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1.
Reprod Domest Anim ; 53(3): 617-623, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29431233

ABSTRACT

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 µg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 µM/µg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.


Subject(s)
Cryopreservation/veterinary , Cryoprotective Agents/pharmacology , Horses , Lactoferrin/pharmacology , Spermatozoa/drug effects , Acrosome Reaction , Animals , Antioxidants , Catalase/pharmacology , Cell Membrane/physiology , Cryopreservation/methods , Male , Semen Analysis/veterinary , Semen Preservation/veterinary , Sperm Motility , Spermatozoa/cytology
2.
Andrologia ; 48(9): 862-868, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27739145

ABSTRACT

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 µg ml-1 , C4) C2 + Lf 500 µg ml-1 and C5) C2 + Lf 1000 µg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 µm per ×106 spermatozoa) compared with the fresh semen (8.6 ± 1.9 µm per ×106 spermatozoa). In contrast, the H2 O2 concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 µm per ×106 spermatozoa) than in the milk extender (430.9 ± 199.8 µm per ×106 spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 µm per ×106 spermatozoa). Caseinate showed to be as efficient as milk to protect equine-cooled spermatozoon.


Subject(s)
Antioxidants , Horses , Semen Preservation/veterinary , Animals , Caseins , Cell Membrane/metabolism , Cold Temperature , Hydrogen Peroxide/metabolism , Insemination, Artificial/methods , Insemination, Artificial/veterinary , Lactoferrin , Male , Milk , Nitrites/metabolism , Semen/metabolism , Semen Preservation/methods , Sperm Motility , Spermatozoa/physiology
3.
Peptides ; 71: 250-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26256416

ABSTRACT

Renin-angiotensin system (RAS) is an important factor in the pathophysiology of hypertension. Mas receptor, Angiotensin-(1-7) [Ang-(1-7)]-activated receptor, is an important RAS component and exerts protective effects in the vasculature. Ang-(1-7) vascular effects and Mas receptor expression in carotid from renovascular hypertensive (2K-1C) rats is not clear. In the present study we investigated Mas receptor vasodilator response activated by Ang-(1-7) in the carotid rings from sham and 2K-1C rats. Changes in isometric tension were recorded on organ chamber. Mas receptors expression was investigated in carotid by Western blot. Nitric oxide production was evaluated by 2,3-diaminonaphthalene (DAN) and eNOS expression and activity by immunofluoresce and western blot, respectively. Ang-(1-7) induced concentration-dependent vasodilator effect in carotid rings from sham and 2K-1C, which the hypertension increased vasodilatation response. In the 2K-1C carotid rings, A-779 (Mas receptor antagonist) reduced but not abolish the vasodilator effect of Ang-(1-7). Corroborating, Mas receptor protein expression was significantly increased in the 2K-1C rats. L-NAME and ibuprofen decreased Ang-(1-7) vasodilator response and L-NAME plus ibuprofen practically abolish the remaining vasodilatation response. Nitric oxide production is increased due increased of eNOS expression and pSer(1177) activity. Our results demonstrated that renovascular hypertension increased Mas receptors expression and nitric oxide production in the rats carotid which, consequently increased Ang-(1-7)-vasorelaxant response.


Subject(s)
Angiotensin I/metabolism , Carotid Arteries/metabolism , Gene Expression Regulation , Hypertension/metabolism , Peptide Fragments/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Renin-Angiotensin System , Vasodilation , Animals , Carotid Arteries/pathology , Carotid Arteries/physiopathology , Hypertension/pathology , Hypertension/physiopathology , Ibuprofen/pharmacology , Male , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type III/biosynthesis , Proto-Oncogene Mas , Rats , Rats, Wistar
4.
J Ethnopharmacol ; 137(1): 709-13, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21756990

