Addition Of Atrial Natriuretic Peptide Research Articles

Hemodynamic effects of atrial natriuretic peptide in ischemia-repertusion injury that occurs after exercise.

Atrial natriureticpeptide (ANP) is known as a protective agent against ischemia-reperfusion injuryfor cardiomyocytes. We compared the hemodynamic effects of ANP and isatin, which is known as an ANP receptor blocker, in ischemia followed by reperfusion in exercised rat hearts with nonexercised ones. Isolated hearts were perfused in 4 exercised (E) groups after a running protocol for 5 days and 4 nonexercised (NE) groups. In the first protocol, ANP was added to the perfusion solution before ischemia in an E and NE group. In the second protocol, different doses of isatin (0.1, 10, 100 µM/L) were added to the perfusion solution before ANP in 3 E and 3 NE groups. Left ventricular developed pressure (LVDP) and maximum and minimum rates of change in left ventricular pressure (dP/dtmax and dP/dtmin) were recorded. Higher LVDP and dP/dtmin values were observed in the E group than the NE group following addition of ANP before ischemia. Values of dP/dtmax were higher in the E group at the first minute of reperfusion period. Hemodynamic difference was not observed between groups given the same amount of isatin before ANP. This study indicated that higher ANP concentrations before ischemia were more effective on the left ventricle contractility and relaxation functions in the hearts that were exposed to exercise.

Modulation of Sarcolemmal ATP-Sensitive Potassium Channels by Atrial Natriuretic Peptide in Ventricular Cardiomyocytes

ATP-sensitive potassium (KATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation in the heart. Natriuretic peptides (NPs) produced by the heart, the vasculature and the kidney exert physiologically important actions such as natriuresis and inhibition of sympathetic tone and cardiomyocyte hypertrophy. Our recent study reveals that the gaseous messenger nitric oxide evokes cardiac KATP channel stimulation via activation of soluble guanylyl cyclase and subsequently a cGMP-dependent signaling pathway. Binding of NPs like atrial natriuretic peptide (ANP) to the type 1 NP receptor (NPR-A) also increases intracellular cGMP level, through activating guanylyl cyclase intrinsic to the receptor; however, how ANP modulates the function of cardiac KATP channels remains to be established. By performing cell-attached patch recordings in ventricular cardiomyocytes acutely isolated from adult rabbits, we found that the single-channel activity of sarcolemmal KATP (sarcKATP) channels induced by sodium azide through metabolic inhibition was potentiated by addition of ANP (100 nM) (n=16), whereas the potentiation by ANP was abolished when the NPR-A antagonist anantin (1 μM) was coapplied (n=5); these findings suggest that the stimulatory action of ANP is mediated by NPR-A activation, which may depend on generation of diffusible cGMP. Indeed, sarcKATP channel stimulation by ANP in intact ventricular myocytes was significantly abated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823 (1 μM) (n=5). Moreover, the ANP effect was prevented by selective suppression of extracellular signal-regulated protein kinase (ERK)1/2 (n=6) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (n=6). Collectively, here we demonstrate that the polypeptide hormone ANP positively modulates ventricular KATP channel function via activation of NPR-A, PKG, ERK1/2 and CaMKII. By opening myocardial KATP channels, this signaling pathway may regulate cardiac excitability and contribute to cytoprotection against ischemia-reperfusion injury.

Atrial Natriuretic Peptide Reduces Vascular Leakage and Choroidal Neovascularization

