名古屋学芸大学健康 ･ 栄養研究所年報

創刊号

２００７年

《Review》

Functional Ca2+-couplings among the mitochondrion, endoplasmic
reticulum and plasmalemma in thermogenic brown adipocytes:
Possible roles in energy consumption
Masako Kuba＊, Yoko Higure, Ryotaro Hayato, Hisashi Susaki and Kenji Kuba
Laboratory of Anatomy and Physiology, School of Nutritional Sciences,
Nagoya University of Arts and Sciences.
57 Takenoyama, Iwasaki-cho, Nissin, Aichi 470-0196, Japan.

Abstract
Obesity is the result of excess energy intake and/or decreased energy consumption. Energy is consumed bybasal metabolic activity, muscle activity and thermogenesis. Adrenergic activations of lipolysis and uncoupling
proteins in brown adipocytes lead to heat production without oxidative phosphorylation. The energy dissipatedby this process is the H+ electrochemical potential across the mitochondrial membrane generated by H+ pumps,
respiratory chains, that require NADH and FADH2 produced by Ca2+-dependent dehydrogenases in TCA cycle.
Thus, this process is strongly affected by the level of intracellular free Ca2+ ([Ca2+]i), which are regulated by
Ca2+ binding to Ca2+ binding proteins, Ca2+ entry and extrusion at the plasma membrane, Ca2+ release anduptake into, and from, mitochondria and the endoplasmic reticulum (ER). We review here how these organelles
and the plasmalemma communicate with each other in regulating [Ca2+]i and discuss how these coupling are
involved in thermogenesis in rat brown adipocytes. Our recent observations suggest the new mechanisms of[Ca2+]i regulation in brown adipocytes: 1) uncoupling of oxidative phosphorylation and the subsequent recoupling activate Ca2+ entry at the plasmalemma that depends on restoration of H+ electrochemical potential
or ATP synthesis, 2) mitochondrial Ca2+ release induces Ca2+ release from the ER, 3) Ca2+ depletion in the ER
via mitochondria-induced Ca2+ release activates store-operated Ca2+ entry (SOC) in a fraction of cells and 4)
Ca2+ depletion in the ER activates Ca2+ release from mitochondria. Since these mechanisms are activated by
the a- and b-actions of noradrenaline, they likely play important roles in thermogenesis.
Obesity that leads to a variety of diseases results from the imbalance of energy uptake and consumption.
Excess energy intake and/or decreased energy consumption are caused by impairment of hypothalamic appetitecontrol, metabolic abnormalities, decreased muscle activity and reduction of nonshivering thermogenesis.
Brown adipose tissue is a thermogenic organ that dissipates energy in response to cold exposure (1, 2). The
b-action of noradrenaline released by sympathetic activity causes lipolysis and enhances production of NADH
and FADH2 via activation of TCA cycle in mitochondria. Free fatty acids produced activates uncoupling proteinsin mitochondria, which shunts H+ electrochemical potential at the inner membrane, generating heat without＊ Correspondence: Masako Kuba at the above address
TEL/FAX: +81-561-75-2559,
kubam@nuas.ac.jp
Key words: Calcium, mitochondria, endoplasmic reticulum, plasmalemmal Ca2+ entry, brown adipocytes41oxidative phosphorylation (3-5). For this process to occur, activation of Ca2+-dependent dehydrogenases isneeded to produce NADH and FADH2 (6, 7). Thus, increases in free Ca2+ concentration in mitochondria is theabsolute requirement for thermogenesis and so energy consumption.
In brown adipocytes, [Ca2+]i is largely increased by the release of Ca2+ from the ER through IP3 receptors(8-11) and the subsequent activation of store-operated Ca2+ entry (SOC:12, 13) in response to the a-action ofnoradrenaline. This a-action could obviously be complementary to the b-action generating heat. Then, it ispossible that these functions of mitochondria and the ER interact with each other via Ca2+ dynamics. We review
here how these organelles and the plasma membrane communicate with each other in regulating intracellular
Ca2+ based on our recent experimental findings and discuss how these couplings are involved in thermogenesis
in rat brown adipocytes. Several lines of evidence suggest the new mechanisms of [Ca2+]i regulation in brown
adipocytes (14). First, uncoupling of oxidative phosphorylation in mitochondria and the subsequent re-coupling
activate a new mode of plasmalemmal Ca2+ entry that depends on restoration of H+ electrochemical potential or
ATP synthesis. Second, Ca2+ release from mitochondria causes Ca2+ release from the ER and the resultant Ca2+
depletion in the ER activates SOC in a fraction of cells. Third, Ca2+ release from the ER results in depletionof Ca2+ in mitochondria as well as activation of SOC. Since these mechanisms are activated by the actions ofnoradrenaline and if one considers the indispensability of intracellular Ca2+ to build up the H+ electrochemical potential (mH) across the mitochondrial membrane, the driving force for heat induction, they likely play
important roles in thermogenesis.

