Ana Carolina de Carvalho Correia

Malignant Hyperthermia: Clinical and Molecular Aspects

CONTENT Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder that affects genetically predisposed individuals. It manifests in susceptible individuals in response to exposure to Inhalant anesthetics, depolarizing muscle relaxants or extreme physical activity in hot environments. During exposure to these triggering agents, there is a rapid and sustained increase of myoplasmic calcium (Ca(2+)) concentration induced by hyperactivation of ryanodine receptor of skeletal muscle (RyR1), causing a profound change in Ca(2+) homeostasis, featuring a hypermetabolic state. RyR1, Ca(2+) release channels of sarcoplasmic reticulum, is the primary locus for MH susceptibility. Several mutations in the gene encoding the protein RyR1 have been identified; however, other genes may be involved. Actually, the standard method for diagnosing MH susceptibility is the muscle contracture test for exposure to halothane-caffeine (CHCT) and the only treatment is the use of dantrolene. However, with advances in molecular genetics, a full understanding of the disease etiology may be provided, favoring the development of an accurate diagnosis, less invasive, with DNA test, and also will provide the development of new therapeutic strategies for treatment of MH. Thus, this brief review aims to integrate molecular and clinical aspects of MH, gathering input for a better understanding of this channelopathy.

Spasmolytic Effect of Caulerpine Involves Blockade of Ca2+ Influx on Guinea Pig Ileum

In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC50 = 7.0 ± 1.9 × 10−5 M), histamine (IC50 = 1.3 ± 0.3 × 10−4 M) and serotonin (IC50 = 8.0 ± 1.4 × 10−5 M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD′2 = 4.48 ± 0.08), shifting the curves to the right with Emax reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC50 = 9.0 ± 0.9 × 10−5 M) and carbachol (EC50 = 4.6 ± 0.7 × 10−5 M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca2+ influx through voltage-gated calcium channels (CaV), since caulerpine slightly inhibited the CaCl2-induced contractions in depolarizing medium without Ca2+, shifting the curves to the right and with Emax reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca2+ influx through CaV. However, other mechanisms are not discarded.

Essential oil from the leaves of Xylopia langsdorfiana (Annonaceae) as a possible spasmolytic agent

Xylopia langsdorfiana A. St.-Hil. &Tul. (Annonaceae) is popularly known in the northeast of Brazil as ‘pimenteira da terra’, and an essential oil (XL-OE) was extracted from its leaves. Since Xylopia species are cited in folk medicine and diterpenes from X. langsdorfiana have spasmolytic activity, this study aimed to investigate a possible spasmolytic action of XL-OE on smooth muscle models. XL-OE (243 and 729 μg/mL) showed low pharmacologic efficacy on guinea pig trachea and rat aorta and uterus. However, in guinea pig ileum, XL-OE (27–729 μg/mL) inhibited carbachol or histamine-induced phasic contractions (1 μM) in a significant and concentration-dependent manner. In addition, XL-OE (81 μg/mL) reduced fluorescence intensity in ileal myocytes stimulated by histamine, indicating a decrease in cytosolic calcium concentration, which could explain the spasmolytic activity. Thus, XL-OE proved to be a promising natural product to be used in gastrointestinal diseases acting by modulating the cytosolic calcium concentration.

Flavonoid galetin 3,6-dimethyl ether attenuates guinea pig ileum contraction through K(+) channel activation and decrease in cytosolic calcium concentration.

Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10(-7) M) than by KCl (EC50=2.6±0.5×10(-6) M) or carbachol (EC50=1.8±0.4×10(-6) M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca(2+)-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10(-6) M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10(-6) M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10(-7) and 1.6±0.3×10(-7) M, respectively), ruling out the involvement of small and big conductance Ca(2+)-activated K(+) channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10(-6) and 1.5±0.5×10(-6) M, respectively), indicating the involvement of voltage- and ATP-sensitive K(+) channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca(2+) concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca(2+) levels.

Mechanisms underlying the relaxant effect of Galetin 3,6- dimethyl ether, from Piptadenia stipulacea (Benth.) Ducke, on guinea-pig trachea.

