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Pulmonary artery tone is partly determined by the
membrane potential (Em) of pulmonary artery smooth muscle cells (PASMCs). The
Kv7 family of K+ channels, encoded by KCNQ genes, has been implicated in the
regulation of Em in rat PASMCs, such that retigabine, an activator of Kv7
channels, promotes pulmonary vasodilation (Joshi et al, 2009). Zinc
pyrithione (ZnPy) is a recently identified Kv7 channel activator with a site
of action distinct from retigabine (Xiong et al, 2008). This study aimed to
determine if ZnPy could activate pulmonary artery Kv7 channels. Its effects
on Em and whole-cell K+ currents were investigated in acutely isolated rat
PASMC using the whole-cell patch clamp technique. Data are given as mean ±
s.e.m. of n cells and were compared using students unpaired t-test, p<0.05
considered significant. At 10 µM, ZnPy consistently hyperpolarized PASMCs by
11 ± 1 mV (n=12, p<0.001). Its effect on Em was unaffected by the presence
of 10μM glibenclamide (11 ± 1 mV, n=60, p<0.001), indicating that it did
not involve activation of KATP channels. In contrast, in the presence of 10
mM tetraethylammonium ions (TEA), ZnPy (10 μM) did not cause significant
hyperpolarisation (n= 10), suggesting that it might involve TEA-sensitive Kv7
or BKCa channels. Involvement of the latter is unlikely, because in the
presence of 50 nM iberiotoxin to selectively block BKCa channels, the
hyperpolarisation induced by ZnPy remained at 14 ± 3mV (n= 5, p=0.01). Kv7
channels are more likely to underlie the response to ZnPy, because it was
abolished by the Kv7 blocker XE991 (10µM, n=10). ZnPy (10 μM) was also found
to activate K+ current, but with different pharmacology. The current
activated at 0 mV, by brief (200ms) steps from a holding potential of -80 mV,
was enhanced nearly 4-fold from 532 ± 84 pA to 2000 ± 331 pA (n= 33, p
<0.001). This increase was unaltered by 50 nM iberiotoxin and 10 μM XE99,
but was greatly reduced by 10 mM TEA: ZnPy increased current from 547 ± 183
pA to only 617 ± 171 pA (n=6). When cells were clamped at 0 mV for at least 5
min to inactivate delayed rectifier K+ channels, ZnPy (10 μM) still induced
outward current, from 41± 6 pA to 261 ± 79 pA (n=31, p<0.01). This effect
was prevented in the presence of either 10 mM TEA (n=5) or 10 μM XE991 (n=4).
Thus ZnPy activated a current with Kv7-like properties, but also induced an
inactivating current mediated by distinct, TEA-sensitive, but
XE991-insensitive channels. Taken together, the results support the idea that
ZnPy hyperpolarises PASMC by opening non-inactivating, Kv7 channels. The
hyperpolarisation is expected to inhibit voltage-gated Ca2+ influx, leading
to muscle relaxation and ultimately pulmonary artery dilation.
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Pulmonary
artery tone is partly determined by the membrane potential (Em) of pulmonary
artery smooth muscle cells (PASMCs). The Kv7 family of K+ channels, encoded
by KCNQ genes, has been implicated in the regulation of Em in rat PASMCs,
such that retigabine, an activator of Kv7 channels, promotes pulmonary
vasodilation (Joshi et al, 2009). Zinc pyrithione (ZnPy) is a recently
identified Kv7 channel activator with a site of action distinct from
retigabine (Xiong et al, 2008). This study aimed to determine if ZnPy could
activate pulmonary artery Kv7 channels. Its effects on Em and whole-cell K+
currents were investigated in acutely isolated rat PASMC using the whole-cell
patch clamp technique. Data are given as mean ± s.e.m. of n cells and were
compared using students unpaired t-test, p<0.05 considered significant. At
10 µM, ZnPy consistently hyperpolarized PASMCs by 11 ± 1 mV (n=12,
p<0.001). Its effect on Em was unaffected by the presence of 10μM
glibenclamide (11 ± 1 mV, n=60, p<0.001), indicating that it did not
involve activation of KATP channels. In contrast, in the presence of 10 mM
tetraethylammonium ions (TEA), ZnPy (10 μM) did not cause significant
hyperpolarisation (n= 10), suggesting that it might involve TEA-sensitive Kv7
or BKCa channels. Involvement of the latter is unlikely, because in the
presence of 50 nM iberiotoxin to selectively block BKCa channels, the
hyperpolarisation induced by ZnPy remained at 14 ± 3mV (n= 5, p=0.01). Kv7
channels are more likely to underlie the response to ZnPy, because it was
abolished by the Kv7 blocker XE991 (10µM, n=10). ZnPy (10 μM) was also found
to activate K+ current, but with different pharmacology. The current
activated at 0 mV, by brief (200ms) steps from a holding potential of -80 mV,
was enhanced nearly 4-fold from 532 ± 84 pA to 2000 ± 331 pA (n= 33, p
<0.001). This increase was unaltered by 50 nM iberiotoxin and 10 μM XE99,
but was greatly reduced by 10 mM TEA: ZnPy increased current from 547 ± 183
pA to only 617 ± 171 pA (n=6). When cells were clamped at 0 mV for at least 5
min to inactivate delayed rectifier K+ channels, ZnPy (10 μM) still induced
outward current, from 41± 6 pA to 261 ± 79 pA (n=31, p<0.01). This effect
was prevented in the presence of either 10 mM TEA (n=5) or 10 μM XE991 (n=4).
Thus ZnPy activated a current with Kv7-like properties, but also induced an
inactivating current mediated by distinct, TEA-sensitive, but
XE991-insensitive channels. Taken together, the results support the idea that
ZnPy hyperpolarises PASMC by opening non-inactivating, Kv7 channels. The
hyperpolarisation is expected to inhibit voltage-gated Ca2+ influx, leading
to muscle relaxation and ultimately pulmonary artery dilation.
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موجز عن المشاركة:
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Pulmonary artery tone is partly determined by the
membrane potential (Em) of pulmonary artery smooth muscle cells (PASMCs). The
Kv7 family of K+ channels, encoded by KCNQ genes, has been implicated in the
regulation of Em in rat PASMCs, such that activators of Kv7 channels promote
pulmonary vasodilation and inhibitors promote constriction. The Kv7 opener
flupirtine was shown to be beneficial in two mouse models of pulmonary
hypertension. This study aimed to investigate the role of Kv7 channels in a
monocrotaline- induced pulmonary hypertension model in Wistar rats.
Contractile studies investigating the effects of various Kv7 modulators were
conducted on pulmonary and mesenteric arteries obtained from monocrotaline
and control rats. Pulmonary arteries from monocrotaline rats showed an
increased sensitivity to Kv7 modulators, not seen in mesenteric arteries.
Also there was a raised intrinsic tone which was abolished by Kv7 activators.
This data suggests that Kv7 channels may play an important role in pulmonary
hypertension and that Kv7 activators may represent a new direction for
treating pulmonary hypertension.
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