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.     

Carvedilol reverses hyperthermia and attenuates rhabdomyolysis induced by 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) in an animal model

OBJECTIVE: Hyperthermia is a potentially fatal manifestation of severe 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) intoxication. No proven effective drug treatment exists to reverse this potentially life-threatening hyperthermia, likely because mechanisms of peripheral thermogenesis are poorly understood. Using a rat model of MDMA hyperthermia, we evaluated the acute drug-induced changes in plasma catecholamines and used these results as a basis for the selection of drugs that could potentially reverse this hyperthermia. DESIGN: Prospective, controlled, randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male, adult Sprague-Dawley rats. INTERVENTIONS: Based on MDMA-induced changes in plasma catecholamine levels, rats were subjected to the nonselective (beta1 + beta2) adrenergic receptor antagonists propranolol or nadolol or the alpha1- + beta1,2,3-adrenergic receptor antagonist carvedilol before or after a thermogenic challenge of MDMA. MEASUREMENT AND MAIN RESULTS: Plasma catecholamines levels 30 mins after MDMA (40 mg/kg, subcutaneously) were determined by high-pressure liquid chromatography and electrochemical detection. Core temperature was measured by a rectal probe attached to a thermocouple. Four hours after MDMA treatment, blood was drawn and serum creatine kinase levels were measured as a marker of rhabdomyolysis using a Vitros analyzer. MDMA induced a 35-fold increase in norepinephrine levels, a 20-fold increase in epinephrine, and a 2.4-fold increase in dopamine levels. Propranolol (10 mg/kg, intraperitoneally) or nadolol (10 mg/kg, intraperitoneally) administered 30 mins before MDMA had no effect on the thermogenic response. In contrast, carvedilol (5 mg/kg, intraperitoneally) administered 15 mins before or after MDMA prevented this hyperthermic response. Moreover, when administered 1 hr after MDMA, carvedilol completely reversed established hyperthermia and significantly attenuated subsequent MDMA-induced creatine kinase release. CONCLUSION: These data show that alpha1 and beta3-adrenergic receptors may contribute to the mediation of MDMA-induced hyperthermia and that drugs targeting these receptors, such as carvedilol, warrant further investigation as novel therapies for the treatment of psychostimulant-induced hyperthermia and its sequelae.     

Timbós: ichthyotoxic plants used by brazilian indians

The pharmacology of serjanosides, active principles isolated from the fishpoison plant Serjania lethalis St. Hil, a Sapindaceae, was investigated by comparing their actions in fishes and mammals with those of rotenone and certain saponins. The ichthyocid activity of the serjanosides was 2.5 times greater than that of the crude plant extract, approximately 10 times lower than the activity of rotenone, but from 10 to 50 times greater than the activity of the other saponins. When injected in mammals, the serjanosides induced deep prostration, dyspnea, cyanosis, ectopic heart beats, cardiovascular failure and respiratory arrest. These effects, leading to death that was not prevented by artificial respiration, indicated several mechanisms for the toxic action of the serjanosides. In vitro studies with these substances have shown that membrane depolarization and muscle contracture were probably due to unspecific surface actions. Rotenone, under the same experimental conditions induced hypotension, bradycardia and respiratory arrest. Death was prevented by artificial respiration. Ectopic foci, membrane depolarization, contractures and neuromuscular block were not observed after rotenone. Apparently, death from rotenone poisoning was a consequence of respiratory failure of central origin.

The serjanosides are rather potent fish poison saponins. Mammals, however, are apparently insensitive to the same specific action since other toxic effects induced by those substances in rats and mice were also observed by employing saponins devoid of fish-killing activity.     

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.     

Cholinesterase activity in developing rat skeletal muscles

The influence of testosterone on the cholinesterase (ChE) activity of a target muscle (levator ani, LA), and of a nontarget muscle (extensor digitorum longus, EDL) was studied in newborn, prepubertal, and adult male rats. From birth to puberty (45 to 60 days of age) the ChE activity was similar in both muscles, either considering the total enzyme activity per muscle or per gram of tissue. After 60 days of age the weight and the ChE activity of the LA muscle increased abruptly, but the enzyme activity per gram of tissue remained constant to 105 days of age. Development of the EDL muscle was proportional to body weight during the entire experimental period. The total ChE activity of the muscle did not increase proportionately, but the ChE activity per gram of tissue decayed with the age of the rat. The activity of the specific cholinesterase, acetylcholinesterase (AChE), which is involved in the cholinergic transmission of the neuromuscular junction, attained maximal values in both muscles at puberty. From puberty to adulthood, AChE activity constituted the major form of ChE in the LA and EDL muscles. We conclude that testosterone stimulates the growth of the LA muscle and the increase of its AChE activity, but it decreases the nonspecific cholinesterase (BuChE) activity. Except for the latter, these effects are probably related to a general increase in protein synthesis in the target muscle fibers of the LA, but not of the EDL, induced by testosterone.     

Rapalogs induce non-apoptotic,autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.