Abstract Galetin 3,6-dimethyl ether (FGAL), a flavonoid from the aerial parts of Piptadenia stipulacea (Benth.) Ducke, was found to exert a relaxant effect on carbachol (CCh)-pre-contracted guinea-pig trachea. Based on cumulative concentration-response curves to CCh, FGAL antagonized muscarinic receptors pseudo-irreversibly and noncompetitively, since it inhibited and shifted these curves towards higher concentrations in a nonparallel manner. In addition, FGAL was more potent in relaxing contractions induced by 18 mM as compared to 60 mM KCl (pD2 = 5:50 ±0:36 and 4.80 ±0.07, respectively), indicating the participation of K+ channels. In the presence of 10 mM tetraethylammonium (TEA+) chloride, a nonselective K+ channel blocker, the relaxant potency of FGAL was reduced (from pD2 = 5:12 ±0:07 to 4.87 ±0.02). Among several selective blockers of K+ channel subtypes, only apamin, an SKCa (small-conductance Ca2+-activated K+ channels) blocker, attenuated the relaxant potency of FGAL (pD2 = 4:85±0:06), suggesting SKCa activation. FGAL was equipotent in relaxing trachea contracted by 60 mM KCl (pD2 =4:80 ±0:07) or 10-6 M CCh (pD2 = 5:02 ±0:07), suggesting CaV (voltage-gated calcium channel), but not ROCs (receptor-operated calcium channels) participation. Furthermore, aminophylline-induced relaxation (pD2 = 4:12 ±0:06) was potentiated around 4-fold (pD2 = 4:80 ±0:44) in the presence of FGAL. Moreover, forskolininduced relaxation (pD2 = 6:51 ±0:06) was potentiated around 2.5-fold (pD2 = 6:90 ±0:05) by FGAL. Conversely, sodium nitroprusside-induced relaxation was unaffected, indicating that the AC/cAMP/PKA pathway, but not the NO pathway, may be modulated by the flavonoid. These results suggest that, in guinea-pig trachea, FGAL induces relaxation by pseudo-irreversible noncompetitive antagonism on muscarinic receptors, modulation of K+ and Ca2+ channels, as well as activation of the AC/cAMP/PKA pathway.

Hipertermia maligna: aspectos moleculares e clínicos

CONTENIDO: La hipertermia maligna (HM) es una enfermedad farmacogenetica potencialmente letal que afecta a individuos geneticamente predispuestos. Se manifiesta en los individuos susceptibles en respuesta a la exposicion a los anestesicos inhalatorios, relajantes musculares despolarizantes o actividad fisica extrema en ambientes calientes. Durante la exposicion a esos agentes desencadenantes, existe un aumento rapido y constante de la concentracion de calcio mioplasmatico (Ca2+) inducido por la hiperactivacion de los receptores de rianodina (RYR1) del musculo esqueletico, causando una alteracion profunda en la homeostasa de Ca2+, y caracterizando un estado hipermetabolico. RYR1, canales de liberacion de Ca2+ del reticulo sarcoplasmatico, es la principal region de susceptibilidad a la HM. Varias mutaciones en el gen que codifica la proteina RYR1 han sido identificadas, pero otros genes pueden estar involucrados tambien. Actualmente, el metodo estandar para el diagnostico de la sensibilidad a la HM es el test de contractura muscular para la exposicion al halotano-cafeina (CHCT) y el unico tratamiento es el uso de dantroleno. Sin embargo, con los avances en el campo de la genetica molecular, un pleno entendimiento de la etiologia de la enfermedad puede ser suministrado, favoreciendo asi el desarrollo de un diagnostico preciso, menos invasivo, con el test de ADN, y tambien proporcionar el desarrollo de nuevas estrategias terapeuticas para el tratamiento de la HM. Por eso, esta breve revision intenta integrar los aspectos clinicos y moleculares de la HM, reuniendo informaciones para lograr una mejor comprension de esa canalopatia.

Relaxant effect of Ent-7α-hydroxytrachyloban-18-oic acid, a trachylobane diterpene from Xylopia langsdorfiana A. St-Hil. & Tul., on tracheal smooth muscle

Ent-7α-hydroxytrachyloban-18-oic acid, a trachylobane diterpene from Xylopia langsdorfiana, has previously been shown to relax the guinea-pig trachea in a concentration-dependent manner. In this study we aimed to elucidate the mechanisms underlying this action and so contribute to the discovery of natural products with therapeutic potential. A possible interaction between diterpene and the Ca2+-calmodulin complex was eliminated as chlorpromazine (10-6 M), a calmodulin inhibitor, did not significantly alter the diterpene-induced relaxation (pD2 = 4.38 ± 0.07 and 4.25 ± 0.07; mean ± S.E.M., n=5). Trachylobane-318 showed a higher relaxant potency when the trachea was contracted by 18 mM KCl than it did with 60 mM KCl (pD2 = 4.90 ± 0.25 and 3.88 ± 0.01, n=5), suggesting the possible activation of K+ channels. This was confirmed, as in the presence of 10 mM TEA+ (a non-selective K+ channel blocker), diterpene relaxation potency was significantly reduced (pD2 = 4.38 ± 0.07 to 4.01 ± 0.06, n=5). Furthermore, K+ channel subtypes KATP, KV, SKCa and BKCa seem to be modulated positively by trachylobane-318 (pD2 = 3.91 ± 0.003, 4.00 ± 0.06, 3.45 ± 0.14 and 3.80 ± 0.05, n=5) but not the Kir subtype channel (pD2 = 4.15 ± 0.10, n=5). Cyclic nucleotides were not involved as the relaxation due to aminophylline (pD2 = 4.27 ± 0.09, n=5) was not altered in the presence of 3 × 10-5 M trachylobane-318 (pD2 = 4.46 ± 0.08, n=5). Thus, at a functional level, trachylobane-318 seems to relax the guinea-pig trachea by positive modulation of K+ channels, particularly the KATP, KV, SKCa and BKCa subtypes.