Blockade of Hemichannels Normalizes the Differentiation Fate of Myoblasts and Features of Skeletal Muscles from Dysferlin-Deficient Mice.

Dysferlinopathies are muscle dystrophies caused by mutations in the gene encoding dysferlin, a relevant protein for membrane repair and trafficking. These diseases are untreatable, possibly due to the poor knowledge of relevant molecular targets. Previously, we have shown that human myofibers from patient biopsies as well as myotubes derived from immortalized human myoblasts carrying a mutated form of dysferlin express connexin proteins, but their relevance in myoblasts fate and function remained unknown. In the present work, we found that numerous myoblasts bearing a mutated dysferlin when induced to acquire myogenic commitment express PPARγ, revealing adipogenic instead of myogenic commitment. These cell cultures presented many mononucleated cells with fat accumulation and within 48 h of differentiation formed fewer multinucleated cells. In contrast, dysferlin deficient myoblasts treated with boldine, a connexin hemichannels blocker, neither expressed PPARγ, nor accumulated fat and formed similar amount of multinucleated cells as wild type precursor cells. We recently demonstrated that myofibers of skeletal muscles from blAJ mice (an animal model of dysferlinopathies) express three connexins (Cx39, Cx43, and Cx45) that form functional hemichannels (HCs) in the sarcolemma. In symptomatic blAJ mice, we now show that eight-week treatment with a daily dose of boldine showed a progressive recovery of motor activity reaching normality. At the end of this treatment, skeletal muscles were comparable to those of wild type mice and presented normal CK activity in serum. Myofibers of boldine-treated blAJ mice also showed strong dysferlin-like immunoreactivity. These findings reveal that muscle dysfunction results from a pathophysiologic mechanism triggered by mutated dysferlin and downstream connexin hemichannels expressed de novo lead to a drastic reduction of myogenesis and favor muscle damage. Thus, boldine could represent a therapeutic opportunity to treat dysfernilopathies.

Relaxant effects of a hydroalcoholic extract of Ruta graveolens on isolated rat tracheal rings.

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

Relaxant effects of a hydroalcoholic extract of Ruta graveolens on isolated rat tracheal rings

BACKGROUND: Ruta graveolens L. (R. graveolens) is a medicinal plant employed in non-traditional medicines that has various therapeutic properties, including anthelmintic, and vasodilatory actions, among others. We evaluated the trachea-relaxant effects of hydroalcoholic extract of R. graveolens against potassium chloride (KCl)- and carbachol-induced contraction of rat tracheal rings in an isolated organ bath. RESULTS: The results showed that the airway smooth muscle contraction induced by the depolarizing agent (KCl) and cholinergic agonist (carbachol) was markedly reduced by R. graveolens in a concentration-dependent manner, with maximum values of 109 ± 7.9 % and 118 ± 2.6 %, respectively (changes in tension expressed as positive percentages of change in proportion to maximum contraction), at the concentration of 45 µg/mL (half-maximal inhibitory concentration IC50: 35.5 µg/mL and 27.8 µg/mL for KCl- and carbachol-induced contraction, respectively). Additionally, the presence of R. graveolens produced rightward parallel displacement of carbachol dose-response curves and reduced over 35 % of the maximum smooth muscle contraction. CONCLUSIONS: The hydroalcoholic extract of R. graveolens exhibited relaxant activity on rat tracheal rings. The results suggest that the trachea-relaxant effect is mediated by a non-competitive antagonistic mechanism. More detailed studies are needed to identify the target of the inhibition, and to determine more precisely the pharmacological mechanisms involved in the observed biological effects.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

Inhibition of PKC-dependent extracellular Ca2+ entry contributes to the depression of contractile activity in long-term pressure-overloaded endothelium-denuded rat aortas.

