[Show abstract][Hide abstract] ABSTRACT: Objectives
We investigated the mechanisms underlying the relaxant effect of adrenomedullin (AM) in the rat carotid artery and verified the expression of AM system components in this tissue.Methods
The carotid artery was isolated from male Wistar rats and immunohistochemical, Western immunoblotting, real-time polymerase chain reaction and functional assays were conducted.Key findingsProtein and mRNA expression of AM, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)1, 2, 3 were detected in carotid segments from male Wistar rats. Immunohistochemical assays showed that AM and CRLR receptors are expressed in the endothelium and smooth muscle cells. Functional assays showed that AM concentration dependently relaxed carotid rings with intact endothelium. Endothelial removal reduced, but not abolished, the relaxation induced by AM. AM22–52 (selective antagonist for AM receptors) and calcitonin gene-related peptide (CGRP)8–37 (selective CGRP receptor antagonist) reduced AM-induced relaxation in endothelium-intact rings. Pre-incubation of endothelium-intact rings with N-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or Rp-8-Bromo-?-phenyl-1,N2-ethenoguanosine 3′,5′cyclic monophosphorothioate reduced AM-induced relaxation. Inhibition of cyclooxygenase-1 and protein kinase A (PKA) reduced AM-induced relaxation. The relaxation induced by AM was attenuated by the K+ channel blockers apamin and glibenclamide. AM increased nitrate levels and 6-keto-prostaglandin F1α (stable product of prostacyclin) in the rat carotid. In endothelium-denuded rings, AM22–52, glibenclamide and PKA inhibition by H89 reduced AM-induced relaxation.Conclusions
The novelty of this work is that it first demonstrated functionally that AM-induced relaxation is mediated by AM and CGRP receptors located on the endothelium and AM receptors located on smooth muscle of rat carotid arteries. AM-induced relaxation involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, the opening of K+ channels and the activation of PKA.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 08/2014; · 1.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After partial hepatectomy (PH), the liver remnant (LR) shows a regenerative response, always keeping a percent relationship with the host. This process has been well described in the literature, but several aspects still need to be understood. There are no studies on hepatic LR regeneration during hypothermic preservation. Thus, the objective of the present study was to analyze LR regeneration after PH under conditions of hypothermal preservation.
[Show abstract][Hide abstract] ABSTRACT: To study the therapeutic application of guanylate cyclase inhibition by methylene blue in an experimental model of acute pancreatitis in pigs.
acute necrotizing pancreatitis was induced in anesthetized pigs by the retrograde infusion of 1 ml/kg of 5% sodium taurocholate and 8 U/kg enterokinase in the pancreatic duct. Three groups were studied (n = 5): control (C), pancreatitis (AP), and MB bolus followed by pancreatitis (MB+P). The data included serum and abdominal fluid enzymes, hemodynamic variables, arterial hemogasometry, abdominal fluid volume, inflammatory markers, plasma nitrite/nitrate (NOx), plasma myeloperoxidase (MPO) and plasma malondialdehyde (MDA). One- and two-way analysis of variance (ANOVA) was performed, followed by the Bonferroni test (p < 0.05).
amylase and lipase were three and 10-fold higher in the AP group. Myeloperoxidase activity was 50% higher in the AP group. The hemodynamic data indicated early hypovolemic shock followed by cardiogenic shock. Severe fluid translocation to the peritoneal cavity was observed. Plasma NOx remained unchanged. The MB+P group had a five-fold increase in MDA compared with the C group.
preemptive application of MB in pigs with AP demonstrated no significant effects on hemodynamic and inflammatory variables. The use of MB is inadequate in cases of exponential NO release, and extreme caution must be exercised, given the increase in lipid peroxidation based on the malondialdehyde dosage.
Revista do Colégio Brasileiro de Cirurgiões 12/2013; 40(6):480-489.
[Show abstract][Hide abstract] ABSTRACT: Damage provoked by ischemia in renal transplants is difficult to quantify. To determine whether a donated organ is fit for transplantation. We sought to correlate the findings of fluorescence spectroscopy (FS) with histologic evidence of ischemic injury and organ viability.
Kidneys of 33 rats were submitted to FS of the upper and lower poles as well as the middle third. Excitation was generated by the laser's wavelengths of 408, 442, and 532 nm. Rats were randomized into groups with the 30, 60, and 120 minutes warm ischemia before analysis by FS, that was repeated at 5 minutes after reperfusion.
