心房钠尿肽（ANP）对大鼠卵巢细胞生成雌激...

Isolated ovarian corpus luteal cells and granulosa cells of rat were employed to investigate the effect of alpha-ANP on the secretion of progesterone and estradiol. The contents of the steroid hormones are determined by RIA. The results showed that 0.1-10 ng/ml ANP promoted progesterone production in a dose dependent manner. alpha-ANP also enhanced progesterone production by granulosa cells, but not estradiol. It seems that the effect of alpha-ANP on ovarian steroidogenesis is a direct one.     

The endocrine heart and natriuretic peptides: histochemistry, cell biology, and functional aspects of the renal urodilatin system

 This review focuses on some selected aspects of the endocrine heart and natriuretic peptides. The endocrine heart is composed of specific myoendocrine cells of the cardiac atria. The myoendocrine cells synthesize and secrete the natriuretic peptide hormones which exhibit natriuretic, diuretic, and vasorelaxant properties. Immunohistochemical analyses show that natriuretic peptides of the A-type and B-type are localized not only in the specific granules of these myoendocrine cells but also in many other organs including the brain, adrenal medulla, and kidney. Also, their receptors are detected in many organs showing the multiple functions of these regulatory peptides. Of the members of the natriuretic peptide family, ANP (ANP for atrial natriuretic peptide; also denominated cardiodilatin, CDD), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and the A-type, including its renal form, urodilatin, are emphasized in this review. Urodilatin is localized in the kidney, differentially processed, and secreted into the urine. The intrarenal synthesis and secretion is the basis for a paracrine system regulating water and sodium reabsorption at the level of the collecting duct. CDD/ANP-1-126, cleaved from a precursor of 126 amino acids in the heart to a 28-amino acid-containing circulating molecular form (CDD/ANP-99-126), and urodilatin (CDD/ANP-95-126) share similar biochemical features and biological functions, but urodilatin may be more involved in the regulation of body fluid volume and water–electrolyte excretion, while circulating CDD/ANP-99-126 is responsible for blood pressure regulation. The physiological and pharmacological properties of these peptides have great clinical impact, and as a consequence urodilatin is involved in drug development for the treatment of acute renal failure, cardiomyopathia, and acute asthma. Accepted: 8 July 1998     

Electronmicroscopical, immunohistochemical, immunocytochemical and biological evidence for the occurrence of cardiac hormones (ANP/CDD) in chondrichthyes

As representatives of the vertebrate class of chondrichthyes the plagostomian species Squalus acanthias, Scyliorhinus canicula and Raja clavata as well as the holocephalan species Chimaera monstrosa were investigated for the presence of cardiac hormones of the atrial natriuretic polypeptide/cardiodilatin- (ANP/CDD-) family. ANP/CDD-immunoreactive cells were detected in the atria and the ventricles of all species studied. While these cells failed to react with antisera raised against the N-terminus of CDD-126 (= gamma-ANP) they reacted with all antisera directed against sequences of the C-terminus of CDD-126 (CDD 99-126) which is identical to alpha-ANP. The ANP/CDD-immunoreactive cells were found in high numbers in all regions of the atria and in moderate density also in the ventricles. In correspondence, in the electron microscope, myoendocrine cells which were characterized by dense-cored secretory granules were identified in the atrial and ventricular myocardium. With the use of the protein A-gold technique, ANP/CDD-immunoreactivity was determined within the secretory granules. Furthermore, in the bioassay, prepurified extracts of the atria and the ventricles of Scyliorhinus and Chimaera exerted dose-dependent relaxations of the pre-contracted mammalian (rabbit) aorta. In both cases the atrial extracts proved to be more potent than the ventricular extracts. The present findings indicate that myoendocrine cells occur in the atria and ventricles of chondrichthyes and that these cells contain homologous cardiac hormones of the ANP/CDD-family in their secretory granules. The results are compared with those obtained earlier for the other vertebrate classes and their phylogenetic and functional significance is discussed.     

