Ca2+ 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

FGF vWF, TromboxanVEGF ThrombomodulinProstaglandin CAMNO ProstacyclinEndothelin Transport proteins

Dysfunction

PLCPIP2

IP3

DAG

ER

NucleusGPCR

RCIC

↑ [Ca2+]

SERCA

P M C A

↑ [Ca2+]

Stretch channels

NCX

SOC

Ca2+ Homeostasis

• Injury results inInjury results in

• Injury impairs endothelial functionInjury impairs endothelial function

• removal of contact inhibitionremoval of contact inhibition• release of paracrine stimulatory signalsrelease of paracrine stimulatory signals• transient increase in intracellular Catransient increase in intracellular Ca2+2+

• disrupting barrier function• enhancing vasoconstriction, coagulation, leukocyte adhesion & smooth muscle cells proliferation

•Normaly occurs in healthy organismsNormaly occurs in healthy organisms

Fura 2/AM16M(1hr)

15 minIn PSSUpright epifluorescence microscope

Excitation = 340 / 380Emmision = 510

Wistar rats

5 sec

340

380

0 250 500 750 1000 1250 1500

1.0

1.2

1.4

1.6

1.8

2.0

Time (s)

F3

40 /

F3

80

510nm

Filter wheel

Ratio

(F340/F380)

Intracellular Ca2+

Concentration

0 250 500 750 10001.0

1.2

1.4

1.6

1.8

Time (s)

(F3

40 /

F3

80

)

A) Injury provokes a Ca2+ wave characterized by two phases, peak and plateau

250 s

R

atio

(0

.1)

Distance from injured zone

B) Calcium signal is larger in the cells next to the injured zone

Plateau

Peak

0 250 500 750 1000

1.0

1.2

1.4

1.6

Control

Ca2+ Free

Time(s)

Rat

ioC) Peak and plateau phases are sensitive to the extracellular concentration of Ca2+

0 400 800 1200 16000.75

0.80

0.85

0.90

0 Ca2+

Time (s)

Rat

io

0 400 800 1200 1600 20000.80

0.85

0.90

0.95

7.5 mM Ca2+

Time (s)

Rat

io

0 100 200 300 400 5000.3

0.5

0.7

0.9 0 Ca2++Suramin

Time (s)

Rat

io

Suramin

Control

500 s

R

atio

(0

.2)

D) ATP signaling pathway is involved in the Ca2+ response to injury

200 600 1000 1400 18000.6

0.7

0.8

0.90 Ca2+ + U73122

Time (s)

Rat

io

P2-R P2Y (Ca2+ Release)

IP3 pathway

SuraminP2X / P2Y receptors antagonist

100 300 500 7000.8

1.0

1.2

1.4

1.6CONTROLMRS2179

Time (s)

Rat

io

100 300 500 7000.8

1.0

1.2

1.4

1.6CONTROL2-MeSAMP

Time (s)

Rat

io

MRS2179P2Y1 Antagonist 2-MeSAMPP2Y12,13 Antagonist

P2 ReceptorsP2Y

P2X

ATP

ADP

(Metabotropic)

(Ionotropic)

MRS2179 2-MeSAMP

a,b-MeATP

100 300 500 700

0.9

1.1

1.3

1.5

CONTROL

,,-MeATP

Time (s)

Ra

tio

P2 ReceptorsP2Y

P2X

ATP

ADP

(Metabotropic)

(Ionotropic)

a,b,-MeATP Preferential agonist of P2X

0 250 500 750 1000 1250

1.0

1.2

1.4

1.6

1.8

BTP-2

Time (s)

Rat

io

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

0 100 200 300 400 500 6000.6

0.8

1.0

1.2

Gd3+

Time (s)

Rat

io

Gd3+Unspecific SOC blocker

BTP-2Specific SOC blocker

0 500 1000 1500 2000 2500

0.9

1.1

1.3

1.51.5mM Ca2+

Ca2+ Free

CPA

Control

BTP-2

Time (s)

Rat

io

0 200 400 600 8000.9

1.1

1.3

1.5

1.7

1.9ATP

CONTROL

BTP-2

Time (s)

Rat

io

0 200 400 600 800 10000.85

1.05

1.25

Palmitoleic acid

Time (s)

Rat

io

50 200 350 500 6500.8

1.0

1.2

1.4

Heptanol

Time (s)

Rat

io

100 300 500 700

1.0

1.1

1.2

1.3

Octanol

Time (s)

Rat

io

100 300 500 700

0.8

1.0

1.2

1.4

1.6

Oleamide

Time (s)

Rat

io

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

Palm

itole

ic A

c.

