibitor alone and after the NEP inhibitor plus ANP. Saralasin augmented the natriuretic response. None of the other protocols influenced sodium excretion. We conclude that angiotensin may modulate either the enzymatic degradation of ANP or influence its renal tubular effects.     

Functional and structural characterization of a novel member of the natriuretic family of peptides from the venom of Pseudocerastes persicus

A novel peptide, PNP (Pseudocerastes persicus natriuretic peptide), was isolated from the venom of the Iranian viper P. persicus. Amino acid sequencing revealed that the 37-residue peptide belongs to the family of natriuretic peptides. The physiological effects of intra-venously PNP infused into anesthetized rats on urine flow, sodium excretion and blood pressure were comparable to those of atrial natriuretic peptide (ANP). In PC12 cells that were treated with either PNP, ANP, or C-type natriuretic peptide, PNP induced a similar cGMP response as ANP. Since PC12 cells only express the natriuretic peptide receptor (NPR)-A receptor we conclude that PNP binds to the NPR-A receptor. The solution conformation of PNP was characterized using (1)H nuclear magnetic resonance spectroscopy and indicates a high degree of conformational flexibility.     

Natriuretic peptides stimulate steroidogenesis in the fetal rat testis

To study the regulation of fetal testicular steroidogenesis in the rat, we examined effects of members of the natriuretic peptide family, that is, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), on testosterone production of dispersed Leydig cells of rat fetuses at Embryonic Day (E) 18.5. All three peptides stimulated testosterone production, with significant effect at concentrations > or =1 x 10(-8) mol/L of ANP, > or =1 x 10(-9) mol/L of BNP, and > or =1 x 10(-6) mol/L of CNP. Likewise, receptors for all three peptides (i.e., NPR-A, NPR-B, and NPR-C) were expressed in the fetal testis as early as E15.5. The natriuretic peptides had no effect on cAMP production by fetal Leydig cells. When tested in combination with two other peptides previously shown to stimulate fetal testicular steroidogenesis, vasoactive intestinal peptide and pituitary adenylate cyclase-stimulating polypeptide (PACAP-27), the combined effects did not differ significantly from the maximum effect with any one of the peptides alone. In conclusion, our present findings provide both functional and molecular evidences for NPR-A, NPR-B, and NPR-C in the fetal testis. Because ANP has previously been detected in fetal plasma and we now demonstrate the expression of BNP and CNP in fetal testes, these findings indicate involvement of the natriuretic peptides in endocrine and paracrine regulation during the early phase of fetal testicular steroidogenesis at E15.5--19.5 (i.e., before the onset of pituitary LH secretion).     

Attenuated diuretic and natriuretic effects of atrial natriuretic peptide in rats with heart failure

Plasma levels of atrial natriuretic peptide (ANP) and renal responses to ANP were examined in rats with chronic cardiac failure produced by coronary artery ligation and in sham-operated controls. Plasma ANP levels were elevated in the rats with severe cardiac failure as compared with the controls (P less than 0.001). ANP injections at the doses of 1, 5, 25 and 50 micrograms/kg increased water and sodium excretion significantly at all but the lowest dose in the controls; only the two largest doses caused clear diuresis and natriuresis in the heart failure group. The diuretic and natriuretic effects of ANP were significantly weaker at the doses of 5 and 25 micrograms/kg in the rats with heart failure as compared with the controls. We conclude, that natriuretic and diuretic effects of ANP are attenuated in this chronic heart failure mode.     

Four-hour atrial natriuretic peptide infusion in conscious rats: effects on urinary volume, sodium, and cyclic GMP

Atrial natriuretic peptide (ANP) is released from the cardiac atria in response to acute volume loads; when infused acutely ANP causes diuresis and natriuresis. Cyclic GMP (cGMP) appears to be the second messenger for ANP in the kidney. The role that ANP plays in the long-term regulation of salt and water balance is unclear, however, since resistance to ANP's natriuretic and diuretic activity develops during prolonged administration. The purpose of the present study is to examine the relationship between the rate of cGMP excretion in response to ANP and the development of resistance to ANP's diuretic and natriuretic activity. Following a 30-min baseline period of infusion of Ringer's solution conscious rats received ANP at 15 micrograms/kg/hr (n = 6) or Ringer's alone (n = 5) for 240 min. ANP-infused rats had a significant diuresis and natriuresis during the first hour of infusion; urinary cGMP excretion also increased compared to baseline. By 120 min after initiating the infusion in ANP-rats urinary volume and sodium excretion had declined to values not significantly different from those of baseline or control. In contrast, urinary cGMP excretion remained elevated for the duration of the ANP infusion, whether compared to baseline values or the control group. Resistance to the diuretic and natriuretic activity of ANP is not a result of mechanisms that involve cGMP generation.     

The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and Bay K8644 and inhibited by 8-bromo-cyclic GMP

The role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release was studied using the isolated perfused rat heart. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the protein kinase C pathway, produced a dose-dependent increase in perfusate ANP immunoreactivity. Bay k8644, a putative calcium channel activator, and forskolin, which stimulates adenylate cyclase, induced a sustained increase in ANP secretory rate. TPA in combination with either Bay k8644 or forskolin induced higher ANP secretion than the calculated additive value for each agent. 8-bromo-cyclic GMP and sodium nitroprusside, when given alone, had no effect on ANP secretion, but delayed the TPA-stimulated increase in perfusate ANP. ANP secretion appears therefore to be mediated both by the phosphoinositide and the cAMP system, whereas the cGMP pathway may be inhibitory.     