Atrial natriuretic peptide (ANP) is a hormone with diuretic, natriuretic, and vasodilatory properties. ANP blocks vascular endothelial growth factor (VEGF) production and signaling in vitro; however, its role in vascular leakage and angiogenesis is unknown. In vitro, retinal barrier permeability (transepithelial electrical resistance (TEER)) was measured in cultured retinal endothelial (HuREC) and retinal epithelial (ARPE-19) cells with VEGF (10 ng/ml), ANP (1 pM to 1 micromol/L), and/or isatin, an ANP receptor antagonist. In vivo, blood-retinal barrier (BRB) leakage was studied using the Evans Blue dye technique in rats treated with intravitreal injections of ANP, VEGF, or vehicle. Choroidal neovascularization was generated by laser injury, and 7 days later, lesion size and leakage was quantitated. ANP significantly reversed VEGF-induced BRB TEER reduction in both HuREC and ARPE-19 cells, modeling the inner and the outer BRB, respectively. Isatin, a specific ANP receptor antagonist, reversed ANP's effect. ANP reduced the response of ARPE-19 cells to VEGF apically but not basolaterally, suggesting polarized expression of the ANP receptors in these cells. ANP's TEER response was concentration but not time dependent. In vivo, ANP significantly reduced VEGF-induced BRB leakage and the size of laser-induced choroidal neovascularization lesions. In sum, ANP is an effective inhibitor of VEGF-induced vascular leakage and angiogenesis in vivo. These results may lead to new treatments for ocular diseases where VEGF plays a central role, such as age-related macular degeneration or diabetic retinopathy.

Natriuretic peptides up-regulate aquaporin 3 in a human colonic epithelial cell line.

Several specialized channels termed aquaporins (AQPs) facilitate water transport in the gastrointestinal tract. AQP3 localizes to epithelial cells in the human small intestine and colon. However, the regulatory mechanisms responsible for AQP3 function in the gastrointestinal tract are not well understood. To characterize the regulation of AQP3 expression by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), we studied mRNA expression by Northern blotting, protein expression by Western blotting and DNA binding activity by electrophoretic mobility shift assay (EMSA) in the human colonic epithelial cell line HT-29. We also used several inhibitors to investigate signal transduction. AQP3 mRNA was up-regulated in addition to ANP (>100 nM) and BNP (>10 nM). The expression of AQP3 protein was enhanced at 1 h after the addition of ANP and BNP. The combination of protein kinase-A (PK-A) and protein kinase-G (PK-G) inhibitors completely inhibited the expression of AQP3 mRNA enhanced by ANP or BNP to its basal level. The EMSA of the cyclic-AMP response element (CRE) in HT-29 cells revealed a single band. These results indicate that ANP and BNP up-regulated the expression of AQP3 mRNA and protein, and both PK-A and PK-G dependent pathways mediated this effect.

Inhibition of leptin release by atrial natriuretic peptide (ANP) in human adipocytes

The addition of atrial natriuretic peptide (ANP) to isolated human adipocytes in primary culture from very obese individuals resulted in an inhibition of leptin release after a 24- or 48-hr incubation. There was also an inhibition of leptin release by isoproterenol (ISO) that was partially reversed by insulin, whereas the inhibition due to ANP was unaffected. Similar results were seen with N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H-89), which is a cell-permeable inhibitor of protein kinase A. H-89 markedly reduced the effects of ISO on both lipolysis and leptin release without affecting the stimulation of lipolysis or the inhibition of leptin release due to ANP. Inhibition of endogenous nitric oxide formation using N ω-nitro- l-arginine resulted in a 20% increase in leptin release over 48 hr, which suggests that the nitric oxide/cyclic GMP pathway might play a small role in the regulation of endogenous leptin release. Similarly, the addition of the nitric oxide donor ( Z)-1-[2-aminoethyl)- N-(2-aminoethyl)diazen-1-ium-1,2-diolate (DETA NONOate) at 0.1 or 1 μM to explants of human adipose tissue enhanced lipolysis by 29%. Our data demonstrate that the lipolytic effect of ANP is probably secondary to stimulation of cyclic GMP accumulation in human adipocytes, and this is accompanied by an inhibition of leptin release.

