ATP and
-adrenergic stimulation enhance voltage-gated K
current inactivation in brown adipocytes
Sean M.
Wilson1,
Sherwin C.
Lee2,
Sheryl
Shook2, and
Pamela A.
Pappone2
2 Section of Neurobiology, Physiology, and Behavior,
Division of Biological Sciences, University of California, Davis,
California 95616; and 1 Department of Physiology and Cell
Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
Sympathetic activation of
brown fat thermogenesis stimulates adrenergic and purinergic receptors.
We examined the effects of extracellular ATP and
-adrenergic
agonists on voltage-activated K currents (IKv) in voltage-clamped rat
brown adipocytes. ATP or the
-adrenergic agonist isoproterenol
increased the development of IKv inactivation during depolarizing
voltage steps in perforated patch-clamped cells. The effects on
inactivation developed slowly in the presence of agonist and continued
to increase for long times following agonist washout. 8-bromo-cAMP or
forskolin had similar effects on IKv inactivation. Development of IKv
inactivation during depolarizations was consistently enhanced by ATP or
-adrenergic stimulation in perforated-patch voltage-clamped cells
but was not altered by these agents in whole cell recordings,
suggesting that cytosolic factors are necessary for inactivation
modulation. In either recording configuration, ATP or isoproterenol
shifted the activation voltage dependence of IKv to more negative
potentials, indicating the activation effect is mediated by a different
pathway. Since both P2 purinergic and
-adrenergic signaling pathways
generate fatty acids, we tested whether fatty acids could reproduce
these modulations of IKv. Linoleic or arachidonic acid applied in whole cell recordings had effects similar to those of ATP or isoproterenol in
perforated-patch experiments. These results are consistent with the
possibility that
-adrenergic and P2 receptor stimulation modulate
IKv through generation of fatty acids.
adenosine triphosphate/pharmacology; brown fat/physiology; electrophysiology; potassium channels/physiology; receptors; purinergic/agonists/metabolism
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INTRODUCTION |
SYMPATHETIC NERVE
STIMULATION in response to cold exposure or overeating potently
enhances the energy-wasting activity of brown fat. Acute sympathetic
stimulation increases brown fat metabolic rate up to ~40 times
resting values through activation of lipolysis and mitochondrial
uncoupling (41). Chronic sympathetic stimulation promotes
hyperplasia, hypertrophy, and differentiation of brown fat cells,
increasing the thermogenic capacity of the tissue (6, 18).
These effects of cold exposure or overeating on brown adipose tissue
cannot be mimicked by adrenergic stimulation in vitro
(17), suggesting that sympathetic nerves may release
trophic factors in addition to norepinephrine. Norepinephrine and ATP
are often colocalized in sympathetic nerve terminals and may be
released simultaneously with neuronal activity (30, 69).
Moreover, brown adipocytes respond to both norepinephrine and ATP.
Adrenergic stimulation increases membrane conductances
(37), elevates cytosolic Ca (32, 70),
and activates thermogenesis (41). P2 receptor stimulation
by ATP also increases membrane conductances and cytosolic Ca but
initiates only small increases in heat production (33). In
addition, ATP but not norepinephrine increases membrane trafficking (48).
Important cellular functions are influenced by voltage-gated K channel
activity. Many nonexcitable cells are unable to divide if their
voltage-gated K currents are blocked (7, 12, 13, 15, 34, 43, 46,
54, 63, 65). Blocking voltage-gated K currents can reduce
cytosolic Ca signals by decreasing the electrochemical gradient for Ca
influx (34). Furthermore, cells that do not have
functioning voltage-gated K currents may not be able to regulate their
volume in response to hyposmotic swelling (14).
Brown adipocytes have voltage-gated K currents that activate with
membrane depolarization (38, 57). Blocking this
voltage-gated K current inhibits brown adipocyte proliferation
(49). Moreover, the amount of activatable voltage-gated K
current is modified by P2 receptor stimulation in whole cell recordings
(72). In the present study, we tested the effects of
adrenergic and purinergic stimulation on voltage-gated K currents in
perforated-patch voltage-clamped brown adipocytes. We found that
-adrenergic agonists and extracellular ATP share similar effects on
IKv. Both kinds of stimulation enhance IKv inactivation rates and shift
the voltage dependence of IKv activation to more negative potentials.
Furthermore, some of these gating changes can be mimicked by exposure
of the cells to fatty acids, suggesting that purinergic and adrenergic
pathways may activate convergent lipid signals in brown adipocytes.
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METHODS |
Cells.
Brown adipocytes were isolated from the interscapular fat pads of 1- to
14-day-old Sprague-Dawley rat pups by collagenase digestion and plated
on collagen-coated glass coverslips as previously described
(38). Cells were incubated at 37°C in 5%
CO2 in Dulbecco's modified Eagle's medium supplemented
with 5% fetal calf/horse serum, 0.2 U/ml insulin, 100 µg/ml
penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. Cells were maintained in culture for 1-14 days before
patch-voltage clamping. Mature brown adipocytes were identified
visually by their many fat droplets.
Electrophysiology.
