145 CHARACTERIZATION OF THE PROTEIN ARGININE METHYLTRANSFERASE-DIMETHYLARGININE DIMETHYLAMINOHYDROLASE-NITRIC OXIDE AXIS DURING PORCINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 185
Author(s):  
K. Tessanne ◽  
B. Redel ◽  
K. Whitworth ◽  
L. Spate ◽  
A. Brown ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryonic Development ◽  
Real Time ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
No Production ◽  
Dimethylarginine Dimethylaminohydrolase ◽  
Porcine Embryo

Transcriptional deep sequencing analysis by Bauer et al. (2010) revealed a significant increase in expression of the arginine transporter SLC7A1 in in vitro–cultured porcine blastocysts compared with those cultured in vivo and this was corrected through supplemental arginine. This indicates an important role for arginine during porcine embryo development. Arginine is the precursor for nitric oxide (NO) production and previous work in mice and cattle has shown decreased development when embryos were cultured with a nitric oxide synthase (NOS) inhibitor. The NOS activity is inhibited by monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) that are released during degradation of proteins methylated by protein arginine methyltransferases (PRMT). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for degrading MMA and ADMA in the cell. Therefore, the goal of this study was to investigate whether this PRMT-DDAH-NO axis exists in pre-implantation porcine embryos. To this end, expression of PRMT1, PRMT3, PRMT5, DDAH1 and endothelial NOS (NOS3) was analysed at different stages of embryonic development using real-time quantitative RT-PCR. In addition, the effect of supplemental arginine (1.69 mM) on the expression of the aforementioned genes was investigated. Production of NO in porcine embryos was also visualised using 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM-DA). In vitro–fertilized porcine embryos were collected at the 4-cell and blastocyst stages. The RNA was isolated from pools of 18 to 20 embryos and cDNA, was synthesised using Superscript III (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was performed and the mean fold change in gene expression from the reference gene YWHAG was analysed by t-test after a log transformation. Expression of PRMT3 and PRMT5 was significantly higher (P < 0.05) in blastocysts versus 4-cell embryos. Expression of PRMT1, however, was higher in 4-cell embryos (P < 0.05). The expression of DDAH1 was detected in 4-cell embryos, but DDAH1 became undetectable by the blastocyst stage. Previous microarray analysis in our laboratory by Whitworth et al. (2005 Biol. Reprod. 72(6), 1437–1451) also revealed a significant up-regulation of DDAH2 expression at the 4-cell stage versus blastocysts. Expression of NOS3 was undetectable in the 4-cell and blastocyst; however, NO was detected in 4-cell and blastocyst stage embryos by using DAF-FM-DA. This suggests that a different NOS may be acting in the porcine embryo. Addition of arginine did not have a significant effect on expression of the analysed genes. These results suggest that PRMT-DDAH regulated NO production may play a role during porcine embryo development. Understanding the PRMT-DDAH-NO axis and its regulation during embryonic development will further our ability to tailor in vitro culture so that it more appropriately mimics that of an in vivo environment. Funding was provided by NIH U42 RR18877.

1 ARGININE SUPPLEMENTATION IN VITRO INCREASES PORCINE EMBRYO DEVELOPMENT AND AFFECTSmRNA TRANSCRIPT EXPRESSION

2011 ◽  
Vol 23 (1) ◽  
pp. 107 ◽  
Author(s):  
B. K. Bauer ◽  
L. D. Spate ◽  
C. N. Murphy ◽  
R. S. Prather
Keyword(s):  
Treatment Group ◽  
Mrna Expression ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Sequencing Analysis ◽  
Treatment Groups ◽  
Porcine Embryo ◽  
Arginine Supplementation