Mechanisms of hepatocyte protection against hypoxic injury by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) reduces ischemia and/or reperfusion damage in several organs, but the mechanisms involved are largely unknown. We used freshly isolated rat hepatocytes to investigate the mechanisms by which ANP enhances hepatocyte resistance to hypoxia. The addition of ANP (1 μmol/L) reduced the killing of hypoxic hepatocytes by interfering with intracellular Na + accumulation without ameliorating adenosine triphosphate (ATP) depletion and pH decrease caused by hypoxia. The effects of ANP were mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (cGMP) and were associated with the activation of cGMP-dependent kinase (cGK), suggesting the involvement of guanylate cyclase–coupled natriuretic peptide receptor (NPR)-A/B ANP receptors. However, stimulating NPR-C receptor with des-(Gln 18, Ser 19,Gly 20,Leu 21,Gly 22)-ANP fragment 4-23 amide (C-ANP) also increased hepatocyte tolerance to hypoxia. C-ANP protection did not involve cGK activation but was instead linked to the stimulation of protein kinase C (PKC)-δ through G i protein– and phospholipase C–mediated signals. PKC-δ activation was also observed in hepatocytes receiving ANP. The inhibition of phospholipase C or PKC by U73122 and chelerythrine, respectively, significantly reduced ANP cytoprotection, indicating that ANP interaction with NPR-C receptors also contributed to cytoprotection. In ANP-treated hepatocytes, the stimulation of both cGK and PKC-δ was coupled with dual phosphorylation of p38 mitogen-activated protein kinase (MAPK). The p38 MAPK inhibitor SB203580 abolished ANP protection by reverting p38 MAPK-mediated regulation of Na + influx by the Na +/H + exchanger. In conclusion, ANP recruits 2 independent signal pathways, one mediated by cGMP and cGK and the other associated with G i proteins, phospholipase C, and PKC-δ. Both cGK and PKC-δ further transduce ANP signals to p38 MAPK that, by maintaining Na + homeostasis, are responsible for ANP protection against hypoxic injury. (H EPATOLOGY 2003;37:277-285.)

Atrial natriuretic peptide inhibits iodide uptake and thyroglobulin messenger ribonucleic acid expression in cultured bovine thyroid follicles.

The involvement of atrial natriuretic peptide (ANP) in the regulation of thyroid gland is supported by the presence of high-affinity ANP receptors and the identification of the peptide in thyroid follicular cells. The aim of this work was to study the action of ANP on parameters of thyroid hormone biosynthesis and analyze the intracellular mechanism of the ANP action in cultured bovine thyroid follicles. The addition of ANP (0.1-10 nM) to the culture medium for 24 h inhibited the TSH (thyroid-stimulating hormone)-stimulated iodide uptake with a maximal inhibition at 1 nM ANP. When thyrocytes were incubated with 10 nM ANP the inhibitory effect slightly increased from 24 to 72 h. Thyroglobulin (Tg) mRNA expression was reduced by 1 and 10 nM ANP. After 24 h of treatment with the cGMP analogue, N(2),2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate [(Bu)(2)cGMP] (0.1 and 1 mM), an inhibition of iodide uptake and Tg mRNA expression was obtained, evidencing a cGMP-mediated inhibitory signal in the thyroid cell. A reduction of the cAMP production was induced by incubation of thyroid follicles with 1 and 10 nM ANP for 24 h. Under a similar treatment the cGMP accumulation was increased only by 10 nM ANP. The inhibitory effect of ANP on Tg mRNA level was reverted in the presence of pertussis toxin, an inhibitor of the G(i)-protein-mediated reduction of the adenylate cyclase activity. These results indicate an inhibitory action of ANP on parameters of thyroid hormone biosynthesis. A G(i)-protein-mediated reduction of the cAMP production seems to be the main factor involved in the ANP action although a role of the cGMP pathway should not be discarded specially at high ANP levels.

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells

Purpose. Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair. Methods. The SV-40 immortalized RCEC were cultured in 24-well plates until they became confluent. Wounds of uniform size (8 mm diameter) were created and the cells allowed to grow in the presence and absence of EGF and/or other agents. At prescribed time intervals, the cells were stained by Giemsa and the wound areas digitized and quantified by Sigma Image Scan System. The cGMP contents in RCEC, treated with or without ANP or SNP, were measured by radioimmunoassay. Results. Addition of EGF (1–100 ng/ml) to RCEC stimulated cell proliferation which significantly reduced the time required for wound closure. Addition of ANP (1 nM to 10 µM) or SNP (10 µM to 1 mM), in the presence of EGF, dose-dependently inhibited the growth factor-stimulated wound closure in RCEC. When added alone to the cells, ANP or SNP increased cGMP accumulation in a dose-dependent manner. Addition of ANP (1 µM) or SNP (1 mM) to primary corneal epithelial cells, in the presence and absence of EGF, also inhibited the wound closure with a corresponding increase in cGMP contents. Treatment of the cells with ODQ (10 nM to 10 µM), a soluble guanylate cyclase inhibitor, dose-dependently decreased the SNP-induced accumulation of cGMP, and reversed the inhibitory effect of SNP on EGF-stimulated wound closure. Addition of membrane-permeable cGMP analog, 8-bromo-cGMP, to RCEC inhibited the EGF-stimulated wound closure in a dose-dependent manner. Treatment of RCEC with mitomycin C (5 µM) exerted a marked inhibitory effect on wound closure in the presence and absence of EGF, and also abrogated the inhibitory effect of 8-bromo-cGMP on wound closure in the EGF-treated and untreated cells. Conclusions. The results demonstrate that ANP and SNP inhibit the EGF-stimulated wound repair in RCEC. The effect of these agents is mediated via activation of guanylate cyclases that generate cGMP. Cyclic GMP appears to exert its inhibitory effect at the level of cell proliferation and not cell migration. The data suggest an important role for cGMP-dependent protein kinase in proliferation of RCEC stimulated by EGF.

