Effect of hyperoxia on NRP-cGMP signaling pathway in brain tissue of newborn rats / 吉林大学学报(医学版)

Objective: To construct the brain tissue damage models of newborn rats induced by hyperoxia, and to explore the effect of hyperoxia on the natriuretic peptide receptor-cyclic guanosine monophosphate (NRP-cGMP) signaling pathway in brain tissue of the newborn rats. Methods: A total of 60 newly born Wistar rats were divided into control group and hyperoxia model group, and there were 30 rats in each group. At the 1st, 3rd and 7th days after hyperoxia exposure, the brain tissues of 10 rats randomly selected from each group were gotten, and the body weights of the rats in two groups were detected. HE staining was used to observe the morphology of brain tissue of the rats in two groups; the ultrastructures of brain tissue of the rats in two groups were observed under transmission electron microscope. Western blotting method was used to detect the expression levels of natriuretic peptide receptor A (NPR-A) and natriuretic peptide receptor B (NPR-B) in brain tissue of the rats in two groups. ELISA method was used to determine the levels of cGMP in brain tissue of the rats in two groups. Results: The body weight of the rats in hyperoxia model group was significantly lower than that in control group at the 1st, 3rd and 7th days after hyperoxia exposure (P<0. 05). The HE staining results showed that at the 3rd day after hyperoxia exposure, the volume of the hippocampal pyramidal cells of the rats in hyperoxia model group was reduced, and the arrangement was sparse; at the 7th day after hyperoxia exposure, the hippocampal cells showed deep staining and shrinkage, and the cell boundaries were not clear. Under electron microscope, at the 3rd day after hyperoxia exposure, the mitochondrial double membrane structure of the rats in hyperoxia model group was destroyed, a few crest disappeared, and the number of mitochondria and synapses was decreased; the above damage conditions were aggravated at the 7th day after birth. The Western blotting results showed that at the 1st, 3rd and 7th days after hyperoxia exposure, the expression level of NPR-A in brain tissue of the rats in hyperoxia model group was higher than that in control group (P<0. 05). But only at the 1st day after hyperoxia exposure, the expression level of NPR-B in brain tissue of the rats in hyperoxia model group was higher than that in control group (P<0. 05). The ELASA results showed that at the 1st, 3rd and 7th days after hyperoxia exposure, the level of cGMP in brain tissue of the rats in hyperoxia model group was higher than that in control group (P

Effect of estrodial on C type natriuretic peptide and insulin like growth factor 1 expression in rat growth plate chondrocytes / 上海交通大学学报（医学版）

Objective · To observe effect of 17β estrodial (17β E2) with different concentrations on C type natriuretic peptide (CNP), insulin like growth factor 1 (IGF1), and natriuretic peptide receptor B (NPR-B) expression and proliferation of growth plate chondrocytes of rats in vitro. Methods · Eight Wistar rats were sacrificed and their epiphyseal cartilages of the upper tibias were separated to obtain chondrocytes on the 14th day after birth. Then chondrocytes were cultured with 17β E2 in different concentrations (10-4、10-6、10-8、10-10 and 10-12 mol/L) for 48 h, while control group was cultured without 17β E2. CCK8 method, ELISA and qRT-PCR were used to analyze the proliferation of chondrocytes, the levels of CNP and IGF1 in culture medium and mRNA levels of CNP, NPR-B and IGF1, respectively. Results · 17β E2 in different concentrations affected the proliferation of growth plate chondrocytes significantly. When the concentration of 17β E2 was 10-8 mol/L, it had the strongest effect on the cell proliferation. When the concentration increased to 10-4 mol/L, the proliferation of chondrocytes was inhibited. With the increasement of 17β E2 concentration, the levels of CNP in the culture medium and the mRNA levels of CNP in the chondrocytes were significantly different. The highest levels of CNP protein and mRNA both appeared in 10-8 mol/L group, while the lowest levels both appeared in 10-4 mol/L group. IGF1 and its mRNA also reached the highest levels in 10-8 mol/L group,but the lowest concentration and mRNA level were in 10-10 mol/L group and 10-12 mol/L, respectively. Both CNP mRNA and protein levels were positive correlated with the proliferation of chondrocytes (P=0.000). Nevertheless, there was no significant correlation between the proliferation of chondrocytes and IGF1 mRNA or protein levels (P>0.05). Conclusion · 17β E2 modulates proliferation of rat growth plate chondrocytes in a dose-effect manner. It enhances proliferation at relatively low concentrations (10-10-10-8 mol/L) and inhibits proliferation at high concentration. This effect is positively related to CNP expression in chondrocytes.

