Matheus Lavorenti Rocha

Electrochemical behavior and determination of major phenolic antioxidants in selected coffee samples

The redox behavior of commercial roasted coffee products were evaluated by electroanalysis, whereas high performance liquid chromatography (HPLC) was used for determination of cinnamic acid markers, the total phenolic content (TPC) was achieved by Folin-Ciocalteu assay, and the traditional DPPH (1-diphenyl-2-picrylhydrazyl) method for antioxidant power determination. In turn, an optimized electrochemical index was proposed to estimate the antioxidant power of different samples and it was found a great correlation between all methods. The voltammetric profile of all coffee samples was quite similar, presenting the same number of peaks at the same potential values. Minor differences on current levels were in agreement with the total phenolic and major markers content, as well as, to the antioxidant power. Therefore, the electrochemical methods showed to be practical, low cost and very useful to evaluate the antioxidant properties of coffee samples, which is a relevant quality parameter of this widely consumed beverage.

Blood pressure variability increases connexin expression in the vascular smooth muscle of rats

AIMS Following sinoaortic denervation (SAD), isolated rat aortas present oscillatory contractions and demonstrate a heightened contraction for alpha-adrenergic agonists. Our aim was to verify the effects of SAD on connexin43 (Cx43) expression and phenylephrine-induced contraction in isolated aortas. METHODS AND RESULTS Three days after surgery (SAD or sham operation), isolated aortic rings were exposed to phenylephrine and acetylcholine (0.1-10 microM) in the presence or absence of the gap junction blocker 18beta-glycyrrhetinic acid (18beta-GA, 100 microM). Vascular reactivity to potassium chloride (KCl, 4.7-120 mM) was also examined. The incidence of rats presenting oscillatory contractions was measured. Effects of SAD on the vascular smooth muscle expression of the Cx43 mRNA by RT-PCR and western blotting for Cx43 protein were examined. Phenylephrine-induced contraction was higher in SAD rat aortas compared with the control. In the presence of 18beta-GA, the response to phenylephrine was similar in both groups. Oscillatory contractions were observed in 10/10 SAD rat aortas vs. 2/10 controls. Relaxing response to acetylcholine was similar in both groups, but in the presence of 18beta-GA, the response to acetylcholine decreased significantly in the sham-operated group (82.7 +/- 7.6% reduction of relaxation), whereas a half-maximal relaxation (reduction of 46.2 +/- 5.3%) took place in SAD rat aortas. KCl-induced contraction was similar in both groups. Following SAD, RT-PCR revealed significantly increased levels of Cx43 mRNA (9.85 fold, P < 0.01). Western blot analysis revealed greater levels of Cx43 protein (P < 0.05). CONCLUSION Blood pressure variability evoked by SAD leads to increased expression of Cx43, which could contribute to enhanced phenylephrine-induced contraction and oscillatory activity in isolated aortas.

A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle.

Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K(+) channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca(2+) sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca(2+) concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (E(max)) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPY-induced relaxation was reduced by tetraethylammonium (K(+) channels blocker) and by pre-contraction with 75mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca(2+) influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca(2+) concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca(2+) and K(+) across the membrane, it is more effective in reducing cytosolic Ca(2+) concentration and inducing relaxation in the rat trachea than the standard drug, SNP.

Relaxation evoked by extracellular Ca2+ in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca2+ channel.

The perivascular nerve network expresses a Ca2+ receptor that is activated by high extracellular Ca2+ concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca2+-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca2+ concentration. We also studied the role of the endothelium on the Ca2+-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenase-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K+ channels and L-type Ca2+ channels on the Ca2+-induced relaxation were evaluated. We have observed that the Ca2+-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF2alpha receptor antagonist) enhance the relaxing response to Ca2+. This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca2+-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca2+. The relaxing response to Ca2+ was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca2+ channels and sarcoplasmic reticulum in this response.

