Atrial Natriuretic Peptide Receptor Research Articles

CNP-induced changes in pHi, cGMP/cAMP and mRNA expression of natriuretic peptide receptors in human trabecular meshwork cells.

It has been demonstrated that natriuretic peptides lower intraocular pressure, however, the underlying cellular mechanism(s) mediating this response remain(s) to be determined. The purpose of this study was to investigate the effects of C-type natriuretic peptide (CNP) on pH(i), cGMP/cAMP and expression of atrial natriuretic peptide receptor (NPR-A), brain natriuretic peptide receptor (NPR-B) and C-type natriuretic peptide receptor (NPR-C), in HTM cells. At concentrations of 10(-7) M, CNP caused an acidification of pH(i). In addition, CNP caused a dose-dependent increase in cGMP formation and inhibition of forskolin-stimulated cAMP accumulation. These changes were not significantly altered in the absence of 10(-3) M isobutylmethylxanthine (IBMX). Treatment with the NPR-A antagonist, anantin, produced no influence on basal cGMP/cAMP levels, the CNP-stimulated cGMP accumulation and CNP-induced inhibition of forskolin-stimulated cAMP accumulation. However, CNP-induced reduction of forskolin-stimulated cAMP accumulation was inhibited by pretreatment with pertussis toxin (PTX). Furthermore, NPRB receptors were predominantly expressed and pretreatment with CNP (10(-7) M, 24hr) enhanced all NPR mRNAs expression which was not altered by higher concentrations or longer incubation. Results demonstrate that NPR-A, NPR-B and NPR-C receptors' expression can be up-regulated by CNP treatment. CNP activates NPR-B receptors preferentially to increase cGMP accumulation and acts through the PTX-sensitive cAMP-signaling pathway leading to a decrease in pH(i).

Crystallization and preliminary X-ray analysis of the atrial natriuretic peptide (ANP) receptor extracellular domain complex with ANP: use of ammonium sulfate as the cryosalt.

Atrial natriuretic peptide (ANP) plays a major role in blood pressure and volume regulation owing to its natriuretic and vasodilatory activities. The ANP receptor is a single-span transmembrane receptor coupled to its intrinsic guanylyl cyclase activity. The extracellular hormone-binding domain of rat ANP receptor (ANPR) was overexpressed by permanent transfection in CHO cells and purified. ANPR complexed with ANP was crystallized at 301 K by the hanging-drop vapor-diffusion method. The crystals were frozen in 3.4 M ammonium sulfate used as a cryoprotectant. The crystals diffracted to 3.1 A resolution using synchrotron radiation and belonged to the hexagonal space group P6(1), with unit-cell parameters a = b = 100.3, c = 258.6 A.

Plasma atrial natriuretic peptide and brain natriuretic peptide are increased in septic shock: impact of interleukin-6 and sepsis-associated left ventricular dysfunction.

Interest has recently focused on the use of neurohormonal markers such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) as indices of left ventricular systolic dysfunction and prognosis in heart failure. Also, peptides belonging to the interleukin-6 (IL-6) family have been shown to induce ANP and BNP secretion. We hypothesized that BNP and ANP spillover in the peripheral circulation reflects left ventricular dysfunction and IL-6 production in septic shock. Retrospective, clinical study in the medical intensive care unit of a university hospital. 17 patients with septic shock and 19 control subjects. Collection of clinical and demographic data in relation to ANP, BNP, IL-6, and soluble TNF receptors (sTNF-R-p55, sTNF-R-p75) in plasma over a period of 4 days. In septic shock we found a significant increase in ANP (82.7+/-9.9 vs. 14.9+/-1.2 pg/ml) and BNP (12.4+/-3.6 vs. 5.5+/-0.7 pg/ml). Plasma ANP peaked together with IL-6. Peaks of ANP and IL-6 were significantly correlated (r=0.73; p<0.01). BNP was inversely correlated to cardiac index (r=-0.56; p<0.05). ANP and BNP increase significantly in patients with septic shock. BNP reflects left ventricular dysfunction. ANP is related to IL-6 production rather than to cardiovascular dysfunction.