In vitro culture systems are suboptimal as compared to in vivo. Previous next-generation sequencing analysis of in vivo fertilized and in vitro cultured (IVC) or in vivo cultured (IVV) porcine blastocyst stage embryos identified an arginine transporter (SLC7A1) expressed 63 fold higher in IVC compared to IVV blastocysts (Bauer et al. 2010 Biol. Reprod. Epub ahead of print). Arginine catabolism may play important roles in placental and conceptus growth and development as it is a substrate for synthesis of nitric oxide synthase and polyamines. The objective of this study was to determine the effects arginine had on both embryo development and mRNA expression in in vitro fertilized embryos. Cumulus–oocyte complexes were matured for 44 h in M199 supplemented with EGF, FSH, and LH. Oocytes with a visible polar body (metaphase II) were selected and fertilized in modified Tris Buffered Medium for 5 h and then placed into one of 5 treatment groups (Porcine Zygote Medium 3 (PZM3) with 0 mM, 0.12 mM (current concentration of arginine in PZM3), 0.36 mM, 0.72 mM, or 1.69 mM arginine). Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25 μL drops of respective culture media and cultured to day 6 in 5% CO2, 5% O2, 90% N2 at 38.5°C. To determine the effect arginine had on development the percent of embryos that made it to the blastocyst stage for each treatment group were analysed using PROC GLM in SAS (SAS Institute, Cary, NC). A least significant difference post test comparison was completed to determine if significant differences existed between treatment groups (a,b,cP < 0.05). The percentage of cleaved embryos on Day 6 that developed to blastocyst was 57.2%b,c, 50.2%c, 67.3%a,b, 67.3%a,b, 70.4%a (N = 147, 163, 150, 120, and 134) in 0 mM, 0.12 mM, 0.36 mM, 0.72 mM, and 1.69 mM arginine, respectively. Real-time PCR was then completed to assess the affect arginine supplementation had on SLC7A1 mRNA expression. Three biological replicates, each containing 10 blastocyst pools to ensure enough starting material, were collected for each treatment group. RNA was isolated from each sample and 5 μL was linearly amplified (NuGEN Ovation Pico WTA System) so multiple genes could be compared and then purified using Bio-Rad MicroSpin Columns. Expression levels were calculated relative to the reference sample and the housekeeping gene, YWHAG. The ΔΔCT values were log-transformed and analysed using PROC GLM in SAS. The expression of SLC7A1 mRNA was decreased (P = 0.0006) compared to PZM3 in the 1.69 mM arginine group. These results illustrate the positive effects that additional arginine may be having on porcine embryo development during culture from the 2-cell to the blastocyst stage. Supplementing arginine to a final concentration of 1.69 mM during culture increases development of porcine embryos to blastocyst compared to PZM3 and also decreases the expression of SLC7A1. Evaluation of the transcriptional profile appears to be a good method of letting the embryo tell us what it needs for development, and in this case arginine. Funded by F21C.

scholarly journals Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

2018 ◽  
Vol 60 (No. 8) ◽  
pp. 359-366
Author(s):  
J. Li ◽  
B. Shi ◽  
S. Yan ◽  
L. Jin ◽  
Y. Guo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Weaned Piglets ◽  
Inos Activity ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 &micro;g chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P &lt; 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P &lt; 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P&nbsp;&lt; 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P &lt; 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

scholarly journals In Vitro Study of Nitric Oxide Metabolites Effects on Human Hydatid ofEchinococcus granulosus

2009 ◽  
Vol 2009 ◽  
pp. 1-7 ◽  
Author(s):  
Razika Zeghir-Bouteldja ◽  
Manel Amri ◽  
Saliha Aitaissa ◽  
Samia Bouaziz ◽  
Dalila Mezioug ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Hydatid Cyst ◽  
Echinococcus Granulosus ◽  
In Vitro Study ◽  
No Production ◽  
In Vitro Effects ◽  
Nitric Oxide Metabolites

Hydatidosis is characterized by the long-term coexistence of larvaEchinococcus granulosusand its host without effective rejection. Previous studies demonstrated nitric oxide (NO) production (in vivo and in vitro) during hydatidosis. In this study, we investigated the direct in vitro effects of NO species: nitrite (NO2−), nitrate (NO3−) and peroxynitrite (ONOO−) on protoscolices (PSCs) viability and hydatid cyst layers integrity for 24 hours and 48 hours. Our results showed protoscolicidal activity ofNO2−andONOO−24 hours and 3 hours after treatment with 320 μM and 80 μM respectively. Degenerative effects were observed on germinal and laminated layers. The comparison of the in vitro effects of NO species on the PSCs viability indicated thatONOO−is more cytotoxic thanNO2−. In contrast,NO3−has no effect. These results suggest possible involvement ofNO2−andONOO−in antihydatic action and point the efficacy of these metabolites as scolicidal agents.

