Neutral Endopeptidase Activity Research Articles

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range.

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.

New Borane-Protected Derivatives of α-Aminophosphonous Acid as Anti-Osteosarcoma Agents: ADME Analysis and Molecular Modeling, In Vitro Studies on Anti-Cancer Activities, and NEP Inhibition as a Possible Mechanism of Anti-Proliferative Activity.

Many organophosphorus compounds (OPs), especially various α-aminophosphonates, exhibit anti-cancer activities. They act, among others, as inhibitors of the proteases implicated in cancerogenesis. Thesetypes of inhibitors weredescribed, e.g., for neutral endopeptidase (NEP) expressed in different cancer cells, including osteosarcoma (OS). The aim of the present study isto evaluate new borane-protected derivatives of phosphonous acid (compounds 1–7) in terms of their drug-likeness properties, anti-osteosarcoma activities in vitro (against HOS and Saos-2 cells), and use as potential NEP inhibitors. The results revealed that all tested compounds exhibited the physicochemical and ADME properties typical for small-molecule drugs. However, compound 4 did not show capability of blood–brain barrier penetration (Lipiński and Veber rules;SwissAdme tool). Moreover, the α-aminophosphonite-boranes (compounds 4–7) exhibited stronger anti-proliferative activity against OS cells than the other phosphonous acid-borane derivatives (compounds 1–3),especially regarding HOS cells (MTT assay). The most promising compounds 4 and 6 induced apoptosis through the activation of caspase 3 and/or cell cycle arrest at the G2 phase (flow cytometry). Compound 4 inhibited the migration and invasiveness of highly aggressive HOS cells (wound/transwell and BME-coated transwell assays, respectively). Additionally, compound 4 and, to a lesser extent, compound 6 inhibited NEP activity (fluorometric assay). This activity of compound 4 was involved in its anti-proliferative potential (BrdU assay). The present study shows that compound 4 can be considered a potential anti-osteosarcoma agent and a scaffold for the development of new NEP inhibitors.

Effect of Qinbai Qingfei Concentrated Pellets on substance P and neutral endopeptidase of rats with post-infectious cough

BackgroundIn recent years, it has been reported that Qinbai Qingfei Concentrated Pellet (QQCP) has the effect of relieving cough and reducing sputum. However, the therapeutic potentials of QQCP on post-infectious cough (PIC) rat models has not been elucidated. So the current study was aimed to scientifically validate the efficacy of QQCP in post infectious cough.MethodsAll rats were exposed to sawdust and cigarette smokes for 10 days, and intratracheal lipopolysaccharide (LPS) and capsaicin aerosols. Rats were treated with QQCP at dose of 80, 160, 320 mg/kg. Cough frequency was monitored twice a day for 10 days after drug administration. Inflammatory cell infiltration was determined by ELISA. Meanwhile, the histopathology of lung tissue and bronchus in rats were evaluated by hematoxylin-eosin staining (H&E). Neurogenetic inflammation were measured by ELISA and qRT-PCR.ResultsQQCP dose-dependently decreased the cough frequency and the release of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8, but exerted the opposite effects on the secretion of anti-inflammatory cytokines IL-10 and IL-13 in BALF and serum of PIC rats. The oxidative burden was effectively ameliorated in QQCP-treated PIC rats as there were declines in Malondialdehyde (MDA) content and increases in Superoxide dismutase (SOD) activity in the serum and lung tissue. In addition, QQCP blocked inflammatory cell infiltration into the lung as evidenced by the reduced number of total leukocytes and the portion of neutrophils in the broncho - alveolar lavage fluid (BALF) as well as the alleviated lung damage. Furthermore, QQCP considerable reversed the neurogenetic inflammation caused by PIC through elevating neutral endopeptidase (NEP) activity and reducing Substance P (SP) and Calcitonin gene related peptide (CGRP) expression in BALF, serum and lung tissue.ConclusionsOur study indicated that QQCP demonstrated a protective role of PIC and may be a potential therapeutic target of PIC.