SPONTANEOUS OSCILLATORY CONTRACTIONS IN AORTAS OF RATS WITH ARTERIAL PRESSURE LABILITY CAUSED BY SINOAORTIC DENERVATION

1 The spontaneous variation of blood pressure is defined as arterial pressure lability. Sinoaortic denervation (SAD) is characterized by arterial pressure lability without sustained hypertension. 2 The phenomenon of spontaneous oscillatory contractions (SOCs) occurs more frequently in the vascular beds of hypertensive animals. In large arteries, such as the aorta, SOCs occur only occasionally or they can be initiated by application of chemical stimuli. 3 In the present study, we investigated whether the arterial pressure lability evoked by SAD could be related to the emergence of SOCs in the aorta of rats submitted to SAD compared with sham‐operated rats (SO). Three days after surgery (SAD or SO), aortic rings were placed in an organ chamber and the incidence (percentage of rats presenting SOCs), frequency (number of SOCs in 10 min) and amplitude (mN) of SOCs were measured. The participation of external Ca2+ and K+ channels in the maintenance of SOCs was also verified. 4 The incidence and frequency of SOCs were higher in endothelium‐denuded aortas from SAD rats (82% and 38 ± 4 SOCs/10 min, respectively) than in aortas from SO rats (40% and 14 ± 2 SOCs/10 min, respectively). In aortas from SAD rats, verapamil (0.2 µmol/L), pinacidil (0.3 µmol/L) and tetraethylammonium (TEA; 5 mmol/L) totally inhibited SOCs, whereas increasing the CaCl2 concentration to 2.0 and 2.5 mmol/L increased the frequency of SOCs. Interestingly, increasing the concentration of CaCl2 to 3.5 mmol/L inhibited these contractions in aortas from SAD rats. 5 These results show that although SAD rats did not become hypertensive, their aortas were capable of initiating SOCs without the application of any chemical stimuli. The SOCs seem to be dependent on Ca2+ influx sensitive to verapamil and also involve K+ channels sensitive to pinacidil and TEA.

Relaxing effect of a new ruthenium complex nitric oxide donor on airway smooth muscle of an experimental model of asthma in rats.

NO is a potent bronchodilator and NO‐donor compounds have demonstrated clinical significance for obstructive airway diseases. This study evaluated the relaxation mechanisms of two NO donors, a ruthenium compound (TERPY), and sodium nitroprusside (SNP), in rat tracheas with ovalbumin‐induced asthma (OVA group) and in another control group. The effect of TERPY and SNP was evaluated in tracheal rings in an isolated organ chamber. The contribution of K+ channels, sGC/cGMP pathway, phosphodiesterases, and extra and intracellular Ca2+ sources were analyzed. The TERPY and SNP‐induced tracheal smooth muscle relaxation in both groups. However, the maximum effect induced by TERPY was higher than that of SNP in both control (110.2 ± 3.2% vs 68.3 ± 3.1%, P < 0.001) and OVA groups (106.1 ± 1.5% vs 49.9 ± 2.7%, P < 0.001). In the control group, TERPY relaxation was induced by the activation of K+ channels and reduction of the calcium influx, while in the OVA group, these same effects were also brought about by TERPY, but with participation of the sGC/cGMP pathway. In both groups, SNP‐induced relaxation occurred through the activation of K+ channels, sGC/cGMP pathway and reduction of calcium influx. However, the activation of sGC pathway and reticular Ca2+‐ATPase seemed to be reduced in the OVA group. Furthermore, TERPY is capable of reversing the contraction of carbachol in asthmatic bronchioles. Finally, TERPY and SNP relaxation mechanisms were modified by asthma. SNP presented less relaxation than TERPY, which induced full relaxation with greater participation of K+ and Ca2+ fluxes through the membrane, thereby making TERPY a promising drug for reversing the narrowing of airways.

