Membrane responses to norepinephrine in cultured brown fat cells.

We used the "perforated-patch" technique (Horn, R., and A. Marty, 1988. Journal of General Physiology. 92:145-159) to examine the effects of adrenergic agonists on the membrane potentials and membrane currents in isolated cultured brown fat cells from neonatal rats. In contrast to our previous results using traditional whole-cell patch clamp, 1-23-d cultured brown fat cells clamped with the perforated patch consistently showed vigorous membrane responses to both alpha- and beta-adrenergic agonists, suggesting that cytoplasmic components essential for the thermogenic response are lost in whole-cell experiments. The membrane responses to adrenergic stimulation varied from cell to cell but were consistent for a given cell. Responses to bath-applied norepinephrine in voltage-clamped cells had three possible components: (a) a fast transient inward current, (b) a slower outward current carried by K+ that often oscillated in amplitude, and (c) a sustained inward current largely by Na+. The fast inward and outward currents were activated by alpha-adrenergic agonists while the slow inward current was mediated by beta-adrenergic agonists. Oscillating outward currents were the most frequently seen response to norepinephrine stimulation. Activation of this current, termed IK,NE, was independent of voltage and seemed to be carried by Ca2(+)-activated K channels since the current oscillated in amplitude at constant membrane potential and gradually decreased when the cells were bathed with calcium-free external solution. IK,NE had a novel pharmacology in that it could be blocked by 4-aminopyridine, tetraethylammonium, apamin, and charybdotoxin. Both IK,NE and the voltage-gated K channels also present in brown fat (Lucero, M. T., and P. A. Pappone, 1989a. Journal of General Physiology. 93:451-472) may play a role in maintaining cellular homeostasis in the face of the high metabolic activity involved in thermogenesis.

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Alpha-adrenergic stimulation activates a calcium-sensitive chloride current in brown fat cells.

The Journal of General Physiology ◽

10.1085/jgp.106.2.231 ◽

1995 ◽

Vol 106 (2) ◽

pp. 231-258 ◽

Cited By ~ 18

Author(s):

P A Pappone ◽

S C Lee

Keyword(s):

Intracellular Calcium ◽

Brown Fat ◽

Chloride Conductance ◽

Free Calcium ◽

Fat Cells ◽

Adrenergic Stimulation ◽

Chloride Currents ◽

Whole Cell ◽

Perforated Patch ◽

Whole Cell Recordings

The first response of brown adipocytes to adrenergic stimulation is a rapid depolarizing conductance increase mediated by alpha-adrenergic receptors. We used patch recording techniques on cultured brown fat cells from neonatal rats to characterize this conductance. Measurements in perforated patch clamped cells showed that fast depolarizing responses were frequent in cells maintained in culture for 1 d or less, but were seen less often in cells cultured for longer periods. Ion substitution showed that the depolarization was due to a selective increase in membrane chloride permeability. The reversal potential for the depolarizing current in perforated patch clamped cells indicated that intracellular chloride concentrations were significantly higher than expected if chloride were passively distributed. The chloride conductance could be activated by increases in intracellular calcium, either by exposing intact cells to the ionophore A23187 or by using pipette solutions with free calcium levels of 0.2-1.0 microM in whole-cell configuration. The chloride conductance did not increase monotonically with increases in intracellular calcium, and going whole cell with pipette-free calcium concentrations > or = 10 microM rapidly inactivated the current. The chloride currents ran down in whole-cell recordings using intracellular solutions of various compositions, and were absent in excised patches. These findings imply that cytoplasmic factors in addition to intracellular calcium are involved in regulation of the chloride conductance. The chloride currents could be blocked by niflumic acid or flufenamic acid with IC50s of 3 and 7 microM, or by higher concentrations of SITS (IC50 = 170 microM), DIDS (IC50 = 50 microM), or 9-anthracene carboxylic acid (IC50 = 80 microM). The chloride conductance activated in whole cell by intracellular calcium had the permeability sequence PNOS > PI > PBr > PCl > Paspartate, measured from either reversal potentials or conductances. Instantaneous current-voltage relations for the calcium-activated chloride currents were linear in symmetric chloride solutions. Much of the current was time and voltage independent and active at all membrane potentials between -100 and +100 mV, but an additional component of variable amplitude showed time-dependent activation with depolarization. Volume-sensitive chloride currents were also present in brown fat cells, but differed from the calcium-activated currents in that they responded to cell swelling, required intracellular ATP in whole-cell recordings, showed no sensitivity to intracellular or extracellular calcium levels, and were relatively resistant to block by niflumic and flufenamic acids. (ABSTRACT TRUNCATED AT 400 WORDS)