Experimental Hypomagnesemia Induces Neurogenic Inflammation and Cardiac Dysfunction

Hypomagnesemia occurs clinically as a result of restricted dietary intake, Mg-wasting drug therapies, chronic disease status and may be a risk factor in patients with cardiovascular disorders. Dietary restriction of magnesium (Mg deficiency) in animal models produced a pro-inflammatory/pro-oxidant condition, involving hematopoietic, neuronal, cardiovascular, renal and other systems. In Mg-deficient rodents, early elevations in circulating levels of the neuropeptide, substance P (SP) may trigger subsequent deleterious inflammatory/oxidative/nitrosative stress events. Evidence also suggests that activity of neutral endopeptidase (NEP, neprilysin), the major SP-degrading enzyme, may be impaired during later stages of Mg deficiency, and this may sustain the neurogenic inflammatory response. In this article, experimental findings using substance P receptor blockade, NEP inhibition, and N-methyl-D-aspartate (NMDA) receptor blockade demonstrated the connection between hypomagnesemia, neurogenic inflammation, oxidative stress and enhanced cardiac dysfunction. Proof of concept concerning neurogenic inflammation is provided using an isolated perfused rat heart model exposed to acute reductions in perfusate magnesium concentrations.

Localization of angiotensin-(1-7) and Mas receptor in the rat ovary throughout the estrous cycle.

We have previously demonstrated the presence of Angiotensin (Ang)-(1-7) in rat ovary homogenates and its stimulatory effect on estradiol and progesterone production. The present study was undertaken to identify the cellular localization of Ang-(1-7) and its receptor Mas in the rat ovary in the different phases of the estrous cycle. Ang-(1-7) and Mas were localized by immunohistochemistry and Mas mRNA expression was assessed by RT-PCR. Immunostaining for both Ang-(1-7) and Mas was found in all phases of the estrous cycle, particularly in the thecal and interstitial cells, as well as in regressing corpora lutea. However, granulosa cells were positive only in antral and preovulatory follicles at proestrus and estrus phases. This pattern contrasted with the distribution of the octapeptide Ang II, which was abundant in granulosa but not in theca cells. In addition, the expression of Mas mRNA was demonstrated in all estrous cycle phases. Angiotensin-converting enzyme activity did not vary between estrous cycle phases, whereas prolyl endopeptidase activity was significantly higher in diestrus and neutral endopeptidase activity was significantly higher in metestrus. These data provide the first evidence that new RAS components are dynamically expressed in the ovary across the rat estrous cycle. Further functional studies should clarify the role of Ang-(1-7) signaling through Mas receptor in the regulation of ovarian physiology.

Combinatorial inhibition of Angiotensin converting enzyme, Neutral endopeptidase and Aminopeptidase N by N-methylated peptides alleviates blood pressure and fibrosis in rat model of dexamethasone-induced hypertension

Angiotensin converting enzyme (ACE), neutral endopeptidase (NEP) and aminopeptidase N (APN) are responsible for generation of vasoactive peptides that regulates vasoconstriction, vasodilation and natriuresis, which altogether regulate blood pressure. Cumulative inhibition of ACE, NEP and APN effectively blocks the progression of respective pathways. In this study, N-methylated peptide inhibitors F-N(Me)H-L, V-N(Me)F-R and R-N(Me)V-Y were synthesized against ACE, NEP and APN respectively, using their respective physiological substrates. F-N(Me)H-L inhibited ACE activity with an IC50 of 83 nmol/L, V-N(Me)F-R inhibited NEP activity with an IC50 of 1.173 μmol/L and R-N(Me)V-Y inhibited APN activity with an IC50 of 3.94 nmol/L respectively. Further, the anti-hypertensive effect of N-methylated peptides was evaluated using rat model of dexamethasone-induced hypertension. Individual peptides and their cocktail treatment were started from day 6 of the study period and blood pressure was measured on every alternate day during 15 day study. Administration of F-N(Me)H-L (138 ± 3 mmHg) and cocktail of all the three peptides at a dose of 100 mg/kg significantly reduced systolic blood pressure (SBP) compared to dexamethasone group (SBP of Groups-dexamethasone; (167 ± 5 mmHg), F-N(Me)H-L (138 ± 3 mmHg), and Cocktail (122 ± 3 mmHg). Anti-hypertensive, anti-hypertrophic and anti-fibrotic effects of N-methylated peptides and cocktail was further reflected by the decreased levels of circulating Ang II and increased ANP levels in sera of hypertensive rats along with decrease in collagen deposition in heart and kidney. Though, ACE inhibition is adequate to reduce SBP, targeting NEP and APN along with ACE is beneficial in tackling hypertension and associated fibrosis of heart.