374 THE EFFECTS OF POLYVINYLPYRROLIDONE ON BOVINE SPERM FUNCTION AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 302 ◽  
Author(s):  
Y. Kato ◽  
M. Fukushima ◽  
A. Kenmotsu ◽  
K. Chikazawa ◽  
Y. Nagao
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Staining Method ◽  
Blastocyst Stage ◽  
Sperm Function ◽  
Bovine Sperm ◽  
Developmental Capacity ◽  
Chromatin Integrity ◽  
Triple Staining

In assisted reproduction by ICSI, PVP has been successfully used to replicate the viscosity of sperm solution, thus facilitating the handling and immobilization of spermatozoa. Sperm is suspended in medium containing polyvinylpyrrolidone (PVP), then injected into the oocytes together with a small amount of the medium in ICSI. However the effects of PVP on sperm function and embryo development have not been investigated in detail. In the present study, we investigated the effects of PVP solution on sperm function and embryonic development. Frozen–thawed spermatozoa from a Japanese Black bull and immature COCs from slaughterhouse bovine ovaries were used for all experiments. In experiment 1, bovine sperm was cultured in SOF or SOF containing 10% PVP. For detection of sperm acrosomal and chromatin integrity, sperm cultured in each medium were stained by the triple staining method and acridine orange after 0, 15, 30, and 60 min of culture. In experiment 2, zygotes were injected with PVP solution and cultured in vitro; subsequent cleavage and development to blastocysts were examined. In experiment 3, zygote injected with PVP solution was fixed by 4% paraformaldehyde after 1–3 h of PVP injection. The location of PVP solution in zygote was observed. In experiment 4, two-cell embryos were microinjected with a solution of dextran conjugated with fluorescein (FITC-dextran) and cultured in vitro. The location of FITC-dextran in the embryo was examined. In experiment 1, acrosome reactions of the sperm were enhanced after 15 min of incubation in PVP solution (P &lt; 0.05), but chromatin integrity of the sperm was not influenced (P &gt; 0.05). In experiment 2, PVP suppressed the development of the zygote to 2-cell, morula and blastocyst (75.0%, 35.1%, and 26.3% vs. 61.3%, 20.2%, and 12.9% for control and PVP group, respectively, P &lt; 0.05). In experiment 3, the locations of PVP solution in the zygote were observed 1–3 h after injection. In experiment 4, FITC-dextran was observed in ICM at the blastocyst stage. These findings suggest that PVP affects the acrosome but not the chromatin of sperm in ICSI. PVP solution exists locally in embryos injected and affects the developmental capacity of the embryos.

35 EFFECTIVENESS OF MICROWELL-BASED IN VITRO CULTURE SYSTEMS FOR BOVINEZONA-FREE CLONED EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 124
Author(s):  
C. Feltrin ◽  
M. Machado ◽  
L. M. V. Queiroz ◽  
M. A. S. Peixer ◽  
P. F. Malard ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Aggregation State ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Vitro Development

