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Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The
vasoconstrictor angiotensin II (ANG II) inhibits several types of
K+ channels. We examined the inhibitory mechanism of ANG II
on voltage-gated K+ (KV) currents
(IKV) recorded from isolated rat
arterial smooth muscle using patch-clamp techniques. Application of 100 nM ANG II accelerated the activation of
IKV but also caused inactivation. These effects were abolished by the AT1 receptor antagonist
losartan. The protein kinase A (PKA) inhibitor Rp-cyclic 3',5'-hydrogen phosphothioate adenosine (100 µM) and an analog of diacylglycerol, 1,2-dioctanyoyl-rac-glycerol (2 µM), caused a significant reduction of IKV. Furthermore, the combination
of 5 µM PKA inhibitor peptide 5-24 (PKA-IP) and 100 µM protein
kinase C (PKC) inhibitor peptide 19-27 (PKC-IP) prevented the
inhibition by ANG II, although neither alone was effective. The ANG II
effect seen in the presence of PKA-IP remained during addition of the
Ca2+-dependent PKC inhibitor Gö6976 (1 µM) but was
abolished in the presence of 40 µM PKC-
translocation inhibitor
peptide. These results demonstrate that ANG II inhibits KV
channels through both activation of PKC- and inhibition of PKA.
potassium channel; activation; inactivation; protein kinase A; protein kinase C
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ANGIOTENSIN II (ANG II) is a potent vasoactive substance known to increase intracellular Ca2+ and the contractile force of vascular smooth muscle cells. Changes in intracellular [Ca2+] play a central role in the control of vascular smooth muscle tone. Contractile agonists cause an elevation in intracellular [Ca2+] by increasing the influx of extracellular Ca2+ through voltage-dependent L-type Ca2+ channels or by release of Ca2+ from intracellular stores. In many tissues, including vascular smooth muscle, ANG II receptors have been reported to couple to several cellular signaling pathways. ANG II binds to angiotensin AT1 receptors, which are coupled via pertussis toxin-insensitive G protein (Gq/11) to the activation of phospholipase C (PLC) (9, 12, 28, 35). PLC hydrolyzes membrane phosphoinositides into diacylglycerol (DAG), which causes activation of protein kinase C (PKC), and inositol phosphates (3, 9, 22, 28, 34). AT1 receptors have also been shown to regulate adenylate cyclase activity, and in most systems AT1 receptors act via Gi to inhibit adenylate cyclase (2, 37).
K+ channels play an important role in regulating the membrane potential of arterial smooth muscle (16, 26). Activation of K+ channels results in hyperpolarization, whereas their inhibition contributes to membrane depolarization, leading to opening of voltage-dependent Ca2+ channels and vasoconstriction. Arterial smooth muscle expresses ATP-dependent K+ channels (KATP), large conductance Ca2+-activated K+ channels (BKCa), inwardly rectifying K+ channels (Kir), and voltage-gated K+ channels (KV), all of which contribute to the resting membrane potential (27, 30). For example, the smooth muscle KV channel inhibitor 4-aminopyridine causes depolarization and constriction in various arteries (14, 18), showing that KV channels provide an important K+ conductance in the physiological membrane potential range.
ANG II has been reported to inhibit several vascular K+ channels, including BKCa channels in the coronary artery (25, 36), KV channels in the rabbit portal vein (5), and KATP channels in the rat mesenteric artery (15, 20). The inhibition of KV current of the portal vein by ANG II involves activation of PKC (5), and we have shown that a component of the action of ANG II on KATP current in the mesenteric artery also occurs via this pathway (15, 20). However, we also showed that a substantial component of the inhibition of KATP current by ANG II resulted from a reduction in steady-state channel activation by protein kinase A (PKA) (15). Because KV channels of the rabbit portal vein and colonic smooth muscle are activated by PKA-induced phosphorylation (1, 19), it is possible that a reduction in PKA activity could also contribute to the inhibitory effect of ANG II on KV current (IKV).
In the present study, we investigated the mechanism by which ANG II
inhibits IKV of rat mesenteric
arterial smooth muscle by using conditions designed to minimize
contamination from other currents. ANG II altered the kinetics of
IKV by speeding up both activation
and inactivation. We show that ANG II inhibits
IKV via binding to AT1
receptors and demonstrate for the first time that, as for
KATP channels, inhibition of KV occurs not only
through activation of PKC but also by inhibition of PKA. Moreover, our results establish that PKC- is the subtype involved in the
inhibition of KV channels.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of vascular smooth muscle cells. Vascular smooth muscle cells from rat mesenteric arteries were dissociated as follows. Male adult Wistar rats were rendered unconscious by exposure to a rising concentration of CO2 and killed by exsanguination. The care of the animal conformed to the requirements of the UK Animals (Scientific Procedures) Act of 1986.
