Mauricio A. Retamal

Cx43 Hemichannels and Gap Junction Channels in Astrocytes Are Regulated Oppositely by Proinflammatory Cytokines Released from Activated Microglia

Astrocytes have a role in maintaining normal neuronal functions, some of which depend on connexins, protein subunits of gap junction channels and hemichannels. Under inflammatory conditions, microglia release cytokines, including interleukin-1β and tumor necrosis factor-α, that reduce intercellular communication via gap junctions. Now, we demonstrate that either conditioned medium harvested from activated microglia or a mixture of these two cytokines enhances the cellular exchange with the extracellular milieu via Cx43 hemichannels. These changes in membrane permeability were not detected in astrocytes cultured from Cx43 knock-out mice and were abrogated by connexin hemichannel blockers, including La3+, mimetic peptides, and niflumic acid. Both the reduction in gap junctional communication and the increase in membrane permeability were mediated by a p38 mitogen-activated protein kinase-dependent pathway. However, the increase in membrane permeability, but not the gap junction inhibition, was rapidly reversed by the sulfhydryl reducing agent dithiothreitol, indicating that final regulatory mechanisms are different. Treatment with proinflammatory cytokines reduced the total and cell surface Cx43 levels, suggesting that the increase in membrane permeability was attributable to an increase in hemichannels activity. Indeed, unitary events of ∼220 pS corresponding to Cx43 hemichannels were much more frequent in astrocytes treated with microglia conditioned medium than under control conditions. Finally, the effect of cytokines enhanced the uptake and reduced the intercellular diffusion of glucose, which might explain changes in the metabolic status of astrocytes under inflammatory conditions. Accordingly, this opposite regulation may affect glucose trafficking and certainly will modify the metabolic status of astrocytes involved in brain inflammation.

Cell membrane permeabilization via connexin hemichannels in living and dying cells

Vertebrate cells that express connexins likely express connexin hemichannels (Cx HCs) at their surface. In diverse cell types, surface Cx HCs can open to serve as a diffusional exchange pathway for ions and small molecules across the cell membrane. Most cells, if not all, also express pannexins that form hemichannels and increase the cell membrane permeability but are not addressed in this review. To date, most characterizations of Cx HCs have utilized cultured cells under resting conditions have and revealed low open probability and unitary conductance close to double that of the corresponding gap junction channels. In addition, the cell membrane permeability through Cx HCs can be markedly affected within seconds to minutes by various changes in the intra and/or extracellular microenvironment (i.e., pH, pCa, redox state, transmembrane voltage and intracellular regulatory proteins) that affect levels, open probability and/or (single channel) permeability of Cx HC. Net increase or decrease in membrane permeability could result from the simultaneous interaction of different mechanisms that affect hemichannels. The permeability of Cx HCs is controlled by complex signaling cascades showing connexin, cell and cell stage dependency. Changes in membrane permeability via hemichannels can have positive consequences in some cells (mainly in healthy cells), whereas in others (mainly in cells affected by acquired and/or genetic diseases) hemichannel activation can be detrimental.

Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential

Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca2+ and enhanced by low Ca2+, positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La3+, by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeant-reducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.

FGF-1 induces ATP release from spinal astrocytes in culture and opens pannexin and connexin hemichannels

Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through “hemichannels” (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X7 receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X7 receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application, but not when given 1 h after FGF-1. We infer that ATP is initially released from vesicles and then it mediates continued release by action on P2X7 receptors and opening of HCs. These changes in HCs and gap junction channels may promote inflammation and deprive neurons of astrocyte-mediated protection in spinal cord trauma and neurodegenerative disease.

Connexin Hemichannel Composition Determines the FGF-1–induced Membrane Permeability and Free [Ca2+]i Responses

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.

Modulation of Cx46 hemichannels by nitric oxide

Gap-junction hemichannels are composed of six protein subunits (connexins). Undocked hemichannels contribute to physiological autocrine/paracrine cell signaling, including release of signaling molecules, cell-volume regulation, and glucose uptake. In addition, hemichannels may be pathologically activated by dephosphorylation and cell-membrane depolarization. Such hemichannel opening may induce and/or accelerate cell death. It has been suggested that connexin43 (Cx43) hemichannels are sensitive to redox potential changes and that one or more intracellular cysteines is/are important for this process. Cx46 is expressed in the lens, and its dysfunction induces cataract formation. It contains six cysteines in the extracellular loops, one in the fourth transmembrane helix, and two in the COOH-terminal domain. The latter may be susceptible to oxidation by nitric oxide (NO), which could be involved in cataract formation through cysteine S-nitrosylation. Here we report studies of the effects of the NO donor S-nitrosoglutathione (GSNO) on the electrical properties and fluorescent-dye permeability of wild-type Cx46 and mutant hemichannels expressed in Xenopus laevis oocytes. GSNO enhanced hemichannel voltage sensitivity, increased tail-current amplitude, and changed activation and closing kinetics in Cx46 and Cx46-CT43 (Cx46 mutant in which the COOH terminus was replaced with that of Cx43), but not in Cx46-C3A (Cx46 in which the intracellular and transmembrane helix 4 cysteines were mutated to alanine). We conclude that Cx46 hemichannels are sensitive to NO and that the NO effects are mediated by modification of one or more intracellular cysteines. However, it is unlikely that NO induces cataract formation due to the hemichannel activation, because at normal resting potential, NO had no major effects on Cx46 hemichannel permeability.