The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct microcystin-sensitive and magnesium-dependent protein phosphatases

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl2 stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 °C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl2 or MnCl2, but not CaCl2, further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl2, EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.

Modulation of ANP-C receptor signaling by arginine-vasopressin in A-10 vascular smooth muscle cells: role of protein kinase C

We have previously shown that pretreatment of A-10 vascular smooth muscle cells (VSMC) with angiotensin II (Ang II) attenuated atrial natriuretic peptide receptor-C (ANP-C)-mediated inhibition of adenylyl cyclase without altering [ 125I]ANP binding. In the present studies, we have investigated the modulation of ANP-C receptor signaling by arginine-vasopressin (AVP). Pretreatment of A-10 VSMC with AVP for 24 h resulted in a reduction in ANP receptor binding activity by about 50% ( B max; control cells, 22.9 ± 2.5 fmol/mg protein, AVP-treated cells, 11.4 ± 1.2 fmol/mg protein). In addition, the expression of ANP-C receptor as determined by immunoblotting was also decreased by about 50% by AVP treatment, which was prevented by GF109203X, an inhibitor of protein kinase C (PKC). The decreased expression of ANP-C receptor was reflected in an attenuation of ANP-C receptor-mediated inhibition of adenylyl cyclase. C-ANP 4–23 [des(Gln 18,Ser 19,Gln 20,Leu 21,Gly 22)ANP 4–23–NH 2], a ring deleted peptide of ANP that interacts specifically with ANP-C receptor, inhibited adenylyl cyclase activity by about 30% in control cells, which was completely attenuated in AVP-treated cells. This attenuated inhibition was significantly restored by GF 109203X. In addition, AVP treatment augmented the levels of Giα-2 and Giα-3 proteins; however, the Gi functions were completely attenuated. The increased expression of Giα proteins induced by AVP was inhibited by GF109203X as well as by actinomycin D treatments. In addition, AVP treatment also enhanced the expression of Gsα protein and Gsα-mediated stimulation of adenylyl cyclase by GTPγS, N-ethylcarboxamide adenosine (NECA), and forskolin (FSK), whereas the levels of Gβ were not altered by AVP treatment. These results indicate that AVP-induced PKC signaling may be responsible for the down-regulation of ANP-C receptor that results in the attenuation of C-ANP 4–23-mediated inhibition of adenylyl cyclase activity, and suggest a cross-talk between vasopressin V 1 and ANP-C receptor-mediated signaling pathways.

Increased Sensitivity to Endothelial Nitric Oxide (NO) Contributes to Arterial Normotension in Mice with Vascular Smooth Muscle-selective Deletion of the Atrial Natriuretic Peptide (ANP) Receptor

Atrial natriuretic peptide (ANP) plays a key regulatory role in arterial blood pressure homeostasis. We recently generated mice with selective deletion of the ANP receptor, guanylyl cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO) mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142-7147). The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of cGMP-dependent protein kinase I, 8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC (sGC), but not of endothelial NO synthase or cGMP-dependent protein kinase I, was significantly increased in SMC GC-A KO mice. Chronic oral treatment with the NO synthase inhibitor N(w)-nitro-l-arginine methyl ester increased arterial blood pressure, the effect being significantly enhanced in SMC GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher vasodilating sensitivity to NO. This can be attributed to an enhanced expression of sGC, whereas the expression and/or activity levels of downstream cGMP-effector pathways are not involved. Increased vasodilating responsiveness to endothelial NO contributes to compensate for the missing vasodilating effect of ANP in SMC GC-A KO mice.

Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A.

Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), alpha-skeletal actin (1.7-fold), and beta-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7-10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.