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Hancornia speciosa Gomes are popularly used in Brazil to treat diabetes and hypertension. Cardiovascular diseases are the main cause of death worldwide and their incidences are increasing in Brazilian population. The present study aimed to investigate the hypotensive effect and the mechanism of action of Hancornia speciosa Gomes. METHODS: A fraction of the ethanolic extract of leaves from Hancornia speciosa (SFH) was obtained and standardized by its content on rutin, bornesitol and quinic acid. Systolic blood pressure (SBP) of normotensive mice was measured by tail plethysmography. SFH was given orally and SBP was monitored for 5h. Angiotensin-converting enzyme (ACE) inhibitor activity of SFH (1mg/kg) or captopril (10mg/kg) was measured by colorimetric methods. Serum nitrite levels were measured by spectrophotometry. RESULTS: SFH induced a dose-dependent hypotensive effect in normotensive mice. The serum activity of ACE and the level of angiotensin II were significantly reduced by SFH and by captopril. Administration of SFH induced a significant increase on plasmatic level of nitrites and the systemic inhibition of nitric oxide synthase by L-NAME (20mg/kg) reduced the hypotensive effect of SFH. CONCLUSIONS: The present work demonstrated that Hancornia speciosa has a potent hypotensive effect in normotensive mice. The inhibition of ACE leading to reduction on angiotensin II and increase on NO levels might account for the hypotensive effect. These results support the use of Hancornia speciosa by traditional medicine as antihypertensive.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Antihypertensive Agents/pharmacology , Apocynaceae , Blood Pressure/drug effects , Nitric Oxide/metabolism , Plant Extracts/pharmacology , Administration, Oral , Angiotensin II/blood , Angiotensin-Converting Enzyme Inhibitors/administration & dosage , Angiotensin-Converting Enzyme Inhibitors/chemistry , Angiotensin-Converting Enzyme Inhibitors/isolation & purification , Animals , Antihypertensive Agents/administration & dosage , Antihypertensive Agents/chemistry , Antihypertensive Agents/isolation & purification , Apocynaceae/chemistry , Captopril/pharmacology , Ethanol/chemistry , Male , Mice , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitrites/blood , Plant Extracts/administration & dosage , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Leaves , Plants, Medicinal , Plethysmography , Solvents/chemistry , Time Factors , Up-Regulation
5.
Br J Pharmacol ; 164(6): 1738-48, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21615722

ABSTRACT

BACKGROUND AND PURPOSE: Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H(2)O(2) is an important endothelium-derived relaxant factor in the mouse aorta. The role of H(2)O(2) and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H(2)O(2) contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE(-/-)). EXPERIMENTAL APPROACH: Changes in isometric tension were recorded on a myograph; simultaneously, NO and H(2)O(2) were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms. KEY RESULTS: Aortas from ApoE(-/-) mice showed impaired vasodilatation paralleled by decreased NO and H(2)O(2) production. Inhibition of nNOS with L-Arg(NO2) -L-Dbu, knockdown of nNOS and catalase, which decomposes H(2)O(2) into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H(2)O(2) in wild-type animals, but had no effect in ApoE(-/-) mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE(-/-) mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE(-/-) mice. CONCLUSIONS AND IMPLICATIONS: Our data show that endothelial nNOS-derived H(2)O(2) production is impaired and contributes to endothelial dysfunction in ApoE(-/-) aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.


Subject(s)
Atherosclerosis/physiopathology , Endothelium, Vascular/physiopathology , Hydrogen Peroxide/metabolism , Nitric Oxide Synthase Type I/physiology , Acetylcholine/pharmacology , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiopathology , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Atherosclerosis/metabolism , Catalase/pharmacology , Disease Models, Animal , Endothelium, Vascular/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type I/deficiency , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type III/deficiency , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/physiology , Vasodilation/drug effects
6.
Br J Pharmacol ; 161(7): 1458-71, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20942845

ABSTRACT

BACKGROUND AND PURPOSE: Vasculopathies represent the main cause of morbidity and mortality in diabetes. Vascular malfunctioning in diabetes is associated with abnormal vasoconstriction and Ca(2+) handling by smooth muscle cells (SMC). Phosphatidylinositol 3-kinases (PI3K) are key mediators of insulin action and have been shown to modulate the function of voltage-dependent L-type Ca(2+) channels (Ca(V) 1.2). In the present work, we investigated the involvement of PI3K signalling in regulating Ca(2+) current through Ca(V) 1.2 (I(Ca,L) ) and vascular dysfunction in a mouse model of type I diabetes. EXPERIMENTAL APPROACH: Changes in isometric tension were recorded on myograph. Ca(2+) currents in freshly dissociated mice aortic SMCs were measured using the whole-cell patch-clamp technique. Antisense techniques were used to knock-down the PI3Kδ isoform. KEY RESULTS Contractile responses to phenylephrine and KCl were strongly enhanced in diabetic aorta independent of a functional endothelium. The magnitude of phenylephrine-induced I(Ca,L) was also greatly augmented. PI3Kδ expression, but not PI3Kα, PI3Kß, PI3Kγ, was increased in diabetic aortas and treatment of vessels with a selective PI3Kδ inhibitor normalized I(Ca,L) and contractile response of diabetic vessels. Moreover, knock-down of PI3Kδin vivo decreased PI3Kδ expression and normalized I(Ca,L) and contractile response of diabetic vessels ex vivo. CONCLUSIONS AND IMPLICATIONS: Phosphatidylinositol 3-kinase δ was essential to the increased vascular contractile response in our model of type I diabetes. PI3Kδ signalling was up-regulated and most likely accounted for the increased I(Ca,L,) leading to increased vascular contractility. Blockade of PI3Kδ may represent a novel therapeutic approach to treat vascular dysfunction in diabetic patients.


