␣1-Adrenoceptor–Mediated Breakdown of
Phosphatidylinositol 4,5-Bisphosphate Inhibits
Pinacidil-Activated ATP-Sensitive Kⴙ Currents in Rat
Ventricular Myocytes
Tetsuya Haruna, Hidetada Yoshida, Tomoe Y. Nakamura, Lai-Hua Xie, Hideo Otani,
Tomonori Ninomiya, Makoto Takano, William A. Coetzee, Minoru Horie
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Abstract—Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates ATP-sensitive K⫹ (KATP) channel activity. Because
phospholipase C (PLC) hydrolyzes membrane-bound PIP2, which in turn may potentially decrease KATP channel activity,
we investigated the effects of the ␣1-adrenoceptor–Gq–PLC signal transduction axis on pinacidil-activated KATP channel
activity in adult rat and neonatal mouse ventricular myocytes. The ␣1-adrenoceptor agonist methoxamine (MTX)
reversibly inhibited the pinacidil-activated KATP current in a concentration-dependent manner (IC50 20.9⫾6.6 ␮mol/L).
This inhibition did not occur when the specific ␣1-adrenoceptor antagonist, prazosin, was present. An involvement of
G proteins is suggested by the ability of GDP␤S to prevent this response. Blockade of PLC by U-73122 (2 ␮mol/L) or
neomycin (2 mmol/L) attenuated the MTX-induced inhibition of KATP channel activity. In contrast, the MTX response
was unaffected by protein kinase C inhibition or stimulation by H-7 (100 ␮mol/L) or phorbol 12,13-didecanoate. The
MTX-induced inhibition became irreversible in the presence of wortmannin (20 ␮mol/L), an inhibitor of
phosphatidylinositol-4 kinase, which is expected to prevent membrane PIP2 replenishment. In excised inside-out patch
membranes, pinacidil induced a significantly rightward shift of ATP sensitivity of the channel. This phenomenon was
reversed by pretreatment of myocytes with MTX. Direct visualization of PIP2 subcellular distribution using a PLC␦
pleckstrin homology domain– green fluorescent protein fusion constructs revealed reversible translocation of green
fluorescent protein fluorescence from the membrane to the cytosol after ␣1-adrenoceptor stimulation. Our data
demonstrate that ␣1-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated
KATP channels. (Circ Res. 2002;91:232-239.)
Key Words: ATP-sensitive K⫹ channels 䡲 phosphatidylinositol 4,5-bisphosphate 䡲 ␣1-adrenoceptors
uring myocardial ischemia, cardiac ATP-sensitive K⫹
(KATP) channels open and shorten the duration of action
potentials. Among numerous modulators of KATP channel
activity, exogenously applied phosphatidylinositol 4,5bisphosphate (PIP2) produces two remarkable effects on both
native and reconstituted KATP channels: (1) it increases the
channel open probability (even that of channels existing in a
rundown state), and (2) it decreases the ability of the KATP
channels to be blocked by cytosolic ATP.1– 4 More recently,
we demonstrated that phosphorylation of membrane phosphoinositol (PI) to PIP2 is an indispensable process in keeping
the channel operative, both for Kir6.2/SUR2A channels
reconstituted in COS-7 cells and for native KATP channels in
guinea pig ventricular myocytes.5–7 Therefore, the membrane
level of PIP2 is a potential key regulator of KATP channel
activity.
D
Levels of PIP2 at the membrane are regulated by a subtle
balance between PIP2 breakdown (hydrolysis by phospholipase C [PLC]) and replenishment (by PI kinases).8 PLC
activity features in the transduction pathways of several
hormonal receptors, including the M1 muscarinic, ␣1adrenergic, endothelin (ET)-1, and angiotensin II (Ang II)
receptors.9 Therefore, we hypothesize that the stimulation of
PLC-linked receptors may change the level of membranebound PIP2 and thereby modulate the activity of KATP channels. In support of this hypothesis, ␣1-adrenoceptor or ET
receptor stimulation has been reported to inhibit inward
rectifier K⫹ channels, including KATP channels.10 –12 We have
recently demonstrated that M1 muscarinic receptor stimulation inhibits the Kir6.2/SUR2A channel current through
depletion of membrane PIP2.6 The physiological relevance
and exact pathway by which ␣1-adrenoceptor–mediated inhi-
Original received March 13, 2001; resubmission received January 28, 2002; revised resubmission received July 8, 2002; accepted July 8, 2002.
From the Departments of Cardiovascular Medicine (T.H., H.Y., H.O., T.N., M.H.) and Physiology and Biophysics (L.-H.X., M.T.), Kyoto University
Graduate School of Medicine, Kyoto, Japan, and Pediatric Cardiology (T.Y.N., W.A.C.), NYU School of Medicine, New York, NY.