In vitro embryo production by handmade cloning (HMC) usually requires individual embryo culture, because zona-free embryos cannot be grouped in standard in vitro culture (IVC) protocols. The aim of this study was to evaluate the developmental potential of bovine embryos produced by HMC (Ribeiro et al. 2009 Cloning Stem Cells 11, 377–386) after in vitro culture (IVC) in 3 microwell (WOW) systems. After in vitro maturation, oocytes were denuded and incubated in demecolcine (Ibáñez et al. 2003 Biol. Reprod. 68, 1249–1258), followed by zona pellucida removal, oocyte bisection, embryo reconstruction, electrofusion, and chemical activation. Cloned embryos were allocated to 1 of 3 IVC groups: cWOW: conventional microwells (250 μm, round; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264); mWOW: modified microwells (130 μm, conical; Feltrin et al. 2006 Reprod. Fert. Dev. 18, 126); and WOW-PDMS: microwells in polydimethylsiloxane chips (170 μm, cylindrical with microchannels); IVF embryos were used as controls (Bertolini et al. 2004 Reproduction 128, 341–354). Cleavage (Day 2), blastocyst (Day 7), and pregnancy (Day 30) rates were analysed by the chi-square test, for P < 0.05. Results are shown in Table 1. Cleavage rates were similar between groups, but development to the blastocyst stage was higher in IVF controls than cloned embryo groups. Among cloned embryo groups, blastocyst rate was higher in the mWOW group than the conventional and the PMDS-based microchannels. Nevertheless, in vivo development to Day 30 of pregnancy was not different between cloned groups. Our results for in vitro embryo development indicated that the mWOW provided more suitable conditions for embryo development to the blastocyst stage when compared with cWOW or even WOW-PDMS. Among some possible reasons include the physical advantage of a smaller microwell that may better mimic the constraining effect of the zona pellucida on the developing embryo. That may also provide greater blastomere stability, favouring the aggregation state during the first rounds of cleavages, also aiding compaction and subsequent cavitation. The narrower microwell system appeared to have promoted better in vitro development than the conventional and the DMPS-based microwell systems, with no impact on subsequent in vivo development. However, the IVC in the WOW-PDMS system supported reasonable rates of development, in accordance with the current literature. Table 1.In vitro development of bovine IVF and cloned embryos produced after the in vitro culture in distinct IVC systems

scholarly journals In vitro and in vivo anti-inflammatory activities of a sterol-enriched fraction from freshwater green alga, Spirogyra sp.

2020 ◽  
Vol 23 (1) ◽  
Author(s):  
Lei Wang ◽  
You-Jin Jeon ◽  
Jae-Il Kim
Keyword(s):  
Nitric Oxide ◽  
Green Alga ◽  
Raw 264.7 Cells ◽  
Ros Generation ◽  
Prostaglandin E ◽  
No Production ◽  
Raw 264.7 ◽  
Anti Inflammatory

Abstract Background Inflammation plays a crucial role in the pathogenesis of many diseases such as arthritis and atherosclerosis. In the present study, we evaluated anti-inflammatory activity of sterol-rich fraction prepared from Spirogyra sp., a freshwater green alga, in an effort to find bioactive extracts derived from natural sources. Methods The sterol content of ethanol extract of Spirogyra sp. (SPE) was enriched by fractionation with hexane (SPEH), resulting 6.7 times higher than SPE. Using this fraction, the in vitro and in vivo anti-inflammatory activities were evaluated in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells and zebrafish. Results SPEH effectively and dose-dependently decreased the production of nitric oxide (NO) and prostaglandin E2 (PGE2). SPEH suppressed the production of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1β through downregulating nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells without cytotoxicity. The in vivo test results indicated that SPEH significantly and dose-dependently reduced reactive oxygen species (ROS) generation, cell death, and NO production in LPS-stimulated zebrafish. Conclusions These results demonstrate that SPEH possesses strong in vitro and in vivo anti-inflammatory activities and has the potential to be used as healthcare or pharmaceutical material for the treatment of inflammatory diseases.

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system

2003 ◽  
Vol 15 (4) ◽  
pp. 249 ◽  
Author(s):  
J. R. Herrick ◽  
M. L. Conover-Sparman ◽  
R. L. Krisher
Keyword(s):  
Embryonic Development ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
High Incidence ◽  
Porcine Embryo ◽  
Medium System ◽  
Epidermal Growth ◽  
Vitro Production ◽  
Single Medium

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL−1 hyaluronan, 0.6 mM cysteine, 10 ng mL−1 epidermal growth factor (EGF), 0.1 U mL−1 porcine LH and FSH, and 1 × Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 × 105 sperm mL−1) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mm bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.

