Acetaminophen treatment evokes anticontractile effects in rat aorta by blocking L-type calcium channels.

BACKGROUND: Acetaminophen (APAP) is the most widely used analgesic and antipyretic in the world. However, in high or continuous doses, it can cause serious side effects including blood pressure variability and cardiovascular injuries, which are barely explored. This study aimed to evaluate the acute effect of APAP treatment on vascular tone focused on the blocking of Ca2+ channels. METHODS: Rats were treated with APAP orally by gavage (500 mg/kg/single dose). After 12 h, the aorta was isolated for vascular reactivity studies in an isolated organ bath. Vascular contraction and relaxation were measured after different stimuli. Moreover, molecular docking studies were performed to evaluate the action of NAPQI (APAP metabolite) on L-type calcium channels. RESULTS: Phenylephrine-induced maximal vascular contraction was reduced in the APAP group (138.4 ± 9.2%) compared to the control group (172.2 ± 11.1%). APAP treatment significantly reduced contraction induced by Ca2+ influx stimulated with phenylephrine or KCl and reduced contraction mediated by Ca2+ released from the sarcoplasmic reticulum induced by caffeine. There was no difference in vascular relaxation induced by acetylcholine or sodium nitroprusside. Computational molecular docking demonstrated that NAPQI is capable of blocking L-type Ca2+ channels (Cav1.2), which would limit the influx of Ca2+. CONCLUSION: These results suggest that APAP treatment causes an anticontractile effect in rat aorta, possibly by blocking the influx of Ca2+ through L-type channels (Cav1.2).

Evaluation of Antioxidant Potential of Commercial Cinnamon Samples and Its Vasculature Effects.

Growing concerns on free radicals are the oxidative processes associated with physiological damage. The consumption of functional foods and use of plants with antioxidant capacity are widespread. Given the importance of determining antioxidant capacity in relation to the therapeutic effect, this study was aimed at evaluating cinnamon extract (Cinnamomum sp.) in commercial samples by spectrophotometric and voltammetric methods and assessing the vascular activity of some samples. The spectrophotometric methods performed were DPPH (1,1-diphenyl-2-picrihydrazine), ABTS (2,21-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)), and Folin-Ciocalteu radical sequestration assays. For the electrochemical experiments, a three-electrode system was used, consisting of carbon paste electrode, platinum wire, and Ag/AgCl/KClsat, representing the working, auxiliary, and reference electrodes, respectively. The electroanalytical methods used were differential pulse, square wave, and cyclic voltammetries. The extracts were prepared in hydroalcoholic solution. A calibration curve with gallic acid was calculated to quantify their equivalent amounts in the analyzed extract. The correlation between the electrochemical approach and the total phenols calculated by the ABTS, DPPH, and Folin-Ciocalteu methods was 0.63, 0.7, and 0.73, respectively, with 1 being an ideal directly proportional correlation. The correlation between spectrophotometric methods was 0.83. A biosensor was developed in a carbon paste electrode using the enzyme laccase, obtained by the fungus Marasmiellus colocasiae. It was observed that the antioxidant profile of the cinnamon samples had an analytical sign improvement of up to 4 times when compared with the electrode without the modification. The samples were analyzed by mass spectrometer, and the main chemical markers found were coumarin, cinnamaldehyde, and eugenol. Pharmacological trials showed that these samples also induce a significant vasorelaxant effect associated to antioxidant potential on vascular injury induced by oxidative stress. Thus, cinnamon showed a high antioxidant capacity, in agreement with the results obtained in other studies, emphasizing its importance as a functional food.

Vascular relaxing effect of Hydrocotyle umbellata L. is mediated by blocking of l-type Ca2+ channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydrocotyle umbellata L. is a medicinal herb for the treatment of some health problems including hypertension, according to traditional medicine. Even so, its vascular effects and the pharmacological action mechanisms have not been analyzed. AIM OF THE STUDY: This experiment aimed to analyze the effects of hydroalcoholic extract of Hydrocotyle umbellata L. (HEHU) on isolated vessels and verify the interaction of hibalactone (chemical marker) against Cav1.2 channels using molecular docking. MATERIALS AND METHODS: Vascular reactivity experiments were performed using rat aortas with (E+) or without endothelium (E-) in an isolated organ bath. Computational molecular docking approaches were used to show the direct effect on L-type Ca2+ Channels. RESULTS: HEHU (0-560 µg/mL) induced relaxation of the pre-contracted arteries in a concentration-dependent manner. The maximum effect was higher in E+ (76.8 ± 4.1%) as compared to E- (47.3 ± 5.5%). Pre-treatment of E+ arteries with L-NAME or ODQ reduced the relaxation to similar level of E- arteries. The treatment of arteries with MDL-12,330 A, diclofenac, propranolol and atropine did not change the relaxation induced by HEHU. The contraction caused by internal Ca2+ release induced by caffeine was reduced after HEHU treatment. Moreover, the HEHU also impaired the contraction induced by Ca2+ influx stimulated with phenylephrine or high KCl. The docking study demonstrated the effectiveness of hibalactone in blocking the Cav1.2 channel. CONCLUSIONS: These findings show that HEHU induces vascular relaxation which is potentiated (but not dependent) by endothelial cells. Blocking of Ca2+ influx seems to be the main mechanism for the vascular effects of HEHU.

Blood pressure variability provokes vascular ß-adrenoceptor desensitization in rats.