Whole cell voltage-clamp currents were measured using either nystatin
perforated-patch (26) or standard whole cell
patch-voltage-clamp techniques (21) as previously
described (37, 38). Thick-walled borosilicate capillaries
(Sutter Instruments, Novato, CA) were used to manufacture pipettes with
typical resistances of 3-4 M
. The voltage offset between the
patch pipette and the bath solution was nulled immediately before patch
formation. Membrane currents were recorded, filtered through a
four-pole Bessel filter at 5 kHz, and membrane capacity currents nulled
with a patch-voltage-clamp amplifier (model 3900A; Dagan, Minneapolis,
MN; model EPC-7; List Electronic, Darmstadt, Germany; or Axopatch 200A;
Axon Instruments, Foster City, CA) connected to a computer via a TL-1
(Axon Instruments), Basic 23 (Indec Systems, Sunnyvale, CA), or ITC-16
interface (Instrutech, Port Washington, NY). Cell capacitance before
agonist exposure ranged from 15 to 92 pF. Access resistance of
perforated-patch recordings ranged from 15 to 80 M
.
Two different pulse protocols were used. The voltage dependence of the
inactivation rate was determined from currents during 400- or 440-ms
step depolarizations from the
60-mV holding potential. The time
course of agonist-induced current modulation was determined from
currents during depolarizations to positive membrane potentials (+40 to
+60 mV) repeated every 15-60 s. Linear leak and membrane capacitative currents were subtracted using a P/4 or P/5 procedure (1, 38). Since IKv is the only voltage-gated current
present in these cells, this procedure isolates IKv (38).
Pulse protocols were delivered and data collected and analyzed using
either pCLAMP, version 6 (Axon Instruments), Pulse, version 8.07 (Instrutech), or software developed by R. S. Lewis (Stanford
University, Palo Alto, CA).
Solutions.
Cells for patch-voltage-clamp experiments were bathed in Krebs-Ringer
solution containing (in mM) 120 NaCl, 4.5 KCl, 0.5 MgCl2, 2 CaCl2, 0.7 Na2HPO4, 1.3 NaH2PO4, 25 NaHCO3, and 10 glucose, pH 7.4, and gassed with 95% O2-5% CO2; normal
Ringer solution (in mM: 135 NaCl, 4 KCl, 2 CaCl2, 0.5 MgCl2, 10 HEPES, pH 7.4); or a low-Cl Ringer solution, in
which the NaCl was replaced with sodium aspartate. Cells were placed in
a 0.5-ml chamber, and the bath solution was either perfused at a rate
of ~1 ml/min or periodically changed without continuous perfusion.
The pipette solution for whole cell experiments was usually (in mM) 100 potassium aspartate, 25 KCl, 10 NaCl, 10 EGTA, 1 CaCl2, and
10 MOPS titrated to pH 7.2 with KOH. The free Ca concentration of this
solution was calculated to be 19 nM with Patchers Power Tools (F. Mendez, Göttingen, Germany). In some whole cell experiments, 0.5 mM MgCl2 and 1 mM Na2ATP were added to the
internal solution. For perforated-patch experiments, the pipette
usually contained (in mM) 115 potassium aspartate, 25 KCl, 10 NaCl, 10 MOPS, ~0.25 mg/ml nystatin, and 0.1% Pluronic F-127 titrated to pH
7.2 with KOH. Osmolarities of the solutions ranged from 270 to 310 mosM. All chemicals applied to cells were mixed with the bath solution
just before application from stock solutions stored at
20°C.
Nystatin (50 mg/ml) and Pluronic F-127 (0.2 g/ml) stock solutions were
made up in DMSO. ATP, norepinephrine, isoproterenol, phenylephrine, and
8-bromoadenosine 3'-5'-cyclic monophosphate (8-Br-cAMP) were made up as
1-100 mM stock solutions in water. Forskolin was made as a 10 mM
stock solution in ethanol. Stock solutions of arachidonic acid and
linoleic acid were either made fresh each day from sealed ampules or
stored under nitrogen gas at
20°C until use. The arachidonic acid
and linoleic acid experimental working solutions were stored at room temperature in the dark until use. Arachidonic acid was made as a 100 mM stock solution in ethanol. Linoleic acid was made as either a 10 or
100 mM stock solution in DMSO.
Chemicals.
Pluronic F-127 is a product of BASF (Mount Olive, NJ), nystatin
dihydrate and EGTA were from Fluka Chemical (Ronkonkoma, NY), and
apamin was from Alamone Labs (Jerusalem, Israel). All other chemicals
were from Sigma Chemical (St. Louis, MO).
Analysis.
A number reflecting both the rate and extent of IKv inactivation was
determined from the percentage of the peak current still active after
400 ms of depolarization. The time course of IKv activation was
quantified as the time required to reach one-half of the peak current
from the start of the depolarizing pulse. The voltage dependence of K
current activation was determined from peak conductances (g)
calculated from peak current values at each potential, assuming that
the reversal potential was equal to EK. The
conductance values were fitted with the Boltzmann relation
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(1)
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where gmax was the fitted maximum
conductance value, E1/2 the half-maximal
activation voltage, Em the membrane voltage, and k reflects the steepness of the voltage dependence. Averages
in the figures are given as means ± SE. As appropriate,
Student's t-test or an analysis of variance were performed
using a P < 0.05 criterion to test for significance.
