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

Vascular Endothelium

Angiogenesis and vasculogenesis Blood pressure regulation (Vascular Tone) Haemostasis (Anticoagulant barrier) Inflamation (immunological responses) Transport function (paracellular permeability) Vascular Endothelium Angiopathies Hypertension Hypotension Thromboses Atherosclerosis Factors released FGFvWF, Tromboxan VEGFThrombomodulin ProstaglandinCAM NOProstacyclin EndothelinTransport proteins Dysfunction

PLC PIP2 IP 3 DAG ER Nucleus GPCR RCIC ↑ [Ca 2+ ] SERCA P M C A ↑ [Ca 2+ ] Stretch channels NCX SOC Ca 2+ Homeostasis

Injury results in Injury results in Injury impairs endothelial function Injury impairs endothelial function removal of contact inhibition removal of contact inhibition release of paracrine stimulatory signals release of paracrine stimulatory signals transient increase in intracellular Ca 2+ transient increase in intracellular Ca 2+ disrupting barrier function enhancing vasoconstriction, coagulation, leukocyte adhesion & smooth muscle cells proliferation Normaly occurs in healthy organismsNormaly occurs in healthy organisms

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

5 sec nm Filter wheel Ratio (F 340 /F 380) Intracellular Ca 2+ Concentration

A) Injury provokes a Ca 2+ wave characterized by two phases, peak and plateau 250 s  Ratio (0.1)  Distance from injured zone B) Calcium signal is larger in the cells next to the injured zone Plateau Peak

C) Peak and plateau phases are sensitive to the extracellular concentration of Ca 2+

500 s  Ratio (0.2) D) ATP signaling pathway is involved in the Ca2+ response to injury P2-R P2Y (Ca2+ Release) IP 3 pathway Suramin  P2X / P2Y receptors antagonist

MRS2179  P2Y 1 Antagonist2-MeSAMP  P2Y 12,13 Antagonist P2 Receptors P2Y P2X ATP ADP (Metabotropic) (Ionotropic) MRS MeSAMP a,b-MeATP

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

E) Ca 2+ influx during plateau phase occurs through GAP juctions Gd 3+  Unspecific SOC blocker BTP-2  Specific SOC blocker

E) Ca 2+ influx during plateau phase occurs through GAP juctions

1.CCE (SOC) 2.GAP Junction 3.Stretch channels Possible ways of Ca 2+ entry ? ~80% P2Y 1 (~19%) P2Y 12,13 (~22%) P2Y Sensitive to suramin P2X (~15%)

Capacitative Calcium Entry (SOC) Stretch channels

Gap Juntions

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

[Ca 2+ ] i L-Arginine Nitric Oxide Citruline NOS e DAF  Fluorescent dye Indicator of NO production Emission: 510nm Excitation: 490nm

Nitric Oxide production

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.