Mibefradil improves skeletal muscle mass, function and structure in obese mice / 南方医科大学学报

OBJECTIVE@#To observe the effect of mibefradil on skeletal muscle mass, function and structure in obese mice.@*METHODS@#Fifteen 6-week-old C57BL/6 mice were randomized equally into normal diet group (control group), high-fat diet (HFD) group and high-fat diet +mibefradil intervention group (HFD +Mibe group). The grip strength of the mice was measured using an electronic grip strength meter, and the muscle content of the hindlimb was analyzed by X-ray absorptiometry (DXA). Triglyceride (TG) and total cholesterol (TC) levels of the mice were measured with GPO-PAP method. The cross-sectional area of the muscle fibers was observed with HE staining. The changes in the level of autophagy in the muscles were detected by Western blotting and immunofluorescence assay, and the activation of the Akt/mTOR signaling pathway was detected with Western blotting.@*RESULTS@#Compared with those in the control group, the mice in HFD group had a significantly greater body weight, lower relative grip strength, smaller average cross sectional area of the muscle fibers, and a lower hindlimb muscle ratio (P < 0.05). Immunofluorescence assay revealed a homogenous distribution of LC3 emitting light red fluorescence in the cytoplasm in the muscle cells in HFD group and HFD+Mibe group, while bright spots of red fluorescence were detected in HFD group. In HFD group, the muscular tissues of the mice showed an increased expression level of LC3 II protein with lowered expressions of p62 protein and phosphorylated AKT and mTOR (P < 0.05). Mibefradil treatment significantly reduced body weight of the mice, lowered the expression level of p62 protein, and increased forelimb grip strength, hindlimb muscle ratio, cross-sectional area of the muscle fibers, and the expression levels of LC3 II protein and phosphorylated AKT and mTOR (P < 0.05).@*CONCLUSION@#Mibefradil treatment can moderate high-fat diet-induced weight gain and improve muscle mass and function in obese mice possibly by activating AKT/mTOR signal pathway to improve lipid metabolism and inhibit obesityinduced autophagy.

Mibefradil reduces blood glucose concentration in db/db mice

OBJECTIVE: Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca2+]i) is a common defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanism in the defective pancreatic β-cell function in type 2 diabetes. T-type Ca2+ channel antagonists lower blood glucose in type 2 diabetes, but the mechanism remains unknown. METHODS: We examined the effect of the Ca2+ channel antagonist mibefradil on blood glucose in male db/db mice and phenotypically normal heterozygous mice by intraperitoneal injection. RESULTS: Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from 430.92±20.46 mg/dl to 285.20±5.74 mg/dl in three days. The hypoglycemic effect of mibefradil was reproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca2+ channels, but not by the specific L-type Ca2+ channel blocker nicardipine. Mibefradil did not show such hypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake were significantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis, immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-type Ca2+ channel α-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those from heterozygous animals. CONCLUSIONS: Collectively, these results suggest that T-type Ca2+ channels are potential therapeutic targets for antidiabetic drugs. .

Intracisternal Administration of Voltage Dependent Calcium Channel Blockers Attenuates Orofacial Inflammatory Nociceptive Behavior in Rats

Voltage dependent calcium channel (VDCC), one of the most important regulator of Ca2+ concentration in neuron, play an essential role in the central processing of nociceptive information. The present study investigated the antinociceptive effects of L, T or N type VDCC blockers on the formalin-induced orofacial inflammatory pain. Experiments were carried out on adult male Sprague-Dawley rats weighing 220-280 g. Anesthetized rats were individually fixed on a stereotaxic frame and a polyethylene (PE) tube was implanted for intracisternal injection. After 72 hours, 5% formalin (50 microL) was applied subcutaneously to the vibrissa pad and nociceptive scratching behavior was recorded for nine successive 5 min intervals. VDCC blockers were administered intracisternally 20 minutes prior to subcutaneous injection of formalin into the orofacial area. The intracisternal administration of 350 or 700 microg of verapamil, a blocker of L type VDCC, significantly decreased the number of scratches and duration in the behavioral responses produced by formalin injection. Intracisternal administration of 75 or 150 microg of mibefradil, a T type VDCC blocker, or 11 or 22 microg of cilnidipine, a N type VDCC blocker, also produced significant suppression of the number of scratches and duration of scratching in the first and second phase. Neither intracisternal administration of all VDCC blockers nor vehicle did not affect in motor dysfunction. The present results suggest that central VDCCs play an important role in orofacial nociceptive transmission and a targeted inhibition of the VDCCs is a potentially important treatment approach for inflammatory pain originating in the orofacial area.

