Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

Effect of native and synthetic atrial natriuretic factor on cyclic GMP

Mammalian atrial cardiocyte granules contain a potent natriuretic and diuretic peptide. Since cGMP appears to be involved in the modulation of cholinergic and toxin-induced sodium transport, we examined the effect of atrial natriuretic factor (ANF) on this nucleotide. Atrial but not ventricular extracts elicited approximately a 28-fold increase of urinary cGMP excretion parallel to the natriuresis and diuresis. The atrial extracts also elevated cGMP levels in kidney slices and primary cultures of renal tubular cells. The effect of ANF on cGMP appeared to be specific since antibodies which were capable of inhibiting the ANF-induced diuresis also suppressed cGMP excretion. Furthermore, during the course of ANF purification, the ANF-induced increase of cGMP production by kidney cells paralleled the heightened specific natriuretic activity of the atrial factor. A synthetic peptide (8-33)-ANF similarly increased urinary plasma and kidney tubular cGMP levels. The exact mechanism of action of ANF on cGMP remains to be elucidated, but indirect inhibition of cGMP phosphodiesterase appears to participate in its effect.     

Identification in rat atrial tissue of multiple forms of natriuretic polypeptides of about 3,000 daltons

Rat atrial natriuretic peptides of relatively low molecular weight have been isolated from the alpha-component of rectum relaxant activity corresponding to about 3,000 daltons, which was obtained as a side fraction in our previous isolation of beta-rat atrial natriuretic polypeptide (beta- rANP ). In contrast to the same fraction from human atria, the rat atrial alpha-component was found to contain six or more distinct but related peptides, eliciting a potent natriuretic activity. Six of them (B-II, C, D, E, B-I and A), containing 35, 33, 32, 31, 28 and 25 amino acid residues, respectively, have been purified to homogeneity and sequenced. All these peptides were found to correspond to the C-terminal sequence of beta- rANP composed of 48 residues, with varying N-terminal elongations. This indicates that these peptides are derived from beta- rANP . Peptide B-I, composed of 28 residues, is identical to alpha-human atrial polypeptide(alpha- hANP ), with a single replacement of Ile for Met at position 12.     

C-type natriuretic peptide induces human colonic myofibroblast relaxation

Intestinal response to injury requires coordinated regulation of the tension exerted by subepithelial myofibroblasts (SEM). However, the signals governing relaxation of intestinal SEM have not been investigated. Our aim was to test the hypothesis that signal transduction pathways initiated by C-type natriuretic peptide (CNP) induce intestinal SEM relaxation. We directly quantified the effects of CNP on isometric tension exerted by cultured human colonic SEM. We also measured the effects of CNP on cGMP content, myosin regulatory light chain (MLC) phosphorylation, and cytosolic Ca2+ concentration. CNP induced relaxation of SEM within 10 s. By 10 min, relaxation reached a plateau that was sustained for 2 h. CNP-induced relaxation was saturable, with a maximal decrease in tension (51.7 +/- 3.8 dyn) observed at 250 nM. SEM relaxation in response to CNP constituted approximately 23% of total basal tension. CNP increased intracellular cGMP content and reduced MLC phosphorylation. Effects of CNP on cGMP and MLC exhibited the same dose dependence as CNP-induced relaxation. MLC phosphorylation decreased within 2 min of CNP exposure and was sustained for at least 45 min. CNP also stimulated a large transient increase in cytosolic Ca2+ concentration that occurred within 30 s and was nearly complete by 1 min. We also observed that calyculin-A, a potent inhibitor of MLC phosphatase, completely abolished the reduction in MLC phosphorylation induced by CNP. These results suggest that CNP induces intestinal SEM relaxation through cGMP-associated reductions in MLC phosphorylation. Moreover, these findings raise the possibility that CNP plays a role in intestinal wound healing.     

C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells.

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than alpha-human atrial natriuretic peptide (alpha-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than alpha-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than alpha-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.     