Influence of atrial natriuretic peptide on thymocyte development in fetal thymic organ culture

The aim of the present study was to examine the effect of atrial natriuretic peptide (ANP) on T-cell development. The addition of ANP (10 −6–10 −10 M, 7 days) to organ cultures of 14 day old fetal mouse thymuses (FTOC) resulted in a decreased cell yield (up to 65% of the amount in control cultures). Flow cytometric analysis revealed a decrease (up to 35%) in the percentage of the CD4 +CD8 + thymocytes accompanied by an increase (100%) of the CD4 −CD8 − cell population in FTOC incubated with ANP. Exposure of FTOC to ANP furthermore decreases the percentage of CD5 expressing cells and increases the mean size of thymocytes estimated by monitoring their forward scatter intensity (flow cytometry). Thus, ANP seems to affect maturation and differentiation of thymocytes.

Effect of efflux of guanosine 3′,5′ cyclic monophosphate (cGMP) on the regulation of intracellular levels of cGMP in the inner medullary collecting duct

Guanosine 3′,5′ cyclic monophosphate (cGMP) acts as a second messenger in the inner medullary collecting duct (IMCD) where it inhibits sodium transport; therefore, it is important to investigate processes that regulate intracellular cGMP levels. We hypothesized that efflux of cGMP is a major mechanism in this process. IMCDs were isolated from rat kidneys and exposed to atrial natriuretic peptide (ANP) for 0, 3, and 20 min in buffer with or without isobutyl methylxanthine (IBMX), a phosphodiesterase (PDE) inhibitor. Extracellular and intracellular cGMP levels were measured by radioimmunoassay. After cGMP production was stimulated by addition of ANP (10 −7 M), cGMP efflux was 3.29 ± 0.60 fmol/μg · min at 3 min (P = 0.016) and 0.51 ± 0.25 fmol/μg · min at 20 min (NS). Intracellular cGMP peaked at 3 min at 26.66 ± 4.84 fmol/μg (P = 0.017) and decreased to 12.98 ± 2.76 fmol/μg at 20 min (NS). Since PDEs were inhibited, these data suggest that efflux regulates intracellular cGMP. Efflux was correlated with intracellular cGMP levels (r = 0.97). After 3 min of stimulation with 10 −9 M ANP, efflux was 2.0 ± 0.3 fmol/μg · min, while intracellular cGMP content was 13.8 ± 3.6 fmol/μg. With 10 −8 M ANP, efflux was 3.5 ± 0.7 fmol/μg · min, while intracellular content was 20.5 ± 7.6 fmol/μg; and at 10 −7 M ANP, efflux was 5.1 ± 0.6 fmol/μg · min and intracellular content was 26.6 ± 8.0 fmol/μg. By 20 min, efflux and intracellular levels had returned to control values. Finally, we measured efflux and PDE activity in the absence of IBMX. Efflux was ≈15% of PDE activity (N = 7). We conclude that cGMP efflux is concentration-dependent and, under some circumstances, may be an important regulator of intracellular cGMP levels in isolated IMCDs.

A mutation of the atrial natriuretic peptide (guanylyl cyclase-A) receptor results in a constitutively hyperactive enzyme.

