Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP > CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.     

Natriuretic peptide C receptor signalling in the heart and vasculature

Natriuretic peptides (NPs), including atrial, brain and C-type natriuretic peptides (ANP, BNP and CNP), bind two classes of cell surface receptors: the guanylyl cyclase-linked A and B receptors (NPR-A and NPR-B) and the C receptor (NPR-C). The biological effects of NPs have been mainly attributed to changes in intracellular cGMP following their binding to NPR-A and NPR-B. NPR-C does not include a guanylyl cyclase domain. It has been denoted as a clearance receptor and is thought to bind and internalize NPs for ultimate degradation. However, a substantial body of biochemical work has demonstrated the ability of NPR-C to couple to inhibitory G proteins (Gi) and cause inhibition of adenylyl cyclase and activation of phospholipase-C. Recently, novel physiological effects of NPs, mediated specifically by NPR-C, have been discovered in the heart and vasculature. We have described the ability of CNP, acting via NPR-C, to selectively inhibit L-type calcium currents in atrial and ventricular myocytes, as well as in pacemaker cells (sinoatrial node myocytes). In contrast, our studies of the electrophysiological effects of CNP on cardiac fibroblasts demonstrated an NPR-C-Gi-phospholipase-C-dependent activation of a non-selective cation current mediated by transient receptor potential (TRP) channels. It is also known that CNP and BNP have important anti-proliferative effects in cardiac fibroblasts that appear to involve NPR-C. In the mammalian resistance vessels, including mesenteric and coronary arteries, CNP has been found to function as an NPR-C-dependent endothelium-derived hyperpolarizing factor that regulates local blood flow and systemic blood pressure by hyperpolarizing smooth muscle cells. In this review we highlight the role of NPR-C in mediating these NP effects in myocytes and fibroblasts from the heart as well as in vascular smooth muscle cells.     

Expression and glucocorticoid regulation of natriuretic peptide clearance receptor (NPR-C) mRNA in rat brain and choroid plexus

The natriuretic peptide clearance receptor (NPR-C) binds atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide with high affinity. This receptor lacks an intracellular guanylate cyclase domain, and is believed to exert biological actions by sequestration of released natriuretic peptides and/or inhibition of adenylate cyclase. The present report summarizes the first detailed mapping of NPR-C mRNA in rat brain. In situ hybridization analysis revealed high levels of NPR-C mRNA expression in frontal and retrosplenial granular cortices, medial preoptic nucleus, ventral cochlear nucleus and choroid plexus. NPR-C mRNA expression was also observed in deep layers of neocortex and limbic cortex, posterior cortical amygdala, ventral subiculum, amygdalohippocampal area, and dentate gyrus. Positive hybridization signal was observed in both anterior and intermediate lobes of the pituitary gland. Regulatory studies indicated that expression of NPR-C mRNA was increased in the medial preoptic nucleus of adrenalectomized rats, suggesting negative glucocorticoid regulation. No changes in NPR-C mRNA expression were observed in frontal cortex or choroid plexus. These results suggest a role for the NPR-C in modulation of natriuretic peptide availability and/or adenylate cyclase activity in a subset of central natriuretic peptide circuits concerned with cortical, olfactory and neuroendocrine functions. Response of the NPR-C gene to changes in circulating hormones suggests the capacity for glucocorticoid modulation of natriuretic peptide action at the receptor level.     

NPR-C protects embryonic stem cells from apoptosis by regulating p53 levels

The identification of intrinsic factors required for propagation of self-renewing embryonic stem (ES) cells is important to improve the efficiency of expansion of ES cells for therapeutic purposes. Here, we report a novel role for natriuretic peptide receptor-C (NPR-C) in the survival of murine ES cells. We found that NPR-C was highly expressed in ES cells and was downregulated during ES cell differentiation. Knockdown of NPR-C in ES cells by using a small-interfering RNA resulted in apoptotic cell death, and the induction of p53 protein expression. Conversely, chemical inhibition of p53 by α-pifithrin significantly reduced apoptosis in NPR-C-deficient cells. cANF((4-23)), a selective NPR-C agonist, protected ES cells against oxidative stress-induced apoptosis, and blocked activation of p53 and Nanog suppression in the presence of DNA-damaging agents. Thus, NPR-C is required to control DNA damage-induced p53 levels to maintain ES cell self-renewal.     