Thermogenesis in brown adipocytes
Brown adipose tissues are locatedmainly in interscapular regions and fragmentally scattered in other body areas ofrodents and infants of human. They areheavily supplied with blood vessels anddense sympathetic innervation. Brown
adipocytes are endowed with numerous
mitochondria and fragmented lipid droplets, but not a single large lipid drop (1,15). Dense blood supplies and numerous
mitochondria render the brown adipose
tissue brownish appearance. Exposure tocold environment enhances the activityof neurons in the hypothalamus. This
activates sympathetic nervous systems
via the midbrain and eventually leads
to the release of noradrenaline towardbrown adipocytes (16-18). The activation of b3-adrenergic receptor on the cellFig. 1. The mechanisms of thermogenesis in brown adipocytes. See text.

membrane of brown adipocyte enhanceshydrolysis of neutral lipids, which leads to production of NADH and FADH2 via activation of Ca2+-dependent
dehydrogenases. Free fatty acids produced by lipolysis activate the type 1 uncoupling protein (2, 19) that42

Mitochondrion-ER-plasmalemma couplings in adipocytes

Fig. 2. Ca2+ signaling in brown adipocytes. Ps is Ca2+-binding proteins, PIP2 phosphatidylinositol 4,5-bisphosphate, PLC
Phospholipase C, G GTP-binidng protein, R receptor for neurotransmitter or hormone, SERCA sarcoplasmic/endoplasmic Ca2+
pump. CRAC Ca2+-release activated cation channel is one of the channels involved in SOC. Stim1 is a protein that normally sits
in the membrane of the ER and binds with Ca2+. When the free Ca2+ level is decreased in the lumen of the ER, Stim1 migrates tothe plasmalemma and activates SOC. Permeability transition pore in the inner mitochondrial membrane is a large conductancechannel that allows to pass many species of ions and opens usually when apoptosis starts to take place. Presumably it also
operates in response to various conditional changes under the physiological conditions. See text.

shuttles H+ across the mitochondrial inner membrane, shunting mH that is increased by enhanced productionof NADH and FADH2 (3, 4: Fig. 1). On the other hand, a1-activation of IP3 receptors by noradrenaline causes
release of Ca2+ from the ER (8-11). The resultant depletion of Ca2+ in the ER activates SOC (13: Fig. 2). These
increases in [Ca2+]i by the a-actions would also contribute to thermogenesis.
2+

Regulation and functions of intracellular free Ca in general
To facilitate the understanding of this review by the readers outside the fields of physiology and cell biology, how [Ca2+]i is regulated and what functions it exerts in general are described in some details (Fig. 2). Free
Ca2+ concentration in the cytosol is normally set at less than 100 nM, one ten thousandth of the extracellular
Ca2+, while a large amount of Ca2+ is bound to Ca2+-binding proteins and stored in Ca2+-storing organelles(Ca2+-stores), such as ER and mitochondria.
The extremely low level of [Ca2+]i, which is prerequisite for intracellular Ca2+ signaling for various cellular functions, is achieved by powerful Ca2+ extrusion at the plasma membrane via activity of Ca2+ pumps andNa+/Ca2+ exchangers (20, 21: Fig. 1). Ca2+ that is released from Ca2+-stores or enters at the plasmalemma into
the cytosol is buffered by binding to Ca2+-binding proteins, Ca2+ extrusion at the cell membrane and Ca2+ uptake