Statview version 4.5 (Abacus, Berkeley, CA) or Microsoft Excel, version
5 (Redmond, WA) software were used for statistical analyses.
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RESULTS |
Extracellular ATP modifies IKv gating.
Extracellular ATP increases the amount of IKv inactivation during
depolarizing voltage pulses. Figure
1A shows IKv currents measured at +40 mV in a perforated-patch recording. Before ATP exposure, only 6% of the peak IKv inactivated during the 400-ms pulse.
After four exposures to 5 µM ATP, 58% of the peak IKv inactivated in
400 ms, showing that the rate and/or extent of inactivation at this
membrane potential had increased. The intermittent brief ATP exposures
in this experiment initiated a slow and continuous increase in IKv
inactivation. Figure 1B shows the time course of the change
in IKv inactivation, measured as the percent peak current remaining at
the end of the pulse. The slow constant rate of change in IKv
inactivation suggests that ATP exposure activates a long-lasting signal
in these cells. The extent of IKv inactivation measured at 400 ms was
independent of the membrane potential both before and after ATP
exposure as shown in Fig. 1C. ATP exposure decreased the
percent of noninactivated IKv by similar amounts at all membrane
potentials. ATP decreased the amount of noninactivated IKv at 400 ms in
perforated-patch experiments on average by 30 ± 5%
(n = 13). As summarized in Fig. 7A, ATP
exposure had less effect on IKv inactivation in whole cell recordings,
decreasing the noninactivated IKv by only 8 ± 2%
(n = 24). The attenuated effects of ATP in whole cell
experiments suggests that the sustained modulation activated by ATP
requires cytosolic components.

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Fig. 1.
ATP alters voltage-activated K current (IKv) inactivation
(A-C) and activation (D-F) in a
perforated-patch recording. A: IKv currents recorded during
depolarizations to +40 mV from the 60 mV holding potential before
(1) and after (2) ATP. Before ATP, peak IKv was
1.4 nA, and 94% of the current was still present at 400 ms. After ATP,
peak IKv was still 1.4 nA, but only 42% of the peak current had not
inactivated by 400 ms. B: percent noninactivated IKv at +40
mV measured every 20 s. In this and subsequent figures, ATP (5 µM in this experiment) was present only during the times indicated by
the bars. Cells were either perfused continually with ATP-free or
ATP-containing solution, or the chamber was flushed with the
appropriate solution. Data from numbered time points 1 and
2 were used in the other panels of this figure.
C: percent noninactivated IKv at 400 ms as a function of
membrane potential before (1, ) and after
(2, ) ATP in the same cell. D:
IKv currents shown on an expanded time scale during depolarizations to
0 and +60 mV before (1, dotted lines) and after ATP
(2, solid lines). Before ATP, one-half of the peak current
activated in 31 ms (time to half peak IKv, T1/2
) at 0 mV, and after ATP half activation occurred in only 18 ms.
E: T1/2-voltage relation before
(1, ) and after (2,
) ATP. F: normalized peak IKv
conductance-voltage relations before (1, )
and after (2, ) ATP. Before ATP,
E1/2 = 2 mV, and after ATP,
E1/2 = 3 mV (Eq. 1). A slope
factor, k, of 19 mV was used throughout; g, peak
conductance; Em, membrane voltage.
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Extracellular ATP also affected IKv activation. Figure 1D
shows IKv on an expanded time scale recorded in the same perforated patch-clamped cell before and after ATP exposure. ATP increased the
rate of IKv activation during depolarizations to membrane potentials
less than or equal to +10 mV, but not at more positive potentials (Fig. 1E). The time required for one-half the
peak current to activate, T1/2, following
depolarization to 0 mV, decreased from 31 to 18 ms following ATP
stimulation. ATP also induced a 5-mV hyperpolarizing shift in the
voltage dependence of peak IKv activation (Fig. 1F).
Activation rate increased following ATP exposure in both
perforated-patch and whole cell recordings, as summarized in Fig.
7B. ATP decreased T1/2 at 0 mV by
~40% (average of 12 ± 3 ms) in 13 perforated-patch experiments
and by ~24% (17 ± 2 ms) in 22 whole cell recordings. The
T1/2 values measured at +60 mV were similar
(within ~6%) before and after ATP exposure in both perforated-patch
and whole cell recordings. As summarized in Table
1, the hyperpolarizing shifts in
the voltage dependence of activation were also present in both
perforated-patch and whole cell recordings following ATP. The
activation voltage dependence shifted negative by 7 ± 2 mV in 16 perforated-patch and by 13 ± 2 mV in 35 whole cell recordings. No
significant shifts in the activation voltage dependence were seen in
either perforated-patch (
E1/2 = treated
E1/2
control E1/2 = 0 ± 2 mV, n = 10) or whole cell
(
E1/2 =
2 ± 2 mV,
n = 8) recordings of untreated cells over similar time
periods. The fact that IKv activation was modulated similarly by ATP in
perforated-patch and whole cell recordings, while the effects of ATP on
IKv inactivation depended on the recording configuration, suggests that
purinergic stimulation alters IKv inactivation and activation through
distinct mechanisms.
