Vasorelaxant effects of ellagitannins isolated from Cuphea carthagenensis.

Cuphea carthagenensis (Jacq.) J. F. Macbr. is a popular plant in Brazilian folk medicine owing to its hypotensive and central nervous system depressant effects. This study aimed to validate the hypotensive effect of the plant's aqueous extract (AE) in rats and examine the vascular actions of three hydrolyzable tannins, oenothein B, woodfordin C, and eucalbanin B, isolated from AE. Systolic blood pressure in unanesthetized rats was determined using the non-invasive tail-cuff method. Oral treatment of normotensive rats with 0.5 and 1.0 g/kg/day AE induced a dose-related hypotensive effect after 1 week. In rat aortic rings pre-contracted with noradrenaline, all ellagitannins (20 - 180 µM) induced a concentration-related vasorelaxation. This effect was blocked by either removing the endothelium or pre-incubating with NG-nitro-l-arginine methyl ester (10 µM), an inhibitor of nitric oxide (NO) synthase. In KCl-depolarized rat portal vein preparations, the investigated compounds did not affect significantly the maximal contractile responses and pD2 values of the concentration-response curves to CaCl2. Our results demonstrated the hypotensive effect of C. carthagenensis AE in unanesthetized rats. All isolated ellagitannins induced vasorelaxation in vitro via activating NO synthesis/NO release from endothelial cells, without altering the Ca2+ influx in vascular smooth muscle preparations. Considering the low oral bioavailability of ellagitannins, the determined in vitro actions of these compounds are unlikely to account for the hypotensive effect of AE in vivo. It remains to be determined the role of the bioactive ellagitannin-derived metabolites in the hypotensive effect observed after oral treatment of unanesthetized rats with the plant extract.

The mechanisms of calcium mobilization by procyanidins, flavonols and flavonoids from Cecropia glaziovii Sneth in pulmonary endothelial cell cultures endorse its popular use as vasodilator phytomedicine.

The hypotensive and antihypertensive activities of the aqueous extract (AE) and butanolic fraction (ButF) isolated from Cecropia glaziovii Sneth have been demonstrated in previous studies in animal models. This study aimed to evaluate the molecular mechanism of action responsible for the vasodilatory effect of procyanidins, flavanols, and flavonoids found in C. glaziovii in endothelial cell culture. For this purpose, we analyzed the effect of procyanidin B2 and B3 compounds, catechin, epicatechin, orientin, isoorientin, and isovitexin in the mobilization of Ca2+ in rat endothelial cell cultures. Parallel associations with different antagonists were examined by considering the following in vivo hypotensive mechanisms: blockage of L-type calcium channels, action on ß-2 adrenergic receptors, and vasodilation via the nitric oxide pathway. All measurements of calcium mobilization were carried out by using the fluorescence measurement methodology in a Flexstation M3 spectrophotometer. The results indicate that some of the compounds have mixed actions, acting through different calcium mobilization pathways. The mobilization induced by such compounds significantly decreased when they were incubated with their corresponding antagonists. Taken together, our data suggest that the beneficial effects seen with the popular use of Cecropia glaziovii Sneth in pathological conditions, such as systemic arterial hypertension, seem to be related to the plant's hypotensive effect, very probably promoted by the actions of flavonols, flavonoids, and procyanidins, by different pathways of calcium mobilization.

Altered release and uptake of gamma-aminobutyric acid in the cerebellum of dystrophin-deficient mice.

Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABAARs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [3H]-GABA release, induced by nicotinic receptor (nAChR) activation or K+ depolarization, and [3H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K+ solutions led to a concentration-dependent and Ca2+-dependent [3H]-GABA release in control and mdx synaptosomes. [3H]-GABA release induced by 10 µM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K+-induced [3H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and ß2-containing nAChRs were involved in nicotine-induced [3H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [3H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t1/2) and increased (44%) rate of [3H]-GABA uptake (Vmax) compared to controls. The apparent transporter affinity (Km) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABAAR in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD.