43into Ca2+-stores so that a rise in [Ca2+]i became slower in time course and smaller in amplitude. The membraneof the ER and sarcoplasmic reticulum (SR) are endowed with sarcoplasmic/endoplasmic Ca2+ pumps (SERCA)
for taking up Ca2+ into the lumen and also with Ca2+ release channels, IP3 receptors or ryanodine receptors forreleasing Ca2+ into the cytosol. The former channel is activated by IP3 and/or Ca2+, while the latter is by Ca2+
and/or the electrical coupling with voltage-gated Ca2+ channel at the plasma membrane. In the lumen of the
ER and SR, Ca2+ is largely bound by Ca2+-binding proteins enabling a large amount of Ca2+ stored. Thus, theER and also SR are the important source and store of Ca2+ for Ca2+ signaling (Fig. 2).
Mitochondria is also the important Ca2+ source and store.. Mitochondria take up Ca2+ into the lumen
through a Ca2+ uniporter driven by the large inside-negative mH (22-24). The uniporter is now identified asa Ca2+ permeable channel (25). Mitochondria releases Ca2+ through Na+/Ca2+ exchangers, Na+-independent
Ca2+ transporters, permeability transition pores and even Ca2+ uniportetrs, once the [Ca2+]i became lower than
the threshold for Ca2+ uptake (Fig. 2). In the matrix of mitochondria, Ca2+ is largely bound to phosphates sothat acidification raises the free Ca2+ concentration in the matrix. Furthermore, recent studies suggest that the
intimate interaction of mitochondria with the ER maintains the activation of SOC by releasing Ca2+ from theER (26-31).
A rise in [Ca2+]i via Ca2+ entry through voltage-gated or transmitter-operated Ca2+ channels and/or Ca2+
release from the sarcoplasmic or ER causes a variety of functions in excitable cell: muscle contraction, therelease of neurotransmitter, the modulation of synaptic transmission, the formation of memory, gene activation,
growth and others in neurons (31-34). Rises in [Ca2+]i by Ca2+ release from the ER via inositol 1,4,5-trisphosphate (IP3) receptors (35) and Ca2+ entry through the plasma membrane as a result of depletion of Ca2+ inthe ER (SOC:12, 36: Fig. 1) results in many functions of nonexcitable cells , including cell growth, exo- andendocytosis, gene expression, malignant formation and others.

2+

Ca coupling of mitochondria to the ER in brown adipocytes
Functional coupling of mitochondria to the ER in respect to Ca2+ dynamics can be revealed by uncouplingof oxidative phosphorylation in mitochondria by the action of a protonophore, carbonyl cyanide p-(trifluorome
thoxy)phenylhydrazone (FCCP). FCCP (2 mM) applied for 3 min produces the di- or tri-phasic rises in [Ca2+]i(Fig. 3A and B). Similar di- or tri-phasic rises were induced by another uncoupler, dinitrophenol, (DNP), ora blocker of electron transport, rotenone. The first quickly-rising transient phase of FCCP-induced rises is accompanied by the depolarization of mitochondrial membrane and is not affected by a Ca 2+-free, EGTA solution
with or without EGTA (Fig. 3B) and in a Na+ deficient or free solution. Thus, mitochondrial depolarization
caused by FCCP releases Ca2+ from mitochondria under the increased concentration of free Ca2+ in the matrix
by acidification, constituting the initial phase of FCCP-induced rises. Since it is not affected by applying a
blocker of Ca2+ uniporter, ruthenium red, an inhibitor of permeability transition pores, cyclosporine A (37),
and a Na+ free solution, which inhibits Na+/Ca2+ exchanger, Ca2+ release occurs through a Na+-independent
Ca2+ pathway at the mitochondrial membrane.
The second spiky rise in [Ca2+]i is partially abolished in a nominally Ca2+ free or Ca2+ free, EGTA solution,leaving a small external Ca2+-independent component (Fig. 3B). Thus, the second phase of FCCP-induced
rises in [Ca2+]i consists of two components; one is external Ca2+ dependent, while another is independent of
extracellular Ca2+ (Fig. 4). Accordingly, the uncoupling of oxidative phosphorylation that causes depolarizationand the resultant Ca2+ release from mitochondria activate a transient Ca2+ entry at the plasmalemma and Ca2+
release from a Ca2+ store other than mitochondria. Aside from the mechanism of Ca2+ entry, which is described

