Rosely Oliveira Godinho

Skeletal muscle expresses the extracellular cyclic AMP-adenosine pathway

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.

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-1α 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.

Increase in parasympathetic tone by pyridostigmine prevents ventricular dysfunction during the onset of heart failure

Heart failure (HF) is characterized by elevated sympathetic activity and reduced parasympathetic control of the heart. Experimental evidence suggests that the increase in parasympathetic function can be a therapeutic alternative to slow HF evolution. The parasympathetic neurotransmission can be improved by acetylcholinesterase inhibition. We investigated the long-term (4 wk) effects of the acetylcholinesterase inhibitor pyridostigmine on sympathovagal balance, cardiac remodeling, and cardiac function in the onset of HF following myocardial infarction. Myocardial infarction was elicited in adult male Wistar rats. After 4 wk of pyridostigmine administration, per os, methylatropine and propranolol were used to evaluate the cardiac sympathovagal balance. The tachycardic response caused by methylatropine was considered to be the vagal tone, whereas the bradycardic response caused by propranolol was considered to be the sympathetic tone. In conscious HF rats, pyridostigmine reduced the basal heart rate, increased vagal, and reduced sympathetic control of heart rate. Pyridostigmine reduced the myocyte diameter and collagen density of the surviving left ventricle. Pyridostigmine also increased vascular endothelial growth factor protein in the left ventricle, suggesting myocardial angiogenesis. Cardiac function was assessed by means of the pressure-volume conductance catheter system. HF rats treated with pyridostigmine exhibited a higher stroke volume, ejection fraction, cardiac output, and contractility of the left ventricle. It was demonstrated that the long-term administration of pyridostigmine started right after coronary artery ligation augmented cardiac vagal and reduced sympathetic tone, attenuating cardiac remodeling and left ventricular dysfunction during the progression of HF in rats.

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

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.

Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment

This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor‐dependent stimulation of adenylyl cyclase (AC). Isoprenaline, calcitonin gene‐related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. Conversely, the forskolin‐dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor‐induced activation of AC, was followed by an increase in the extracellular cyclic AMP. This effect was independent on PDEs, since it was obtained in the presence of 3‐isobutyl‐1‐methylxanthine (IBMX). Besides, in isoprenaline treated cells, the beta‐adrenoceptor antagonist propranolol reduced both intra‐ and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. Together our data show that direct or receptor‐dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid.

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: A1, A2A, A2B, and A3. 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.

Short‐ and long‐term influences of calcitonin gene‐related peptide on the synthesis of acetylcholinesterase in mammalian myotubes

The present study analyses the short‐ (15 min – 2 h) and long‐term (24 – 48 h) influences of calcitonin gene‐related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. To assess the effect of CGRP on AChE synthesis, myotubes were pre‐exposed to the irreversible AChE inhibitor diisopropyl fluorophosphate (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. The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 – 30 μM 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. Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. These findings 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 fibre in response to long‐term changes in neuromuscular transmission.

Contribution of the Extracellular cAMP-Adenosine Pathway to Dual Coupling of β2-Adrenoceptors to Gs and Gi Proteins in Mouse Skeletal Muscle

β2-Adrenoceptor (β2-AR) agonists increase skeletal muscle contractile force via activation of Gs protein/adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of β2-AR to Gs and Gi proteins and the influence of the β2-AR/Gs-Gi/cAMP signaling cascade on skeletal muscle contraction. Assuming that the increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of the extracellular cAMP-adenosine pathway on the β2-AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (β2-AR agonists), forskolin (AC activator), cAMP/8-bromo-cAMP, and adenosine were evaluated on isometric contractility of mouse diaphragm muscle induced by supramaximal direct electrical stimulation (0.1 Hz, 2 ms duration). Clenbuterol/fenoterol (10–1000 μM), 1 μM forskolin, and 20 μM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10 to 20% of peak levels in 30 min. The late descending phase of the β2-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX) (Gi signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of β2-AR to Gi protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β2-AR inotropic effect was inhibited by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5′-phosphodiesterase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt, indicating that β2-AR coupling to Gi is indirect and dependent on A1 receptor activation. The involvement of the extracellular cAMP-adenosine pathway in β2-AR signaling would provide a negative feedback loop that may limit stimulatory G protein-coupled receptor positive inotropism and potential deleterious effects of excessive contractile response.

Increased expression of acetylcholine receptors in the diaphragm muscle of mdx mice

The absence of dystrophin in Duchenne muscular dystrophy (DMD) and in the mutant mdx mouse causes muscle degeneration and disruption of the neuromuscular junction. Based on evidence from the denervation‐like properties of these muscles, we assessed the ligand‐binding constants of nicotinic acetylcholine receptors (nAChRs) and the mRNA expression of individual subunits in membrane preparations of diaphragm muscles from adult (4‐month‐old) and aged (20‐month‐old) control and mdx mice. The concentration of nAChRs as determined by the maximal specific [125I]‐α‐bungarotoxin binding (Bmax) in the muscle membranes did not change with aging in both animal strains. When compared to age‐matched control groups, the Bmax in mdx muscles was increased by 65% in adults, and by 103% in aged mice with no alteration of toxin affinity for nAChRs. Reverse‐transcription polymerase chain reaction assays showed that mRNA transcripts for the nAChR α1, γ, α7, and β2, but not the ϵ subunits, were more abundant in mdx than in control muscles. The results indicate increased expression of extrajunctional nAChRs in the mdx diaphragm and reflect impairment of nAChR regulation in dystrophin‐deficient muscles. These observations may be related to the resistance to nondepolarizing muscle relaxants and the high sensitivity to depolarizing agents reported in DMD patients. Muscle Nerve 38: 1585–1594, 2008

Heparin Induces Rat Aorta Relaxation via Integrin-Dependent Activation of Muscarinic M 3 Receptors

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 M3 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 (EC50: 100±10 &mgr;mol/L; Emax: 41±3%). Atropine and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide inhibitors reduced the heparin-dependent relaxation, indicating that M3 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 M3 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 M3 receptor to integrin. Taken together, these data demonstrate the participation of M3 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.