Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5 0 -AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5 0 -AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications:Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine.
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.
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The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10 6 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 α-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 µm; N = 213), the diameter of ectopic fibers (59.1 µm; 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.
Timely detection, staging, and treatment initiation are pertinent to controlling HIV infection. CD4+ cell-based point-of-care (POC) devices offer the potential to rapidly stage patients, and decide on initiating treatment, but a comparative evaluation of their performance has not yet been performed. With this in mind, we conducted a systematic review and metaanalyses. For the period January 2000 to April 2014, 19 databases were systematically searched, 6619 citations retrieved, and 25 articles selected. Diagnostic performance was compared across devices (i.e., PIMA, CyFlow, miniPOC, MBioCD4 System) and across specimens (i.e., capillary blood vs. venous blood). A Bayesian approach was used to meta-analyze the data. The primary outcome, the Bland-Altman (BA) mean bias (which represents agreement between cell counts from POC device and flow cytometry), was analyzed with a Bayesian hierarchical normal model. We performed a headto-head comparison of two POC devices including the PIMA and PointCareNOW CD4. PIMA appears to perform better vs. PointCareNOW with venous samples (BA mean bias: -9.5 cells/μL; 95% CrI: -37.71 to 18.27, vs. 139.3 cells/μL; 95% CrI: -0.85 to 267.4, mean difference = 148.8, 95% CrI: 11.8, 285.8); importantly, PIMA performed well when used with capillary samples (BA mean bias: 2.2 cells/μL; 95% CrI: -19.32 to 23.6). Sufficient data were available to allow pooling of sensitivity and specificity data only at the 350 cells/μL cutoff. For PIMA device sensitivity 91.6 (84.7-95.5) and specificity was 94.8 (90.1-97.3), respectively. There were not sufficient data to allow comparisons between any other devices. PIMA device was comparable to flow cytometry. The estimated differences between the CD4+ cell counts of the device and the reference was small and best estimated in capillary blood specimens. As the evidence stands, the PointCareNOW device will need to improve prior to widespread use and more data on MBio and MiniPOC are needed. Findings inform implementation of PIMA and improvements in other CD4 POC device prior to recommending widespread use.
Timely detection, staging, and treatment initiation are pertinent to controlling HIV infection. CD4+ cell-based point-of-care (POC) devices offer the potential to rapidly stage patients, and decide on initiating treatment, but a comparative evaluation of their performance has not yet been performed. With this in mind, we conducted a systematic review and metaanalyses. For the period January 2000 to April 2014, 19 databases were systematically searched, 6619 citations retrieved, and 25 articles selected. Diagnostic performance was compared across devices (i.e., PIMA, CyFlow, miniPOC, MBioCD4 System) and across specimens (i.e., capillary blood vs. venous blood). A Bayesian approach was used to meta-analyze the data. The primary outcome, the Bland-Altman (BA) mean bias (which represents agreement between cell counts from POC device and flow cytometry), was analyzed with a Bayesian hierarchical normal model. We performed a headto-head comparison of two POC devices including the PIMA and PointCareNOW CD4. PIMA appears to perform better vs. PointCareNOW with venous samples (BA mean bias: -9.5 cells/μL; 95% CrI: -37.71 to 18.27, vs. 139.3 cells/μL; 95% CrI: -0.85 to 267.4, mean difference = 148.8, 95% CrI: 11.8, 285.8); importantly, PIMA performed well when used with capillary samples (BA mean bias: 2.2 cells/μL; 95% CrI: -19.32 to 23.6). Sufficient data were available to allow pooling of sensitivity and specificity data only at the 350 cells/μL cutoff. For PIMA device sensitivity 91.6 (84.7-95.5) and specificity was 94.8 (90.1-97.3), respectively. There were not sufficient data to allow comparisons between any other devices. PIMA device was comparable to flow cytometry. The estimated differences between the CD4+ cell counts of the device and the reference was small and best estimated in capillary blood specimens. As the evidence stands, the PointCareNOW device will need to improve prior to widespread use and more data on MBio and MiniPOC are needed. Findings inform implementation of PIMA and improvements in other CD4 POC device prior to recommending widespread use.
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