Effect of natriuretic peptides on in vitro stimulated adrenocorticotropic hormone release and pro-opiomelanocortin mRNA expression by the fetal rat pituitary gland in late gestation

OBJECTIVE AND METHODS: We investigated the effects of individual natriuretic peptides (atrial natriuretic peptide, ANP; brain natriuretic peptide, BNP, and C-type natriuretic peptide, CNP) on rat corticotropin-releasing factor stimulated adrenocorticotropic hormone (ACTH) secretion by the pituitary gland of 21-day-old rat fetuses in vitro and on pro-opiomelanocortin gene expression using in situ hybridization. RESULTS: Graded concentrations of ANP, BNP, or CNP (10(-10), 10(-9), and 10(-8) mol/l) induced a log dose dependent inhibition of ACTH secretion induced by rat corticotropin-releasing factor (10(-10) mol/l). These natriuretic peptides showed equipotent effects on a molar basis. Moreover, ANP, BNP, or CNP at 10(-10) mol/l reduced significantly the pituitary pro-opiomelanocortin mRNA expression. In addition, the immunoreactive ANP, BNP, and CNP cells were localized in the anterior lobe, but not in the intermediate lobe of the fetal pituitary gland. CONCLUSIONS: These data suggest that the fetal pituitary gland may be both a source and a target for natriuretic peptides that might control ACTH synthesis and release via an endocrine and/or paracrine mechanism. The natriuretic peptides could participate, as well as glucocorticoids, in the control of the corticotropin-stimulating activity of the fetal rat in late gestation.     

Atrial natriuretic peptide prohormone gene expression: hormones and diseases that upregulate its expression

Atrial natriuretic peptides consist of a family of peptide hormones that are synthesized by three separate genes and then stored as three different prohormones (i.e., 126-amino acid [a.a.]) atrial natriuretic peptide (ANP), 108-a.a. brain natriuretic peptide (BNP), and 126-aa. C-natriuretic peptide (CNP) prohormones. The gene encoding for the synthesis of the atrial natriuretic peptide prohormone (proANP) consists of three exons and two introns. Exon 1 encodes the signal peptide and the first 16 aa. of the ANP prohormone. These 16 a.a. form the N-terminus of a peptide hormone named long-acting natriuretic hormone (LANH). A valine-to-methionine substitution in LANH results in a 2-fold increased incidence of strokes in humans. Exon 2 of the proANP gene encodes for three peptide hormones, i.e., vessel dilator, kaliuretic hormone, and ANP. Each of the proANP gene products have vasodilatory, diuretic, natriuretic, and/or kaliuretic properties. Stretch, glucocorticoids, thyroid hormone(s), mineralocorticoids, and calcium enhance proANP gene expression. Enhanced proANP gene expression is found in congestive heart failure, hypertension, and cirrhosis with ascites. The proANP gene is present with invertebrates and plants as well as in humans and other vertebrates.     

Functional expression of components of the natriuretic peptide system in human ocular nonpigmented ciliary epithelial cells

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP > ANP >/= BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65-75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4-23 elicited an inhibitory effect (30-37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75-85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.     

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.     

Characterization of immunoreactive human C-type natriuretic peptide in brain and heart.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.     

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

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

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

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

Effects of natriuretic peptides (ANP, BNP, CNP) on catecholamine synthesis and TH mRNA levels in PC12 cells

Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are present in adrenal chromaffin cells, and are co-secreted with catecholamines suggesting that these natriuretic peptides (NPs) may modulate functions of chromaffin cells in an autocrine and/or paracrine manner. Therefore, we investigated the effects of NPs on tyrosine hydroxylase (TH: a rate-limiting enzyme in biosynthesis of catecholamine) mRNA in rat pheochromocytoma PC12 cells. It was also determined whether the cyclic GMP/cGMP-dependent protein kinase (cGMP/PKG) pathway was involved in theses effects. Finally, we examined the effects of NPs on intracellular catecholamine content to confirm increase of catecholamine synthesis following TH mRNA induction. NPs (0.1 microM) induced significant increases of the TH mRNA (ANP= BNP> CNP). Also, the effects of NPs on TH mRNA were mimicked by 8-bromo cyclic GMP (1mM), and were blocked by KT5823 (1 microM) (inhibitor PKG) or LY83583 (1 microM) (guanylate cyclase inhibitor). Moreover, NPs were shown to induce significant increases of intracellular catecholamine contents (ANP= BNP> CNP). These findings suggest that NPs induced increases of TH mRNA through cGMP/PKG dependent mechanisms, which, in turn, resulted in stimulation of catecholamine synthesis in PC12 cells.     