Subject(s)
Calcium Channels, L-Type/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Aorta/physiopathology , Calcium/metabolism , Class I Phosphatidylinositol 3-Kinases , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 1/physiopathology , Male , Mice , Mice, Inbred C57BL , Muscle Contraction , Muscle, Smooth, Vascular/physiopathology , Myocytes, Smooth Muscle/physiology , Patch-Clamp Techniques , Phosphoinositide-3 Kinase Inhibitors , Receptors, Adrenergic, alpha-1/metabolism , Signal Transduction , Up-Regulation , Vasoconstriction , Vasodilation
7.
Phytomedicine ; 17(8-9): 690-2, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20097048

ABSTRACT

In the present work we investigated the mechanism involved in the vasodilator effect induced by euxanthone in rat small mesenteric arteries. We observed that euxanthone induced concentration-dependent vasodilatation in arteries by a mechanism independent on the release of endothelial factors, such as nitric oxide (NO) and cyclooxygenase-derived factors. In addition our results also suggest that euxanthone induced its vasodilator effect through inhibition of calcium-sensitive mechanisms activated by protein kinase C, rather than by inhibition of contractions dependent on the release of the intracellular calcium stores or by inhibition of voltage-operated calcium channels.


Subject(s)
Calcium/metabolism , Clusiaceae/chemistry , Plant Extracts/pharmacology , Protein Kinase C/metabolism , Vasodilation/physiology , Vasodilator Agents/pharmacology , Xanthones/pharmacology , Animals , Calcium Channels/metabolism , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Male , Mesenteric Arteries/drug effects , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Rats , Rats, Wistar , Signal Transduction/drug effects , Vasodilation/drug effects
8.
Phytomedicine ; 17(5): 383-7, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19879122

ABSTRACT

Erythroxylum species have several traditional uses in different countries, including the treatment of hypertension. The ethanol extract from E. gonocladum aerial parts, a species endemic to the Brazilian cerrado, elicited a concentration-dependent inhibition of angiotensin converting enzyme (ACE) (pIC(50)=4.53+/-0.05). Extract fractionation led to the isolation of two compounds, whose structures were assigned by spectrometric data as astilbin and beta-sitosterol, along with a mixture of palmitic, stearic and linolenic acids. This is the first report on the occurrence of these compounds on E. gonocladum. Astilbin promoted significant ACE inhibition in vitro (pIC(50)=5.86+/-0.33) and its activity did not differ from captopril, when both compounds were assayed at 10 microM concentration.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Antihypertensive Agents/pharmacology , Erythroxylaceae/chemistry , Flavonols/pharmacology , Peptidyl-Dipeptidase A/metabolism , Plant Extracts/pharmacology , Angiotensin-Converting Enzyme Inhibitors/isolation & purification , Animals , Antihypertensive Agents/isolation & purification , Brazil , Captopril , Dose-Response Relationship, Drug , Fatty Acids/isolation & purification , Flavonols/isolation & purification , Plant Components, Aerial , Plant Extracts/chemistry , Rats , Sitosterols/isolation & purification
9.
Am J Physiol Heart Circ Physiol ; 295(6): H2503-11, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18952716

ABSTRACT

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.


