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Research Paper

Free Access

Expression of Pannexin Isoforms in the Systemic Murine Arterial Network

Lohman A.W.a, b · Billaud M.a · Straub A.C.a · Johnstone S.R.a · Best A.K.a · Lee M.c · Barr K.d · Penuela S.e · Laird D.W.e · Isakson B.E.a, b

Author affiliations

aRobert M. Berne Cardiovascular Research Center, and bDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Va., and cDepartment of Pharmacology, Yale University, New Haven, Conn., USA; Departments of dPhysiology and Pharmacology, and eAnatomy and Cell Biology, University of Western Ontario, London, Ont., Canada

Corresponding Author

Dr. Brant E. Isakson

Robert M. Berne Cardiovascular Research Center, University of Virginia

PO Box 801394

Charlottesville, VA 22908 (USA)

Tel. +1 434 924 2093, E-Mail brant@virginia.edu

Related Articles for ""

J Vasc Res 2012;49:405–416

Abstract

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.

© 2012 S. Karger AG, Basel


Introduction

Pannexins (Panx) are a class of glycoproteins that oligomerize to form channels at the plasma membrane [1,2,3]. Panx proteins are found in 3 different isoforms (Panx1, Panx2 and Panx3) in cultured cells and in vivo. To date, Panx1 has been characterized extensively and has been shown to be ubiquitously expressed. Conversely, Panx2 expression has been found primarily in the central nervous system [4,5,6] and Panx3 is mainly expressed in the skin, osteoblasts and chondrocytes [7,8,9]. While Panx proteins have been shown to possess a similar membrane topology to the vertebrate gap junction proteins, the connexins, there are key differences in their respective functions within the cell. One of the most important differences is that connexins allow for a direct communication between 2 cells by forming gap junction channels through the docking of 2 connexin hemichannels, or connexons, whereas the formation of gap junctions by the docking of 2 Panx channels has never been demonstrated in vivo [10]. Another key difference is the ability of Panx channels to open and release ATP into the extracellular space under physiological extracellular calcium concentration, whereas hexameric connexin hemichannels present at the plasma membrane have been shown to be closed under these conditions and open when the extracellular calcium concentration is reduced [11,12,13]. Therefore, since their first description in 2000, Panx channels have been suggested to act as paracrine release channels that are strongly implicated in the release of purine nucleotides from cells [3,14,15,16,17].

The role of extracellular purines, including ATP, in the systemic circulation has been shown to be important for several vascular functions including the regulation of vascular tone [18,19], reactive hyperemia during contraction of skeletal muscle [20,21] and hypoxia-induced vasodilation [22,23,24]. Although there are well-described reports of ATP release occurring from both circulating erythrocytes and sympathetic nerves innervating vascular smooth muscle cells (VSMC) [16,23,25,26,27,28,29,30,31], there is accumulating evidence which indicates that endothelial cells (EC) and smooth muscle cells of the vascular wall can also release ATP [24,32,33,34,35,36,37]. The conduit for ATP release from these cells continues to be investigated, but several reports suggest that Panx channels may be an important candidate. Indeed, we have recently demonstrated that both smooth muscle and EC in small arteries express Panx1 and our results showed that VSMC can release ATP through Panx1 channels [36]. However, the distribution of Panx isoforms across the vasculature is not yet known. This is an important omission considering the potential role these channels may have in the vasculature, whereas for decades ATP and its breakdown products have been documented to have tremendous physiological importance [21,30,38,39,40,41,42,43,44,45,46,47]. Importantly, Panx channels in EC could play essential roles in ATP signaling in the blood vessel lumen which could potentially include vasodilation and monocyte recruitment. Alternatively, expression of Panx channels in smooth muscle cells may be involved in purinergic signaling such as the regulation of vasoconstriction or VSMC proliferation [48,49]. Together, the expression of these channels in vascular cells could play important roles in regulating a number of physiological processes.

As blood vessels across the arterial tree experience a variety of different environments, identification of how ATP is released from the EC and smooth muscle cells of different arterial beds is essential for understanding how purinergic signaling is regulated in the control of vascular tone in both the regulation of blood pressure and the maintenance of proper organ physiology. Therefore, as Panx proteins have been strongly implicated in ATP release from cells, we sought to characterize the expression of the different Panx isoforms across the vasculature to help provide a more detailed understanding of the potential ATP release mechanisms in these cells.

Methods

HEK293T Cells. Human embryonic kidney cells 293T (HEK293T) were used under passage 20 and grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine and 1% nonessential amino acids. Cells were transfected with mouse Panx1 (pcDNA3.1), mouse Panx2 (pEGFP-N1 [50]) or mouse Panx3 (pEGFP-N1 [50]) plasmids at 750 ng/ml using 8 µg Lipofectamine 2000 on 6 × 105 cells for 15 h. Cells were washed with PBS and were either fixed with 4% paraformaldehyde before being processed for immunocytochemistry (as previously described [51]) or protein was extracted for Western blot analysis.

Western Blot. Western blots were performed as described previously [51]. For all experiments, 50 µg of protein was loaded into each well.

qRT-PCR. qRT-PCR was performed as described previously [52] with primers for mouse and human Panx1 (mouse forward 5′-TAAGCTGCTTCTCCCCGAGT-3′, mouse reverse 5′-TGGCAAACAGCAGTAGGATG-3′; human forward 5′-TGCAGAGCGAGTCTGGAAAC-3′, human reverse 5′-CAGCCTTAATTGCACGGTTG-3′) mouse and human Panx2 (mouse forward 5′-AAGCATACCCGCCACTTCTC-3′, mouse reverse 5′-GGGGTACGGGATTTCCTTCT-3′; human forward 5′-GTCACCCTGGTCTTCACCAA-3′, human reverse 5′-GCAGGAACTTGTGCTCAAACA-3′) or mouse and human Panx3 (mouse forward 5′-CCCATTCTCAGCAGCATCAT-3′, mouse reverse 5′-ACTCCTGGGCGAAAGCTAGA-3′; human forward 5′-CTCAAAGGACTGCGTCTGGA-3′, human reverse 5′-CTGCCGGATGCTGAAGTTAC-3′). Transcripts were quantified by comparing to the housekeeping gene β2 microglobulin (B2M) [53].

Animals. All mice were male, 10–14 weeks of age, on a C57Bl/6 genetic background and were cared for under the provisions of the University of Virginia Animal Care and Use Committee. The Panx1–/– mice were kind gifts of Genentech Corporation and were cared for under the provisions of the University of Western Ontario Animal Care and Use Committee. The Panx1–/– mouse tissue samples were harvested at the University of Western Ontario and sent to the University of Virginia for analysis. All experiments were performed on a minimum of 3 mice.