Mutation of an invariant glutamate residue found within the catalytic domain of guanylyl cyclases resulted in a dramatic 14-fold increase in the activity of the guanylyl cyclase-A receptor. Even in the presence of Mn2+/Triton X-100, a treatment previously thought to yield hormone-independent and maximum cyclase activity, the mutant enzyme remained 7-fold more active; to our knowledge, this is the first example of a protein modification or of an added agent that significantly increases cyclase activity in the presence of Mn2+/Triton X-100. Intracellular concentrations of cGMP in cells expressing the mutant (E974A) cyclase were only marginally elevated by the addition of atrial natriuretic peptide, and in broken-cell preparations, the mutant enzyme also was relatively insensitive to ligand/regulatory nucleotide. The marked increase in cyclase activity was not due to a relief of protein kinase domain inhibition, since the point mutation caused 7- to 13-fold elevations in guanylyl cyclase-A activity when the protein kinase homology domain was deleted. The E974A mutation also altered the kinetics from positive cooperative to linear with respect to MnGTP, suggesting disruption of subunit-subunit interactions. Thus, a single point mutation within the catalytic domain of a guanylyl cyclase results in a constitutively hyperactive enzyme that is independent of protein kinase domain regulation.

Effect of luminal angiotensin II and ANP on early and late cortical distal tubule HCO3- reabsorption.

Bicarbonate reabsorption was evaluated by stationary microperfusion "in vivo" early distal (ED) and late distal (LD) segments of at kidney. Intratubular pH was recorded by double-barreled of H+ exchange resin/reference (1 M KCl) microelectrodes for the determination of HCO3- reabsorption. In the presence of angiotensin II (ANG II) (10(-12) M), a significant increase in HCO3- reabsorption was observed both in ED (from 0.930 +/- 0.060 to 2.64 +/- 0.210 nmol.cm-2.s-1 in luminally perfused tubules and from 0.850 +/- 0.040 to 2.03 +/- 0.210 nmol.cm-2.s-1 during capillary perfusion) and LD segments from 0.310 +/- 0.130 to 2.16 +/- 0.151 nmol.cm-2.s-1 during luminal perfusion and from 0.530 +/- 0.031 to 2.16 +/- 0.211 nmol.cm-2.s-1 with capillary perfusion). The addition of the AT1-receptor antagonist losartan (10(-6) M) to luminal perfusion blocked luminal ANG II-mediated stimulation in ED and LD segments. 5-(N,N-hexamethylene)amiloride (10(-4) M) added to luminal perfusion inhibited luminal ANG II-mediated stimulation in ED (by 81%) and LD (by 54%) segments. The addition of bafilomycin A1 (2 x 10(-7) M) to luminal perfusion does not affect luminal ANG II-mediated stimulation in ED segments but reduces it in LD segments (by 33%). During the addition of atrial natriuretic peptide (ANP) (10(-6) M) or ANG II plus ANP in both segments, no significant differences in HCO3- reabsorption were observed. Our results indicate that luminal ANG II acts to stimulate Na+/H+ exchange in ED and LD segments via activation of AT1 receptors, as well as the vacuolar H(+)-adenosinetriphosphatase in LD segments. ANP does not affect HCO3- reabsorption in either ED or LD segments and does not impair the stimulation caused by ANG II.

Atrial natriuretic peptide inhibits mesangial cell proliferation induced by early postnephrectomy serum.

Rat kidney mesangial or tubular cells were grown in tissue culture medium supplemented with sera of 20 sham-operated vs. 20 nephrectomized Charles River rats. Each cell sample was grown with or without addition of atrial natriuretic peptide (ANP) to the medium. ANP completely abolished the growth stimulatory effect of postnephrectomy serum [i.e., effect of renotropic factor(s) present in the serum] on mesangium cells. Tubular cell proliferation was unchanged. We propose that interaction between renotropin and atrial natriutetic peptide activity might be one of the possible mechanisms regulating compensatory renal growth following nephrectomy.

ANP has no postsynaptic effect on autonomic regulation of cardiac pacemaker rate in the rat.