Correspondence to Dr Minoru Horie, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin
Kawahara-cho Sakyo-ku, Kyoto 606-8507, Japan. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000029971.60214.49
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Haruna et al
PIP2 and KATP Channels
233
bition occurs remains unknown. Therefore, we used rat
ventricular myocytes to examine whether ␣1-adrenoceptor
stimulation modulates native KATP channel activity. We used
a pharmacological approach to delineate the pathway through
which this occurs. In addition, we directly measured PIP2
levels using a fluorescent PIP2 reporter protein. Our data
show that (as is the case for reconstituted channels) the
␣1-adrenoceptor pathway inhibits cardiac KATP channels
through a reduction of membrane PIP2 levels.
Materials and Methods
Single-Cell Preparation and Electrophysiology
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All experimental protocols were performed in accordance with
institutional animal guidelines. Ventricular myocytes were isolated
from the hearts of adult Sprague-Dawley rats (Shimizu Experimental
Animal Supply, Kyoto, Japan) using a collagenase perfusion method.
Single ventricular myocytes were placed in a recording chamber and
superfused with Tyrode’s solution containing (mmol/L) NaCl 143,
NaH2PO4 0.3, KCl 5.4, MgCl2 0.5, CaCl2 1.8, and HEPES/NaOH 5,
pH 7.4, adjusted by NaOH. The pipette solution for whole-cell patch
clamping contained (mmol/L) aspartic acid 110, NaCl 10, MgCl2 1,
K2ATP 4, GTP 0.1, BAPTA 10, and HEPES 5, pH 7.4, with KOH.
In some experiments, the GTP was replaced with GDP␤S (1 mmol/
L). KATP currents were usually evoked by extracellular application of
the KATP channel opener pinacidil (100 ␮mol/L) and were recorded
by applying ramp pulses (⫾100 mV/s) every 6 seconds from a
⫺40-mV holding potential to obtain quasi-instantaneous currentvoltage (I-V) relations (from 20 to ⫺120 mV). Membrane current
was recorded using an amplifier (Axopatch 200A, Axon Instruments), low-pass–filtered (Bessel response, 1 kHz) before being
acquired online (pClamp 6.1 version, Axon), and displayed on a
chart recorder (Linerecorder, WR 3320, Graphtec).
Single KATP channel activities were recorded in the inside-out
mode. Glucose-free Tyrode’s solution was used as a pipette solution.
For the internal solution bathing the cytoplasmic surface of the patch
membrane, a K⫹-rich solution was used. Its composition was
(mmol/L) KCl 150, EGTA 0.5, and HEPES 5, pH 7.4, adjusted by
KOH. Patch pipettes were prepared by pulling borosilicate glass
capillaries (Hilgenberg) at 2 to 3 M⍀ for whole-cell recording and at
5 to 7.5 M⍀ for single-channel recording when they were filled with
each pipette solution.
Cell Culture and Transfection
Ventricular myocytes were isolated from 2- to 3-day-old neonatal
Swiss Webster mice (Taconic, Germantown, NY) and dissociated
into single isolated cells by trypsinization, as described previously.13
To exclude nonmyocytes, cells were preplated at 37°C for 45
minutes in culture medium (DMEM containing 10% FBS and
antibiotics). The cell suspension was passed through a 100-␮m nylon
mesh (Falcon) and plated (1⫻105 per dish) onto a collagen-coated
(type VI, 100 ␮g/mL, Sigma Chemical Co) glass coverslip attached
to the bottom of a Petri dish (glass bottom microwells, Corning). One
day after incubation at 37°C, myocytes were transfected with a PLC␦
pleckstrin homology domain– green fluorescent protein (PH-GFP)
fusion construct (1 ␮g)14 using FuGENE6 reagent (Boehringer).
COS-7 cells were plated onto poly-L-lysine (100 ␮g/mL)– coated
glass coverslips and cultured for 1 day. Cells were transfected with
the PH-GFP construct and ␣1c-adrenoceptors (or pCDNA3 as a
control) (1 ␮g each) as described above. One day after transfection,
the culture medium was changed to the serum-free DMEM to
prevent possible contamination of receptor agonists that might be
present in the serum.
Confocal Microscopy
Cells on glass coverslips were transferred in a recording chamber
continuously perfused with Ca2⫹-free Tyrode’s solution at 31°C to
33°C (by temperature controller TC-324B, Warner Instruments
Corp). Images were obtained with a confocal microscope (Carl Zeiss
Figure 1. ␣1-Adrenoreceptor agonists inhibit KATP current repeatedly and reversibly. A, Whole-cell KATP current was activated by
100 ␮mol/L pinacidil. The clamping protocol consisted of ramp
voltage pulses (⫾100 mV/s, ⫺120 to 20 mV) applied from a
holding potential of ⫺40 mV, which were repeated every 6 seconds. The arrow indicates zero current level. The horizontal bars
above the recording indicate the onset and duration of test
solutions. B, Current was measured from the declining portion
of the ramp and plotted against the corresponding voltage to
obtain quasi-instantaneous I-V relations. Shown are I-V relations, obtained at various time points, indicated by letters a
through g on the current recording. C, Difference I-V relationships were obtained by digital subtraction. We show difference
I-V relations for pinacidil-induced current (b-a), phenylephrinesensitive current (b-c), MTX-sensitive current (d-e), and
glibenclamide-sensitive current (f-g).