FS results in the reperfusion phase correlated with ischemia time and degree of histologic injury. After 60 or 120 minutes of ischemia, the excitation lasers of 532 and 442 nm resented a significant negative correlation coefficient with the histological grade (r = -0.61 and r = -0.73, respectively).
There was a strong correlation between FS and histologic changes only in the reperfusion phase after renal ischemia. The method was thus unable to assess the viability of organs before transplantation.
[Show abstract][Hide abstract] ABSTRACT: Renal puncture biopsies are directed at the lower poles of the organ to decrease the risk of hemorrhage and complications.
To evaluate by fluorescence spectroscopy (FS) the most appropriate renal region (in terms of metabolic changes) to obtain a biopsy.
The kidneys of 33 Rattus norvegicus rats were submitted to FS detection in the upper and lower poles and in the middle third. Excitations were generated with lasers at wavelengths of 408, 442, and 532 nm. Animals were divided at random into groups of warm ischemia (30, 60, and 120 minutes), whose kidneys were again analyzed by FS, as well as after 5 minutes of reperfusion using the same excitation beams in the same renal regions. Then the kidneys underwent histologic preparation and examination.
The middle third area of the rat's kidneys proved to be significantly more sensitive to ischemic and reperfusion changes than the renal poles, as determined by FS (P < .001).
The middle third of the kidney was the most appropriate site for a renal biopsy to monitor a transplanted organ.
[Show abstract][Hide abstract] ABSTRACT: Photodynamic therapy (PDT) is known to be limited to applications in large volume tumors due to its limited penetration. Therefore, a combination of PDT and carbon dioxide (CO2) laser ablation may constitute a potential protocol to destroy bulk tumors because it involves an association of these two techniques allowing the removal of visible lesions with a high selectivity of destruction of remnant tumors. The main aim of this study is to investigate the most appropriate procedure to combine use of a CO2 laser and PDT on livers of healthy rats, and to analyze different techniques of this treatment using three types of photosensitizers (PSs). Forty eight animals were separated to form six groups: (1) only CO2 laser ablation, (2) drug and CO2 laser ablation, (3) only PDT, (4) drug and light (PDT) followed by CO2 laser ablation, (5) ablated with CO2 laser followed by PDT, and (6) drug followed by CO2 laser ablation and light. For each group, three types of photosensitization were used: topical 5-aminolevulinic acid (ALA), intravenous ALA and intravenous Photogem®. Thirty hours after the treatments, the animals were sacrificed and the livers removed. The depth of necrosis was analyzed and measured, considering microscopic and macroscopic aspects. The results show that the effects of the PDT were considerably enhanced when combined with CO2 laser ablation, especially when the PDT was performed before the CO2 laser ablation.
[Show abstract][Hide abstract] ABSTRACT: Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 01/2013; · 1.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the expression of anti-apoptotic genes (XIAP and Bcl-2) and apoptotic genes (cytochrome c, caspase-9, Apaf-1) in tissue samples of patients with superficial bladder cancer. Thirty-two bladder cancer tissue samples (8 papillary urothelial neoplasm of low malignant potential, 10 low-grade, and 14 high-grade) and 8 normal bladder tissue samples from necropsy were used for the study of gene expression by real-time PCR analysis. Analysis of the expression of apoptotic gene constituents of an apoptosome demonstrated an increase in Apaf-1 expression in the three tumor grades when compared with the control (P < 0.01, P < 0.05, and P < 0.01), low expression of caspase-9 in all groups (P < 0.05), and an increase in cytochrome c expression in all tumor grades in relation to the control, although without statistically significant difference. The expression of anti-apoptotic genes revealed an increase in XIAP expression in all tumor grades in relation to the control, although without statistically significant difference, and low expression of Bcl-2 in all tumor grades and the control (P < 0.05). The results proved that there is low evidence of apoptotic activity by the intrinsic pathway, demonstrated by the low expression of caspase-9 and considerable increase in XIAP expression, which may render these genes potential therapeutic targets in bladder cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Objective:This study was carried out to determine high pressure and pulsatile flow perfusion effects on human saphenous vein (HSV) segments obtained from diabetic and non-diabetic patients.Methods:The veins were perfused with oxygenated Krebs solution for 3 h, with a pulsatile flow rate of 100 mL/min and pressures of 250 × 200 or 300 × 250 mmHg. After perfusion, veins were studied by light microscopy; nitric oxide synthase (NOS) isoforms, CD34 and nitrotyrosine immunohistochemistry and tissue nitrite/nitrate (NO(x)) and malondialdehyde (MDA) quantification.Results:Light microscopy revealed endothelial denuding areas in all HSV segments subjected to 300 × 250 mmHg perfusion pressure, but the luminal area was similar. The percentage of luminal perimeter covered by endothelium decreased as perfusion pressures increased, and significant differences were observed between groups. The endothelial nitric oxide synthase (eNOS) isoform immunostaining decreased significantly in diabetic patients' veins independent of the perfusion pressure levels. The inducible NOS (iNOS), neuronal NOS (nNOS) and nitrotyrosine immunostaining were similar. Significant CD34 differences were observed between the diabetic 300 × 250 mmHg perfusion pressure group and the non-diabetic control group. Tissue nitrite/nitrate and MDA were not different among groups.Conclusions:Pulsatile flow and elevated pressures for 3 h caused morphological changes and decreased the eNOS expression in the diabetic patients' veins.
