Miranda E. Good

Emerging concepts regarding pannexin 1 in the vasculature

Pannexin channels are newly discovered ATP release channels expressed throughout the body. Pannexin 1 (Panx1) channels have become of great interest as they appear to participate in a multitude of signalling cascades, including regulation of vascular function. Although numerous Panx1 pharmacological inhibitors have been discovered, these inhibitors are not specific for Panx1 and have additional effects on other proteins. Therefore, molecular tools, such as RNA interference and knockout animals, are needed to demonstrate the role of pannexins in various cellular functions. This review focuses on the known roles of Panx1 related to purinergic signalling in the vasculature focusing on post-translational modifications and channel gating mechanisms that may participate in the regulated release of ATP.

Pannexin channel and connexin hemichannel expression in vascular function and inflammation.

Control of blood flow distribution and tissue homeostasis depend on the tight regulation of and coordination between the microvascular network and circulating blood cells. Channels formed by connexins or pannexins that connect the intra- and extracellular compartments allow the release of paracrine signals, such as ATP and prostaglandins, and thus play a central role in achieving fine regulation and coordination of vascular function. This review focuses on vascular connexin hemichannels and pannexin channels. We review their expression pattern within the arterial and venous system with a special emphasis on how post-translational modifications by phosphorylation and S-nitrosylation of these channels modulate their function and contribute to vascular homeostasis. Furthermore, we highlight the contribution of these channels in smooth muscle cells and endothelial cells in the regulation of vasomotor tone as well as how these channels in endothelial cells regulate inflammatory responses such as during ischemic and hypoxic conditions. In addition, this review will touch on recent evidence implicating a role for these proteins in regulating red blood cell and platelet function.

Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone

Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. Methods and Results: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited &agr;-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX)

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named Hb&agr;X) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, Hb&agr;X administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II–induced hypertensive states. Hb&agr;X acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O2 binding in red blood cells. In human vasculature, Hb&agr;X blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, Hb&agr;X modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature.

Endothelial cell Pannexin1 modulates severity of ischemic stroke by regulating cerebral inflammation and myogenic tone

Ischemic stroke is a leading cause of morbidity and mortality in the US; however, there currently exists only one effective acute pharmacological therapeutic intervention. Purinergic signaling has been shown to regulate vascular function and pathological processes, including inflammation and arterial myogenic reactivity, and plays a role in ischemic stroke outcome. Purinergic signaling requires extracellular purines; however, the mechanism of purine release from cells is unclear. Pannexin1 (Panx1) channels are potentially novel purine release channels expressed throughout the vascular tree that couples regulated purine release with purinergic signaling. Therefore, we examined the role of smooth muscle and endothelial cell Panx1, using conditional cell type-specific transgenic mice, in cerebral ischemia/reperfusion injury outcomes. Deletion of endothelial cell Panx1, but not smooth muscle cell Panx1, significantly reduced cerebral infarct volume after ischemia/reperfusion. Infiltration of leukocytes into brain tissue and development of cerebral myogenic tone were both significantly reduced when mice lacked endothelial Panx1. Panx1 regulation of myogenic tone was unique to the cerebral circulation, as mesenteric myogenic reactivity and blood pressure were independent of endothelial Panx1. Overall, deletion of endothelial Panx1 mitigated cerebral ischemic injury by reducing inflammation and myogenic tone development, indicating that endothelial Panx1 is a possible novel target for therapeutic intervention of ischemic stroke.

Endothelial cell α-globin and its molecular chaperone α-hemoglobin–stabilizing protein regulate arteriolar contractility