LSM 510 and a Leica DM-IRE2 inverted microscope fitted with a
TCS SP2 scan head) equipped with a plan-apochromat ⫻60 oil
objective lens. Fluorescence was detected using an argon laser
(488-nm line). Series of confocal images were taken at 10- to
30-second intervals. The line intensity profiles and the ratios of the
averaged fluorescence signals from membrane and cytosol area were
analyzed with Leica confocal software.
Drugs
Methoxamine (MTX), phenylephrine, prazosin (Sigma), and neomycin (Nakalai) were freshly prepared in various test solutions immediately before each experiment. Propranolol (1 ␮mol/L) was present
throughout each experiment to eliminate possible ␤-adrenergic
actions of the agonists used. Wortmannin, U-73122, pinacidil glibenclamide (Sigma), and bimakalim (a generous gift from Merck,
Darmstadt, Germany) were dissolved in dimethyl sulfoxide at 10 or
100 mmol/L (stock solution). Phorbol 12,13-didecanoate (PDD) or
H-7 was also dissolved in dimethyl sulfoxide as a stock solution
before being diluted to its final concentration in the experimental
solution.
Results
Stimulation of ␣1-Adrenoceptor Inhibits
Pinacidil-Activated KATP Current
Pinacidil15 (100 ␮mol/L) consistently elicited an outward
current at a holding potential of ⫺40 mV (Figure 1A, a3b).
I-V relationships (Figure 1B, 1) show that the pinacidilinduced current reverses at ⫺90 mV and is inhibited by
glibenclamide (1 ␮mol/L, Figure 1A, f3g). Difference currents for pinacidil-induced and glibenclamide-sensitive components exhibit reversal potentials of ⬇⫺85 mV, which is
close to the estimated equilibrium potential for K⫹ under
these experimental conditions (Figure 1C, b-a and f-g). These
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Figure 2. MTX inhibits KATP current in concentration-dependent
manner. A, Three different concentrations of MTX were applied
in a cumulative manner. B, Concentration-inhibition relationship
for MTX is shown. Percentage of MTX-induced inhibition was
determined as described in the text. Each data point at the various MTX concentrations represents mean⫾SEM inhibition
observed in at least in 5 myocytes.
findings suggest that the pinacidil-induced current is caused
by the activity of KATP channels.
In the continued presence of propranolol (1 ␮mol/L), bath
application of the ␣1-adrenoceptor agonist phenylephrine
(100 ␮mol/L) or MTX (100 ␮mol/L) reversibly suppressed
the pinacidil-induced current (Figure 1A, b3c and d3e),
without altering its reversal potential (Figure 1B, b-2 and
b-3). The current component that is sensitive to the ␣1adrenoceptor agonists (Figure 1C, b-c and d-e) also had a
reversal potential close to the equilibrium potential for K⫹.
Thus, agents that stimulate ␣1-adrenoceptors cause a reversible inhibition of the pinacidil-activated KATP channel current.
The dose dependence of MTX on pinacidil-activated KATP
channel current is shown in Figure 2. Three different concentrations of MTX caused a cumulative inhibition of the
pinacidil-induced KATP current (Figure 2A). We quantified the
percentage of MTX-induced current inhibition (at steady
state) as follows: 100⫻(IMTX/IKATP), where IMTX denotes the
current component (at 0 mV) that is blocked by MTX (a and
b in Figure 2A), and IKATP is defined as the glibenclamidesensitive current component (a through c in Figure 2A). The
MTX concentration-inhibition relationship was compiled
from measurements made in different cells (n⫽35, Figure
2B), and the data points were fitted to a Hill equation as
follows: % inhibition⫽100/{1⫹(IC50/[MTX])h}, where IC50
indicates the concentration for the half-maximal inhibition
(20.9⫾6.6 ␮mol/L), [MTX] indicates MTX concentration,
and h indicates the Hill coefficient (0.68⫾0.15).
Involvement of ␣1-Adrenoceptors and Role of G
Proteins in MTX-Induced Inhibition
The inhibition of pinacidil-induced current by 100 ␮mol/L
MTX was largely prevented by pretreatment with prazosin
(2.5 ␮mol/L), which is a specific ␣1-adrenoceptor antagonist
(Figure 3A). Replacement of GTP in the pipette solution with
GDP␤S (1 mmol/L), a nonspecific inhibitor of G proteins,
also prevented the MTX-induced inhibition (Figure 3B).
