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+).     

C-type natriuretic peptide system in rabbit colon

C-type natriuretic peptide (CNP) is mainly distributed in the brain and vascular endothelium and is considered to act as a local regulator in many tissues. The present study was aimed to determine the presence of CNP system and its biological function in rabbit colon. The serial dilution curves of tissue extracts were parallel to the standard curve of CNP-22. With gel permeation chromatography and reverse-phase HPLC, the major immunoreactive peak of CNP was observed at the same elution time corresponding to the synthetic CNP-53. The concentration of CNP in the mucosal layer of colon was 212.49 ± 30.44 pg/g tissue wet weight (n = 7), which was significantly higher than that in the muscular layer. The presence of CNP mRNA was also detected by RT-PCR and Southern blot analysis. Production of cGMP by the activation of particulate guanylyl cyclase stimulated by BNP and CNP was higher in membranes obtained from the muscular layer than from mucosal layer. More cGMP was produced by CNP than by ANP. Both natriuretic peptide receptor-A and -B mRNAs were detected by RT-PCR and specific binding sites to ¹²⁵I-[Tyr⁰]-CNP-22 were mainly localized to the muscular layer. Synthetic CNP inhibited basal tension, frequency and amplitude of basal motility of taenia coli of the right colon. This study showing the presence of CNP system and its biological function in colon suggests that endogenous CNP synthesized in the mucosal layer may have a paracrine function as a local regulator of colonic motility.     

MAPK and PI3K pathways regulate hypoxia-induced atrial natriuretic peptide secretion by controlling HIF-1 alpha expression in beating rabbit atria

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are pivotal and intensively studied signaling pathways in hypoxic conditions. However, the roles of MAPK and PI3K in the regulation of hypoxia-induced atrial natriuretic peptide (ANP) secretion are not well understood. The purpose of the present study was to investigate the mechanism by which the MAPK/ERK (extracellular signal-regulated kinase) and PI3K signaling pathways regulate the acute hypoxia-induced ANP secretion in isolated beating rabbit atria. An acute hypoxic perfused beating rabbit atrial model was used. The ANP levels in the atrial perfusates were measured by radioimmunoassay, and the hypoxia-inducible factor-1α (HIF-1α) mRNA and protein levels in the atrial tissue were determined by RT-PCR and Western blot. Acute hypoxia significantly increased ANP secretion and HIF-1α mRNA and protein levels. Hypoxia-induced ANP secretion was markedly attenuated by the HIF-1α inhibitors, rotenone (0.5 μmol/L) and CAY10585 (10 μmol/L), concomitantly with downregulation of the hypoxia-induced HIF-1α mRNA and protein levels. PD098059 (30 μmol/L) and LY294002 (30 μmol/L), inhibitors of MAPK and PI3K, markedly abolished the hypoxia-induced ANP secretion and atrial HIF-1α mRNA and protein levels. The hypoxia-suppressed atrial dynamics were significantly attenuated by PD098059 and LY294002. Acute hypoxia in isolated perfused beating rabbit atria, markedly increased ANP secretion through HIF-1α upregulation, which was regulated by the MAPK/ERK and PI3K pathways. ANP appears to be part of the protective program regulated by HIF-1α in the response to acute hypoxic conditions.     

C-type natriuretic peptide system in rabbit oviduct.

C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is known to be distributed mainly in brain and vascular endothelium and is considered to act as a local regulator in many tissues. The purpose of this study was to determine the presence of CNP system and its biological function in rabbit oviduct. The serial dilution curve of tissue extracts was parallel to the standard curve of CNP((1-22)) and a major peak of molecular profile of tissue extracts by HPLC was CNP((1-53)). mRNA of CNP which was the same size as positive control was also detected by Southern blot analysis. CNP increased the production of 3',5'-cyclic guanosine monophosphate (cGMP) in the purified membrane of oviduct, which was more in membranes derived from the isthmic portion than in the ampullar portion. The presence of mRNAs of natriuretic peptide receptor-A (NPR-A) and NPR-B was demonstrated by RT-PCR. Synthetic CNP((1-22)) inhibited both frequency and amplitude of basal motility of oviduct in a dose-dependent manner. The inhibitory effect of CNP on the basal motility was more potent in the isthmic portion than in the ampullar portion. These results demonstrate the presence of CNP system in the oviduct and regional differences in motility inhibition by CNP between isthmic and ampullar portions. Therefore, these findings suggest the possible existence of a CNP system that may exert a local regulator of basal motility, either alone or in concert with other hormones.     