We examined the contractile responsiveness of rat thoracic aortas under pressure overload after long-term suprarenal abdominal aortic coarctation (lt-Srac). Endothelium-dependent angiotensin II (ANG II) type 2 receptor (AT2R)-mediated depression of contractions to ANG II has been reported in short-term (1 week) pressure-overloaded rat aortas. Contractility was evaluated in the aortic rings of rats subjected to lt-Srac or sham surgery (Sham) for 8 weeks. ANG I and II levels and AT2R protein expression in the aortas of lt-Srac and Sham rats were also evaluated. lt-Srac attenuated the contractions of ANG II and phenylephrine in the aortas in an endothelium-independent manner. However, lt-Srac did not influence the transient contractions induced in endothelium-denuded aortic rings by ANG II, phenylephrine, or caffeine in Ca2+-free medium or the subsequent tonic constrictions induced by the addition of Ca2+ in the absence of agonists. Thus, the contractions induced by Ca2+ release from intracellular stores and Ca2+ influx through stored-operated channels were not inhibited in the aortas of lt-Srac rats. Potassium-elicited contractions in endothelium-denuded aortic rings of lt-Srac rats remained unaltered compared with control tissues. Consequently, the contractile depression observed in aortic tissues of lt-Srac rats cannot be explained by direct inhibition of voltage-operated Ca2+ channels. Interestingly, 12-O-tetradecanoylphorbol-13-acetate-induced contractions in endothelium-denuded aortic rings of lt-Srac rats were depressed in the presence but not in the absence of extracellular Ca2+. Neither levels of angiotensins nor of AT2R were modified in the aortas after lt-Srac. The results suggest that, in rat thoracic aortas, lt-Srac selectively inhibited protein kinase C-mediated activation of contraction that is dependent on extracellular Ca2+ entry.

Some current challenges in research on air pollution and health / Desafíos actuales de la investigación sobre contaminación del aire y salud

This commentary addresses some of the diverse questions of current interest with regard to the health effects of air pollution, including exposure-response relationships, toxicity of inhaled particles and risks to health, multipollutant mixtures, traffic-related pollution, accountability research, and issues with susceptibility and vulnerability. It considers the challenges posed to researchers as they attempt to provide useful evidence for policy-makers relevant to these issues. This commentary accompanies papers giving the results from the ESCALA project, a multi-city study in Latin America that has an overall goal of providing policy-relevant results. While progress has been made in improving air quality, driven by epidemiological evidence that air pollution is adversely affecting public health, the research questions have become more subtle and challenging as levels of air pollution dropped. More research is still needed, but also novel methods and approaches to address these new questions.

Este comentario aborda algunos de los temas de interés actual en relación con los efectos de la contaminación del aire sobre la salud, tales como las relaciones exposición-respuesta, la toxicidad y riesgos para la salud de las partículas inhaladas, las mezclas de contaminantes múltiples, la contaminación relacionada con el tráfico, la investigación sobre responsabilidad, y los problemas de susceptibilidad y vulnerabilidad. Considera los retos que se presentan a los investigadores que intentan proporcionar evidencia para los responsables políticos en estas cuestiones. Este texto acompaña otros trabajos con resultados del proyecto ESCALA, un estudio en varias ciudades de América Latina que tiene como objetivo general proporcionar resultados relevantes para la política pública. Aunque ha habido avances para mejorar la calidad del aire, gracias a la evidencia epidemiológica de que la contaminación aérea está afectando negativamente a la salud pública, las preguntas de investigación se han vuelto más sutiles y difíciles a medida que los niveles de contaminación se reducen. Se necesita más investigación, pero también nuevos métodos y enfoques capaces de enfrentar estas preguntas.

Synergistic relaxing effect of the paracetamol and pyrilamine combination in isolated human myometrium.

OBJECTIVE: To evaluate the interaction type of the human uterine relaxant effect of the paracetamol-pyrilamine combination (PPC) in vitro. STUDY DESIGN: Uterine strips were contracted with KCl (60 mM) and treated with vehicle or increasing concentrations of paracetamol (100-3200 µM), pyrilamine (3.2-100 µM) or the PPC. The relaxing effects of the drugs alone and in combination were measured. Isobolographic analysis was used to determine the pharmacologic interaction type. RESULTS: Paracetamol, pyrilamine and the PPC produced a significant relaxing effect on non-pregnant human uterine strips pre-contracted with KCl (60 mM). The EC30 values for paracetamol and pyrilamine on the uterine contraction were 2391.3±595.3 µM and 14.7±1.7 µM, respectively. The derived experimental EC30 for the PPC was 401.8±129.8 µM. This value was significantly lower (p<0.05) than the theoretical EC30 expected for a purely additive interaction, which was 1203.0±297.7 µM for the PPC. The interaction index (γ) was 0.33±0.14 for PPC, being statistically different from unity. CONCLUSION: Data suggest that low doses of the PPC can interact synergistically and therefore this drug association may represent a therapeutic advantage for the clinical treatment of dysmenorreic pain.