Embedding and Immunocytochemistry. Prior to tissue harvesting, fixation was performed by perfusing room temperature 4% paraformaldehyde (PFA) made in PBS through the heart. The specific tissues were immediately removed from the animal and placed in 4% PFA for 30 min before being placed in 70% ethanol for paraffin embedding. Paraffin sections (4–5 µm in thickness) were deparaffinized and processed for immunocytochemistry as previously described [36].

Imaging. All images were obtained with an Olympus Fluoview 1000. For each vessel bed, a minimum of 5 sections from each mouse (with at least 3 mice) were evaluated. Figures are representative of composite z-stacks. For z-stacks, a minimum of 5 images at a depth of 0.5 µm/image were compiled. Although each of the different Panx isoforms was scanned at different settings, the individual ones were scanned at the same settings across tissue beds.

Antibodies. Each of the Panx antibodies was made in rabbit and initially described in [50]. The Panx1 antibodies were made against amino acids 247–265 (SIKSGVLKNDSTIPDRFQC) in the extracellular loop (anti-Panx1 EL [50]) or amino acids 395–409 (QRVEFKDLDLSSEAA) in the carboxyl tail (anti-Panx1 CT [50]) The Panx2 antibody was made against amino acids 494–508 (ASEKKHTRHFSLDVH) and the Panx3 antibody was made against amino acids 379–392 (KPKHLTQHTYDEHA). AlexaFluor 594 donkey anti-rabbit secondary antibodies were used to observe all primary antibodies. Nuclei were stained with DAPI (Invitrogen). At least 5 observations were made per mouse, per experiment.

Electron Microscopy. All tissues were processed and stained for immunogold-transmission electron microscopy as previously described [54]. Quantification of gold beads in EC, at the myoendothelial junction (MEJ) or VSMC was performed as previously described [54] on 5 TEM images per artery that were sectioned 10 µm apart. Using Metamorph analysis software, areas of EC, MEJ and VSMC were determined in µm2 and the number of gold beads in each area was counted. Measurements are representative of the average number of gold beads per µm2 ± SE. For each vessel bed, a minimum of 4 observations from each mouse, with at least 3 mice, were evaluated.

En face Imaging of MEJ. The thoracodorsal artery (TDA) was maximally dilated by whole-mouse perfusion with Krebs-HEPES buffer (in mM: 118.4 NaCl, 4.7 KCl, 1.2 MgSO4, 4 NaHCO3, 1.2 KH2PO4, 2 CaCl2, HEPES and 6 D-glucose, pH 7.4 with NaOH) containing sodium nitroprusside followed by fixation with 4% PFA. The arteries were removed, cut longitudinally and conventional immunocytochemistry was performed (as described above). The arteries were mounted in DAPI and coverslip-sealed to provide a flat surface for confocal imaging. Autofluorescence of the internal elastic lamina (IEL) between the endothelium and smooth muscle was readily apparent with ‘holes’ between the 2 layers of cells which has been extensively documented (e.g. [55,56,57]). In our study, the detection of punctate fluorescence in >25% of the ‘holes’ was considered indicative of protein localization to MEJ.

Results

Initially HEK293T cells were tested for endogenous Panx expression by quantitative RT-PCR for detection of mRNA (fig. 1a) or by Western blot for detection of protein (fig. 1b). There was no detectable Panx3 mRNA, with minimal amounts of Panx1 and Panx2 mRNA compared to the housekeeping gene B2M [53]; however, no endogenous protein was detected for any of the Panx isoforms in these cells. We therefore transfected the HEK293T cells with plasmids encoding murine Panx1, Panx2 or Panx3. Western blot analysis revealed specific bands for each Panx isoform when probed with the respective Panx antibody (fig. 1b). Using immunocytochemistry, we found expression of each transfected Panx isoform at the plasma membrane (fig. 1c–e), as well as intracellular staining for Panx2 which has been shown in a number of cell types, again demonstrating the specificity of the Panx antibodies.

Fig. 1

Specificity of Panx antibodies in cultured cells. HEK293T cells were tested for endogenous Panx expression by mRNA via RT-PCR (a) and for protein via Western blot (b). a mRNA expression was normalized to the housekeeping gene β2 microglobulin (B2M). The HEK cells were transfected with Panx1, Panx2 or Panx3 plasmids, stained using anti-Panx1 CT antibody (Ab), anti-Panx1 EL antibody, anti-Panx2 antibody or anti-Panx3 antibody and detected with anti-rabbit IRDye 800CW (LiCOR) for Western blots (b) or anti-rabbit Alexa 594 for immunofluorescence (c–e). In addition, for immunofluorescence, the antibody corresponding to the transfected Panx isoform was incubated with its respective blocking peptide for negative controls. c–e DAPI-stained nuclei (blue) and expression of each Panx (red). Scale bar: 10 µm (in unstained images) and 5 µm (in stained images).

http://www.karger.com/WebMaterial/ShowPic/206393

Next, we tested the specificity of the Panx1 antibodies on TDA, as we previously reported that this is the only isoform expressed in the TDA wall [36]. Using TDA harvested from C57Bl/6 mice, we found consistent staining in both EC and VSMC using both the anti-Panx1 CT and anti-Panx1 EL antibodies (fig. 2a). We could not detect any Panx1 in TDA isolated from the Panx1–/– mouse (fig. 2b), again demonstrating the specificity of the Panx1 antibodies.

Fig. 2

Specificity of Panx1 antibodies in arteries. The anti-Panx1 antibodies were tested on mouse TDA from C57Bl/6 (a) and Panx1–/– mice (b). DAPI-stained nuclei (blue), autofluorescence of IEL (green) and Panx staining (red). Scale bar: 20 µm. Asterisks indicate the lumen of the artery.

http://www.karger.com/WebMaterial/ShowPic/206392

Because we determined the Panx antibodies to be specific and Panx1 to be endogenously expressed in the vascular cells of the TDA, we tested for Panx isoform expression through the systemic arterial tree, beginning in the aorta (fig. 3a) and going on to the carotid artery (fig. 3b), the femoral artery (fig. 3c), the renal artery (fig. 3d), the TDA (fig. 3e), the abdominal arteries (fig. 3f), the arterioles in the spinotrapezius muscle (fig. 3g), and finally the cremasteric arterioles (fig. 3h). Throughout the arterial tree, Panx1 was the only protein detected and was consistently expressed in the endothelium regardless of artery size. However, the expression of Panx1 in smooth muscle cells was poorly detectable in the larger conduit arteries (aorta/carotid/femoral). Some expression of Panx1 in smooth muscle cells was detected in the renal arteries, whereas in the smaller arteries including the TDA, the abdominal artery and the spinotrapezius and cremasteric arterioles, Panx1 was found throughout the smooth muscle. Similar to the smaller systemic arteries, both small (luminal diameter of 20–90 µm) and large (luminal diameter of 100–250 µm) coronary arteries also expressed Panx1 in endothelium and smooth muscle cells (fig. 4a, b), but there was no Panx2 expression (fig. 4c, d). Interestingly, in coronary arteries with a luminal diameter less than 100 µm, Panx3 was detected in endothelium and smooth muscle (fig. 4e, f).