Ouabain stimulates atrial natriuretic peptide secretion via the endothelin-1/ET(B) receptor-mediated pathway in beating rabbit atria

AimsOuabain has been reported to increase the secretion of atrial natriuretic peptide (ANP) in vitro. However, the mechanism by which ouabain increases ANP secretion is not well known. Therefore, the purpose of the present study was to investigate the underlying mechanism of ouabain-stimulated ANP secretion.Main methodsA perfused beating rabbit atrial model was used. The ANP and ET-1 levels in the atrial perfusates were measured by radioimmunoassays.Key findingsOuabain (1.0, 3.0 and 6.0 μmol/L) significantly increased atrial ANP secretion in a dose-dependent manner, while the endothelin (ET)-1 levels were increased by the higher doses (3.0 and 6.0 μmol/L) of ouabain. Ouabain-increased atrial ET-1 release was blocked by PD98059 (30.0 μmol/L), an inhibitor of mitogen-activated protein kinase (MAPK). Nifedipine (1.0 μmol/L), an inhibitor of L-type Ca²⁺ channels, completely abolished ouabain-increased ANP secretion without changing the ouabain-induced atrial dynamics. KB-R7943 (3.0 μmol/L), an inhibitor of Na⁺–Ca²⁺ exchangers, completely blocked the effects of ouabain-increased atrial dynamics, but did not modulate ouabain-increased ANP secretion. ET-1 significantly stimulated atrial ANP release in a dose-dependent manner. The effects of ET-1 and ouabain on ANP secretion were completely blocked by BQ788 (0.3 μmol/L), an inhibitor of ET-1 type B (ET_B) receptors, but not by BQ123 (0.3 μM), an inhibitor of ET-1 type A receptors. Ouabain-increased atrial ANP secretion was blocked by PD98059 and indomethacin (30.0 μmol/L), an inhibitor of cyclooxygenase.SignificanceOuabain significantly stimulated atrial ANP secretion via an ET-1-ET_B receptor-mediated pathway involving MAPK signaling pathway activation and prostaglandin formation.     

Accentuation of ursolic acid on muscarinic receptor-induced ANP secretion in beating rabbit atria

Aims

Ursolic acid has recently been reported to increase both atrial natriuretic peptide (ANP) secretion and mechanical dynamics in rabbit atria.

Main methods

The present study was designed to clarify the regulatory effects of ursolic acid on the β-adrenergic or muscarinic receptor-mediated changes in ANP secretory and contractile function allowing measurement of atrial dynamics such as pulse pressure, stroke volume, and cAMP efflux in isolated perfused beating rabbit atria.

Key findings

Pretreatment with ursolic acid significantly attenuated the isoproterenol (β-adrenergic agonist)-induced decrease in ANP secretion and increases in cAMP levels and atrial dynamics. Interestingly, ursolic acid concentration-dependently accentuated the acetylcholine-induced increase in ANP secretion and decrease in pulse pressure in the presence of isoproterenol (p < 0.001). These findings indicate that acetylcholine-induced increase in ANP secretion is potentiated by ursolic acid; furthermore, acetylcholine-induced decrease in atrial dynamics is also potentiated by ursolic acid, suggesting that ursolic acid regulates muscarinic receptor-mediated secretory and contractile responses in perfused beating rabbit atria.

Significance

This implicates for the beneficial effects of ursolic acid in the regulation of cardiovascular and body fluid homeostasis.     

Increase of atrial ANP release by 2,3-butanedione monoxime in beating rabbit atria

2,3-Butanedione monoxime (BDM) is a chemical phosphatase and has been known to dissociate mechanical contraction in the excitation–contraction coupling via inhibition of myofibrillar ATPase. BDM has also been found to decrease sarcolemmal L-type Ca²⁺ channel activity and intracellular Ca²⁺ in cardiac myocytes. It has been shown that Ca²⁺ entry via L-type Ca²⁺ channels decreased atrial myocyte atrial natriuretic peptide (ANP) release. The purpose of the present study was to address the effects of BDM in the regulation of ANP release. Experiments were performed in perfused beating rabbit atria. BDM accentuated atrial myocyte ANP release concomitantly with a decrease in atrial stroke volume and pulse pressure in a concentration-dependent manner. The BDM-induced activation of ANP release was attenuated by the treatment with nifedipine, an inhibitor of L-type Ca²⁺ channels. BDM further decreased atrial stroke volume and pulse pressure in the presence of nifedipine. Blockade of function of the sarcoplasmic reticulum with thapsigargin plus ryanodine slightly but not significantly attenuated the BDM-induced activation of ANP release. These data show that BDM is a potent stimulator for the ANP release and also suggest that the mechanism by which BDM activates atrial myocyte ANP release is related to inhibition of the L-type Ca²⁺ channel activity. The present finding also suggests that the effects of ANP released may be considered in an occasion of uncoupling by BDM of the excitation–contraction coupling of cardiomyocytes.     

