Although we have recently demonstrated that plasma catecholamines induce antiproteolytic effects on skeletal muscle (Graça FA, Gonçalves DAP, Silveira WA, Lira EC, Chaves VE, Zanon NM, Garófalo MAR, Kettelhut IC, Navegantes LCC. Am J Physiol Endocrinol Metab. 305: E1483-E1494, 2013), the role of the muscle sympathetic innervation and, more specifically, norepinephrine (NE) in regulating the ubiquitin (Ub)-proteasome system (UPS) remains unknown. Based on previous findings that chemical sympathectomy acutely reduces UPS activity, we hypothesized that muscle NE depletion induces adrenergic supersensitivity in rat skeletal muscles. We report that surgical sympathetic denervation (SDEN), a condition in which only muscle NE from both hindlimbs is depleted, transiently reduced the overall proteolysis and the UPS activity (∼25%) in both soleus and extensor digitorum longus muscles. This antiproteolytic response was accompanied by increased activity of adenylyl cyclase (112%), levels of cyclic adenosine monophosphate (cAMP; 191%), and the serine phosphorylation of cAMP response element-binding protein (32%). In extensor digitorum longus from normal rats, NE (10(-4) M) in vitro increased the levels of cAMP (115%) and the serine phosphorylation of both cAMP response element-binding protein (2.7-fold) and forkhead box class O1 transcription factor. Similar effects were observed in C2C12 cells incubated with forskolin (10 μM). In parallel, NE significantly reduced the basal UPS (21%) activity and the mRNA levels of atrophy-related Ub-ligases. Similar responses were observed in isolated muscles exposed to 6-BNZ-cAMP (500 μM), a specific PKA activator. The phosphorylation levels of Akt were not altered by SDEN, NE, forskolin or 6-BNZ-cAMP. Our results demonstrate that SDEN induces muscle adrenergic supersensitivity for cAMP leading to the suppression of UPS, and that the suppressive effects of NE on UPS activity and expression of Ub-ligases can be mediated by the activation of cAMP/PKA signaling, with the inhibition of forkhead box class O1 transcription factor.

Abstract-Previous reports have shown that heparin may promote human hypotension and vascular relaxation by elevation of NO levels through unclear mechanisms. We hypothesized that endothelial muscarinic M 3 receptor activation mediates the heparin-induced vasodilation of rat aortic rings. The experiments were carried out using unfractionated heparin extracted from bovine intestinal mucosa, which elicited an endothelium and NO-dependent relaxation of aortic segments with maximal potency and efficacy (EC 50 : 100Ϯ10 mol/L; E max : 41Ϯ3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M 3 muscarinic receptor is involved in this phenomenon. However, no direct binding of heparin to muscarinic receptors was observed.More importantly, studies performed using the arginine-glycine-aspartic acid peptide and 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine, an Src family inhibitor, reduced by 51% and 73% the heparin-dependent relaxation, respectively, suggesting the coupling of heparin and M 3 receptor through extracellular matrix molecules and integrin. Furthermore, unfractionated heparin induced activation of focal adhesion protein kinase, Src, and paxillin. Finally, fluorescence resonance energy transfer approach confirmed the interaction of the M 3 receptor to integrin. Taken together, these data demonstrate the participation of M 3 receptor and integrin in heparin-dependent relaxation of vascular smooth muscle. These results provide new insights into the molecular mechanism and potential pharmacological action of heparin in vascular physiology. (Hypertension. 2010;56:713-721.)Key Words: heparin Ⅲ muscarinic receptors Ⅲ M 3 receptor Ⅲ integrin Ⅲ smooth muscle relaxation Ⅲ aorta T he successful use of heparin in the treatment of thromboembolic diseases has been associated with numerous pharmacological actions of this complex polysaccharide. Alterations in the blood vessel and associated cells, such as platelet hyperactivity, inflammatory processes, endothelial dysfunction, and angiogenesis, lead to the development of diseases related to clot formation, which can be efficiently modulated by heparin and its derivatives. [1][2][3] Heparin is a glycosaminoglycan composed of repeating 13 4-linked ␣-D-glucosamine, mainly N-sulfated, and uronic acid, either ␤-D-glucuronic acid or ␣-L-iduronic acid; also, O-sulfation occurs at different positions of the disaccharide units. 4,5 Although heparin usually acts as an anticoagulant, 6 other biological activities have also been described, including antiviral, 7 antibacterial, 8 and antithrombotic effects. This antithrombotic action has been related, at least in part, to the increased production of a peculiar heparan sulfate proteoglycan produced by the endothelium. 9 -11 In addition, heparin modulates the function of many proteins, for example, the myosin from skeletal muscle, 12 the inositol 1,4,5-trisphosphate receptor inhibition, 13 and the activation of Na ϩ /Ca 2ϩ ...

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