C-type Natriuretic Peptide Peptide Research Articles

Abstract P371: Fusion Of C-type Natriuretic Peptide With Elastin-like Polypeptide For Enhanced Pharmacological Activity

C-type natriuretic peptide (CNP) is a pluripotent molecule produced by the endothelium and myocardium with therapeutic potential in pulmonary hypertension, myocardial infarction, and heart failure. However, the native peptide’s short elimination half-life of under 3 minutes limits its use clinically. To improve the pharmacokinetic profile of CNP we recombinantly expressed the CNP peptide as a fusion with an elastin-like polypeptide (ELP). ELPs are biopolymers that undergo reversible temperature-dependent phase separation and form a subcutaneous depot upon injection. CNP-ELP fusion proteins were expressed in E. coli , purified, and showed in vitro potency 2-4 fold less than native CNP. When administered subcutaneously in mice CNP-ELP showed a significantly enhanced PK profile due to the slow release of the fusion into the circulation with detectable fusion protein 24 hours after a single injection. In addition, the CNP-ELP increased growth velocity in mice significantly greater than that of native peptide indicating an in vivo activation of the CNP receptor, NPR-B. These findings represent a new method of administering a long acting CNP peptide for treatment of different cardiovascular diseases.

C-type natriuretic peptide enhances mouse preantral follicle growth.

Compared to ovarian antral follicle development, the mechanism underlying preantral follicle growth has not been well documented. Although C-type natriuretic peptide (CNP) involvement in preantral folliculogenesis has been explored, its detailed role has not been fully defined. Here, we used mouse preantral follicles and granulosa cells (GCs) as a model for investigating the dynamic expression of CNP and natriuretic peptide receptor 2 (NPR2) during preantral folliculogenesis, the regulatory role of oocyte-derived growth factors (ODGFs) in natriuretic peptide type C (Nppc) and Npr2 expression, and the effect of CNP on preantral GC viability. Both mRNA and protein levels of Nppc and Npr2 were gradually activated during preantral folliculogenesis. CNP supplementation in culture medium significantly promoted the growth of in vitro-cultured preantral follicles and enhanced the viability of cultured GCs in a follicle-stimulating hormone (FSH)-independent manner. Using adult and prepubertal mice as an in vivo model, CNP pre-treatment via intraperitoneal injection before conventional superovulation also had a beneficial effect on promoting the ovulation rate. Furthermore, ODGFs enhanced Nppc and Npr2 expression in the in vitro-cultured preantral follicles and GCs. Mechanistic study demonstrated that the regulation of WNT signaling and estrogen synthesis may be implicated in the promoting role of CNP in preantral folliculogenesis. This study not only proves that CNP is a critical regulator of preantral follicle growth, but also provides new insight in understanding the crosstalk between oocytes and somatic cells during early folliculogenesis.

Human genotyping and an experimental model reveal NPR-C as a possible contributor to morbidity in coarctation of the aorta.

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C (NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca2+]i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca2+]i activity. Genotyping of NPR-C variants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function.

C-type natriuretic peptide: a link between hyperandrogenism and anovulation in a mouse model of polycystic ovary syndrome.

The polycystic ovary (PCO) syndrome (PCOS) is the most common cause of anovulatory infertility in women and is associated with several clinical disorders. Despite the great amount of research in the area, mechanisms involved in the genesis of this syndrome remain poorly understood. In a recent issue of Clinical Science (vol. 132, issue 7, 759-776), Wang and colleagues, highlight the important role of overactivated C-type natriuretic peptide (CNP) and natriuretic peptide receptor 2 (CNP/NPR2) system in preventing oocyte maturation and ovulation in PCOS mice model induced by androgen. Dehydroepiandrosterone (DHEA) treatment caused anovulation, high levels of androgen and estrogen receptors (AR and ER) in the ovary, high expression of CNP and natriuretic peptide receptor 2 (NPR2) in granulosa cells (GC), and an increase in testosterone and estradiol (E2) levels in sera. The high level of CNP/NPR2 was associated with oocyte meiotic arrest and very low ovulation rate. Treatment with human chorionic gonadotropin (hCG) or inhibitors of AR or ER reduced the level of CNP/NPR2, which resulted in meiotic resumption and ovulation. The article provided important information for understanding the effect of ovarian steroids on control of oocyte maturation and fertility and highlighted CNP/NPR2 as a specific pathway that is potentially involved in the ovulatory disruption in PCOS.