Fig. 3

Panx expression in the murine systemic arterial network. a–h Each Panx antibody was tested on arteries of progressively decreasing size. DAPI-stained nuclei (blue), autofluorescence of IEL (green) and Panx staining (red). Scale bar: 10 µm (representative for the row of staining). Asterisks indicate the lumen of the artery. Cre. = Cremasteric; Spino. = spinotrapezius.

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Fig. 4

Panx expression in the coronary arteries. Coronary arteries that were 100–250 µm in diameter (a, c, e) or 20–90 µm in diameter (b, d, f) were assessed for expression of Panx1 (a, b), Panx2 (c, d) or Panx3 (e, f). DAPI-stained nuclei (blue), autofluorescence of the IEL (green) and Panx staining (red). Scale bar: 5 µm. Asterisks indicate the artery lumen.

http://www.karger.com/WebMaterial/ShowPic/206390

Next we tested whether Panx were expressed at MEJ, where endothelium and smooth muscle make contact in small arteries and arterioles [58,59]. Using immuno-TEM, we quantified the amount of each Panx isoform expressed in the coronary, cremasteric and TDA and found little detectable Panx expression at MEJ in selected arteries (fig. 5a–d). While immuno-TEM can be a valuable method for quantifying the distribution of proteins at distinct subcellular locations within tissues at high resolution, this method has its limitations and results can potentially be confounded by nonuniform antigen distribution across sections. Therefore, we also analyzed Panx isoform expression at the MEJ by performing whole-mount immunocytochemistry on isolated TDA that were fixed in a maximally dilated state to facilitate visualization of MEJ at IEL holes. This method allows for visualization of large areas of antigen distribution. In agreement with our immuno-TEM data, there were no Panx isoforms detectable; however, Cx43 (previously found to be located at MEJ via quantified immuno-TEM [60]; as used above) was localized to these holes (fig. 5e).

Fig. 5

Minimal Panx expression at MEJ. a–c The number of gold beads after staining with Panx1, Panx2 or Panx3 per µm2 in EC, MEJ and VSMC was quantified in cremaster arterioles (20–40 µm; a), coronary arteries (50–100 µm; b) and TDA (200 µm; c). d Representative immuno-TEM from coronary arterioles is shown with gold beads representing Panx1. Asterisks indicate the artery lumen and arrows indicate representative location of gold beads. Scale bar: 0.5 µm. e Holes in the IEL from TDA, corresponding to possible MEJ, were examined for protein expression. Autofluorescence of the IEL (green) and protein of interest (red). Only when Cx43 antibody was used could punctate fluorescence be detected in the holes. Each image is 15 µm × 15 µm.

http://www.karger.com/WebMaterial/ShowPic/206389

We also examined Panx expression in the arterial network of the kidney (fig. 6). We did not detect any Panx1 or Panx2 in the juxtaglomerular apparatus (fig. 6a, b), but Panx3 was found throughout the unit in very distinct punctate stains (fig. 6c), but was not detectable when incubated with the Panx3 peptide (fig. 6d). This same pattern of expression was found in the arterioles of the cortical kidney with Panx3 being detectable only in the endothelium, with more limited expression in smooth muscle cells (fig. 6e–g), which was again absent when incubated with the Panx3 peptide (fig. 6h).

Fig. 6

Panx expression in the kidney. a–d The glomerulus of the kidney was stained for Panx1 (a), Panx2 (b) and Panx3 (c), with Panx3 peptide competition (d). e–h Arterioles of the cortical kidney were stained for Panx1 (e), Panx2 (f) and Panx3 (g), with Panx3 peptide competition (h). Panx (red) and DAPI-stained nuclei (blue). Scale bar: 20 µm (representative for all images).

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Lastly, as a contrast to the systemic circulation, we examined Panx expression in the pulmonary circulation (fig. 7). The first-order intrapulmonary artery had positive staining for Panx1 in the endothelium and smooth muscle. There was some Panx1 staining in the IEL; it is not clear why, but the most likely reason is z-stack compression. Panx2 was expressed in the smooth muscle cells with some detectable Panx2 in EC (fig. 7a, b). Peptide competition eliminated the Panx2 staining (fig. 7c). The expression of Panx3 was predominantly in the endothelium, but some detectable expression in the smooth muscle cells remained (fig. 7d). In the distal lung, Panx1 and Panx3 were the dominant isoforms expressed, with some detectable Panx2 (fig. 7e–h). Of particular note, it appeared that in the alveoli the Panx could also be localized around the nuclei.

Fig. 7

Panx expression in the pulmonary artery and lung alveoli. a–d The pulmonary artery (lumen diameter approx. 400 µm) was stained for each of the Panx isoforms. c Panx2 peptide competition. Asterisks indicate the luminal side of the arterial wall. e–h The distal lung alveoli were also stained with each of the Panx isoforms, with Panx2 peptide competition (g). In all images, green shows autofluorescence of matrix proteins and red blood cells (e–h), the Panx isoform of interest is red and DAPI-stained nuclei are blue. Scale bars: 20 µm.

http://www.karger.com/WebMaterial/ShowPic/206387

Discussion

Panx form plasma membrane channels capable of releasing ATP that provide key physiological functions including the regulation of apoptosis and with a potential role in the regulation of vasoconstriction by ATP release mechanisms [36,61]. For this reason, we sought to identify whether the various Panx isoforms were expressed across the arterial vasculature and to describe their expression pattern in several vascular beds. We began by extensively testing each of the Panx antibodies on HEK cells which do not endogenously express Panx proteins, although some residual Panx mRNA was detectable. After transfection of each Panx isoform in HEK cells, we found reactivity for each Panx isoform with corresponding Panx antibodies by Western blotting and immunocytochemistry.

In the blood vessel wall, EC and VSMC are in constant communication both within and often between cell types through multiple signaling mechanisms. One such signaling molecule is ATP, which has been implicated in the control of vasoconstriction and vasodilation [41,42,44,62]. Subsequently, ATP-sensitive purinergic receptors in vascular EC and VSMC have been extensively characterized [62,63,64,65,66]. In EC, ATP binding to P2Y purinergic receptors has been shown to mediate vasodilation, whereas in VSMC, ATP-sensitive P2X purinergic receptors have been shown to control vasoconstriction [62,64,65,66]. While much work has been done characterizing the functional role of extracellular ATP in the vasculature, the conduit for its release from the cells in the arterial wall is a current topic of investigation. As early studies on Panx channels identified the potential for these membrane proteins to release ATP into the extracellular space, their expression in vascular cells may provide critical insight into the regulation of purinergic signaling at the level of purine release from these cells.