Subject(s)
Aorta, Thoracic/enzymology , Biological Factors/metabolism , Endothelium, Vascular/enzymology , Endothelium-Dependent Relaxing Factors/metabolism , Hydrogen Peroxide/metabolism , Nitric Oxide Synthase Type I/metabolism , Vasodilation , Acetylcholine/pharmacology , Animals , Aorta, Thoracic/drug effects , Catalase/metabolism , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Hydrazines/pharmacology , Imidazoles/pharmacology , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase Type I/antagonists & inhibitors , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type III/metabolism , Nitroarginine/pharmacology , Nitroprusside/pharmacology , Oligonucleotides, Antisense/metabolism , RNA, Messenger/metabolism , Vasodilation/drug effects , Vasodilator Agents/pharmacology
10.
Toxicon ; 51(7): 1197-206, 2008 Jun 01.
Article in English | MEDLINE | ID: mdl-18397797

ABSTRACT

The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.


Subject(s)
Neuropeptides/pharmacology , Neurotoxins/pharmacology , Penile Erection/drug effects , Penis/drug effects , Spider Venoms/pharmacology , Spiders , Animals , Disease Models, Animal , Drug Synergism , Electric Stimulation , Erectile Dysfunction/complications , Erectile Dysfunction/drug therapy , Erectile Dysfunction/physiopathology , Hypertension/chemically induced , Hypertension/complications , Hypertension/physiopathology , Male , NG-Nitroarginine Methyl Ester/pharmacology , Neuropeptides/isolation & purification , Neurotoxins/isolation & purification , Nitric Oxide/metabolism , Penile Erection/physiology , Penis/innervation , Penis/metabolism , Rats , Rats, Wistar
11.
Br J Pharmacol ; 150(8): 1075-83, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17339838

ABSTRACT

BACKGROUND AND PURPOSE: Aortic complications account for the major mortality in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in FBN1 encoding fibrillin-1. We hypothesized that MFS impaired endothelial function and nitric oxide (NO) production in the aorta. EXPERIMENTAL APPROACH: Mice (at 3, 6, 9 and 12 months of age) heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1 (C1039G/+), Marfan mice, n=75), the most common class of mutation in MFS, were compared with age-matched control littermates (n=75). Thoracic and abdominal aortas from the two groups were studied. KEY RESULTS: Isometric force measurements revealed that relaxation to ACh (but not to sodium nitroprusside) was diminished in the phenylephrine-precontracted Marfan thoracic aorta at 6 months of age (pEC(50)=6.12+/-0.22; maximal response, E(max)=52.7+/-6.8%; control: pEC(50)=7.34+/-0.19; E(max)=84.8+/-2.2%). At one year, both inhibition of NO production with N(omega)-nitro-L-arginine methyl ester, or denudation of endothelium increased the phenylephrine-stimulated contraction in the control thoracic aorta by 35%, but had no effect in the Marfan aorta, indicating a loss of basal NO production in the Marfan vessel. From 6 months, a reduced phosphorylation of endothelial NOS (eNOS)(Ser1177) and Akt(Thr308) detected by Western blotting was observed in the Marfan thoracic aorta, which was accompanied by decreased levels of cGMP. Expressions of Akt and eNOS in the abdominal aorta were not different between the two groups. CONCLUSIONS AND IMPLICATIONS: MFS impairs endothelial function and signaling of NO production in the thoracic aorta, suggesting the importance of NO in the age-related progression of thoracic aortic manifestations.


Subject(s)
Aorta, Thoracic/metabolism , Endothelium, Vascular/metabolism , Marfan Syndrome/metabolism , Microfilament Proteins/metabolism , Nitric Oxide Synthase Type II/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Vasodilation , Acetylcholine/pharmacology , Age Factors , Animals , Aorta, Abdominal/metabolism , Aorta, Abdominal/physiopathology , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiopathology , Calcium/metabolism , Cyclic GMP/metabolism , Disease Models, Animal , Disease Progression , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiopathology , Enzyme Inhibitors/pharmacology , Fibrillin-1 , Fibrillins , Marfan Syndrome/physiopathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/genetics , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide Synthase Type III , Phosphorylation , Signal Transduction/drug effects , Vasodilation/drug effects , Vasodilator Agents/pharmacology
12.
Phytomedicine ; 14(7-8): 473-8, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17174539