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca2+ entry through the voltage-dependent Ca2+ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca2+ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca2+ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca2+]i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca2+ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 micrometer) of nifedipine, a L-type Ca2+ channel blocker, produced a decrease in the amplitude of the spontaneous Ca2+ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (alpha1G, alpha1H) of the T-type Ca2+ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca2+ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca2+ transients consistent with the reduction of the frequency. It is concluded that T-type Ca2+ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.

Antihyperalgesic Effects of Ethosuximide and Mibefradil, T-type Voltage Activated Calcium Channel Blockers, in a Rat Model of Postoperative Pain / 대한통증학회지

BACKGROUND: A correlation between a T-type voltage activated calcium channel (VACC) and pain mechanism has not yet been established. The purpose of this study is to find out the effect of ethosuximide and mibefradil, representative selective T-type VACC blockers on postoperative pain using an incisional pain model of rats. METHODS: After performing a plantar incision, rats were stabilized on plastic mesh for 2 hours. Then, the rats were injected with ethosuximide or mibefradil, intraperitoneally and intrathecally. The level of withdrawal threshold to the von Frey filament near the incision site was determined and the dose response curves were obtained. RESULTS: After an intraperitoneal ethosuximide or mibefradil injection, the dose-response curve showed a dose-dependent increase of the threshold in a withdrawal reaction. After an intrathecal injection of ethosuximide, the threshold of a withdrawal reaction to mechanical stimulation increased and the increase was dose-dependent. After an intrathecal injection of mibefradil, no change occurred in either the threshold of a withdrawal reaction to mechanical stimulation or a dose-response curve. CONCLUSIONS: The T-type VACC blockers in a rat model of postoperative pain showed the antihyperalgesic effect. This effect might be due to blockade of T-type VACC, which was distributed in the peripheral nociceptors or at the supraspinal level. Further studies of the effect of T-type VACC on a pain transmission mechanism at the spinal cord level would be needed.

Effects of calcium channel blocker, mibefradil, and potassium channel opener, pinacidil, on the contractile response of mid-pregnant goat myometrium.

The present study was undertaken to investigate the in vitro influence of mibefradil, a calcium channel blocker, and pinacidil, a potassium channel opener, on pregnant goat myometrial spontaneous rhythmic contractility and contractions induced with the agonist, oxytocin. Longitudinal strips from the distal region of uterus, collected from goats at midgestation, were mounted in an organ bath for recording isometric contractions. Mibefradil (10(-8)-10(-4) M) or pinacidil (10(-10)-10(-4) M), added cumulatively to the bath at an increment of 1 log unit, caused concentration-dependent inhibition of the spontaneous rhythmic contractions of isolated uterine strips. The rhythmic contraction was, respectively, abolished at 100 and 10 microM concentrations of mibefradil and pinacidil. In a concentration-dependent manner, mibefradil (1 and 10 microM) antagonized the contractions elicited with oxytocin (10(-5)-10(-2) IU). Pretreatment of uterine strips with glibenclamide (10 microM), a selective KATP channel blocker, caused a rightward shift of the concentration-response curve of pinacidil with a concomitant decrease in its pD2 value. Pinacidil (0.3, 1 and 3 microM), in a concentration-related manner, antagonized the oxytocin (10(-5)-10(-2) IU)-induced contractile response. The inhibition of spontaneous rhythmic contractions and antagonism of oxytocin-induced contraction by mibefradil in the pregnant goat myometrium may be related to the antagonism of voltage-dependent Ca2+ channels, while by pinacidil suggests that KATP channel could be a therapeutic target for tocolysis.

Blood pressure response to antihypertensive agents related to baseline blood pressure.

