Kailash N. Pandey

Biology of natriuretic peptides and their receptors.

Increasing evidence suggests that natriuretic peptides (NPs) play diverse roles in mammals, including renal hemodynamics, neuroendocrine, and cardiovascular functions. Collectively, NPs are classified as hypotensive hormones; the main actions of NPs are implicated in eliciting natriuretic, diuretic, steroidogenic, antiproliferative, and vasorelaxant effects, important factors in the control of body fluid volume and blood pressure homeostasis. One of the principal loci involved in the regulatory actions of NPs is their cognate plasma membrane receptor molecules, which are activated by binding with specific NPs. Interaction of NPs with their receptors plays a central role in physiology and pathophysiology of hypertension and cardiovascular disorders. Gaining insight into the intricacies of NPs-specific receptor signaling pathways is of pivotal importance for understanding both hormone-receptor biology and the disease states arising from abnormal hormone receptor interplay. During the last decade there has been a surge in interest in NP receptors; consequently, a wealth of information has emerged concerning molecular structure and function, signaling mechanisms, and use of transgenics and gene-targeted mouse models. The objective of this present review is to summarize and document the previous findings and recent discoveries in the field of the natriuretic peptide hormone family and receptor systems with emphasis on the structure-function relationship, signaling mechanisms, and the physiological and pathophysiological significance in health and disease.

Involvement of the NF-κB/Matrix Metalloproteinase Pathway in Cardiac Fibrosis of Mice Lacking Guanylyl Cyclase/Natriuretic Peptide Receptor A

Mice carrying a targeted disruption of the Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor A (NPRA)) exhibit increased blood pressure, cardiac hypertrophy, and congestive heart failure, similar to untreated human hypertensive patients. The objective of this study was to determine whether permanent ablation of NPRA signaling in mice alters the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), leading to myocardial collagen remodeling. Here, we report that expression levels of the MMP-2 and MMP-9 genes were increased by 3-5-fold and that the expression of the TNF-α gene was enhanced by 8-fold in Npr1 homozygous null mutant (Npr1-/-) mouse hearts compared with wild-type (Npr1+/+) control mouse hearts. Myocardial fibrosis, total collagen, and the collagen type I/III ratio (p < 0.01) were dramatically increased in adult Npr1-/- mice compared with age-matched wild-type counterparts. Hypertrophic marker genes, including the β-myosin heavy chain and transforming growth factor-β1, were significantly up-regulated (3-5-fold) in both young and adult Npr1-/- mouse hearts. NF-κB binding activity in ventricular tissues was enhanced by 4-fold with increased translocation of the p65 subunit from the cytoplasmic to nuclear fraction in Npr1-/- mice. Our results show that reduced NPRA signaling activates MMP, transforming growth factor-β1, and TNF-α expression in Npr1-/- mouse hearts. The findings of this study demonstrate that disruption of NPRA/cGMP signaling promotes hypertrophic growth and extracellular matrix remodeling, leading to the development of cardiac hypertrophy, myocardial fibrosis, and congestive heart failure.

Hypertension Associated with Decreased Testosterone Levels in Natriuretic Peptide Receptor-A Gene-Knockout and Gene-Duplicated Mutant Mouse Models*

Mice lacking the gene (Npr1) encoding the natriuretic peptide receptor A (NPRA) have hypertension with elevated blood pressure and cardiac hypertrophy. In particular, Npr1 gene-deficient male mice exhibit lethal vascular events similar to those seen in untreated human hypertensive patients. Serum testosterone levels tend to be lower in hypertensive male humans than in normal males without hypertension, but the genetic basis for this tendency remains unknown. To determine whether Npr1 gene function affects the testosterone level, we measured serum testosterone in male hypertensive mice lacking a functional Npr1 gene, wild-type animals with two copies, and the gene-duplicated littermates expressing four copies of the gene. In the Npr1 gene-knockout (zero-copy) mice, the serum testosterone level was 62% lower than that in the two-copy control mice (80 ± 10 vs. 120 ± 14 ng/ml, respectively; P < 0.005). Serum testosterone in the four-copy mice was 144% (P < 0.005) of that in the two-copy wild-type control mice...