The neuroendocrinology of stress and the pathophysiology and therapy of depression and anxiety

Clinical and preclinical studies have gathered substantial evidence that stress response alterations play a major role in the development of major depression, panic disorder, and post-traumatic stress disorder. The stress response, the hypothalamic pituitary adrenocortical (HPA) system and its modulation by corticotropin-releasing hormones (CRH),corticosteroids,and their receptors, and the roles of natriuretic peptides and neuroactive steroids are described. We review the role of the HPA system in major depression, panic disorder, and post-traumatic stress disorder and its possible relevance for treatment. Impaired glucocorticoid receptor function in major depression is associated with an excessive release of neurohormones such as CRH, to which a number of signs and symptoms characteristic of depression can be ascribed. In panic disorder, a role of central CRH in panic attacks has been suggested. Atrial natriuretic peptide (ANP) is causally involved in sodium lactate-induced panic attacks. Furthermore, preclinical and clinical data on its anxiolytic activity suggest that nonpeptidergic ANP receptor ligands may be potentially useful in the treatment of anxiety disorders. Post-traumatic stress disorder is characterized by a peripheral hyporesponsive HPA system and elevated CRH concentrations in the CSF. This dissociation is probably related to an increased risk of this disorder. We further review recent data that describe an important role of GABA(A)-receptor modulatory,3 alpha-reduced neuroactive steroids in major depression, anxiety, and its treatment. Antidepressants are effective in both depression and anxiety disorders and have major effects on the HPA system,especially on glucocorticoid and mineralocorticoid receptors. Normalization of HPA system abnormalities is a strong predictor of the clinical course, at least in major depression and panic disorder. Currently,CRH-R1 or glucocorticoid receptor antagonists and ANP receptor agonists are being studied and may provide future treatment options more closely related to the pathophysiology of these disorders.

Atrial Natriuretic Peptides Elevate Cyclic GMP Levels in Primary Cultures of Rat Ependymal Cells

The aim of this study was to examine the effect of atrial natriuretic peptides on primary cultures of ependymal cells, as measured by changes in intracellular levels of cyclic GMP. Incubation of ependymal cells with rat atrial natriuretic peptide-(1-28) (rANP) elicited a 30-fold increase in ependymal cGMP content within 1 min and more than a 100-fold increase within 10 min to a plateau value of approximately 30 pmol/mg protein. The C-type natriuretic peptide (CNP) elicited a similar increase in cGMP levels; however the maximal effect was observed within 1 min and the levels subsequently dropped by 90% to a low plateau within 10 min. A comparison of the concentration-response curves for rANP, human ANP-(1-28) (hANP) and CNP showed that rANP, hANP and CNP had similar effects, with regards to elevation of cGMP levels at high concentrations, but with differing EC50 values. These results demonstrate the presence of a heterogenous population of functional ANP receptors i n cultured ependymalcells suggesting that ANP may regulate specific ependymal cell activity.

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.)

Guanylyl cyclase receptors mediate cardiopulmonary vagal reflex actions of ANP.

Atrial natriuretic peptide (ANP) potentiates vagal cardiopulmonary reflexes due to chemosensory (Bezold-Jarisch [B-J] reflex) or mechanosensory (ramp baroreflex) activation. The ANP receptor mediating these actions is unknown. We examined the role of particulate guanylyl-cyclase (pGC) receptors in ANP-induced enhancement of cardiopulmonary vagal reflexes. Cardiopulmonary baroreceptor reflex function was assessed by bradycardic responses to ramp blood pressure rises after rapid intravenous methoxamine (100 micro g/kg bolus dose). The B-J reflex was evoked by 3 intravenous doses of serotonin (1 to 10 micro g/kg). In conscious, chronically instrumented rats (n=9), these tests were performed on each animal during randomized infusions of rat ANP (150 ng/kg per minute IV), saline (270 micro L/h IV), the pGC receptor antagonist HS-142-1 (3 mg/kg IV), or combined HS-142-1+ANP treatment. HS-142-1 alone attenuated normal B-J reflex (by 33+/-8%, P<0.05) but not ramp baroreflex responses. As we showed previously, ANP enhanced baroreflex and B-J reflex bradycardia (by approximately 140% and approximately 30%, respectively, P<0.05), compared with saline infusion. These ANP effects were completely blocked by HS-142-1, demonstrating that the cardiopulmonary vagal reflex actions of ANP occurred through pGC natriuretic peptide receptors. Additionally, we have provided evidence for the first time that pGC natriuretic peptide receptors are essential for the full expression of the B-J reflex but not for that of cardiopulmonary vagal baroreflexes. This tonic interaction between pGC natriuretic peptide receptors and cardiopulmonary chemosensitive receptors may be important during pathophysiological activation of B-J reflex, such as with myocardial infarction.

