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Thus, H89 inhibition of Rho kinase, which is downstream of RhoA and PKA
(Qiao et al.
2008
), may lead to decreased permeability and may not rule out the
possible role of PKA in decreasing permeability. Kooistra et al. demonstrated the
role of Epac in regulating endothelial cell permeability via the use of RNAi
(Kooistra et al.
2005
). Kooistra et al. also used Epac-specific analogues in combina-
tion with RNAi knockdown of Epac to demonstrate the role of Epac in regulating
endothelial cell permeability. RhoA seems to be regulated by both PKA and Epac.
The use of PKI, a specific inhibitor of PKA, was shown to partially reverse the
inhibitory effect of cAMP on RhoA (Qiao et al.
2003
). On the other hand, it was
shown that an Epac-specific cAMP analogue, 8-pCPT-2
0
O-Me-cAMP, could mimic
completely the inhibitory effects of forskolin- and rolipram (PDE4-specific inhibitor)-
induced cAMP on RhoA, suggesting that the effects of cAMP are via Epac (Cullere
et al.
2005
). Further studies are needed to clarify the relative roles of PKA and Epac
in mediating the inhibitory effects of cAMP on RhoA. Unpublished data from our
laboratory suggest that the barrier-enhancing effects of cAMP may be due to both
PKA and Epac. We found that inhibition of PKA by Rp-8-Br-cAMP increased
thrombin-induced permeability, suggesting a role for PKA in preventing decreases
in barrier function (Fig.
1
). However, forskolin was still able to attenuate thrombin-
induced permeability, and low doses of ANP, via inhibition of PDE3, were able to
potentiate this effect, suggesting that another effector molecule also mediates
the barrier-enhancing effect of cAMP (Surapisitchat et al.
2007
). We also found
that the activation of Epac was able to decrease thrombin-induced permeability using
the Epac-specific activator, 8-CPT-2-O-ME-cAMP (Fig.
2
). These results suggest
that there may be different pools of cAMP mediating the effects of PKA and Epac on
permeability. A recent report has demonstrated that cAMP can act via both PKA and
Vehicle
Rp-8-Br-cAMP
200
175
150
125
100
75
50
25
0
Thrombin
Forskolin
-
-
-
+
-
-
+
+
-
+
+
+
-
-
-
+
-
-
+
+
-
+
+
+
ANP
Fig. 1 PKA mediates some of the effects of cAMP on endothelial permeability. Permeability
assays with thrombin, forskolin, and 0.3 nM ANP
100
m
M Rp-8-Br-cAMP were performed as
described previously (Surapisitchat et al.
2007
). Rp-8-Br-cAMP was added 15 min prior to
stimulation with thrombin, forskolin, and ANP. Data represent mean (mean
SEM) from two
independent experiments using HUVEC isolated from two different umbilical cords