Arteriolar endothelial cell–expressed (EC-expressed) &agr;-globin binds endothelial NOS (eNOS) and degrades its enzymatic product, NO, via dioxygenation, thereby lessening the vasodilatory effects of NO on nearby vascular smooth muscle. Although this reaction potentially affects vascular physiology, the mechanisms that regulate &agr;-globin expression and dioxygenase activity in ECs are unknown. Without &bgr;-globin, &agr;-globin is unstable and cytotoxic, particularly in its oxidized form, which is generated by dioxygenation and recycled via endogenous reductases. We show that the molecular chaperone &agr;-hemoglobin–stabilizing protein (AHSP) promotes arteriolar &agr;-globin expression in vivo and facilitates its reduction by eNOS. In Ahsp−/− mice, EC &agr;-globin was decreased by 70%. Ahsp−/− and Hba1−/− mice exhibited similar evidence of increased vascular NO signaling, including arteriolar dilation, blunted &agr;1-adrenergic vasoconstriction, and reduced blood pressure. Purified &agr;-globin bound eNOS or AHSP, but not both together. In ECs in culture, eNOS or AHSP enhanced &agr;-globin expression posttranscriptionally. However, only AHSP prevented oxidized &agr;-globin precipitation in solution. Finally, eNOS reduced AHSP-bound &agr;-globin approximately 6-fold faster than did the major erythrocyte hemoglobin reductases (cytochrome B5 reductase plus cytochrome B5). Our data support a model whereby redox-sensitive shuttling of EC &agr;-globin between AHSP and eNOS regulates EC NO degradation and vascular tone.

Pannexin-1 channels on endothelial cells mediate vascular inflammation during lung ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-CreERT2+/Panx1fl/fl mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.

Small Interfering RNA-Mediated Connexin Gene Knockdown in Vascular Endothelial and Smooth Muscle Cells

Global knockout of vascular connexins can result in premature/neonatal death, severe developmental complications, or compensatory up-regulation of different connexin isoforms. Thus, specific connexin gene knockdown using RNAi-mediated technologies is a technique that allows investigators to efficiently monitor silencing effects of single or multiple connexin gene products. The present chapter describes the transient knockdown of connexins in vitro and ex vivo for cells of the blood vessel wall. In detail, different transfection methods for primary endothelial cells and ex vivo thoracodorsal arteries are described. Essential controls for validating transfection efficiency as well as targeted gene knockdown are explained. These protocols provide researchers with the ability to modify connexin gene expression levels in a multitude of experimental setups.

Response by Good et al to Letter Regarding Article, “Pannexin-1 Channels as an Unexpected New Target of the Antihypertensive Drug Spironolactone”

In our recent publication, we reported the discovery that spironolactone—an antihypertensive—is also a new Panx1 (pannexin 1) inhibitor. In addition, we found that spironolactone interferes with α1AR (α1 adrenoceptor)-mediated vasoconstriction of resistance vessels and acutely lowers blood pressure in mice; importantly, these effects require Panx1 channel expression in vascular smooth muscle cells but are independent of the MR (mineralocorticoid receptor)—the traditional target of spironolactone. Based on the accumulated evidence, we proposed that Panx1 is a novel target of spironolactone that, in combination with MR-dependent actions, may contribute to the beneficial blood pressure-lowering effects of spironolactone that are especially relevant for treatment of resistant hypertensive patients. We are pleased that Drs Wright and Angus recognize that this novel action adds to the well-accepted MR-dependent antihypertensive effects of spironolactone and that this unveils a potentially important mechanism with translational clinical implications. In addition, however, these authors raise some points of contention with our studies that can be summarized in 2 general arguments: (1) based on perceived differences with their own pharmacological reports, they insist that additional pharmacological studies are necessary to support the fundamental underlying mechanism (ie, that Panx1-mediated ATP release contributes to α1AR-mediated vasoconstriction) and (2) that spironolactone acts less potently at Panx1 than at MR and thus may require concentrations not achieved clinically. Below, we address both of these points. Despite the narrow focus of their argument on work involving a single Panx1 inhibitor (mefloquine)1 and a single P2X1 blocker (NF449),2 there is now substantial pharmacological and, importantly, genetic evidence supporting a role for Panx1-mediated ATP release in α1AR-mediated vasoconstriction. For Panx1, this …

Non–Endoplasmic Reticulum–Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function

Objective— In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. Approach and Results— We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr &Dgr;/&Dgr;). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr &Dgr;/&Dgr; mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr &Dgr;/&Dgr; had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. Conclusions— Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.