Angiotensin II-mediated negative regulation of Npr1 promoter activity and gene transcription.

Atrial natriuretic peptide receptor A (NPRA) plays important role(s) in the control of extracellular fluid volume and blood pressure homeostasis. We have determined and analyzed the functional promoter region of Npr1 gene (coding for NPRA) and studied the effect of angiotensin (Ang) II on its promoter activity and expression in cultured mouse mesangial cells. The promoter analysis of Npr1 gene revealed the presence of positive regulatory cis-elements in the regions -1982 to -1841 bp and -916 to -496 bp and of the repressor elements in the regions -1841 to -916 bp and 56 to 382 bp relative to transcription start site. The Ang II pretreatment of cultured mouse mesangial cells transiently transfected with the promoter construct pNPRA-luc1 significantly inhibited the promoter activity in a time- and dose-dependent manner, with a maximum inhibition at 24 hours. The Ang II-dependent repression of Npr1 promoter activity was partially blocked by both angiotensin type 1 and type 2 antagonists candesartan and PD 123,319, respectively. The mRNA level of NPRA was also downregulated by Ang II treatment as determined by semiquantitative reverse transcriptase-polymerase chain reaction assay. The deletion analysis showed that the promoter region approximately 916 bp upstream of transcription start site contains the cis-elements involved in Ang II-mediated repression of transcription of Npr1 gene. The present study thus reveals the presence of functional cis-regulatory elements in the promoter region of the murine Npr1 gene and its transcriptional downregulation by vasoactive peptide Ang II.

Genistein Potentiates the ANP Effect on a K+-Conductance in HEK-293 Cells

HEK-293 cells are known to reflect many features of the late distal tubule. Furthermore, they have the ability to release urodilatin, the structural analog to ANP. RT-PCR was performed to test for the expression of natriuretic peptide receptors. While the mRNA for the human ANP receptor (NPR-A, GC-A) could be amplified, the CNP-specific receptor NPR-B (GC-B) and the receptor specific for guanylins, GC-C, could not be detected. In patch clamp experiments the effects of ANP (10 nM) on membrane voltage (V_m) were monitored and HEK-293 cells depolarized by 2.3 ± 0.5 mV (n=14). In the presence of the EGF receptor blocker genistein (10 µM) the effect of ANP was increased by 65% to 3.9 ± 0.8 mV (n=14). After removal of genistein the ANP-mediated depolarization further increased by 147% to 5.7 ± 1.0 mV (n=14). ANP given repetitively without genistein had no increasing depolarizing effect in HEK-293 cells with time. The ANP effect could be fully blocked by 1 mM Ba²⁺ and by 1 µM of the specific PKG inhibitor KT5823 indicating that ANP inhibits a K⁺-conductance via a cGMP-dependent protein kinase. Genistein itself hyperpolarized the membrane voltage of HEK-293 cells by –3.9 ± 0.6 mV (n=11) and this effect could also be fully blocked by Ba²⁺ (-0.3 ± 0.1 mV, n=5), indicating that genistein activates a K⁺-conductance which contributes significantly to the membrane potential of HEK-293 cells.

Effects of dehydration on endocrine regulation of the electrolyte and fluid balance and atrial natriuretic peptide-binding sites in perinatally malnourished adult male rats.