Antinociceptive activity of atranorin in mice orofacial nociception tests.

Physicochemical characterization and antinociceptive and anti-inflammatory activities of atranorin (AT) extracted from Cladina kalbii Ahti in formalin- and capsaicin-induced orofacial pain and anti-inflammatory tests in rodents were studied. Physicochemical characterization showed that AT has the general formula C19H18O8. Male Swiss mice were pretreated with AT (100, 200, and 400 mg/kg, i.p.), morphine (3 mg/kg, i.p.), or vehicle (0.9% saline with two drops of 0.2% Tween 80) before formalin (20 microl, 2%) or capsaicin (20 microl, 2.5 microg) were injected into the right vibrissa. Our results showed that i.p. treatment with AT displayed marked inhibitory effects in different orofacial pain tests in mice. AT (400 mg/kg, i.p.) was effective in reducing the nociceptive face-rubbing behavioural response in both phases of the formalin test, which was also naloxone-sensitive. Additionally, AT produced a significant antinociceptive effect at all doses in the capsaicin test. Such results were unlikely to be provoked by motor abnormality, since AT-treated mice exhibited no performance alteration on the rota rod apparatus. AT exhibited significant anti-inflammatory activity in the acute model of inflammation (leukocyte migration to the peritoneal cavity), carrageenan- and arachidonic acid-induced hind paw edema in rats. Additionally, AT exhibited a dose-dependent antioxidant activity in vitro, as assessed by total radical-trapping antioxidant parameter and total antioxidant reactivity assays. All these findings suggest that AT might represent an important tool for the management of orofacial pain and/or inflammatory disorders.

[Evaluation of tracheal intubation conditions in morbidly obese patients: a comparison of succinylcholine and rocuronium]. / Evaluación de las condiciones de intubación traqueal en pacientes obesos mórbidos: succinilcolina frente a rocuronio.

OBJECTIVE: To compare tracheal intubation conditions in morbidly obese patients 60 seconds after administration of succinylcholine or rocuronium doses based on real weight or ideal weight. METHOD: We evaluated patients with a body mass index (BMI) of 40 kg x m(-2) or more but no other indications of difficult-intubation risk. Induction was performed under fentanyl and propofol at doses calculated based on real weight. Patients were assigned to 1 of 4 groups for tracheal intubation. Group 1 received 1 mg of succinylcholine per kilogram of ideal weight, group 2 received 1 mg of succinylcholine per kilogram of real weight, group 3 received 0.6 mg of rocuronium per kilogram of ideal weight, and group 4 received 0.6 mg of rocuronium per kilogram of real weight. Tracheal intubation was performed 60 seconds later and intubation conditions were recorded using a clinical scoring system. RESULTS: Eighty patients with a mean BMI of 47.5 kg x m(-2) were enrolled. The difficult intubation rate was 3.75%. All patients were intubated. Laryngoscopy conditions and position and movement of vocal cords were similar in all 4 groups. Reaction to cuff inflation revealed intergroup differences; group 3 presented slight limb movements, diaphragm movement, and sustained cough for more than 10 seconds. CONCLUSION: These results suggest that administration of succinylcholine calculated based on real weight or ideal weight and rocuronium based on real weight can provide clinically acceptable conditions for tracheal intubation in morbidly obese patients with no other difficult-intubation criteria.

[Interaction between mivacurium and succinylcholine from a different point of view]. / Interacción entre mivacurio y succinilcolina: vista desde otra perspectiva.