We studied whether atrial natriuretic peptide (ANP) influences sinoatrial node pacemaker activity or whether it modifies the response to activation of postsynaptic autonomic receptors. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium (45 mg/kg). Their hearts were removed quickly and placed in physiological salt solution. The atria were isolated; the right intra-atrial chamber was exposed to allow intracellular recording from sinoatrial node pacemaker cells. The tissue was placed in a temperature-regulated recording chamber and superfused with warmed oxygenated physiological salt solution. With use of standard microelectrode recording techniques, action potentials were recorded from spontaneously depolarizing cells in the presence of muscarine (62.5-500 nM) or norepinephrine (0.1 and 1.0 microM). Muscarine reduced the frequency of action potentials dose dependently, whereas norepinephrine increased their frequency. The addition of ANP (0.1-100 nM) to the superfusion had no effect on the frequency of action potentials during the superfusion of physiological salt solution or in the presence of either muscarine or norepinephrine. We conclude that ANP does not act on cardiac pacemaker cells to modulate the effect of neurotransmitters.

ANP inhibits NaCl absorption and elicits Cl secretion in porcine colon: evidence for cGMP and Ca mediation.

The effects of atrial natriuretic peptide (ANP) on ion transport were examined in the isolated, short-circuited proximal colon epithelium of the pig. Addition of ANP to the serosal solution decreased the rate of neutral Na and Cl absorption and elicited electrogenic Cl secretion. Amiloride, at a concentration that inhibits Na-H exchange, produced an identical inhibition of Na transport and abolished the ANP-induced decrease in Na absorption. In contrast, serosal addition of bumetanide, an inhibitor of Na-K-2Cl cotransport, partially inhibited the short-circuit current (Isc) response to ANP. Whereas 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) produced qualitatively similar effects as ANP, relatively high concentrations of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) or prostaglandin E2 were required to alter NaCl transport. Furthermore, incubation of colonic mucosa with ANP induced a threefold increase in cGMP content, whereas cAMP was unaffected. The ANP-induced Cl secretion and Isc were diminished with tetrodotoxin and verapamil, whereas these agents were without effect on the ANP-induced inhibition of Na and Cl absorption. Results indicate that ANP inhibits net colonic absorption of ions by antiabsorptive and secretory mechanisms that are dependent on both cGMP and Ca.

Colocalization of receptors for vasoactive peptides on astrocytes of cultured rat spinal cord and brain stem: electrophysiological effects of atrial and brain natriuretic peptide, neuropeptide Y and bradykinin

Electrophysiological studies have been made of the actions of the vasoactive peptides atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), neuropeptide Y (NPY) and bradykinin (BK) on astrocytes in explant cultures of rat brain stem and spinal cord. Addition of ANP, BNP and NPY to the bathing solution at concentrations of 10 −8 and 10 −7 M caused depolarizations of the majority of astrocytes studied, some cells were hyperpolarized and on a small number of cells the peptides had no action. BK (10 −8 M) only induced depolarizations of almost all astrocytes studied. Our electrophysiological data provide strong evidence for the presence of receptors for these vasoactive peptides on astrocytes. When ANP, BNP, NPY and BK were tested on the same cell, all peptides were effective, suggesting a coexistence of peptidergic receptors on astrocytes.

Dephosphorylation of the guanylyl cyclase-A receptor causes desensitization.

Atrial natriuretic peptide (ANP) binds to the guanylyl cyclase-A (GC-A) receptor found in tissues such as the kidney and adrenal gland, resulting in marked elevations of the intracellular signaling molecule, cGMP. Here, GC-A is shown to exist as a phosphoprotein when expressed in human embryonic 293 cells. The 32P is principally associated with phosphoserine, with only trace amounts of phosphothreonine. The addition of ANP causes a time-dependent dephosphorylation of the receptor, as well as desensitization, which is not due to an ANP-mediated decrease in the amount of receptor protein. The mobility of GC-A on sodium dodecyl sulfate-polyacrylamide gel electrophoresis increases after treatment of cells with ANP, and protein phosphatase 2A induces the same mobility shift. The protein phosphatase also catalyzes dephosphorylation of GC-A, and this is directly correlated with decreases in ANP-stimulatable guanylyl cyclase activity. Okadaic acid, an inhibitor of protein phosphatase 2A, blocks both the dephosphorylation and the desensitization. Therefore, in contrast to many other cell surface receptors, GC-A is desensitized by ligand-induced dephosphorylation.