Ligand-regulated Internalization, Trafficking, and Down-regulation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A in Human Embryonic Kidney 293 Cells

We examined the kinetics of internalization, trafficking, and down-regulation of recombinant guanylyl cyclase/natriuretic peptide receptor-A (NPRA) utilizing stably transfected 293 cells expressing a very high density of receptors. After atrial natriuretic peptide (ANP) binding to NPRA, ligand-receptor complexes are internalized, processed intracellularly, and sequestered into subcellular compartments, which provided an approach to examining directly the dynamics of metabolic turnover of NPRA in intact cells. The translocation of ligand-receptor complexes from cell surface to intracellular compartments seems to be linked to ANP-dependent down-regulation of NPRA. Using tryptic proteolysis of cell surface receptors, it was found that ∼40–50% of internalized ligand-receptor complexes recycled back to the plasma membrane with an apparent t 1 2 = 8 min. The recycling of NPRA was blocked by the lysosomotropic agent chloroquine, the energy depleter dinitrophenol, and also by low temperature, suggesting that recycling of the receptor is an energy- and temperature-dependent process. Data suggest that ∼70–80% of internalized 125I-ANP is processed through a lysosomal degradative pathway; however, 20–25% of internalized ligand is released intact into the cell exterior through an alternative mechanism involving an chloroquine-insensitive pathway. It is implied that internalization and processing of bound ANP-NPRA complexes may play an important role in mediating the biological action of hormone and the receptor protein. In retrospect, this could occur at the level of receptor regulation or through the initiation of ANP mediated signals. It is envisioned that the endocytotic pathway of ligand-receptor complexes of ANP-NPRA would lead to termination and/or diminished responsiveness of ANP in target cells.

Emerging roles of natriuretic peptides and their receptors in pathophysiology of hypertension and cardiovascular regulation

Thus far, three related natriuretic peptides (NPs) and three distinct receptors have been identified, which have advanced our knowledge towards understanding the control of high blood pressure, hypertension, and cardiovascular disorders to a great extent. Biochemical and molecular studies have been advanced to examine receptor function and signaling mechanisms and the role of second messenger cGMP in pathophysiology of hypertension, renal hemodynamics, and cardiovascular functions. The development of gene-knockout and gene-duplication mouse models along with transgenic mice have provided a framework for understanding the importance of the antagonistic actions of natriuretic peptides receptor in cardiovascular events at the molecular level. Now, NPs are considered as circulating markers of congestive heart failure, however, their therapeutic potential for the treatment of cardiovascular diseases such as hypertension, renal insufficiency, cardiac hypertrophy, congestive heart failure, and stroke has just begun to unfold. Indeed, the alternative avenues of investigations in this important are need to be undertaken, as we are at the initial stage of the molecular therapeutic and pharmacogenomic implications.

Expression of atrial natriuretic peptide receptor-A antagonizes the mitogen-activated protein kinases (Erk2 and P38MAPK) in cultured human vascular smooth muscle cells.

To understand the signaling mechanisms of atrial natriuretic peptide (ANP) receptor-A (NPRA), we studied the effect of the ANP/NPRA system on mitogen-activated protein kinases (MAPKs), with particular emphasis on the extracellular-regulated kinase (Erk2) and stress-activated protein kinase (p38MAPK) in cultured human vascular smooth muscle cells (HVSMC). Angiotensin II (ANG II) and platelet-derived growth factor (PDGF) stimulated the immunoreactive Erk2 and p38MAPK activities and their protein levels by 2–4 fold. The pretreatment of cells with ANP significantly inhibited the agonist-stimulated Erk2 and p38MAPK activities and protein expression by 65–75% in HVSMC transiently transfected with NPRA, as compared with only 18–22% inhibition in vector-transfected cells. The pretreatment of cells with KT5823, an inhibitor of cGMP-dependent protein kinase (PKG), reversed the inhibitory effects of ANP on MAPK activities and protein expression by 90–95%. PD98059, which inhibits Erk2 by directly inhibiting the MAPK-kinase (MEK), and SB202192, a selective antagonist of p38MAPK, blocked the Erk2 and p38MAPK activities, respectively. Interestingly, ANP stimulated the MAPK-phosphatase-3 (MKP-3) protein levels by more than 3-fold in HVSMC over-expressing NPRA, suggesting that ANP-dependent inhibition of MAPKs may also proceed by stimulating the phosphatase cascade. These present findings provide the evidence that ANP exerts inhibitory effects on agonist-stimulated MAPKs (Erk2 and p38MAPK) activities and protein levels in a 2-fold manner: by antagonizing the upstream signaling pathways and by activation of MKP-3 to counter-regulate MAPKs in a cGMP and PKG-dependent manner. Our results identify a signal transduction pathway in HVSMC that could contribute to vascular remodeling and structural changes in human hypertension.

