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.

1 The present study analyses the short-(15 min ± 2 h) and long-term (24 ± 48 h) in¯uences of calcitonin gene-related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. 2 To assess the eect of CGRP on AChE synthesis, myotubes were pre-exposed to the irreversible AChE inhibitor diisopropyl¯uorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 ± 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP. 3 The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 ± 30 mM forskolin. In contrast, pre-treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment. 4 Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. 5 These ®ndings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time-dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP-dependent mechanism. The decreased AChE synthesis observed after long-term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down-regulation, that might function as an important adaptative mechanism of the muscle ®bre in response to long-term changes in neuromuscular transmission.

Unique Objects (UNOs) are relevant for real-world applications such as anti-counterfeiting systems. In this work, cork is demonstrated as a UNO, part of the Physical Unclonability and Disorder (PUD) system. An adequate measurement kit (illumination device) and recognition method are also devised and evaluated. Natural hills and valleys of the cork are enhanced using the illumination device and the overall robustness of the recognition application inherent to UNOs is presented. The lighting device is based on grazing light and the recognition task is based on a local feature detector and descriptor called ORB - Oriented FAST (Features from Accelerated Segment Test) and Rotated BRIEF (Binary Robust Independent Elementary Features). The performance evaluation utilizes a private cork database (1500 photos of 500 cork stoppers) and three public iris databases. In the tests carried out on the illumination device, the results clearly show the success of capturing stable/repeatable features needed for the recognition task in the cork database. This achievement is also reflected in the perfect recognition score achieved in the cork database, in the intra-distance measure μ i n t r a , which gives the notion of average noise between measures, and in the inter-distance μ i n t e r which provides hints about the randomness/uniqueness of a cork. Regarding the recognition application, its effectiveness is further tested using the iris databases. Regardless of the fact that the recognition algorithm was not designed for the iris recognition problem, the results show that the proposed approach is capable of competing with the techniques found in the literature specially designed for iris recognition. Furthermore, the evaluation shows that the three requirements that constitute a UNO (Disorder, Operability, and Unclonability) are fulfilled, thus supporting the main assertion of this work: that cork is a UNO.

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