Comparison of the efficacy of biodegradable and non-biodegradable scintillation liquids on the counting of tritium- and [14C]-labeled compounds

The widespread use of ³H and 14C in research has generated a large volume of waste mixed with scintillation liquid, requiring an effective control and appropriate storage of liquid radioactive waste. In the present study, we compared the efficacy of three commercially available scintillation liquids, Optiphase HiSafe 3, Ultima-Gold AB (biodegradable) and Insta-Gel-XF (non-biodegradable), in terms of [14C]-glucose and [³H]-thymidine counting efficiency. We also analyzed the effect of the relative amount of water (1.6 to 50%), radioisotope concentration (0.1 to 100 nCi/ml), pH (2 to 10) and color of the solutions (samples containing 0.1 to 1.0 mg/ml of Trypan blue) on the counting efficiency in the presence of these scintillation liquids. There were few significant differences in the efficiency of 14C and ³H counting obtained with biodegradable or non-biodegradable scintillation liquids. However, there was an 83 and 94% reduction in the efficiency of 14C and ³H counting, respectively, in samples colored with 1 mg/ml Trypan blue, but not with 0.1 mg/ml, independent of the scintillation liquid used. Considering the low cost of biodegradable scintillation cocktails and their efficacy, these results show that traditional hazardous scintillation fluids may be replaced with the new safe biodegradable fluids without impairment of ³H and 14C counting efficiency. The use of biodegradable scintillation cocktails minimizes both human and environmental exposure to hazardous solvents. In addition, some biodegradable scintillation liquids can be 40% less expensive than the traditional hazardous cocktails.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Unidade de Proteção RadiológicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de FarmacologiaUNIFESP, EPM, Unidade de Proteção RadiológicaUNIFESP, EPM, Depto. de FarmacologiaSciEL

Nitric Oxide in Skeletal Muscle: Role on Mitochondrial Biogenesis and Function

Nitric oxide (NO) has been implicated in several cellular processes as a signaling molecule and also as a source of reactive nitrogen species (RNS). NO is produced by three isoenzymes called nitric oxide synthases (NOS), all present in skeletal muscle. While neuronal NOS (nNOS) and endothelial NOS (eNOS) are isoforms constitutively expressed, inducible NOS (iNOS) is mainly expressed during inflammatory responses. Recent studies have demonstrated that NO is also involved in the mitochondrial biogenesis pathway, having PGC-1 alpha as the main signaling molecule. Increased NO synthesis has been demonstrated in the sarcolemma of skeletal muscle fiber and NO can also reversibly inhibit cytochrome c oxidase (Complex IV of the respiratory chain). Investigation on cultured skeletal myotubes treated with NO donors, NO precursors or NOS inhibitors have also showed a bimodal effect of NO that depends on the concentration used. the present review will discuss the new insights on NO roles on mitochondrial biogenesis and function in skeletal muscle. We will also focus on potential therapeutic strategies based on NO precursors or analogs to treat patients with myopathies and mitochondrial deficiency.Fundacao de Amparo a Pesquisa de São PauloConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Paulista Sch Med, Dept Neurol & Neurosurg, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Paulista Sch Med, Dept Pharmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Paulista Sch Med, Dept Neurol & Neurosurg, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Paulista Sch Med, Dept Pharmacol, BR-04044020 São Paulo, BrazilWeb of Scienc

New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway

G protein coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases, which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A(1), A(2)A, A(2B), and A(3). Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response.PapespConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Farmacol, Disciplina Farmacol Celular, BR-04044020 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Farmacol, Disciplina Farmacol Celular, BR-04044020 São Paulo, SP, BrazilPapesp: 2011/01519-4Papesp: 2012/20148-0Web of Scienc

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (106 cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodaminelabeled alpha-bungarotoxin. the development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. the presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 +/- 1.0 mu m; N = 213), the diameter of ectopic fibers (59.1 mu m; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.UNIFESP, EPM, Dept Farmacol, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Neurol & Neurocirurgia, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Farmacol, BR-04044020 São Paulo, BrazilUNIFESP, EPM, Dept Neurol & Neurocirurgia, BR-04044020 São Paulo, BrazilWeb of Scienc

The lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes

Bergantin LB, Figueiredo LB, Godinho RO. the lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes. J Appl Physiol 111: 1710-1718, 2011. First published September 15, 2011; doi:10.1152/japplphysiol.00586.2011.-The molecular regulation of skeletal muscle proteolysis and the pharmacological screening of anticatabolic drugs have been addressed by measuring tyrosine release from prepubertal rat skeletal muscles, which are thin enough to allow adequate in vitro diffusion of oxygen and substrates. However, the use of muscle at accelerated prepubertal growth has limited the analysis of adult muscle proteolysis or that associated with aging and neurodegenerative diseases. Here we established the adult rat lumbrical muscle (4/hindpaw; 8/rat) as a new in situ experimental model for dynamic measurement of skeletal muscle proteolysis. By incubating lumbrical muscles attached to their individual metatarsal bones in Tyrode solution, we showed that the muscle proteolysis rate of adult and aged rats (3-4 to 24 mo old) is 45-25% of that in prepubertal animals (1 mo old), which makes questionable the usual extrapolation of proteolysis from prepubertal to adult/senile muscles. While acute mechanical injury or 1- to 7-day denervation increased tyrosine release from adult lumbrical muscle by up to 60%, it was reduced by 20-28% after 2-h incubation with beta-adrenoceptor agonists, forskolin or phosphodiesterase inhibitor IBMX. Using inhibitors of 26S-proteasome (MG132), lysosome (methylamine), or calpain (E64/leupeptin) systems, we showed that ubiquitin-proteasome is accountable for 40-50% of total lumbrical proteolysis of adult, middle-aged, and aged rats. in conclusion, the lumbrical model allows the analysis of muscle proteolysis rate from prepubertal to senile rats. By permitting eight simultaneous matched measurements per rat, the new model improves similar protocols performed in paired extensor digitorum longus (EDL) muscles from prepubertal rats, optimizing the pharmacological screening of drugs for anticatabolic purposes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Div Cellular Pharmacol, Dept Pharmacol, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Div Cellular Pharmacol, Dept Pharmacol, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 05/59006-1FAPESP: 08/55988-2CNPq: 304602/2008-6Web of Scienc

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo

Pires-Oliveira M, Maragno AL, Parreiras-E-Silva LT, Chiavegatti T, Gomes MD, Godinho RO. Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo. J Appl Physiol 108: 266-273, 2010. First published November 19, 2009; doi:10.1152/japplphysiol.00490.2009.-Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. in the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. in addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Dept Biochem & Immunol, Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04044020 São Paulo, BrazilFAPESP: 05/59006-1FAPESP: 2006/58629-8Web of Scienc

Regulação androgênica da acetilcolinesterase de músculos esqueléticos do rato

BV UNIFESP: Teses e dissertaçõe

Influências hormonais e nervosas na atividade acetilcolinesterásica da musculatura esquelética do rato

BV UNIFESP: Teses e dissertaçõe

GLOBULAR AND ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE IN THE RAT LEVATOR ANI MUSCLE

The endplate (+EP) and non-endplate (-EP) distribution of molecular forms of acetylcholinesterase (AChE) was compared in the dimorphic levator ani and diaphragm muscles from adult male rats. Enzyme activity was measured by the thiocholine method and AChE forms were separated on the basis of solubility in sodium phosphate buffer of different ionic strength. For the dimorphic levator ani muscle, total AChE activity was 342.6 +/- 18.9 nmol ASCh hydrolyzed min-1 muscle-1, 90% of which was globular and predominated in the -EP region (78%). The asymmetric forms were almost exclusively detected in the +EP region (9%). In diaphragm muscle, total AChE activity was 176.7 +/- 11.0 units; 66% was mainly globular and located in the -EP region (56%); the asymmetric forms (34%) were either in -EP (11%) or +EP (23%) regions. Thus, a greater proportion of globular form was present in the dimorphic levator ani muscle than in diaphragm muscle. In view of the control exerted by testosterone on dimorphic muscles, it is suggested that the greater synthesis of the globular form in the levator ani occurs under the trophic influence of testosterone.ESCOLA PAULISTA MED SCH,DEPT FARMACOL,DISCIPLINA FARMACOL CELULAR,RUA 3 MAIO 100,BR-04044 SAO PAULO,BRAZILESCOLA PAULISTA MED SCH,DEPT FARMACOL,DISCIPLINA FARMACOL CELULAR,RUA 3 MAIO 100,BR-04044 SAO PAULO,BRAZILWeb of Scienc