Comparative biological activities of alpha-rat and alpha-human atrial natriuretic peptide in the inhibition of arginine vasopressin release in conscious rats

The comparative biological activities of intracerebroventricular (icv) injection of alpha-rat and alpha-human atrial natriuretic peptide (rANP and hANP, respectively) in the arginine vasopressin (AVP) release in conscious rats and the binding properties of these peptides to their specific receptors have been investigated. An icv injection of 5 micrograms rANP inhibited the AVP release induced by osmotic and hemorrhagic stimuli. In contrast, 20 micrograms of hANP was needed to exert an inhibitory effect on the AVP release. The receptor binding studies were carried out by using rat hypothalamic membrane preparations. The binding studies revealed that the potency of rANP was greater than that of hANP in displacing radioligand from its binding sites. Scatchard analysis revealed that the dissociation constant for rANP was significantly lower than that for hANP (0.52 +/- 0.04 vs 1.20 +/- 0.16 nM, P less than 0.01). The binding capacity of these peptides was similar. These results suggest that the greater biological potency of rANP compared with hANP in the inhibition of AVP release is caused by the difference in the binding potency of these peptides.     

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

AC-NP: a novel chimeric peptide with natriuretic and vasorelaxing actions

The aim of this study was to evaluate the cardiovascular and renal activities of a newly designed natriuretic peptide (NP). Here, we engineered a novel 28-amino acid chimeric peptide, termed AC-NP that combined the 17-amino acid ring of C type natriuretic peptide (CNP) with the 6-amino acid N-terminus and 5-amino acid C-terminus of atrial natriuretic peptide (ANP). Both in vitro and in vivo experiments were performed to determine the actions of AC-NP. In normal rats, AC-NP proved to be more potentially diuretic, natriuretic and hypotensive compared with other NPs, such as ANP, CNP and vasonatrin peptide (VNP), which is another man-made NP. In relaxation of isolated abdominal aorta from rat, AC-NP was equally effective to ANP, CNP and VNP. Elevated levels of 3',5'-guanosine monophosphate (cGMP) in plasma and urine cGMP excretion indicated the participation of cGMP in the functions of AC-NP. Taken together, innovative designed AD-NP might be a new candidate therapeutic peptide against cardiorenal disorders.     

Atrial natriuretic peptide provokes a dramatic increase in cyclic GMP formation and markedly inhibits muscarinic-stimulated Ca2+ mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca2+]i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca+1]i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 microM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca2+]i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca2+]i mobilisation with IC50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with EC50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>CNP>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant (Kd) and total receptor density (Bmax) values of the natriuretic peptide receptor for [125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic responses in the iris sphincter smooth muscle cells, and SV-CISM-2 cells can serve as an excellent model for investigating the cross talk between cGMP and the Ca2+ signalling system.     

Altered role of C-type natriuretic peptide-activated pGC-cGMP-PDE3-cAMP signaling in hyperthyroid beating rabbit atria

The role of C-type natriuretic peptide (CNP) in the pathophysiology of atrial function in hyperthyroidism has not been defined. This study was to define the role of CNP-activated particulate (p) guanylyl cyclase (GC)-cGMP-phosphodiesterase (PDE)3 signaling in the regulation of cAMP levels and contractile and secretory functions in the atria from hyperthyroid rabbits. Experiments were performed in perfused beating rabbit atria. CNP was used to activate pGC. In euthyroid atria from sham-treated rabbits, CNP (100 nM) increased cGMP and cAMP efflux by 176.7+/-17.7 and 55.3+/-10.0%, respectively. CNP decreased stroke volume and pulse pressure and ANP release by 51+/-7 and 41+/-2 and 60.4+/-3.2%, respectively. Pretreatment with milrinone blocked the CNP-induced increase of cAMP but without significant changes in decrease of atrial dynamics and ANP release. In hyperthyroid atria, CNP-induced increase of cGMP levels was accentuated, while CNP-induced increase of cAMP was attenuated. The gain of cAMP, i.e., change in cAMP efflux concentration in terms of cGMP was attenuated in the hyperthyroid compared to euthyroid atria. CNP rather increased atrial dynamics in hyperthyroid atria instead of decrease. CNP-induced decrease in atrial ANP release was attenuated. Pretreatment with milrinone blocked the CNP-induced increase of cAMP levels concomitantly with a decrease of atrial dynamics. The present study demonstrates that altered role of CNP-activated pGC-cGMP-PDE3-cAMP signaling is involved in the pathophysiology of hyperthyroid heart.     