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The effect of β-adrenergic agonists on the membrane potential of fat-cell mitochondria in situ

Biochemical Journal ◽

10.1042/bj2060611 ◽

1982 ◽

Vol 206 (3) ◽

pp. 611-618 ◽

Cited By ~ 11

Author(s):

R J Davis ◽

B R Martin

Keyword(s):

Membrane Potential ◽

Fat Cells ◽

Adrenergic Agonists ◽

Fat Cell ◽

Beta Adrenergic ◽

Fatty Acid Binding ◽

Beta Adrenergic Agonists ◽

Adrenergic Antagonist ◽

Beta Adrenergic Agonist

1. The accumulation of [3H]methyltriphenylphosphonium by isolated fat-cells was used to estimate the membrane potential of mitochondria in situ. 2. Adrenaline caused a large decrease in the accumulation of [3H]methyltriphenylphosphonium. Mitochondria in fat-cells incubated in the presence of adrenaline had a very low calculated membrane potential. This effect was also given by isoprenaline (a beta-adrenergic agonist) and was blocked by propranolol (a beta-adrenergic antagonist). 3. The effect of isoprenaline could be partially antagonized by the use of media with high albumin concentrations. Addition of sodium oleate to saturate the fatty acid-binding sites on the albumin reversed this antagonism. 4. It is proposed that the decrease in the calculated mitochondrial membrane potential is due to the uncoupling effect of the non-esterified fatty acids released by the stimulation of lipolysis observed in the presence of beta-adrenergic agonists.

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Potassium channel block does not affect metabolic responses of brown fat cells

AJP Cell Physiology ◽

10.1152/ajpcell.1992.262.3.c678 ◽

1992 ◽

Vol 262 (3) ◽

pp. C678-C681 ◽

Cited By ~ 14

Author(s):

P. A. Pappone ◽

M. T. Lucero

Keyword(s):

Potassium Channels ◽

Metabolic Response ◽

Brown Fat ◽

Neonatal Rat ◽

K Channel ◽

Fat Cells ◽

Adrenergic Stimulation ◽

K Channels ◽

Voltage Gated ◽

Calcium Activated Potassium Channels

Hormonally stimulated brown fat cells are capable of extremely high metabolic rates, making them an excellent system in which to examine the role of plasma membrane ion channels in cell metabolism. We have previously shown that brown fat cell membranes have both voltage-gated and calcium-activated potassium channels (Voltage-gated potassium channels in brown fat cells. J. Gen. Physiol. 93: 451-472, 1989; Membrane responses to norepinephrine in cultured brown fat cells. J. Gen. Physiol. 95: 523-544, 1990). Currents through both the voltage-activated potassium channels, IK,V, and the calcium-activated potassium channels, IK,Ca, can be blocked by the membrane-impermeant K channel blocker tetraethylammonium (TEA). We used microcalorimetric measurements from isolated neonatal rat brown fat cells to assess the role these potassium conductances play in the metabolic response of brown fat cells to adrenergic stimulation. Concentrations of TEA as high as 50 mM, sufficient to block approximately 95% of IK,V and 100% of IK,Ca, had no effect on norepinephrine-stimulated heat production. These results show that neither voltage-gated nor calcium-activated K channels are necessary for a maximal thermogenic response in brown fat cells and suggest that K channels are not involved in maintaining cellular homeostasis during periods of high metabolic activity.