ABSTRACT

The vasodilator effect of the ethanolic extract of leaves from Hancornia speciosa Gomes (HSE) was evaluated in superior mesenteric artery rings. HSE produced a concentration-dependent vasodilation (IC50 = 10.8 +/- 4.0 microg/mL) in arterial rings pre-contracted with phenylephrine, which was completely abolished in endothelium-denuded vessels. Endothelium-dependent vasodilation induced by HSE was strongly reduced by L-NAME (100 microM), a nitric oxide (NO) synthase inhibitor, but neither by atropine, a muscarinic receptor antagonist (1 microM), nor by indomethacin (10 microM), a cyclooxygenase inhibitor. In rings pre-contracted with 80 mM KCl, the vasodilator effect of HSE was shifted to the right and was completely abolished in the presence of L-NAME (100 microM). Similar effects were obtained in mesenteric rings pre-contracted with phenylephrine in the presence of KCl 25 mM alone or in addition to 100 microM L-NAME. In addition, BaCl2 (1 mM) dramatically reduced the vasodilation induced by HSE. Together, these findings led us to conclude that HSE induces an endothelium-dependent vasodilation in rat mesenteric artery, by a mechanism dependent on NO, on the activation of potassium channels and endothelium-derived hyperpolarizing factor release. Rutin, identified as a major peak in the HPLC fingerprint obtained for HSE, might contribute for the observed vasodilator effect, since it was able to induce an endothelium-dependent vasodilation in rat superior mesenteric arteries.


Subject(s)
Apocynaceae/chemistry , Endothelium/drug effects , Mesenteric Arteries/drug effects , Plant Extracts/pharmacology , Vasodilation/drug effects , Animals , Dose-Response Relationship, Drug , Male , Plant Extracts/chemistry , Plant Leaves/chemistry , Rats , Rats, Wistar
13.
Peptides ; 28(3): 702-7, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17129638

ABSTRACT

We have recently described, in the mouse aorta, the vasodilator effect of angiotensin-(1-7) (Ang-(1-7)) was mediated by activation of the Mas Ang-(1-7) receptor and that A-779 and D-Pro7-Ang-(1-7) act as Mas receptor antagonists. In this work we show pharmacological evidence for the existence of a different Ang-(1-7) receptor subtype mediating the vasodilator effect of Ang-(1-7) in the aorta from Sprague-Dawley (SD) rats. Ang-(1-7) induced an endothelium-dependent vasodilator effect in aortic rings from SD rats which was inhibited by removal of the endothelium and by L-NAME (100 microM) but not by indomethacin (10 microM). The Ang-(1-7) receptor antagonist D-Pro7-Ang-(1-7) (0.1 microM) abolished the vasodilator effect of the peptide. However, the other specific Ang-(1-7) receptor antagonist, A-779 in concentrations up to 10 microM, did not affect vasodilation induced by Ang-(1-7). The Ang II AT1 and AT2 receptors antagonists CV11974 (0.01 microM) and PD123319 (1 microM), respectively, the bradykinin B2 receptor antagonist HOE 140 (1 microM) and the inhibitor of ACE captopril (10 microM) did not change the effect of Ang-(1-7). Our results show that in the aorta of SD rats, the vasodilator effect of Ang-(1-7) is dependent on endothelium-derived nitric oxide. This effect is mediated by the activation of Ang-(1-7) receptors sensitive to D-Pro7-Ang-(1-7), but not to A-779, which suggests the existence of a different Ang-(1-7) receptor subtype.


Subject(s)
Angiotensin I/metabolism , Aorta, Thoracic/metabolism , Peptide Fragments/metabolism , Receptors, Angiotensin/classification , Receptors, Angiotensin/metabolism , Angiotensin I/antagonists & inhibitors , Angiotensin I/pharmacology , Angiotensin II/analogs & derivatives , Angiotensin II/pharmacology , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , In Vitro Techniques , Indomethacin/pharmacology , Male , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Angiotensin/drug effects , Vasodilation/drug effects
14.
Phytomedicine ; 13(6): 442-5, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16716915

