Luis A. Cea

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium.

L‐Arginine transport and nitric oxide (NO) synthesis (L‐arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D‐glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D‐glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2‐, but lower hENT1‐mediated transport in normal D‐glucose. Insulin increased hENT2 protein abundance in normal or high D‐glucose, but reduced hENT1 protein abundance in normal D‐glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D‐glucose. Insulin effect on hENT1 mRNA expression in normal D‐glucose was blocked by NG‐nitro‐L‐arginine methyl ester (L‐NAME, NO synthase inhibitor) and mimicked by S‐nitroso‐N‐acetyl‐L,D‐penicillamine (SNAP, NO donor). L‐NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO‐independent mechanism. These findings could be important in hyperglycemia‐associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development. J. Cell. Physiol. 209: 826–835, 2006.

Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis.

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca2+ levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43fl/flCx45fl/fl:Myo-Cre mice was significantly better than that of control mdx Cx43fl/flCx45fl/fl mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects.

Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs.

Dysferlin function in skeletal muscle: Possible pathological mechanisms and therapeutical targets in dysferlinopathies

Mutations in the dysferlin gene are linked to a group of muscular dystrophies known as dysferlinopathies. These myopathies are characterized by progressive atrophy. Studies in muscle tissue from dysferlinopathy patients or dysferlin-deficient mice point out its importance in membrane repair. However, expression of dysferlin homologous proteins that restore sarcolemma repair function in dysferlinopathy animal models fail to arrest muscle wasting, therefore suggesting that dysferlin plays other critical roles in muscle function. In the present review, we discuss dysferlin functions in the skeletal muscle, as well as pathological mechanisms related to dysferlin mutations. Particular focus is presented related the effect of dysferlin on cell membrane related function, which affect its repair, vesicle trafficking, as well as Ca(2+) homeostasis. Such mechanisms could provide accessible targets for pharmacological therapies.

The absence of dysferlin induces the expression of functional connexin-based hemichannels in human myotubes

BackgroundMutations in the gene encoding for dysferlin cause recessive autosomal muscular dystrophies called dysferlinopathies. These mutations induce several alterations in skeletal muscles, including, inflammation, increased membrane permeability and cell death. Despite the fact that the etiology of dysferlinopathies is known, the mechanism that explains the aforementioned alterations is still elusive. Therefore, we have now evaluated the potential involvement of connexin based hemichannels in the pathophysiology of dysferlinopathies.ResultsHuman deltoid muscle biopsies of 5 Chilean dysferlinopathy patients exhibited the presence of muscular connexins (Cx40.1, Cx43 and Cx45). The presence of these connexins was also observed in human myotubes derived from immortalized myoblasts derived from other patients with mutated forms of dysferlin. In addition to the aforementioned connexins, these myotubes expressed functional connexin based hemichannels, evaluated by ethidium uptake assays, as opposed to myotubes obtained from a normal human muscle cell line, RCMH. This response was reproduced in a knock-down model of dysferlin, by treating RCMH cell line with small hairpin RNA specific for dysferlin (RCMH-sh Dysferlin). Also, the presence of P2X7 receptor and the transient receptor potential channel, TRPV2, another Ca2+ permeable channels, was detected in the myotubes expressing mutated dysferlin, and an elevated resting intracellular Ca2+ level was found in the latter myotubes, which was in turn reduced to control levels in the presence of the molecule D4, a selective Cx HCs inhibitor.ConclusionsThe data suggests that dysferlin deficiency, caused by mutation or downregulation of dysferlin, promotes the expression of Cx HCs. Then, the de novo expression Cx HC causes a dysregulation of intracellular free Ca2+ levels, which could underlie muscular damage associated to dysferlin mutations. This mechanism could constitute a potential therapeutical target in dysferlinopathies.

Dexamethasone-induced muscular atrophy is mediated by functional expression of connexin-based hemichannels.

Long-term treatment with high glucocorticoid doses induces skeletal muscle atrophy. However, the molecular mechanism of such atrophy remains unclear. We evaluated the possible involvement of connexin-based hemichannels (Cx HCs) in muscle atrophy induced by dexamethasone (DEX), a synthetic glucocorticoid, on control (Cx43(fl/fl)Cx45(fl/fl)) and Cx43/Cx45 expression-deficient (Cx43(fl/fl)Cx45(fl/fl):Myo-Cre) skeletal myofibers. Myofibers of Cx43(fl/fl)Cx45(fl/fl) mice treated with DEX (5h) expressed several proteins that form non-selective membrane channels (Cx39, Cx43, Cx45, Panx1, P2X7 receptor and TRPV2). After 5h DEX treatment in vivo, myofibers of Cx43(fl/fl)Cx45(fl/fl) mice showed Evans blue uptake, which was absent in myofibers of Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice. Similar results were obtained in vitro using ethidium as an HC permeability probe, and DEX-induced dye uptake in control myofibers was blocked by P2X7 receptor inhibitors. DEX also induced a significant increase in basal intracellular Ca(2+) signal and a reduction in resting membrane potential in Cx43(fl/fl)Cx45(fl/fl) myofibers, changes that were not elicited by myofibers deficient in Cx43/Cx45 expression. Moreover, treatment with DEX induced NFκB activation and increased mRNA levels of TNF-α in control but not in Cx43/Cx45 expression-deficient myofibers. Finally, a prolonged DEX treatment (7days) increased atrogin-1 and Murf-1 and reduced the cross sectional area of Cx43(fl/fl)Cx45(fl/fl) myofibers, but these parameters remained unaffected in Cx43(fl/fl)Cx45(fl/fl):Myo-Cre myofibers. Therefore, DEX-induced expression of Cx43 and Cx45 plays a critical role in early sarcolemma changes that lead to atrophy. Consequently, this side effect of chronic glucocorticoid treatment might be avoided by co-administration with a Cx HC blocker.

On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells

Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.

Sepsis induced channelopathy in skeletal muscles is associated with expression of non selective channels

ABSTRACT Skeletal muscles (∼50% of the body weight) are affected during acute and late sepsis and represent one sepsis associate organ dysfunction. Cell membrane changes have been proposed to result from a channelopathy of yet unknown cause associated with mitochondrial dysfunction and muscle atrophy. We hypothesize that the channelopathy might be explained at least in part by the expression of non-selective channels. Here, this possibility was studied in a characterized mice model of late sepsis with evident skeletal muscle atrophy induced by cecal ligation and puncture (CLP). At day seven after CLP, skeletal myofibers were found to present de novo expression (immunofluorescence) of connexins 39, 43, and 45 and P2X7 receptor whereas pannexin1 did not show significant changes. These changes were associated with increased sarcolemma permeability (∼4 fold higher dye uptake assay), ∼25% elevated in intracellular free-Ca2+ concentration (FURA-2), activation of protein degradation via ubiquitin proteasome pathway (Murf and Atrogin 1 reactivity), moderate reduction in oxygen consumption not explained by changes in levels of relevant respiratory proteins, ∼3 fold decreased mitochondrial membrane potential (MitoTracker Red CMXRos) and ∼4 fold increased mitochondrial superoxide production (MitoSox). Since connexin hemichannels and P2X7 receptors are permeable to ions and small molecules, it is likely that they are main protagonists in the channelopathy by reducing the electrochemical gradient across the cell membrane resulting in detrimental metabolic changes and muscular atrophy.