Epicardial release of immunoreactive atrial natriuretic peptides in inside-out perfused rabbit atria

An easy and convenient isolated atrial perfusion technique was developed. The effect of stretch of the atrial subpericardial myocytes was investigated in the inside-out perfused rabbit atria. Graded distension of the inverted atria was induced by changing the elevation of the atrial catheter tip. Intra-luminal volume expansion resulted in an increase in release of immunoreactive atrial natriuretic peptides (irANPs). The response was volume, or pressure dependent. Distension-induced release of irANPs occurred at the reduction of the distension. IrANPs in epicardial perfusate showed both high and low molecular weights. The major peak of irANP was observed at the corresponding fraction to the rat ANP-(1-28) in the Sephadex G-50 gel chromatography. The data suggest that the epicardial release of irANP is stretch-induced response and that the release may be involved in the regulation of cardiac function.     

The positive inotropic effect of the aqueous extract of Convallaria keiskei in beating rabbit atria

The positive inotropic effect of the aqueous extract of Convallaria keiskei (ACK) and the possible mechanisms responsible for this effect were investigated in beating rabbit atria. ACK significantly increased atrial stroke volume, pulse pressure, and cAMP efflux in beating rabbit atria. The effects were not altered by pre-treatment with staurosporine and diltiazem, a non-selective protein kinase inhibitor and an L-type Ca2+ channel blocker, respectively. In addition, ACK markedly increased the K+ concentration in the beating atria-derived perfusate. Convallatoxin, a well-known digitalis-like cardiac glycosidic constituent of ACK, also increased atrial stroke volume and pulse pressure but did not alter the cAMP efflux level. The increases in atrial stroke volume and pulse pressure induced by convallatoxin were not also altered by pre-treatment with diltiazem. These results suggest that the ACK-induced positive inotropic effect in beating rabbit atria may, at least in part, be due to the digitalis-like activity of convallatoxin.     

Characteristics of distension-induced release of immunoreactive atrial natriuretic peptide in isolated perfused rabbit atria

Atrial pressure- or distension-induced release of atrial natriuretic peptide (ANP) has been considered as an important regulatory mechanism of ANP release in cardiac atria. A new technique to permit graded continuous atrial distension has been developed in an isolated perfused single rabbit atrium. Graded atrial distension was induced by changing the elevation of the outflow catheter tip. Intra-atrial volume expansion resulted in an increase in immunoreactive ANP release. The graded increase in atrial distension from 43.9 +/- 10.2 to 207.7 +/- 29.1 microliter resulted in 6.2-27.1-fold increases in volume-dependent immunoreactive ANP release. A rise in immunoreactive ANP release induced by increasing atrial distension did not occur in the state of atrial distension but occurred only after return to the reduced distension. However, in the case of atrial distension with pacing, an increase in immunoreactive ANP release was observed during atrial distension with pacing and after return to the basal level. The present study shows that the new technique is applicable to the study of the 'stretch-secretion coupling' mechanism of ANP release in vitro, and that the more important factor involved in the release of immunoreactive ANP induced by atrial distension may be the atrial reduction to basal level after distension rather than the stretch itself.     

Postanoxic recovery of spontaneously beating isolated atria: pH related role of adenylate kinase

The functional activity, adenine nucleotides, and creatine phosphate content of spontaneously beating isolated rabbit atria were measured prior to anoxia, after 1 hr anoxia, and at the end of 1 hr reoxygenation at pH 6.7 and 7.2 During anoxia at pH 7.2 there was 13.3% loss of adenine nucleotides pool, 35.2% loss of ATP, 36.2% increase in ADP, 200% increase in AMP, and a decrease to 8.8% of CP assayed to the beating atria in oxygen. At pH 6.7 there was almost the same decrease in CP, about 10% decrease in ATP, no change in total adenine nucleotides, no change in AMP and a higher increase in ADP (88.7%). The postanoxic recovery was much more complete when the pH was 6.7 during anoxia, and the first 40 min of reoxygenation. The extent of recovery of functional activity correlated well with the level of ATP in all cases not CP. Since the adenylate kinase and ATPase activity both decrease at acidic pH, their combined diminution would tend to preserve the adenine nucleotide pool and thus the better recovery at pH 6.7, because of a decrease in energy demand and unavailability of AMP for the degradation process. This study also supports the notion of compartmented adenine nucleotides connected by the creatine phosphate-creatine energy shuttle.     