OBJECTIVES: Succinylcholine (SCH) may first be used and continue with mivacurium (MIV). MIV has been suggested as a pretreatment. Conflicting results arises from studies on SCH-MIV interaction. The following trial revisits this interaction. PATIENTS AND METHODS: The patients were intubated after randomized administration of 100 microg x Kg(-1) of mivacurium (group 1) or 1 mg x Kg(-1) of succinylcholine and, after 50% recovery, 100 microg x Kg(-1) of mivacurium (group 2). A third group received the same regimen as group 2, preceded by pretreatment with 10 microg x Kg(-1) of mivacurium. Maximum effect (MAX), onset time, the 10%-25% recovery index, and duration of effect of mivacurium were determined by electromyography. In groups 2 and 3, the corrected MAX was defined as the difference between the actual MAX effect and the residual block after administration of succinylcholine, and speed of action was defined as the ratio between MAX or corrected MAX and onset time. Data were subjected to analysis of variance and Student-Newman-Keuls and t tests for bivariate comparisons. A value of P less than 0.05 was considered significant. RESULTS: Groups 2 and 3 had significantly greater MAX effects (97% and 98%, respectively) in comparison with group 1 (93%), shorter onset times (135 and 158 seconds in groups 2 and 3 vs 279 seconds in group 1), and greater speed of action without changes in duration of effect. MAX was halved when corrected (to 47% and 49% in groups 2 and 3, respectively), and speed of action was significantly reduced (from 1.34 and 1.62 seconds/% in groups 2 and 3 respectively, to 2.69 and 3.36 seconds/%). Mivacurium pretreatment did not produce relevant clinical changes. CONCLUSIONS: When mivacurium is used before the effects of succinylcholine disappear, a residual effect is not usually taken into consideration. This study corrected MAX and calculated speed of action, demonstrating a reduction in net block and speed of action, consistent with an antagonistic action when the 2 blockers are administered sequentially.

Comienzo bifásico de acción de la succinilcolina / Biphasic onset of succinylcholine's action

Introducción: Anteriormente hemos descrito y comprobado los cambios de velocidad durante la instalación del bloqueo neuromuscular producido por el rocuronio. El propósito de la presente investigación es estudiar el período de comienzo de acción de la succinilcolina utilizando los índices diseñados en aquella oportunidad y basados en la relación entre el bloqueo fraccionado y el tiempo de instalación, que permiten determinar su velocidad y rapidez. Material y métodos: Después de su consentimiento, dos grupos iguales de pacientes (n = 20 c/u) sin contraindicación aparente para su empleo fueron intubados directamente con succinilcolina o precurarizados con rapacuronio. Por electromiografía se determinaron los tiempos para alcanzar un bloqueo del 80 por ciento, el efecto final y el máximo bloqueo. Utilizando los índices respectivos se calcularon la rapidez (seg/por ciento) y velocidad (por ciento/seg). Resultados: La precurarización con rapacuronio alargó el tiempo para alcanzar un bloqueo del 80 por ciento y el máximo efecto, disminuyendo este último. La velocidad y rapidez inicial fueron mayores que al final y menores en el grupo precurarizado. Conclusión: Los resultados obtenidos aplicando esta metodología indican que la succinilcolina, lo mismo que el rocuronio y el vecuronio, tiene un tiempo de comienzo bifásico con una fase inicial casi cuatro veces más rápida y veloz que la final. La precurarización con rapacuronio alarga el tiempo parcial y total de instalación del efecto de la succinilcolina; no modifica su carácter bifásico pero vuelve exageradamente lenta su fase final. Se comenta la posibilidad de hacer uso de la brevedad inicial de su acción para realizar una intubación traqueal precoz, ya que con el efecto periférico mostrado la relajación laríngea debe ser completa. Se hacen especulaciones sobre las supuestas causas responsables de esta peculiar manera de instalarse el bloqueo neuromuscular.

Comienzo bifásico de acción de la succinilcolina / Biphasic onset of succinylcholines action