ABSTRACT

The vascular effects of 7-epiclusianone on the rat aorta were investigated. In the rat aortic rings with functional endothelia, 7-epiclusianone up to 10microM induced a concentration-dependent vasodilatation of the sustained contractions induced by phenylephrine (0.3microM). At concentrations higher than 10microM, 7-epiclusianone induced a concentration-dependent contraction in the aortic rings. The vasodilator effect of 7-epiclusianone was drastically decreased with L-NAME (100microM) as well as in endothelium-denuded aortic rings. Moreover, indomethacin (10microM) induced a significant shift to the left in the vasodilator but did not modify the vasoconstrictor effect of 7-epiclusianone. In arteries without pre-contraction, 7-epiclusianone (3-100microM) induced concentration-dependent contraction only in endothelium-intact and in the presence of L-NAME (100microM). This effect was inhibited by indomethacin (10microM) and ZM230487 (1microM), selective inhibitors of cyclooxygenase and of 5-lipoxygenase, respectively. We can conclude that at low concentrations 7-epiclusianone induces an endothelium-dependent vasodilator effect in rat aortic rings. At higher concentrations and in conditions where NO synthase was inhibited, 7-epiclusianone induces a vasocontractile effect. Nitric oxide seems to participate in the vasodilatation, while endothelial cyclooxygenase- and 5-lipoxygenase-derived products play a role in the vasoconstrictor effect.


Subject(s)
Aorta/drug effects , Benzophenones/pharmacology , Benzoquinones/pharmacology , Clusiaceae , Phytotherapy , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Animals , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Male , Protein Prenylation , Rats , Rats, Wistar
15.
J Ethnopharmacol ; 102(2): 137-42, 2005 Nov 14.
Article in English | MEDLINE | ID: mdl-16054792

ABSTRACT

Ierobina is a Brazilian phytopharmaceutical product indicated for the treatment of dyspepsia. It contains the hydroethanolic extracts of Solanum paniculatum L. (Solanaceae), Remijia ferruginea D.C. (Rubiaceae), Jacaranda caroba D.C. (Bignoniaceae) and Erythraea centaurium (L.) Borkh. (Gentianaceae), species traditionally used to treat gastrointestinal disorders. The effect of Ierobina on the digestive system was investigated in rats fed with normal or high-fat (HF) diets, at doses of 2.16, 4.32 and 8.64 mg/kg. The product did not affect the plasmatic levels of glucose, total cholesterol and HDL-cholesterol in the evaluated doses, whereas the triacylglycerol (TAG) concentration showed a dose-dependent increase in HF-fed animals. TAG-rich lipoprotein uptake, estimated by measuring total lipoprotein lipase activity in epididymal adipose tissue, was accompanied by TAG increase in HF-fed rats, after Ierobina administration. The product also induced a concentration-dependent relaxant effect on spontaneous ileum contractions and on the rat ileum pre-contracted with carbachol. Together, these results support the indication of Ierobina as an anti-dyspeptic agent.


Subject(s)
Dyspepsia/drug therapy , Intestines/drug effects , Lipid Metabolism/drug effects , Phytotherapy , Plant Extracts/therapeutic use , Animals , Blood Glucose/analysis , Brazil , Carbachol/pharmacology , Dose-Response Relationship, Drug , In Vitro Techniques , Intestines/physiology , Lipoprotein Lipase/metabolism , Male , Rats , Triglycerides/blood
16.
Phytomedicine ; 12(6-7): 424-32, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16008118

ABSTRACT

A new method for the in vitro screening of plant extracts with potential angiotensin-converting enzyme (ACE) inhibitory activity is proposed. The method is based on the cleavage of the substrate hippuryl-glycyl-glycine by ACE and subsequent reaction with trinitrobenzenesulfonic acid to form 2,4,6-trinitrophenyl-glycyl-glycine, whose absorbance is determined at 415 nm in a microtitre plate reader. Rabbit lung dehydrated by acetone was employed as an enzyme source. Validation of the method showed satisfactory intra-day (CV = 7.63%) and inter-day precision (CV = 13.61%), recovery (97-102.1%), sensitivity (IC50 = 14.1 nmol/l) and linearity in the range 7.5-120 mmol/l of glycyl-glycine (r2 = 0.9921). Besides, the method showed good correlation with a HPLC assay already established for the screening of ACE inhibitors (r = 0.9935 and 0.9034, respectively, for captopril solutions and for plant extracts). The method involves only inexpensive reagents and apparatus.