Our observation that Panx1 is expressed in EC across the arterial tree may suggest that this protein is intimately involved in the release of ATP into the blood vessel lumen and thus may be involved in modulating the extent of vasodilation in response to stimuli such as increases in blood flow and hypoxia, which are known to cause ATP release from EC [22,24,35,67]. At the smooth muscle cell axis, we have previously suggested a pivotal role for ATP release from Panx1 channels in regulating vasoconstriction in response to stimulation of α1D-adrenergic receptors [36]. The observation that Panx isoforms are highly expressed in the smooth muscle cells of small arteries and arterioles, versus selected larger conduit arteries, may suggest a role for these channels in the regulation of peripheral resistance by ATP release mechanisms, because small arterioles have the highest resistance and contribute the most to total peripheral resistance [68]. While we detected very little Panx expression in smooth muscle cells in large arteries, we did find expression of Panx1 in the EC layer of these arteries. It is possible that Panx1 channels in EC across the vasculature may also play a role in mediating the inflammatory response, where ATP released into the vascular lumen has been shown to promote monocyte and macrophage recruitment and migration into the vascular intima [69,70,71,72]. With the possibility that Panx channels in the vasculature may play dual roles in regulating blood pressure as well as pathological events such as inflammation, it will be important to identify potential binding partners and regulatory mechanisms that control the gating of these channels and thus ATP release from vascular cells.

Although the main arteries of the systemic arterial vascular tree had only Panx1 expression, the arteries in the specialized organs had more variability in terms of the other Panx isoforms expressed. For example, while Panx1 was the main isoform expressed in the coronary circulation, we observed the expression of Panx3 in coronary arteries with an internal diameter <100 µm. This is the first evidence for Panx3 expression in vascular cells and may suggest that this isoform plays a special role in cardiac physiology. The coronary arteries play an important role in oxygen delivery to cardiac tissue which has an extremely high metabolic demand, and changes in coronary blood flow and thus changes in oxygen delivery to myocardial tissue can lead to alterations in myocardial metabolism and ultimately cardiac output. Therefore, changes in coronary blood flow could have profound effects on blood pressure and it is possible that these arteries may require more dynamic ATP release mechanisms to facilitate rapid changes in the blood flow to cardiac tissue to match oxygen demand. It will be interesting in the future to determine whether coronary blood flow may be influenced by the expression of the different Panx isoforms in these arteries. Along with Panx1, Panx3 has also been shown to release ATP and expression of this isoform along with Panx1 in critically small arterioles in the coronary circulation may provide additional channels for ATP release. This observation warrants further investigation into the role of Panx channels in the regulation of coronary blood flow, the dynamics of channels composed of Panx3 and whether heteromeric channels can be formed by Panx1 and Panx3 [6,73].

In the renal vasculature, we observed expression of Panx3 only, with no detection of Panx1 and Panx2, in both the juxtaglomerular apparatus as well as in the endothelium of arteries in the cortical kidney. This was the sole vascular bed in which we did not observe Panx1 expression, which may suggest that the different Panx isoforms may play specific roles in different vascular beds and that the renal vasculature may utilize purinergic signaling cascades distinct from other arterial beds across the arterial tree. This observation may also suggest, as with the coronary circulation, that the different Panx isoforms in the arteries supplying these tissues may play distinct roles in regulating ATP release in different physiological environments that are subject to unique interstitial and intraluminal pressures.

When we examined the pulmonary circulation, we observed expression of Panx1 in both the EC and smooth muscle cells of the first-order intrapulmonary artery, while Panx2 was found primarily in the smooth muscle and Panx3 primarily in the endothelium. In the distal lung, we observed expression of Panx1 and Panx3 in the alveoli with Panx2 expressed in a perinuclear fashion.

Lastly, it should be noted that all of the images described in this manuscript were from paraffin-embedded tissue blocks. This presents its own unique set of potential issues, including the well-described lack of good signal-to-noise for immunofluorescence and poor antigenicity (as compared to unfixed frozen sections). Although these may be issues, the embedding allowed us to directly compare all the tissue beds and have a consistent amount of antibody applied to each section and consistent setting on the microscope. While these conditions can also be met with frozen tissue sections, the morphology of the tissue is then often compromised.

In conclusion, the consistent observation from our data was (1) Panx are expressed throughout the arterial tree, (2) Panx1 is the predominant isoform in the arterial network, (3) Panx1 is predominantly expressed in the endothelium throughout the arterial network and (4) smooth muscle cells in large conduit arteries express less Panx as compared to the small arteries, where Panx1 is found to be abundantly expressed. Further studies on the ways in which these channels are utilized should provide fascinating new data on the regulation of ATP release in the vasculature.

Acknowledgments

We thank the University of Virginia Histology Core for sectioning and Jan Redick and Stacey Guillot at the University of Virginia Advanced Microscopy Core and Genentech Inc. for the use of the Panx1–/– mice. This work was supported by a National Institutes of Health grant HL088554 (B.E.I.), an 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.), a National Institutes of Health Cardiovascular Training grant (A.W.L.) and the Canadian Institutes of Health Research (D.W.L.).