Involvement of atrial natriuretic peptide in blood pressure reduction induced by estradiol in spontaneously hypertensive rats

The aim of the present study was to determine the involvement of atrial natriuretic peptide (ANP) in blood pressure (BP) alterations induced by estradiol treatment. Spontaneously hypertensive rats (SHR) and Wistar rats (WR) were ovariectomized and, after 3 weeks, were injected daily for 4 days with estradiol benzoate (E2; 5 microg/100 g/day) or a vehicle. One day after the last injection, the animals were decapitated, blood was collected, and both right and left atrial appendages were quickly removed for determination of ANP by radioimmunoassay (RIA), or used for ANP mRNA determination. Estradiol treatment induced a significant reduction of blood pressure in SHR, but not in WR. This reduction was correlated with the increase of plasma ANP levels that were significantly increased in E2-treated, compared with vehicle-treated, SHR. E2-treated SHR showed significant increases in ANP concentration in the right and left atria compared to the vehicle-treated animals. These observations were confirmed by ANP mRNA. In summary, the present study shows that short-term estradiol treatment reduces the blood pressure of ovariectomized SHR, but not of WR. This reduction was highly correlated with increased plasma estradiol and ANP levels. These results suggest that ANP is involved in mediating the effect of estradiol on blood pressure reduction.     

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101-Arg102-Ser103-Ser104. Cyclic AMP-dependent phosphorylation of Ser104 of atrial peptides with [gamma-32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD-28/alpha-hANP) is dramatically altered compared to the unphosphorylated peptide: the vaso-relaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.     

Amiloride differentially modulates ANP binding in human thyroid cells and bovine endothelial cells.

The diuretic and sodium channel inhibitor, amiloride, has been shown to increase atrial natriuretic peptide (ANP) binding several fold in certain cell types, but in other tissues it causes only marginal increases in specific ANP binding. In the present report we compare the effects of amiloride on ANP binding in bovine endothelial cells and human thyroid-derived cells, two cell types which differ in their predominant ANP receptor subtype. We found that amiloride (10(-3) M) increased specific [125I]ANP binding to 750% above control in endothelial cells, but among several thyroid cultures tested the maximal increase in ANP binding with amiloride was only 23% above control. Moreover, most of the thyroid cultures showed decreased ANP binding in the presence of amiloride. The increased ANP binding in endothelial cells exposed to amiloride is best explained by an increased affinity of the receptor for its ligand since the drug lowered the Kd of ANP binding from 0.73 nM to 0.16 nM without affecting the receptor binding capacity. The degree of amiloride enhancement of ANP binding in endothelial cells is increased with time in culture (200% above control at 5 days, 750% above at 30 days) suggesting the increase of an amiloride-sensitive receptor relative to an amiloride-insensitive receptor. The fact that the amiloride-induced decrease in ANP binding in thyroid cells was not exacerbated by pre-incubation with amiloride suggested that the observed amiloride effect was not due to increased receptor internalization with the drug. These results support a hypothesis that ANP receptor subtypes associated with separate signal transduction mechanisms might be modulated in an opposite manner by the binding of amiloride.     

Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells.

Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.     

Human plasmacytoid dendritic cells express an atrial natriuretic peptide receptor, guanylyl cyclase-A

Atrial natriuretic peptide is a cardiovascular hormone secreted mainly by the cardiac atria and regulates the volume–pressure homeostasis. The action of ANP is mediated by GC-A. We previously reported that human monocyte-derived dendritic cells express GC-A and respond to ANP with polarization toward a Th2-inducing phenotype. In the present study, we explored the possibility that pDC are subjected to immunoregulation via the ANP/GC-A system. We examined GC-A expression on blood pDC and found that GC-A was not expressed on fresh pDC but was induced after stimulation with CpG-oligodeoxynucleotide AAC-30, IL-3, or interleukin-3 plus CD40 ligand. Activated pDC responded to ANP with an increase in cGMP production, indicating that GC-A expressed on pDC was functional. We investigated whether tonsillar pDC express GC-A by immunohistochemistry and immunofluorescence staining. We found that GC-A⁺ HLA-DR⁺ cells were present in the T-cell areas and the perivascular areas. Flow cytometric analysis with tonsillar cells confirmed that lineage⁻ CD123^high pDC express GC-A. These results indicate that the ANP/GC-A system is involved in immune regulation through pDC in secondary lymphoid organs.     