scholarly journals Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall

2008 ◽  
Vol 295 (2) ◽  
pp. H499-H508 ◽  
Author(s):  
Wael F. Alzawahra ◽  
M. A. Hassan Talukder ◽  
Xiaoping Liu ◽  
Alexandre Samouilov ◽  
Jay L. Zweier
Keyword(s):  
Nitric Oxide ◽  
Heme Proteins ◽  
Soluble Guanylyl Cyclase ◽  
Vascular Wall ◽  
Rat Aorta ◽  
No Production ◽  
Paramagnetic Resonance ◽  
Physiological Importance

Nitric oxide (NO) has been shown to be the endothelium-derived relaxing factor (EDRF), and its impairment contributes to a variety of cardiovascular disorders. Recently, it has been recognized that nitrite can be an important source of NO; however, questions remain regarding the activity and mechanisms of nitrite bioactivation in vessels and its physiological importance. Therefore, we investigated the effects of nitrite on in vivo hemodynamics in rats and in vitro vasorelaxation in isolated rat aorta under aerobic conditions. Studies were performed to determine the mechanisms by which nitrite is converted to NO. In anesthetized rats, nitrite dose dependently decreased both systolic and diastolic blood pressure with a threshold dose of 10 μM. Similarly, nitrite (10 μM-2 mM) caused vasorelaxation of aortic rings, and NO was shown to be the intermediate factor responsible for this activity. With the use of electrochemical as well as electron paramagnetic resonance (EPR) spectroscopy techniques NO generation was measured from isolated aortic vessels following nitrite treatment. Reduction of nitrite to NO was blocked by heating the vessel, suggesting that an enzymatic process is involved. Organ chamber experiments demonstrated that aortic relaxation induced by nitrite could be blocked by both hemoglobin and soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). In addition, both electrochemical and EPR spin-trapping measurements showed that ODQ inhibits nitrite-mediated NO production. These findings thus suggest that nitrite can be a precursor of EDRF and that sGC or other heme proteins inhibited by ODQ catalyze the reduction of nitrite to NO.

scholarly journals The effects of disodium pamidronate on human polymorphonuclear leukocytes and platelets: An in vitro study

2009 ◽  
Vol 14 (3) ◽  
Author(s):  
Eleonora Salvolini ◽  
Monia Orciani ◽  
Arianna Vignini ◽  
Roberto Primio ◽  
Laura Mazzanti
Keyword(s):  
Nitric Oxide ◽  
In Vitro Study ◽  
Protective Mechanism ◽  
No Production ◽  
Myeloperoxidase Activity ◽  
Inflammatory Processes ◽  
Anti Inflammatory ◽  
Constitutive Nitric Oxide Synthase

AbstractRecent reports have indicated that, as well as having antiresorptive effects, bisphosphonates could have an application as anti-inflammatory drugs. Our aim was to investigate whether this anti-inflammatory action could be mediated by the nitric oxide (NO) released by the leukocytes migrating to the site of inflammation. In particular, we investigated in vitro the intracellular calcium concentration ([Ca2+]i), the level of NO released by PMN and platelets, and the PMN myeloperoxidase activity after incubation with disodium pamidronate, since there was a postulated modulatory effect of this aminosubstituted bisphosphonate on leukocytes both in vitro and in vivo. Our data shows that the pamidronate treatment provoked a significant increase in the [Ca2+]i parallel to the enhancement in NO release, suggesting a possible activation of constitutive nitric oxide synthase, while the myeloperoxidase activity was significantly reduced. In conclusion, we hypothesized that treatment with pamidronate could stimulate NO-production by cells present near the bone compartment, thus constituting a protective mechanism against bone resorption occurring during inflammation. In addition, PMN- and platelet-derived NO could act as a negative feed-back signal to restrict the inflammatory processes.

scholarly journals Crambescin C1 Acts as A Possible Substrate of iNOS and eNOS Increasing Nitric Oxide Production and Inducing In Vivo Hypotensive Effect

2021 ◽  
Vol 12 ◽  
Author(s):  
Juan A. Rubiolo ◽  
Emilio Lence ◽  
Concepción González-Bello ◽  
María Roel ◽  
José Gil-Longo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Active Site ◽  
In Silico ◽  
Hypotensive Effect ◽  
Docking Studies ◽  
No Production ◽  
Guanidine Alkaloids ◽  
Endogenous Substrate

Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.

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