References

  1. Panchin YV: Evolution of gap junction proteins – the pannexin alternative. J Exp Biol 2005;208:1415–1419.
  2. Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O, Tiunova A, Born TL, Usman N, Staroverov D, Lukyanov S, Panchin Y: The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 2004;83:706–716.
  3. Bao L, Locovei S, Dahl G: Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett 2004;572:65–68.
  4. Ray A, Zoidl G, Weickert S, Wahle P, Dermietzel R: Site-specific and developmental expression of pannexin1 in the mouse nervous system. Eur J Neurosci 2005;21:3277–3290.
  5. Santiago MF, Veliskova J, Patel NK, Lutz SE, Caille D, Charollais A, Meda P, Scemes E: Targeting pannexin1 improves seizure outcome. PloS One 2011;6:e25178.
  6. Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H: Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci USA 2003;100:13644–13649.
  7. Barbe MT, Monyer H, Bruzzone R: Cell-cell communication beyond connexins: the pannexin channels. Physiology (Bethesda) 2006;21:103–114.
  8. Bond SR, Lau A, Penuela S, Sampaio AV, Underhill TM, Laird DW, Naus CC: Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes. J Bone Miner Res 2011;26:2911–2922.
  9. Penuela S, Celetti SJ, Bhalla R, Shao Q, Laird DW: Diverse subcellular distribution profiles of pannexin 1 and pannexin 3. Cell Commun Adhes 2008;15:133–142.
  10. Dahl G, Locovei S: Pannexin: To gap or not to gap, is that a question? IUBMB Life 2006;58:409–419.
  11. Pfahnl A, Dahl G: Gating of cx46 gap junction hemichannels by calcium and voltage. Pflugers Arch 1999;437:345–353.
  12. Quist AP, Rhee SK, Lin H, Lal R: Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 2000;148:1063–1074.
  13. Li H, Liu TF, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG: Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J Cell Biol 1996;134:1019–1030.
  14. Qiu F, Dahl G: A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. Am J Physiol Cell Physiol 2009;296:C250–C255.
  15. Ransford GA, Fregien N, Qiu F, Dahl G, Conner GE, Salathe M: Pannexin 1 contributes to ATP release in airway epithelia. Am J Resp Cell Mol Biol 2009;41:525–534.
  16. Sridharan M, Adderley SP, Bowles EA, Egan TM, Stephenson AH, Ellsworth ML, Sprague RS: Pannexin 1 is the conduit for low oxygen tension-induced ATP release from human erythrocytes. Am J Physiol Heart Circ Physiol 2011;299:H1146–H1152.
    External Resources
  17. Woehrle T, Yip L, Elkhal A, Sumi Y, Chen Y, Yao Y, Insel PA, Junger WG: Pannexin-1 hemichannel-mediated ATP release together with p2x1 and p2x4 receptors regulate T-cell activation at the immune synapse. Blood 2010;116:3475–3484.
  18. Kauffenstein G, Drouin A, Thorin-Trescases N, Bachelard H, Robaye B, D’Orleans-Juste P, Marceau F, Thorin E, Sevigny J: NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse. Cardiovasc Res 2010;85:204–213.
  19. Kauffenstein G, Furstenau CR, D’Orleans-Juste P, Sevigny J: The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation. Br J Pharmacol 2010;159:576–585.
  20. Forrester T, Lind AR: Identification of adenosine triphosphate in human plasma and the concentration in the venous effluent of forearm muscles before, during and after sustained contractions. J Physiol 1969;204:347–364.
  21. Mortensen SP, Gonzalez-Alonso J, Bune LT, Saltin B, Pilegaard H, Hellsten Y: ATP-induced vasodilation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins, and adenosine. Am J Physiol Regul Integr Comp Physiol 2009;296:R1140–R1148.
  22. Bodin P, Burnstock G: Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells. Experientia 1995;51:256–259.
  23. Bergfeld GR, Forrester T: Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovasc Res 1992;26:40–47.
  24. Bodin P, Bailey D, Burnstock G: Increased flow-induced ATP release from isolated vascular endothelial cells but not smooth muscle cells. Br J Pharmacol 1991;103:1203–1205.
  25. Sneddon P, Burnstock G: ATP as a co-transmitter in rat tail artery. Eur J Pharmacol 1984;106:149–152.
  26. Sprague RS, Ellsworth ML, Stephenson AH, Kleinhenz ME, Lonigro AJ: Deformation-induced ATP release from red blood cells requires CFTR activity. Am J Physiol 1998;275:H1726–H1732.
  27. Sneddon P, Burnstock G: Inhibition of excitatory junction potentials in guinea-pig vas deferens by alpha, beta-methylene-ATP: further evidence for ATP and noradrenaline as cotransmitters. Eur J Pharmacol 1984;100:85–90.
  28. Burnstock G: Noradrenaline and ATP as cotransmitters in sympathetic nerves. Neurochem Int 1990;17:357–368.
  29. Sneddon P, Westfall DP: Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens. J Physiol 1984;347:561–580.
  30. Sprague RS, Hanson MS, Achilleus D, Bowles EA, Stephenson AH, Sridharan M, Adderley S, Procknow J, Ellsworth ML: Rabbit erythrocytes release ATP and dilate skeletal muscle arterioles in the presence of reduced oxygen tension. Pharmacol Rep 2009;61:183–190.
  31. Lew MJ, White TD: Release of endogenous ATP during sympathetic nerve stimulation. Br J Pharmacol 1987;92:349–355.
  32. Bodin P, Burnstock G: ATP-stimulated release of ATP by human endothelial cells. J Cardiovasc Pharmacol 1996;27:872–875.
  33. Moser TL, Kenan DJ, Ashley TA, Roy JA, Goodman MD, Misra UK, Cheek DJ, Pizzo SV: Endothelial cell surface f1-f0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proc Natl Acad Sci USA 2001;98:6656–6661.
  34. Bodin P, Burnstock G: Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol 2001;38:900–908.
  35. Yamamoto K, Shimizu N, Obi S, Kumagaya S, Taketani Y, Kamiya A, Ando J: Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells. Am J Physiol Heart Circ Physiol 2007;293:H1646–H1653.
  36. Billaud M, Lohman AW, Straub AC, Looft-Wilson R, Johnstone SR, Araj CA, Best AK, Chekeni FB, Ravichandran KS, Penuela S, Laird DW, Isakson BE: Pannexin1 regulates alpha1-adrenergic receptor-mediated vasoconstriction. Circ Res 2011;109:80–85.
  37. Goedecke S, Roderigo C, Rose CR, Rauch BH, Goedecke A, Schrader J: Thrombin-induced ATP release from human umbilical vein endothelial cells. Am J Physiol Cell Physiol 2012;302:C915–C923.