Figure 3. MTX inhibits pinacidil-activated KATP channel current
through an ␣1-adrenoceptor and G-protein–mediated pathway.
A, Effect of prazosin, a specific ␣1-adrenergic antagonist, on
MTX-induced inhibition. B, Effect of intracellular GDP␤S
(1 mmol/L), a nonspecific GTP-binding protein inhibitor. C, Summary of magnitudes of MTX-induced inhibition (expressed as
percentage of maximal KATP channel current) for the control,
prazosin, and GDP␤S groups. Numerals in parentheses indicate
number of observations per group. Data are expressed as
mean⫾SEM. *P⬍0.05 compared with control.
Figure 3C summarizes the effects of these two interventions.
The blockade of ␣1-adrenoceptors and G proteins significantly suppressed the MTX-induced current inhibition from
75.0⫾3.4% (control) to 17.8⫾5.4% or 22.7⫾1.7%, respectively (P⬍0.05). These findings suggest that the MTXinduced inhibition of the pinacidil response occurs via the
␣1-adrenoceptor pathway and illustrates an involvement of G
proteins in this process.
PLC Activity Is Required for MTX-Induced Inhibition
Stimulation of Gq proteins activates PLC activity.16 To
examine the involvement of PLC activity in the inhibition of
KATP channel activity by MTX, we used two types of PLC
inhibitors, U-73122 and neomycin. After the reversible inhibition of pinacidil-induced current by MTX was confirmed in
a given myocyte (Figure 4A), we applied U-73122 (2 ␮mol/
L), which by itself caused a slight increase of the pinacidilactivated current. In the continued presence of U-73122, a
subsequent application of MTX caused comparatively little
inhibition of the pinacidil-activated current (Figure 4A).
These data are summarized in Figure 4C, in which the
magnitude of the first MTX-induced inhibition is compared
with that of a second application (the latter being made after
U-73122 treatment for ⬎3 minutes). Treatment with U-73122
significantly attenuated the MTX-induced inhibition (from
68.0⫾5.0% to 40.1⫾4.9%, P⬍0.05). Using another PLC
blocker (neomycin, 2 mmol/L), we essentially obtained an
identical result (Figures 4B and 4C), except that neomycin
produced a mild decrease in the pinacidil-induced current by
itself. Neomycin attenuated the second MTX-induced inhibition from 78.4⫾4.5% (in the absence of the drug) to
39.5⫾6.4% (P⬍0.05, n⫽5). These findings demonstrate an
Haruna et al
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Figure 4. MTX triggers receptor-stimulated pathway in which
activation of PLC is a key step. A, Effect of U-73122, an inhibitor of PLC, on MTX-induced current inhibition. B, Effect of neomycin, another PLC inhibitor, on MTX-induced inhibition. C,
Summary of magnitudes of MTX-induced inhibition of pinacidilactivated KATP current before and after treatment of myocytes
with U-73122 and neomycin. Numerals in parentheses indicate
number of observations in each experimental group. Data are
expressed as mean⫾SEM. #P⬍0.05 compared with MTX
application.
involvement of PLC activation in the effects of ␣1-receptors
on the MTX inhibition of the pinacidil-activated KATP current.
Lack of Involvement of PKC
PLC-induced hydrolysis of PIP2 produces diacylglycerol
(DAG) and inositol 1,4,5-phosphate (IP3).8 DAG, in turn,
activates protein kinase C (PKC) and may increase cytosolic
Ca2⫹ levels.9 Therefore, it was possible that activation of PKC
(or increases in cytosolic Ca2⫹) may have mediated the
MTX-induced inhibition of the current. An involvement of
intracellular Ca2⫹ is unlikely in our experiments because the
myocytes were dialyzed with pipette solutions containing
10 mmol/L BAPTA, which is expected to chelate the cytosolic [Ca2⫹] to subnanomolar levels. To exclude the possibility of an involvement of PKC activation, we used two types
of PKC modulators. First, H-7, a nonspecific inhibitor of
various PKC isoforms, did not affect the MTX-induced
inhibition (Figure 5A). Second, we used a phorbol ester
(PDD, 2 ␮mol/L), which activates PKC. PDD by itself had no
statistically significant effect on the pinacidil-induced KATP
current (n⫽6, 116.5⫾10.2% of control at 0 mV after 3
minutes). PDD pretreatment (for ⬎3 minutes) did not prevent
the MTX-induced inhibition of the KATP current (Figure 5B).