-Adrenergic stimulation has similar effects on IKv gating.
-Adrenergic receptor stimulation produced changes in IKv
inactivation and activation that were similar to those produced by ATP
exposure. IKv inactivation during depolarizations was increased by
-adrenergic stimulation in perforated-patch but not in whole cell
recordings. Figure 2A shows
scaled IKv recorded at +50 mV before and after a 2-min exposure to 10 µM isoproterenol in a perforated patch-clamped cell. Before
isoproterenol exposure, only 6% of the peak IKv inactivated in 400 ms,
while after isoproterenol, 72% of IKv inactivated in this time. Like
ATP, the effects of isoproterenol on IKv inactivation developed slowly
and continued to increase long after washout of the isoproterenol (Fig.
2B). As with ATP, IKv inactivation was increased to a
similar extent at all membrane potentials following isoproterenol (Fig.
2C). The magnitude of the increases in inactivation with
-adrenergic and ATP stimulation were similar, as summarized in Fig.
7A. Norepinephrine or isoproterenol exposure for 2-30
min diminished the percent noninactivated IKv by an average of 26 ± 4% in 22 perforated-patch experiments. Like ATP,
-adrenergic
agonists did not significantly alter IKv inactivation in whole cell
recordings (Ref. 38 and data not shown).

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Fig. 2.
-Adrenergic stimulation alters IKv inactivation
(A-C) and activation (D-F) in a
perforated-patch recording. A: IKv currents scaled to have
the same peak amplitude activated by a depolarization to +50 mV from
the 60-mV holding potential (HP) before (1) and after
(2) exposure to 10 µM isoproterenol. Control peak IKv was
0.8 nA, and 94% of the current was still present at 400 ms. After
isoproterenol, peak IKv was 0.5 nA, and only 28% of the peak current
remained at 400 ms. B: percent noninactivated IKv over time.
Data from numbered time points 1 and 2 were used
in the other panels of this figure. Isoproterenol was present for 2 min, during the time shown by the bar. C: percent
noninactivated IKv after 400 ms at the membrane voltages shown before
(1, ) and after (2,
) isoproterenol. D: IKv currents scaled to
have the same peak amplitude recorded during depolarizations to 0 and
+60 mV before (1, dotted lines) and after (2,
solid lines) isoproterenol exposure. T1/2 was 48 ms at 0 mV in 1 and 30 ms in 2. E:
T1/2-voltage relation before (1,
) and after (2, )
isoproterenol. F: normalized peak IKv conductance-voltage
relations before (1, ) and after
(2, ) isoproterenol exposure.
E1/2 = 11 mV for 1 and 5 mV
for 2 (Eq. 1). A slope factor, k, of
12 mV was used throughout.
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-Adrenergic agonists had effects similar to ATP on IKv activation in
perforated-patch recordings. Figure 2D shows activation of
IKv on an expanded time scale, illustrating that isoproterenol increased the rate of IKv activation. As shown in Fig. 2E,
the activation rate was increased at membrane potentials less than or
equal to +10 mV, but was less affected at more positive potentials. The
voltage dependence of IKv activation was also shifted toward more
hyperpolarized potentials following
-adrenergic stimulation (Fig.
2F). The effects of
-adrenergic stimulation and ATP are both qualitatively and quantitatively similar. As shown in Fig. 7B, norepinephrine or isoproterenol decreased the
T1/2 by 9 ± 3 ms at 0 mV in
perforated-patch recordings but did not significantly alter the average
activation rate at +60 mV. As shown in Table 1, norepinephrine or
isoproterenol induced a significant negative shift of 7 ± 1 mV in
the voltage dependence of activation in 11 perforated-patch
experiments. Because ATP and
-adrenergic stimulation cause similar
changes in IKv activation and inactivation, they may mediate their
effects through similar signaling pathways.
-Adrenergic stimulation does not affect IKv gating.
In contrast to the effects of ATP and
-adrenergic stimulation,
-adrenergic stimulation did not affect the inactivation of IKv in
perforated-patch recordings. As summarized in Fig. 7A, phenylephrine exposure resulted in only a 1 ± 1% decrease in the percent noninactivated current in five perforated-patch experiments.
Activation of cAMP-mediated pathways reproduces the effects of ATP
and
-adrenergic stimulation on IKv gating.
8-Br-cAMP, a membrane-permeant cAMP analog, mimics some of the effects
of
-adrenergic stimulation on IKv gating. Figure
3A shows scaled IKv currents
at +50 mV before and during exposure to 1 mM 8-Br-cAMP. Like
isoproterenol and ATP, 8-Br-cAMP enhanced IKv inactivation. Before
8-Br-cAMP treatment, 30% of the IKv current inactivated in 400 ms,
whereas, in the presence of 8-Br-cAMP, 75% of the current inactivated.