Hepta

nol

Oleam

ide

Octan

ol0

25

50

75

100

n = 65 n = 51 n = 41n = 47

% o

f in

hib

itio

n(P

late

au a

mp

litu

de)

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

PSS F

ree

2+

Ca Suram

in

Suram

in in

0 C

a2+

MRS 2

179

2-M

eSAM

P

0

25

50

75

100

125

n= 231

n = 76n = 33

n = 33n = 29

**

***

** ***

29

***

% o

f in

jury

res

po

nse

1. CCE (SOC)2. GAP Junction3. Stretch channels

Possible ways of Ca2+ entry

?

~80%P2Y1 (~19%)

P2Y12,13 (~22%)

P2Y

Sensitive to suramin

P2X (~15%)

100 300 500 700

0.9

1.1

1.3

1.5

1.7CONTROLBTP-2

Time(s)

Rat

io

Capacitative Calcium Entry(SOC)

Stretch channels

100 300 500 7000.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7 ControlStretching

Time (s)

Rat

io

100 300 5000.8

1.0

1.2

1.4

1.6CONTROLOCTANOL

Time (s)

Rat

io

100 300 500 700

0.9

1.1

1.3

1.5

1.7

1.9CONTROLOLEAMIDE

Time (s)R

atio

Gap Juntions

PSS F

ree

2+

Ca Suram

in

,-MeA

TP

,

BTP2

Oleam

ide

Octan

ol

Stretc

hing

0

25

50

75

100

125

231

76

29

5280

32 4843

***

***

** *****

***% o

f in

jury

res

po

nse

GAP Juntions (~20-25%)

P2X channels (~15%)

Stretch channels (~6%)

~40%

[Ca2+]i

L-Arginine

Nitric Oxide

Citruline

NOSe

DAF Fluorescent dyeIndicator of NO production Emission: 510nmExcitation: 490nm

200 500 800 1100 1400 1700-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

ATP

CONTROL

L-NAME

Time (s)

Flu

ore

scen

cein

crea

se

CONTROL

L-NAM

E

0.00

0.01

0.02

0.03

41

41***F

luo

resc

ence

incr

ease

Nitric Oxide production

0 500 1000 1500-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

CONTROL

L-NAME

Time (s)

Flu

ore

scen

cein

crea

se

Nitric Oxide production

0 500 1000 1500-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

CONTROL

Ca2+ FREE

Time (s)

Flu

ore

scen

cein

crea

se

0 500 1000 1500-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

CONTROL

THAPSIGARGIN

Time (s)F

luo

res

ce

nce

incr

ease

Nitric Oxide production

0 500 1000 1500-0.01

0.00

0.01

0.02

0.03

0.04

0.05

0.06

CONTROL

OCTANOL

Time (s)

Flu

ore

scen

cein

crea

se

Nitric Oxide production

PSS

L-NAM

E F

ree

2+

Ca

Thapsi

gargin

Oct

anol

0.00

0.01

0.02

0.03

0.04

233

151

50

107 135

******

*

***

Flu

ore

scen

ce i

ncr

ease

(a.u

)

Nitric Oxide production

Conclusions

These results suggest that endothelium scraping: 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.

0 500 1000 1500 2000 2500

0.9

1.1

1.3

1.51.5mM Ca2+

Ca2+ Free

CPA

Control

BTP-2

Time (s)

Rat

io

0 200 400 600 8000.9

1.1

1.3

1.5

1.7

1.9ATP

CONTROL

BTP-2

Time (s)

Rat

io

0 200 400 600 800 10000.9

1.1

1.3

1.5

ATP ATP

, -MeSATP

Time (s)

Rat

io

0 250 500 750 10000.40

0.45

0.50

0.55

0.60

Time (s)

F3

40

0 250 500 750 10000.3

0.4

0.5

F38

0

0 250 500 750 10001.0

1.2

1.4

1.6

1.8

Time (s)

F3

40 /

F3

80

0 400 800 12000.50

0.75

1.00Suramin

Ach + Suramin

Time (s)

Rat

io