Prevalence of white-coat hypertension varies approximately 20 per cent among mild hypertensives. When white-coat hypertensives are prescribed antihypertensive medication, there is usually a decrease in clinic blood pressure (BP), but little or no change in 24 hours blood pressure (ABPM). The objective of the study was to test the hypothesis that efficacy of medication therapy for hypertension is identical in any grading of severity of baseline blood pressure. The authors retrospectively analysed ABPM data from mild to moderate hypertensive patients. Efficacy in decreasing blood pressure by antihypertensives has linear relation to baseline blood pressure. Response to antihypertensive agents in white-coat hypertension is minimal but a significant effect still persists and the possibility of hypotensive adverse events from medication in the case cannot be overlooked.

Vasodilatadores en arterias radiales humanas aisladas / Vasodilators in isolated human radial arteries

Este trabajo se diseñó para evaluar la efectividad de diversos vasodilatadores aplicados tópicamente para prevenir la hiperreactividad de las arterias radiales (AR) implantadas como bypass aortocoronario. De cada uno de los remanentes de AR provenientes de 20 pacientes operados se obtuvieron 4 anillos que se incubaron por 30 minutos en condiciones control (n = 20) o en presencia de 30 µM de diltiazem (DILT, n = 6), mibefradil (MIBE, n = 4) o mezcla de verapamil + nitroglicerina (VP-NTG, n = 6). La subsiguiente exposición a CIK 80 mM (en ausencia de vasodilatadores) provocó una contracción sostenida en los anillos control, que fue atenuada en un 35 ñ 9 por ciento por DILT, 48 ñ 13 por ciento por VP-NTG y 69 ñ 20 por ciento por MIBE (p < 0.05). La preincubación con vasodilatadores provocó también la disminución de frecuencia e intensidad de contracciones rítmicas espontáneas de la AR. En anillos almacenados en frío por 24 hs y luego reestimulados con CIK 80 mM el efecto depresor fue aun evidente: DILT 53 ñ 6 por ciento, VP-NTG 46 ñ 14 por ciento y MIBE 61 ñ 9 por ciento (p < 0.05). El efecto del MIBE fue más intenso y persistente que el de DILT o VP-NTG, aún a concentraciones que provocan un igual efecto depresor inicial. Se concluye que la exposición a vasodilatadores durante un período equivalente a la duración de la preparación de la AR a implantar produce una atenuación de la reactividad arterial que proporcionaría una protección adicional contra el espasmo durante el postoperatorio inmediato.

Observation of the Incidence of Acrosome Reaction in Human Spermatozoa Treated with Mibefradil as a T-type Ca2+i Channels Inhibitor / 대한불임학회지

OBJECTIVE : The sperm acrosome reaction is a Ca2+ -dependent exocytotic event that is triggered by adhesion to the mammalian egg's zona pellucida. Previous studies suggested a role of Ca2+ channels in acrosome reactions. This study was conducted to investigate the T-type calcium channel is operated in acrosome reaction of human spermatozoa. METHOD : Human semen samples were obtained from healthy donors with nomal criteria. The spermatozoa were divided into five groups: Group 1 were non-treated as a control; Group 2 where spermatozoa were exposed to 5 micrometer Ca2+ A23187 (Ca2+i); Group 3 where spermatozoa were exposed 5 micrometer Ca2+i and mibefradil; Group 4 where spermatozoa were exposed 5 micrometer Ca2+i and nifedipine, and Group 5 where spermatozoa were treated with 5 micrometer Ca2+i and both of mibefradil and nifedipine. Spermatozoa in all groups were retrieved after incubation for 15 and 30 minutes at 37degrees C. After staining with PSA-FITC, fluorescence was observed under a fluorescence microscope, and AR was evaluated on a total >100 spermatozoa/side. RESULT AND CONCLUSION : We observed on acrosome reaction inhibition rate in human spermatozoa the various of concentration of mibefradil, nifedipine. Maximum response was noted with 1.0 micrometer mibefradil and the decrease of acrosome reaction inhibition rate 45%. Nifedipine in acrosome reaction inhibition rate was only about 25%. The Ca2+i-induced AR of spermatozoa was significantly suppressed by mibefradil. Incidence of the suppression was depending on concentration of mibefradil. Results from the present study suggest that the human spermatozoa possess T-type channel. The observation that reversible inhibitor of T channels in male germ cells provides a new mechanism of contraceptive action.