Introducción: Anteriormente hemos descrito y comprobado los cambios de velocidad durante la instalación del bloqueo neuromuscular producido por el rocuronio. El propósito de la presente investigación es estudiar el período de comienzo de acción de la succinilcolina utilizando los índices diseñados en aquella oportunidad y basados en la relación entre el bloqueo fraccionado y el tiempo de instalación, que permiten determinar su velocidad y rapidez. Material y métodos: Después de su consentimiento, dos grupos iguales de pacientes (n = 20 c/u) sin contraindicación aparente para su empleo fueron intubados directamente con succinilcolina o precurarizados con rapacuronio. Por electromiografía se determinaron los tiempos para alcanzar un bloqueo del 80 por ciento, el efecto final y el máximo bloqueo. Utilizando los índices respectivos se calcularon la rapidez (seg/por ciento) y velocidad (por ciento/seg). Resultados: La precurarización con rapacuronio alargó el tiempo para alcanzar un bloqueo del 80 por ciento y el máximo efecto, disminuyendo este último. La velocidad y rapidez inicial fueron mayores que al final y menores en el grupo precurarizado. Conclusión: Los resultados obtenidos aplicando esta metodología indican que la succinilcolina, lo mismo que el rocuronio y el vecuronio, tiene un tiempo de comienzo bifásico con una fase inicial casi cuatro veces más rápida y veloz que la final. La precurarización con rapacuronio alarga el tiempo parcial y total de instalación del efecto de la succinilcolina; no modifica su carácter bifásico pero vuelve exageradamente lenta su fase final. Se comenta la posibilidad de hacer uso de la brevedad inicial de su acción para realizar una intubación traqueal precoz, ya que con el efecto periférico mostrado la relajación laríngea debe ser completa. Se hacen especulaciones sobre las supuestas causas responsables de esta peculiar manera de instalarse el bloqueo neuromuscular. (AU)

The anthelmintic pyrantel acts as a low efficacious agonist and an open-channel blocker of mammalian acetylcholine receptors.

Pyrantel is an anthelmintic which acts as an agonist of nicotinic receptors (AChRs) of nematodes and exerts its therapeutic effects by depolarizing their muscle membranes. Here we explore at the single-channel level the action of pyrantel at mammalian muscle AChR. AChR currents are elicited by pyrantel. However, openings do not appear in clearly identifiable clusters over a range of pyrantel concentrations (1-300 microM). The mean open time decreases as a function of concentration, indicating an additional open-channel block. Single-channel recordings in the presence of high ACh concentrations and pyrantel demonstrate that the anthelmintic acts as a high-affinity open-channel blocker. When analyzed in terms of a sequential blocking scheme, the calculated forward rate constant for the blocking process is 8x10(7) M(-1) x s(-1), the apparent dissociation constant is 8 microM at a membrane potential of -70 mV and the process is voltage dependent. Pyrantel displaces alpha-bungarotoxin binding but the concentration dependence of equilibrium binding is shifted towards higher concentrations with respect to that of ACh binding. Thus, by acting at the binding site pyrantel activates mammalian AChRs with low efficacy, and by sterical blockade of the pore, the activated channels are then rapidly inhibited.

Ocho años de experiencia en el diagnóstico y manejo de pacientes susceptibles a hipertermia maligna en el Hospital "J. M. de Los Ríos" / Eight years of experience in the management and diagnosis of the malignant hypertermia susceptible patients in the Hospital \"J. M. de Los Ríos\"

La hipertermia maligna es un síndrome farmacogenético que se presenta cuando una persona susceptible se pone en contacto con agentes anestésicos inhalatorios o relajantes musculares despolarizantes. Puede tener una evolución fulminante y su único tratamiento es el dantrolone sódico. En los momentos actuales Venezuela no cuenta ni con los medios para el diagnóstico ni con el tratamiento, por lo que el reconocimiento temprano de la enfermedad es vital. En 1984 el Hospital "J.M de los Ríos" reportó su primer caso y hasta 1992 se diagnosticaron 49 casos, basados en esta experiencia se propuso la siguiente investigación retrospectiva con el objetivo de estudiar las características clínicas, distribución por sexo, consanguinidad, tipos de anestesia, de los niños que durante ese tiempo tuvieron biopsias positivas. La edad promedio fue de 6,85 años, mientras que los varones ocuparon el 72,59 por ciento de la muestra, Un 27,50 por ciento tenían consanguinidad, el 70 por ciento recibió anestésicos desencadenantes, los signos clínicos más relevantes fueron la taquicardia y el trismo: Nuestra investigación demostró que hay un carácter hereditario, los agentes antes descritos guardan relación con el desencadenamiento de la enfermedad

Role of protein kinase C in the release of [3H]acetylcholine from myenteric plexus treated with vesamicol.

The present experiments investigated the release of [3H]acetylcholine ([3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na+-K+ ATPase inhibitor, released [3H]ACh synthesised in the presence of (-)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca2+-dependent and in the presence of (-)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (-)-vesamicol from interfering with the release of [3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [3H]ACh induced by ouabain in the presence of (-)-vesamicol depends on PKC activation.