Effects of the butanol-containing fraction from leaves of Calea prunifolia H.B.K. on blood pressure and smooth muscle responses of Wistar rats / Efecto de la fracción butanólica de las hojas de Calea prunifolia H.B.K. sobre la presión arterial y la relajación muscular en ratas wistar

The butanol-containing fraction from leaves of Calea prunifolia H.B.K. was obtained in order to examine cardiovascular effects on two distinct rat preparations. The responses elicited in isolated aorta and isolated vas deferens, were examined, as were the changes in blood pressure in anesthetized and conscious Wistar rats. In addition, the effects on angiotensin-converting enzyme activity in plasma and the effects on cytosolic calcium in cardiomyocytes and uterine cells were measured. Results show that the butanol-containing fraction from C. prunifolia (0.1-100 µg/mL) relaxes phenylephrine-induced contractions in isolated aorta (CI50: 58 µg/mL), decreases the contractile response induced by noradrenaline in vas deferens, and shows a competitive antagonism profile (pA2: 4.48, m: 1024). The butanol-containing fraction from C. prunifolia also decreases blood pressure in anesthetized rats in a dose-dependent manner (1-100 mg/kg, i.v.), yet is devoid of hypotensive effects in normotensive, conscious rats. In addition, C. prunifolia does not modify the hypertensive effects induced by the nitric oxide synthase inhibitor L-NAME, and it also does not affect the angiotensin-converting enzyme plasma activity nor the cytosolic calcium in cardiomyocytes and uterine cells. According to these results, C. prunifolia relaxes smooth muscle and vascular tone, favoring the decrease in blood pressure in rats via mechanisms related to alpha adrenergic inhibition.

Se evaluó el efecto cardiovascular en ratas inducido por la fracción butanólica de las hojas de Calea prunifolia H.B.K., examinando el efecto generado en preparaciones de anillos aislados de aorta y conducto deferente, y los cambios de presión arterial en ratas wistar anestesiadas y despiertas. También se cuantificó el efecto sobre la enzima convertidora de angiotensina (ECA) en plasma y sobre el calcio citosólico en cardiomiocitos y uteromiocitos. Los resultados mostraron que la fracción butanólica de C. prunifolia (0,1-100 µg/mL) relajaba anillos de aorta contraídos con fenilefrina (CI50: 58 µg/mL) y disminuía la contracción del conducto deferente inducido por noradrenalina con un perfil de inhibición competitiva (pA2: 4.48, m: 1.024). La fracción butanólica de C. prunifolia disminuye la presión sanguínea en ratas anestesiadas en función de la dosis (1-100 mg/kg, i.v.), si bien estuvo desprovista de efectos hipotensores en ratas despiertas. C. prunifolia tampoco modificó el efecto hipertensor inducido por el inhibidor de óxido nítrico sintetasa L-NAME, ni los niveles de ECA, ni la concentración de calcio citosólico en cardio y uteromiocitos. De acuerdo con estos resultados, C. prunifolia relaja el músculo liso y disminuye el tono vascular favoreciendo la disminución de la presión arterial en ratas por medio de mecanismos vinculados con la inhibición alfa adrenérgica.

A comparative study of two clerodane diterpenes from Baccharis trimera (Less.) DC. on the influx and mobilization of intracellular calcium in rat cardiomyocytes.

Baccharis trimera (Less.) D.C. (Asteraceae) is a medicinal species native to South America and used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. The aqueous extract (AE) of the aerial parts of this species presented two mainly constituents: the ent-clerodane diterpene (Fig. 1) and the neo-clerodane diterpene (Fig. 2). The objective of this work was to study their activities on the blockade of Ca(2+)-induced contractions in KCL-depolarized rat portal vein preparations, and on the influx and mobilization of cytosolic calcium in rat cardiomyocytes by fluorescence measurements. The results showed that both the neo- and the ent-clerodane diterpenes reduced the maximal contractions induced by CaCl2, in KCl depolarized rat portal vein preparations, without modifying the EC50. The data on the concentration of cytosolic calcium ([Ca(2+)]c) showed that, while the neo-clerodane diterpene stimulates the mobilization of [Ca(2+)]c in rat cardiomyocytes, this effect was not observed with the ent-clerodane diterpene. On the other hand, the influx of calcium was not altered by the neo-clerodane diterpene, but was reduced in the presence of the ent-clerodane diterpene, indicating that this compound induces a blockade of the voltage-dependent calcium channels.

Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors.

Gyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. The serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. In the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. The kcat/KM values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. For the sh and lg PAR-2 mimetic peptides the kcat/KM values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. The kcat/KM values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-1, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. The determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects.

Quantitative changes of nicotinic receptors in the hippocampus of dystrophin-deficient mice.

Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, ß2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4ß2) and [¹²5I]-α-bungarotoxin ([¹²5I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²5I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²5I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, ß2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients.

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom.

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.

Antisecretory actions of Baccharis trimera (Less.) DC aqueous extract and isolated compounds: analysis of underlying mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models. AIM OF THE STUDY: To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera. MATERIALS AND METHODS: AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H(+), K(+)-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions. RESULTS: Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4°C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 µM) and F9 (10-300 µg/ml) decreased the basal [(14)C]-AP uptake by 50 and 60% of control (Ratio=6.2±1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 µg/ml) decreased the [(14)C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [(14)C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [(14)C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [(14)C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H(+), K(+)-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro. CONCLUSIONS: The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract.

Bradykinin-related peptides in the venom of the solitary wasp Cyphononyx fulvognathus.

Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine(6)-bradykinin (Thr(6)-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr(6)-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B(2) receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr(6)-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B(1) receptor antagonist, while the effect of both cyphokinin and Thr(6)-BK was reversed by B(2) antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms.

Natural compounds endowed with cholinergic or anticholinergic activity. Enhancement of acetylcholine release by a quaternary derivative of L-hyoscyamine.

New compounds that target nicotinic receptors (nAChRs) have been sought to correct disorders affecting cholinergic transmission in central and peripheral synapses. A quaternary derivate of l-hyoscyamine, phenthonium (Phen), was shown by our group to enhance the spontaneous acetylcholine (ACh) release without altering the nerve-induced transmitter release at the neuromuscular junction. The effect was unrelated to membrane depolarization, and was not induced by an increase of calcium influx into the nerve terminal. Phen also presented a competitive antimuscarinic activity and blocked noncompetitively the neuromuscular transmission. In this work we re-examined the mechanisms underlying the facilitatory actions of Phen on [(3)H]-ACh release in isolated ganglia of the guinea pig ileal myenteric plexus. Exposure of the preparations to Phen (10-50 microM) increased the release of [(3)H]-ACh by 81 to 68% over the basal. The effect was not affected by the ganglionic nAChR antagonist hexamethonium (1 nM) at a concentration that inhibited the increase of [(3)H]-ACh release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP, 30 microM). Association of Phen (10 microM) with DMPP potentiated the facilitatory effect of Phen. [(3)H]-ACh release was not altered by the muscarinic antagonists atropine (1 nM) or pirenzepine (1 microM). However, both antagonists inhibited the release of [(3)H]-ACh induced by either the muscarinic M1 agonist McN-343 (10 microM) or Phen (20 microM). The facilitatory effect of Phen was not altered by CdCl(2) (50 mM), but it was potentiated in the presence of tetraethylammonium (40 mM). The results indicate that the facilitatory action of Phen appears to be mediated by an increase of the inwardly rectifying potassium channels conductance probably related to the compound antimuscarinic activity.

Allosteric interaction of the anticholinergic drug [N-(4-phenyl)-phenacyl-l-hyoscyamine] (Phenthonium) with nicotinic receptors of post-ganglionic sympathetic neurons of the rat vas deferens.

Phenthonium (Phen), a quaternary analog of hyoscyamine, is a blocker of muscarinic activity and an allosteric blocker of alpha(1)2betagammaepsilon nicotinic receptors. Specifically, Phenthonium increases the spontaneous release of acetylcholine at the motor endplate without depolarizing the muscle or inhibiting cholinesterase activity. This paper compares Phenthonium's effects on sympathetic transmission and on ganglionic nicotinic receptor activation. Neurotransmitter release and twitch of the rat vas deferens were induced either by electrical stimulation or by 1,1-dimethyl-4-phenylpiperazine (DMPP) activation of nicotinic receptors. Contractions independent of transmitter release were induced by noradrenaline and adenosine 5'-triphosphate (ATP). Phenthonium inhibited transmitter release and depressed twitch without changing the responsiveness to noradrenaline or ATP. Twitch depression did not occur after K(+)-channel blockade with 4-aminopyridine (4-AP) or charybdotoxin. DMPP had a similar effect, but high concentrations induced contraction of non-stimulated organs. Incubation of Phenthonium inhibited further DMPP twitch depression and non-competitively depressed the contractile responses elicited by DMPP. Furthermore, mecamylamine, but neither methyllycaconitine nor atropine, blocked the contraction elicited by DMPP. Phenthonium and DMPP are K(+)-channel openers that primarily inhibit sympathetic transmission. Contraction induced by DMPP was probably mediated by neuronal nicotinic receptor other than the alpha7 subtype. The blockade of DMPP contractile response was unrelated to Phenthonium's antimuscarinic or K(+)-channel opening activities. Since Phenthonium's quaternary chemical structure limits its membrane diffusion, the non-competitive inhibition of DMPP excitatory responses should be linked to allosteric interaction with neuronal nicotinic receptors that putatively qualify Phenthonium as a novel modulator of cholinergic synapses.