Differential diagnostic significance of epithelial and stromal CD10 expression in tumors of trichogenic origin.

The histological differential diagnosis between benign trichogenic skin tumors and basal cell carcinoma may be challenging. We therefore set out to investigate whether expression of CD10, a cell surface protein with neutral endopeptidase activity, might be a suitable marker for the histological differential diagnosis of trichogenic skin tumors. We immunohistochemically analyzed 119 cases of benign trichogenic skin tumors and basal cell carcinoma. These included 28 nodular and 15 sclerosing basal cell carcinomas, 21 Pinkus tumors, 20 trichoblastomas, nine trichofolliculomas, eleven trichoepitheliomas, five desmoplastic trichoepitheliomas, and ten seborrheic keratoses. The majority of nodular basal cell carcinomas expressed CD10 in tumor cells at the peripheral (22/28 [75%]). On the other hand, trichoblastomas revealed peripheral CD10 expression in only 10% (2/20) of cases, whereas 50% showed central expression (10/20). Peripheral epithelial expression of CD10 was also found in Pinkus tumors (9/21 [42.9%]) and trichoepithelioma (4/11 [36.4%]). Desmoplastic trichoepithelioma showed no tumoral CD10 expression at all (0/5 [0%]), while the majority of sclerosing basal cell carcinomas was positive for CD10 (13/15 [86.7%]). Our findings suggest that epithelial expression of CD10 -and not peritumoral stromal CD10 expression, as has been postulated -may well be of differential diagnostic significance. The pattern of distribution of CD10-positive neoplastic cells in particular can be useful in the diagnosis of trichogenic tumors.

MODULATION WAY CLASSICAL AND ALTERNATIVES OF RENIN ANGIOTENSIN SYSTEM (RAS) IN MESANGIAL CELLS AFTER EXPOSURE TO FRUCTOSE.

BackgroundOur group demonstrated previously that rats subjected to a high fructose diet showed increased activity of kidney ACE. In order to understand the effect of fructose in the forming process of angiotensin II (Ang II) and angiotensin 1–7 (Ang1–7) affecting the levels of these effectors, the aim of this study was to evaluate the activities of the RAS enzymes responsible for the formation of Ang1–7 and Ang II of immortalized human mesangial cells (IMCH)exposed to low and high concentration of fructose.MethodsIMCH were divided into control (low glucose medium), Fructose 5 mM(medium + 5 mM Fructose) and Fructose 30mM(medium + 30 mM Fructose) groups. The enzymatic activity of ACE, chymase, cathepsin D, Neutral Endopeptidase (NEP) and ACE2 were evaluated using Z‐Phe‐His‐Leu, Abz‐AIKFFSAQ‐EDDnp, Abz‐AIAFFSRQ‐EDDnp, Abz(d)‐Arg‐Gly‐Leu, Mca‐APK (DNP) as substrates, respectively. Data were analyzed using ANOVA one way.ResultsAmong the AngII forming enzymes, fructose decreased ACE activity (CG:0,044±0,01 vs.F5:0,016±0,004* vs. F30:0,008±0,002* mU/mg protein) in cell lysate. Both fructose concentrations increased chymase (CG:0,85±0,03 vs. F5:1,07±0,07* vs. F30:0,99±0,05* nM/min/mL) in the culture medium. No effect was observed on the activity of cathepsin D. When evaluating the activities of peptidases forming Ang‐1–7, NEP activity was decreased in lysate cells in F30 group and decreased in F5 group in culture medium. We did not detect differences in ACE2 activity.ConclusionThe results suggest fructose modulates renal and systemic RAS in IMCH acting in the classical and alternative pathways of Ang II formation. Additionally, this sugar was able to decrease NEP activity, forming Ang‐1–7. Quantitation of angiotensin remains necessary to better understand how the end effector is available in IMCH and to evaluate possible effects on glomerular physiology.Support or Funding InformationCapesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling sex differences in the renin angiotensin system and the efficacy of antihypertensive therapies

