Antinociceptive activity of sulfated carbohydrates from the red algae Bryothamnion seaforthii (Turner) Kütz. and B. triquetrum (S.G. Gmel.) M. Howe

We report the antinociceptive activity, determined by the writhing, formalin and hot-plate tests in mice, of crude (F0/60), lectin and carbohydrate fractions isolated by ammonium sulfate precipitation (0 to 60%) from Bryothamnion seaforthii and B. triquetrum, species of red algae. Not only fraction F0/60 but also lectins from both species significantly inhibited acetic acid-induced abdominal contractions after intraperitoneal or oral administrations. In the formalin test, lectins (1 and 5 mg/kg, ip, and 5 to 20 mg/kg, po) inhibited the 1st and 2nd phases (5 and 20 min, respectively), but the effect occurred predominantly on the 2nd phase. The effects of the lectins were totally or partially reversed by naloxone (2 mg/kg, sc) in the 1st and 2nd phases, respectively. Experiments performed with lectins in the absence and presence of avidin (1 mg/kg, ip) and D-mannose (1 mg/kg, ip) showed that avidin did not interfere with the effect of B. seaforthii lectin but partially reversed the effect of B. triquetrum lectin. D-Mannose completely reversed the effects of both species. F0/60 fractions from both algae significantly increased the latency time in response to thermal stimuli, and naloxone reversed antinociception, indicating the involvement of the opioid system in both the peripheral and central effects of the fractions. In the writhing test, the carbohydrate fractions were the most active, inhibiting the contractions by 71 and 79% (B. triquetrum) and by 46 and 69% (B. seaforthii) at doses of 1 and 5 mg/kg, ip, respectively. Sulfated carbohydrate fractions of B. seaforthii and B. triquetrum, containing only about 5% protein as contaminants, are probably responsible for the antinociceptive effects of these red algae.

Gender differences in vascular expression of endothelin and ET A/ET B receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

High concentrations of KCl release noradrenaline from noradrenergic neurons in the rat anococcygeus muscle

The aim of the present study was to investigate the effects of high concentrations of KCl in releasing noradrenaline from sympathetic nerves and its actions on postsynaptic alpha-adrenoceptors. We measured the isotonic contractions induced by KCl in the isolated rat anococcygeus muscle under different experimental conditions. The contractile responses induced by KCl were inhibited by alpha-adrenoceptor antagonists in 2.5 mM Ca2+ solution. Prazosin reduced the maximum effect from 100 to 53.9 ± 10.2% (P<0.05) while the pD2 values were not changed. The contractile responses induced by KCl were abolished by prazosin in Ca2+-free solution (P<0.05). Treatment of the rats with reserpine reduced the maximum effect induced by KCl as compared to the contractile responses induced by acetylcholine from 339.5 ± 157.8 to 167.3 ± 65.5% (P<0.05), and increased the pD2 from 1.57 ± 0.01 to 1.65 ± 0.006 (P<0.05), but abolished the inhibitory effect of prazosin (P<0.05). In contrast, L-NAME increased the contractile responses induced by 120 mM KCl by 6.2 ± 2.3% (P<0.05), indicating that KCl could stimulate the neurons that release nitric oxide, an inhibitory component of the contractile response induced by KCl. Our results indicate that high concentrations of KCl induce the release of noradrenaline from noradrenergic neurons, which interacts with alpha1-adrenoceptors in smooth muscle cells, producing a contractile response in 2.5 mM Ca2+ (100%) and in Ca2+-free solution, part of which is due to a direct effect of KCl on the rat anococcygeus muscle.