Role of C-type natriuretic peptide in the function of normal human sperm / 亚洲男科学杂志(英文版)

C-type natriuretic peptide (CNP) is a newly discovered type of local regulatory factor that mediates its biological effects through the specic, membrane-bound natriuretic peptide receptor-B (NPR-B). Recent studies have established that CNP is closely related to male reproductive function. The aims of this study were to determine the distribution of CNP/NPR-B in human ejaculated spermatozoa through different methods (such as immunolocalization, real time polymerase chain reaction and Western Blot), and then to evaluate the influence of CNP on sperm function i n vitro, such as motility and acrosome reaction. Human semen samples were collected from consenting donors who met the criteria of the World Health Organization for normozoospermia. Our results show that the specic receptor NPR-B of CNP is localized in the acrosomal region of the head and the membrane of the front-end tail of the sperm, and there is no signal of CNP in human sperm. Compared with the control, CNP can induce a signicant dose-dependent increase in spermatozoa motility and acrosome reaction. In summary, CNP/NPR-B can affect sperm motility and acrosome reaction, thus regulating the reproductive function of males. CNP may be a new key factor in regulating sperm function.

Expression of CNP/NPR-B axis and NPR-C/NEP elimination pathways in rats with unilateral ureteral obstruction / 临床儿科杂志

Objectives To study the expression features of C-type natriuretic peptide (CNP)/natriuretic peptide receptor-B (NPR-B) axis and two parallel elimination pathways, natriuretic peptide receptor-C (NPR-C) and neutral endopeptidase (NEP) in unilateral ureteral obstruction (UUO) rats. Methods CNP, NPR-B, NPR-C, NEP, Col-IV and type IV collagen (Col-IV) mRNA and proteins were determined by in situ hybridization, real-time PCR, immunohistochemistry and western blot in UUO rats at 24h, 72h, 1w, 2w, 3w, 1m, 2m and 3m. Results CNP expression tended to be higher immediately after ligation and de-clined along with the progression of disease, occurring predominantly in tubular epithelial cells. A high-level CNP may attribute to the elevated expression of NPR-B in the early phase of UUO. Conclusions NEP and NPR participate in the regulation of CNP expression in tubulointerstitial fibrosis. The gradual increased expression of NPR-C and NEP may cause the subsequent de-cline of CNP.

Expression of natriuretic peptide receptor A in the developing retina of the mouse / 解剖学报

Objective Our purpose is to investigate the expression of natriuretic peptide receptor A (NPR-A) in the retina and to understand the NPR-A’ s functions during the mouse development .Methods Mice eyes were harvested from E16 ( embryonic day 16 ) to P90 ( postnatal day 90 ) . Total of 127 eyes were used in the study . Immunohistochemistries of NPR-A were carried out .Results During development , NPR-A was widely expressed in the retinal neurons .In the outer nuclear layer , NPR-A began to appear in the inner and outer projections of cone and rod cells at P7, but decreased at P14.From P30 afterward, it continued to express weakly .In the inner nuclear layer , NPR-A expressed in the dendrites of bipolar cells weakly from P 7 to adulthood , whereas no expression in horizontal cells .In the ganglion cell layer, NPR-A started highly to express in the ganglion cell bodies at E 16, and in the meantime, in the nerve fiber layer , ganglion cell axons , NPR-A was expressed highly from embryonic to adult .In the inner and outer plexiform layers, NPR-A was highly expressed at P14, but decreased gradually after P30.In addition, NPR-A also widely expressed in the inner protrusions of Müller cells.Conclusion NPR-A participates in the development of the retina , and may be the key molecule in the developing retinal neurons .Moreover, it plays an important regulatory role in the functional activity of Müller cells .

Angiotensin-II down-regulates cardiac natriuretic peptide receptor-A mediated anti-hypertrophic signaling in experimental rat hearts.