The increase in inactivation was slow to develop (Fig. 3B)
and was similar at all membrane potentials (Fig. 3C). As
summarized in Fig. 7A, the 25 ± 4% decrease in
noninactivated IKv was similar in magnitude to that caused by
-adrenergic stimulation. Thus exposure of adipocytes to this cAMP
analog reproduces all of the effects on inactivation seen with
-adrenergic stimulation.

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Fig. 3.
8-Br-cAMP affects IKv inactivation (A, B, C;
D and E: activation) in a perforated-patch
recording. A: scaled IKv currents activated by a
depolarization to +50 mV from the 60 mV HP before (1,
dotted line) and during (3, solid line) exposure to 1 mM
8-Br-cAMP. Before 8-Br-cAMP, peak IKv was 0.5 nA, and 70% of the peak
IKv was still present at 400 ms. During 8-Br-cAMP exposure, peak IKv
was 0.7 nA, and only 25% of the peak current remained at 400 ms.
B: percent noninactivated IKv at +50 mV over time. Data from
numbered time points 1-3 were used in the other panels
of this figure. 8-Br-cAMP (1 mM) was present during the time shown by
the bar. C: percent noninactivated current after 400 ms at
the membrane potentials shown before (1, )
and during (2, ) 8-Br-cAMP exposure.
D: scaled IKv currents recorded during depolarizations to 0 mV and +60 mV before (1, dotted lines) and during 8-Br-cAMP
exposure (2, solid lines). Before 8-Br-cAMP,
T1/2 was 27 ms at 0 mV, and during 8-Br-cAMP
exposure it was 26 ms. E:
T1/2-voltage relation before (1,
) and during (2, ) exposure
to 8-Br-cAMP.
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Although 8-Br-cAMP and
-adrenergic stimulation have similar effects
on IKv inactivation, their effects on IKv activation differ. Whereas
isoproterenol exposure significantly increased IKv activation rate,
8-Br-cAMP had minimal effects. This distinction is illustrated in the
scaled current traces and T1/2- voltage relations in Fig. 3. Figure 7B summarizes the 8-Br-cAMP
effects on activation, showing that 8-Br-cAMP did not appreciably
decrease the average T1/2 at 0 or +60 mV.
Forskolin, which increases cellular cAMP levels by activating adenylate
cyclase, also produces some of the same effects as
-adrenergic
stimulation. Figure 4A shows
scaled IKv currents before, during, and after a 7-min exposure to 5 µM forskolin. In the presence of forskolin IKv inactivated rapidly,
within 50 ms in this example. Forskolin washout removed the rapid
inactivation, suggesting it is due to a direct forskolin block of IKv,
as has been described in other voltage-gated K currents (29,
64). Forskolin also increased the extent of IKv inactivation
measured at the end of the pulse, and this effect continued to develop long after forskolin was removed (Fig. 4B). Forskolin
increased IKv inactivation similarly at positive and negative membrane
potentials (Fig. 4C). As summarized in Fig. 7A,
the 25 ± 5% decrease in the noninactivated IKv following
forskolin was similar in magnitude to that caused by isoproterenol.
Neither the voltage dependence nor the rate of IKv activation were
measured in the presence of forskolin.

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Fig. 4.
Forskolin alters IKv inactivation in a perforated-patch
recording. A: IKv currents scaled to have the same peak
amplitude recorded during depolarization to +50 mV from the 60 mV HP
before (1), during (2), and after (3)
exposure to 5 µM forskolin. Before forskolin (1), peak IKv
was 0.8 nA, and 93% of the current remained at 400 ms. During
forskolin exposure (2), peak IKv was 0.5 nA, and 51% of the
current was present at 400 ms. After forskolin washout (3),
peak IKv was 1.5 nA, and 61% of the current was still present at 400 ms. B: percent noninactivated IKv over time. Data from
numbered time points 1-3 were used in the other panels
of this figure. Forskolin (5 µM) was present for 7 min during the
time shown by the bar. C: percent noninactivated current at
400 ms at 0 and +60 mV before (1, ) and
after (3, ) forskolin.
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Isoproterenol, 8-Br-cAMP, and forskolin all caused slowly developing
increases in IKv inactivation in perforated-patch experiments, suggesting that this effect could be mediated through activation of a
common pathway. Furthermore, because 8-Br-cAMP does not increase IKv
activation rate as does isoproterenol and because modulation of IKv
activation is insensitive to cytosolic washout, IKv inactivation and
activation may be modulated through stimulation of separate pathways.
Fatty acids modify IKv inactivation in whole cell recordings.
The changes in IKv inactivation following exposure to adrenergic and
purinergic agonists were slow to develop and seemed to result in
gradual modification of the properties of all IKv channels present
rather than producing populations of channels with differing properties. Such continuous evolution in gating properties of all
channels is more consistent with changes in the environment seen by
channel gates than with discrete modulations such as phosphorylation. We therefore explored the possibility that the agonist effects were
mediated by changes in the lipids present in the cells. Both
-adrenergic and P2 receptor stimulation can produce fatty acids. In
brown fat cells,
-adrenergic stimulation activates hormone-sensitive lipase generating metabolic fatty acids (41). Brown fat
cells have P2 purinergic receptors that are activated by extracellular ATP (48). The signaling pathways activated by ATP in brown
fat are not known, but P2 receptor stimulation activates phospholipases that liberate signaling lipids in other cells (5, 23, 39). We therefore tested whether the similar effects of ATP and
-adrenergic agonists on IKv might be mediated by fatty acids. We
tested two such fatty acids, arachidonic acid, a product of
phospholipase activation, and linoleic acid, a product of
hormone-sensitive lipase activity.
