José L. Vega

Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress

Background: The gap junction protein Cx43 is implicated in maintaining anti-oxidative defense in astrocytes. Results: In contrast to hypoxia/reoxygenation, oxidative stress induced by H2O2 triggers more astrocytic death in the absence of Cx43 channels. Conclusion: Gap junction intercellular communication is required for Cx43-mediated resistance to H2O2. Significance: An altered Cx43 phosphorylation state in response to cellular stress may be critical for Cx43-mediated cell death or recovery. Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.

Equilibrative Nucleoside Transporters in Fetal Endothelial Dysfunction in Diabetes Mellitus and Hyperglycaemia

Diabetes mellitus types 1 and 2, and gestational diabetes are characterized by abnormal D-glucose metabolism and hyperglycaemia, and induce foetal endothelial dysfunction with implications in adult life increasing the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (i.e. the L-arginine/NO signalling pathway) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pathological pregnancies, including pregnancies with pre-established diabetes mellitus or in gestational diabetes. The mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), amino acid transporters and NO synthases (NOS). Modulation of ENTs and NOS expression and activity in endothelium involves several signalling molecules, including protein kinase C, mitogen-activated protein kinases p42 and p44, calcium and phosphatidyl inositol 3 kinase. Elevated extracellular D-glucose and diabetes alters human endothelial function. However, information regarding modulation the transport capacity as well as expression of ENTs is limited. This review focuses on the effect of diabetes mellitus and gestational diabetes, and hyperglycaemia on the reported mechanisms described for transcriptional and post-transcriptional regulation of ENTs, and the potential consequences for foetal endothelial function in these pathologies. Recent available information regarding functional consequences of an abnormal environment on the functionality of the endothelium from microvasculature of the human placenta is mentioned. The available information is scarce, but it could contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in this pathological conditions, emphasizing the key role of this type of epithelium in fetal-placental function and the normal foetal development and growth.

Modulation of gap junction channels and hemichannels by growth factors

Gap junction hemichannels and cell-cell channels have roles in coordinating numerous cellular processes, due to their permeability to extra and intracellular signaling molecules. Another mechanism of cellular coordination is provided by a vast array of growth factors that interact with relatively selective cell membrane receptors. These receptors can affect cellular transduction pathways, including alteration of intracellular concentration of free Ca(2+) and free radicals and activation of protein kinases or phosphatases. Connexin and pannexin based channels constitute recently described targets of growth factor signal transduction pathways, but little is known regarding the effects of growth factor signaling on pannexin based channels. The effects of growth factors on these two channel types seem to depend on the cell type, cell stage and connexin and pannexin isoform expressed. The functional state of hemichannels and gap junction channels are affected in opposite directions by FGF-1 via protein kinase-dependent mechanisms. These changes are largely explained by channels insertion in or withdrawal from the cell membrane, but changes in open probability might also occur due to changes in phosphorylation and redox state of channel subunits. The functional consequence of variation in cell-cell communication via these membrane channels is implicated in disease as well as normal cellular responses.

TGF-β1 Inhibits Expression and Activity of hENT1 in a Nitric Oxide-dependent Manner in Human Umbilical Vein Endothelium

AIMS We studied whether transforming growth factor beta1 (TGF-beta1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-beta1 in this cell type. TGF-beta1 increases NO synthesis via activation of TGF-beta receptor type II (TbetaRII), and NO inhibits hENT1 expression and activity in HUVECs. METHODS AND RESULTS HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-beta1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-beta1 receptor type II (TTbetaRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-beta1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-beta1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-beta1 was ineffective in cells expressing TTbetaRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-beta1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385. CONCLUSION hENT1 is down-regulated by activation of TbetaRII by TGF-beta1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-beta1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.

