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.

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.

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.

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)