Possible involvement of different connexin43 domains in plasma membrane permeabilization induced by ischemia-reperfusion

In vitro and in vivo studies support the involvement of connexin 43-based cell-cell channels and hemichannels in cell death propagation induced by ischemia-reperfusion. In this context, open connexin hemichannels in the plasma membrane have been proposed to act as accelerators of cell death. Progress on the mechanisms underlying the cell permeabilization induced by ischemia-reperfusion reveals the involvement of several factors leading to an augmented open probability and increased number of hemichannels on the cell surface. While open probability can be increased by a reduction in extracellular concentration of divalent cations and changes in covalent modifications of connexin 43 (oxidation and phosphorylation), increase in number of hemichannels requires an elevation of the intracellular free Ca2+ concentration. Reversal of connexin 43 redox changes and membrane permeabilization can be induced by intracellular, but not extracellular, reducing agents, suggesting a cytoplasmic localization of the redox sensor(s). In agreement, hemichannels formed by connexin 45, which lacks cytoplasmic cysteines, or by connexin 43 with its C-terminal domain truncated to remove its cysteines are insensitive to reducing agents. Although further studies are required for a precise localization of the redox sensor of connexin 43 hemichannels, modulation of the redox potential is proposed as a target for the design of pharmacological tools to reduce cell death induced by ischemia-reperfusion in connexin 43-expressing cells.

Nitric oxide signaling in the retina: What have we learned in two decades?

Two decades after its first detection in the retina, nitric oxide (NO) continues to puzzle visual neuroscientists. While its liberation by photoreceptors remains controversial, recent evidence supports three subtypes of amacrine cells as main sources of NO in the inner retina. NO synthesis was shown to depend on light stimulation, and mounting evidence suggests that NO is a regulator of visual adaptation at different signal processing levels. NO modulates light responses in all retinal neuron classes, and specific ion conductances are activated by NO in rods, cones, bipolar and ganglion cells. Light-dependent gap junction coupling in the inner and outer plexiform layers is also affected by NO. The vast majority of these effects were shown to be mediated by activation of the NO receptor soluble guanylate cyclase and resultant cGMP elevation. This review analyzes the current state of knowledge on physiological NO signaling in the retina.

Adenosine triphosphate-induced peripheral nerve discharges generated from the cat petrosal ganglion in vitro

Since nucleotides have been postulated as transmitters between glomus cells and chemosensory nerve endings in the carotid body, we studied the effects of their application to the petrosal ganglion, where the perikarya of carotid (sinus) nerve are located. Cat petrosal ganglia were superfused in vitro, while electrical activities of their peripheral processes (carotid nerve and glossopharyngeal branch) were recorded simultaneously. Adenosine triphosphate (ATP) evoked dose-dependent bursts of impulses in carotid nerve, while those in glossopharyngeal branch were less intense and consistent. Adenosine monophosphate was less effective than ATP. ATP-induced carotid nerve responses presented no temporal desensitization and persisted after applying P(2Y) receptor blocker Reactive Blue 2 to the ganglion. The results indicate that ATP has an excitatory effect on the perikarya of the population of petrosal ganglion neurons projecting peripherally through the carotid nerve.

Diseases associated with leaky hemichannels.

Hemichannels (HCs) and gap junction channels (GJCs) formed by protein subunits called connexins (Cxs) are major pathways for intercellular communication. While HCs connect the intracellular compartment with the extracellular milieu, GJCs allow the interchange of molecules between cytoplasm of two contacting cells. Under physiological conditions, HCs are mostly closed, but they can open under certain stimuli allowing the release of autocrine and paracrine molecules. Moreover, some pathological conditions, like ischemia or other inflammation conditions, significantly increase HCs activity. In addition, some mutations in Cx genes associated with human diseases, such as deafness or cataracts, lead to the formation of more active HCs or “leaky HCs.” In this article we will revise cellular and molecular mechanisms underlying the appearance of leaky HCs, and the consequences of their expression in different cellular systems and animal models, in seeking a common pattern or pathological mechanism of disease.