Arteries were removed and cleaned of blood and connective tissue in ice-cold solution containing (in mM) 137 NaCl, 5.6 KCl, 0.42 Na2HPO4, 0.44 NaH2PO4, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, adjusted with NaOH to pH 7.4. After dissection, the arteries were transferred to the same solution except that CaCl2 was reduced to 0.1 mM (low-calcium solution) for 10 min, and warmed to 35°C in a water bath. First, arteries were incubated for 30-35 min in low-calcium solution containing (in mg/ml) 0.9 albumin, 1.4 papain, and 0.9 dithioerythritol. The mesenteric artery was then further digested for 16-20 min in low-calcium solution containing (in mg/ml) 0.9 albumin, 1.4 collagenase type F (Sigma), and 0.9 hyaluronidase type I-S (Sigma). Arteries were then transferred to low-calcium solution containing albumin (1 mg/ml). Single smooth muscle cells were obtained by gentle trituration with a wide-bore pipette, stored at 4°C, and used on the day of preparation.Solutions and chemicals. For conventional whole cell recordings, the intracelluar solution contained (in mM) 110 KCl, 30 KOH, 10 HEPES, 10 EGTA, 1 MgCl2, 1 CaCl2, 3.0 Na2ATP, and 0.5 GTP, adjusted to pH 7.2. To minimize the activity of KATP channels, a high concentration of ATP (3.0 mM) was used. The free [Ca2+], calculated using the program Maxchelator (http://www.stanford.edu/%7Ecpatton/maxc. html), was 20 nM. The 6 mM K+ extracellular solution contained (in mM) 134 NaCl, 6 KCl, 1 MgCl2, 0.1 CaCl2, 10 HEPES, and 10 glucose, adjusted to pH 7.4. To minimize the activity of BKCa channels, external Ca2+ was lowered to 0.1 mM to reduce Ca2+ influx, and intracellular Ca2+ was buffered to a low level with EGTA. For single channel experiments, the pipette contained (in mM) 140 KCl, 10 HEPES, 1 CaCl2, and 1 MgCl2 (pH 7.4), and the bath solution contained (in mM) 110 KCl, 30 KOH, 10 EGTA, 10 HEPES, 1 MgCl2, and 1 Na2ATP (pH 7.2).
The external solution was changed by continuous perfusion of the experimental chamber (volume, 0.4 ml). ANG II and 1,2-dioctanyoyl-rac-glycerol (DiC8) were purchased from Sigma. Gö6976, Rp-adenosine 3',5'-cyclic monophosphothioate, triethylammonium salt (Rp-cAMPS), PKA inhibitor peptide 5-24 (PKA-IP), myristoylated PKC inhibitor peptide 19-27 (PKC-IP), and PKC- translocation inhibitor peptide were obtained from Calbiochem.
Losartan was kindly provided by Dr. A. Stanley, Department of Medicine,
University of Leicester. DiC8 and Gö6976 were dissolved in DMSO.
The final concentration of DMSO was <0.2%.
Data recording and analysis.
Whole cell and single channel currents were recorded from single smooth
muscle cells using the patch-clamp technique (13). Currents were recorded using an Axopatch 200 amplifier (Axon
Instruments). Patch pipettes were made from thin-walled borosilicate
glass (Clark Electromedical) using a pp-83 vertical puller
(Narishige; Tokyo, Japan) and coated with sticky wax (Kemdent) to
reduce capacitance. Whole cell currents were filtered at 5 kHz, and
single channel currents were filtered at 2 kHz (
3 dB). Electrode
resistance before sealing was 3-5 M
and after sealing was >10
G. All experiments were done at 20-25°C.
70 and +40 mV. Tail current amplitude was measured as its initial value after the voltage pulse. Activation and inactivation time constants were fitted using custom software. Data are expressed as
means ± SE. Intergroup differences were analyzed by analysis of
variance followed by Duncan's multiple range test or Student's t-test as appropriate. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Properties of IKV of rat mesenteric artery
smooth muscle.
Whole cell patch-clamp recordings from rat mesenteric artery smooth
muscle cells revealed the presence of
IKV, which was activated by
depolarizing steps to potentials more positive than approximately 30
mV. The current activated relatively slowly, and under control conditions little or no inactivation was observed during 400-ms pulses.