Subject(s)
Angiotensin-Converting Enzyme Inhibitors/chemistry , Phytotherapy , Plant Extracts/chemistry , Plants, Medicinal , Animals , Brazil , Colorimetry , Dose-Response Relationship, Drug , Humans , Medicine, Traditional , Oligopeptides/chemistry , Rabbits , Reproducibility of Results , Sensitivity and Specificity , Trinitrobenzenesulfonic Acid/chemistry
17.
Br J Pharmacol ; 133(6): 849-58, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11454658

ABSTRACT

The hypotensive and vasorelaxant effect of dioclein in resistance mesenteric arteries was studied in intact animals and isolated vessels, respectively. In intact animals, initial bolus administration of dioclein (2.5 mg kg(-1)) produced transient hypotension accompanied by an increase in heart rate. Subsequent doses of dioclein (5 and 10 mg kg(-1)) produced hypotensive responses with no significant change in heart rate. N(G)-nitro-L-arginine methyl ester (L-NAME) did not affect the hypotensive response. In endothelium-containing or -denuded vessels pre-contracted with phenylephrine, dioclein (5 and 10 mg kg(-1) produced a concentration-dependent vasorelaxation (IC(50)=0.3+/-0.06 and 1.6+/-0.6 microM, respectively) which was not changed by 10 microM indomethacin. L-NAME (300 microM) produced a shift to the right. Dioclein was without effect on contraction of vessels induced by physiological salt solution (PSS) containing 50 mM KCl and the concentration dependence of dioclein's effect on phenylephrine induced contraction was shifted to the right in vessels bathed in PSS containing 25 mM KCl. Tetraethylammonium (10 mM) and BaCl(2) (1 mM) increased the IC(50) for dioclein-induced vasorelaxation without affecting the maximal response (E(max)). Charybdotoxin (100 nM), 4-aminopyridine (1 mM) and iberiotoxin (100 nM) increased the IC(50) and reduced the E(max). Apamin (1 microM) reduced the E(max) without affecting the IC(50). Dioclein produced a hyperpolarization in smooth muscle of mesenteric arteries with or without endothelium (7.7+/-1.4 mV and 12.3+/-3.6 mV, respectively). In conclusion dioclein lowered arterial pressure probably through a decrease in peripheral vascular resistance. The underling mechanism implicated in the vasorelaxant effect of dioclein appears to be the opening of K(Ca) and Kv channels and subsequent membrane hyperpolarization.


Subject(s)
Analgesics/pharmacology , Flavanones , Flavonoids/pharmacology , Hypotension/chemically induced , Mesenteric Arteries/drug effects , Potassium Channels/drug effects , Vasodilation/drug effects , 4-Aminopyridine/pharmacology , Animals , Apamin/pharmacology , Barium Compounds/pharmacology , Charybdotoxin/pharmacology , Chlorides/pharmacology , Consciousness , Dose-Response Relationship, Drug , Endothelium, Vascular/physiology , Enzyme Inhibitors/pharmacology , Glyburide/pharmacology , Hemodynamics/drug effects , In Vitro Techniques , Male , Membrane Potentials/drug effects , Mesenteric Arteries/physiology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Peptides/pharmacology , Potassium Channel Blockers , Potassium Channels/metabolism , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Tetraethylammonium/pharmacology , Time Factors , Vascular Resistance
18.
Phytomedicine ; 7(1): 63-7, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10782492

ABSTRACT

In the present work the effect of the aqueous fraction of the ethanolic extract of the leaves (AFL) of Cissampelos sympodialis Eichl. was investigated in the rat aorta. In the presence of functional endothelium, AFL produced concentration-dependent contractions (EC50 value of 76.6 +/- 17.8 micrograms/ml). In the absence of functional endothelium, the concentration-response curves to AFL were significantly shifted to the left (EC50 values of 1.3 +/- 0.9 micrograms/ml) without modification of its maximal contractile effect. In the presence of L-NAME (300 microM) and of indomethacin (10 mM), the concentration-response curves produced by AFL were also shifted to the left (EC50 values of 21.8 +/- 6.2 and 24.3 +/- 13.2 micrograms/ml, respectively). The treatment of the aortas with L-NAME (300 microM) plus indomethacin (10 microM) produced a significant shift to the left of the concentration-dependent curves of AFL (EC50 value of 4.9 +/- 2.2 micrograms/ml), similar to that observed in the absence of the vascular endothelium. In addition, AFL-induced contraction was abolished in the presence of prazosin (1 microM), and significantly shifted to the right in the presence of yohimbine (EC50 value of 723.6 +/- 76.4 micrograms/ml). Thus, based on these results, it can be concluded that contractions induced by AFL in the rat aorta were due to activation of alpha-adrenoceptors. Furthermore, these results also showed that the AFL-induced contractions were modulated by the endothelium, via the release of NO and of a cyclooxygenase-derived relaxant product. Finally, it can be concluded that the contractile effects of AFL on vascular smooth muscle may play an important role in the hypertensive effects of this plant in vivo.