These data are summarized in Figure 5C. Under control
conditions, MTX inhibited the pinacidil response by
75.3⫾3.8%. This value was unchanged by H-7 (88.0⫾1.9%,
n⫽4) or PDD (82.5⫾4.5%, n⫽6). Taken together, these data
suggest that PKC activation is not involved in the response of
pinacidil-activated KATP channel activity to ␣1-receptor
stimulation.
Recovery From MTX-Induced Inhibition Is
Prevented by Wortmannin
After depletion of PIP2 by PLC activation, membrane PIP2
levels were restored principally by de novo synthesis, which
PIP2 and KATP Channels
235
in turn was mediated by PI kinases (PI-4 and PIP kinases).8
Wortmannin (an antifungal antibiotic isolated from a culture
of a fungus, Penicillium) blocks PI-3 kinase17 but also inhibits
some PI-4 kinase isoforms at higher concentrations.18,19 We
used wortmannin as a PI-4 kinase inhibitor to examine
whether replenishment of the membrane PIP2 is involved in
the response of pinacidil-activated KATP channel activity to
␣1-receptor stimulation.
Pinacidil was again used to evoke the KATP current, and as
before, we used a double application of MTX (100 ␮mol/L)
to compare the first response with the second. The magnitudes of MTX-induced inhibition of pinacidil-activated current by both MTX applications were comparable, and both
were readily reversible on MTX washout (Figure 6A). Double applications of MTX inhibited pinacidil-activated current
by similar amounts. We then examined the effect of wortmannin at three different concentrations (2, 10, and 20
␮mol/L) (Figures 6B through 6D). After the myocytes were
exposed to ⬎10 ␮mol/L wortmannin, however, the current
did not return fully to the control level but instead declined
gradually even in the maintained presence of pinacidil, after
the second washout of MTX (Figures 6C and 6D). In contrast,
at 2 ␮mol/L, it did not affect the recovery after MTX washout
(Figure 6B).
We grouped data from several experiments and plotted the
current amplitude after the MTX applications as a percentage
of the amplitude before MTX treatment (Figure 6E). In the
absence of wortmannin (filled circles in Figure 6E), the
current recovered up to 91.6⫾4.5% within 4 minutes after
MTX washout (n⫽10). Similar recovery was observed in the
presence of 2 ␮mol/L wortmannin (filled triangles, n⫽6).
But, at higher wortmannin concentrations, the current level
did not recover after MTX washout, and mean percent
recovery at 3 minutes washout was 59.0⫾5.4% in 10 ␮mol/L
wortmannin (open squares, n⫽5) and 42.2⫾2.9% in 20
␮mol/L wortmannin (open circles, n⫽4).
Figure 5. MTX-induced inhibition of pinacidil-activated KATP current is independent of PKC. A, Effect of H-7, a PKC inhibitor, on
MTX-induced inhibition. B, Effect of PDD, a PKC activator, on
MTX-induced inhibition. C, Summary of magnitudes in MTXinduced inhibition of KATP current for control, H-7, and PDD.
Numerals in parentheses indicate number of observations. Data
are expressed as mean⫾SEM.
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Circulation Research
August 9, 2002
Figure 6. MTX-induced inhibition becomes irreversible after blockade of PI-4 kinase with wortmannin. A through D, Typical traces of whole-cell
current depicting MTX-induced current inhibition in
the absence (A) and presence of various concentrations of wortmannin (B, 2 ␮mol/L; C, 10 ␮mol/L;
and D, 20 ␮mol/L) that was applied ⬇3 minutes
before the second exposure to MTX. E, Summary
of data, illustrating recovery time course of KATP
channel current after MTX washout. Current is
expressed as percentage of current recorded at 0
mV immediately before the second application of
MTX. Shown are recovery profiles in the absence
(filled circles) and presence of 2 ␮mol/L (filled triangles), 10 ␮mol/L (open squares), and 20 ␮mol/L
(open circles) wortmannin. Open triangles indicate
time course after application of wortmannin alone.
Data are expressed as mean⫾SEM. *P⬍0.05
between control and wortmannin.
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Open triangles in the graph (Figure 6E) indicate the time
course of relative current amplitude at 0 mV in the continued
presence of 20 ␮mol/L wortmannin without MTX (n⫽5). The
compound alone reduced the pinacidil-induced current slightly,
suggesting the inhibition of basal level of PIP2 recruitment, but
its suppression was statistically not significant. Thus, PI-4 kinase
activity and membrane PIP2 replenishment are necessary for the
recovery from the MTX-induced inhibition of the pinacidil-activated KATP current in rat ventricular myocytes.