Increased expression of acetylcholine receptors in the diaphragm muscle of MDX mice.

The absence of dystrophin in Duchenne muscular dystrophy (DMD) and in the mutant mdx mouse causes muscle degeneration and disruption of the neuromuscular junction. Based on evidence from the denervation-like properties of these muscles, we assessed the ligand-binding constants of nicotinic acetylcholine receptors (nAChRs) and the mRNA expression of individual subunits in membrane preparations of diaphragm muscles from adult (4-month-old) and aged (20-month-old) control and mdx mice. The concentration of nAChRs as determined by the maximal specific [(125)I]-alpha-bungarotoxin binding (Bmax) in the muscle membranes did not change with aging in both animal strains. When compared to age-matched control groups, the Bmax in mdx muscles was increased by 65% in adults, and by 103% in aged mice with no alteration of toxin affinity for nAChRs. Reverse-transcription polymerase chain reaction assays showed that mRNA transcripts for the nAChR alpha1, gamma, alpha7, and beta2, but not the epsilon subunits, were more abundant in mdx than in control muscles. The results indicate increased expression of extrajunctional nAChRs in the mdx diaphragm and reflect impairment of nAChR regulation in dystrophin-deficient muscles. These observations may be related to the resistance to nondepolarizing muscle relaxants and the high sensitivity to depolarizing agents reported in DMD patients.

In vivo inhibition of gastric acid secretion by the aqueous extract of Scoparia dulcis L. in rodents.

The freeze-dried aqueous extract (AE) from the aerial parts of Scoparia dulcis was tested for its effects on experimental gastric hypersecretion and ulcer in rodents. Administration of AE to animals with 4h pylorus ligature potently reduced the gastric secretion with ED(50)s of 195 mg/kg (rats) and 306 mg/kg (mice). The AE also inhibited the histamine- or bethanechol-stimulated gastric secretion in pylorus-ligated mice with similar potency suggesting inhibition of the proton pump. Bio-guided purification of the AE yielded a flavonoid-rich fraction (BuF), with a specific activity 4-8 times higher than the AE in the pylorus ligature model. BuF also inhibited the hydrolysis of ATP by H(+),K(+)-ATPase with an IC(50) of 500 microg/ml, indicating that the inhibition of gastric acid secretion of Scoparia dulcis is related to the inhibition of the proton pump. Furthermore, the AE inhibited the establishment of acute gastric lesions induced in rats by indomethacin (ED(50)=313 mg/kg, p.o.) and ethanol (ED(50)=490 mg/kg, p.o.). No influence of the AE on gastrointestinal transit allowed discarding a possible CNS or a cholinergic interaction in the inhibition of gastric secretion by the AE. Collectively, the present data pharmacologically validates the popular use of Scoparia dulcis in gastric disturbances.

Inhibition of the gastric H+,K+ -ATPase by plectrinone A, a diterpenoid isolated from Plectranthus barbatus Andrews.

