Pannexin1 Regulates α1-Adrenergic Receptor– Mediated VasoconstrictionNovelty and Significance
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 α1D-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.
In resistance arteries, the coordination of vascular smooth muscle cell (VSMC) constriction helps to regulate blood flow and peripheral resistance and thus overall blood pressure.1 Although it has been hypothesized that gap junctions link VSMCs in resistance arteries to provide this coordination, the gap junctional coupling of VSMCs is controversial,2,3 and the presence of gap junctions and the gap junction proteins (connexins) is difficult to observe.4 Thus, VSMCs may use other mechanisms to communicate and coordinate their responses.
Pannexins (Panx) are tetra-spanning membrane proteins that mediate paracrine intercellular communication via release of purines such as ATP or UTP.5,6 The physiological function of Panx remains poorly documented in the vasculature, where there is currently no indication of Panx expression or function. We thus hypothesized that pannexins may coordinate VSMC constriction through the release of purines and activation of purinergic receptors.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Male mice (12 to 16 weeks) were used according to the University of Virginia Animal Care and Use Committee guidelines.
Thoracodorsal resistance arteries (TDAs) were immunolabeled with Panx1 antibody and 10-nm gold beads and imaged with a JEOL 6400 scanning electron microscope.
The VSMCs of TDAs were transfected with either a plasmid that contained Panx1-green fluorescent protein or Panx1 small interfering RNA and were cultured for 16 to 18 hours.
Panx1 or α1D-adrenergic receptor antibody was conjugated to Dynabeads, incubated with TDA lysates, and run on an SDS-PAGE gel.
One-way or 2-way ANOVA, followed by a Bonferroni test, was used for comparisons between treatments. P<0.05 was significant.
Panx1 Is Expressed in VSMCs of TDAs
Using transmission electron microscopy, we could not observe gap junctions between VSMCs in TDAs but could clearly identify tight junctions between endothelial cells (ECs; Figure 1A). The absence of membrane apposition was confirmed in other resistant arteries, whereas gap junctions could be observed between VSMCs in mouse aorta (Online Figure I). Therefore, we tested for the presence of the 3 Panx isoforms in TDA. Using Western blot (Figure 1B) and immunolabeling, we could only identify Panx1, both in ECs and VSMCs and between VSMCs (Figures 1C–E). In mouse aorta, we could not detect any Panx isoform in the VSMCs (Online Figure I). Immuno-scanning electron microscopy on TDAs with an extracellular loop Panx1 antibody revealed the presence of the protein on the VSMC plasma membrane (Figure 1F).
Phenylephrine-Induced Constriction Is Mediated by Panx
The data above clearly demonstrate that Panx1 is expressed in VSMCs; however, its functional role in the resistance arteries is unknown. We therefore constructed dose-response curves using the α1-adrenergic receptor agonist phenylephrine in the presence of 3 Panx inhibitors: mefloquine7 (Figure 2A), 10Panx1 peptide8,9 (Figure 2B), and probenecid6,9 (Figure 2C). Panx inhibitors all significantly reduced phenylephrine-induced vasoconstriction (Figures 2A–C; Online Figures II and III) but did not affect ATP-induced vasoconstriction (Online Figure IV).
The Panx channels release purines,5,6 which induce constriction when applied to TDA (Online Figure IV). We thus tested the effect of apyrase, an ectonucleotidase that degrades purines. Both 1 and 10 U/mL significantly reduced TDA constriction in response to phenylephrine (Figure 2D; Online Figures II and III). Purines such as ATP and UTP are known to bind purinergic receptors present on VSMCs; therefore, we treated the vessel with suramin, a purinergic receptor inhibitor (Figure 2E; Online Figures II and III), and reactive blue-2 (Figure 2F; Online Figures II and III), which inhibits the P2Y subfamily of purinergic receptors. Both antagonists significantly inhibited phenylephrine-induced constriction. To test whether phenylephrine-induced constriction was due to Panx1 localized on ECs, we perfused probenecid, apyrase, and reactive blue-2 in the lumen of TDAs and found no changes in phenylephrine responses, a result consistent with experiments on endothelium-denuded TDAs in which probenecid inhibited phenylephrine-induced constriction to the same extent as observed on intact TDAs (Online Figure V). Lastly, cultured VSMCs from coronary resistance arteries (expressing both α1D-adrenergic receptor and Panx1) had significant increases in ATP release after phenylephrine stimulation that was inhibited by 10Panx1, whereas cultured aortic VSMCs (expressing only α1D-adrenergic receptor) and normal rat kidney cells (which do not express α1D-adrenergic receptor or Panx1) did not have any increase in ATP release after phenylephrine stimulation (Online Figure VI).
Modulation of Panx1 Expression Modifies Phenylephrine-Induced Constriction of TDAs
TDAs were transfected either with Panx1-green fluorescent protein or with Panx1 small interfering RNA (Online Figure VII). When Panx1 was overexpressed, the response of TDAs to phenylephrine was increased by approximately 30%, whereas underexpression of Panx1 induced a 45% decrease in constriction in response to phenylephrine (Figure 3A; Online Figure VIII). Transfection of TDAs with control small interfering RNA did not affect phenylephrine-induced constriction (Online Figure VII). Immunolabeling of transfected TDAs revealed that Panx1 expression was modified only in VSMCs and not in ECs (Figure 3B), which was confirmed with an anti-green fluorescent protein antibody (Online Figure VII). Endothelial and smooth muscle function were not altered by transfection (Online Figure IX).