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 (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.     

C-Type Natriuretic Peptide Is a Potent Stimulator of Cyclic GMP Production in Cultured Mouse Astrocytes

The effect of C-type natriuretic peptide (CNP), a novel member of the natriuretic peptide family, on cyclic GMP (cGMP) generation was studied in primary cultures of mouse astrocytes. CNP stimulated cGMP production by mouse astrocytes in a dose-dependent fashion, with an EC50 of 32 nM and a maximal stimulatory concentration of greater than 1 microM, which induced a rise of cGMP level from a baseline of 1.0 +/- 0.1 pmol/mg of protein to 196.2 +/- 22.0 pmol/mg of protein. Compared with our previously reported atrial and brain natriuretic peptide-induced cGMP responses, CNP had a lower EC50 and was 10-20 times more efficacious in its maximal effect on cGMP stimulation. These data lend support to the concept of a significant role of CNP in neuromodulation/neurotransmission.     

Biological effects of C-type natriuretic peptide in human myofibroblastic hepatic stellate cells.

During chronic liver diseases, hepatic stellate cells (HSC) acquire a myofibroblastic phenotype, proliferate, and synthetize fibrosis components. Myofibroblastic HSC (mHSC) also participate to the regulation of intrahepatic blood flow, because of their contractile properties. Here, we examined whether human mHSC express natriuretic peptide receptors (NPR). Only NPR-B mRNA was identified, which was functional as demonstrated in binding studies and by increased cGMP levels in response to C-type natriuretic peptide (CNP). CNP inhibited mHSC proliferation, an effect blocked by the protein kinase G inhibitor 8-(4 chlorophenylthio)-cGMP and by the NPR antagonist HS-142-1 and reproduced by analogs of cGMP. Growth inhibition was associated with a reduction of extracellular signal-regulated kinase and c-Jun N-terminal kinase and with a blockade of AP-1 DNA binding. CNP and cGMP analogs also blunted mHSC contraction elicited by thrombin, by suppressing calcium influx. The relaxing properties of CNP were mediated by a blockade of store-operated calcium channels, as demonstrated using a calcium-free/calcium readdition protocol. These results constitute the first evidence for a hepatic effect of CNP and identify mHSC as a target cell. Activation of NPR-B by CNP in human mHSC leads to inhibition of both growth and contraction. These data suggest that during chronic liver diseases, CNP may counteract both liver fibrogenesis and associated portal hypertension.     

Metabolic clearance rate and plasma half life of alpha-human atrial natriuretic peptide in man

The disappearance and metabolic clearance rate (MCR) of alpha human atrial natriuretic peptide (alpha h-ANP) has been studied in normal man by radioimmunoassay of the atrial peptide in plasma and plasma extracts. After an intravenous (iv) bolus injection of 100 micrograms alpha h-ANP, levels of immunoreactive alpha h-ANP (IR-alpha hANP) in unextracted plasma fell rapidly and exponentially during the first 10 min (t1/2 = 2.5 min), after which levels declined more slowly to reach basal values 30 min after injection. Venous plasma extracts, purified by Sep Pak cartridges, were used to calculate the MCR of IR-alpha hANP under steady state conditions of constant iv infusion (200 micrograms over 60 min) in healthy volunteers. Calculated MCR from venous samples was 2.4 L/min and volume of distribution 10.7 L. After cessation of infusions, the disappearance rate (rapid phase) of IR-alpha hANP was 3.1 min. These studies show that alpha h-ANP is rapidly metabolized at rates similar to other vasoactive hormones such as angiotensin II and vasopressin.     

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.     

Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes

In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.