This work assessed the mechanism underlying the antisecretory gastric acid effect of Plectranthus barbatus Andrews (Lamiaceae) and active constituents. Popularly known as "false-boldo", this plant is used in Brazilian folk medicine to treat gastrointestinal and hepatic ailments. The plant aqueous extract (AE) and isolated compounds were assayed in vivo in pylorus-ligated mice, and in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands and gastric H(+),K(+)-ATPase preparations. Injected into the duodenal lumen, the AE of the plant leaves (0.5 and 1.0 g/kg) decreased the volume (62 and 76%) and total acidity (23 and 50%) of gastric acid secretion in pylorus-ligated mice. Bioguided purification of the AE yielded an active fraction (IC(50)=24 microg/ml) that inhibited acid secretion in rabbit gastric glands with a potency 10 to 18 times greater than that of the originating extract, on both the basal and stimulated acid secretion by histamine (His) (1 microM) or bethanechol (100 microM). At the same concentrations the gastric H(+),K(+)-ATPase activity was also inhibited. The active constituent was chemically identified as the abietanoid dienedione plectrinone A which reduced the H(+),K(+)-ATPase activity with IC(50)=171 microM. The results indicate that inhibition of the gastric proton pump by this diterpenoid may account for the antisecretory acid effect and reputed anti ulcer activity of Plectranthus barbatus.

Calcium handling in a testosterone responsive skeletal muscle of the MDX mouse

Muscle necrosis in Duchenne muscle dystrophy (DMD) and in the mdx mouse has been related to abnormal calcium homeostasis associated with the lack of dystrophin. We have previously shown that the testosterone-dependent levator ani (LA) muscle of the mdx mouse develops a mild muscle wasting and fiber degeneration compared to the less hormone sensitive diaphragm (DIA) muscle, suggesting a protective effect of androgens. This study assessed the calcium handling mechanisms and cytosolic calcium concentration ([Ca2+]i) in LA muscles of mdx mice at critical stages of muscle disease. Muscle contractures induced by caffeine and 4-chloro-m-cresol (4-CmC), two activators of ryanodine channels, were recorded in LA and DIA muscles of prepubertal (1 month-old), adult (4 month-old) and aged (18 month-old) wild-type (wt) and mdx mice. [Ca2+]i was estimated with the fura-2 fluorescent dye in enzymatically dissociated LA muscle fibers of the same wt and mdx groups. Tetanus tension (TT) in the LA increased proportionately to the muscle weight (4 to 5-fold), but specific TT (TT/mg) did not differ among age-matched wt and mdx groups. Muscle contractures induced by caffeine (3-100 mM) or 4-CmC (0.1-5.0 mM) in the LA were greater in prepubertal than in adult and aged mice, but they did not differ among age-matched wt and mdx groups. The resting [Ca2+]i in mdx LA muscle fibers was not significantly affected at any age. Comparatively, dystrophic DIA presented reduced muscle strength in adult (40%) and aged (45%) mice, whereas the muscle responses to caffeine increased with age (63 to 82%), indicating changes in the Ca2+ handling mechanisms. The results indicated that muscle strength and calcium homeostasis in dystrophic LA muscle fibers were not significantly altered, confirming previous evidence of androgens’ beneficial effects on hormone-sensitive skeletal muscles.

Behavioral effects of a neurotoxic compound isolated from Clibadium surinamense L (Asteraceae).

Clibadium surinamense L, popularly known as cunambi, is a native plant from the Northern region of Brazil illegally used for predatory fishing. Previous results from our laboratory have demonstrated that the oral treatment of mice with the ethanolic extract (EE) of the leaves of the plant induced generalized tonic-clonic seizures followed by death within 30 min. The aims of the present paper were to characterize the convulsant effect of the hexanic extract (HE) of the stems and leaves of C. surinamense and, by bioguided purification, to identify the active principle and its mechanism of action. The leaves and stems were extracted with hexane (100 g/L) in Soxhlet for 36 h (yield of 2.4%), the solvent was evaporated and the powder dissolved in 1.5% saline/Tween 80. Male mice (30-35 g) treated with HE (22.5-360 mg/kg, p.o.) showed behavioral alterations consistent with CNS stimulation. The intensity and duration of the effect were proportional to the administered doses. The behavioral alterations, measured with a graded score of seizure severity, revealed that pretreatment with carbamazepine (30 mg/kg, i.p., 60 min) or phenytoin (50 mg/kg, i.p., 30 min) did not alter the HE convulsive effect. In contrast, phenobarbital (30 mg/kg, i.p., 60 min) or diazepam (2 mg/kg, i.p., 30 min) reduced the HE effect, increasing the ED(50) for clonic seizures from 64.4 to 89.8 mg/kg and 168.9 mg/kg, respectively. Purification of the HE in a silica gel column eluted with a hexane/ethyl acetate gradient yielded a single fraction with convulsant effect in which cunaniol acetate was identified by (1)H NMR as the main active compound. These results indicated that inhibition of GABAergic transmission by cunaniol acetate might be responsible for the convulsant effects of C. surinamense L in mice, but do not exclude a direct cunaniol action labilizing neuronal excitability.