Distribution and characterization of immunoreactive porcine C-type natriuretic peptide.

C-type natriuretic peptide (CNP) is a new member of the natriuretic peptide family recently identified in porcine brain (1). We raised an antiserum against porcine CNP and set up a radioimmunoassay (RIA) for CNP. Using this RIA system, distribution of immunoreactive (ir-) CNP in porcine tissue was measured and compared with that of ir-atrial natriuretic peptide (ANP) and ir-brain natriuretic peptide (BNP). Tissue concentration of ir-CNP in brain was the highest of the three natriuretic peptides at about 0.79 pmol/g wet wt. CNP was present in medulla-pons in high concentration, with a significant concentration detected in cerebellum. In contrast, ir-CNP was not detected in peripheral tissue, including heart, in a significant concentration. These data demonstrated sharp contrasts in the distribution of the three natriuretic peptides, suggesting that CNP is a natriuretic peptide functioning in the central nervous system.     

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.     

C-type natriuretic peptide is a potent activator of guanylate cyclase in endothelial cells from brain microvessels.

An exposure of endothelial cells from rat brain microvessels to C-type natriuretic peptide (CNP) resulted in a rapid and large increase in cGMP formation. The action of CNP did not require inhibitors of phosphodiesterases to be observed and occurred at nanomolar concentrations. Other natriuretic peptides (ANP and BNP) also stimulated cGMP formation in endothelial cells from brain microvessels but with a potency that was at least 100 times less than that of CNP. In contrast, endothelial cells from the aorta showed large cGMP responses to low concentrations of ANP and BNP but were unresponsive to CNP up to concentrations as large as 100 nM. It is concluded that endothelial cells from brain microvessels and from aorta express different receptors subtypes for natriuretic peptides. Endothelial cells from brain microvessels express CNP specific ANPB receptors; aortic endothelial cells express ANP (and BNP) specific ANPA receptors. CNP may play an important role in the regulation of water and electrolyte movements across the blood brain barrier.     

Natriuretic peptides: a new lipolytic pathway in human adipocytes.

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.     

C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain

Two types of natriuretic peptide, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated "C-type natriuretic peptide (CNP)", the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension.     

Discovery of a natriuretic peptide family and their clinical application

The identification of atrial natriuretic peptide (ANP) induced an explosive series of studies on the new peptide involved in control of the circulation, both in the basic and clinical fields. During the first decade of ANP research surprising progress has been made, revealing that the heart is an endocrine organ regulating the circulation system. ANP has been developed as a diagnostic tool and as a therapeutic drug for cardiac failure. In the second decade, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were identified, unveiling new profiles of this peptide family. Although BNP is also a circulating hormone that shares a common receptor with ANP, it is different from ANP in its' synthesis and secretion. Plasma concentration of BNP reflects the severity of heart failure in patients in a dramatic fashion, much moreso than ANP. Thus, BNP has been developed as a powerful diagnostic tool for cardiovascular diseases. The third congener, CNP, having a receptor of its own, was initially thought to function only in the brain. CNP was subsequently found to be produced from vascular endothelial cells and macrophages, indicating that CNP is a local regulator and also an antiproliferative factor in the vascular cell system, rather than a circulating hormone. Trials for the clinical application of CNP have also been discussed.     

