α1-Adrenergic Stimulation Potentiates the Thermogenic Action of β3-Adrenoreceptor-generated cAMP in Brown Fat Cells

The activity of the uncoupling protein in brown fat mitochondria is enhanced at alkaline pH, leading to the hypothesis that changes in intracellular pH (pHi) may modulate the thermogenic response to sympathetic stimulation. We employed ratio imaging of the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi in acutely isolated single brown fat cells from hamster and neonatal rat and in cultured rat cells. Basal pHi averaged approximately 7.2 in HCO3- media and 0.1-0.15 pH units lower in nominally HCO3(-)-free media in all cell types. In both HCO3- and HCO3(-)-free media, stimulation with norepinephrine (NE) typically caused an alkalinization of approximately 0.05-0.1 pH units, which was followed by a smaller net acidification occurring primarily after NE was removed. Alkalinization seemed to be mediated predominantly by alpha-adrenergic stimulation, while acidification most often followed beta-adrenergic activation. Similar pHi changes were elicited by NE in rat and hamster cells, but responses were more frequent in hamster cells. Assays of recovery from ammonium prepulse-induced acid loads indicated that rat and hamster cells have both Na(+)-H+ and Na(+)- and HCO3(-)-dependent regulatory systems, while hamster cells have, in addition, a Na(+)-independent recovery mechanism activated at acid pHi. We conclude that alpha-adrenergic alkalinization of brown fat may contribute to the control of thermogenesis.

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Effects of P2 purinergic receptor stimulation in brown adipocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1997.273.2.c679 ◽

1997 ◽

Vol 273 (2) ◽

pp. C679-C686 ◽

Cited By ~ 30

Author(s):

S. C. Lee ◽

P. A. Pappone

Keyword(s):

Heat Production ◽

Purinergic Receptor ◽

Cytosolic Calcium ◽

Extracellular Atp ◽

Brown Fat ◽

Brown Adipocyte ◽

Fat Cells ◽

Sympathetic Stimulation ◽

Adrenergic Stimulation ◽

Brown Adipocytes

Sympathetic stimulation of brown adipocytes plays a major role in body energy homeostasis by activating energy-wasting pathways. Sympathetic neuronal input initiates a variety of metabolic, developmental, and membrane responses in brown fat cells. Many of these actions are mediated by adrenergic pathways mobilized by released norepinephrine. However, since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. Micromolar concentrations of extracellular ATP had a number of effects on brown adipocytes. We have shown previously that ATP elicits substantial (average of approximately 30%) increases in cell membrane capacitance (P. A. Pappone and S. C. Lee, J. Gen. Physiol. 108: 393-404, 1996). Here, we show that cytosolic calcium levels were increased by ATP, both through release from intracellular stores and through influx, as assessed by fura 2 imaging. In addition, ATP indirectly activated a nonselective cation conductance that was independent of cytosolic calcium levels in patch voltage-clamped brown fat cells. Similar calcium, conductance, and capacitance responses could be activated by 2-methylthio-ATP and ADP, consistent with mediation by a P2 type purinergic receptor. Calorimetric measurements from cell suspensions showed that ATP increased basal heat production of isolated brown fat cells by approximately 40% but had no effect on the greater than fivefold increase in heat production seen with maximal adrenergic stimulation. These myriad responses to extracellular ATP suggest that P2 receptor-mediated signaling is important in brown adipocyte physiology and that sympathetic stimulation may normally activate purinergic as well as adrenergic pathways in brown fat.

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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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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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Distinction between mechanisms underlying alpha 1- and beta-adrenergic respiratory stimulation in brown fat cells

AJP Cell Physiology ◽

10.1152/ajpcell.1987.253.2.c301 ◽

1987 ◽

Vol 253 (2) ◽

pp. C301-C308 ◽

Cited By ~ 33

Author(s):

N. Mohell ◽

E. Connolly ◽

J. Nedergaard

Keyword(s):

Large Fraction ◽

Atp Synthesis ◽

Brown Fat ◽

Fat Cells ◽

Adrenergic Stimulation ◽

Experimental Conditions ◽

Beta Adrenergic ◽

Carbonyl Cyanide ◽

High Concentrations ◽

Respiratory Stimulation

Experimental conditions are described for selective alpha 1- and beta-adrenergic stimulation of the respiration of brown fat cells. The dual agonist norepinephrine was unsuitable as a selective alpha 1-agonist, since unacceptably high concentrations of propranolol were needed to abolish the beta-response. Phenylephrine at 50 microM, in the presence of 5 microM dl-propranolol, was shown to lead to a maximal, selective alpha 1-stimulation, whereas maximal, selective beta-stimulation was achieved with 1 microM isoproterenol in the presence of 5 microM prazosin. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to further increase respiration that was already maximally alpha 1-stimulated, but when added before the alpha 1-stimulation, FCCP totally abolished the response. In contrast, FCCP had no effect on the beta-stimulated response. Similarly, oligomycin (an inhibitor of mitochondrial ATP synthesis) inhibited alpha 1-respiration but had a much smaller effect on beta-respiration. Ouabain (an inhibitor of the Na+-K+-ATPase) halved alpha 1-respiration but only induced a small inhibition of beta-respiration. It is concluded that only a small fraction of thermogenesis from beta-adrenergic processes is due to oxidative phosphorylation, whereas alpha 1-respiration is largely due to the oxygen cost of mitochondrial ATP synthesis, and a large fraction of this ATP is apparently used for the restoration of ion gradients.

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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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