44

Mitochondrion-ER-plasmalemma couplings in adipocytes

Fig. 3. Rises in [Ca2+]i and mitochondrial membrane depolarization induced by FCCP and effects of a Ca2+ free, EGTA
solution. A: Triphasic rises in [Ca2+]i with mitochondrial membrane depolarization and effects of a Ca2+ free, EGTA solution
on the third phase of rises. The lower graph indicates the time course of changes in fluorescence of rhodamine 123 (F480) thatreflects a change in mitochondrial membrane potential. An increase in F480 implies a decrease in membrane potential. A solution
containing FCCP (2 mM) or a Ca2+ free, EGTA solution was superfused to the bath during a period indicated by a black or grayhorizontal bar, respectively. B: Diphasic rises in [Ca2+]i with membrane depolarization and effects of a Ca2+ free, EGTA solution.
Explanations are similar to those in A. Reproduced from Kuba et al. (14) by permission of Am. J. physiol. Methods: Rats (Wistar
ST or WKAH) kept at 4°C for 5 to 7 hours were killed, the procedures approved by Animals Ethics Committee of Nagoya
University (Nagoya, Japan). Brown adipocytes were isolated and cultured for 2 to 7 days (34). Adipocytes were loaded withfura-2/AM (5 mM) or Oregon Green 488 BAPTA-1/AM (OGB-1: 5 mM) . Changes in [Ca2+]i was measured with conventionalratio-imaging systems using fura-2 in most experiments or single wavelength-imaging using Oregon Green 488 BAPTA-1
in some experiments. Changes in mitochondrial membrane potential were measured by recording changes in fluorescence of
rhodamine 123 loaded together with fura-2/AM.

in detail below, Ca2+ release caused by mitochondrial uncoupling appears to take place from the ER. If so, it isexpected that once the ER is depleted of Ca2+ by inhibiting Ca2+ uptake, the Ca2+ release-dependent componentof the second phase would disappear. This is indeed the case. The application of cyclopiazonic acid (CPA: 10mM), a blocker of the SERCA, eliminated this Ca2+ release component with the marked depression of the first45

component (for Ca2+ release frommitochondria activated by Ca 2+
release from the ER: see below for
the mechanism: Fig. 5A). Similar
abolition occurs after the application of an irreversible blocker of
SERCA, thapsigargin (Fig. 6).
Alternatively, the CPA-induced
rise in [Ca2+]i in a Ca2+ free solution was markedly decreased after
the application of FCCP (Fig. 5B).
Thus, it is of no doubt that Ca2+
release from the ER is involvedin the second phase. Ca2+ releasefrom the ER by the Ca2+ liberated
from mitochondria would occur via
a Ca2+-induced Ca2+ release (CICR)
mechanism through IP3 receptors
or ryanodine receptors (35, 38, 39).
The former is the case, as shown
below.
The involvement of PLC in theactivation of Ca2+ release from the
ER by mitochondrial Ca2+ releasecan be assessed by the action of a
blocker of phospholipase C (PLC),
U-73122. In the presence of theFig. 4. Comparison of the time courses of the Ca2+ entry-dependent componentof the second spiky rise in [Ca2+]i and mitochondrial depolarization induced
by FCCP. The Ca2+ entry-dependent component of the second spiky rise (lightgray trace) was isolated by subtracting the FCCP-induced response in a Ca2+
free, EGTA solution (dark gray trace) from that in Krebs solution (black trace).
Changes in mitochondrial membrane potential was shown in the reversed
direction (interrupted trace) and superimposed so that a repolarizing phase canbe compared with the time course of the Ca 2+ entry-dependent component of
the second spiky rise. FCCP (2 mM) was applied for 3 min as shown by a black
horizontal bar. The panels in A and B are the analyses from two different cells.
Reproduced from Kuba et al. (14) by permission of Am. J. physiol.

