Connexin hemichannels-mediated Ca 2+ entry results in nitric oxide production in in situ injured...
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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
Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s
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
Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s
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
.)
Connexin mimetic peptides reduce both injury-induced Ca2+ elevation and NO production in ra aortic EC’s
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