Extracellular application of either fatty acid had effects similar to
ATP and
-adrenergic stimulation. Arachidonic acid and linoleic acid
each increased IKv inactivation during depolarizations, as shown in
Figs. 5 and
6. On average, arachidonic acid
decreased noninactivated IKv by 32 ± 8% and linoleic acid
decreased noninactivated IKv by 49 ± 7%, as summarized in Fig.
7A. As with ATP or
isoproterenol, the increase in IKv inactivation with arachidonic acid
or linoleic acid was similar at all membrane potentials (Figs.
5C and 6C).

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Fig. 5.
Arachidonic acid effects on IKv in a whole cell recording
(A-C: inactivation; D-F: activation).
A: IKv currents scaled to have the same peak amplitude
recorded during a depolarization to +40 mV from a 60 mV HP in the
absence (1) and presence (2) of 100 µM
arachidonic acid (AA). The control peak IKv was 1.5 nA, 88% of which
was still present at 400 ms. In the presence of arachidonic acid, peak
IKv was 0.8 nA, and 41% of the peak current remained at 400 ms.
B: percent noninactivated IKv after 400 ms at +40 mV
measured every 20 s. Data from numbered time points 1 and 2 were used in the other panels of this figure.
Arachidonic acid (100 µM) was present during the times shown by the
bars. C: percent noninactivated current as a function of the
membrane potential before (1, ) and during
(2, ) arachidonic acid exposure.
D: IKv currents scaled to have the same peak amplitude
recorded during depolarizations to 0 and +60 mV before (1,
dotted lines) and during exposure to arachidonic acid (2,
solid lines). T1/2 was 23 ms at 0 mV in
1 and 13 ms in 2. E:
T1/2-voltage relation before (1,
) and during (2, ) exposure
to arachidonic acid. F: normalized peak IKv
conductance-voltage relations before (1, )
and during (2, ) exposure to arachidonic
acid. E1/2 = 5 mV, k = 12 mV for 1; and E1/2 = 6 mV,
k = 16 mV for 2 (Eq. 1).
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Fig. 6.
Linoleic acid alters IKv inactivation
(A-C) and activation (D-F) in a whole
cell recording. A: IKv currents scaled to have the same peak
amplitude activated by depolarizations to +40 mV from the 60 mV HP
before (1, dotted line), during (3, solid line),
and following (4, solid line) linoleic acid exposure. Before
linoleic acid, the peak IKv was 3.4 nA, and 97% had not inactivated in
400 ms. In the presence of 200 µM linoleic acid, peak IKv was 1.7 nA,
and only 49% of the peak current remained at 400 ms. Following washout
of the linoleic acid peak, IKv was 2.3 nA, and 95% of the peak current
remained at 400 ms. B: percent noninactivated IKv after 400 ms at +40 mV measured every 20 s. Data from numbered time points
1-4 were used in the other panels of this figure.
Linoleic acid (LA; 20 or 200 µM) or 0.2% BSA were present during the
times shown by the bars. C: percent noninactivated IKv at
400 ms as a function of the membrane potential in the absence
(1, ) and presence (2,
) of linoleic acid. D: IKv currents scaled
to have the same peak amplitude recorded during depolarizations to 0 and + 60 mV in the absence (1, dotted lines) and
presence (2, solid lines) of linoleic acid.
T1/2 at 0 mV was 57 ms in 1 and 26 ms
in 2. E: T1/2-voltage
relation before (1, ) and during
(2, ) linoleic acid exposure. F:
normalized peak IKv conductance-voltage relations before (1,
) and during (2, ) exposure
to linoleic acid. Control E1/2 = 17 mV, and
linoleic acid E1/2 = 14 mV (Eq. 1). A slope factor k of 14 mV was used throughout.
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Fig. 7.
Summarized effects of modulators of IKv gating. A
(inactivation): average change in the percent peak IKv that was
not inactivated after 400 ms depolarizations to +40, +50, or +60 mV
after exposure to modulator compared with control values. Solid bars,
cells were exposed to 0.01-100 µM ATP for 30 s to 10 min in
perforated-patch (ATP-PP) or whole cell recordings (ATP-WC). Hatched
bars: adrenergic and cAMP stimulation in perforated patch-clamped
cells. Cells were exposed to 1-20 µM isoproterenol (Iso) or
norepinephrine (NE) for 2-30 min, 1 mM 8-Br-cAMP for 12-60
min, 5-10 µM forskolin for 7 min, or to 20-125 µM
phenylephrine for 2 min. Open bars: fatty acid effects in whole cell
recordings. Cells were exposed to 1-100 µM arachidonic acid for
10-20 min or 100-200 µM linoleic acid for 10-12 min.