  38. Mustafa SJ, Morrison RR, Teng B, Pelleg A: Adenosine receptors and the heart: role in regulation of coronary blood flow and cardiac electrophysiology. Handb Exp Pharmacol 2009:161–188.
    External Resources
  39. Berne RM: Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow. Am J Physiol 1963;204:317–322.
  40. Burnstock G: Control of vascular tone by purines and pyrimidines. Br J Pharmacol 2010;161:527–529.
  41. Winbury MM, Papierski DH, Hemmer ML, Hambourger WE: Coronary dilator action of the adenine-ATP series. J Pharmacol Exp Ther 1953;109:255–260.
  42. Burnstock G: Dual control of vascular tone and remodelling by ATP released from nerves and endothelial cells. Pharmacol Rep 2008;60:12–20.
  43. Jones RD, Berne RM: Evidence for a metabolic mechanism in autoregulation of blood flow in skeletal muscle. Circ Res 1965;17:540–554.
  44. Drury AN, Szent-Gyorgyi A: The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol 1929;68:213–237.
  45. Rubio R, Berne RM: Release of adenosine by the normal myocardium in dogs and its relationship to the regulation of coronary resistance. Circ Res 1969;25:407–415.
  46. Dobson JG Jr, Rubio R, Berne RM: Role of adenine nucleotides, adenosine, and inorganic phosphate in the regulation of skeletal muscle blood flow. Circ Res 1971;29:375–384.
  47. Nishiyama A, Rahman M, Inscho EW: Role of interstitial ATP and adenosine in the regulation of renal hemodynamics and microvascular function. Hypertens Res 2004;27:791–804.
  48. Erlinge D: Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol 1998;31:1–8.
  49. Wang DJ, Huang NN, Heppel LA: Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells. J Cellul Physiol 1992;153:221–233.
  50. Penuela S, Bhalla R, Nag K, Laird DW: Glycosylation regulates pannexin intermixing and cellular localization. Mol Biol Cell 2009;20:4313–4323.
  51. Johnstone SR, Ross J, Rizzo MJ, Straub AC, Lampe PD, Leitinger N, Isakson BE: Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation. Am J Pathol 2009;175:916–924.
  52. Lee MY, Garvey SM, Baras AS, Lemmon JA, Gomez MF, Schoppee Bortz PD, Daum G, LeBoeuf RC, Wamhoff BR: Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells. Hum Mol Genet 2010;19:468–479.
  53. Kadl A, Meher AK, Sharma PR, Lee MY, Doran AC, Johnstone SR, Elliott MR, Gruber F, Han J, Chen W, Kensler T, Ravichandran KS, Isakson BE, Wamhoff BR, Leitinger N: Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via NRF2. Circ Res 2010;107:737–746.
  54. Heberlein KR, Straub AC, Best AK, Greyson MA, Looft-Wilson RC, Sharma PR, Meher A, Leitinger N, Isakson BE: Plasminogen activator inhibitor-1 regulates myoendothelial junction formation. Circ Res 2010;106:1092–1102.
  55. Haddock RE, Grayson TH, Brackenbury TD, Meaney KR, Neylon CB, Sandow SL, Hill CE: Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40. Am J Physiol Heart Circ Physiol 2006;291:H2047–H2056.
  56. Sandow SL, Neylon CB, Chen MX, Garland CJ: Spatial separation of endothelial small- and intermediate-conductance calcium-activated potassium channels (K(Ca)) and connexins: possible relationship to vasodilator function? J Anat 2006;209:689–698.
  57. Sandow SL, Haddock RE, Hill CE, Chadha PS, Kerr PM, Welsh DG, Plane F: What’s where and why at a vascular myoendothelial microdomain signalling complex. Clin Exp Pharmacol Physiol 2009;36:67–76.
  58. Heberlein KR, Straub AC, Isakson BE: The myoendothelial junction: breaking through the matrix? Microcirculation 2009;16:307–322.
  59. Isakson BE, Duling BR: Heterocellular contact at the myoendothelial junction influences gap junction organization. Circ Res 2005;97:44–51.
  60. Straub AC, Billaud M, Johnstone SR, Best AK, Yemen S, Dwyer ST, Looft-Wilson R, Lysiak JJ, Gaston B, Palmer L, Isakson BE: Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall. Arterioscler Thromb Vasc Biol 2011;31:399–407.
  61. Chekeni FB, Elliott MR, Sandilos JK, Walk SF, Kinchen JM, Lazarowski ER, Armstrong AJ, Penuela S, Laird DW, Salvesen GS, Isakson BE, Bayliss DA, Ravichandran KS: Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 2010;467:863–867.
  62. Hopwood AM, Burnstock G: ATP mediates coronary vasoconstriction via P2X-purinoceptors and coronary vasodilatation via P2Y-purinoceptors in the isolated perfused rat heart. Eur J Pharmacol 1987;136:49–54.
  63. Ralevic V, Mathie RT, Alexander B, Burnstock G: Characterization of P2X- and P2Y-purinoceptors in the rabbit hepatic arterial vasculature. Br J Pharmacol 1991;103:1108–1113.
  64. Kennedy C, Delbro D, Burnstock G: P2-purinoceptors mediate both vasodilation (via the endothelium) and vasoconstriction of the isolated rat femoral artery. Eur J Pharmacol 1985;107:161–168.
  65. Buvinic S, Briones R, Huidobro-Toro JP: P2Y(1) and P2Y(2) receptors are coupled to the NO/CGMP pathway to vasodilate the rat arterial mesenteric bed. Br J Pharmacol 2002;136:847–856.
  66. Raqeeb A, Sheng J, Ao N, Braun AP: Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells. Cell Calcium 2011;49:240–248.
  67. Woodward HN, Anwar A, Riddle S, Taraseviciene-Stewart L, Fragoso M, Stenmark KR, Gerasimovskaya EV: Pi3k, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells. Am J Physiol Lung Cell Mol Physiol 2009;297:L954–L964.
  68. Mohrman DE, Heller LJ: Cardiovascular Physiology, ed 3. New York, McGraw Hill, 1991, pp 175–188.
  69. Kukulski F, Ben Yebdri F, Lecka J, Kauffenstein G, Levesque SA, Martin-Satue M, Sevigny J: Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration. Cytokine 2009;46:166–170.
  70. Bours MJ, Dagnelie PC, Giuliani AL, Wesselius A, Di Virgilio F: P2 receptors and extracellular ATP: a novel homeostatic pathway in inflammation. Front Biosci (Schol Ed) 2011;3:1443–1456.
    External Resources
  71. Erlinge D, Burnstock G: P2 receptors in cardiovascular regulation and disease. Purinergic Signal 2008;4:1–20.
  72. Bours MJ, Swennen EL, Di Virgilio F, Cronstein BN, Dagnelie PC: Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol Ther 2006;112:358–404.
  73. Bruzzone R, Barbe MT, Jakob NJ, Monyer H: Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in xenopus oocytes. J Neurochem 2005;92:1033–1043.