Protein kinase-dependent and Ca(2+)-independent cAMP inhibition of ANP release in beating rabbit atria

Regulation of atrial release of atrial natriuretic peptide (ANP) is coupled to changes in atrial dynamics. However, the mechanism by which mechanical stretch controls myocytic ANP release must be defined. The purpose of this study was to define the mechanism by which cAMP controls myocytic ANP release in perfused, beating rabbit atria. The cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX) inhibited myocytic ANP release. The activation of adenylyl cyclase with forskolin inhibited ANP release, which was a function of an increase in cAMP production. Inhibitors for L-type Ca(2+) channels and protein kinase A (PKA) attenuated a minor portion of the forskolin-induced inhibition of ANP release. G?-6976 and KN-62, which are specific inhibitors for protein kinase C-alpha and Ca(2+)/calmodulin kinase, respectively, failed to modulate forskolin-induced inhibition of ANP release. The nonspecific protein kinase inhibitor staurosporine blocked forskolin-induced inhibition of ANP release in a dose-dependent manner. Staurosporine but not nifedipine shifted the relationship between cAMP and ANP release. Inhibitors for L-type Ca(2+) channels and PKA and staurosporine blocked forskolin-induced accentuation of atrial dynamics. These results suggest that cAMP inhibits atrial myocytic release of ANP via protein kinase-dependent and L-type Ca(2+)-channel-dependent and -independent signaling pathways.     

Effects of lidocaine in rabbit atria

Standard microelectrode recordings were obtained from rabbit right and left atria. Lidocaine (1 X 10(-5) M) had no effect on these, but 5 X 10(-5) M lidocaine significantly slowed rate and Vmax. This concentration had no effect on the duration of the action potential, a result clearly different from the effect of this drug in Purkinje tissue. Lidocaine had much less effect on the 'steady-state' relation of membrane potential to Vmax of phase 0 of the action potential than on the 'membrane responsiveness curve' obtained by the extra stimulus technique. We have demonstrated time-related recovery from sodium inactivation in rabbit left atria and have shown that lidocaine slows recovery in this tissue as it does in Purkinje fibres.     

Endothelin-3 reduces C-type natriuretic peptide-induced cyclic GMP formation in C6 glioma cells

The effect of endothelin-3 (ET-3) on C-type natriuretic peptide (CNP)-induced guanosine 3′,5′-cyclic monophosphate (cGMP) was examined in C6 glioma cells, CNP-induced cGMP formation was both time- and dose-dependent, with an EC₅₀ value of about 10 nM. While ET-3 and phorbol 12-myristate 13-acetate (PMA) had no effect on basal cGMP production, both compounds were potent inhibitors of CNP-induced cGMP formation, with IC₅₀ values of approximately 10 and 2 nM, respectively. Although protein kinase C (PKC) inhibitors had no effect on basal cGMP formation, Ro 31-8220, a PKC inhibitor, reversed the ET-3 inhibition on CNP-induced cGMP formation by 63% and that of PMA almost completely. Our findings suggest that stimulation of cGMP formation by CNP in C6 glioma cells is negatively modulated by PKC activation, and that the inhibitory action of ET-3 on CNP-stimulated cGMP formation is mediated partly by PKC.     

Isolation and identification of C-type natriuretic peptide in chicken brain

C-type natriuretic peptide (CNP) has recently been identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system for porcine CNP, we found a significant concentration of immunoreactive (ir-) CNP in chicken brain, from which a new peptide was isolated. By microsequence analysis, the amino acid sequence of the peptide was determined to be Gly-Leu-Ser-Arg-Ser-Cys-Phe- Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys. Based on its high homology to porcine CNP, the peptide was designated chicken C-type natriuretic peptide. Chicken CNP also elicits pharmacological effects highly similar to porcine CNP, suggesting that CNP functions as a neuropeptide in the chicken central nervous system.     