*Significant difference from control using paired t-test
(P < 0.05). B (activation): change in the
time to reach half maximum IKv, T1/2, after
exposure to modulators compared with control values at membrane
potentials of 0 and 60 mV for a subset of the above experiments. Solid
bars: ATP stimulation in perforated-patch or whole cell recordings.
Hatched bars: -adrenergic stimulation in perforated-patch
experiments. Open bars: fatty acid effects in whole cell recordings.
These values correspond to a 14% decrease in
T1/2 for 8-Br-cAMP and a 52% decrease in
T1/2 in linoleic acid; n = number of cells. *Significant difference in the
T1/2 of peak IKv activation compared with
control values at membrane potentials of 0 and 60 mV using paired
t-test (P < 0.001). Error bars are ± SE. No comparison was made between purinergic, adrenergic, and fatty
acid groups.
|
|
The effects of arachidonic acid and linoleic acid on IKv inactivation
differed from those of ATP or isoproterenol in their cytosolic
requirements and reversibility. Arachidonic acid and linoleic acid
increased inactivation in whole cell recordings, while ATP and
-adrenergic stimulation were only effective in perforated-patch
recordings. IKv inactivation increases were slow in onset and
irreversible when induced by ATP or isoproterenol, whereas arachidonic
acid and linoleic acid increased IKv inactivation rapidly and their
effects were readily reversed on washout (Figs. 5B and
6B). These results suggest that fatty acids may be
downstream effectors of slow and sustained signals induced by the agonists.
Fatty acids also increased IKv activation rates as shown in Figs.
5D and 6D. Activation rates were increased
substantially at membrane potentials less than or equal to +10 mV, and
the effects were smaller at more positive potentials (Figs.
5E and 6E). The decrease in
T1/2 in the presence of arachidonic acid was
similar in magnitude to those induced by ATP or
-adrenergic receptor stimulation, as summarized in Fig. 7B. In contrast to ATP or
-adrenergic agonists, arachidonic acid and linoleic acid did not
affect the voltage dependence of activation in whole cell recordings
(Figs. 5F and 6F and Table 1). These results
suggest that fatty acids could be the signaling agents responsible for
the
-adrenergic- and ATP-induced increases in IKv inactivation and
activation. However, because neither arachidonic acid nor linoleic acid
caused hyperpolarizing shifts in the voltage dependence of activation, other fatty acids or other signaling agents may be involved in this
modulation of IKv gating.
 |
DISCUSSION |
We found that stimulation of brown adipocyte
-adrenergic
receptors and exposure to extracellular ATP similarly modulate the inactivation and activation of voltage-gated K currents, IKv. In
perforated patch-clamped cells, the rate of IKv decline during depolarizations increased and the voltage dependence of IKv activation was shifted to more negative membrane potentials by
-adrenergic or
ATP stimulation. The onset of the effects on IKv inactivation were
delayed and continued to develop long after removal of the agonist.
Similar alterations of IKv gating properties were seen when cells were
exposed to micromolar arachidonic acid or linoleic acid. In contrast to
the agonist-induced changes in IKv inactivation, the effects of the
exogenous fatty acids were rapid in onset and readily reversed on
washout. These results suggest that
-adrenergic and P2 purinergic
signaling pathways converge in generating fatty acids that modulate K
channel function in brown fat cells.
Previous work has shown that various types of K channel activity
increases or decreases following fatty acid exposure (22, 45, 50,
67). Increased inactivation of voltage-gated K currents resembling our results have been reported in several preparations. Arachidonic acid increases the inactivation rate of Kv1.1 transfected into Sf9 cells (20), A-type K currents in smooth muscle
(40) or expressed in oocytes (66), Kv1.5 in
cardiomyocytes (25), and unspecified voltage-gated K
currents in oesteoblasts (11) and carotid body neurons
(24). Linoleic acid and other polyunsaturated fatty acids
can similarly increase the inactivation rate of a delayed rectifier K
current in neuroblastoma cells (56). Thus fatty acid
modulation of voltage-gated K currents is common and may also underlie
the IKv modulations seen here in brown fat.
Possible sources of fatty acids in brown fat.
It is well established that
-adrenergic stimulation of brown
adipocytes generates fatty acids through cAMP-mediated activation of
hormone-sensitive lipase (41). The particular fatty acids liberated from triglycerides by hormone-sensitive lipase activity depends on a number of factors. The animal's diet (44,
68), environmental temperature (62), metabolic and
hormonal status (19, 53, 58, 68), and level of arousal
from hibernation (8) all can influence the fatty acid
composition and metabolism of the brown fat depots. Cold adaptation
increases the proportion of arachidonic acid (62) and
linoleic acid (8) stored in rat brown fat. Linoleic acid
is preferentially released from brown fat stores during the
-adrenergically activated thermogenesis involved in arousal from
hibernation (8). In addition, normally rare precursors of
arachidonic acid, i.e., homo-
-linoleic acid and eicosadienoic acid,
increase to high levels in the brown fat of hibernating hamsters and
return to near normal levels during arousal (8). Thus
-adrenergic metabolic activation of brown fat can generate linoleic
acid and arachidonic acid production.