Increased activity and expression of Ca2+-dependent NOS in renal cortex of ANG II-infused hypertensive rats

We have previously demonstrated that nitric oxide (NO) exerts a greater modulatory influence on renal cortical blood flow in ANG II-infused hypertensive rats compared with normotensive rats. In the present study, we determined nitric oxide synthase (NOS) activities and protein levels in the renal cortex and medulla of normotensive and ANG II-infused hypertensive rats. Enzyme activity was determined by measuring the rate of formation of L-[(14)C]citrulline from L-[(14)C]arginine. Western blot analysis was performed to determine the regional expression of endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms in the renal cortex and medulla of control and ANG II-infused rats. Male Sprague-Dawley rats were prepared by the infusion of ANG II at a rate of 65 ng/min via osmotic minipumps implanted subcutaneously for 13 days and compared with sham-operated rats. Systolic arterial pressures were 127 +/- 2 and 182 +/- 3 mmHg in control (n = 13) and ANG II-infused rats (n = 13), respectively. The Ca(2+)-dependent NOS activity, expressed as picomoles of citrulline formed per minute per gram wet weight, was higher in the renal cortex of ANG II-infused rats (91 +/- 11) than in control rats (42 +/- 12). Likewise, both eNOS and nNOS were markedly elevated in the renal cortex of the ANG II-treated rats. In both groups of rats, Ca(2+)-dependent NOS activity was higher in the renal medulla than in the cortex; however, no differences in medullary NOS activity were observed between the groups. Also, no differences in medullary eNOS levels were observed between the groups; however, medullary nNOS was decreased by 45% in the ANG II-infused rats. For the Ca(2+)-independent NOS activities, the renal cortex exhibited a greater activity in the control rats (174 +/- 23) than in ANG II-infused rats (101 +/- 10). Similarly, cortical iNOS was greater by 47% in the control rats than in ANG II-treated rats. No differences in the activity were found for the renal medulla between the groups. There was no detectable signal for iNOS in the renal medulla for both groups. These data indicate that there is a differential distribution of NOS activity, with the Ca(2+)-dependent activity and protein expression higher in the renal cortex of ANG II-infused rats compared with control rats, and support the hypothesis that increased constitutive NOS activity exerts a protective effect in ANG II-induced hypertension to maintain adequate renal cortical blood flow.

Angiotensin II–Mediated Negative Regulation of Npr1 Promoter Activity and Gene Transcription

Abstract—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 ≈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.

Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription

Cardiac hormones atrial and brain natriuretic peptides activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which plays a critical role in reduction of blood pressure and blood volume. Currently, the mechanisms responsible for regulating the Npr1 gene (coding for GC-A/NPRA) transcription are not well understood. The present study was conducted to examine the interactive roles of all-trans retinoic acid (ATRA), Ets-1, Sp1, and histone acetylation on the transcriptional regulation and function of the Npr1 gene. Deletion analysis of the Npr1 promoter and luciferase assays showed that ATRA enhanced a 16-fold Npr1 promoter activity and greatly stimulated guanylyl cyclase (GC) activity of the receptor protein in both atrial natriuretic peptide (ANP)-dependent and -independent manner. As confirmed by gel shift and chromatin immunoprecipitation assays, ATRA enhanced the binding of both Ets-1 and Sp1 to the Npr1 promoter. The retinoic acid receptor α (RARα) was recruited by Ets-1 and Sp1 to form a transcriptional activator complex with their binding sites in the Npr1 promoter. Interestingly, ATRA also increased the acetylation of histones H3 and H4 and enhanced their recruitment to Ets-1 and Sp1 binding sites within the Npr1 promoter. Collectively, the present results demonstrate that ATRA regulates Npr1 gene transcription and GC activity of the receptor by involving the interactive actions of Ets-1, Sp1, and histone acetylation.

Enhanced activation of pro-inflammatory cytokines in mice lacking natriuretic peptide receptor-A.

Natriuretic peptide receptor-A (NPRA) is the principal receptor for the cardiac hormones ANP and BNP. Mice lacking NPRA develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for hypertrophic growth in the absence of NPRA signaling are not yet known. In the present study, we determined whether deficiency of NPRA/cGMP signaling alters the cardiac pro-inflammatory cytokines gene expression in Npr1 (coding for NPRA) gene-knockout (Npr1(-/-)) mice exhibiting cardiac hypertrophy and fibrosis as compared with control wild-type (Npr1(+/+)) mice. A significant up-regulation of cytokine genes such as TNF-alpha (five-fold), IL-6 (three-fold) and TGF-beta1 (four-fold) were observed in mutant mice hearts lacking NPRA as compared with the age-matched wild-type mice. In parallel, NF-kappaB binding activity was almost five-fold greater in the nuclear extract of Npr1(-/-) mutant mice hearts as compared with wild-type Npr1(+/+) mice hearts. Guanylyl cyclase (GC) activity and cGMP levels were drastically reduced by 10- and 5-fold, respectively, in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory cytokines, probably via NF-kappaB mediated signaling pathway, and is associated with hypertrophic growth of null mutant mice hearts.