Sustained increases in plasma C-type natriuretic peptides fail to increase concentrations in cerebrospinal fluid: Evidence from pregnant sheep

C-type natriuretic peptide (CNP) is a paracrine growth factor with high abundance in CNS tissues and cerebrospinal fluid (CSF). Consistent with findings of CNP transcripts in the cerebral microvasculature and hypothalamus, CNP increases the permeability of the blood–brain barrier and reduces food intake when administered intracerebroventricularly in rodents. Whether high concentrations of CNP in plasma can affect CSF levels is unknown. Accordingly we have studied changes (days 4, 87 and 116) in concurrent plasma and CSF concentrations of CNP peptides in pregnant sheep – a physiologically unique setting in which plasma CNP is elevated for prolonged periods. Preliminary studies in non pregnant sheep showed stable CNP levels in CSF during repetitive sampling. Compared with values in non pregnant controls, plasma concentrations of CNP peptides were markedly raised (30-fold) at days 87 and 116 in pregnant sheep, yet CSF levels in the two groups did not differ. CNP peptides in CSF decreased from day 4 to day 87 in pregnant sheep, possibly reflecting an adaptive response of the cerebral vasculature to increased hemodynamic load. We conclude that sustained high concentrations of CNP – far exceeding levels encountered in human pathophysiology – fail to affect CNP peptide levels in CSF.

C‐Type Natriuretic Peptide as Mediator of an Endothelial Cell to Mast Cell Communication

Mast cells (MC) play a critical role in leukocyte recruitment to sites of inflammation after ischemia/reperfusion (I/R). They reside in the perivascular tissue and release proteases and chemokines having detrimental effects on their immediate surroundings, such as endothelial cells. The triggers and counterregulators of MC activation are still enigmatic. We investigated whether endothelial C‐type natriuretic peptide (CNP) via its cGMP forming guanylyl cyclase B (GC‐B) receptor modulates the activity of MC and inflammation after I/R.Intravital microscopy was used to visualize the microcirculation within the cremaster muscle of anesthetized mice. Blood flow was temporally blocked for 30 min of ischemia. FITC‐Dextran was used to quantify macromolecule leakage during reperfusion. Ruthenium Red was used to stain perivascular degranulated MC. I/R provoked marked degranulation of perivascular resident MC (~7‐fold vs controls) and prominent leakage of FITC‐Dextran from postcapillary venules to the adjacent interstitial space. Local superfusion of CNP during reperfusion, significantly reduced the number of degranulated MC (to ~1.8‐fold vs controls) and concomitantly prevented vascular leakage. These effects were mimicked by the membrane‐permeable cGMP analog, 8‐bromo cGMP. In cultured murine bone marrow and human MC CNP activated GC‐B/cGMP signaling and cGMP protein kinase I‐dependent phosphorylation of the cytoskeleton‐associated vasoactive stimulated phosphoprotein (VASP) at Ser237.Our study indicates that the endothelial peptide CNP inhibits I/R‐induced degranulation of perivascular MC and thereby attenuates microvascular inflammation.Supported by SFB 688 and CHFC Würzburg

Differential response of C-type natriuretic peptide to estrogen and dexamethasone in adult bone