The renin angiotensin system is a major regulator of blood pressure and a target for many anti-hypertensive therapies; yet the efficacy of these treatments varies between the sexes. We use published data for systemic RAS hormones to build separate models for four groups of rats: male normotensive, male hypertensive, female normotensive, and female hypertensive rats. We found that plasma renin activity, angiotensinogen production rate, angiotensin converting enzyme activity, and neutral endopeptidase activity differ significantly among the four groups of rats. Model results indicate that angiotensin converting enzyme inhibitors and angiotensin receptor blockers induce similar percentage decreases in angiotensin I and II between groups, but substantially different absolute decreases. We further propose that a major difference between the male and female RAS may be the strength of the feedback mechanism, by which receptor bound angiotensin II impacts the production of renin.

RSIY-11: discovery and evaluation of a novel peptide and its relation to sperm motility

Seminal neutral endopeptidase (NEP) has been suggested to regulate sperm motility based on the observation that addition of NEP inhibitors such as opiorphin (OP) to semen increase sperm motility (1). We conducted a peptidomic screen of semen to identify potential endogenous NEP inhibitors and identified a novel peptide, RSIY-11, which based on sequence analysis, may act as an NEP inhibitor. In these studies we evaluated its role as an NEP inhibitor and effects on sperm motility. Prospective laboratory in vitro studies. Peptidomics and mass spectrometry was performed on a semen sample with normal semen parameters. A highly expressed 11 amino acid (AA) peptide was identified, termed RSIY-11 (based on the last 4 AA at the carboxyl terminus) which, based on its AA sequence, could act as an NEP inhibitor. RSIY-11 was synthesized and a colorimetric assay performed with purified NEP to evaluate the inhibitory constant (Ki) of RSIY-11 and compared to that of OP. The effect of preincubation of NEP with RSIY-11 at 8.2μM was examined, comparing 0, 3 and 24 hour (hr) preincubation times. Dixon and Lineweaver Burk (LB) plots determined the inhibitory nature of RSIY-11 on NEP. Microscopy was used to evaluate sperm from 49 patients undergoing a semen analysis for an infertility workup for total motility (TM) and progressive motility (PM) at baseline and after addition of phosphate buffer solution (PBS) (negative control), OP at 200μM (positive control) and RSIY-11 at 0.75, 7.5, and 75μM at 0, 30, and 60 minutes following addition of peptides. TM and PM were done in duplicates and averages were calculated. STATA version 14.0 was used to analyze continuous data related to sperm motility using repeated measures ANOVA. P < 0.05 was considered significant. Data are presented as mean ± standard deviation. RSIY-11 acts as a competitive inhibitor of NEP with Ki of 18.4 ± 1.6 μM; considerably more potent than OP (Ki 264.5 ± 61.5 μM). Pre-incubation of RSIY-11 with NEP for 24 or 3 hr reduced its ability to inhibit NEP activity (26% and 29% inhibition, respectively), compared to no preincubation (47% inhibition), suggesting RSIY-11 is a substrate for NEP. Compared to baseline, OP and RSIY-11 at all concentrations significantly increased TM and PM at 0, 30, and 60 minutes (Table). No significant differences were found in TM and PM between OP and RSIY-11 at all concentrations. RSIY-11 is an endogenous, competitive, NEP inhibitor that when added to semen increases sperm motility. RSIY-11 is a potential biomarker for male factor infertility and a potential therapeutic strategy for treatment of asthenospermia.Tabled 1Motility Following Addition of PeptidesTotal Motility 0 minutes (%)Progressive Motility 0 minutes (%)Total Motility 30 minutes (%)Progressive Motility 30 minutes (%)Total Motility 60 minutes (%)Progressive Motility 60 minutes (%)Baseline49.7 ± 16.143.6 ± 16.449.6 ± 15.645.5 ± 16.148.0 ± 15.542.8 ± 16.0PBS50.8 ± 14.545.3 ± 15.649.8 ± 15.845.9 ± 15.749.5 ± 14.544.5 ± 15.1Opiorphin 200 μM59.1 ± 17.153.6 ± 18.457.6 ± 15.752.6 ± 16.857.1 ± 15.252.3 ± 16.6RSIY-11 0.75 μM60.2 ± 17.354.9 ± 17.657.6 ± 16.352.7 ± 17.054.4 ± 16.350.7 ± 15.7RSIY-11 7.5 μM58.7 ± 17.353.5 ± 17.758.4 ± 15.753.9 ± 16.054.9 ± 17.050.9 ± 16.7RSIY-11 75 μM58.7 ± 16.852.9 ± 17.957.2 ± 16.152.0 ± 17.657.9 ± 14.452.8 ± 14.9 Open table in a new tab

