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Skeletal muscle expresses the extracellular cyclic AMP-adenosine pathway.

ABSTRACT: 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'-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'-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.

SUBMITTER: Chiavegatti T

PROVIDER: S-EPMC2275449 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Project description:Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate (PP(i)), and regulated generation and homeostasis of extracellular PP(i) is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular PP(i) generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular PP(i) mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate (P(i)) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular PP(i). Attenuated PP(i) accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades PP(i). Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular P(i) alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated P(i) induced decreases in the ATP release pathway(s) that supports ENPP1 activity; this resulted in markedly reduced rates of PP(i) accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) alpha-actin, SM22alpha, and calponin. Notably, addition of exogenous ATP (or PP(i) per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular PP(i) generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification.

Project description:Recently we discovered that intact kidneys release into the extracellular compartment 2',3'-cAMP (a positional isomer of 3',5'-cAMP with unknown pharmacology) and metabolize 2',3'-cAMP to 2'-AMP, 3'-AMP, and adenosine. Because adenosine inhibits growth of vascular smooth muscle cells and mesangial cells, we tested the hypothesis that extracellular 2',3'-cAMP attenuates growth of preglomerular vascular smooth muscle and mesangial cells via production of adenosine. For comparison, all of the experiments were performed with both 2',3'-cAMP and 3',5'-cAMP. In study 1, 2',3'-cAMP, 3',5'-cAMP, 5'-AMP, 3'-AMP, or 2'-AMP was incubated with cells and purines measured in the medium by mass spectrometry. Both preglomerular vascular smooth muscle and mesangial cells metabolized 3',5'-cAMP to 5'-AMP and adenosine; 5'-AMP to adenosine; 2',3'-cAMP to 2'-AMP, 3'-AMP, and adenosine; and 2'-AMP and 3'-AMP to adenosine. 3-Isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and 1,3-dipropyl-8-p-sulfophenylxanthine (ecto-phosphodiesterase inhibitor) blocked conversion of 3',5'-cAMP to 5'-AMP and adenosine, and alpha,beta-methylene-adenosine-5'-diphosphate (CD73 inhibitor) blocked conversion of 5'-AMP to adenosine. These enzyme inhibitors had little effect on metabolism of 2',3'-cAMP, 2'-AMP, or 3'-AMP. For study 2, 2',3'-cAMP and 3',5'-cAMP profoundly inhibited proliferation (thymidine incorporation and cell number) of both cell types, with 2',3'-cAMP more potent than 3',5'-cAMP. Antagonism of A(2B) receptors (MRS-1724), but not A(1) (1,3-dipropyl-8-cyclopentylxanthine), A(2A) (SCH-58261), or A(3) (VUF-5574) receptors, attenuated the growth inhibitory effects of 2',3'-cAMP and 3',5'-cAMP. Extracellular 2',3'-cAMP inhibits growth of preglomerular vascular smooth muscle and mesangial cells more profoundly than does 3',5'-cAMP. Although both cAMPs inhibit growth in part via conversion to adenosine followed by A(2B) receptor activation, their metabolism is mediated by different enzymes.

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Skeletal muscle expresses the extracellular cyclic AMP-adenosine pathway.

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