钠尿肽C型受体信号通路与心血管疾病

钠尿肽家族(natriuretic peptides,NPs)主要包括心房钠尿肽(atrial natriuretic peptide, ANP)、脑钠尿肽(brain natriuretic peptide, BNP)和C型钠尿肽(C-typenatriuretic peptide, CNP)3类,新发现的还有曼巴蛇钠尿肽、尿扩张素及在澳大利亚大班蛇毒液中的一类钠尿肽样肽类[1].     

NPR-C: a component of the natriuretic peptide family with implications in human diseases

The natriuretic peptide (NP) family includes atrial natriuretic peptide (ANP), B-type natriuretic peptide, C-type natriuretic peptide and their receptors NPR-A, NPR-B and NPR-C. The effects exerted by this hormonal system in the control of cardiovascular, renal and endocrine functions have been extensively investigated. Moreover, the involvement of NP in the pathogenesis of cardiovascular diseases has been demonstrated. Among the NP components, NPR-C has been described, at the time of its discovery, as the clearance receptor of NP devoid of any physiological functions. Emerging roles of NPR-C, however, have been highlighted over the last few years in relation to its effects on the cardiovascular system and other organs. These effects appear to be directly mediated through distinct cAMP-dependent intracellular mechanisms. Moreover, evidence has been accumulated on a potential pathophysiological role of NPR-C in human diseases. Ongoing studies from our group are revealing its involvement in the mediation of antiproliferative effects exerted on vascular cells by a molecular variant of human ANP. Thus, a new appraisal of NPR-C is overcoming the traditional view of a mere clearance receptor. This review focuses on the most important evidence supporting an involvement of NPR-C in mediating some of the actions of NP and its direct implication in cardiovascular diseases. The current state of knowledge highlights the need of further studies to better clarify the specific roles of NPR-C in pathophysiological processes.     

Brain natriuretic peptide: physiological, biological and clinical aspects

Brain natriuretic peptide is one member of the natriuretic peptide family, including also ANP, CNP, DNP and urodilatin. In human, brain natriuretic peptide is mainly secreted by the cardiac ventricles. BNP is synthetized as pre-proBNP form, secondary cleaved in proBNP, itself equimolarly cleaved in BNP and NT-proBNP. The biological action of BNP is mediated by the NPR-A receptor. This peptide is eliminated from the systemic circulation by a neutral endopeptidase and by a clearance receptor (NPR-C). The BNP and NT-proBNP concentrations are measured using automated rapid immunoassay techniques. Plasma concentrations of the two peptides physiologically increase with age and are found to be higher in women than in men. The action of BNP against fluid expansion is explained by its vascular (vasodilatation), renal (diuretic and natriuretic) and cerebral activities. The measurement of these two peptides contributes to the diagnosis of heart failure. These peptides are prognostic markers both in heart failure and in acute coronary syndromes. In renal insufficiency, the interpretation of the increase in these two peptide concentrations may be difficult, particularly with the NT-proBNP which is mainly excreted by the kidneys.     

CNP/NPR-B/cGMP信号通路调控雌性生殖过程

C型利钠肽(C-type natriuretic peptide,CNP)与其特异性受体利钠肽受体B(natriuretic peptide receptor B,NPR-B)结合并催化下游第二信使环磷酸鸟苷(cyclic guanosinc monophosphate,cGMP)生成,形成CNP/NPR-B/cGMP信号通路,从而介导多种生物学效应.近年来大量研究证实,CNP/NPR-B/cGMP信号通路与卵泡发育、卵母细胞减数分裂与成熟及胚胎着床和发育关系密切,提示此信号通路可能在雌性生殖过程中发挥显著作用.     