Effect of Inhibition or Deletion of Neutral Endopeptidase on Neuropathic Endpoints in High Fat Fed/Low Dose Streptozotocin-Treated Mice.

Previously we demonstrated that a vasopeptidase inhibitor of angiotensin converting enzyme and neutral endopeptidase (NEP), a protease that degrades vaso- and neuro-active peptides, improves neural function in diabetic rodent models. The purpose of this study was to determine whether inhibition or deletion of NEP provides protection from neuropathy caused by diabetes with an emphasis on morphology of corneal nerves as a primary endpoint. Diabetes, modeling type 2, was induced in C57Bl/6J and NEP deficient mice through a combination of a high fat diet and streptozotocin. To inhibit NEP activity, diabetic C57Bl/6J mice were treated with candoxatril using a prevention or intervention protocol. Twelve weeks after the induction of diabetes in C57Bl/6J mice, the existence of diabetic neuropathy was determined through multiple endpoints including decrease in corneal nerves in the epithelium and sub-epithelium layer. Treatment of diabetic C57Bl/6J mice with candoxatril improved diabetic peripheral neuropathy and protected corneal nerve morphology with the prevention protocol being more efficacious than intervention. Unlike C57Bl/6J, mice deficient in NEP were protected from the development of neuropathologic alterations and loss of corneal nerves upon induction of diabetes. These studies suggest that NEP contributes to the development of diabetic neuropathy and may be a treatable target.

Abstract P286: Modulation Way Classical and Alternatives of Renin Angiotensin System (RAS) in Mesangial Cells After Exposure to Fructose

Background: Our group demonstrated previously that rats subjected to a high fructose diet showed increased activity of kidney ACE.In order to understand the effect of fructose in the forming process of angiotensin II (AngII) and angiotensin 1-7 (Ang1-7) affecting the levels of these effectors, the aim of this study was to evaluate the activities of the RAS enzymes responsible for the formation of Ang1-7 and AngIIof immortalized human mesangial cells (IMCH)exposed to low and high concentration of fructose. Methods: IMCH were divided into control (low glucose medium), Fructose 5 mM(medium + 5 mM Fructose) and Fructose 30mM(medium + 30 mM Fructose) groups. The enzymatic activity of ACE, chymase, cathepsin D, Neutral Endopeptidase (NEP) and ACE2 were evaluated using Z-Phe-His-Leu,Abz-AIKFFSAQ-EDDnp, Abz-AIAFFSRQ-EDDnp,Abz(d)-Arg-Gly-Leu,Mca-APK (DNP) as substrates respectively. Data were analyzed using ANOVA one way. Results: Among the AngIIforming enzymes, fructose decreased ACE activity in cell lysate.Both fructose concentrations increased chymase in the culture medium. No effect was observed on the activity of cathepsin D. When evaluating the activities of peptidases forming Ang-1-7,NEP activity was increasedin culture medium.We did not detected differences in ACE2 activity. Conclusion: The results suggest fructose modulates renal and systemic RAS in IMCH acting in the classical andalternative pathways of AngII formation. Additionally this sugar was able to increase NEP activity, forming Ang-1-7. Quantitation of angiotensin remains necessary to better understand how the end effector is available in IMCH and evaluate possible effects on glomerular physiology.