The steady-state activation voltage relation for
IKV was obtained by measuring tail
currents at 20 mV after a range of depolarizing steps from 60 mV
(Fig. 1A). Steady-state
inactivation curves were obtained by varying the holding potential and
measuring the current induced by a step to +20 mV (Fig. 1B).
Plots of the mean steady-state activation and inactivation curves from
six cells were fitted with a Boltzmann distribution, giving voltages for half-maximal activation and inactivation
(V1/2) of 3.5 and 33.9 mV, respectively.
Examination of the conductance data and Boltmann curves shown in Fig. 1
indicates a significant conductance due to KV channels
above 40 mV, which is within the physiological range of membrane
potential in arterial smooth muscle (18, 26).
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ANG II inhibits IKV.
The effect of ANG II on IKV is shown
in Fig. 2. Depolarizing steps to +20 mV
were applied for 400 ms every 1 min from a holding potential of 60 mV
to follow the time course of the effect of ANG II. The runup of
IKV over the first 4 min is most
likely due to the presence of Mg-ATP in the pipette solution leading to
PKA-mediated phosphorylation (1). All experiments were
performed only after a stable amplitude of
IKV was recorded after runup
current. ANG II induced a progressive decline in the amplitude of the
outward current measured at the end of the depolarizing steps. The
effect of ANG II was maximal after a period of 5-20 min and was
reversible upon washout. Similar results were obtained from nine other
cells. To test whether ANG II affected
IKV in a voltage-dependent manner, a
series of voltage pulses was applied (from 70 and +40 mV in 10-mV
intervals), and the currents at the end of the 400-ms pulses were
compared before and after application of 100 nM ANG II (Fig. 2,
C and D). ANG II induced a significant and
similar decrease in the amplitude of steady-state and tail
currents at all potentials where accurate measurements could be
obtained. An interesting feature of the ANG II effect was a speeding up
of IKV activation and the induction
of inactivation (see below). Steady-state activation parameters were
obtained from tail currents at 20 mV after the test pulses. The
V1/2 value for activation in the presence of ANG
II was 7.8 mV compared with the control value of 3.5 mV (see
above), suggesting that ANG II had no significant effect on the voltage
dependence of the steady-state activation of
IKV. A similar result was obtained
by Clément-Chomienne et al. (5) for ANG
II-induced inhibition of IKV in
rabbit portal vein cells.
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Losartan inhibits the action of ANG II.
At least two types of ANG II receptors are currently known,
AT1 and AT2 (37). The selective
AT1 antagonist losartan has been shown to be effective at
inhibiting the reduction in blood flow caused by ANG II
(35). To investigate whether this type of receptor is
involved in KV channel inhibition in rat arterial muscle,
we tested the effect of ANG II in the presence of 1 µM losartan.
Figure 3, A and B,
shows that 10-min pretreatment with losartan prevented
IKV inhibition by ANG II, indicating
that the AT1 receptor mediates this effect.
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ANG II is still effective in the presence of iberiotoxin. Our recording conditions (low intracellular [Ca2+] and only moderate depolarizations) were designed to minimize the activation of BKCa channels, which are prevalent in this tissue (32). To test for a possible involvement of BKCa channels, we used iberiotoxin (IbTX), a potent and selective inhibitor of these channels in smooth muscle (11). A series of recordings from a cell under control conditions, in the presence of 100 nM IbTX, and in the presence of both IbTX and ANG II is shown in Fig. 3C. At voltages above +10 mV there is a slight reduction in current with IbTX, indicating that a small component of current flows through BKCa channels. Application of ANG II, however, still caused a marked reduction in IKV in the presence of IbTX. The mean I-V curve, normalized to the control current at +40 mV, from four cells is shown in Fig. 3D. The mean fractional reduction of the IbTX-resistant current by ANG II was 0.49 ± 0.15 at +40 mV, which is a similar reduction to that seen in the absence of IbTX. These results show that the major effect of ANG II that we describe here is on IKV rather than on BKCa channels, even in the absence of IbTX.
ANG II alters the kinetics of IKV.