Distribution of natriuretic peptide receptor-C immunoreactivity in the rat brainstem and its relationship to cholinergic and catecholaminergic neurons

The natriuretic peptide receptor type C (NPR-C) binds all natriuretic peptides. It is thought to be involved in the clearance of natriuretic peptides and more recently has been defined as essential for the neuromodulatory effects of natriuretic peptides. Although the distribution of NPR-C mRNA has been reported in the rat forebrain, there are no data on the distribution of NPR-C in the brainstem. We report an immunofluorescence study on the distribution of NPR-C immunoreactivity in the rat brainstem, and its presence in cholinergic and catecholaminergic neurons. NPR-C immunoreactivity was detected in several regions, including the periaqueductal gray, oculomotor nucleus, red nucleus and trochlear nucleus of the midbrain; the pontine nucleus, dorsal tegmental nucleus, vestibular nucleus, locus coeruleus, trigeminal motor nucleus, nucleus of the trapezoid body, abducens nucleus and facial nucleus of the pons; and the dorsal motor nucleus of the vagus, hypoglossal nucleus, lateral reticular nucleus, nucleus ambiguus and inferior olivary nucleus of the medulla oblongata. Interestingly, NPR-C immunoreactivity was detected in the cholinergic neurons of the oculomotor nucleus, trochlear nucleus, dorsal tegmental nucleus, motor trigeminal nucleus, facial nucleus, dorsal motor nucleus of the vagus, nucleus ambiguus and hypoglossal nucleus. Furthermore, NPR-C immunoreactivity was detected in several catecholaminergic neuronal groups including the A6, A5, A1, C3 and C1 cell groups. These results are consistent with an important role for natriuretic peptides in neuroendocrine regulation and central cardiovascular integration. The extensive distribution of NPR-C in the brainstem supports the hypothesis that NPR-C is involved in the neuromodulatory effect of natriuretic peptides.     

Elevated C-type natriuretic peptide elicits exercise preconditioning-induced cardioprotection against myocardial injury probably via the up-regulation of NPR-B

To evaluate exercise preconditioning (EP)-induced cardioprotective effects against exercise-induced acute myocardial injury and investigate the alterations of C-type natriuretic peptide (CNP) and its specific receptor, natriuretic peptide receptor B (NPR-B), during EP-induced cardioprotection. Rats were subjected to treadmill exercise as an EP model (4 periods of 10 min each at 30 m/min with intervening periods of rest lasting 10 min). High-intensity exercise was performed 0.5 and 24 h after the EP. EP attenuated high-intensity exercise-induced myocardial injury in both the early and late phases. After EP and high-intensity exercise, CNP and NPR-B levels increased robustly, but no alterations in the plasma CNP were observed. The enhanced NPR-B, plasma and tissue CNP, and its mRNA levels after high-intensity exercise were significantly elevated by EP. These results suggest that cardiac CNP and NPR-B play an important role in EP-mediated cardioprotection against high-intensity exercise-induced myocardial injury in rats.     

Homozygous Sequence Variants in the NPR2 Gene Underlying Acromesomelic Dysplasia Maroteaux Type (AMDM) in Consanguineous Families

Acromesomelic dysplasia Maroteaux type (AMDM) is an autosomal recessive skeletal disorder characterized by disproportionate short stature with shortening of the acromesomelic sections of the limbs. AMDM is caused by mutations in the NPR2 gene located on chromosome 9p21‐p12. The gene encodes the natriuretic peptide receptor B (NPR‐B) that acts as an endogenous receptor for C‐type natriuretic peptide (CNP). Both CNP and NPR‐B are considered as important regulators of longitudinal growth. The study presented here investigated three consanguineous families (A, B, C) segregating AMDM in an autosomal recessive manner. Linkage in the families was established to the NPR2 gene on chromosome 9p12‐21. Sequence analysis of the gene revealed two novel missense variants (p.Arg601Ser; p.Arg749Trp) in two families and a previously reported splice site variant (c.2986+2T>G) in the third family.     

C-type natriuretic peptide modulates bidirectional plasticity in hippocampal area CA1 in vitro