Mild dystrophic damage in the androgen-sensitive levator ani muscle of the mdx mouse.

The time course of muscular dystrophy on the androgen-sensitive levator ani muscle was compared to that of the diaphragm of dystrophic (mdx) mice aged 1-20 months. Muscle growth, isometric contractile properties and caffeine-induced contractures were determined to assess the hormone myotrophic effect, muscle strength and sarcoplasmic reticulum function, respectively, of both control and dystrophic muscles. Histological analysis of mdx muscles showed variable fiber size, centronucleated cells, infiltration of connective tissue, and necrosis which was less severe in the levator ani than in the diaphragm muscle. Tetanic tension per unit weight in the mdx levator ani was reduced (29%) after aging, while the contraction time remained unchanged. The tetanic tension of the mdx diaphragm muscle decreased with age from 3 to 20 months (20-64%), and the relaxation time was prolonged after aging (22%). Gonadectomy of young adult mdx mice caused atrophy of the levator ani muscle, accelerated muscle wasting, reduced the tetanic force (31%), but it did not affect the contraction time and caffeine responses. The results showed that testosterone does not prevent the progress of muscle disease in the mdx levator ani, but androgen withdrawal accelerates muscle wasting suggesting a normonal beneficial effect.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles

The mechanisms underlying the muscle relaxant of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05 - 1 g/kg,i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragmand toad sartorius muscles, BB depressed the indirectly elicited muscles twitches (IC50:228 muM and 5.4 muM, respectively, at 22 degree) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reserved by neostigmine (10 muM). High concentrations of BB (170 and 340 muM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 muM)increaded the twitch/tetanus ratio (46 percent and 125 percent) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potasium conductance. BB (0.1 - 340 muM) reduced the binding of [1251]alpha- -bungarotoxin to the junctional AACh receptor of the rat diaphragm (IC50:47.7 muM, at 37 degree. At low concentrations BB (1.5 - 15 muM) induced either opening or blockade of the Ach receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the Ach recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery.

Natural probes for cholinergic sites: L-bebeerine actions on the neuromuscular transmission, the nicotinic receptor/ionic channel complex, and contraction of skeletal muscles

The mechanisms underlying the muscle relaxant of 1-bebeerine (BB), a tertiary alkaloid isolated from the roots of Chondrodendron platyphyllum, were examined in mammalian and amphibian skeletal muscles. Injections of BB (0.05 - 1 g/kg,i.p.) in rats caused a dose-related flaccid paralysis and respiratory arrest at high doses. In isolated rat diaphragmand toad sartorius muscles, BB depressed the indirectly elicited muscles twitches (IC50:228 muM and 5.4 muM, respectively, at 22 degree) and blocked the nerve-elicited muscle action potential. The neuromuscular blockade was not reserved by neostigmine (10 muM). High concentrations of BB (170 and 340 muM) caused muscle contracture unrelated to the junctional blockade, and intensified by increasing the bath temperature. Analysis of the contraction properties showed that BB (40 and 80 muM)increaded the twitch/tetanus ratio (46 percent and 125 percent) and prolonged the relaxation time; the falling phase of the directly elicited action potential in toad sartorius muscle fibers was slower probably by a decreased potasium conductance. BB (0.1 - 340 muM) reduced the binding of [1251]alpha- -bungarotoxin to the junctional AACh receptor of the rat diaphragm (IC50:47.7 muM, at 37 degree. At low concentrations BB (1.5 - 15 muM) induced either opening or blockade of the Ach receptor-ionic channel. The results showed that BB blocked noncompetitively the neuromuscular transmission through a mechanism that affects the Ach recognition site and the ionic channel properties. The alkaloid also produced muscle contracture and changed the contractile properties through its extra-junctional action at the calcium handling by the sarcoplasmic reticulum or the contractile machinery. (AU)