Substance P and thiorphan synergically enhance angiogenesis in wound healing.

Impaired angiogenesis is a common pathological characteristic of chronic wounds. Therefore, the regulation of angiogenesis is important for proper tissue repair. It was reported that substance P (SP) accelerates wound healing in a skin injury model. SP is degraded by neutral endopeptidase (NEP). Our study shows that systemic co-treatment of SP and thiorphan, an inhibitor of NEP synergically increased the number of α-smooth muscle actin positive-blood vessels in skin wounds. However, there was no synergic improvement in wound contraction and extracellular matrix deposition. Therefore, inhibition of endogenous NEP activity by thiorphan treatment might modulate the effects of SP treatment specifically on accelerating angiogenesis during wound healing. However, the molecular mechanism(s) of the synergic increase in angiogenesis by SP and thiorphan treatment is still unknown.

PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.

The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. PnPP-19 (5, 10 and 20 μg per paw) induced peripheral antinociception in rats. Specific antagonists of μ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 μmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, μ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.

A Study of Pro-Bnp Compared to Procalcitonin in Patient With Severe Sepsis As a Marker of Diagnosis of Sepsis in Critically Ill Patients

Early diagnosis of sepsis is the key for improving the survival, therefore biomarkers have an important place in this process because they can indicate the presence or absence of sepsis [1]. Procalcitonin aids in diagnosis and risk stratification of bacterial sepsis [2].Using pro-BNP as inflammatory marker in diagnosing severe sepsis and septic shock is based on cytokines production independent of myocardial dysfunction. Bacterial endotoxins secreted from Gram-negative organisms and Staphylococcus aureus alpha toxin increase the BNP messenger RNA expression. Also the activity of Neutral endopeptidase 24.11 the enzyme responsible for degradation of proBNP decreases in septic shock. Finally, proBNP clearance decreases due to renal dysfunction [3].

Peptides in seminal fluid and their role in infertility: a potential role for opiorphin inhibition of neutral endopeptidase activity as a clinically relevant modulator of sperm motility: a review.

Infertility is a devastating medical condition that adversely affects emotional health and well-being of couples who desire pregnancy and parenthood. The overall demographic data suggest that the indication for more than one-third of assisted reproductive technology cycles performed in the United States includes male factor infertility. There is increasing recognition of the role that peptides present in seminal plasma have in determining sperm motility. Several recent studies suggest that peptidases, such as neutral endopeptidase (NEP) and aminopeptidase N (APN), impose significant adverse effects on sperm motility. Interestingly, several recent studies demonstrate that there is an endogenous NEP/APN inhibitor peptide called opiorphin in human seminal plasma. Our pilot studies suggest opiorphin promotes sperm motility and may positively influence sperm motility parameters in some cases of males infertility characterized by asthenozoospermia.

Downregulation of natriuretic peptide system and increased steroidogenesis in rat polycystic ovary

Atrial natriuretic peptide (ANP) is known to regulate ovarian functions, such as follicular growth and steroid hormone production. The aim of the present study was to investigate the natriuretic peptide system in a rat model of chronic anovulation, the rat polycystic ovary. Adult female Wistar rats received a single subcutaneous injection of 2mg estradiol valerate to induce polycystic ovaries, while the control group received vehicle injection. Two months later, their ovaries were quickly removed and analyzed. Polycystic ovaries exhibited marked elevation of testosterone and estradiol levels compared to control ovaries. The levels of ANP and the expression of ANP mRNA were highly reduced in the polycystic ovaries compared to controls. By immunohistochemistry, polycystic ovaries showed weaker ANP staining in stroma, theca cells and oocytes compared to controls. Polycystic ovaries also had increased activity of neutral endopeptidase, the main proteolytic enzyme that degrades natriuretic peptides. ANP receptor C mRNA was reduced and ANP binding to this receptor was absent in polycystic ovaries. Collectively, these results indicate a downregulation of the natriuretic peptide system in rat polycystic ovary, an established experimental model of anovulation with high ovarian testosterone and estradiol levels. Together with previous evidence demonstrating that ANP inhibits ovarian steroidogenesis, these findings suggest that low ovarian ANP levels may contribute to the abnormal steroid hormone balance in polycystic ovaries.