Vasorelaxant and Hypotensive Effects of Jaboticaba Fruit (Myrciaria cauliflora) Extract in Rats

This studys aim was to determine the effect of hydroalcoholic extract of M. cauliflora (HEMC) on vascular tension and blood pressure in rats. In our in vitro studies using precontracted isolated aortas from rats, HEMC and acetylcholine (positive control) induced relaxation only in vessels with endothelium. Pretreatment with L-NAME (NO synthase inhibitor) or ODQ (soluble guanylyl cyclase (sGC) inhibitor) abolished the HEMC-induced relaxation. The treatment with MDL-12,330A (adenylyl cyclase (AC) inhibitor) or diclofenac (COX inhibitor) reduced HEMC-induced vasorelaxation. The blockade of muscarinic and β-adrenergic receptors (by atropine and propranolol, resp.) did not promote changes in HEMC-induced vasorelaxation. In our in vivo studies, catheters were inserted into the right femoral vein and artery of anesthetized rats for HEMC infusion and the measurement of blood pressure, heart rate, and aortic blood flow. The intravenous infusion of HEMC produced hypotension and increased aortic blood flow with no changes in heart rate. These findings showed that HEMC induces endothelium-dependent vascular relaxation and hypotension with no alteration in heart rate. The NO/sGC/cGMP pathway seems to be the main cellular route involved in the vascular responsiveness.

Radical Scavenger Capacity of Jabuticaba Fruit (Myrciaria cauliflora) and Its Biological Effects in Hypertensive Rats

Jabuticaba is an exotic fruit native to Brazil that has been arousing medicinal interest. Using chemical (HPLC-PDA, resonance mass spectra, and NMR), electroanalytical (differential pulse voltammetry, radical scavenging assay), and pharmacological (in vivo and in vitro) approaches, we have identified its bioactive compounds and hypotensive effects on hypertensive rats. The hydroalcoholic extract of jabuticaba (HEJ) presents a great quantity of phenolic compounds, and several molecules with hydroxyl groups present high efficiency as an antioxidant. The treatment with HEJ (100 and 300 mg/kg/day, for four weeks) presented hypotensive effects on L-NAME-induced hypertensive rats, possibly improving the nitric oxide bioavailability because of its high antioxidant potential. Furthermore, renal and cardiac hypertrophies were also attenuated after the HEJ treatment. Moreover, the vascular responses to contractile and dilating agonists were improved with the HEJ treatment, which is also able to induce nitric oxide production in endothelial cells.

Diuretic effects and urinary electrolyte excretion induced by Aspidosperma subincanum Mart. and the involvement of prostaglandins in such effects.

ETHNOPHARMACOLOGICAL RELEVANCE Aspidosperma subincanum Mart. is a medicinal herb known for its diuretic properties and used for the treatment of cardiovascular-related illnesses. Although our earlier study has shown that the ethanol extract of Aspidosperma subincanum (EEAS) induces hypotension and vasodilation, no scientific data have been recorded to evaluate the diuretic effects of this Brazilian medicinal plant. The aim of this study was to evaluate the diuretic activity of EEAS, and possible mechanism of action, using Wistar rats. MATERIAL AND METHODS EEAS (60 and 120mg/kg), furosemide (20mg/kg) or saline (control) were orally administered to rats individually held in metabolic cages for urine collection 1, 2, 4, 6, 8, 12 and 24h after treatment. In order to evaluate the involvement of prostaglandins in the diuretic action of EEAS, the animals received piroxicam (5mg/kgi.p.), a nonselective inhibitor of cyclooxygenase, before treatment with EEAS at 120mg/kg. The control groups received only saline (NaCl, 0.9%), or saline and piroxicam. Urinary volume, electrolyte excretion and pH were measured. RESULTS Oral administration of EEAS 60 and 120mg/kg significantly increased diuresis and electrolyte excretion of Na(+) and K(+) on a continuous basis throughout the study period. Both EEAS 60 and 120mg/kg caused a relative increase of around 77% and 142%, respectively, in cumulative diuresis compared with the control group. From 4th hour until the end of the experiment, the group treated with EEAS 120mg/kg provided a greater excretion of Na(+) than the furosemide group. The diuretic effects of EEAS were neutralized by piroxicam between 4 and 8h after treatment. CONCLUSION The results suggest that EEAS could present compound(s) responsible for diuretic activities, and the mechanism could involve the prostaglandin system.

Aortas Isolated from Sinoaortic‐Denervated Rats Exhibit Rhythmic Contractions That Are Regulated by Pharmacologically Distinct Calcium Sources

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca(2+) sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca(2+) released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca(2+) entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca(2+)-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca(2+) release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca(2+) entry through L-type Ca(2+).