MTX Reverses Pinacidil-Induced Alteration of
ATP Sensitivity
Pinacidil is known to activate the KATP channel by altering its
sensitivity to ATP.15 In the reconstituted channel experiment
with Kir6.2 and SUR, PIP2 was shown to interact with the
Kir6.2 subunit and to alter its ATP sensitivity.2 Taken
together, the MTX-stimulated reduction in membrane PIP2
level might modulate the pinacidil-induced shift of ATP
sensitivity of KATP channel in native cardiac myocytes too. In
the excised patch experiment, we studied the hypothesis. As
shown on the left of Figure 7A, on the formation of this mode
in the artificial internal solution containing no ATP, vigorous
KATP channel activities could be recorded as an upward
deflection. They were completely inhibited by 2 mmol/L ATP
(Figure 7A) or 1 ␮mol/L glibenclamide (data not shown),
confirming that the single-channel events were indeed openings of KATP channels.
K2ATP (100 ␮mol/L) applied to the inner surface largely
suppressed mean patch currents from 17.7 to 1.1 pA. The
inhibitory effect of K2ATP was determined as a relative
current that was calculated by normalizing mean patch
currents in K2ATP to the control measured immediately
Figure 7. MTX reverses pinacidil-induced
change of ATP sensitivity. A, Representative single-channel recording. Three
bars above the graph indicate application of 2 mmol/L or 100 ␮mol/L K2ATP
and exposure of 100 ␮mol/L pinacidil. B,
ATP concentration– channel inhibition
relationship measured at 2 different
times: open circles, immediately before
application of 100 ␮mol/L pinacidil (at
baseline); filled circles, after 3-minute
exposure of 100 ␮mol/L pinacidil. Symbols and bars indicate mean⫾SEM.
Smooth curves were best fits to the Hill
equation. C, Representative singlechannel recording from an excised
patch. The myocyte was pretreated with
50 ␮mol/L MTX, and the pipette solution
contained the same concentration of
MTX. D, ATP concentration– channel inhibition relationship after MTX treatment in
2 different conditions: open and filled
circles are as in panel B.
Haruna et al
PIP2 and KATP Channels
237
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Figure 8. Direct visualization of membrane PIP2. A, COS7 cells were transiently transfected with PH-GFP alone (top) or cotransfected with ␣1cadrenoceptor cDNA (bottom). Confocal images depict GFP fluorescence obtained before (left) and 2 to 3 minutes after application of phenylephrine
(100 ␮mol/L, right). Line intensity profiles across the cell width are shown below each image. B, On the left are 2 confocal images depicting PH-GFP
fluorescence, obtained from neonatal mouse ventricular cells before (top) and after a 1-minute application of MTX (bottom). On the right are 2 time
courses of GFP fluorescent signals measured at the cell membrane (filled squares) and the cytosol (filled circles). Their ratios are also plotted underneath the graphs. Bars above the graphs indicate extracellular applications of bimakalim, prazosin, and MTX.
before its application. Relative currents thus calculated were
0.06. Subsequent application of 100 ␮mol/L pinacidil gradually opened the channel, even in the continued presence of
K2ATP, increasing the relative currents to 0.96.
To produce the breakdown of PIP2, 50 ␮mol/L MTX was
added to the pipette solution, and myocytes were preincubated with bathing solution containing 50 ␮mol/L MTX for
⬇60 seconds before the formation of the inside-out patch. In
the presence of MTX (Figure 7B), the stimulatory action of
100 ␮mol/L pinacidil was blunted: relative currents were not
significantly increased by pinacidil (from 0.05 to 0.16).
Direct application of MTX (50 ␮mol/L) to the inner surface
of the patch membrane was without effect (data not shown).
Figures 7C and 7D summarizes the inhibitory action of
K2ATP before (open circles) and after a 3-minute application
of pinacidil (filled circles). Relative currents at a given
concentration of K2ATP were averaged and are plotted as a
function of ATP concentrations. Four smooth curves in the
graphs are best fitted to the Hill equation as follows: relative
current⫽1/{1⫹([K2ATP]/IC50)h}, where IC50 is the K2ATP
concentration ([K2ATP]) for the half-maximal inhibition, and
h is the Hill coefficient. In the control condition (Figure 7C),
pinacidil significantly increased the IC50 from 49.0⫾0.5 to
264.7⫾11.1 ␮mol/L. However, after MTX treatment (Figure
7D), these values were not changed (47.5⫾0.4 and 49.0⫾0.5
␮mol/L in the absence and the presence of 100 ␮mol/L
pinacidil, respectively). Thus, MTX abolished the pharmacological action of pinacidil.