C-type natriuretic peptide (CNP) and the natriuretic peptide receptor B (NPR-B) are expressed throughout the hippocampus. We tested whether CNP affected long-term potentiation (LTP) or long-term depression (LTD) in area CA1. Field potentials (FP) were simultaneously recorded in stratum pyramidale (SP) and stratum radiatum (SR) of area CA1 in rat hippocampal slices. To induce LTD and LTP stimulation was applied to SR in area CA1 at 1 and 5 Hz and 30–100 Hz, respectively. CNP (100 nM) increased LTD magnitude while LTP induction was impeded. Thus, in the presence of CNP the threshold for LTP induction was shifted to higher stimulus frequencies, a modulation that showed layer-specific differences in area CA1. Effects of CNP were prevented by the NPR-B antagonist HS-142-1. In the presence of the GABA_A receptor blocker bicuculline (BMI, 5 μM), CNP-mediated effects were attenuated in SP and SR. Intracellular recordings under this condition revealed that CNP significantly reduced number of action potentials generated during depolarizing current steps. The input resistance of CA1 cells and amplitude of isolated excitatory postsynaptic potential (EPSPs) were significantly increased by CNP whereas these changes were not observed in the absence of BMI. 100 Hz stimulation induced stable potentiation of the EPSP amplitude in CA1 pyramidal cells while this effect was strongly attenuated by CNP. This effect was prevented by BMI. Immunohistochemistry indicated that the peptide binds to receptors expressed on pyramidal cells and GAD_65/67-immunopositive interneurons. 20 Hz stimulation, applied for 30 s, induced LTP in SR and SP. CNP attenuated LTP in SP and reversed LTP into LTD in SR. These effects were mimicked by low-dose dl-2-amino-5-phosphonopentanoic acid (dl-APV) (10 μM) suggesting partial N-methyl d-aspartate (NMDA) receptor dependency of CNP-mediated effects. Together, our data suggest that CNP is involved in the regulation of bidirectional plasticity in area CA1 potentially by modulating GABA_A-mediated inhibition and NMDA receptors.     

C-型利钠利尿多肽在大鼠血管球囊损伤中作用

目的和方法:本实验在大鼠主动脉球囊剥脱模型上,观察血浆和主动脉组织C-型利钠利尿肽(CNP)的动态变化及外源性CNP对大鼠球囊损伤后内膜生成的影响,以探讨CNP在再狭窄中的作用。结果:发现大鼠球囊损伤后血浆CNP的浓度升高,内膜剥脱后第3 d、10 d、21 d,血浆CNP水平分别增高80.7%(P＜0.01),43.5%(P＜0.05)和27.5%(P＜0.05),而主动脉组织CNP含量在损伤后第3 d下降46.6%(P＜0.05),第10 d,21 d和28 d CNP含量分别增加2.8倍(P＜0.01)、1.6倍(P＜0.05)和0.82倍(P＜0.05)。外源性CNP显著抑制球囊损伤后第7 d和21 d新生内膜的形成,内膜/中膜比值分别降低22%(P＜0.05)和20%(P＜0.05)。结论:CNP参与血管球囊损伤后的修复过程,外源性CNP可抑制内膜增厚。     

失血性休克患者血浆C型利钠多肽含量变化的研究

目的：观察失血性休克患者血浆Ｃ型利钠多肽的含量变化,探讨其在休克发病机制中的作用。方法：采用放射免疫法测定了３０例健康人,２５例轻度休克、２６例中度休克患者身浆Ｃ型利钠多肽含量。结果：轻度休克患者血浆ＣＮＰ含量明显高于正常对照组（Ｐ〈０．０１）;中度休克患者血浆ＣＮＰ含量明显高于正常对照组（Ｐ〈０．０１）,也明显高于轻度休克组（Ｐ〈０．０１）。结论：血浆ＣＮＰ含量增高与失血性休克的严于正常对照组（     

C-type natriuretic peptide decreases hippocampal network oscillations in adult rats in vitro

C-type natriuretic peptide (CNP) is an abundant neuropeptide in the human brain and the cerebrospinal fluid. CNP is involved in anxiogenesis and exerts its effects through the natriuretic peptide receptor B (NPR-B), which is expressed in the hippocampus. Hippocampal network oscillations of distinct frequency bands like gamma (γ)-oscillations and sharp wave-ripple complexes (SPW-Rs) are likely involved in various cognitive functions such as the storage of information and memory consolidation in vivo. Here, we tested the effects of CNP on distinct network oscillations in horizontal slices of rat hippocampus. We found that CNP decreased the power of stimulus- and ACh/physostigmine-induced γ-oscillations. In contrast to stimulus-induced γ-oscillations, CNP increased the frequency of ACh-induced, persistent network oscillations. Moreover, the peptide hormone reduced the incidence of LTP-associated SPW-Rs in area CA3 and CA1. Immunohistochemistry indicates that the peptide binds to receptors expressed on a subset of GAD 65–67-immunopositive cells in addition to binding to principal and other presumably non-neuronal cells. CNP caused a hyperpolarization of CA3 neurons increased their input resistance and decreased inhibitory conductance. Together, our data suggest that the effects of CNP on synchronized hippocampal network oscillations might involve effects on hippocampal interneurons.     