Molecular cloning of natriuretic peptide receptor A from bullfrog (Rana catesbeiana) brain and its functional expression

A comparative study of natriuretic peptide receptor (NPR) was performed by cloning the NPR-A receptor subtype from the bullfrog (Rana catesbeiana) brain and analyzing its functional expression. Like other mammalian NPR-A receptors, the bullfrog NPR-A receptor consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain, a kinase-like domain and a guanylate cyclase domain. Sequence comparison among the bullfrog and mammalian receptors revealed a relatively low ( approximately 45%) similarity in the extracellular domain compared to a very high similarity ( approximately 92%) in the cytoplasmic regulatory and catalytic domains. Expression of NPR-A mRNA was detected in various bullfrog tissues including the brain, heart, lung, kidney and liver; highest levels were observed in lung. Functional expression of the receptor in COS-7 cells revealed that frog atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elicited cyclic guanosine 3'5'-monophosphate production by stimulating the receptor in a dose-dependent manner from 10(-10) M concentrations. Rat ANP was also effective in stimulating the frog receptor whereas rat BNP and porcine BNP were less responsive to the receptor. On the other hand, frog C-type natriuretic peptide (CNP) as well as porcine CNP stimulated the receptor only at high concentrations (10(-7) M). This clearly indicates that the bullfrog receptor is a counterpart of mammalian NPR-A, and is specific for ANP or BNP but not for CNP.     

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.     

ANP, BNP, and CNP enhance bradycardic responses to cardiopulmonary chemoreceptor activation in conscious sheep

We demonstrated previously that atrial natriuretic peptide (ANP) enhances reflex bradycardia to intravenous serotonin [5-hydroxytryptamine (5-HT)] (von Bezold-Jarisch reflex) in rats. To determine whether 1) ANP affects this cardiopulmonary vagal reflex in another species and 2) B-type (BNP) and C-type (CNP) natriuretic peptides share with ANP the ability to modulate this reflex, we used intravenous phenylbiguanide (PBG), a 5-HT(3) agonist, as the stimulus to evoke a von Bezold-Jarisch reflex (dose-related, reproducible bradycardia) in conscious adult sheep (n = 5). Three doses of PBG (13 +/- 3, 20 +/- 3, and 31 +/- 4 microg/kg) injected into the jugular vein caused reflex cardiac slowing of -7 +/- 1, -15 +/- 2, and -36 +/- 3 beats/min, respectively, under control conditions. These doses of PBG were repeated during infusions of ANP, BNP, or CNP (10 pmol. kg(-1). min(-1) iv), or vehicle (normal saline). Each of the natriuretic peptides significantly (P < 0.05) enhanced the sensitivity of bradycardic responses to PBG by 94 +/- 8% (ANP), 142 +/- 55% (BNP), and 61 +/- 16% (CNP). Thus not only did ANP sensitize cardiopulmonary chemoreceptor activation in a species with resting heart rate close to that in humans, but BNP and CNP also enhanced von Bezold-Jarisch reflex activity in conscious sheep.     

Natriuretic peptides cause relaxation of human esophageal mucosal muscle

Natriuretic peptides have been demonstrated to cause relaxation of the human gallbladder muscle through interaction with natriuretic peptide receptor-B (NPR-B/NPR2). Effects of natriuretic peptides in the human esophageal muscle were unknown. To investigate the effects of natriuretic peptides in the human esophagus, we measured relaxation of muscularis mucosae strips isolated from the human esophagus caused by C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. In endothelin-1 or carbachol-contracted mucosal muscle strips, CNP caused moderate, sustained and concentration-dependent relaxation. BNP caused a very mild relaxation whereas ANP and cANP(4-23) did not cause any relaxation. CNP was much more potent than BNP and ANP in causing relaxation. These suggest the existence of NPR-B mediating relaxation. The CNP-induced relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted esophageal strips and not by tetrodotoxin in carbachol-contracted strips, indicating a direct effect of CNP on the human esophageal muscularis mucosae. Taken together, these results demonstrate that natriuretic peptides cause relaxation of the muscularis mucosae of the human esophagus and suggest that the relaxation is through interaction with NPR-B. Natriuretic peptides may play an important role in the control of human esophageal motility.     

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

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

Natriuretic peptides stimulate steroidogenesis in the fetal rat testis

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