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.

Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to α-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca²⁺ signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca²⁺ concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca²⁺ or a Ca²⁺-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca²⁺ increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells.

Significance: Vascular oxidative stress and endothelial cell dysfunction contribute to various human pathologies. Here, we show that, in vascular endothelial cells, a key mitochondrial antioxidant enzyme, namely thioredoxin reductase 2 (TrxR2), plays a central role in the control of vascular integrity. Its targeted loss is associated with disruption of both nitric oxide (·NO) and redox homeostasis in vivo. Impaired TrxR2 activity leads to elevated steady-state levels of peroxynitrite, reflecting the decreased bioavailability of ·NO by its O ₂ ^{· −} mediated oxidative inactivation. Thus, TrxR2 represents a central control point of peroxynitrite levels by providing reducing equivalents to mitochondrial peroxiredoxins that, in turn, catalytically reduce peroxynitrite to nitrite. The data additionally support pharmacological approaches for mitochondrial peroxynitrite detoxification under conditions of vascular oxidative stress.

A cytosolic calcium (Ca²⁺_i) increase is an important activation signal for the endothelium. We investigated whether interendothelial spreading of the Ca²⁺ signal via gap junctions (GJs) plays a role for the overall Ca²⁺_i increase in response to vasoactive agonists. In human umbilical vein endothelial cells (HUVECs), a Ca²⁺_i increase (Fura2) in response to histamine or ATP occurred initially only in about 30% of the cells (initially responding cells) reflecting the cell fraction expressing H₁ or purinergic receptors (FACS/immunohistochemistry). In the remaining adjacent cells, Ca²⁺_i increases occurred only after a delay of up to 5 s. Blockade of GJ communication (meclofenamic acid and heptanol, or H₂O₂; verified by dye injection) did not affect responses in the initially responding cells but abolished the delayed Ca²⁺_i response of the remaining adjacent cells. The resulting reduction in the global endothelial Ca²⁺_i response significantly reduced the nitric oxide synthesis (assessed as cGMP levels). Similar Ca²⁺_i results were obtained in the endothelium of freshly isolated mouse (C57BL/6) aortas stimulated with ATP. The receptor-independent Ca²⁺_i response to ionomycin occurred simultaneously in all cells, regardless of GJ inhibition. In separate experiments, inhibition of the IP₃ receptor (xestospongin-C; 40, µmol/L) but not of the ryanodine receptor (ryanodine, 250 µmol/L) reduced the spread of the Ca²⁺_i signal into adjacent cells over longer distances. The global Ca²⁺_i response of the endothelium to agonists is determined decisively by the functionality of GJs, thus establishing a new role for GJs in controlling endothelial activity and vasomotor function.

Gap junctional calcium signal propagation (transfer of calcium or a calcium releasing messenger via gap junctions) between vascular cells has been shown to be involved in the control of vascular tone. We have shown before that nitric oxide (NO) inhibits gap junctional communication in HeLa cells exclusively expressing connexin 37 (HeLa-Cx37) but not in HeLa-Cx40 or HeLa-Cx43. Here we studied the effect of NO on the gap junctional calcium signal propagation in endothelial cells which, in addition to Cx37, also express Cx40 and Cx43. Furthermore, we analyzed the impact of NO on intermuscle and on myoendothelial gap junction-dependent calcium signal propagation. Since specific effects of NO at one of these three junctional areas (interendothelial/ myoendothelial/ intermuscle) may depend on a differential membrane localization of the connexins, we also studied the distribution of the vascular connexins in small resistance arteries.

During acute inflammation, the recruitment of leukocytes from the blood stream into the inflamed tissue is a well-described mechanism encompassing the interaction of endothelial cells with leukocytes allowing leukocytes to reach the site of tissue injury or infection where they can fulfill their function such as phagocytosis. This process requires a fine-tuned regulation of a plethora of signaling cascades, which are still incompletely understood. Here, connexin 43 (Cx43) and pannexin 1 (Panx1) are known to be pivotal for the correct communication of endothelial cells with leukocytes. Pharmacological as well as genetic approaches provide evidence that endothelial Cx43-hemichannels and Panx1-channels release signaling molecules including ATP and thereby regulate vessel function and permeability as well as the recruitment of leukocytes during acute inflammation. Furthermore, Cx43 hemichannels and Panx1-channels in leukocytes release signaling molecules and can mediate the activation and function of leukocytes in an autocrine manner. The focus of the present review is to summarize the current knowledge of the role of Cx43 and Panx1 in endothelial cells and leukocytes in the vasculature during acute inflammation and to discuss relevant molecular mechanisms regulating Cx43 and Panx1 function.