C-type natriuretic peptide (CNP) is crucial in promoting endochondral bone growth in mammals including humans but whether this paracrine hormone participates in maintaining bone integrity in the mature skeleton is unknown. Accordingly we studied changes in plasma and bone tissue CNP in anoestrus adult ewes receiving short term anabolic (estrogen) or catabolic (dexamethasone) treatment for 7days. CNP and the aminoterminal fragment of the CNP prohormone (NTproCNP) were measured in plasma and extracts of cancellous bone excised from vertebral, iliac, tibial and marrow tissues. Concentrations of CNP peptides were much higher in vertebral and iliac extracts than those of tibial or marrow. Both plasma CNP and NTproCNP increased rapidly after estrogen followed by a later rise in bone alkaline phosphatase. Vertebral and iliac (but not tibial or marrow) CNP peptide content were significantly increased by estrogen. Consistent with a skeletal source, plasma NTproCNP was significantly associated with vertebral tissue CNP. In contrast, bone tissue CNP peptide content was unaffected by dexamethasone despite suppression of plasma CNP peptides and bone alkaline phosphatase. We postulate that increases in trabecular bone CNP reflect new endosteal bone formation in these estrogen responsive tissues whereas reduced plasma CNP peptides after dexamethasone, without change in cancellous bone content, reflects reductions in cortical bone turnover.

C-Type Natriuretic Peptide (CNP) Levels Are Altered in Boys with Klinefelter Syndrome

B-type natriuretic peptide (BNP) expression in vitro is up-regulated by the protein coded by the short stature homeobox gene (SHOX). C-type natriuretic peptide (CNP) is a paracrine regulatory factor of the growth plate that plays a key role in endochondral growth and shares clearance pathways with BNP. We explored the possibility that alterations in natriuretic peptide regulation may play a role in the overgrowth of boys with Klinefelter syndrome. The objectives of the study were to document the blood levels of amino-terminal propeptide of B-type natriuretic peptide (NTproBNP), CNP, and its amino-terminal propeptide (NTproCNP) in boys with Klinefelter syndrome and compare values with age- and height-matched control subjects. This was a prospective, case-controlled, observational study. Participants were 24 healthy boys with Klinefelter syndrome between 4 and 14 yr of age. Data from sex-, age-, and height-matched healthy controls were obtained from subjects participating in a previously described study. Plasma levels of NTproBNP and CNP were lower, whereas levels of NTproCNP were higher in boys with Klinefelter syndrome compared with published reference ranges. In addition, CNP levels were lower and NTproCNP levels higher than in sex-, age-, and height-matched controls. In contrast to plasma NTproBNP, CNP production and clearance are increased in boys with Klinefelter syndrome. Together these findings argue against an interaction between BNP with CNP in the growth plate. Why CNP peptide levels are altered in Klinefelter syndrome remains to be explored.

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.

Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides

The mammalian natriuretic peptide family consists of atrial (ANP), brain [B-type; BNP] and C-type natriuretic peptide (CNP) and three receptors, natriuretic receptors-A (NPR-A), -B (NPR-B) and -C (NPR-C). Both ANP and BNP are abundantly expressed in the heart and are secreted mainly from the atria and ventricles, respectively. By contrast, CNP is mainly expressed in the central nervous system, bone and vasculature. Plasma concentrations of both ANP and BNP are elevated in patients with cardiovascular disease, though the magnitude of the increase in BNP is usually greater than the increase in ANP. This makes BNP is a clinically useful diagnostic marker for several pathophysiological conditions, including heart failure, ventricular remodeling and pulmonary hypertension, among others. Recent studies have shown that in addition to BNP-32, proBNP-108 also circulates in human plasma and that levels of both forms are increased in heart failure. Furthermore, proBNP-108 is O-glycosylated and circulates at higher levels in patients with severe heart failure. In this review we discuss recent progress in our understanding of the biochemistry, molecular biology and clinical relevance of the natriuretic peptide system.