Atrial natriuretic peptide (ANP) exerts anti-hypertrophic effects in the heart via natriuretic peptide receptor-A (NPR-A). However, ANP mediated anti-hypertrophic activity is decreased in the cardiomyopathic conditions. In the present investigation the in vivo effects of angiotensin II (Ang II), a hypertrophic agonist have been studied on the ventricular expression level of NPR-A in Wistar rat hearts. NPR-A expression at the protein and mRNA levels were found to be markedly reduced by 5-fold respectively in Ang II infused rats heart as compared with sham rat hearts. Moreover, cGMP production in response to ANP was reduced by 77% in the isolated cardiac membrane preparation from the Ang II infused rat hearts. Losartan treatment reversed NPR-A expression and responsiveness to ANP. This study suggests that Ang II down regulates cardiac NPR-A activity by suppressing Npr1 gene transcription.

Selective mastoporan inhibition of muscarinic activation of bovine tracheal smooth muscle

Muscarinic activation of bovine tracheal smooth muscle (BTSM) leading to smooth muscle contraction involves the generation of two cGMP signals (20 and 60 s), being 20s peak associated with soluble (sGC) and the second (60s) to membrane-bound Natriuretic Peptide- receptor-Guanylylcy clases (NPR-GC). In this study, we showed that pre-incubation of isolated BTSM strips with mastoparan and superactive mastoparan (mastoparan 7) decreased significantly the muscarinic dependent contractile smooth muscle responses in dose-dependent and non-competitive manner. Moreover, mastoparan (50 nM) inhibited completely the BTSM-muscarinic contractile responses and affected dramatically the carbachol-dependent cGMP signals being the first cGMP signal inhibited in a 63 ± 5%, whereas the second signal disappeared. Mastoparan inhibition of muscarinic activation is specific since other spasmogens as serotonin and histamine fully contracted these BTSM strips under mastoparan treatment. Cyclic GMP levels were evaluated by exposing BTSM strips to activators of NO-sensitive sGC as Sodium Nitroprussiate (SNP) and Natriuretic Peptides as CNP-53 for membrane-bound NPR-GC. Thus, SNP and CNP increased in a binary way, in more than 20 fold cGMP levels at 30-40 s being both increments inhibited by mastoparan. Furthermore, the Gi/o-protein involvement on mastoparan inhibition of cGMP elevations induced by CNP and SNP is suggested by Pertussis toxin pre-treatment, which reversed mastoparan effects. These results indicate that muscarinic signal transduction cascades leading to airway smooth muscle contractions involved two different guanylyl cyclases being both regulated by mastoparan-sensitive G-proteins. ANP, Natriuretic Peptide type A; ASM, Airway Smooth Muscle; BTSM, Bovine Tracheal Smooth Muscle; CNP-53, Natriuretic Peptide type C-53; GPCR, G-Protein Coupled Receptor; Gq16, Heterotrimeric G protein subtype 16; Gi/o, Heterotrimeric G protein subtype...

La activación muscarínica del músculo liso de las vías aéreasrelacionada a la contracción de dicho músculo liso esta asociada a la generación de dos señales de GMPc (20 y 60 s), siendo la señal de los 20s relacionado a la activación de la guanililciclasa soluble mientras que el pico de los 60s a la guanililciclasa unida membranas y sensible a péptidos natriuréticos (NPR-GC). En este trabajo, nosotros mostramos que la pre-incubación de fragmentos del músculo liso traqueal de bovino (BTSM) con mastoparan y su análogo superactivo (mastoparan 7), en una forma dosis dependiente, son capaces de disminuir de manera significativa la actividad contráctil dependiente de agentes muscarinicos. Adicionalmente, mastoparan (50 nM) inhibió completamente la respuesta contráctil muscarinica del BTSM y afectó dramáticamente los picos de GMPc asociados a la activación muscarinica siendola primera señal inhibida en un 63 ± 5%, mientras que la segunda señal desapareció completamente. Esta inhibición del mastoparan de la activación muscarínica es especifica ya que otros espamogenos como la serotonina y la histamina fueron capaces de inducir respuestas máximas en presencia del mastoparan y su análogos. Este efecto del mastoparan sobre los niveles del GMPc fue evaluado en presencia de otros agentes generadores de este segundo mensajero como son el nitroprusiato de sodio (SNP) que activa la guanililciclasa soluble sensible a NO y los péptidos natriureticos como el CNP-53 (CNP) activador de la NPR-GC asociada a membranas plasmáticas. Tanto, el SNP como el CNP aumentaronen mas de 50 veces los niveles de GMPc a los 30-40 s en forma bifasica, siendo estos incrementos inhibidos de manera significativa por el mastoparan. Ademas, se sugiere la participación de proteínas Gi/o en los efectos inhibitoriosdel mastoparan, porque la Toxina pertussis revertió los efectos inhibitorios. Estos resultados indican que la cascada de activación muscarinica que conduce...

