Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a fluid mechanical force generated by flowing blood but, although its release has a crucial role in controlling a variety of vascular functions by activating purinergic receptors, the mechanism of ATP release has never been established. To analyze the dynamics of ATP release, we developed a novel chemiluminescence imaging method by using cell-surface-attached firefly luciferase and a CCD camera. Upon stimulation of shear stress, cultured human pulmonary artery ECs simultaneously released ATP in two different manners, a highly concentrated, localized manner and a less concentrated, diffuse manner. The localized ATP release occurred at caveolin-1-rich regions of the cell membrane, and was blocked by caveolin-1 knockdown with siRNA and the depletion of plasma membrane cholesterol with methyl-β-cyclodexrin, indicating involvement of caveolae in localized ATP release. Ca2+ imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca2+ concentration and the subsequent Ca2+ wave that originated from the same sites as the localized ATP release. These findings suggest that localized ATP release at caveolae triggers shear-stress-dependent Ca2+ signaling in ECs.

[1]  J. Ando,et al.  Cell-surface-localized ATP detection with immobilized firefly luciferase. , 2006, Analytical biochemistry.

[2]  P. D. Bell,et al.  Macula densa cell signaling involves ATP release through a maxi anion channel , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  P. Davies,et al.  Flow-mediated endothelial mechanotransduction. , 1995, Physiological reviews.

[4]  Z Wang,et al.  Direct observation of calcium-independent intercellular ATP signaling in astrocytes. , 2000, Analytical chemistry.

[5]  J. Ando,et al.  Fluid Shear Stress Activates Ca2+ Influx Into Human Endothelial Cells via P2X4 Purinoceptors , 2000, Circulation research.

[6]  G. Dubyak,et al.  Colocalization of ATP Release Sites and Ecto-ATPase Activity at the Extracellular Surface of Human Astrocytes* , 2003, Journal of Biological Chemistry.

[7]  G. Dubyak,et al.  Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase. , 1999, American journal of physiology. Cell physiology.

[8]  J. Ando,et al.  Fluid Shear Stress Activates Ca 2 1 Influx Into Human Endothelial Cells via P 2 X 4 Purinoceptors , 2000 .

[9]  T. Takano,et al.  Intercellular calcium signaling mediated by point-source burst release of ATP , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Seiko F. Okada,et al.  Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia*♦ , 2006, Journal of Biological Chemistry.

[11]  M. Drab,et al.  Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels. , 2006, The Journal of clinical investigation.

[12]  J. Ando,et al.  Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells. , 2007, American journal of physiology. Heart and circulatory physiology.

[13]  G Burnstock,et al.  Evidence That Release of Adenosine Triphosphate From Endothelial Cells During Increased Shear Stress Is Vesicular , 2001, Journal of cardiovascular pharmacology.

[14]  Sonja Hatz,et al.  Microelectrode biosensor for real-time measurement of ATP in biological tissue. , 2005, Analytical chemistry.

[15]  G. Burnstock,et al.  Endothelial cells cultured from human umbilical vein release ATP, substance P and acetylcholine in response to increased flow , 1990, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[16]  B. Helmke,et al.  Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells. , 2003, Biophysical journal.

[17]  H. Jo,et al.  Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase. , 2000, American journal of physiology. Heart and circulatory physiology.

[18]  J. Ando,et al.  Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Kimiko Yamamoto,et al.  Vascular mechanobiology: endothelial cell responses to fluid shear stress. , 2009, Circulation journal : official journal of the Japanese Circulation Society.

[20]  Richard G. W. Anderson,et al.  Role of plasmalemmal caveolae in signal transduction. , 1998, American journal of physiology. Lung cellular and molecular physiology.

[21]  G. Yegutkin.  Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. , 2008, Biochimica et biophysica acta.

[22]  Amal K. Dutta,et al.  Detecting ATP Release by a Biosensor Method , 2004, Science's STKE.

[23]  F. Di Virgilio,et al.  A novel recombinant plasma membrane-targeted luciferase reveals a new pathway for ATP secretion. , 2005, Molecular biology of the cell.

[24]  J. Ando,et al.  P2X4 receptors mediate ATP-induced calcium influx in human vascular endothelial cells , 2000 .

[25]  D. Benos,et al.  CFTR is a conductance regulator as well as a chloride channel. , 1999, Physiological reviews.

[26]  D. Adriaensen,et al.  Purinergic signaling in the pulmonary neuroepithelial body microenvironment unraveled by live cell imaging , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[27]  C. Naus,et al.  Intercellular Calcium Signaling in Astrocytes via ATP Release through Connexin Hemichannels* , 2002, The Journal of Biological Chemistry.

[28]  P. Oh,et al.  In Situ Flow Activates Endothelial Nitric Oxide Synthase in Luminal Caveolae of Endothelium with Rapid Caveolin Dissociation and Calmodulin Association* , 1998, The Journal of Biological Chemistry.

[29]  J. Ando,et al.  Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice , 2006, Nature Medicine.

[30]  B. Jena,et al.  Continuous detection of extracellular ATP on living cells by using atomic force microscopy. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Paul A Insel,et al.  A novel method using fluorescence microscopy for real-time assessment of ATP release from individual cells. , 2007, American journal of physiology. Cell physiology.

[32]  Y. Okada,et al.  Cell surface measurements of ATP release from single pancreatic β cells using a novel biosensor technique , 1998, Pflügers Archiv.

[33]  Geoffrey Burnstock,et al.  The double life of ATP. , 2009, Scientific American.

[34]  E. Gratton,et al.  Visualizing lipid structure and raft domains in living cells with two-photon microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Sabirov,et al.  Wide nanoscopic pore of maxi-anion channel suits its function as an ATP-conductive pathway. , 2004, Biophysical journal.

[36]  Kimiko Yamamoto,et al.  Endogenously released ATP mediates shear stress-induced Ca2+ influx into pulmonary artery endothelial cells. , 2003, American journal of physiology. Heart and circulatory physiology.