Everardo Avelino

Connexin hemichannels-mediated Ca2+ entry results in nitric oxide production in in situ injured

endothelial cells

Everardo Avelino Secondo annoCiclo XXII

Time

R

atio

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

PEAK

~ 60% > intracellular stores depletion (P2Y1,2,12,13)

~ 40 > Ca2+ influx (P2X and Gap junctions)

PLATEAU

Ca2+ influx (Gap junctions)

Introduction

Endothelium functions:

• Angiogenesis and vasculogenesis

• Preserve a surface unreactive toward

circulating cells

• Mantain thromboresistence

• Inhibit vascular tone

• Inhibit cells growth in outer layer of the

vesell wall.

Introduction

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07

L-NAME

CONTROL

ATP 300 µM

Time (s)

F

luo

resc

ence

(a.

u.)

400 900 1400 1900 2400 2900 3400-0.01

0.01

0.03

0.05

0.07 SNAP

Time (s)

F

luo

resc

ence

(a.

u.)

ATP

ATP + L

-NAM

E

0.00

0.01

0.02

0.03

170

192***

F

luo

resc

ence

(a.

u)

(20

min

aft

er a

pp

lica

tio

n)

Nitric oxide measurement

DAF FM DA

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

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07

CONTROL

Time (s)

F

luo

resc

ence

(a.

u.)

Injury augments nitric oxide production in rat aortic endothelium

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07

L-NAME

CONTROL

Time (s)

F

luo

resc

ence

(a.

u.)

Injury augments nitric oxide production in rat aortic endothelium

Injury induced NOP* depend on extracellular Ca2+

* Nitric oxide production

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07

Ca2+ Free

CONTROL

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500-0.02

0.00

0.02

0.04

0.06

CONTROL

THAPSIGARGIN

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500-0.02

0.00

0.02

0.04

0.06

CONTROL

BTP2

Time (s)

F

luo

resc

ence

(a.

u.)

Capacitative Ca2+ entry does not sustain injury-induced NOP

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07ATP

CONTROL

BTP-2

Time (s)

F

luo

resc

ence

(a.

u.)

ATP

ATP + B

TP-20.00

0.01

0.02

0.03

170

170***

F

luo

resc

ence

(a.

u)

(20

min

aft

er a

pp

lica

tio

n)

0 500 1000 1500 2000 2500-0.02

0.00

0.02

0.04

0.06CONTROL

PALMITOLEIC AC.

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500-0.02

0.00

0.02

0.04

0.06CONTROL

OLEAMIDE

Time (s)

F

luo

resc

ence

(a.

u.)

Putative blockers of CxHcs* reduce NO synthesis induced by Ca2+ entry in injured endothelium

*Connexin hemichannels

0 500 1000 1500 2000 2500-0.02

0.00

0.02

0.04

0.06CONTROL

OCTANOL

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500-0.02

0.00

0.02

0.04

0.06CONTROL

HEPTANOL

Time (s)

F

luo

resc

ence

(a.

u.)

Putative blockers of CxHcs* reduce NO synthesis induced by Ca2+ entry in injured endothelium

*Connexin hemichannels

Control Gap 27Cx 37,430

25

50

75

100

125

*

n = 25

n = 31% o

f p

eak

Ca2

+

resp

on

se t

o i

nju

ry

0 200 400 600 800 1000 12000.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

CONTROL

GAP 27Cx 37,43

Time (s)

R

atio

Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s

Control Gap 27Cx37,430

25

50

75

100

125

n= 31n=25

Per

cen

tag

e o

fA

rea

un

der

cu

rve

Pre-incubation

Control Gap 27Cx400

25

50

75

100

125

**

n = 38n = 44

Per

cen

tig

e o

fA

rea

un

der

cu

rve

0 250 500 750 1000 12500.9

1.0

1.1

1.2

1.3

1.4

1.5

CONTROL

Gap 27Cx 40

Time (s)

R

atio

Control Gap 27Cx400

25

50

75

100

125

n = 44 n = 38

% o

f p

eak

Ca2

+

resp

on

se t

o i

nju

ry


Pre-incubation

Cx 37-43 Cx 400

20

40

60

80

100

n = 31n = 52% o

f d

ecre

ase

of

the

dec

ay p

has

e

200 600 1000 1400 18000.95

1.05

1.15

1.25

1.35

1.45

Cx 40 La3+

Time (s)

R

atio

200 600 1000 1400 18000.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

Cx 37,43 La3+

Time (s)

R

atio


Accute effect

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07

CONTROL

Cx37,43 BLOCKER

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500

-0.01

0.01

0.03

0.05

0.07

CONTROL

Cx40 BLOCKER

Time (s)

Flu

ore

scen

ce (

a.u

.)


CONTROL

BTP-2

PALMIT

OLEIC. A

C.

OLEAMID

E

OCTANOL

HEPTANOL

Cx 37

,43

Cx 40

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

39198

86 134 156145 63 89

*** ******

****** ***

F

luo

resc

ence

(a.

u)

(20

afte

r in

jury

)

CONTROL

L-NAM

E

Thapsi

gargin

Fre

e

2+

Ca

-0.0050.0000.0050.0100.0150.0200.0250.0300.0350.0400.045

391

118

90

170

******

F

luo

resc

ence

(a.

u)

(20

min

aft

er i

nju

ry)

Summary

Cx 37-43 Cx 400

20

40

60

80

100

n = 31n = 52% o

f d

ecre

ase

of

the

dec

ay p

has

e

Nucleus

Intracellular Ca2+ stores

Ca2+ ATPase

Gap

Gap

ATP

ATP

ATP

TR

PV

4

IP3R

PGPLC

IP3

Ca2+

eNOS

CaM

Inhibit CCE

Ca2+

Ca2+

Ca2+

ActivatesNCCE

?

Summary

0 200 400 600 800 1000 1200 14000.8

0.9

1.0

1.1

1.2

Control

Time (s)

R

atio

0 250 500 750 10000.7

0.8

0.9

1.0

1.1L-NAME

Time (s)

R

atio

0 200 400 600 800 1000 1200 14000.8

0.9

1.0

1.1

1.2SNAP

Time (s)

R

atio

Control

L-NAM

E

SNAP0.0

0.1

0.2

0.3

****

n=73n=58 n=35

Rat

io

Preeliminar results

0 200 400 600 8000.80

0.85

0.90

0.95

SNAP

(n = 114)

Time (s)

R

atio

0 200 400 600 8000.80

0.85

0.90

0.95

SNAP

Ca2+ Free

Time (s)

R

atio

(n = 57)

Preeliminar results

0 400 800 1200 1600 2000 2400 2800-0.04

-0.03

-0.02

-0.01

0.00

0.01

Me-CyD

n = 165

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500 3000 3500-0.04

-0.03

-0.02

-0.01

0.00

0.01

Me-CyD

ATP

n = 85

Time (s)

F

luo

resc

ence

(a.

u.)

0 500 1000 1500 2000 2500 3000 3500-0.04

-0.03

-0.02

-0.01

0.00

0.01

Me-CyD

Injury

n = 80

Time (s)

F

luo

resc

ence

(a.

u.)

0 200 400 600 800 1000 1200 14000.8

0.9

1.0

1.1

1.2

Time (s)

R

atio

Me--CyD

Preeliminar results

Documents

Everardo Avelino