Altered regulation of phosphatidylinositol 3-kinase signaling in cathepsin D-deficient brain

Cathepsin D (CD) is an essential lysosomal protease and mice lacking this enzyme exhibit neuropathology similar to that observed in brains of patients with neuronal ceroid lipofuscinosces (NCL/Batten disease), a group of autosomal recessive pediatric neurodegenerative diseases. CD-deficient (CD-/-) brains exhibit a dramatic induction of autophagic stress as defined by the aberrant accumulation of autophagosomes, which is concomitant with markers of apoptosis. However, the signaling abnormalities which lead to CD deficiency-induced neurodegeneration are poorly defined. Since phosphatidylinositol-3 kinase (PI3-K) is known to regulate both apoptosis and autophagy, PI3-K-mediated signaling events were assessed in CD-/- brain at P14 and P25-26. Compared to WT littermate controls, CD-/- cortical neurons exhibited a widespread decrease in phosphorylation of Akt (inactivation) and GSK3beta (disinhibition) at P25-26, while levels of total Akt and GSK3beta remained unchanged. This P25-26-specific decrease in phosphorylation of Akt and GSK-3beta in CD-/- brain coincided temporally with markers of apoptosis but followed the induction of autophagic stress observed at both P14 and P25-26. In addition, levels and/or activation of mTOR and Beclin were not affected by CD deficiency, suggesting that the accumulation of autophagosomes is not due to an increased synthesis of autophagosomes but rather from an inhibition of autophagosome recycling, due most likely to a compromise in lysosome function. Together these observations indicate a pronounced decrease in pro-survival PI3-K signaling in CD-/- brain that may contribute to autophagic stress-induced and apoptotic neuropathology.     

mRNA quantification of C-type natriuretic peptide in brain areas of rodents

C-type natriuretic peptide (CNP) is regarded as an endothelium-derived vasodilator and might therefore have an important role in controlling vascular tone and remodeling. Because CNP also is expressed in the brain, it is considered to be a neurotransmitter. The present study compares expression levels of CNP mRNA in distinct areas of the mouse brain with the expression pattern in the rat brain. A distinct expression of CNP was found in all investigated areas with the exception of the mouse striatum. In both rodents, high CNP expression was detected in the tegmentum.     

Characteristics of accumulation of ephedrine in rabbit atria.

The characteristics of uptake of (not equal to)-(beta-14C)ephedrine were studied in isolated rabbit atria. Ephedrine was rapidly accumulated against the concentration gradient. From 5 X 10-7 to 10-2 M, uptake occurred at a uniform initial rate. Uptake was slightly inhibited by high concentrations of ouabain, cocaine, desipramine, lidocaine and phenethylamines, and by a reduction in the external Na+ concentration. Uptake was not, however, reduced by omission of K+ from the medium, by metabolic inhibitors or by a variety of drugs known to inhibit the extraneuronal uptake and binding of noradrenaline. Pretreatment of animals with 6-hydroxydopamine very significantly reduced the uptake of (not equal to)-(3H)metaraminol, but did not alter the uptake of ephedrine. It was concluded that the uptake of ephedrine in rabbit atria occurred predominantly in extraneuronal tissues possibly as a result of passive diffusion followed by binding.     

Radioimmunoassay of atrial natriuretic factor (ANF) in rat atria

We describe a solid phase radioimmunoassay for atrial natriuretic factor (ANF) and its application for measurement of this peptide in homogenates of rat atria. The method uses a synthetic 26 amino-acid fragment (8-33 ANF) of the native peptide. Sample (or standard) are incubated with the rabbit anti-8-33 ANF antiserum in peptide (8-33 ANF)-coated wells. Then an excess of I125 goat anti-rabbit IgG is added. The radioactivity bound is directly proportional to the amount of ANF present. The concentration of immunoreactive ANF has been found to be about 4 times higher in the right atrium than in the left atrium of the rat.     

Distribution of atrial natriuretic factor in fetal rat atria and ventricles

Summary An immunohistochemical study of rat fetal hearts at 20 days of gestation revealed the presence of immunoreactive atrial natriuretic factor (ANF) in cardiocytes of the left and right atria as well as in certain cells is the left and right ventricles. In the atria, cells of the adluminal pectinate muscles appear more densely labeled than the more peripheral mural cells. In the ventricles, immunoreactive cells were found only in adluminal cardiocytes of the presumptive trabeculae and papillary muscles. The results indicate that ANF is synthesized in the perinatal heart, and that the presence of this hormone in the ventricular cardiocytes may be of only temporary nature during certain stages of pre- and postnatal development.Supported by Miami Valley Chapter of American Heart Association MVH-86-019 and MVH-86-010