Anti-inflammatory and antispasmodic activity of Ipomoea imperati (Vahl) Griseb (Convolvulaceae)

Ipomoea imperati (Convolvulaceae) lives on the sandy shores of the Brazilian coast and in other areas of the world. The anti-inflammatory activity of a methanol-water extract of the leaves of I. imperati was investigated in experimental models of acute and subchronic inflammation. Topical application of the extract (10 mg/ear) inhibited mouse ear edema induced by croton oil (89.0 ± 1.3% by the lipid fraction with an IC50 of 3.97 mg/ear and 57.0 ± 1.3% by the aqueous fraction with an IC50 of 3.5 mg/ear) and arachidonic acid (42.0 ± 2.0% with an IC50 of 4.98 mg/ear and 31.0 ± 2.0% with an IC50 of 4.72 mg/ear). Phospholipase A2, purified from Apis mellifera bee venom, was also inhibited by the extract (5.0 mg/ml lipid and aqueous fraction) in vitro in a dose-dependent manner (85% by the lipid fraction with an IC50 of 3.22 mg/ml and 25% by the aqueous fraction with an IC50 of 3.43 mg/ml). The methanol-water extract of I. imperati (1000 mg/kg) administered by the oral route also inhibited the formation of cotton pellet-induced granulomas (73.2 ± 1.2% by the lipid fraction and 56.14 ± 2.7% by the aqueous fraction) and did not cause gastric mucosal lesions. I. imperati extracts (10 mg/ml) also inhibited in a dose-dependent manner the muscle contractions of guinea pig ileum induced by acetylcholine and histamine (IC50 of 1.60 mg/ml for the lipid fraction and 4.12 mg/ml for the aqueous fraction). These results suggest the use of I. imperati as an anti-inflammatory and antispasmodic agent in traditional medicine.

Functional changes of human quadriceps muscle injured by eccentric exercise

The present study evaluated functional changes of quadriceps muscle after injury induced by eccentric exercise. Maximal isometric torque of quadriceps and the surface electromyography (root mean square, RMS, and median frequency, MDF) of the vastus medialis oblique (VMO) and vastus lateralis (VL) muscles were examined before, immediately after and during the first 7 days after injury. Serum creatine kinase (CK) levels and magnetic resonance imaging (MRI) were used to identify muscle injury. The subject was used as her own control and percent refers to pre-injury data. Experiments were carried out with a sedentary 23-year-old female. Injury was induced by 4 bouts of 15 maximal isokinetic eccentric contractions (angular velocity of 5º/s; range of motion from 40º to 110º of knee flexion). The isometric torque of the quadriceps (knee at 90º flexion) decreased 52% immediately after eccentric exercise and recovered on the 5th day. The highest reduction of RMS occurred on the 2nd day after injury in both VL (63%) and VMO (66%) and only VL recovered to the pre-injury level on the 7th day. Immediately after injury, the MDF decreased by 5 and 3% (VMO and VL, respectively) and recovered one day later. Serum CK levels increased by 109% on the 2nd day and were still increased by 32% on the 7th day. MRI showed large areas of injury especially in the deep region of quadriceps. In conclusion, eccentric exercise decreased the isometric torque and electromyographic signals of quadriceps muscle, which were recovered in one week, despite the muscle regeneration signals.

4-Aminopyridine inhibits the neuromuscular effects of nitric oxide and 8-Br-cGMP

The effects induced by nitric oxide (NO) in different tissues depend on direct and/or indirect interactions with K+ channels. The indirect interaction of NO is produced by activation of guanylyl cyclase which increases the intracellular cGMP. Since NO, cGMP and 4-aminopyridine alone induce tetanic fade and increase amplitude of muscular contractions in isolated rat neuromuscular preparations, the present study was undertaken to determine whether or not the neuromuscular effects of NO and 8-Br-cGMP can be modified by 4-aminopyridine. Using the phrenic nerve and diaphragm muscle isolated from male Wistar rats (200-250 g), we observed that L-arginine (4.7 mM) and 8-Br-cGMP (18 µM), in contrast to D-arginine, induced an increase in the amplitude of muscle contraction (10.5 ± 0.7%, N = 10 and 8.0 ± 0.7%, N = 10) and tetanic fade (15 ± 2.0%, N = 8 and 11.6 ± 1.7%, N = 8) at 0.2 and 50 Hz, respectively. N G-nitro-L-arginine (4 mM, N = 8 and 8 mM, N = 8) antagonized the effects of L-arginine. 4-Aminopyridine (1 and 10 µM) caused a dose-dependent increase in the amplitude of muscle contraction (15 ± 1.8%, N = 9 and 40 ± 3.1%, N = 10) and tetanic fade (17.7 ± 3.3%, N = 8 and 37.4 ± 1.3%, N = 8). 4-Aminopyridine (1 µM, N = 8) did not cause any change in muscle contraction amplitude or tetanic fade of preparations previously paralyzed with d-tubocurarine or stimulated directly. The effects induced by 4-aminopyridine alone were similar to those observed when the drug was administered in combination with L-arginine or 8-Br-cGMP. The data suggest that the blockage of K+ channels produced by 4-aminopyridine inhibits the neuromuscular effects induced by NO and 8-Br-cGMP. Therefore, the presynaptic effects induced by NO seem to depend on indirect interactions with K+ channels.