Diabetes & Vascular Disease Research 11/2012; · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the neuroprotection of mild hypothermia, applied in different moments, in temporary focal cerebral ischemia in rats.
Rats was divided into Control (C), Sham (S), Ischemic-control(IC), Pre-ischemic Hypothermia (IH1), Intra-ischemic Hypothermia (IH2), and Post-ischemic Hypothermia (IH3) groups. Morphometry was performed using the KS400 software (Carl Zeiss®) in coronal sections stained by Luxol Fast Blue. Ischemic areas and volumes were obtained.
Statistically, blue areas showed difference for C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.01; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH vs. IH2 (p=0.39; p=0.85; p=0.63). Red areas showed difference between C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.009; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH1 vs. IH2 (p=0.48; p=0.27; p=0.68). Average ischemic areas and ischemic volumes showed difference between IC vs. IH1 and IC vs. IH2 (p=0.0001 and p=0.0011), and no difference between IC vs. IH3 and IH1 vs. IH2 (p=0.57; p=0.79).
Pre-ischemic and intra-ischemic hypothermia were shown to be similarly neuroprotective, but this was not true for post-ischemic hypothermia.
Arquivos de neuro-psiquiatria 08/2012; 70(8):609-16. · 0.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AIM: On the average, 15% to 25% of peripheral grafts and 10% to 30% of coronary grafts fail within 5 years. Changes in mechanical forces to which the vein is subjected could be an explanation for this phenomenon. We submitted human saphenous vein segments to non-pulsatile ex vivo perfusion with crescent pressures and evaluated morphology, nitric oxide synthase immunohistochemical expression; tissue levels of nitrite/nitrate and oxidative stress products. METHODS: Intact segments of human saphenous veins were obtained from 30 patients submitted to elective coronary artery bypass graft surgery. Ex vivo perfusion was performed during 3 hours, using oxygenated Krebs solution, flow of 100 mL/min and pressures of 0, 50, 100, 200 and 300 mmHg, defining five groups. RESULTS:Optical microscopy showed that veins of groups perfused with 200 and 300 mmHg presented increased luminal area and endothelial denuding. Electron microscopy transmission showed alterations in veins perfused with 200 and 300 mmHg. Immunohistochemical expression of the three nitric oxide synthase isoforms was observed in all vein layers, without significant difference among groups. Tissue levels of nitrite/nitrate were not significantly different among distinctive perfusion. Nitrotyrosine was not immunohistochemically expressed in all veins and malondialdehyde tissue levels were not different among groups. CONCLUSION: Non-pulsatile ex vivo perfusion during 3h caused morphological alterations in human saphenous veins (HSVs), which were not accompanied by immunohistochemical and biochemical alterations. Even with mechanical lesions, HSVs maintained the ability of express nitric oxide synthase (NOS) and release nitric oxide.
The Journal of cardiovascular surgery 05/2012; · 1.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We tested the effects of liver reperfusion in the immunohistochemical expression of nitric oxide synthase on the thoracic aorta and the heart.
We randomized 24 male Wistar rats into 3 groups: (1) control; (2) R2 group, with 60 minutes of partial (70%) liver ischemia and 2 hours of global liver reperfusion; (3) and R6 group, with 60 minutes of partial liver ischemia and 6 hours of global liver reperfusion.
