Alexander W. Lohman

Endothelial cell expression of haemoglobin α regulates nitric oxide signalling

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe3+ state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe2+ state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.

Differentiating connexin hemichannels and pannexin channels in cellular ATP release

Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally‐designed drugs for modulation of Panx and Cx activity, respectively.

Pannexin1 Regulates α1-Adrenergic Receptor– Mediated Vasoconstriction

Rationale: The coordination of vascular smooth muscle cell constriction plays an important role in vascular function, such as regulation of blood pressure; however, the mechanism responsible for vascular smooth muscle cell communication is not clear in the resistance vasculature. Pannexins (Panx) are purine-releasing channels permeable to the vasoconstrictor ATP and thus may play a role in the coordination of vascular smooth muscle cell constriction. Objective: We investigated the role of pannexins in phenylephrine- and KCl-mediated constriction of resistance arteries. Methods and Results: Western blot, immunohistochemistry, and immunogold labeling coupled to scanning and transmission electron microscopy revealed the presence of Panx1 but not Panx2 or Panx3 in thoracodorsal resistance arteries. Functionally, the contractile response of pressurized thoracodorsal resistance arteries to phenylephrine was decreased significantly by multiple Panx inhibitors (mefloquine, probenecid, and 10Panx1), ectonucleotidase (apyrase), and purinergic receptor inhibitors (suramin and reactive blue-2). Electroporation of thoracodorsal resistance arteries with either Panx1-green fluorescent protein or Panx1 small interfering RNA showed enhanced and decreased constriction, respectively, in response to phenylephrine. Lastly, the Panx inhibitors did not alter constriction in response to KCl. This result is consistent with coimmunoprecipitation experiments from thoracodorsal resistance arteries, which suggested an association between Panx1 and &agr;1D-adrenergic receptor. Conclusions: Our data demonstrate for the first time a key role for Panx1 in resistance arteries by contributing to the coordination of vascular smooth muscle cell constriction and possibly to the regulation of blood pressure.

Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca2+-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg2+ or a Ca2+-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

Posttranslational Modifications in Connexins and Pannexins

Posttranslational modification is a common cellular process that is used by cells to ensure a particular protein function. This can happen in a variety of ways, e.g., from the addition of phosphates or sugar residues to a particular amino acid, ensuring proper protein life cycle and function. In this review, we assess the evidence for ubiquitination, glycosylation, phosphorylation, S-nitrosylation as well as other modifications in connexins and pannexin proteins. Based on the literature, we find that posttranslational modifications are an important component of connexin and pannexin regulation.

S-Nitrosylation Inhibits Pannexin 1 Channel Function

Background: Pannexin 1 contains multiple cysteine residues and is highly expressed in cell types rich in nitric oxide species. Results: S-Nitrosylation of pannexin 1 on cysteines 40 and 346 inhibits channel currents and ATP release. Conclusion: Pannexin 1 channel function can be regulated by S-nitrosylation. Significance: Our results provide the first evidence of a reversible post-translational modification on pannexin 1 to regulate channel activity. S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1C40A, Panx1C346A, and Panx1C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.

Expression of Pannexin Isoforms in the Systemic Murine Arterial Network

Aims: Pannexins (Panx) form ATP release channels and it has been proposed that they play an important role in the regulation of vascular tone. However, distribution of Panx across the arterial vasculature is not documented. Methods: We tested antibodies against Panx1, Panx2 and Panx3 on human embryonic kidney cells (which do not endogenously express Panx proteins) transfected with plasmids encoding each Panx isoform and Panx1–/– mice. Each of the Panx antibodies was found to be specific and was tested on isolated arteries using immunocytochemistry. Results: We demonstrated that Panx1 is the primary isoform detected in the arterial network. In large arteries, Panx1 is primarily in endothelial cells, whereas in small arteries and arterioles it localizes primarily to the smooth muscle cells. Panx1 was the predominant isoform expressed in coronary arteries, except in arteries less than 100 µm where Panx3 became detectable. Only Panx3 was expressed in the juxtaglomerular apparatus and cortical arterioles. The pulmonary artery and alveoli had expression of all 3 Panx isoforms. No Panx isoforms were detected at the myoendothelial junctions. Conclusion: We conclude that the specific localized expression of Panx channels throughout the vasculature points towards an important role for these channels in regulating the release of ATP throughout the arterial network.

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by a plethora of signalling processes influencing cell-to-cell interactions between the vascular endothelial cells (ECs) in post-capillary venules and circulating leukocytes. Recently, ATP-sensitive P2Y purinergic receptors have emerged as downstream regulators of EC activation in vascular inflammation. However, the mechanism(s) regulating cellular ATP release in this response remains elusive. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by TNF-α. This process involves activation of type-1 TNF receptors, recruitment of Src family kinases (SFK) and SFK-dependent phosphorylation of Panx1. Using an inducible, EC-specific Panx1 knockout mouse line, we report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation, placing Panx1 channels at the centre of cytokine crosstalk with purinergic signalling in the endothelium.

A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells

The ATP-releasing channel Panx1 is specifically involved in blood pressure regulation by adrenergic signaling. Regulating blood pressure with ATP Blood pressure is dynamically regulated to enable rapid responses to changes in position and physical or emotional stress, such as exercise or anger and fear. Many signals that activate G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) control vascular tone, including norepinephrine (also known as noradrenaline) released by the sympathetic nervous system, which increases blood pressure. Billaud et al. report that the α1 adrenoreceptor (α1AR)—but not the endothelin-1 or serotonin receptor, which are also Gαq-coupled GPCRs and stimulate vasoconstriction—is specifically coupled to activation of the ATP (adenosine 5′-triphosphate)–releasing channel pannexin1 (Panx1). Mice lacking Panx1 in smooth muscle cells were hypotensive, specifically during their active period of the day. Isolated arteries from these mice did not release ATP and contracted less in response to adrenoreceptor stimulation. Thus, ATP release through Panx1 channels specifically contributes to blood pressure regulation by the sympathetic nervous system. Both purinergic signaling through nucleotides such as ATP (adenosine 5′-triphosphate) and noradrenergic signaling through molecules such as norepinephrine regulate vascular tone and blood pressure. Pannexin1 (Panx1), which forms large-pore, ATP-releasing channels, is present in vascular smooth muscle cells in peripheral blood vessels and participates in noradrenergic responses. Using pharmacological approaches and mice conditionally lacking Panx1 in smooth muscle cells, we found that Panx1 contributed to vasoconstriction mediated by the α1 adrenoreceptor (α1AR), whereas vasoconstriction in response to serotonin or endothelin-1 was independent of Panx1. Analysis of the Panx1-deficient mice showed that Panx1 contributed to blood pressure regulation especially during the night cycle when sympathetic nervous activity is highest. Using mimetic peptides and site-directed mutagenesis, we identified a specific amino acid sequence in the Panx1 intracellular loop that is essential for activation by α1AR signaling. Collectively, these data describe a specific link between noradrenergic and purinergic signaling in blood pressure homeostasis.