Effect of cyproterone acetate on alpha1-adrenoceptor subtypes in rat vas deferens

Gonadal hormones regulate the expression of alpha1-adrenoceptor subtypes in several tissues. The present study was carried out to determine whether or not cyproterone acetate, an anti-androgenic agent, regulates the alpha1-adrenoceptor subtypes that mediate contractions of the rat vas deferens in response to noradrenaline. The actions of subtype selective alpha1-antagonists were investigated in vas deferens from control and cyproterone acetate-treated rats (10 mg/day, sc, for 7 days). Prazosin (pA2 ~9.5), phentolamine (pA2 ~8.3) and yohimbine (pA2 ~6.7) presented competitive antagonism consistent with activation of alpha1-adrenoceptors in vas deferens from both control and treated rats. The pA2 values estimated for WB 4101 (~9.5), benoxathian (~9.7), 5-methylurapidil (~8.5), indoramin (~8.7) and BMY 7378 (~6.8) indicate that alpha1A-adrenoceptors are involved in the contractions of the vas deferens from control and cyproterone acetate-treated rats. Treatment of the vas deferens from control rats with the alpha1B/alpha1D-adrenoceptor alkylating agent chloroethylclonidine had no effect on noradrenaline contractions, supporting the involvement of the alpha1A-subtype. However, this agent partially inhibited the contractions of vas deferens from cyproterone acetate-treated rats, suggesting involvement of multiple receptor subtypes. To further investigate this, the actions of WB 4101 and chloroethylclonidine were reevaluated in the vas deferens from rats treated with cyproterone acetate for 14 days. In these organs WB 4101 presented complex antagonism characterized by a Schild plot with a slope different from unity (0.65 ± 0.05). After treatment with chloroethylclonidine, the complex antagonism presented by WB 4101 was converted into classical competitive antagonism, consistent with participation of alpha1A-adrenoceptors as well as alpha1B-adrenoceptors. These results suggest that cyproterone acetate induces plasticity in the alpha1-adrenoceptor subtypes involved in the contractions of the vas deferens.

Spectral analysis of the electromyograph of the erector spinae muscle before and after a dynamic manual load-lifting test

The aim of the present study was to assess the spectral behavior of the erector spinae muscle during isometric contractions performed before and after a dynamic manual load-lifting test carried out by the trunk in order to determine the capacity of muscle to perform this task. Nine healthy female students participated in the experiment. Their average age, height, and body mass (± SD) were 20 ± 1 years, 1.6 ± 0.03 m, and 53 ± 4 kg, respectively. The development of muscle fatigue was assessed by spectral analysis (median frequency) and root mean square with time. The test consisted of repeated bending movements from the trunk, starting from a 45º angle of flexion, with the application of approximately 15, 25 and 50% of maximum individual load, to the stand up position. The protocol used proved to be more reliable with loads exceeding 50% of the maximum for the identification of muscle fatigue by electromyography as a function of time. Most of the volunteers showed an increase in root mean square versus time on both the right (N = 7) and the left (N = 6) side, indicating a tendency to become fatigued. With respect to the changes in median frequency of the electromyographic signal, the loads used in this study had no significant effect on either the right or the left side of the erector spinae muscle at this frequency, suggesting that a higher amount and percentage of loads would produce more substantial results in the study of isotonic contractions.