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Norepinephrine-induced sustained inward current in brown fat cells: α1-mediated by nonselective cation channels

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.5.e963 ◽

2000 ◽

Vol 279 (5) ◽

pp. E963-E977 ◽

Cited By ~ 5

Author(s):

Ari Koivisto ◽

Detlef Siemen ◽

Jan Nedergaard

Keyword(s):

Fold Increase ◽

Brown Fat ◽

Cation Channels ◽

Current Response ◽

Fat Cells ◽

Adrenergic Stimulation ◽

Open Probability ◽

Inward Current ◽

Nonselective Cation Channels ◽

Sustained Inward Current

The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the α1-agonist cirazoline but not by the β3-agonist CGP-12177A. Reduction of extracellular Cl− concentration had no effect, but omission of extracellular Ca2+ or Na+ totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of ≈30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from ≈0.002 to ≈0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the α1-agonist phenylephrine than with the β3-agonist CGP-12177A, and with the Ca2+ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of ≈30 pS nonselective cation channels via α1-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca2+ concentration.

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Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study

AJP Cell Physiology ◽

10.1152/ajpcell.1992.263.4.c780 ◽

1992 ◽

Vol 263 (4) ◽

pp. C780-C787 ◽

Cited By ~ 26

Author(s):

D. C. Devor ◽

M. E. Duffey

Keyword(s):

Patch Clamp ◽

T84 Cells ◽

Patch Clamp Technique ◽

Whole Cell ◽

Inward Current ◽

Perforated Patch ◽

Ionic Conductances ◽

Inward Currents ◽

K Current ◽

Perforated Patch Clamp

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

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Adrenergically activated Ca2+ increases in brown fat cells: effects of Ca2+, K+, and K channel block

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.1.c217 ◽

1993 ◽

Vol 264 (1) ◽

pp. C217-C228 ◽

Cited By ~ 33

Author(s):

S. C. Lee ◽

R. Nuccitelli ◽

P. A. Pappone

Keyword(s):

Calcium Influx ◽

Brown Fat ◽

Neonatal Rat ◽

K Channel ◽

Initial Increase ◽

Fat Cells ◽

Adrenergic Stimulation ◽

K Channels ◽

High Concentrations ◽

Calcium Cells

We measured intracellular calcium concentration ([Ca2+]i) during adrenergic stimulation using fura-2 ratio imaging of individual cultured neonatal rat brown fat cells. One micromolar norepinephrine (NE) increased [Ca2+]i from an average resting value of 105 nM to 555 nM in approximately 30 s. [Ca2+]i remained elevated as long as NE was present but returned to resting levels within 2-3 min after NE removal. The response was half maximal at approximately 50 nM NE and was primarily alpha-adrenergic. The sustained, but not the initial, increase in [Ca2+]i required extracellular calcium. Cells stimulated in high-K media had [Ca2+]i responses like those in 0 Ca2+, suggesting that depolarization abrogates calcium influx. Parallel perforated-patch recordings showed that the increase in [Ca2+]i activates a calcium-activated K conductance. Blocking K channels with moderate concentrations of tetraethylammonium (TEA) had only small effects on NE-induced changes in [Ca2+]i, but high concentrations of TEA significantly reduced the response. We conclude that cytoplasmic calcium is modulated by fluxes from both intracellular and extracellular sources and that K channels may not be required for normal short-term [Ca2+]i responses to hormone.

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Voltage-gated potassium channels in brown fat cells.

The Journal of General Physiology ◽

10.1085/jgp.93.3.451 ◽

1989 ◽

Vol 93 (3) ◽

pp. 451-472 ◽

Cited By ~ 33

Author(s):

M T Lucero ◽

P A Pappone

Keyword(s):

Single Channel ◽

Reversal Potential ◽

Brown Fat ◽

Membrane Currents ◽

Delayed Rectifier ◽

Fat Cells ◽

K Channels ◽

Voltage Gated ◽

K Currents ◽

External K

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.

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