Do natriuretic peptides modify arterial baroreflexes in sheep?

While atrial and B-type natriuretic peptides (ANP and BNP) have been shown to enhance reflex responses attributed to cardiac vagal afferents, their effects on arterial baroreceptor reflex function remain controversial. The actions of C-type natriuretic peptide (CNP) in this regard are unknown. To clarify their actions on arterial baroreflexes, we tested whether i.v. infusions of ANP, BNP or CNP at 10 pmol kg(-1) min(-1) modified the steady-state mean arterial blood pressure-heart rate (MAP-HR) relationship in conscious sheep. At this dose, all three natriuretic peptides are known to enhance the cardiac chemoreflex response to phenylbiguanide (Bezold-Jarisch reflex). Sigmoid MAP-HR relationships were constructed from the steady-state responses to alternating injections of vasopressor (phenylephrine, 1-15 microg kg(-1)) and vasodepressor agents (nitroprusside, 1-15 microg kg(-1)) in the absence and presence of infused ANP, BNP or CNP (tested in random order at least 1 week apart). No parameter of the steady-state baroreflex relationship was significantly altered by infusion of any of the three natriuretic peptides. We conclude that in conscious sheep, normal arterial baroreceptor-HR reflex function prevails in the presence of moderate doses of ANP, BNP or CNP.     

Membrane-bound guanylate cyclase as the receptor of natriuretic peptides. A minireview

A minireview is presented on the ultracytochemical localization of membrane-bound guanylate cyclase (GC) in various tissues and in cultured cells after activation with three natriuretic peptides, the atrial natriuretic factor (ANF), the brain natriuretic peptide (BNP), and the C-type natriuretic peptide (CNP). GC, two subtypes of which have been recently identified, is the receptor for these peptides. The GC isoforms are differently stimulated by ANF, BNP and CNP. Under our experimental conditions, the natriuretic peptides were strong activators of GC since samples incubated without natriuretic peptides do not reveal any cyclase activity. The natriuretic peptide-stimulated GC activity was studied in rat kidney, lung, adrenal gland and neurohypophysis, in rabbit platelets, in lamb olfactory mucosa, and in rat C6 glioma cells. On the basis of the subcellular GC localization some additional functions of peptides are hypothesized.     

Effects of dendroaspis natriuretic peptide on calcium-activated potassium current and its mechanism

In this study, we sought to investigate the effect of dendroaspis natriuretic peptide (DNP) on calcium-activated potassium current (I K(Ca)) and its mechanism in gastric antral circular smooth muscle cells (SMCs) using the whole-cell patch-clamp technique. DNP concentration-dependently increased macroscopic I K(Ca) and spontaneous transient outward currents (STOCs) in freshly isolated guinea pig gastric antral circular SMCs. The effects of DNP on I K(Ca) and/or STOCs were not blocked by applying calcium-free bath solution or the ryanodine receptor (RyR) antagonist ryanodine (10 microM), but they were inhibited by the inositol triphosphate receptor (IP3R) inhibitor heparin or the guanylate cyclase inhibitor LY83583. Moreover, a DNP-induced increase in STOCs was potentiated by the cyclic guanosine monophosphate (cGMP)-sensitive phosphoesterase inhibitor zaprinast. In conclusion, our results suggest that DNP increases I K(Ca) in gastric antral circular SMCs by increasing cGMP production and activating IP3Rs.     

HS-142-1, a novel non-peptide ANP antagonist, blocks the cyclic GMP production elicited by natriuretic peptides in PC12 and NG108-15 cells.

HS-142-1 is a novel non-peptide antagonist for atrial natriuretic peptide (ANP) receptor. The effect of HS-142-1 on the cyclic GMP production elicited by natriuretic peptides in neuronal cell lines, PC12 and NG108-15 was examined. Natriuretic peptides such as ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) enhanced cyclic GMP production in a dose-dependent manner. HS-142-1 inhibited cyclic GMP accumulation elicited by natriuretic peptides in a dose-dependent fashion in both cells. The results suggest that HS-142-1 will be an important tool for identification and understanding of the mechanisms by which natriuretic peptides act in nervous systems.