Stimulation of ␣1-Adrenoceptor Reduces
Membrane PIP2 Levels
Our data presented until now are consistent with the notion
that ␣1-adrenoceptor stimulation causes PIP2 breakdown (hy-
drolyzed by PLC) and that the decreased PIP2 levels are
responsible, in part, for the inhibition of the pinacidil-activated KATP channel current. To confirm that ␣1-adrenoceptor
stimulation indeed leads to decreases in membrane PIP2
levels in cardiac myocytes, we used a tool that visualizes PIP2
subcellular compartmentalization. To this end, we used the
PLC␦ PH-GFP construct, which has been previously used
with success for this purpose.14 To characterize experimental
conditions, pilot studies were performed in which COS cells
were transiently transfected with PH-GFP cDNA alone or
were cotransfected with ␣1c-receptor cDNA (Figure 8A). In
both cases, strong GFP fluorescence was detected at the cell
periphery. Bath application of phenylephrine (100 ␮mol/L)
led to a rapid translocation of the fluorescent signal from the
plasma membrane to the cytosol, but only in cells that
coexpressed the ␣1c-receptor.
To examine whether PIP2 translocation occurs in cardiac
myocytes, we used mouse neonatal ventricular myocytes,
which (unlike adult myocytes) can readily be transfected
using commercial reagents (Figure 8B). Using patch-clamp
techniques, we verified that pinacidil-activated KATP channels
are present in this preparation and that phenylephrine blocks
this pinacidil response (data not shown). On the left, Figure
8B shows two images of a myocyte that was transfected with
the PH-GFP construct. As was the case in COS cells, GFP
fluorescence was localized at the cell surface (top) and
rapidly translocated to the cytosol after application of MTX
(bottom). On the upper right of Figure 8B is the time course
of the GFP signal at two regions of interest (cell membrane
and cytosol), and the graph on the bottom indicates the
temporal change of fluorescence ratios (PM/Cyt). PM/Cyt
was obtained as the ratio of averaged fluorescence signals
238
Circulation Research
August 9, 2002
from plasma membrane (PM) against those from cytosol
(Cyt) areas. Note that this phenomenon was fully reversible
and that prazosin (2.5 ␮mol/L) completely inhibited the
MTX-induced mobilization of GFP signal (bottom right). The
percent PM/Cyt ratio measured 1 minute after MTX was
reduced by 70.5⫾7.2% (n⫽9) and was 102.3⫾7.5% (n⫽2) in
the presence of prazosin. These data are consistent with the
idea that ␣1-adrenoceptor stimulation reduces membrane PIP2
levels and that membrane PIP2 levels are replenished after the
cessation of receptor stimulation.
Discussion
MTX-Induced Inhibition of KATP Current Is Due
to Activation of PLC
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Stimulation of ␣1-adrenoceptors with MTX led to a rapid and
reversible inhibition of the KATP channel current that was
activated by pinacidil. We found that activation of PLC is a
key step in this pathway, inasmuch as inhibition of PLC with
neomycin or U-73122 significantly attenuated the MTXinduced inhibition of the current. Because PLC-mediated PIP2
hydrolysis produces DAG and IP3 (and possible increase in
cytosolic Ca2⫹), it is possible that KATP channels may have
been modulated by ⱖ1 of these events. It seems very unlikely
that an increase in cytosolic Ca2⫹ activity was responsible for
this effect of MTX, because myocytes were dialyzed with
high concentrations of a high-affinity Ca 2⫹ chelator
(BAPTA). It is also unlikely that DAG mediates the MTX
response, inasmuch as we found DAG to be without effect on
reconstituted KATP channels (Kir6.2/SUR2A).6 Other studies
have similarly found exogenous IP3 also to be without effect
on KATP channel activity.3 Given the sensitivity of the MTX
response to pharmacological interventions affecting PLC
(and the comparative insensitivity to manipulation of the
PKC pathway), we conclude that PLC activation is a key step
in the ␣1-adrenoceptor pathway and in the effect that it has on
cardiac KATP channels.
Effect of PLC Activation Is Mediated Through
Alterations in Membrane PIP2 Levels
Our data suggest that the mechanism by which the ␣1adrenoceptor pathway inhibits KATP channel activity involves
alterations in membrane PIP2 levels. A proposed mechanism
is that PLC-induced PIP2 hydrolysis leads to lower membrane
PIP2 levels that, in turn, may inhibit KATP channel activity.
This concept is supported by two main lines of complementary evidence. First, we directly demonstrated that ␣1adrenoceptor stimulation produces a prompt depletion of the
membrane PIP2 levels in cardiac myocytes, as visualized
using a PLC␦ PH-GFP fusion protein as a PIP2 indicator. This
fusion protein has been widely used in the past by others to
examine the subcellular distribution of PIP2 in a variety of
cells.14,20 We have also characterized the use of this reporter
protein in COS cells and found that ␣1-adrenoceptor stimulation altered only the subcellular fluorescence distribution
when cells express the ␣1c-receptor.