The first aim of this work was to investigate, under basal conditions in adult male rats, the long-term consequences of perinatal maternal food restriction on the plasma concentrations of vasopressin (VP), aldosterone and atrial natriuretic peptide (ANP) and on plasma renin activity (PRA). Furthermore, under these same conditions, the hypothalamic VP gene expression as well as the density (B(max)), affinity (K(d)) and gene expression of ANP receptors were determined in kidneys and adrenals. The second aim of this work was to examine the responsiveness to dehydration in perinatally malnourished rats. Thus, the latter parameters were studied in these rats after 72 h water deprivation. This study was conducted on 4-Month-old male rats from mothers exposed to 50% food restriction (FR50) during the last week of gestation and lactation and on age-matched control animals (C). At this stage, both C and FR50 rats were killed by decapitation between 0900 h and 1000 h in order to determine parameters under basal conditions or after 72 h water deprivation. Plasma VP, ANP and aldosterone levels and PRA were determined by radioimmunoassay. Hypothalamic VP gene expression was determined in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization. The B(max) and K(d) values of ANP receptors were evaluated from Scatchard plots. ANP receptor gene expression was determined by Northern blot analysis. Under basal conditions, perinatal malnutrition reduced body weight, absolute weight of kidneys and adrenals, and haematocrit. Compared with control rats, FR50 rats had significantly greater plasma VP and aldosterone levels but PRA, plasma ANP levels, plasma osmolality and hypothalamic VP gene expression were not significantly different. Perinatal malnutrition did not significantly affect glomerular ANP receptor density, but in adrenals it decreased both B(max) and K(d) values of ANP-B receptors (biological receptors) and increased B(max) of ANP-C receptors (clearance receptors). ANP-B(A) (receptor subtype A of ANP-B receptors) receptor gene expression was not significantly affected, whereas ANP-C receptor gene expression was enhanced in both adrenals and kidneys in FR50 rats. After 72 h dehydration, control rats showed a significant rise in haematocrit, plasma osmolality, PRA, circulating levels of VP and aldosterone and VP gene expression in both PVN and SON but showed a decrease in plasma ANP levels. B(max) of ANP-B receptors was decreased whereas B(max) of ANP-C receptors was enhanced in both adrenals and kidneys. ANP-B(A) receptor gene expression was not significantly affected in either kidneys or adrenals in dehydrated control rats. Similarly, ANP-C receptor gene expression was unaffected in kidneys whereas it was significantly enhanced in adrenals. In FR50 rats, the effects of water deprivation were qualitatively similar to those reported in controls concerning plasma parameters except for plasma VP levels which tended to rise (not significant) but this increase was only very slight compared with controls. Moreover, unlike controls, VP gene expression in both PVN and SON was not enhanced after dehydration in FR50 rats. In kidneys, dehydrated FR50 rats, like controls, upregulated ANP-C receptors, but they were unable to downregulate ANP-B receptors. In adrenals, unlike controls, FR50 rats enhanced ANP-B receptor density whereas they decreased both ANP-C receptor density and expression after 72 h dehydration. Similar to controls, the expression of ANP-B(A) receptors in both kidneys and adrenals as well as the expression of ANP-C receptors in kidneys, were unaffected in dehydrated FR50 rats. Perinatal malnutrition had long-lasting effects on regulation of the fluid and electrolyte balance under basal conditions. The main effects were a significant rise in circulating levels of VP and aldosterone, and changes in density of adrenal ANP-binding sites and ANP-C receptor gene expression in both adrenals and kidneys. Perinatal malnutrition also affects the responsiveness to water deprivation with alterations in both hypothalamic VP gene expression and regulation of ANP-binding sites.

TA repeat variation, Npr1 expression, and blood pressure: impact of the Ace locus.