Application of ANG II accelerated the activation process and caused a
partial time-dependent inactivation of
IKV during a 400-ms pulse. The
relative amount of the inactivating component varied between cells. To
quantify these alterations in kinetics, we fitted exponential functions
to activation and inactivation in the absence and presence of ANG II
and to inactivation in the presence of ANG II only, because in its
absence IKV did not inactivate during the duration of the pulses. Examples of the currents induced by
pulses to +40 mV showing the alteration in activation and inactivation induced by ANG II and of the exponential fits are shown in Fig. 4, A and C. Plots
of the activation and inactivation time constants are shown in Fig. 4,
B and D, respectively. The mean activation time
constants at +40 mV were 29.8 ± 6.2 and 12.2 ± 1.8 ms
(n = 5) in the absence and presence of 100 nM ANG II,
respectively. The mean inactivation time constant induced by ANG II was
85.6 ± 6.2 ms (n = 5).
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IKV is reduced by DiC8 and Rp-cAMPS. The signal transducation events stimulated by binding of ANG II to AT1 receptors include activation of PLC, activation of PKC, and inhibition of adenylyl cyclase (2, 9, 12, 28, 35, 37). We therefore investigated the possibility that the ANG II-induced inhibition of IKV occurred by a dual mechanism involving activation of PKC and inhibition of background PKA activity.
As shown in Fig. 5, A and B, application of 2 µM DiC8, a membrane-permeant analog of DAG, significantly reduced IKV. It is also evident in this example that DiC8 caused a marked inactivation of the current. This is consistent with activation of PKC mediating at least part of the ANG II-induced inhibition of IKV. ANG II-induced inhibition of IKV via PKC has also been reported in rabbit portal vein smooth muscle (5), although in that tissue no inactivation was seen. Their results, however, do not provide any evidence for the involvement of the adenylyl cyclase-PKA system in the inhibitory action of ANG II. Because activation of adenylyl cyclase, and so PKA, has been shown to activate IKV (1), it is possible that inhibition of background adenylyl cyclase activity mediates part of the inhibitory action of ANG II on IKV, as we have shown to be the case for ANG II-induced inhibition of KATP channels (15). To investigate this possibility, we inhibited PKA by application of the cAMP analog Rp-cAMPS, which inactivates PKA by binding to its regulatory subunit (31). Figure 5, C and D, shows that 100 µM Rp-cAMPS inhibited IKV significantly. DiC8 (2 µM) reduced IKV at +40 mV, normalized to its value before addition of the agent, to 0.32 ± 0.03 of control value (n = 12), whereas 100 µM Rp-cAMPS reduced IKV to 0.48 ± 0.03 of control value (n = 8; Fig. 5E).
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The change in kinetics induced by DiC8 is similar to that produced
by ANG II.
Application of DiC8 induced a similar change in the kinetics of
IKV as did ANG II. We fitted both
activation and inactivation time courses with a single exponential
function, as for the ANG II data. Plots of the activation time
constants in the presence and absence of DiC8 and of the inactivation
time constants in the presence of DiC8 are shown in Fig.
6, A and B,
respectively. The values are comparable with those shown in Fig. 4 for
the effect of ANG II. At +40 mV, activation time constants were
19.3 ± 2.5 and 8.0 ± 1.3 ms (n = 4) in
control and 2 µM DiC8, respectively. ANG II (100 nM) and DiC8 (2 µM) both reduced the activation time constants to 41% of the control
value. The inactivation time constant in the presence of 2 µM DiC8
was 67.5 ± 11.9 ms (n = 4), which was again
comparable with that measured in the presence of ANG II. We considered
the possibility that DiC8 and ANG II, through activation of PKC, were
activating an additional A-type K+ channel. Under control
conditions, an A-type K+ current was not seen, even when
the holding potential was increased to 90 or 100 mV to remove any
possible steady-state inactivation, which is characteristic of A-type
currents (see Fig. 1B, for example). Because A-type currents
usually have a relatively negative steady-state inactivation curve
(4, 6), we were interested in measuring this parameter for
the current in the presence of DiC8. We obtained the
steady-state inactivation curve by varying the holding potential, measuring the peak current induced by a step to +20 mV, and normalizing this to the maximum current measured (Fig. 6, C and
D). V1/2 for steady-state
inactivation in the presence of DiC8 was 33.3 mV, almost identical to
that in its absence (33.9 mV), arguing against the possibility that
activation of PKC induces an A-type current.
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The combination of PKC and PKA inhibitors blocks ANG II-induced
inhibition.