Inhibition of human neutrophils NEP activity, CD11b/CD18 expression and elastase release by 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde, oleacein

Polyphenols, such as oleacein (3,4-DHPEA-EDA; 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde), are believed to play a role in the prevention of cardiovascular diseases. Due to an increase of neutrophil mediators in acute myocardial infarction the aim of this study was to establish the effect of oleacein on neutral endopeptidase (NEP) activity and other functions of human neutrophils, such as elastase, MMP-9 and IL-8 production. The effect on CD62L and CD11b/CD18 expression on neutrophils was also determined.Oleacein with a concentration of 100μM inhibited NEP activity, elastase, MMP-9 and IL-8 release from neutrophils by 77.7±2.7%, 21.3±7.8%, 22.7±4.2% and 25.2±5.6%, respectively. Oleacein with a concentration of 50μM suppressed CD11b/CD18 expression by 63.6±3.1% and to a lesser extent, increased CD62L expression by 27.3±8.3% on the surface of neutrophils, in comparison with stimulated cells.Oleacein by inhibiting NEP activity, adhesion molecules expression and elastase release might play a role in the protective effects of olive oil against endothelial injuries.

Effects of four antitussives on airway neurogenic inflammation in a guinea pig model of chronic cough induced by cigarette smoke exposure

The effects of four antitussives, including codeine phosphate (CP), moguisteine, levodropropizine (LVDP) and naringin, on airway neurogenic inflammation and enhanced cough were investigated in guinea pig model of chronic cough. Guinea pigs were exposed to CS for 8weeks. At the 7th and 8th week, the animals were treated with vehicle, CP (4.8mg/kg), moguisteine (24mg/kg), LVDP (14mg/kg) and naringin (18.4mg/kg) respectively. Then the cough and the time-enhanced pause area under the curve (Penh-AUC) during capsaicin challenge were recorded. The substance P (SP) content, NK-1 receptor expression and neutral endopeptidase (NEP) activity in lung were determined. Chronic CS exposure induced a bi-phase time course of cough responsiveness to capsaicin. Eight weeks of CS exposure significantly enhanced the airway neurogenic inflammation and cough response in guinea pigs. Two weeks of treatment with CP, moguisteine, LVDP or naringin effectively attenuated the chronic CS-exposure enhanced cough. Only naringin exerted significant effect on inhibiting Penh-AUC, SP content and NK-1 receptor expression, as well as preventing the declining of NEP activity in lung. Chronic CS-exposed guinea pig is suitable for studying chronic pathological cough, in which naringin is effective on inhibiting both airway neurogenic inflammation and enhanced cough.

Effects of pirfenidone on increased cough reflex sensitivity in guinea pigs

Pirfenidone, an antifibrotic drug with anti-inflammatory and antioxidant effects, delays fibrosis in idiopathic pulmonary fibrosis (IPF). Patients with IPF have a greater cough reflex sensitivity to inhaled capsaicin than healthy people, and cough is an independent predictor of IPF disease progression; however, the effects of pirfenidone on cough reflex sensitivity are unknown.After challenge with an aerosolized antigen in actively sensitized guinea pigs, pirfenidone was administered intraperitoneally, and the cough reflex sensitivity was measured at 48 h after the challenge. Bronchoalveolar lavage (BAL) was performed, and the tracheal tissue was collected.Pirfenidone suppressed the capsaicin-induced increase in cough reflex sensitivity in a dose-dependent manner. Additionally, increased levels of prostaglandin E2, substance P, and leukotriene B4, but not histamine, in the BAL fluid were dose dependently suppressed by pirfenidone. The decrease in neutral endopeptidase activity in the tracheal tissue was also alleviated by pirfenidone treatment. The total number of cells and components in the BAL fluid was not influenced.These results suggest that pirfenidone ameliorates isolated cough based on increased cough reflex sensitivity associated with allergic airway diseases, and potentially relieve chronic cough in IPF patients who often have increased cough reflex sensitivity. Prospective studies on cough-relieving effects of pirfenidone in patients with IPF are therefore warranted.