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

2000 ◽  
Vol 279 (5) ◽  
pp. E963-E977 ◽  
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.

Regulation of the Activity of 27 pS Nonselective Cation Channels in Excised Membrane Patches from Rat Brown-Fat Cells

1998 ◽  
Vol 8 (5) ◽  
pp. 231-245 ◽  
Author(s):  
Ari Koivisto ◽  
Andreas Klinge ◽  
Jan Nedergaard ◽  
Detlef Siemen
Keyword(s):  
Brown Fat ◽  
Cation Channels ◽  
Fat Cells ◽  
Nonselective Cation Channels

Adrenergic modulation of intracellular pH in isolated brown fat cells from hamster and rat

1994 ◽  
Vol 267 (2) ◽  
pp. C349-C356 ◽  
Author(s):  
S. C. Lee ◽  
J. S. Hamilton ◽  
T. Trammell ◽  
B. A. Horwitz ◽  
P. A. Pappone
Keyword(s):  
Intracellular Ph ◽  
Uncoupling Protein ◽  
Cell Types ◽  
Brown Fat ◽  
Neonatal Rat ◽  
Fat Cells ◽  
Adrenergic Stimulation ◽  
Regulatory Systems ◽  
Ratio Imaging ◽  
Free Media

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.

Nonselective Cation Channels in Brown and White Fat Cells

1993 ◽  
pp. 201-211 ◽  
Author(s):  
Ari Koivisto ◽  
Elisabeth Dotzler ◽  
Ulrich Ruß ◽  
Jan Nedergaard ◽  
Detlef Siemen
Keyword(s):  
Cation Channels ◽  
Fat Cells ◽  
Nonselective Cation Channels ◽  
White Fat

scholarly journals Membrane responses to norepinephrine in cultured brown fat cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 523-544 ◽  
Author(s):  
M T Lucero ◽  
P A Pappone
Keyword(s):  
Brown Fat ◽  
Fat Cells ◽  
Adrenergic Agonists ◽  
Whole Cell ◽  
K Channels ◽  
Inward Current ◽  
General Physiology ◽  
Perforated Patch ◽  
Beta Adrenergic Agonists ◽  
Outward Currents

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.

Effects of P2 purinergic receptor stimulation in brown adipocytes

1997 ◽  
Vol 273 (2) ◽  
pp. C679-C686 ◽  
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.

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

1995 ◽  
Vol 106 (2) ◽  
pp. 231-258 ◽  
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)

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

1992 ◽  
Vol 262 (3) ◽  
pp. C678-C681 ◽  
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.

scholarly journals Cold-induced reduction in Giα proteins in brown adipose tissue. Effects on the cellular hypersensitization to noradrenaline caused by pertussis-toxin treatment

1996 ◽  
Vol 314 (3) ◽  
pp. 761-768 ◽  
Author(s):  
Petr SVOBODA ◽  
Lena UNELIUS ◽  
Andrea DICKER ◽  
Barbara CANNON ◽  
Graeme MILLIGAN ◽  
...  
Keyword(s):  
Cold Acclimation ◽  
Pertussis Toxin ◽  
Fold Increase ◽  
Brown Fat ◽  
Fat Cells ◽  
Brown Adipose ◽  
Increased Sensitivity ◽  
Gi Proteins ◽  
Thermogenic Response ◽  
In Cells

The significance of Gi proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of Gi function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis-toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the Gi system in these cells. Therefore the content of Gi proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of Gi1α and Gi2α, as well as of the β-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [3H]ouabain-binding (Na+/K+-ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that Gi proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the Gi pathway is reduced rather than augmented during cold acclimation, Gi activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced Gi content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold.

Norepinephrine does not stimulate protein and UCP synthesis in brown adipocytes of golden Syrian hamsters

1993 ◽  
Vol 265 (1) ◽  
pp. R103-R110 ◽  
Author(s):  
M. Desautels ◽  
R. A. Dulos
Keyword(s):  
Prolonged Exposure ◽  
Uncoupling Protein ◽  
Calf Serum ◽  
Fold Increase ◽  
Brown Fat ◽  
Brown Adipocyte ◽  
Fat Cells ◽  
Cell Recovery ◽  
Succinate Dehydrogenase Activity ◽  
Brown Adipocytes

Hamster brown adipocytes were incubated for up to 24 h with or without norepinephrine (NE) in Dulbecco's modified Eagle's medium supplemented with bovine serum albumin, calf serum, and antibiotics. Brown fat cells were viable for 24 h as defined by their ability to respond to NE by a 10-fold increase in oxygen consumption. However, prolonged exposure of the cells to NE led to a decline in NE-stimulated rates of O2 consumption, which was not the result of loss of cell thermogenic capacity. Brown fat cells incubated for 24 h with or without NE showed no significant change in succinate dehydrogenase activity or uncoupling protein (UCP) content. However, cell recovery after 24 h was significantly reduced in the absence of NE. In brown adipocytes isolated from rat, NE increased [35S]methionine incorporation into cell proteins and UCP. In contrast, [35S]methionine incorporation in hamster brown adipocyte proteins and UCP was greater than in rat brown fat cells and was not increased by NE. These results indicate that although NE may be required for cell survival, it does not stimulate protein and UCP synthesis in hamster brown fat cells.

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