To test the relative contribution of PKA inhibition and PKC activation
to the inhibition of IKV by ANG II,
we pretreated smooth muscle cells with either PKC-IP or PKA-IP. PKA-IP
(5 µM) was applied intracellularly by inclusion in the patch pipette
solution, and PKC-IP (100 µM) was applied extracellularly in its
myristoylated cell-permeant form at least 10 min before recording. We
examined the specificity of these inhibitors previously by testing
their effectiveness at inhibiting dibutyryl cAMP- induced activation of KATP currents (15). Although either PKC-IP
or PKA-IP decreased the effect of ANG II, they failed to induce
complete inhibition of the ANG II effect (Fig.
7). However, after pretreatment with both
PKC-IP and PKA-IP, ANG II no longer affected the amplitude or kinetics
of IKV. These results suggest that
the mechanism for inhibition of IKV
by ANG II occurs through a dual pathway, simultaneous activation of PKC
and inhibition of PKA.
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PKC- is the subtype involved in inhibition by ANG II.
The Ca2+-dependent isoforms of PKC, PKC-
and PKC-
,
and the Ca2+-independent isoforms, PKC- and PKC-
,
have been shown to be expressed in vascular smooth muscle (5, 8,
23). Although previous reports (5, 20) suggest that
the isoform involved in the ANG II pathway is Ca2+
independent, the PKC isoform that is involved in KV channel
inhibition is not known.
and PKC-, are present in smooth muscle, PKC- is
more likely to be involved in the regulation of KV channels
by ANG II because PKC- is insensitive to DAG analogs (29) and the DAG analog DiC8 can inhibit the current (Fig.
5A). We therefore examined the effect of pretreatment with
PKC- translocation inhibitor peptide (40 µM) in the presence of 5 µM PKA-IP (as before). The effect of ANG II on
IKV under these conditions was
completely abolished (Fig. 8), as expected if its effects occurred
solely through PKC-.
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The PKA catalytic subunit directly activates KV
channels.
If part of the inhibition of IKV by
ANG II is through a reduction in PKA activity, then, unless basal
channel activation by PKA is maximal, increasing phosphorylation by PKA
should enhance IKV. We tested this
by applying the catalytic subunit of PKA to excised inside-out patches
and analyzed the ensemble currents produced from ~50 consecutive
depolarizing pulses to +60 mV first in the absence and then in the
presence of the PKA catalytic subunit. In five of eight patches tested,
the PKA catalytic subunit (100 U/ml) increased the activity of single
KV channels. Examples of individual traces and their
ensemble averages obtained in the absence and presence of the PKA
catalytic subunit are shown in Fig. 9.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, the effect of ANG II on whole cell
IKV of enzymatically isolated rat
mesenteric artery smooth muscle cells was investigated using
patch-clamp techniques. We constrained our recording conditions to
minimize current flow through channels other than KV
channels by buffering internal Ca2+ with 10 mM EGTA and
using low external [Ca2+] (0.1 mM) to minimize changes in
internal [Ca2+]. An internal ATP of 3 mM ensured very low
activity of KATP channels. Our results show for the first
time that ANG II, by binding to the AT1 receptor, reduced
IKV through a dual signaling
mechanism involving not only activation of PKC but also inhibition of
PKA. Furthermore, we provide evidence that the PKC component of the action of ANG II is mediated by the -isozyme.
Signaling mechanisms involved in inhibition of
IKV by ANG II.
ANG II has been shown to modulate several types of ionic conductance in
vascular smooth muscle cells and appears to do so by a number of
different pathways. Thus ANG II inhibits BKCa channels by a
PKC-independent mechanism in cultured porcine coronary artery smooth
muscle cells (25), whereas the mechanism by which ANG II
activates a nonselective cation channel in the rabbit ear artery is not
known but is likely to occur without a rise in
D-myo-inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]
(17). We have recently shown that ANG II inhibits
KATP channels through a dual mechanism involving activation
of PKC and inhibition of PKA (15). In rabbit portal vein
smooth muscle cells, ANG II induces inhibition of IKV, which is associated with
activation of PKC (5). This effect was abolished by the
PKC inhibitors calphostin C and chelerythrine (5).
However, in arterial smooth muscle, we find that the effect of ANG II
on IKV is not completely blocked by
PKC-IP but is abolished only in the presence of both PKC-IP and PKA-IP.
The discrepancy between their findings and ours could be due to a
difference in the KV channels involved. The
V1/2 of IKV in
this study was 3.5 mV compared with 24.6 mV as measured by
Clément-Chomienne et al. (5), suggesting that the
K+ channels found in the rabbit portal vein are composed of
different Kv subunits to those found in rat artery smooth muscle. We
obtained recordings of 20-pS channels corresponding to KV
channels from inside-out patches that were activated by application of
the catalytic subunit of PKA (with MgATP), as shown in Fig. 9. A
channel with similar properties (termed KDR1) that has been
described in colonic smooth muscle was also found to be activated by
PKA (19).