Distribution of six genes of essential hypertension In Yunnan Han healthy population / 中国综合临床

Objective To explore the distribution of RAS,AGT,ACE,eNOS,ET-2,ANP and NPRC of es-sential hypertension in Yunnan Han healthy population.Methods Gene chip technology was used to detect the pol-ymorphism of AGT M235T (MM, MT,TT), ACE I/D (II, ID, DD ), eNOS Glu298Asp (EE, ED, DD), ET-2 A985G (AA,AG,GG) ,ANP T2238C(TT,TC,CC) and NPRC A-55C(AA,AC,CC) in 97 health subjects.Results The MM,MT and TT genotype frequency of AGT M235T was 0.052,0.381 and 0.567 ,alle frequency of M and T was 0.242 and 0.758 in 97 healthy subjects of Yunnan population;The II, ID and DD frequency of ACE I/D was 0.340, 0.598 and 0.062, alle frequency of I and D was 0.680 and 0.320 in 97 healthy subjects of Yunnan population ;EE, ED and DD frequency of eNOS Glu 298 Asp was 0.845,0.144 and 0.011 ,alle frequency of E and D was 0.918 and 0.082 in 97 healthy subjects of Yannan population;AA,AG,GG frequency of ET-2 A985G was 0.020,0.258 and 0.722, alle frequency of A and G was 0.149 and 0.851 in 97 healthy subjects of Yunnan population;TT and TC fre-quency of ANP T2238C was 0.959 and 0.041 ,and CC was not detected;alle frequency of T and C was 0.979 and 0.021 in 97 healthy subjects of Yunnan population;AC and CC frequency of NPRC A-55C was 0.763 and 0.237, and no AA was detected,alle frequency of A and C was 0.381 and 0.619 in 97 healthy subjects of Yunnan popula-tion.Conclusion The polymorpbism of ACT M235T,ACE I/D,eNOS Glu298Asp,ET-2 A985G,ANP T2238C and NPRC A-55C is locally distributed in Yunnan Han healthy population.

Investigation of the Association between Normal-tension Glaucoma and Single Nucleotide Polymorphisms in Natriuretic Peptide Gene

PURPOSE: The expression of natriuretic peptides in the neural bundles of the anterior portion of the optic nerves and their functions in regulating vessel tone and blood flow may suggest a possible role in the pathogenesis of glaucoma. The purpose of this study was to investigate the association between normal-tension glaucoma and the genetic variations of atrial natriuretic peptide (Nppa) and natriuretic peptide receptor A (Npr1) gene. METHODS: Sixty-seven Korean normal-tension glaucoma (NTG) patients and 100 healthy subjects (as normal controls) were enrolled. DNA from peripheral blood leukocytes was extracted, and the genotypes of five polymorphisms (c.94G>A, c.454T>C, IVS1+16C>T, IVS2+701G>A, and c.-764C>G) in the Nppa gene and one polymorphism (c.1023G>C) in the Npr1 gene were determined using the restriction fragment length polymorphism and the SNaPshot methods. The genotype and allele frequencies of these polymorphisms in patients with NTG and normal controls were compared using the Fisher's exact test and the chi-square test. RESULTS: In both groups, the genotype distributions were in accordance with the Hardy-Weinberg equilibrium. There was no significant difference in the frequency of the Nppa and Npr1 alleles or genotypes in the normal-tension glaucoma group as compared to the control group. CONCLUSIONS: Nppa and Npr1 gene polymorphisms are not associated with normal-tension glaucoma, suggesting that this gene does not have an important role in the pathogenesis of optic neuropathy in this disease.