The Aging Heart, Myocardial Fibrosis, and its Relationship to Circulating C-Type Natriuretic Peptide

Myocardial aging is characterized by left ventricular (LV) fibrosis leading to diastolic and systolic dysfunction. Studies have established the potent antifibrotic and antiproliferative properties of C-type natriuretic peptide (CNP); however, the relationship between circulating CNP, LV fibrosis, and associated changes in LV function with natural aging are undefined. Accordingly, we characterized the relationship of plasma CNP with LV fibrosis and function in 2-, 11-, and 20-month-old male Fischer rats. Further in vitro, we established the antiproliferative actions of CNP and the participation of the clearance receptor using adult human cardiac fibroblasts. Here we establish for the first time that a progressive decline in circulating CNP characterizes natural aging and is strongly associated with a reciprocal increase in LV fibrosis that precedes impairment of diastolic and systolic function. Additionally, we demonstrate in cultured adult human cardiac fibroblasts that the direct antiproliferative actions of high-dose CNP may involve a non-cGMP pathway via the clearance receptor. Together, these studies provide new insights into myocardial aging and the relationship to the antifibrotic and antiproliferative peptide CNP.

Neuroprotective Effects of C-Type Natriuretic Peptide on Rat Retinal Ganglion Cells

PURPOSE. To evaluate the potential neuroprotective effects of C-type natriuretic peptide (CNP) on rat retinal ganglion cells (RGCs). METHODS. Cultured adult rat retinal cells were treated with vehicle, CNP, or atrial natriuretic peptide (ANP), followed by cytotoxic insults (glutamate, TNFalpha, or withdrawal of trophic factor). RGC survival was analyzed by counting Thy-1-positive cells in each well. For in vivo evaluation, N-methyl-d-aspartate (NMDA) with or without CNP was injected intravitreally into rat eyes. At various time points after injection, retinal cross-sections were analyzed for thickness changes in the retinal layers, and retinal flat mounts were assessed by counting cresyl violet-labeled or TUNEL-positive cells. Expressions of natriuretic peptide receptor-B (NPRB) and apoptosis-related genes in retina, including Bcl-xL, BAX, and micro-calpain, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS. At 50 and 500 nM, CNP, but not ANP, significantly (P < 0.05) protected against glutamate-insult and trophic factor withdrawal-induced RGC death in vitro. Neither peptide significantly affected TNFalpha-induced cytotoxicity. Intravitreal injection of NMDA (20 nanomoles) significantly (P < 0.05) decreased the thickness of the inner plexiform layer (IPL), induced cell loss, increased the number of TUNEL-positive cells in the RGC layer, and upregulated the expression of Bcl-xL, BAX, and micro-calpain. All these effects were significantly (P < 0.05) alleviated by concomitant injection of CNP (4.5 nmol, 10 microg). The neuroprotective effects of CNP were maintained up to 14 days after CNP injection. CONCLUSIONS. CNP protects rat RGCs against the apoptotic damage induced by insults such as excitatory amino acid, both in vitro and in vivo.

C-type natriuretic-peptide-potentiated relaxation response of gastric smooth muscle in streptozotocin-induced diabetic rats

To study the sensitivity of gastric smooth muscle to C-type natriuretic peptide (CNP) in streptozotocin (STZ)-induced diabetic rats. The spontaneous contraction of a gastric smooth muscle strip was recorded by using physiological methods in rats. The expressions of CNP and natriuretic peptide receptor-B (NPR-B) in gastric tissue were examined by using immunohistochemistry techniques in the diabetic rat. At 4 wk after injection of STZ and vehicle, the frequency of spontaneous contraction of gastric smooth muscle was significantly reduced in diabetic rats, and the frequency was decreased from 3.10 +/- 0.14 cycle/min in controls to 2.23 +/- 0.13 cycle/min (n = 8, P < 0.01). However, the amplitude of spontaneous contraction was not significant different from the normal rat. CNP significantly inhibited spontaneous contraction of gastric smooth muscle in normal and diabetic rats, but the inhibitory effect was significantly potentiated in the diabetic rats. The amplitudes of spontaneous contraction were suppressed by 75.15% +/- 0.71% and 58.92% +/- 1.32% while the frequencies were decreased by 53.33% +/- 2.03% and 26.95% +/- 2.82% in diabetic and normal rats, respectively (n = 8, P < 0.01). The expression of CNP in gastric tissue was not changed in diabetic rats, however the expression of NPR-B was significantly increased in diabetic rats, and the staining indexes of NPR-B were 30.67 +/- 1.59 and 17.63 +/- 1.49 in diabetic and normal rat, respectively (n = 8, P < 0.01). The results suggest that CNP induced an inhibitory effect on spontaneous contraction of gastric smooth muscle, potentiated in diabetic rat via up-regulation of the natriuretic peptides-NPR-B-particulate guanylyl cyclase-cyclic GMP signal pathway.