The activity of the atrial natriuretic peptide receptor (Npr1) is altered in spontaneously hypertensive rats (SHR) in relation to its mRNA levels, suggesting abnormal transcriptional control in hypertension. A single-stranded conformational polymorphism caused by a repetitive dinucleotide segment of 10 TA in BN-Lx and of 40 TA in SHR was localized at position -943 relative to the transcription start site of the Npr1 gene, downstream of a putative cGMP-regulatory region, and was the only sequence difference noted between the two strains. Transient transfections of -1520 to -920 Npr1 promoter-SV40-luciferase fusion vector showed that the construct from BN-Lx stimulated the SV40 promoter, whereas that from SHR slightly inhibited it. In contrast to the BN-Lx construct, the activity of the SHR fragment was refractory to downregulation by atrial natriuretic peptide. Genotype-phenotype correlation studies in recombinant inbred strains (RIS) derived from BN-Lx and SHR crosses revealed significant correlations of the TA repeat with basal guanylyl cyclase activity and Npr1 mRNA levels. The correlations were heightened by a locus on chromosome 10 containing the Ace gene. The highest basal guanylyl cyclase activity and Npr1 mRNA values were found in RIS with both genes (Npr1/Ace) of BN genotypes, whereas the lowest were recorded in RIS, with the SHR genotypes at both loci. This was inversely correlated with diastolic blood pressure in these strains. In conclusion, the longer TA repeat unit in the promoter of Npr1 of SHR, in tandem with a putative cGMP responsive element, regulates the transcription of the Npr1 gene with consequences on diastolic blood pressure.

Left but not right cardiac hypertrophy in atrial natriuretic peptide receptor-deficient mice is prevented by angiotensin type 1 receptor antagonist losartan.

Mice with a genetic deletion of the atrial natriuretic peptide (ANP) receptor, guanylyl cyclase A (GC-A -/-), have chronic arterial hypertension and cardiac hypertrophy from the first day of life. To characterize the role of the angiotensin II and endothelin systems in the development of this cardiovascular phenotype, the effects of chronic treatment with either the angiotensin type I (AT1) receptor antagonist losartan or the endothelin A receptor antagonist BSF208075 were tested. Losartan almost completely reversed systemic arterial hypertension and left ventricular hypertrophy of GC-A -/- mice. This was accompanied by a marked regression of the left ventricular mRNA expression of cardiac hypertrophy markers such as ANP and brain natriuretic peptide and a significant reduction of left ventricular and pulmonary interstitial collagen accumulation. BSF208075 had no effect on any of these cardiovascular parameters. Intriguingly, GC-A -/- mice also showed a very marked right ventricular hypertrophy, which was not reversed by losartan or BSF208075 treatment. Analyses of components of the renin-angiotensin system (RAS) revealed an inhibition of renal and systemic RAS contrasting with increased local left ventricular angiotensin II levels in GC-A -/- mice. Collectively, the results suggest that RAS plays a more important role than the endothelin system in the pathogenesis of arterial hypertension as well as left ventricular hypertrophy and fibrosis in GC-A gene-disrupted mice.

Characterization of VIP and PACAP receptors in cultured rat penis corpus cavernosum smooth muscle cells and their interaction with guanylate cyclase-B receptors.

Penile corpus cavernosum smooth muscle relaxation can be induced by both cyclic AMP and cyclic GMP-elevating agents, but possible interactions between these two signalling pathways are still poorly understood. Using in vitro cultured rat penile corpus cavernosum smooth muscle (CCSM) cells, we have characterized the local expression and functional activities of receptors for the cAMP-elevating peptides, PACAP and VIP, and for the cGMP-elevating peptides, CNP and ANP. Stimulation of the cells with various concentrations of PACAP(-27/-38) or VIP resulted in rapid and dose-dependent increases in cyclic AMP levels. RT-PCR analyses revealed gene expression of PAC(1) and VPAC(2) but not of VPAC(1) receptors in the cells. The natriuretic peptide, CNP, and the nitric oxide donor, sodium nitroprusside, were capable of enhancing cyclic GMP formation, indicating the presence of membrane-associated in addition to soluble guanylate cyclase (sGC) activities in these cells. Findings that cyclic GMP formation was preferentially activated by CNP but not by the related peptide, ANP, were consistent with RT-PCR analyses, demonstrating gene expression of the CNP receptor, GC-B, but not of the ANP receptor, GC-A, in these cells. Prior exposure of the cells to 10(-8) M PACAP resulted in a marked down-regulation of GC-B activity, whereas sGC was not affected. These findings provide functional and molecular evidence for the presence of three receptors, PAC(1), VPAC(2) and GC-B, involved in cyclic nucleotide signalling in penile CCSM cells. The observed cross-talk of the PACAP/VIP receptors with GC-B but not with sGC may have implications for the therapy of erectile dysfunction.