Eucalyptol, an essential oil, reduces contractile activity in rat cardiac muscle

Eucalyptol is an essential oil that relaxes bronchial and vascular smooth muscle although its direct actions on isolated myocardium have not been reported. We investigated a putative negative inotropic effect of the oil on left ventricular papillary muscles from male Wistar rats weighing 250 to 300 g, as well as its effects on isometric force, rate of force development, time parameters, post-rest potentiation, positive inotropic interventions produced by Ca2+ and isoproterenol, and on tetanic tension. The effects of 0.3 mM eucalyptol on myosin ATPase activity were also investigated. Eucalyptol (0.003 to 0.3 mM) reduced isometric tension, the rate of force development and time parameters. The oil reduced the force developed by steady-state contractions (50% at 0.3 mM) but did not alter sarcoplasmic reticulum function or post-rest contractions and produced a progressive increase in relative potentiation. Increased extracellular Ca2+ concentration (0.62 to 5 mM) and isoproterenol (20 nM) administration counteracted the negative inotropic effects of the oil. The activity of the contractile machinery evaluated by tetanic force development was reduced by 30 to 50% but myosin ATPase activity was not affected by eucalyptol (0.3 mM), supporting the idea of a reduction of sarcolemmal Ca2+ influx. The present results suggest that eucalyptol depresses force development, probably acting as a calcium channel blocker.

Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 µM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 µM L-NOARG from 73 ± 5.1 to 19 ± 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

Effect of unsaturated fatty acids on myocardial performance, metabolism and morphology

Diets rich in saturated fatty acids are one of the most important causes of atherosclerosis in men, and have been replaced with diets rich in unsaturated fatty acids (UFA) for the prevention of this disorder. However, the effect of UFA on myocardial performance, metabolism and morphology has not been completely characterized. The objective of the present investigation was to evaluate the effects of a UFA-rich diet on cardiac muscle function, oxidative stress, and morphology. Sixty-day-old male Wistar rats were fed a control (N = 8) or a UFA-rich diet (N = 8) for 60 days. Myocardial performance was studied in isolated papillary muscle by isometric and isotonic contractions under basal conditions after calcium chloride (5.2 mM) and ß-adrenergic stimulation with 1.0 µM isoproterenol. Fragments of the left ventricle free wall were used to study oxidative stress and were analyzed by light microscopy, and the myocardial ultrastructure was examined in left ventricle papillary muscle. After 60 days the UFA-rich diet did not change myocardial function. However, it caused high lipid hydroperoxide (176 ± 5 vs 158 ± 5, P < 0.0005) and low catalase (7 ± 1 vs 9 ± 1, P < 0.005) and superoxide-dismutase (18 ± 2 vs 27 ± 5, P < 0.005) levels, and discrete morphological changes in UFA-rich diet hearts such as lipid deposits and mitochondrial membrane alterations compared to control rats. These data show that a UFA-rich diet caused myocardial oxidative stress and mild structural alterations, but did not change mechanical function.

Noninvasive prognostic markers for cardiac death and ventricular arrhythmia in long-term follow-up of subjects with chronic Chagas' disease

The objective of the present study was to investigate clinical, echocardiographic and electrocardiographic (12-lead resting ECG, 24-h ambulatory ECG monitoring and signal-averaged ECG (SAECG)) parameters in subjects with chronic Chagas' disease in a long-term follow-up as prognostic markers for adverse outcomes. Fifty adult outpatients (34 to 74 years old, 31 females) staged according to Los Andes class I, II or III and complaining of palpitation were enrolled in a longitudinal study. SAECG was analyzed in time and frequency domains and the endpoint was a composite of cardiac death and ventricular tachycardia. During a follow-up of 84.2 ± 39.0 months, 34.0% of the patients developed adverse outcomes (9 cardiac deaths and 11 episodes of ventricular tachycardia). After optimal dichotomization, in a stepwise multivariate Cox-hazard regression model, apical aneurysm (HR = 3.7; 95% CI = 1.2-1.3; P = 0.02), left ventricular ejection fraction <62% (HR = 4.60; 95% CI = 1.39-15.24; P = 0.01) and incidence of ventricular premature contractions >614 per 24 h (hazard ratio = 6.1; 95% CI = 1.7-22.6; P = 0.006) were independent predictors of the composite endpoint. Although a high frequency content in SAECG demonstrated association with the presence of left ventricular dysfunction and myocardial fibrosis, its predictive value for the composite endpoint was not significant. Apical aneurysms, reduced left ventricular function and a high incidence of ventricular ectopic beats over a 24-h period have a strong predictive value for a composite endpoint of cardiac death and ventricular tachycardia in subjects with chronic Chagas' disease.

Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

Lead (Pb2+) poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g) before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM) to the bath. Changes in rate of stimulation (0.1-1.5 Hz), relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM), and the effect of isoproterenol (20 ng/mL) were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz) and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

Circadian cardiac autonomic function in perinatally HIV-infected preschool children

The 24-h heart rate variability and QT-interval adaptation was investigated in perinatally HIV-infected preschool children classified according to immunological status in order to assess autonomic function at early stages of infection. Thirty-five perinatally HIV-infected and clinically stable children (4.8 ± 0.3 years) were enrolled after approval of the study by the University Hospital Pedro Ernesto Ethics Committee and written informed parental consent was obtained. The children were classified according to peripheral CD4+ count (cells/µL) as follows: group 1, N = 11 (≥1000); group 2, N = 7 (≥500 and <1000); group 3, N = 17 (<500). Left ventricular ejection fraction (>55%), 24-h RR interval variability (RRV) indexes (NN, SDANN, SDNN index, r-MSSD) and 24-h QT and Bazett-corrected QT (QTc) were determined, and groups were matched for age, body surface area, and left ventricular ejection fraction, reducing biases in RRV. The peak differences (∆) between the highest and lowest RRV and QT indexes were extracted from nocturnal (1 am-6 am) and daytime (1 pm-6 pm) hourly assessed segments, respectively. Pearson’s correlation (r) and Kruskal-Wallis ANOVA were used to compare groups. CD4+ count correlated positively with ∆NN (r = 0.45; P = 0.003). There were no significant differences in daytime NN among groups. Nighttime SDNN index (P = 0.01), nighttime r-MSSD (P = 0.003), ∆NN (P = 0.01), ∆SDNN index (P = 0.03) and ∆r-MSSD (P = 0.004) were significantly lower in group 3 than in the other groups. Expected nighttime QTc-interval lengthening was not observed in all groups. In perinatally HIV-infected preschool children with preserved left ventricular systolic function, parasympathetic-mediated autonomic dysfunction parallels immune status, impairing both RRV and circadian QTc interval adaptation.

Cardiovascular activity of the n-butanol fraction of the methanol extract of Loranthus ferrugineus Roxb.

We investigated the vascular responses and the blood pressure reducing effects of different fractions obtained from the methanol extract of Loranthus ferrugineus Roxb. (F. Loranthaceae). By means of solvent-solvent extraction, L. ferrugineus methanol extract (LFME) was successively fractionated with chloroform, ethyl acetate and n-butanol. The ability of these LFME fractions to relax vascular smooth muscle against phenylephrine (PE)- and KCl-induced contractions in isolated rat aortic rings was determined. In another set of experiments, LFME fractions were tested for blood pressure lowering activity in anesthetized adult male Sprague-Dawley rats (250-300 g, 14-18 weeks). The n-butanol fraction of LFME (NBF-LFME) produced a significant concentration-dependent inhibition of PE- and KCl-induced aortic ring contractions compared to other fractions. Moreover, NBF-LFME had a significantly higher relaxant effect against PE- than against high K+-induced contractions. In anesthetized Sprague-Dawley rats, NBF-LFME significantly lowered blood pressure in a dose-dependent manner and with a relatively longer duration of action compared to the other fractions. HPLC, UV and IR spectra suggested the presence of terpenoid constituents in both LFME and NBF-LFME. Accordingly, we conclude that NBF-LFME is the most potent fraction producing a concentration-dependent relaxation in vascular smooth muscle in vitro and a dose-dependent blood pressure lowering activity in vivo. The cardiovascular effects of NBF-LFME are most likely attributable to its terpenoid content.