Design, Synthesis, and Actions of a Novel Chimeric Natriuretic Peptide: CD-NP

Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides. Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP. Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP. Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation. The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides.

Discordant expression of pro-B-type and pro-C-type natriuretic peptide in newborn infants of mothers with type 1 diabetes

Background Maternal diabetes increases the risk of hypertrophic cardiomyopathy in the fetus. As signaling via the C-type natriuretic peptide (CNP) specific receptor protects against cardiac hypertrophy, we examined whether maternal type 1 diabetes affects the plasma concentrations of proCNP-derived peptides in newborn infants. Methods Plasma concentrations of proCNP-derived peptides were measured in umbilical cord plasma and human placental tissue extracts using sequence-specific radioimmunoassays raised against N-terminal and C-terminal proCNP regions, respectively. Results The median proCNP concentrations were similar in umbilical cord plasma from pregnant women with and without type 1 diabetes (17 pmol/L vs. 19 pmol/L, P not significant) and did not correlate with the proBNP concentrations in the same samples. However, the molar ratio between the proCNP and the CNP peptide was increased in umbilical cord plasma compared to adult plasma (4.6 vs. 1.1), which parallels our earlier findings for proBNP and BNP peptides. Conclusions There is a discordant expression of CNP and BNP peptides in newborn infants of mothers with diabetes. Moreover, fetal metabolism of proCNP and CNP appears to differ from healthy adults. The precise mechanism underlying these differences warrants further investigation.

Does the C-Type Natriuretic Peptide Have Prognostic Value in Chagas Disease and Other Dilated Cardiomyopathies?

Atrial natriuretic peptides (ANP) and brain natriuretic peptides (BNP) are powerful neurohormonal indicators of left-ventricular function and prognosis in heart failure (HF). Chagas disease (CD) caused by the protozoan Trypanosoma cruzi remains a major cause of HF in Latin America. We assessed whether the plasma concentration of the third natriuretic peptide, C-type natriuretic peptide (CNP), also has diagnostic and prognostic properties in patients with CD or other dilated cardiomyopathies (DCM). Blood samples were obtained from 66 patients with CD, 50 patients with DCM from other causes, and 30 gender- and age-matched healthy subjects. Patients were subdivided according to the New York Heart Association (NYHA) class. The CNP concentration was determined by radioimmunoassay (Immundiagnostik, Bensheim, Germany). The main duration of follow-up was 31.4 months (range 13 to 54 months); 19 patients had died and 11 patients received a heart transplant. CNP concentrations were only significantly altered in patients with DCM or CD of the NYHA classes III and IV (P < 0.05). The Pearson correlation of echocardiographic data with CNP revealed an association only with the left-ventricular end systolic volume (P = 0.03) in patients with DCM. Furthermore, CNP did not predict mortality or the necessity for heart transplant. Our data are the first to demonstrate the raised levels of the third natriuretic peptide CNP in CD and other DCM. Whereas ANP and BNP have a high predictive value for mortality in both diseases, CNP is without any predictive potency.

Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide.