Characterization of Differential Gene Expression in Quiescent and Invasive Human Arterial Smooth Muscle Cells

Proliferation, migration and invasion of smooth muscle cells (SMCs) are essential pathogenic processes in the development of a broad spectrum of cardiovascular disorders, like arteriosclerosis, restenosis after percutaneous transluminal angioplasty and stent implantation as well as transplant vessel disease. As an in vitro model mimicking these processes, the Boyden chamber was employed to characterize the diverging migratory and invasive potentials of proliferating and nonproliferating human arterial SMCs (haSMCs). Using this model, differential gene expression of both phenotypes was analyzed by a cDNA array system (Clontech human cardiovascular array). With these arrays, 558 cardiovascular-associated genes could be compared. Further, gene expression was exactly quantified by real-time RT-PCR. Protein expression was analyzed by ELISA and Western blotting. In total, 47 genes were differentially expressed more than 1.5 times. Most of the differentially regulated genes in this study were associated with the extracellular matrix (ECM) and cell motility. In detail, the respective groups were matrix-organizing proteins, ECM proteins, cell adhesion proteins, extracellular communication and cytoskeleton motility proteins. Genes known to be differentially regulated during haSMC migration and invasion, like TIMP 2, TIMP 3, and MMP 3, were confirmed by the array data. Reduced expression of several cytoskeletal proteins, like vimentin, fibronectin, cytokeratins and β1 integrin, was shown in the invasive phenotype. Further, angio-associated protein, alpha E-catenin and atrial brain natriuretic peptide receptor were downregulated whereas TFPI 2 was strongly upregulated in invasive haSMCs. In conclusion, several relevant potential candidate genes for the quiescent and the invasive SMC phenotype were identified and genes already known to be differentially regulated by previous analysis were confirmed.

Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure.

Atrial natriuretic peptide (ANP) is an important regulator of arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve the inhibition of the sympathetic and renin-angiotensin-aldosterone (RAA) systems, increased diuresis/natriuresis, vasodilation, and enhanced vascular permeability. In particular, the contribution of the direct vasodilating effect of ANP to the hypotensive actions remains controversial, because variable levels of the ANP receptor, guanylyl cyclase A (GC-A), are expressed in different vascular beds. The objective of our study was to determine whether a selective deletion of GC-A in vascular smooth muscle would affect the hypotensive actions of ANP. We first created a mutant allele of mouse GC-A by flanking a required exon with loxP sequences. Crossing floxed GC-A with SM22-Cre transgene mice expressing Cre recombinase in smooth muscle cells (SMC) resulted in mice in which vascular GC-A mRNA expression was reduced by approximately 80%. Accordingly, the relaxing effects of ANP on isolated vessels from these mice were abolished; despite this fact, chronic arterial blood pressure of awake SMC GC-A KO mice was normal. Infusion of ANP caused immediate decreases in blood pressure in floxed GC-A but not in SMC GC-A knockout mice. Furthermore, acute vascular volume expansion, which causes release of cardiac ANP, did not affect resting blood pressure of floxed GC-A mice, but rapidly and significantly increased blood pressure of SMC GC-A knockout mice. We conclude that vascular GC-A is dispensable in the chronic and critical in the acute moderation of arterial blood pressure by ANP.

The Atrial Natriuretic Peptide Receptor (NPR-A/GC-A) Is Dephosphorylated by Distinct Microcystin-sensitive and Magnesium-dependent Protein Phosphatases

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl(2) stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 degrees C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl(2) or MnCl(2), but not CaCl(2), further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl(2), EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.