Author Contacts

Dr. Brant E. Isakson

Robert M. Berne Cardiovascular Research Center, University of Virginia

PO Box 801394

Charlottesville, VA 22908 (USA)

Tel. +1 434 924 2093, E-Mail brant@virginia.edu


Article / Publication Details

First-Page Preview
Abstract of Research Paper

Received: December 31, 2011
Accepted: April 04, 2012
Published online: June 26, 2012
Issue release date: August 2012

Number of Print Pages: 12
Number of Figures: 7
Number of Tables: 0

ISSN: 1018-1172 (Print)
eISSN: 1423-0135 (Online)

For additional information: https://www.karger.com/JVR


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References

  1. Panchin YV: Evolution of gap junction proteins – the pannexin alternative. J Exp Biol 2005;208:1415–1419.
  2. Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O, Tiunova A, Born TL, Usman N, Staroverov D, Lukyanov S, Panchin Y: The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins. Genomics 2004;83:706–716.
  3. Bao L, Locovei S, Dahl G: Pannexin membrane channels are mechanosensitive conduits for ATP. FEBS Lett 2004;572:65–68.
  4. Ray A, Zoidl G, Weickert S, Wahle P, Dermietzel R: Site-specific and developmental expression of pannexin1 in the mouse nervous system. Eur J Neurosci 2005;21:3277–3290.
  5. Santiago MF, Veliskova J, Patel NK, Lutz SE, Caille D, Charollais A, Meda P, Scemes E: Targeting pannexin1 improves seizure outcome. PloS One 2011;6:e25178.
  6. Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H: Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci USA 2003;100:13644–13649.
  7. Barbe MT, Monyer H, Bruzzone R: Cell-cell communication beyond connexins: the pannexin channels. Physiology (Bethesda) 2006;21:103–114.
  8. Bond SR, Lau A, Penuela S, Sampaio AV, Underhill TM, Laird DW, Naus CC: Pannexin 3 is a novel target for Runx2, expressed by osteoblasts and mature growth plate chondrocytes. J Bone Miner Res 2011;26:2911–2922.
  9. Penuela S, Celetti SJ, Bhalla R, Shao Q, Laird DW: Diverse subcellular distribution profiles of pannexin 1 and pannexin 3. Cell Commun Adhes 2008;15:133–142.
  10. Dahl G, Locovei S: Pannexin: To gap or not to gap, is that a question? IUBMB Life 2006;58:409–419.
  11. Pfahnl A, Dahl G: Gating of cx46 gap junction hemichannels by calcium and voltage. Pflugers Arch 1999;437:345–353.
  12. Quist AP, Rhee SK, Lin H, Lal R: Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol 2000;148:1063–1074.
  13. Li H, Liu TF, Lazrak A, Peracchia C, Goldberg GS, Lampe PD, Johnson RG: Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells. J Cell Biol 1996;134:1019–1030.
  14. Qiu F, Dahl G: A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. Am J Physiol Cell Physiol 2009;296:C250–C255.
  15. Ransford GA, Fregien N, Qiu F, Dahl G, Conner GE, Salathe M: Pannexin 1 contributes to ATP release in airway epithelia. Am J Resp Cell Mol Biol 2009;41:525–534.
  16. Sridharan M, Adderley SP, Bowles EA, Egan TM, Stephenson AH, Ellsworth ML, Sprague RS: Pannexin 1 is the conduit for low oxygen tension-induced ATP release from human erythrocytes. Am J Physiol Heart Circ Physiol 2011;299:H1146–H1152.
    External Resources
  17. Woehrle T, Yip L, Elkhal A, Sumi Y, Chen Y, Yao Y, Insel PA, Junger WG: Pannexin-1 hemichannel-mediated ATP release together with p2x1 and p2x4 receptors regulate T-cell activation at the immune synapse. Blood 2010;116:3475–3484.
  18. Kauffenstein G, Drouin A, Thorin-Trescases N, Bachelard H, Robaye B, D’Orleans-Juste P, Marceau F, Thorin E, Sevigny J: NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse. Cardiovasc Res 2010;85:204–213.
  19. Kauffenstein G, Furstenau CR, D’Orleans-Juste P, Sevigny J: The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation. Br J Pharmacol 2010;159:576–585.
  20. Forrester T, Lind AR: Identification of adenosine triphosphate in human plasma and the concentration in the venous effluent of forearm muscles before, during and after sustained contractions. J Physiol 1969;204:347–364.
  21. Mortensen SP, Gonzalez-Alonso J, Bune LT, Saltin B, Pilegaard H, Hellsten Y: ATP-induced vasodilation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins, and adenosine. Am J Physiol Regul Integr Comp Physiol 2009;296:R1140–R1148.
  22. Bodin P, Burnstock G: Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells. Experientia 1995;51:256–259.
  23. Bergfeld GR, Forrester T: Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia. Cardiovasc Res 1992;26:40–47.
  24. Bodin P, Bailey D, Burnstock G: Increased flow-induced ATP release from isolated vascular endothelial cells but not smooth muscle cells. Br J Pharmacol 1991;103:1203–1205.
  25. Sneddon P, Burnstock G: ATP as a co-transmitter in rat tail artery. Eur J Pharmacol 1984;106:149–152.
  26. Sprague RS, Ellsworth ML, Stephenson AH, Kleinhenz ME, Lonigro AJ: Deformation-induced ATP release from red blood cells requires CFTR activity. Am J Physiol 1998;275:H1726–H1732.
  27. Sneddon P, Burnstock G: Inhibition of excitatory junction potentials in guinea-pig vas deferens by alpha, beta-methylene-ATP: further evidence for ATP and noradrenaline as cotransmitters. Eur J Pharmacol 1984;100:85–90.
  28. Burnstock G: Noradrenaline and ATP as cotransmitters in sympathetic nerves. Neurochem Int 1990;17:357–368.
  29. Sneddon P, Westfall DP: Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens. J Physiol 1984;347:561–580.
  30. Sprague RS, Hanson MS, Achilleus D, Bowles EA, Stephenson AH, Sridharan M, Adderley S, Procknow J, Ellsworth ML: Rabbit erythrocytes release ATP and dilate skeletal muscle arterioles in the presence of reduced oxygen tension. Pharmacol Rep 2009;61:183–190.
  31. Lew MJ, White TD: Release of endogenous ATP during sympathetic nerve stimulation. Br J Pharmacol 1987;92:349–355.
  32. Bodin P, Burnstock G: ATP-stimulated release of ATP by human endothelial cells. J Cardiovasc Pharmacol 1996;27:872–875.
  33. Moser TL, Kenan DJ, Ashley TA, Roy JA, Goodman MD, Misra UK, Cheek DJ, Pizzo SV: Endothelial cell surface f1-f0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin. Proc Natl Acad Sci USA 2001;98:6656–6661.
  34. Bodin P, Burnstock G: Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol 2001;38:900–908.
  35. Yamamoto K, Shimizu N, Obi S, Kumagaya S, Taketani Y, Kamiya A, Ando J: Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells. Am J Physiol Heart Circ Physiol 2007;293:H1646–H1653.
  36. Billaud M, Lohman AW, Straub AC, Looft-Wilson R, Johnstone SR, Araj CA, Best AK, Chekeni FB, Ravichandran KS, Penuela S, Laird DW, Isakson BE: Pannexin1 regulates alpha1-adrenergic receptor-mediated vasoconstriction. Circ Res 2011;109:80–85.