D-glucose stimulation of L-arginine transport and nitric oxide synthesis results from activation of mitogen-activated protein kinases p42/44 and Smad2 requiring functional type II TGF-β receptors in human umbilical vein endothelium

Elevated extracellular D‐glucose increases transforming growth factor β1 (TGF‐β1) release from human umbilical vein endothelium (HUVEC). TGF‐β1, via TGF‐β receptors I (TβRI) and TβRII, activates Smad2 and mitogen‐activated protein kinases p44 and p42 (p42/44mapk). We studied whether D‐glucose‐stimulation of L‐arginine transport and nitric oxide synthesis involves TGF‐β1 in primary cultures of HUVEC. TGF‐β1 release was higher (∼1.6‐fold) in 25 mM (high) compared with 5 mM (normal) D‐glucose. TGF‐β1 increases L‐arginine transport (half maximal effect ∼1.6 ng/ml) in normal D‐glucose, but did not alter high D‐glucose‐increased L‐arginine transport. TGF‐β1 and high D‐glucose increased hCAT‐1 mRNA expression (∼8‐fold) and maximal transport velocity (Vmax), L‐[3H]citrulline formation from L‐[3H]arginine (index of NO synthesis) and endothelial NO synthase (eNOS) protein abundance, but did not alter eNOS phosphorylation. TGF‐β1 and high D‐glucose increased p42/44mapk and Smad2 phosphorylation, an effect blocked by PD‐98059 (MEK1/2 inhibitor). However, TGF‐β1 and high D‐glucose were ineffective in cells expressing a truncated, negative dominant TβRII. High D‐glucose increases L‐arginine transport and eNOS expression following TβRII activation by TGF‐β1 involving p42/44mapk and Smad2 in HUVEC. Thus, TGF‐β1 could play a crucial role under conditions of hyperglycemia, such as gestational diabetes mellitus, which is associated with fetal endothelial dysfunction. J. Cell. Physiol. 212:626–632, 2007.

Role of Gap Junctions and Hemichannels in Parasitic Infections

In vertebrates, connexins (Cxs) and pannexins (Panxs) are proteins that form gap junction channels and/or hemichannels located at cell-cell interfaces and cell surface, respectively. Similar channel types are formed by innexins in invertebrate cells. These channels serve as pathways for cellular communication that coordinate diverse physiologic processes. However, it is known that many acquired and inherited diseases deregulate Cx and/or Panx channels, condition that frequently worsens the pathological state of vertebrates. Recent evidences suggest that Cx and/or Panx hemichannels play a relevant role in bacterial and viral infections. Nonetheless, little is known about the role of Cx- and Panx-based channels in parasitic infections of vertebrates. In this review, available data on changes in Cx and gap junction channel changes induced by parasitic infections are summarized. Additionally, we describe recent findings that suggest possible roles of hemichannels in parasitic infections. Finally, the possibility of new therapeutic designs based on hemichannel blokers is presented.

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.

Trypanosoma cruzi Infection Induces Pannexin-1 Channel Opening in Cardiac Myocytes

Trypanosoma cruzi, the etiological agent of Chagas diseases, invades the cardiac tissue causing acute myocarditis and heart electrical disturbances. In T. cruzi invasion, the parasite induces [Ca2+]i transients in the host cells, an essential phenomenon for invasion. To date, knowledge on the mechanism that elicits transients of [Ca2+]i during the infection of cardiac myocytes has not been fully characterized. Pannexin1 (Panx1) channel are poorly selective channels found in all vertebrates that serve as a pathway for ATP release. In this article, we demonstrate that T. cruzi infection results in the opening of Panx1 channels in cardiac myocytes. We show that pharmacological blockade of Panx1 channels inhibits T. cruzi-induced [Ca2+]i transients and invasion in cardiac myocytes. Our results indicate that opening of Panx1 channels are required for T. cruzi invasion in cardiac myocytes, and we propose that targeting Panx1 channel could provide new potential therapeutic approaches to treat Chagas disease.

Mutations of Cx43 that affect B cell spreading in response to BCR signaling

ABSTRACT The gap junction (GJ) protein connexin 43 (Cx43) is both necessary and sufficient for B cell receptor (BCR)-mediated cell spreading. To address how Cx43 mediates this effect, we blocked its function genetically, by expressing mutants of Cx43, and pharmacologically, by using chemical inhibitors. While various point mutations of Cx43 inhibited B cell spreading, treatment with channel blocking drugs did not, suggesting that this response was independent of channel function. The critical region of Cx43 appears to be the cytoplasmic carboxyl-terminal (CT) domain, which has previously been shown to be important for B cell spreading. Consistent with this, mutations of either tyrosine 247 or 265 found in the CT were sufficient to inhibit spreading. Thus Cx43 may influence B cell spreading by mechanisms requiring protein binding to, or modification of, these sites in the CT tail.