The role of mast cells in atrial natriuretic peptide-induced cutaneous inflammation

Atrial natriuretic peptide (ANP) is widely distributed throughout the heart, skin, gastrointestinal and genital tracts, and nervous and immune systems. ANP acts to mediate vasodilation and induces mast cell activation in both human and rats in vitro. However, the mechanisms of ANP-induced mast cell activation, the extent to which ANP can induce tissue swelling, mast cell degranulation, and granulocyte infiltration in mouse skin are not fully understood. This issue was investigated by treatment with ANP in rat peritoneal mast cells (RPMCs) and mouse peritoneal mast cells (MPMCs) in vitro and by injection of ANP into the skin of congenic normal WBB6F1/J-Kit+/Kit+ +/+, genetically mast cell-deficient WBB6F1/J-Kit(W)/Kit(W-v) (W/W(v)) and mast cell-engrafted W/W(v) (BMCMC→W/W(v)) mice in vivo. ANP induced the release of histamine and TNF-α from RPMCs and enhanced serotonin release from MPMCs, in a dose-dependent fashion, as well as reduced cAMP level of RPMCs in vitro. In +/+ mice, ANP induced significant tissue swelling, mast cell degranulation, and granulocyte infiltration in a dose-dependent manner, whereas not in genetically mast cell-deficient W/W(v) mice. However, ANP-induced cutaneous inflammation has been restored in BMCMC→W/W(v) mice. These data indicate that mast cells play a key role in the ANP-induced cutaneous inflammation.     

Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade ¹²⁵I-ANP in the presence of EDTA. In this study, we have characterized this degradation of ¹²⁵I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a K_m of 320 ± 60 nM and V_max of 35 ± 14 pmol of ANP degraded/10 min/10⁵ cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0–8.5. Potato carboxypeptidase inhibitor and bestatin inhibited ¹²⁵I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when ¹²⁵I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe²⁶ and tyr²⁸ were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC₅₀ of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.     

Estradiol, progesterone and testosterone exposures affect the atrial natriuretic peptide gene expression in vivo in rats.

To clarify the effects of sex hormones on the expression of atrial natriuretic peptide (ANP), ovariectomized and intact female rats were subcutaneously injected with estradiol, progesterone, a mixture of them or olive oil solvent; castrated and untouched male rats were subcutaneously injected with estradiol, testosterone or olive oil, once a day for 7 days. The relative rANP-mRNA contents of rat atrial were measured by molecular hybridization. rANP-cDNA was labeled with 32P as a probe. The results revealed that estradiol and progesterone increased ANP gene expression. Furthermore their effects were associated with administration dose of these hormones and it was shown that they are probably coordinated. The physiological amounts of estradiol and progesterone may maintain suitable levels of rANP-mRNA and androgen may also increase the ANP gene expression in vivo. These experiments suggested that female sex hormone may have a dual purpose in fluid balance.     

Presence and release of immunoreactive atrial natriuretic peptide in granulosa cells of the pig ovarian follicle.