  37. Goedecke S, Roderigo C, Rose CR, Rauch BH, Goedecke A, Schrader J: Thrombin-induced ATP release from human umbilical vein endothelial cells. Am J Physiol Cell Physiol 2012;302:C915–C923.
  38. Mustafa SJ, Morrison RR, Teng B, Pelleg A: Adenosine receptors and the heart: role in regulation of coronary blood flow and cardiac electrophysiology. Handb Exp Pharmacol 2009:161–188.
    External Resources
  39. Berne RM: Cardiac nucleotides in hypoxia: possible role in regulation of coronary blood flow. Am J Physiol 1963;204:317–322.
  40. Burnstock G: Control of vascular tone by purines and pyrimidines. Br J Pharmacol 2010;161:527–529.
  41. Winbury MM, Papierski DH, Hemmer ML, Hambourger WE: Coronary dilator action of the adenine-ATP series. J Pharmacol Exp Ther 1953;109:255–260.
  42. Burnstock G: Dual control of vascular tone and remodelling by ATP released from nerves and endothelial cells. Pharmacol Rep 2008;60:12–20.
  43. Jones RD, Berne RM: Evidence for a metabolic mechanism in autoregulation of blood flow in skeletal muscle. Circ Res 1965;17:540–554.
  44. Drury AN, Szent-Gyorgyi A: The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. J Physiol 1929;68:213–237.
  45. Rubio R, Berne RM: Release of adenosine by the normal myocardium in dogs and its relationship to the regulation of coronary resistance. Circ Res 1969;25:407–415.
  46. Dobson JG Jr, Rubio R, Berne RM: Role of adenine nucleotides, adenosine, and inorganic phosphate in the regulation of skeletal muscle blood flow. Circ Res 1971;29:375–384.
  47. Nishiyama A, Rahman M, Inscho EW: Role of interstitial ATP and adenosine in the regulation of renal hemodynamics and microvascular function. Hypertens Res 2004;27:791–804.
  48. Erlinge D: Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol 1998;31:1–8.
  49. Wang DJ, Huang NN, Heppel LA: Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells. J Cellul Physiol 1992;153:221–233.
  50. Penuela S, Bhalla R, Nag K, Laird DW: Glycosylation regulates pannexin intermixing and cellular localization. Mol Biol Cell 2009;20:4313–4323.
  51. Johnstone SR, Ross J, Rizzo MJ, Straub AC, Lampe PD, Leitinger N, Isakson BE: Oxidized phospholipid species promote in vivo differential cx43 phosphorylation and vascular smooth muscle cell proliferation. Am J Pathol 2009;175:916–924.
  52. Lee MY, Garvey SM, Baras AS, Lemmon JA, Gomez MF, Schoppee Bortz PD, Daum G, LeBoeuf RC, Wamhoff BR: Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells. Hum Mol Genet 2010;19:468–479.
  53. Kadl A, Meher AK, Sharma PR, Lee MY, Doran AC, Johnstone SR, Elliott MR, Gruber F, Han J, Chen W, Kensler T, Ravichandran KS, Isakson BE, Wamhoff BR, Leitinger N: Identification of a novel macrophage phenotype that develops in response to atherogenic phospholipids via NRF2. Circ Res 2010;107:737–746.
  54. Heberlein KR, Straub AC, Best AK, Greyson MA, Looft-Wilson RC, Sharma PR, Meher A, Leitinger N, Isakson BE: Plasminogen activator inhibitor-1 regulates myoendothelial junction formation. Circ Res 2010;106:1092–1102.
  55. Haddock RE, Grayson TH, Brackenbury TD, Meaney KR, Neylon CB, Sandow SL, Hill CE: Endothelial coordination of cerebral vasomotion via myoendothelial gap junctions containing connexins 37 and 40. Am J Physiol Heart Circ Physiol 2006;291:H2047–H2056.
  56. Sandow SL, Neylon CB, Chen MX, Garland CJ: Spatial separation of endothelial small- and intermediate-conductance calcium-activated potassium channels (K(Ca)) and connexins: possible relationship to vasodilator function? J Anat 2006;209:689–698.
  57. Sandow SL, Haddock RE, Hill CE, Chadha PS, Kerr PM, Welsh DG, Plane F: What’s where and why at a vascular myoendothelial microdomain signalling complex. Clin Exp Pharmacol Physiol 2009;36:67–76.
  58. Heberlein KR, Straub AC, Isakson BE: The myoendothelial junction: breaking through the matrix? Microcirculation 2009;16:307–322.
  59. Isakson BE, Duling BR: Heterocellular contact at the myoendothelial junction influences gap junction organization. Circ Res 2005;97:44–51.
  60. Straub AC, Billaud M, Johnstone SR, Best AK, Yemen S, Dwyer ST, Looft-Wilson R, Lysiak JJ, Gaston B, Palmer L, Isakson BE: Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall. Arterioscler Thromb Vasc Biol 2011;31:399–407.
  61. Chekeni FB, Elliott MR, Sandilos JK, Walk SF, Kinchen JM, Lazarowski ER, Armstrong AJ, Penuela S, Laird DW, Salvesen GS, Isakson BE, Bayliss DA, Ravichandran KS: Pannexin 1 channels mediate ‘find-me’ signal release and membrane permeability during apoptosis. Nature 2010;467:863–867.
  62. Hopwood AM, Burnstock G: ATP mediates coronary vasoconstriction via P2X-purinoceptors and coronary vasodilatation via P2Y-purinoceptors in the isolated perfused rat heart. Eur J Pharmacol 1987;136:49–54.
  63. Ralevic V, Mathie RT, Alexander B, Burnstock G: Characterization of P2X- and P2Y-purinoceptors in the rabbit hepatic arterial vasculature. Br J Pharmacol 1991;103:1108–1113.
  64. Kennedy C, Delbro D, Burnstock G: P2-purinoceptors mediate both vasodilation (via the endothelium) and vasoconstriction of the isolated rat femoral artery. Eur J Pharmacol 1985;107:161–168.
  65. Buvinic S, Briones R, Huidobro-Toro JP: P2Y(1) and P2Y(2) receptors are coupled to the NO/CGMP pathway to vasodilate the rat arterial mesenteric bed. Br J Pharmacol 2002;136:847–856.
  66. Raqeeb A, Sheng J, Ao N, Braun AP: Purinergic P2Y2 receptors mediate rapid Ca(2+) mobilization, membrane hyperpolarization and nitric oxide production in human vascular endothelial cells. Cell Calcium 2011;49:240–248.
  67. Woodward HN, Anwar A, Riddle S, Taraseviciene-Stewart L, Fragoso M, Stenmark KR, Gerasimovskaya EV: Pi3k, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells. Am J Physiol Lung Cell Mol Physiol 2009;297:L954–L964.
  68. Mohrman DE, Heller LJ: Cardiovascular Physiology, ed 3. New York, McGraw Hill, 1991, pp 175–188.
  69. Kukulski F, Ben Yebdri F, Lecka J, Kauffenstein G, Levesque SA, Martin-Satue M, Sevigny J: Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration. Cytokine 2009;46:166–170.
  70. Bours MJ, Dagnelie PC, Giuliani AL, Wesselius A, Di Virgilio F: P2 receptors and extracellular ATP: a novel homeostatic pathway in inflammation. Front Biosci (Schol Ed) 2011;3:1443–1456.
    External Resources
  71. Erlinge D, Burnstock G: P2 receptors in cardiovascular regulation and disease. Purinergic Signal 2008;4:1–20.
  72. Bours MJ, Swennen EL, Di Virgilio F, Cronstein BN, Dagnelie PC: Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol Ther 2006;112:358–404.
  73. Bruzzone R, Barbe MT, Jakob NJ, Monyer H: Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in xenopus oocytes. J Neurochem 2005;92:1033–1043.
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