A second main line of evidence comes from the wortmannin data. We found that preventing PIP2 regeneration by
blockade of key PI-4 kinases by wortmannin prevents the
KATP channel recovery after ␣1-adrenoceptor–mediated inhi-
bition. Thus, we conclude that ␣1-adrenoceptor stimulation
led to temporal reversible alterations in membrane PIP2
levels, which led to the observed changes in KATP channel
activity. In addition, U-73122, a PLC inhibitor, produced a
slight increase in basal pinacidil-induced KATP current. This
would reflect the increase in baseline PIP2 level as a result of
a subtle balance between its degradation by PLC and recruitment by PI kinases. In contrast, neomycin, another PLC
inhibitor, reduced the pinacidil-induced current, suggesting
that it has an additional action, ie, that of blocking the KATP
channel as an aminoglycoside polycation.
Our data are fully consistent with the available literature.
Using radioactivity measurements, another study found that
in rat heart, the ␣1-adrenoceptor agonist stimulates PIP2
hydrolysis to a maximal rate within ⬇60 seconds of application and that the total PIP2 content recovers to ⬇70% of
control values within 3 minutes of washout.17 With confocal
microscopic techniques and PIP2 visualization (using the
PLC␦ PH-GFP construct also used in the present study), it has
been directly demonstrated that PLC activation causes membrane PIP2 breakdown in living cells.18,19 The time course of
membrane PIP2 breakdown reported in the latter study corresponds well with that of PIP2 hydrolysis as measured by
radioactive techniques.18,19 These time courses were also very
similar to those that we observed during the MTX-induced
inhibition and the recovery of the KATP current in rat ventricular myocytes. Thus, taken altogether, our data are in full
support of the idea that ␣1-adrenoceptor stimulation inhibits
pinacidil-activated KATP channel current through a pathway
mediated by decreased sarcolemmal PIP2 levels and the
resultant change in ATP sensitivity of the channel.
Limitations of the Study
In the present study, we always used K⫹ channel openers to
evoke the KATP channel activation. As suggested by our
single-channel data in excised patches, pinacidil artificially
opened the channel by altering its sensitivity to ATP. Therefore, it remains to be determined whether the PIP2 pathway is
also involved in the inhibition of naturally opened currents.
Stimulation of several membrane receptors activates events in
diverse pathways that also involve the activation of PLC.9 We
previously demonstrated in guinea pig ventricular myocytes21,22 that both ET-1 and Ang II inhibit the KATP channels
activated by metabolic inhibition. We proposed that ET-1 and
Ang-II inhibited KATP channel activity through an increased
subsarcolemmal ATP concentration, an event that is mediated
by blockade of adenylate cyclase and pertussis toxin–sensitive G proteins (Gi). However, both of these receptor pathways are also linked to PLC activation. Do ET-1 and Ang II
also exert their modulation on KATP channel activity through
PIP2 metabolism? Prevailing data argue against this possibility. In our previous studies, we used guinea pig ventricular
myocytes and found that ET-1 and Ang II inhibited KATP
channels only when they were activated by metabolic inhibition and not when they were activated by the KATP channel
opener cromakalim.21,22 In contrast, we saw a reduction in
pinacidil-activated current by ET-1 in rat ventricular myocytes. Thus, an involvement of PIP2 metabolism appears to
Haruna et al
depend on the species and the dominance of the type of G
proteins through which each receptor is coupled.
In conclusion, we have demonstrated that PIP2 metabolism
potentially plays a key role in regulating the activity of native
KATP channels in cardiac myocytes. Hormonal regulation of
KATP channel activity may occur under physiological conditions but is expected to have particular relevance to cardiac
ischemia, in which drastic hormonal changes occur. PIP2
levels also regulate the activity of other types of inward
rectifier K⫹ channels as well as the Na⫹-Ca2⫹ exchanger,1,23,24
and receptor stimulation may lead to fundamental alterations
in excitability under these conditions. Further investigation
into the role of membrane PIP2 metabolism may lead to
important new insights into ischemia-related phenomena.
Acknowledgments
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Dr Horie was supported by research grants from the Ministry of
Education, Science, Sports, and Culture of Japan, by the Takeda
Foundation for Science Promotion, and by a Japan Heart Foundation/
Pfizer grant for cardiovascular disease research. Dr Takano was
supported by a grant from the Japan Cardiovascular Research
Foundation. The authors are grateful to K. Tsuji for technical
assistance.
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α1-Adrenoceptor-Mediated Breakdown of Phosphatidylinositol 4,5-Bisphosphate Inhibits
Pinacidil-Activated ATP-Sensitive K + Currents in Rat Ventricular Myocytes
Tetsuya Haruna, Hidetada Yoshida, Tomoe Y. Nakamura, Lai-Hua Xie, Hideo Otani, Tomonori
Ninomiya, Makoto Takano, William A. Coetzee and Minoru Horie
Circ Res. 2002;91:232-239; originally published online July 18, 2002;
doi: 10.1161/01.RES.0000029971.60214.49
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