Subject(s)
Aorta/drug effects , Magnoliopsida/chemistry , Muscle Contraction/drug effects , Plants, Medicinal/chemistry , Adrenergic alpha-Antagonists/pharmacology , Alcohols/chemistry , Animals , Cyclooxygenase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Indomethacin/pharmacology , Male , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Plant Extracts/pharmacology , Plant Leaves/chemistry , Prostaglandin-Endoperoxide Synthases/drug effects , Rats , Rats, Wistar , Receptors, Adrenergic, alpha/drug effects
19.
J Pharm Pharmacol ; 52(11): 1431-4, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11186253

ABSTRACT

We have investigated the endothelium-independent vasorelaxant effect of the new flavonoid dioclein (5,2',5'-trihydroxy-6-7-dimethoxyflavanone) in the rat aorta. In endothelium-denuded vessels, dioclein induced a concentration-dependent relaxation of aortic rings precontracted with noradrenaline (IC50 = 3.5+/-0.89 x 10(-4) M and KCl (IC50 = 5.2+/-1.2 x 10(-4) M). In the absence of extracellular calcium, dioclein reduced the contraction induced by noradrenaline (maximal reduction approximately 33%) but not that induced by caffeine. Dioclein also produced a concentration-dependent inhibition of the sustained contractions induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in normal (IC50 = 4.0+/-0.2 x 10(-4) M) and Ca2+-free solution (IC50 = 4.0+/-0.3 x 10(-4) M). The results indicate that the endothelium-independent vasorelaxant effect of dioclein may be explained by inhibition of contractions dependent on activation of protein kinase C, voltage-dependent Ca2+ influx and on the release of intracellular Ca2+ stores sensitive to noradrenaline.


Subject(s)
Analgesics/pharmacology , Aorta/drug effects , Flavanones , Flavonoids/pharmacology , Vasodilator Agents/pharmacology , Animals , Aorta/physiology , Calcium Channels/drug effects , Calcium Channels/physiology , Culture Techniques , Dose-Response Relationship, Drug , Endothelium/physiology , Norepinephrine/pharmacology , Protein Kinase C/drug effects , Protein Kinase C/metabolism , Rats , Vasodilation
20.
Eur J Pharmacol ; 386(1): 41-6, 1999 Dec 10.
Article in English | MEDLINE | ID: mdl-10611462

ABSTRACT

In the present work, the vasorelaxant effect of dioclein, a new flavonoid isolated from Dioclea grandiflora (Leguminoseae), was investigated in the rat aorta. Dioclein induced a concentration-dependent relaxation in vessels pre-contracted with phenylephrine (IC(50)=1.3+/-0.3 microM), a response which was abolished after endothelium removal. Neither indomethacin (10 microM), an inhibitor of cyclo-oxygenase, nor atropine (1 microM), an antagonist of muscarinic receptors, modified the effect of dioclein. Dioclein (30 microM) induced a significant increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels in aortic rings with endothelium. The nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine-methyl-ester (L-NAME, 300 microM), strongly inhibited or abolished the relaxing effect and rise in cyclic GMP levels induced by dioclein. Furthermore, dioclein (30 microM) had no effect on the endothelium-independent relaxation produced by the NO donor, 3-morpholino-sydnonimine (SIN-1), while superoxide dismutase (100 U ml(-1)) significantly potentiated it. These results indicate that, in the rat aorta, dioclein induces a NO- and endothelium-dependent vasorelaxant effect, which is associated with cyclic GMP elevation. This vasorelaxation likely results from enhanced synthesis of NO rather than enhanced biological activity of NO.


Subject(s)
Endothelium, Vascular/physiology , Flavanones , Flavonoids/pharmacology , Nitric Oxide/physiology , Vasodilator Agents/pharmacology , Animals , Aorta, Thoracic/drug effects , Brazil , Cyclic GMP/metabolism , Endothelium, Vascular/drug effects , In Vitro Techniques , Male , Molsidomine/analogs & derivatives , Molsidomine/pharmacology , Muscle Relaxation/drug effects , Phenylephrine/pharmacology , Plant Roots/chemistry , Plants, Medicinal/chemistry , Rats , Rats, Wistar , Vasoconstrictor Agents/pharmacology
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