blocker, no second component of
FCCP-induced rises is seen in a Ca2+ free solution (Fig. 7A and B). The blocking action of U-73122 on thesecond component is seen even in the presence of external Ca2+ as the depression of the “valley” between thefirst and second phases of FCCP-induced rises in [Ca2+]i (Fig. 7C and D).

2+

Coupling of mitochondrial activity to two modes of Ca entry at the plasma membrane
One mode of Ca2+ entry activated by mitochondrial activity is the activation of SOC in response to Ca2+
depletion in the ER as the result of mitochondria-induced CICR. This produces the late slow rise of FCCPinduced rises in [Ca2+]i in some fractions of cells as evidenced by its marked reduction in a Ca2+ free solution(Fig. 3A). Another mode of Ca2+ entry induced by mitochondrial activity is reflected in the external Ca2+dependent component of the second phase of FCCP-induced rises in [Ca2+]i. The mechanism of this Ca2+ entry
is likely to involve a process or a product related to restoration of mH or ATP synthesis at the mitochondrial
membrane after the subsidence of H+ shunt for the three lines of evidence. First, the rising phase of this Ca2+
entry is closely associated with the time course of mitochondrial repolarization after washout of FCCP (Fig. 4).
Second, this Ca2+ entry component is accompanied by a rise in intracellular Mg2+ concentration (Fig. 7A). This46

Mitochondrion-ER-plasmalemma couplings in adipocytes

Fig. 5. Effects of the prior application of CPA on FCCP-induced rises in [Ca2+]i in a Ca2+ free, EGTA solution and effects ofprior application of FCCP on CPA-induced rises in [Ca2+]i in a Ca2+ free, EGTA solution. A: Effects of the preceding applicationof CPA on FCCP-induced rises in [Ca2+]i in a Ca2+ free, EGTA solution. FCCP (2 mM) was applied for 3 min as shown by ablack horizontal bar. A Ca2+ free, EGTA solution was applied for the period indicated by a light gray bar, while CPA (10 mM)
was for 8 min as indicated by a dark gray bar. The lower trace showing relative changes in F480 to the initial fluorescence reflects
those in mitochondrial membrane potential. B: Effects of the preceding application of FCCP on CPA-induced rises in [Ca2+]i ina Ca2+ free, EGTA solution. Explanations are similar to those in A. Reproduced from Kuba et al. (14) by permission of Am. J.
physiol.

implies hydrolysis of ATP, which releases Mg2+ from Mg2+-ATP. Third, the second phase of FCCP-induced riseis blocked by a blocker of ATP synthase (Fig. 8B). This mode of Ca2+ entry, tentatively called mH-dependent
Ca2+ entry, however, does not take place through a channel involved in SOC, because it is not affected by
Na+ deficient solution, while SOC is markedly reduced. It is to be noted that either mode of Ca2+ entry is notcaused by the inhibition of Ca2+ extrusion via activity of Na+/Ca2+ exchangers at the plasma membrane, since
the mH-dependent Ca2+ entry is generated in a Na+ free solution, while SOC is markedly depressed by theremoval of external Na+. Furthermore, both the modes are not the result of the inhibition of Ca2+ pump at theplasmalemma. Raising the external pH did not affect the mH-dependent Ca2+ entry, but it enhanced SOC.