Changes in Renal Expression of Natriuretic Peptides and Their Receptors mRNA Induced by Head-down Suspension in Rats

BACKGROUND: Head-down suspension (HDS) of rats has been used as a model for the simulation of a microgravity environment. Atrial natriuretic peptide (ANP), C-type natriuretic peptides (CNP) and their receptors are found in the kidney, suggesting that these peptides could play a significant physiological role in the kidney. Therefore, this study was investigated the changes in the adaptations of renal natriuretic peptides and their receptors syntheses after 4 weeks of HDS in rats. METHODS: Unanesthetized, unrestrained, male Sprague-Dawley rats were subjected to either a horizontal position (control rats) or a -45degreeshead-down tilt using the tail-traction technique (HDS rats). This study observed the renal syntheses of natriuretic peptides as a expression of ANP and CNP mRNA, and also determined the expression of A-type natriuretic peptide receptor (NPR-A) mRNA and B-type NPR (NPR-B) mRNA. The expressions of natriuretic peptide and NPR mRNA were measured by reverse transcription-polymerase chain reaction with [(32)P]-dCTP following 4 weeks of HDS in the kidney of both control and HDS rats. RESULTS: After 4 weeks of HDS, the expression of ANP mRNA significantly (P<0.01) decreased, while CNP mRNA expression was showed the non-significant increasing trend in the kidney of HDS rats. NPR-A, which binds with ANP, was significantly (P<0.001) decreased in renal mRNA expression of HDS rats compared with controls. Expression in mRNA of NPR-B, which binds with CNP, showed a slightly decreasing trend in the kidney of rats following HDS. CONCLUSION: These results suggest that the renal adaptation following 4 weeks of HDS exerts to maintain the blood volume and electrolyte balance through attenuation of syntheses in the natriuretic peptide and its binding receptor, especially in ANP rather than in CNP systems.

Altered Atrial Natriuretic Peptide System in the Kidney of Bilateral Obstructive Uropathic Rats / 대한신장학회잡지

BACKGROUND: Although being associated with an elevated plasma atrial natriuretic peptide(ANP), its precise role in the postobstructive diuresis has not been fully understood. Evidence has been provided suggesting that the locally-synthesized ANP in the kidney contributes to the regulation of urinary sodium excretion. The present study was aimed to investigate whether an altered regulation of local ANP system is involved in the postobstructive diuresis. METHODS: Male Sprague-Dawley rats were used. Both proximal ureters were ligated for 48 hours, after which the kidneys were taken without releasing the ligature, being designated as bilateral ureteral obstruction(BUO) group; or the ligature was released and 4 or 24 hr later, urinary data were collected, being designated as BUR-4 or BUR-24, respectively. Sham operated rats were used as control. Plasma ANP levels were determined by radioimmunoassay. The expression of ANP and natriuretic peptide receptor(NPR)-A mRNAs was determined by reverse transcription-polymerase chain reaction(RT-PCR). To further examine whether the altered renal ANP system, if any, was associated with an altered biological effects of guanylyl cyclase, ANP-stimulated cGMP accumulation was determined in membrane preparations of the glomeruli and papillae by radioimmunoassay. RESULTS: The plasma ANP level was increased in BUO group compared with that in the control(260.5+/-32.5 vs. 133.3+/-23.5pg/mL, p<0.05), decreased in BUR-4 group(3.6+/-0.5 vs. 143.5+/-42.8pg/mL, p<0.01), while not significantly different in BUR-24 group. In BUR-4. the urinary flow rate increased compared with that in the control(1598+/-370 vs. 215+/-34 microL/hr, p<0.01), along with increases of FENa(11.5+/-4.1 vs. 0.25+/-0.02%, p<0.05) and UNaV (153.7+/-23.7 vs. 36.5+/-9.3microEq/hr, p<0.01). In BUR-24, the urinary parameters were normalized. Renal tissue expression of ANP mRNA was increased in BUO as well as in BUR-4, while not changed in BUR-24. NPR-A mRNA expression was decreased in the kidney of BUO. The ANP-stimulated accumulation of cGMP in the isolated glomeruli and papillae in BUO was significantly reduced. The guanylyl cyclase activities were partly recovered in BUR-4 and completely in BUR-24. CONCLUSION: An enhanced local activity of ANP in the kidney may be causally related to the postobstructive diuresis.