Atrial natriuretic peptide (ANP) has been reported to be locally synthesized in the ovary although its physiological roles are still unknown. To define the origin of ovarian ANP, we demonstrated the presence and release of immunoreactive (ir) ANP in pig granulosa cells and characterized its biochemical properties. Serial dilution curves made with the extracts of pig granulosa cells, their perfusates and follicular fluid were paralleled to the standard curve of ANP. The amount of irANP in the granulosa cell was 2 fg/cell. The total amount of irANP in granulosa cells significantly correlated with the levels of irANP in follicular fluid. Additionally, the total content of irANP in the follicle negatively correlated with the follicular size. On reverse phase HPLC, the major form of irANP in granulosa cells and follicular fluid was high molecular weight but that in perfusate was low molecular weight. In Northern blot analysis, ANP mRNA was detected in the pig granulosa cells. Immunohistochemistry showed ANP prohormone location in granulosa cells of rat ovary. These data strongly suggest that the granulosa cells synthesize and secrete ANP.     

Atrial granules in the dog heart after cardiac denervation

Summary Because the increase in sodium excretion during left atrial distension in conscious dogs is abolished after chronic cardiac denervation, we have investigated whether this is a result of the disappearance of specific atrial granules. Electron microscopy and light-microscopical and ultrastructural immunohistochemistry of canine atria show that atrial granules displaying immunoreactivity for cardiac hormones of the cardiodilatin/atrial natriuretic polypeptide (CDD/ANP) family are still present in denervated left and right atria, although reduced in quantity. It is concluded that the atrial-induced natriuresis is not only related to the existence of specific atrial granules. The functional link between atrial-induced natriuresis provoked by atrial distension and the release of atrial polypeptide hormones remains uncertain because the denervated heart can secrete CDD although the diuretic-natriuretic effect is altered.     

Natriuretic peptide receptor-A negatively regulates mitogen-activated protein kinase and proliferation of mesangial cells: role of cGMP-dependent protein kinase

We have examined the effect of atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65-75% in cells overexpressing NPRA, whereas in vector-transfected cells, its inhibitory effect was only 18-20%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF-stimulated [(3)H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only 20-25% inhibition in vector-transfected cells. These results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.     

In vitro circadian ANP secretion by gene transferring cells encapsulated in polycaprolactone tubes: gene chronotherapy

A new insofar as chronobiologic therapeutic approach by atrial natriuretic peptide (ANP) for hypertension and/or congestive heart failure (CHF) is based on the release of ANP from ANP cDNA transfected Chinese Hamster Ovary (CHO) cells encapsulated in polycaprolactone (PCL) tubes. ANP secretion was maintained for at least 6 months. The encapsulated cells remained viable during culturing. Control cells without transferred ANP cDNA were negative. ANP secretion is circadian periodic, peaking around 04:18, shifted to around 07:56 by melatonin treatment. The encapsulation technique, based on principles of chronotherapy, may provide a more efficient gene therapy, applicable for eventual human implantation of gene transferred cells.     

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 ± 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4–23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.     

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.     

Response of cardiac mast cells to atrial natriuretic peptide

Previously, our laboratory demonstrated that cardiac mast cell degranulation induces adverse ventricular remodeling in response to chronic volume overload. The purpose of this study was to investigate whether atrial natriuretic peptide (ANP), which is known to be elevated in chronic volume overload, causes cardiac mast cell degranulation. Relative to control, ANP induced significant histamine release from peritoneal mast cells, whereas isolated cardiac mast cells were not responsive. Infusion of ANP (225 pg/ml) into blood-perfused isolated rat hearts produced minimal activation of cardiac mast cells, similar to that seen in the control group. ANP also did not increase matrix metalloproteinase-2 activity, reduce collagen volume fraction, or alter diastolic or systolic cardiac function compared with saline-treated controls. In a subsequent study to evaluate the effects of natriuretic peptide receptor antagonism on volume overload-induced ventricular remodeling, anantin was administered to rats with an aortocaval fistula. Comparable increases of myocardial MMP-2 activity in treated and untreated rats with an aortocaval fistula were associated with equivalent decreases in ventricular collagen (P < 0.05 vs. sham-operated controls). Cardiac functional parameters and left ventricular hypertrophy were unaffected by anantin. We conclude that ANP is not a cardiac mast cell secretagogue and is not responsible for the cardiac mast cell-mediated adverse ventricular remodeling in response to volume overload.