Vein graft disease limits the late results of coronary revascularization. C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells. Given the effects of CNP on cGMP cascade, we hypothesized that transfected CNP genes modulate endothelial repair and thrombogenicity in the vein graft. Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of adenovirus vectors containing CNP gene (Ad.CNP) or Escherichia coli lac Z gene (Ad.LacZ) and then interposed in the carotid artery. Reendothelialization, mural thrombi formation, and intima/media ratio were evaluated on the 14th and 28th postoperative days. More reendothelialization was seen in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts both at 14 days (0.81+/-0.05 versus 0.30+/-0.14, P<0.01) and at 28 days (0.96+/-0.01 versus 0.45+/-0.08, P<0.001). The mural thrombus area was smaller in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts. Neointimal thickening was significantly suppressed in the Ad.CNP group. The in vitro wound assay with human coronary artery endothelial cells revealed significant potentiation of the wound repair process by CNP and atrial natriuretic peptide administration. Infected Ad.CNP accelerated reendothelialization and suppressed thrombosis and neointimal hyperplasia. The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization.

C-type natriuretic peptide (CNP) regulates cocaine-induced dopamine increase and immediate early gene expression in rat brain.

The neuropeptide C-type natriuretic peptide (CNP) is the primary biologically active natriuretic peptide in brain. Using in situ hybridization, the present report demonstrates that CNP regulates egr-1, c-fos and junB immediate early gene expression in rat brain. In the frontal cortex, CNP induced immediate early gene expression whereas it inhibited dose-dependently the cocaine-induced early gene expression in the dopaminergic projection fields nucleus accumbens and caudate-putamen. CNP may produce its effect directly on dopaminergic neurons because we found that its receptor, guanylyl cyclase GC-B, was expressed in the mesencephalon where dopaminergic neurons originate, as well as in their projection fields. The inhibition by CNP of the early gene expression elicited by cocaine in the caudate-putamen is correlated with a CNP-evoked decrease in cocaine-induced rise in extracellular dopamine, measured by in vivo microdialysis experiments. The significance of the inhibition of cocaine-induced dopamine release and early gene induction by the endogenous peptide CNP is demonstrated by data indicating that CNP reduced the cocaine-induced spontaneous locomotor activation. By inhibiting dopaminergic neuronal activity, CNP represents a potential negative regulator of related behavioural effects of cocaine.

Two N-terminally truncated forms of C-type natriuretic peptide from habu snake venom

Two N-terminally truncated forms of the C-type natriuretic peptide (CNP) were isolated from the venom of habu snake, Trimeresurus flavoviridis, and their structures were determined by EMI-MS spectrometry and amino acid sequencing. Tf-CNP(6–22), the shorter peptide retaining the 17-membered ring structure formed by an intra-molecular disulfide bridge, has a vasorelaxant activity in rat aortic strips and a diuretic potency in anesthetized rats. Tf-CNP(3–22), the other 20 amino acid residues peptide, also comprised the 17- membered ring with a short N-terminal extention of 3 amino acid residues. Tf-CNP(6–22), the ring, is the shortest naturally occurring CNP peptide identified so far, and as potent as Tf-CNP(1–22), the supposedly intact CNP of 22 amino acid residues.

C-type natriuretic peptide-like immunoreactivity in the rat inner ear

C-type natriuretic peptide (CNP) is a member of the atrial natriuretic peptide family (ANP family). The family also includes ANP and brain natriuretic peptide (BNP). These peptides regulate the homeostasis of body fluid and blood pressure as a neuropeptide in the central nervous system as well as a cardiac hormone in the periphery. We have recently reported the expression of CNP mRNA in the inner ear. To assess the possible physiological role of CNP in the inner ear, we investigated the localization of CNP peptide in the rat inner ear by immunohistochemistry at the light and electron microscopic level. CNP-like immunoreactivity was widely distributed in the secretory and the neuronal portion of the inner ear, i.e. the spiral ligament, the dark cell region of the utriculus, the epithelium of the endolymphatic sac, the spiral ganglion cells and the vestibular ganglion cells. The results suggest that CNP may play a role in the homeostasis of the perilymph and endolymph and may also influence nerve activities in the inner ear.