Spontaneous variation in blood pressure is defined as 'blood pressure variability' (BPV). Sinoaortic denervation (SAD) is characterized by BPV without sustained hypertension. In the present study, we investigated whether BPV could be related to vascular ß-adrenoceptor desensitization in rats. Three days after surgery (SAD and control), aortic rings were placed in an organ chamber and the relaxation stimulated by ß-adrenoceptor agonists, isoprenaline, terbutaline, BRL37344 and cyanopindolol was verified. The participation of intracellular nucleotides signaling pathways was also verified using forskolin, sodium nitroprusside and acetylcholine to induce relaxation. The effects of BPV on the increase in endothelial cytosolic Ca(2+) concentration stimulated by the ß2-adrenoceptor agonist was examined by confocal microscopy. In addition, the vascular expression of the ß2-adrenoceptor was also examined by immunohistochemistry. The results show that isoprenaline and terbutaline-induced relaxation was lower in the aortas of rats with BPV. Relaxation responses to other vasorelaxant compounds were similar in both groups of rats. Histological analysis revealed a lower level of ß2-adrenoceptor and confocal microscopy showed minor cytosolic Ca(2+) concentration in endothelial cells stimulated by the ß2-adrenoceptor agonist in rats with BPV. In conclusion, BPV leads to desensitization of the ß2-adrenoceptor, which could contribute to worse ß-adrenoceptor agonist-induced relaxation in isolated aortas.

Vasorelaxant activity of 7-ß-O-glycosides biosynthesized from flavonoids.

In this work we report the vasorelaxant activity of 7-ß-O-glycosides obtained with biosynthesis of naringenin-7-ß-O-glycoside (3) and quercetin-7-ß-O-glycoside (4). These compounds were obtained from naringenin (1) and quercetin (2) glycosylation catalyzed by Beauveria bassiana ATCC 7159. Screening of the best strain as a catalyst for glycosylation was carried out and the reaction conditions established. Cultures were grown in PDSM medium for 7 days at 27 °C. After purification by reverse-phase preparative HPLC, naringenin-7-ß-O-glycoside (3) and quercetin-7-ß-O-glycoside (4) were identified by (1)H and (13)C NMR. The right position and ß-configuration of the glucose was determined through HSQC and HMBC experiments. The vasorelaxation potential of naringenin, quercetin and its glycosylated derivatives was evaluated using isolated aorta in vitro models. Interestingly, results suggest that vasorelaxation properties of naringenin, rutin and its glycosides are due to different pathways.

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.

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.

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+).

Endothelial nitric oxide has inhibitory effects on rhythmic contractions in the aortas of sinoaortic deafferented rats.

Sinoaortic deafferentation (SAD) is characterized by arterial pressure lability, without sustained hypertension. Although SAD rats did not become hypertensive, their isolated aortas exhibit RCs. We have investigated whether these RCs are influenced by endothelium. Aortic rings were placed in an organ chamber, and the frequency and amplitude of the RCs were measured in SAD rat aortas with intact and denuded endothelium. Moreover, the participation of endothelial NO and cGMP pathways on the RCs were analyzed through the use of such drugs as L-NAME, acetylcholine, ODQ, L-arginine, and oxyhemoglobin. Indomethacin was used to evaluate the influence of prostanoids on the RCs. Under phenylephrine stimulus, 100% of SAD rat aortas presented RCs, with higher frequency and amplitude in denuded endothelium (8.5 +/- 0.50 cycles/2 min and 0.465 +/- 0.05 g, respectively) compared with intact endothelium (4.5 +/- 0.50 cycles/2 min and 0.311 +/- 0.04 g, respectively). In intact-endothelium aortas, L-NAME, ODQ, and oxyhemoglobin raised the frequency and amplitude of the RCs to values similar to those found in denuded endothelium. L-arginine and indomethacin did not alter the RCs. In conclusion, SAD rat aortas present rhythmic contractions, which are negatively modulated by endothelial NO. Our results indicate that endothelium-derived NO (and not prostanoids), acting through the cGMP pathway, has an inhibitory effect on the oscillations.

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 Ca(2+) 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 micromol/L), pinacidil (0.3 micromol/L) and tetraethylammonium (TEA; 5 mmol/L) totally inhibited SOCs, whereas increasing the CaCl(2) concentration to 2.0 and 2.5 mmol/L increased the frequency of SOCs. Interestingly, increasing the concentration of CaCl(2) 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 Ca(2+) influx sensitive to verapamil and also involve K(+) channels sensitive to pinacidil and TEA.

Importance of the sarcoplasmic reticulum and adrenergic stimulation on the cardiac contractility of the neotropical teleost Synbranchus marmoratus under different thermal conditions.

Experiments were carried out to investigate the heart rate of Synbranchus marmoratus after changing the temperature of the water contained in the experimental chamber of the acclimated fish (from 25 to 35 degrees C and from 25 to 15 degrees C). Then, an isometric cardiac muscle preparation was used to test the relative importance of Ca(2+) released from the sarcoplasmic reticulum and Ca(2+) influx across the sarcolemma for the cardiac performance under different thermal conditions: 25 degrees C (acclimation temperature), 15 and 35 degrees C. Adrenaline and ryanodine were used to modulate the Ca(2+) flux through the sarcolemma and the sarcoplasmic reticulum, respectively. Ryanodine reduced the peak tension by approximately 47% at 25 degrees C, and by 53% at 35 degrees C; however, it had no effect at 15 degrees C. A high adrenaline concentration was able to ameliorate the negative effects of ryanodine. Despite increasing the peak tension, adrenaline increased the times necessary for contraction and relaxation. We conclude that the sarcoplasmic reticulum is active in contributing Ca(2+) to the development of tension at physiological contraction frequencies. The adrenaline-stimulated Ca(2+) influx is able to increase the peak tension, even after addition of ryanodine, at physiologically relevant temperatures and pacing frequencies.