,
has been shown to be translocated in response to ANG II stimulation of
cultured canine pulmonary artery smooth muscle cells (7).
In the present study, we showed that the PKC- translocation
inhibitor peptide abolished the PKC component of the ANG II effect on
IKV, indicating that PKC- is the
isoform involved in the ANG II-induced inhibition of
IKV.
The change in kinetics of IKV induced by
ANG II.
An intriguing effect of ANG II on
IKV was a change in kinetics. In
many cells, ANG II produced a significant inactivation of
IKV such that the current now
resembled an A-type K+ current. This effect was also very
pronounced when DiC8 was applied to the cells. In cells pretreated with
PKC-IP, however, very little or no inactivation was detected in the
presence of ANG II, even though ANG II reduced the total K+
current. A similar change in kinetics was observed by Smirnov and
Aaronson (33) when they applied
1-oleoyl-2-acetyl-sn-glycerol (OAG) to rat pulmonary
arterial myocytes. In control conditions, we could not detect a
fast inactivating component of IKV
even when the holding potential was set to 100 mV to remove any
possible steady-state inactivation known to occur with A-type currents (4). It is possible that ANG II (and DiC8) activates an
A-type current that is quiescent under normal conditions. Given the
similarity of the inactivation curves of the inactivating and control
components shown in Fig. 6, another possibility is a
phosphorylation-dependent interaction of KV -subunits
with the -subunits involved in generating the
IKV seen in our experiments. Kwak et
al. (21) recently reported protein kinase-induced
interactions between recombinant KV channel - and
-subunits, raising the possibility that this might also occur for
native channels. Such an effect could explain the similarity between
the steady-state inactivation parameters of control current and the
inactivating current seen with ANG II or DiC8.
The physiological role of inhibition of IKV by ANG II. In addition to increasing intracellular Ca2+ of vascular smooth muscle by activating membrane Ca2+-permeable channels and causing intracellular Ca2+ release, ANG II inhibits several K+ channels, including KATP, BKCa, and KV channels. Such K+ channel inhibition will serve to enhance the depolarizing actions of ANG II, increasing voltage-dependent Ca2+ entry and vasoconstriction. Under normal conditions, KV channels, through their steep voltage dependence of activation, might be expected to provide a negative feedback system that tends to limit the magnitude of depolarization. Inhibition of KATP channels by ANG II would depolarize the cell, and the simultaneous inhibition of KV channels would decrease the limit on depolarization imposed by KV channels, thus allowing greater depolarization. In the present study, we were concerned with the mechanisms by which ANG II inhibits a particular K+ channel, KV. We therefore buffered intracellular Ca2+ to a low level in our experiments to minimize the activation of Ca2+-activated channels, and thus we have not investigated a possible direct inhibition of KV channels by intracellular Ca2+, as reported by Gelband and Hume (10) for canine renal artery KV channels. However, if such Ca2+-dependent inhibition does occur, it would enhance the kinase-dependent inhibition of KV channels that we describe here. Clearly, inhibition of KV channels forms just one component of the complex network of signaling pathways by which ANG II leads to depolarization, increased intracellular Ca2+, and vasoconstriction. Because of the interdependence of several of these pathways, dissection of the relative functional importance of any single component is a daunting task. However, the fact that ANG II has been shown to inhibit each of the known K+ currents of vascular smooth muscle, with the exception of inward rectifiers, suggests that K+ channel inhibition is likely to make an important contribution to its vasoconstrictor effects.
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ACKNOWLEDGEMENTS |
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We thank Diane Everitt for skilled technical assistance.
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FOOTNOTES |
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This study was supported by The Wellcome Trust and the British Heart Foundation.
Address for reprint requests and other correspondence: N. W. Davies, Ion Channel Group, Dept. of Cell Physiology and Pharmacology, Univ. of Leicester, PO Box 138, Leicester LE1 9HN, UK (E-mail: nwd{at}le.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 May 2001; accepted in final form 7 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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; ±SE, n = 10),
and for inactivation the current amplitudes at +20 mV were normalized
to the maximum current (
; ±SE, n = 4).
The solid lines are fits to the Boltzmann distribution (see Fig. 
), 100 nM IbTX (
activation; ±SE) from 5 cells in control
(