The slow onset and lack of reversal of ATP effects seen in our
experiments suggest that ATP acts through one or more P2 receptors rather than affecting K channels directly or through ectokinase activity. Brown fat cells express at least four different P2Y receptors
(Lee Pappone, unpublished observations), and one or more P2 receptors
alter the voltage dependence of IKv inactivation in these cells
(72). Nothing is currently known about P2
receptor-mediated lipid signaling in brown adipocytes.
Although P2 receptors are known to influence cAMP signaling in other
cell types (27, 28, 51, 52), it is unlikely that ATP
activates hormone-sensitive lipase in brown fat cells. Fatty acids
generated by cAMP-activated hormone-sensitive lipase activity are
thought to provide both the substrate and the trigger for the
thermogenic response to adrenergic stimulation (36).
However, exposure of brown fat cells to extracellular ATP neither
stimulates thermogenesis on its own nor alters norepinephrine-activated
heat production (33). These considerations indicate that
actions on cAMP signaling do not mediate the effects of ATP on IKv in brown fat cells.
Arachidonic acid can be generated in many cells by P2Y
receptor-mediated activation of phospholipase C and phospholipase
A2 (PLA2) (2, 3, 9, 10, 16, 23, 31, 35,
74). P2 receptor stimulation releases Ca from intracellular
stores in brown adipocytes (33), suggesting that
phospholipase C is activated by ATP exposure. We tried testing whether
inhibiting PLA2 prevented ATP from affecting IKv
inactivation, but the results were inconclusive.
-Adrenergic
stimulation activates both PLC and PLA2 and releases
arachidonic acid in brown adipocytes (59, 60), but the
lack of effect of
-adrenergic stimulation on IKv inactivation seen
in our experiments suggests that brief activation of PLC or
PLA2 is not sufficient to elicit changes in IKv
inactivation. In contrast to the rapidly reversible effects of
-adrenergic stimulation, repeated exposures to ATP can irreversibly
activate a Ca-permeable cation current (Ref. 33 and
Pappone, unpublished observations). Repeated ATP exposures were also
required to produce IKv inactivation effects in the present study. Thus
sustained Ca influx through this pathway may be required to support
continued generation of arachidonic acid by ATP, suggesting that brown
fat's Ca-sensitive PLA2 may be involved (60).
Possible functions of IKv modulation.
Potassium channel activity has been shown to affect mitogenesis, volume
regulation, and intracellular Ca signaling in many cells. Functional
voltage-gated K currents are required for the proliferation of many
nonexcitable cells (7, 12, 13, 15, 73), including brown
adipocytes (49). Proliferation of brown preadipocytes in
vivo (18) and in vitro (4) is promoted by
-adrenergic stimulation, and P2 receptor stimulation similarly increases mitogenesis of cultured brown preadipocytes
(71). These results suggest that modulation of IKv by ATP
and norepinephrine may play a role in preadipocyte proliferative
responses. In support of this possibility, the modulation of IKv by
extracellular ATP parallels the effects of ATP on cell proliferation.
Low concentrations of ATP promote preadipocyte proliferation
(71) and can increase activated IKv by shifting the
voltage dependence for IKv activation to more hyperpolarized potentials
(72). Prolonged exposure to ATP or stimulation with high
concentrations of ATP decrease IKv by enhancing inactivation
(72), and higher concentrations of ATP inhibit
preadipocyte proliferation (71).
IKv may affect cell proliferation through actions on cell volume.
Voltage-gated K currents can act in concert with volume-sensitive Cl
channels to effect regulatory volume decreases in many nonexcitable cells (7, 13, 14, 42, 55), and decreases in cell volume promote cell proliferation (54). Brown adipocytes express
volume-activated Cl currents that can depolarize the cells to
potentials that activate IKv (47), making it likely that
IKv contributes to their volume regulatory responses. Adrenergic and P2
receptor stimulation activates Ca-sensitive Cl currents
(47) that could similarly activate IKv and initiate cell
volume decreases.
The findings presented here suggest that P2 receptor and
-adrenergic
receptor stimulation may converge in altering the activity of
voltage-gated K currents in brown adipocytes through the generation of
fatty acids. The effects of ATP and
-adrenergic agonists on IKv
parallel their effects on brown adipocyte proliferation. Hence regulation of IKv activity by P2 purinergic or
-adrenergic receptor stimulation could modulate adipose tissue growth. Thus, determining the
function and mechanism of P2 purinergic and
-adrenergic modulation of IKv gating in brown adipocytes could be important for understanding and controlling obesity.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Martin Wilson, Martha O'Donnell, and Eric Leaver for
critical review of the manuscript; Ken Kunisaki, Jeff Pham, Joel Isaac
Barthelow, and Christina Stroup for culturing cells, and Jock Hamilton
for technical assistance.
 |
FOOTNOTES |
This work was supported by National Institute of General Medical
Sciences Grant GM-44840.
Address for reprint requests and other correspondence: P. A. Pappone, Section of Neurobiology, Physiology, and Behavior, One Shields Ave., Univ. of California at Davis, Davis, CA 95616 (E-mail: papappone{at}ucdavis.edu).
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 14 October 1998; accepted in final form 11 July 2000.
 |
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