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Ca 2+ signaling in injured in situ endothelium of rat aorta

Ca 2+ signaling in injured in situ endothelium of rat aorta. Vascular Endothelium. Vascular Endothelium. Angiogenesis and vasculogenesis Blood pressure regulation (Vascular Tone) Haemostasis (Anticoagulant barrier) Inflamation (immunological responses)

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Ca 2+ signaling in injured in situ endothelium of rat aorta

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  1. Ca2+ signaling in injured in situ endothelium of rat aorta

  2. Vascular Endothelium

  3. Vascular Endothelium • Angiogenesis and vasculogenesis • Blood pressure regulation (Vascular Tone) • Haemostasis (Anticoagulant barrier) • Inflamation (immunological responses) • Transport function (paracellular permeability) Dysfunction Factors released FGF vWF, Tromboxan VEGF Thrombomodulin Prostaglandin CAM NO Prostacyclin Endothelin Transport proteins • Angiopathies • Hypertension • Hypotension • Thromboses • Atherosclerosis

  4. SERCA SOC P M C A ↑ [Ca2+] DAG NCX PIP2 PLC ER IP3 Nucleus GPCR ↑ [Ca2+] RCIC Stretch channels Ca2+ Homeostasis

  5. Normaly occurs in healthy organisms • Injury results in • removal of contact inhibition • release of paracrine stimulatory signals • transient increase in intracellular Ca2+ • Injury impairs endothelial function • disrupting barrier function • enhancing vasoconstriction, coagulation, • leukocyte adhesion & smooth muscle cells • proliferation

  6. Wistar rats Fura 2/AM 16mM (1hr) 15 min In PSS Upright epifluorescence microscope Excitation = 340 / 380 Emmision = 510

  7. Ratio (F340/F380) Intracellular Ca2+ Concentration 510nm 340 5 sec 380 Filter wheel

  8. A) Injury provokes a Ca2+ wave characterized by two phases, peak and plateau D Ratio (0.1) 250 s Peak Plateau B) Calcium signal is larger in the cells next to the injured zone D Distance from injured zone

  9. C) Peak and plateau phases are sensitive to the extracellular concentration of Ca2+

  10. D Ratio (0.2) 500 s D) ATP signaling pathway is involved in the Ca2+ response to injury P2Y (Ca2+ Release) P2-R SuraminP2X / P2Y receptors antagonist IP3 pathway

  11. (Metabotropic) ATP ADP P2Y P2 Receptors P2X (Ionotropic) MRS2179P2Y1 Antagonist 2-MeSAMPP2Y12,13 Antagonist MRS2179 2-MeSAMP a,b-MeATP

  12. (Metabotropic) ATP ADP P2Y P2 Receptors P2X (Ionotropic) a,b,-MeATP Preferential agonist of P2X

  13. E) Ca2+ influx during plateau phase occurs through GAP juctions Gd3+Unspecific SOC blocker BTP-2Specific SOC blocker

  14. E) Ca2+ influx during plateau phase occurs through GAP juctions

  15. E) Ca2+ influx during plateau phase occurs through GAP juctions

  16. ? P2X (~15%) ~80% P2Y1 (~19%) P2Y12,13 (~22%) P2Y Sensitive to suramin Possible ways of Ca2+ entry • CCE (SOC) • GAP Junction • Stretch channels

  17. Capacitative Calcium Entry (SOC) Stretch channels

  18. Gap Juntions

  19. Stretch channels (~6%) ~40% GAP Juntions (~20-25%) P2X channels (~15%)

  20. Citruline NOSe L-Arginine Nitric Oxide [Ca2+]i DAF Fluorescent dye Indicator of NO production Emission: 510nm Excitation: 490nm

  21. Nitric Oxide production

  22. Nitric Oxide production

  23. Nitric Oxide production

  24. Nitric Oxide production

  25. Nitric Oxide production

  26. Conclusions These results suggest that endothelium scraping: • i) causes a short-lasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells. • ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca2+ entry. • iii) increase the nitric oxide production due to calcium influx through gap junctions.

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