ca 2+ signaling in injured in situ endothelium of rat aorta
DESCRIPTION
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) - PowerPoint PPT PresentationTRANSCRIPT
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Ca2+ signaling in injured in situ endothelium of rat aorta
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Vascular Endothelium
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•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
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PLCPIP2
IP3
DAG
ER
NucleusGPCR
RCIC
↑ [Ca2+]
SERCA
P M C A
↑ [Ca2+]
Stretch channels
NCX
SOC
Ca2+ Homeostasis
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• 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
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Fura 2/AM16M(1hr)
15 minIn PSSUpright epifluorescence microscope
Excitation = 340 / 380Emmision = 510
Wistar rats
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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%)
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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
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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
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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%
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[Ca2+]i
L-Arginine
Nitric Oxide
Citruline
NOSe
DAF Fluorescent dyeIndicator of NO production Emission: 510nmExcitation: 490nm
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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
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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
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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
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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
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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
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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.
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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
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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
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0 400 800 12000.50
0.75
1.00Suramin
Ach + Suramin
Time (s)
Rat
io