| pA2
online © Copyright 2003 The British Pharmacological Society |
009P
University of Surrey Summer Meeting June 2003 |
Role
of PKC in
the pulmonary vasoconstrictor effects of thromboxane A2
|
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Thromboxane A2
(TXA2) has been involved in several forms
of pulmonary hypertension (Christman et al., 1992). Our preliminary
results indicated that the contraction induced by TXA2
is mediated, at least partly, through the inhibition of voltage-gated
K+ (KV) channels in rat pulmonary
arteries (PA) (Cogolludo et al., 2002). In the present study, we
have analysed the signalling pathways involved in the KV
channel inhibition and the contraction induced by the TXA2
analogue U46619.
Second to third-order branches of the PA (
600
µm) isolated from male Wistar rats (250-300 g) were used in this
study. Arteries were mounted in Krebs solution for isometric tension recording
and stimulated with U46619 (0.1 µM). PA smooth muscle cells (PASMC)
were enzymatically isolated and currents were recorded using the whole-cell
configuration of the patch-clamp technique. Western blot analysis was
performed with 10 µg of protein from PA per lane.
The pulmonary vasoconstriction induced by U46619 (185 ± 23 mg,
n = 10), was abolished by the TXA2 receptor
antagonist SQ-29548 (3 µM) and markedly inhibited (46 ± 6
% of control, n = 7, P < 0.01) by the L-type Ca2+
channel blocker nifedipine (0.1 µM). Furthermore, the nonselective
PKC inhibitors staurosporine (0.01 µM) and calphostin C (1 µM)
markedly attenuated the response to the TXA2
analogue (45 ± 5 % and 55 ± 5 % of control, respectively;
n = 8; P < 0.01) whereas the inhibitors of tyrosine kinase (genistein,
10 µM) or Rho kinase (Y-27632, 1 µM) did not modify the contraction
tothis agonist (90 ± 4 % and 90 ± 7 % of control, respectively;
n = 8-10; P > 0.05). The responses to U46619 were sensitive
to Gö-6983 (0.01 µM, which preferentially inhibits conventional
or cPKC, some novel or nPKC and PKC
but not PKCµ) (46 ± 8 % of control; n = 6; P <
0.01) but insensitive to bisindolylmaleimide I (1 µM, which shows
selectivity for cPKC isoforms and for the nPKC isoforms 
and 
) and Gö-6976 (0.01
µM, which inhibits cPKC isoforms and PKCµ) (103 ± 11
% and 86 ± 6 % of control, respectively; n = 5-9; P >
0.05). Based on the specificity of these drugs we suggested a role for
PKCin the vasoconstriction
elicited by U46619. Accordingly, a PKC
pseudosubstrate inhibitor (10 µM, Shizukuda & Buttrick, 2002)
prevented U46619-induced contraction (41 ± 6 % of control; n =
5; P < 0.01) and inhibition of KV
channels from 36 ± 5 % to 4 ± 3 % (n = 5-7; P <
0.05). Western blots of homogenates from rat PA using anti-C-terminal
peptide of PKC antibody
recognized two bands of approximately 81 and 75 kDa as previously reported
(Allen et al., 1994). A transient translocation of PKC
from the cytosolic to the particulate fraction upon stimulation with U46619
was observed.
In conclusion, TXA2, via activation of
TP receptors, inhibits KV channels, and this may activate L-type Ca2+
channels, and induces vasoconstriction. PKC
seems to play a major role as a link between TP receptor activation and
KV channel inhibition.
Allen, B.G. et al., (1994) J. Biol. Chem. 269, 29288-98.
Christman, B.W. et al., (1992) N. Engl. J. Med. 327,
70-75.
Cogolludo, A. et al., (2002) J. Hypertension. 20
(suppl. 4), S281.
Shizukuda & Buttrick, (2002) Am. J. Physiol. 282, H320-327.