Differential effects of atrial natriuretic peptide and dopamine on urinary protein excretion in chronic glomerulonephritis

1. To examine whether or not atrial natriuretic peptide-induced proteinuria simply results from increases in urine flow or glomerular filtration rate, we infused dopamine (1 microgram min-1 kg-1) and alpha-human atrial natriuretic peptide (0.025 microgram min-1 kg-1) into nine patients with chronic glomerulonephritis and nine essential hypertensive patients without renal damage, and compared the effects of the two agents on renal function and urinary protein excretion. 2. In patients with chronic glomerulonephritis, dopamine infusion significantly increased urinary sodium excretion (+59%), renal blood flow (+20%) and creatinine clearance (+14%). However, urinary protein excretion was not changed. Addition of atrial natriuretic peptide to the dopamine infusion further increased urinary sodium excretion and maintained creatinine clearance at the same level. In contrast to the infusion of dopamine alone, atrial natriuretic peptide markedly increased urinary protein excretion (77 versus 229 mg min-1 m2, P less than 0.02). Furthermore, the addition of atrial natriuretic peptide elevated the urinary protein/creatinine ratio (1.55 versus 5.35, P less than 0.05), while dopamine alone did not (1.55 versus 1.45, not significant). 3. In essential hypertensive patients, dopamine and dopamine plus ANP showed renal effects similar to those of chronic glomerulonephritis; however, the urinary excretion of protein was not changed significantly. 4. These results suggest that atrial natriuretic peptide may increase urinary protein excretion mainly by increasing the permeability of the damaged glomeruli to protein rather than by simply increasing urine flow or glomerular filtration. Possible mechanisms underlying the proteinuria-increasing effects of atrial natriuretic peptide are discussed.

Effects of atrial natriuretic peptide on renal arterioles: morphometric analysis using microvascular casts

In normal rat kidneys, the effect of atrial natriuretic peptide (ANP) on the diameter of the arterioles was evaluated by scanning electron microscopy of vascular casts. Acryl resin was infused into rat kidneys during the administration of ANP, either alone or with norepinephrine (NE). ANP infusion constricted the proximal efferent arteriole in the superficial cortex. Although NE constricted the proximal and distal segments of the afferent arteriole in the superficial cortex, the addition of ANP reversed the constriction and further constricted the efferent arteriole. In the deep cortex, only the proximal segment of the afferent arteriole was dilated by ANP when infused with NE. In a separate set of experiments, ANP increased both the glomerular filtration rate (GFR) and urinary sodium excretion (UNaV), and NE decreased the renal blood flow (RBF). However, administration of ANP after NE recovered RBF and increased GFR as well as UNaV. Results indicate that ANP increases GFR and natriuresis by constricting the efferent arteriole. NE appears to decrease RBF by constricting the afferent arteriole. ANP antagonizes the renal effects of NE primarily by dilating afferent arterioles.

Renal Effects of Atrial Natriuretic Peptide During Dopamine Infusion

To study whether the renal effects of atrial natriuretic peptide (ANP) are different from those of dopamine, we compared the effects of dopamine and dopamine plus ANP on renal circulation. Dopamine was infused at 1 microgram/kg/min for 120 min into 7 patients with essential hypertension (EH) and 5 normotensive subjects (NT). After 40 min of dopamine infusion, ANP infusion at 25 ng/kg/min was added to dopamine for 40 min. Before, during and after the infusion, renal function and nephrogenous cGMP were determined. Dopamine did not influence blood pressure, but increased urinary Na excretion (UNaV) by 100% in EH and NT. Addition of ANP further increased UNaV by 90%, but increases in UNaV were greater in EH than in NT. Renal blood flow was increased only by dopamine, while glomerular filtration rate (GFR) was increased by both dopamine (+8%) and dopamine plus ANP (+7%) as a whole, resulting in a significant increase in filtration fraction by the addition of ANP. Plasma and urinary cGMP and nephrogenous cGMP were elevated only during ANP infusion. These results suggest that the effects of ANP and dopamine on both GFR and UNaV were additive. However, in contrast with dopamine, ANP increased efferent resistance and nephrogenous cGMP, suggesting that the renal effects of ANP are different from those of dopamine.