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.

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.

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

1993 ◽  
Vol 264 (1) ◽  
pp. C217-C228 ◽  
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.

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)

Normal and Neoplastic Cells of Brown Adipose Tissue Express the Adhesion Molecule CD31

2006 ◽  
Vol 130 (4) ◽  
pp. 480-482 ◽  
Author(s):  
Renato Rosso ◽  
Marco Lucioni
Keyword(s):  
Adipose Tissue ◽  
Adhesion Molecule ◽  
Myxoid Liposarcoma ◽  
Cell Types ◽  
Brown Fat ◽  
Fat Cells ◽  
Neoplastic Cells ◽  
Brown Adipose ◽  
Formalin Fixed Paraffin Embedded ◽  
Well Differentiated

Abstract Context.—CD31 (platelet-endothelial cell adhesion molecule-1; PECAM-1), an adhesion molecule involved in the process of angiogenesis, is used as a marker of normal and neoplastic vascularization. During the assessment of angiogenesis and vascular invasion in a thymic carcinoid tumor, we observed unexpected immunostaining for CD31 in perithymic brown fat nests. Objective.—To determine whether CD31 is expressed by normal and neoplastic cells of brown fat, a tissue whose thermogenetic activity depends heavily on high perfusion. Design.—Formalin-fixed, paraffin-embedded archival tissues were immunostained by the labeled avidin-biotin method using antibodies against CD31 (clones JC70A and 1A10) after retrieval of heat-induced epitopes. Archival tissues included perithymic, periadrenal, axillary, and neck adipose tissue in which were embedded nests of brown fat (n = 15), hibernoma (n = 3), lipoma (n = 6), well-differentiated liposarcoma (n = 4), and myxoid liposarcoma (n = 4). Results.—Invariably, multivacuolated and univacuolated adipocytes of normal brown fat and hibernomas were intensely positive for the CD31 antigen. The immunostaining “decorated” cell membranes and the membranes of intracytoplasmic vacuoles. No expression of CD31 was found in normal adipocytes of white fat, in neoplastic cells of lipomas, or in multivacuolated lipoblasts of well-differentiated and myxoid liposarcomas. Conclusions.—The spectrum of cell types that express CD31 is expanded to include normal and neoplastic brown fat cells. We speculate that the expression of CD31 may play a role in the development and maintenance of the vascular network characterizing this specialized adipose tissue. Moreover, CD31 may inhibit the Bax-mediated apoptosis of brown fat cells. For practical purposes, CD31 may be used as an immunohistochemical marker for distinguishing between white and brown fat and for diagnosing hibernoma in paraffin sections.

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.

Characterization of norepinephrine-stimulated protein synthesis in rat brown adipocytes

1992 ◽  
Vol 263 (5) ◽  
pp. R1003-R1012
Author(s):  
D. Waldbillig ◽  
M. Desautels
Keyword(s):  
Protein Synthesis ◽  
Uncoupling Protein ◽  
Calf Serum ◽  
Brown Fat ◽  
Mitochondrial Proteins ◽  
Cell Protein ◽  
Fat Cells ◽  
Brown Adipocytes ◽  
Rna Transcription

Rat brown adipocytes were incubated for 24 h with or without norepinephrine (NE) in Dulbecco's modified Eagle's medium with albumin, calf serum, and antibiotics. Brown fat cells were viable as defined by unchanged cell morphology, ATP content, or basal and NE-stimulated respiration. However, a 24-h exposure to NE led to a decline in NE-stimulated respiration that was not due to loss of thermogenic capacity. Brown fat cells incubated with or without NE had similar protein, succinate dehydrogenase, and uncoupling protein (UCP) content. These results differ from those observed after food deprivation in rats where loss of mitochondrial proteins occurs within 24 h, suggesting that reduced exposure to NE is not the only factor responsible for brown fat atrophy. NE increased [35S]methionine incorporation into cellular proteins, mitochondrial proteins, and UCP. The effect of NE on cell protein synthesis was inhibited by propranolol but not by prazosin. It was also inhibited 95% by cycloheximide but only partially (50%) by actinomycin D in contrast to NE stimulation of UCP labeling, which required RNA transcription. Chloramphenicol-sensitive protein synthesis was stimulated by NE. These results indicate a trophic action of NE in brown adipocytes exerted both at the level of RNA transcription and translation.

Blockers of voltage-gated K channels inhibit proliferation of cultured brown fat cells

1993 ◽  
Vol 264 (4) ◽  
pp. C1014-C1019 ◽  
Author(s):  
P. A. Pappone ◽  
S. I. Ortiz-Miranda
Keyword(s):  
Cell Proliferation ◽  
Potassium Channels ◽  
Potassium Channel ◽  
Culture Conditions ◽  
Brown Fat ◽  
Neonatal Rat ◽  
Channel Blockers ◽  
Continuous Exposure ◽  
Fat Cells ◽  
Voltage Gated

Cultured brown fat cells have both voltage- and Ca(2+)-activated potassium channels. We tested whether potassium channel activity is necessary for brown fat proliferation by growing adipocytes and preadipocytes from neonatal rat brown fat in the presence of potassium channel blockers. Whole cell patch-clamp experiments showed that verapamil, nifedipine, and quinine block the voltage-gated potassium current (IK,V) with micromolar affinity. Ca(2+)-activated currents (IK,NE) could be activated by micromolar intracellular Ca2+ concentrations and were blocked by nanomolar concentrations of apamin. Both IK,V and IK,NE are blocked by millimolar concentrations of tetraethylammonium (TEA). Under standard culture conditions, the number of cells showing the multilocular morphology characteristic of brown fat cells doubled in 3-5 days. Continuous exposure to 100 nM norepinephrine had no effect on this process. Cell proliferation was inhibited by TEA, quinine, or verapamil. The inhibition was dose dependent, with concentrations for half-block of cell proliferation similar to the Kd values for block of IK,V. Apamin, which selectively blocks IK,NE, had no effect on cell growth. These results suggest that functional voltage-gated potassium channels, but not Ca(2+)-activated potassium channels, may be necessary for the normal proliferation of brown fat cells in culture.

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.

Sign in / Sign up

Export Citation Format

Share Document