Involvement of drinking and intestinal sodium absorption in hyponatremic effect of atrial natriuretic peptide in seawater eels

Atrial natriuretic peptide (ANP) decreases plasma Na+ concentration and promtes seawater (SW) adaptation in eels. The hyponatremia may most probably be caused by increased branchial extrusion of Na+, but the mechanism has not been determined yet. The present study examined initially the effects of ANP on branchial Na+ efflux in vivo using isotopic 22Na. However, the efflux rate was not altered by infusion of a hyponatremic dose of ANP (5 pmol.kg(-1).min(-1)). Therefore, we sought to examine whether the ANP-mediated hyponatremia is caused by a decrease in the uptake of Na+ from the environment. Since a decrease in drinking was highly correlated with a degree of hyponatremia, conscious SW eels were infused with dilute SW into the stomach at a normal drinking rate to offset the antidipsogenic effect of ANP. Under this regimen, the hyponatremic effect of ANP was abolished. Then, we examined the site of Na+ absorption in the alimentary tract by measuring the changes in ion composition of intraluminal fluid along the tract. Since Na+ was absorbed at the esophagus and anterior/middle intestine, a sac was prepared at each site and the effects of ANP were examined in situ in conscious SW eels. ANP infusion did not alter Na+ absorption at the esophagus, but it profoundly reduced the absorption at the intestine. Together with our previous finding that ANP does not alter renal Na+ excretion, we propose that ANP reduces plasma Na+ concentration in SW eels by inhibiting drinking and subsequent absorption of Na+ by the intestine.     

ANP stimulates hepatocyte Ca2+ efflux via plasma membrane recruitment of PKGIalpha

In rat hepatocytes, atrial natriuretic peptide (ANP) elevates cGMP through activation of particulate guanylyl cyclase and attenuates Ca²⁺ signals by stimulating net plasma membrane Ca²⁺ efflux. We show here that ANP-stimulated hepatocyte Ca²⁺ efflux is mediated by protein kinase G (PKG) isotype I. Furthermore, we show that ANP recruits endogenous PKGIα, but not PKGIβ, to the plasma membrane. These effects are mimicked by 8-bromo-cGMP, but not by the soluble guanylyl cyclase activators, sodium nitroprusside and YC-1. We propose that ANP, through localized cGMP elevation, promotes plasma membrane recruitment of PKGIα, which, in turn, stimulates Ca²⁺ efflux.     

Cardiac natriuretic peptides and pancreatic islet blood flow in anesthetized rats.

The aim of the study was to evaluate effects of cardiac natriuretic peptides on splanchnic circulation, especially to the pancreatic islets. Pentobarbital-anesthetized rats were infused intravenously (0.01 ml/min for 20 min) with saline, atrial natriuretic peptide (ANP; 0.25 or 0.5 microg/kg BW/min), brain natriuretic peptide (BNP; 0.5 microg/kg BW/min) or C-type natriuretic peptide (CNP; 0.5 or 2.0 microg/kg BW/min). Splanchnic blood perfusion was then measured with a microsphere technique. Mean arterial blood pressure was decreased by ANP and BNP, but not by CNP. The animals given the highest dose of ANP became markedly hypoglycemic, whilst no such effects were seen in any of the other groups of animals. Total pancreatic blood flow was decreased by the highest dose of CNP, whereas no change was seen after administration of the other peptides. Islet blood flow was increased by the highest dose of ANP. Neither BNP nor CNP affected islet blood flow. None of the natriuretic peptides influenced duodenal, colonic or arterial hepatic blood flow. It is concluded that cardiac natriuretic peptides exert only minor effects on splanchnic blood perfusion in anesthetized rats. However, islet blood perfusion may be influenced by ANP.     

Sodium intake influences the effects of atriopeptin on blood pressure and transcapillary fluid shift in the rat

The influence of chronic changes in sodium intake on the acute effects of atrial natriuretic peptide (ANP) on arterial pressure and fluid translocation was assessed in acutely binephrectomized rats. After 3 weeks of either low sodium or high sodium diet, animals were administered ANP at doses of 0.1 and 1 microgram/kg/min. A marked and irreversible hypotensive response to ANP was observed with the higher infusion rate in the low sodium group, whereas blood pressure did not change significantly in the other groups. The effect of ANP on plasma protein concentration was less marked than that on hematocrit in all groups and was not significantly affected by sodium intake. The effect of both doses of ANP on hematocrit was enhanced in the high sodium group, indicating that the fluid shift out of the intravascular compartment was magnified by high sodium intake.     

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.     

Renal effects of dopamine and ANP in high volume expanded rats

Both dopamine (DA) and atrial natriuretic peptide (ANP) have been postulated to exert similar effects on the kidney, participating in the regulation of body fluid and sodium homeostasis. In the present study, experiments were performed in anesthetized and isotonic sodium chloride volume expanded rats. After acute volume expansion at 15 % of body weight during 30 min, glomerular filtration rate, urine output, sodium excretion, fractional sodium excretion, proximal and distal sodium excretion and blood pressure were measured. In additional groups we administered ANP or haloperidol or the combination of both to volume expanded animals. Blockade of DA receptors with haloperidol, attenuated diuretic and natriuretic responses to volume load. Proximal sodium excretion was not modified by haloperidol in all experimental groups of rats. Reduction in distal tubular excretion was induced by haloperidol in saline infusion expanded rat but not in ANP treated expanded animals. In conclusion, when exaggerated volume expansion is provoked, both DA and ANP exert renal tubular events, but ANP have a major central role in the regulation of renal sodium handling.