Panx1 Is Associated With α1-Adrenergic Receptor and Is Not Involved in KCl Constriction
We investigated whether Panx inhibitors could alter a receptor-independent stimulus such as KCl. We tested the 3 different Panx inhibitors and the purinergic inhibitors and found that none of them affected TDA constriction in response to KCl (Figure 4A). We thus hypothesized that a specific interaction may occur between Panx1 and α1D-adrenergic receptor, the primary adrenergic receptor responsible for VSMC constriction (Online Figure X). Immunolabeling of TDAs with Panx1 and α1D-adrenergic receptor antibodies revealed colocalization of the 2 proteins (Figures 4B and 4C). The association between Panx1 and α1D-adrenergic receptor was confirmed by coimmunoprecipitation from TDA lysates (Figure 4D).
In the microcirculation, coordination of VSMC contraction is essential for the regulation of blood flow distribution and peripheral vascular resistance. Gap junctions are likely responsible for VSMC coordination in conduit arteries; however, their role in VSMC coordination in resistance arteries remains unclear.4 Our transmission electron microscopy results indicate that VSMCs of TDAs are unlikely to be coupled exclusively via gap junctions, because the VSMCs are separated by relatively large intercellular spaces. The close apposition of EC plasma membranes, characteristic of gap junctions, indicates that the intercellular space observed between VSMCs was not an artifact of fixation. Our demonstration of Panx1 expression could suggest a mechanism that allows VSMCs to coordinate their responses independent of gap junctions.
Pannexin channels release purines in response to different stimuli.5 In the vasculature, the concentration of purines is finely regulated by ectonucleotidase at the surface of VSMCs and contributes to local regulation of vascular tone.10 Our findings suggest that Panx1, purines, and purinergic receptors are involved in TDA constriction in response to phenylephrine. However, we also found staining for Panx1 in ECs, which are known to release ATP,11 although our data show that the inhibition of phenylephrine responses was not due to Panx1 in ECs. Nevertheless, Panx1 expression in ECs suggests that Panx1 could be involved in EC functions such as vasodilation or inflammatory cell adhesion.
The present data imply that Panx1 is specifically involved in phenylephrine-induced, but not KCl-induced constriction. This suggests that Panx1 is not activated solely by the rise of intracellular calcium concentrations or mechanical stretch.5 Reports from cell culture studies have suggested that purine release through Panx1 may occur after activation of Gq/11-coupled α1-adrenergic receptor but not after stimulation of Gi/o-coupled α2-adrenergic receptors or Gs-coupled β-adrenergic receptors.12 Thus, Panx1 could also be involved in TDA constriction in response to other Gq/11-coupled receptor agonists, such as serotonin, endothelin-1, or angiotensin II. However, our evidence indicates that Panx1 and α1D-adrenergic receptor are closely associated at the protein level, which suggests that Panx1 and the α1D-adrenergic receptor may be part of a signaling microdomain.
The present data provide the first demonstration of Panx expression in the vasculature and of its function in the control of vasoconstriction. Our results suggest that the release of purines through Panx1 channels on VSMCs is triggered by phenylephrine stimulation and participates in control of vascular tone through purinergic receptors (Figure 4D). Thus, Panx1 could contribute to the coordination of VSMC constriction and the regulation of blood pressure via catecholamines released by sympathetic nerves.
Sources of Funding
This work was supported by National Institutes of Health grant HL088554 (B.E.I.), American Heart Association Scientist Development Grant (B.E.I.), an American Heart Association postdoctoral fellowship (M.B., S.R.J.), a National Research Science Award postdoctoral fellowship from the National Institutes of Health (A.C.S.), National Institutes of Health grant R15 HL102742-01 (R.L.W.), and the Canadian Institutes of Health Research (D.W.L.).
We thank Doug Bayliss and Brian Duling for critical reading of the manuscript; Allison Armstrong for assistance with the ATP assay; the University of Virginia Histology Core for sectioning; Anita Impagliazzo for the illustration; and Jan Redick and Stacey Guillot at the University of Virginia Advanced Microscopy Core.
In March 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days.
Non-standard Abbreviations and Acronyms
- thoracodorsal artery
- Received November 23, 2010.
- Revision received April 21, 2011.
- Accepted April 25, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Intercellular communication between smooth muscle cells (SMCs) within the vascular wall is essential for the control of vasoreactivity, but the mechanism for this remains unclear.
Pannexin channels participate in intercellular communication through the release of purines such as ATP, a potent vasoconstrictor.
What New Information Does This Article Contribute?
Pannexin1 (Panx1) is present in the SMCs of resistant arteries and plays a role in phenylephrine-induced vasoconstriction.
Panx1 and the α1D-adrenergic receptor are part of the same protein complex.
When phenylephrine binds to the α1D-adrenergic receptor, Panx1 opens to release purines that can act on purinergic receptors present on SMCs to enhance phenylephrine-induced vasoconstriction.
Intercellular communication between SMCs serves to regulate the vasoconstriction of resistance arteries, a fundamental process for the control of blood flow and peripheral resistance; however, the exact mechanisms of SMC communication remain unclear. Here, we focused on Panx1, a protein known to participate in the release of ATP that has not been described in the vasculature. Our data indicate that Panx1 releases ATP during contraction of resistance arteries in response to phenylephrine, thereby controlling the intensity of vasoconstriction. These data are the first to describe the presence of, and a role for, Panx1 in the vascular wall, providing a novel mechanism for SMC communication.