A quantized mechanism for activation of pannexin channels

Chiu, Yu‐Hsin; Jin, Xueyao; Medina, Christopher B.; Leonhardt, Susan A.; Kiessling, Volker; Bennett, Brad C.; Shu, Shaofang; Tamm, Lukas K.; Yeager, Mark; Ravichandran, Kodi S.; Bayliss, Douglas A.

doi:10.1038/ncomms14324

Cited by 127 publications

(196 citation statements)

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“…Cells were stimulated with control buffer (Ctrl), 5 μmol L −1 phenylephrine (PE), 10 μmol L −1 forskolin (FSK) and 1 μmol L −1 ionomycin (Iono). 27,31 In order to further assess Panx1 activity as a 'pore', 9,33 we monitored ethidium bromide (EtBr) uptake after adrenergic stimulation. C, Quantification of ATP release is in mature adipocytes is shown as a difference between the basal extracellular ATP (before stimulation) and the peak release with the indicated agonist.…”

Section: Mature Adipocytes Express Panx1 That Forms Functional Poresmentioning

confidence: 99%

“…3 There are two main types of ATP release mechanisms. 7,9,10 Reversibility of Panx1 acti vation and ATP-induced inhibition of Panx1 would mediate physiological functions. 4 Another type of ATP release mechanism includes plasma membrane ion channels and transporters such as pannexin-1, connexin hemichannels, cystic fibrosis trans membrane conductance regulator (CFTR), cell volumesensitive anion channels and maxi-anion channels and even the P2X7 receptors.…”

Section: Introductionmentioning

confidence: 99%

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Pannexin‐1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

2019

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Aim: Extracellular ATP signalling is involved in many physiological and patho physiological processes in several tissues, including adipose tissue. Adipocytes have crucial functions in lipid and glucose metabolism and they express purinergic recep tors. However, the sources of extracellular ATP in adipose tissue are not well charac terized. In the present study, we investigated the mechanism and regulation of ATP release in white adipocytes, and evaluated the role of extracellular ATP as potential autocrine and paracrine signal. Methods: Online ATP release was monitored in C3H10T1/2 cells and freshly iso lated murine adipocytes. The ATP release mechanism and its regulation were tested in cells exposed to adrenergic agonists, insulin, glucose load and pharmacological inhibitors. Cell metabolism was monitored using Seahorse respirometry and expres sion analysis of pannexin-1 was performed on pre-and mature adipocytes. The ATP signalling was evaluated in live cell imaging (Ca 2+ , pore formation), glycerol release and its effect on macrophages was tested in co-culture and migration assays. Results: Here, we show that upon adrenergic stimulation white murine adipocytes release ATP through the pannexin-1 pore that is regulated by a cAMP-PKA-depend ent pathway. The ATP release correlates with increased cell metabolism and is sensi tive to glucose. Extracellular ATP induces Ca 2+ signalling and lipolysis in adipocytes and promotes macrophage migration. Importantly, ATP release is markedly inhibited by insulin, which operates via the activation of phosphodiesterase 3. Conclusions: Our findings reveal an insulin-pannexin-1-purinergic signalling cross talk in adipose tissue and we propose that deregulation of this signalling may contri bute to adipose tissue inflammation and type 2 diabetes. K E Y W O R D Sinflammation, obesity, P2X7 receptor, phosphodiesterase 3, purinergic signalling, type 2 diabetes This article was first published as a preprint: Tozzi M, Hansen JB, Novak I (2018) Pannexin1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration. bioRxiv. https ://doi. org/10.1101/380469 2 | RESULTS | Adrenergically stimulated adipocytes release ATP through Panx1To determine whether adipocytes are capable of releas ing ATP, we used a well-established murine cell line C3H10T1/2, as well as primary adipocytes (see below). Differentiated, mature C3H10T1/2 adipocytes were stimu lated with various agonists: α-and β-adrenergic agonists

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Section: Mature Adipocytes Express Panx1 That Forms Functional Poresmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Pannexin‐1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

2019

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“…; Chiu et al . ). Panxs have been proposed to be involved in multiple (patho)physiological processes, i.e.…”

Section: Introductionmentioning

confidence: 97%

Pannexin 1 activation and inhibition is permeant‐selective

Nielsen

Toft-Bertelsen

Lolansen

et al. 2020

The Journal of Physiology

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Key points The large‐pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C‐terminal cleavage led to a Panx1 open‐state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1‐mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. Abstract Pannexin 1 (Panx1) is a large‐pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant‐selectivity of Panx1 activation and inhibition, we employed Panx1‐expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C‐terminal truncation or cell shrinkage elevated Panx1‐mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K+]o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl−, lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1‐mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant‐selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.

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“…Recent studies from several research groups conducted in vitro and in genetically modified animals have provided evidence that stimulation of α-adrenoceptors leads to opening of the Panx1 (pannexin-1) channel and efflux of ATP from VSMCs. 3–7 ATP released through Panx1 induces vasoconstriction in an autocrine manner via purinergic receptors, thereby regulating the effect of norepinehrine. 5,6 This vasoconstrictor effect of ATP acting directly on VSMCs is in contrast to the endothelium-dependent vasodilator effect of intravascular ATP mediated via formation of NO and prostanoids and activation of inwardly rectifying potassium channels.…”

mentioning

confidence: 99%

Probenecid Inhibits α-Adrenergic Receptor–Mediated Vasoconstriction in the Human Leg Vasculature

et al. 2018

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Coordination of vascular smooth muscle cell tone in resistance arteries plays an essential role in the regulation of peripheral resistance and overall blood pressure. Recent observations in animals have provided evidence for a coupling between adrenoceptors and Panx1 (pannexin-1) channels in the regulation of sympathetic nervous control of peripheral vascular resistance and blood pressure; however, evidence for a functional coupling in humans is lacking. We determined Panx1 expression and effects of treatment with the pharmacological Panx1 channel inhibitor probenecid on the vasoconstrictor response to α1- and α2-adrenergic receptor stimulation in the human forearm and leg vasculature of young healthy male subjects (23±3 years). By use of immunolabeling and confocal microscopy, Panx1 channels were found to be expressed in vascular smooth muscle cells of arterioles in human leg skeletal muscle. Probenecid treatment increased (P<0.05) leg vascular conductance at baseline by ≈15% and attenuated (P<0.05) the leg vasoconstrictor response to arterial infusion of tyramine (α1- and α2-adrenergic receptor stimulation) by ≈15%, whereas the response to the α1-agonist phenylephrine was unchanged. Inhibition of α1-adrenoceptors prevented the probenecid-induced increase in baseline leg vascular conductance, but did not alter the effect of probenecid on the vascular response to tyramine. No differences with probenecid treatment were detected in the forearm. These observations provide the first line of evidence in humans for a functional role of Panx1 channels in setting resting tone via α1-adrenoceptors and in the constrictive effect of noradrenaline via α2-adrenoceptors, thereby contributing to the regulation of peripheral vascular resistance and blood pressure in humans.

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A quantized mechanism for activation of pannexin channels

Cited by 127 publications

References 40 publications

Pannexin‐1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

Pannexin‐1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration

Pannexin 1 activation and inhibition is permeant‐selective

Probenecid Inhibits α-Adrenergic Receptor–Mediated Vasoconstriction in the Human Leg Vasculature

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