In the heart, there was little, diffuse immunohistochemical endothelial staining; immunohistochemical inducible nitric oxide synthase staining was expressed in the adventitia layer of intramyocardial vessels in both cases, with a time-dependent but not statistically significant increase. In the thoracic aorta, a time-dependent decrease in endothelial nitric oxide synthase expression in the muscular layer after reperfusion, which was statistically significant in R6 versus the control. Positive immunostaining for inducible nitric oxide synthase was seen in the muscular and endothelial layers, and this varied from moderate in the control group, to light in the endothelium in groups R2 and R6.
We observed changes that may be implicated in heart injury and impairment of aortal tone after liver ischemia and reperfusion injury.
Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation. 02/2012; 10(1):43-8.
[Show abstract][Hide abstract] ABSTRACT: The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase-2 (MMP-2) were studied in rat kidney.
Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. MMP-2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated.
Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol-treated rats showed increased activity of MMP-2. Histopathological investigation of kidneys from ethanol-treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol-treated rats.
Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS-derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol-treated rats might contribute to progressive renal damage.
The Journal of pharmacy and pharmacology. 01/2012; 64(1):68-76.
[Show abstract][Hide abstract] ABSTRACT: The experimental investigation was performed to study the effects of methylene blue (MB) on hemodynamic, biochemical, and tissue changes among rabbits undergoing liver ischemia and reperfusion (IR). Twenty-four rabbits were randomized into 5 groups: 1, SHAM, control; 2, MB infusion bolus (3 mg/kg); 3, IR, hepatic ischemia for 60 minutes followed by 120 minutes of reperfusion; 4, MB-R, undergoing ischemia that had received an MB bolus infusion (3 mg/kg) prior to reperfusion; 5, R-MB, undergoing ischemia and MB bolus infusion after hemodynamic instability caused by reperfusion. The analysis included continuous recording of vital signs. Blood samples were collected at 0, 60, and 180 minutes of IR to determine blood gases as well as biochemical markers of liver function, nitric oxide, lipid peroxidation, and neutrophil activity. At the end of each experiment, liver tissue samples were collected for histological evaluation of parenchymae markers. Statistical analysis used two-way analysis of variance (ANOVA) tests with significance set at P<.05. Vital signs significantly improved with MB infusion, irrespective of whether it was applied before or after reperfusion. Blood gas data revealed different patterns among the SHAM, MB, IR, MB-R, and R-MB groups, without statistical significance, except for favorable lactate results in the R-MB group (P<.01), which displayed greater survival. Biochemical tests did not show significant differences among the groups, whereas histological analysis revealed favorable appearances for the MB-R and R-MB groups. The MB effect lasted long after reperfusion, suggesting that improvement in the hemodynamic parameters was not based on liver integrity, but rather was possibly related to endothelial function.
[Show abstract][Hide abstract] ABSTRACT: In the present work we evaluated the effect of ethanol consumption in histopathological liver changes and several biochemical biomarkers employed in the detection of hepatic dysfunction. Male Wistar rats were treated with ethanol 20% (vol/vol) for 6 weeks. Histopathological investigation of livers from ethanol-treated animals revealed steatosis. Indices of hepatic function (transaminases) and mitochondrial respiration were not altered in ethanol-treated rats. Chronic ethanol consumption did not alter malondialdehyde (MDA) levels in the liver. Ethanol consumption induced a significant increase on hepatic nitrite and nitrate levels. Treatment with ethanol increased both mRNA expression and immunostaining of iNOS, but not eNOS. Finally, ethanol consumption did not alter hepatic levels of metalloproteinase (MMP)-2 and MMP-9. We conclude that alterations on biochemical biomarkers (nitrite and nitrate levels) and histopathology occurred in ethanol-treated rats, supporting the practice of including both types of evaluation in toxicity studies to detect potential ethanol-related hepatic effects. In our model of ethanol consumption, histopathological liver changes were accompanied by elevation in nitrite and nitrate levels indicating increased nitric oxide (NO) generation. Since iNOS-derived NO contributes to hepatic injury, the increased levels of NO described in our study might contribute to a progressive hepatic damage. Therefore, increases in NO generation may be an early indicator of ethanol-induced liver damage.
Tissue and Cell 09/2011; 43(6):384-91. · 1.05 Impact Factor