ATP and beta -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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sympathetic activation of brown fat thermogenesis stimulates adrenergic and purinergic receptors. We examined the effects of extracellular ATP and beta -adrenergic agonists on voltage-activated K currents (IKv) in voltage-clamped rat brown adipocytes. ATP or the beta -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 beta -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 beta -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 beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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 MOmega .

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
g=g<SUB>max</SUB><IT>/</IT>{<IT>1+</IT>exp[(<IT>E<SUB>1/2</SUB>−E</IT><SUB>m</SUB>)<IT>/k</IT>]} (1)
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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, open circle ) 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, open circle ) and after (2, ) ATP. F: normalized peak IKv conductance-voltage relations before (1, open circle ) 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.

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 (Delta E1/2 = treated E1/2 - control E1/2 = 0 ± 2 mV, n = 10) or whole cell (Delta 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.

                              
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Table 1.   Voltage dependence of IKv activation

beta -Adrenergic stimulation has similar effects on IKv gating. beta -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 beta -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 beta -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, beta -adrenergic agonists did not significantly alter IKv inactivation in whole cell recordings (Ref. 38 and data not shown).


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Fig. 2.   beta -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, open circle ) 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, open circle ) and after (2, ) isoproterenol. F: normalized peak IKv conductance-voltage relations before (1, open circle ) 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.

beta -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 beta -adrenergic stimulation (Fig. 2F). The effects of beta -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 beta -adrenergic stimulation cause similar changes in IKv activation and inactivation, they may mediate their effects through similar signaling pathways.

alpha -Adrenergic stimulation does not affect IKv gating. In contrast to the effects of ATP and beta -adrenergic stimulation, alpha -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 beta -adrenergic stimulation on IKv gating. 8-Br-cAMP, a membrane-permeant cAMP analog, mimics some of the effects of beta -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 beta -adrenergic stimulation. Thus exposure of adipocytes to this cAMP analog reproduces all of the effects on inactivation seen with beta -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, open circle ) 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, open circle ) and during (2, ) exposure to 8-Br-cAMP.

Although 8-Br-cAMP and beta -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 beta -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, open circle ) and after (3, ) forskolin.

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 beta -adrenergic and P2 receptor stimulation can produce fatty acids. In brown fat cells, beta -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 beta -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 beta -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, open circle ) 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, open circle ) and during (2, ) exposure to arachidonic acid. F: normalized peak IKv conductance-voltage relations before (1, open circle ) 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, open circle ) 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, open circle ) and during (2, ) linoleic acid exposure. F: normalized peak IKv conductance-voltage relations before (1, open circle ) 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: beta -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 beta -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 beta -adrenergic receptor stimulation, as summarized in Fig. 7B. In contrast to ATP or beta -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 beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We found that stimulation of brown adipocyte beta -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 beta -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 beta -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 beta -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 beta -adrenergically activated thermogenesis involved in arousal from hibernation (8). In addition, normally rare precursors of arachidonic acid, i.e., homo-gamma -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 beta -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. alpha -Adrenergic stimulation activates both PLC and PLA2 and releases arachidonic acid in brown adipocytes (59, 60), but the lack of effect of alpha -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 alpha -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 beta -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 beta -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 beta -adrenergic agonists on IKv parallel their effects on brown adipocyte proliferation. Hence regulation of IKv activity by P2 purinergic or beta -adrenergic receptor stimulation could modulate adipose tissue growth. Thus, determining the function and mechanism of P2 purinergic and beta -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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