47

Fig. 6. Functional Ca2+-couplings between mitochondria and the ER. A: A Ca2+-coupling of mitochondria to the ER. FCCP
produces triphasic rises in [Ca2+]i. Thapsigargin had no effects on [Ca2+]i during the third phase of rises in [Ca2+]i, while the third
phase was completely abolished by the removal of the external Ca2+. FCCP (2 mM) and thapsigargin (1 mM) were applied during
the period indicated by a black and dark gray bar, respectively. A Ca2+ free, EGTA solution was superfused to the bath during aperiod indicated by a light gray bar. B: Bidirectional Ca2+-couplings between mitochondria and the ER. FCCP produces triphasic
rises in [Ca2+]i. Thapsigargin had again no effects on [Ca2+]i during the third phase of FCCP-induced rises in [Ca2+]i, while thethird phase was completely abolished by the removal of the external Ca2+ and transiently by the second application of FCCP.
Other explanations are the same as those in A. Reproduced from Kuba et al. (14) by permission of Am. J. physiol.

2+

Ca coupling of the ER to mitochondria
Ca2+ release from the ER activates mitochondrial Ca2+ release or uptake. FCCP (2 mM) applied during
the sustained rise in [Ca2+]i by thapsigargin produces two types of actions depending on the cell. In a fraction of cell, Ca2+ uptake takes place in response to Ca2+ release from the ER, as evidenced by a sharp risein [Ca2+]i during the sustained rise by blocking SERCA at the ER membrane. The extent of Ca2+ uptake into
mitochondria, however, varies among cells. The greater Ca2+ uptake occurs for the increased level of [Ca2+]iunder the effect of thapsigargin in a fraction of cells (Figs. 6A and 9B), while the smaller Ca2+ uptake takes
place even at a high level of [Ca2+]i in other fraction of cells. This implies that a fraction of Ca2+ stored in mitochondria is released in this type of cells. In another type of cells, complete release of Ca2+ from mitochondria48

Mitochondrion-ER-plasmalemma couplings in adipocytes

Fig. 7. Effects of a blocker of PLC on FCCP-induced rises in [Ca2+]i and associated changes in mitochondrial membranepotential in Krebs and Ca2+ free, EGTA solutions. A: Effects of U-73122 (2 mM) on FCCP-induced rises in [Ca2+]i andmitochondrial membrane potential changes in a Ca2+ free, EGTA solution. FCCP (2 mM) was applied for 3 min as shown by ablack horizontal bar. A solution containing U-73122 (2 mM) was applied for the period indicated by a dark gray bar, a Ca2+ free,
EGTA solution was applied for the period indicated by a light gray bar. Ba: Superposed traces of FCCP-induced rises in [Ca2+]iin the absence and presence of U-73122 (2 mM) and the U-73122-sensitive component in a Ca2+ free solution shown in A. Bb:
Averaged U-73122-sensitive component (mean ± SE of mean) obtained from 10 cells. C: Effects of U-73122 (2 mM) on FCCPinduced rises in [Ca2+]i and mitochondrial membrane potential changes in Krebs solution. Explanations are the same as thosein A except that U-73122 was applied in Krebs solution. Da: Superposed traces of FCCP-induced rises in [Ca2+]i in the absence
and presence of U-73122 (2 mM) and the U-73122-sensitive component of FCCP-induced response in Krebs solution shown in C.
Db: Superposed traces of control responses and that in the presence of U-73122 (2 mM) and the U-73122-sensitive component
in Krebs solution shown in C. A part of the trace in the presence of U-73122 indicated by a light gray bar in Da was deleted toeliminate the delay in the onset of the second component of FCCP-induced rises. Dc: Averaged U-73122-sensitive component(mean ± SE of mean) obtained from 13 cells, in which a part of the trace in the presence of U-73122 was deleted. Reproduced
from Kuba et al. (14) by permission of Am. J. physiol.

occurs in response to the Ca2+ released from the ER via CICR (Figs. 6B and 9A). The Ca2+ release channel
involved in CICR at the mitochondrial membrane in brown adipocytes, however, is not yet identified, although
Ca2+ uniporters or permeability transition pores are suggested in other cells (24, 40).

49

Fig. 8. Effects of a Ca2+ free, Mg2+ free, EGTA solution on FCCP-induced changes in Magfura-2 fluorescence ratio and effectsof oligomycin on FCCP-induced rises in [Ca2+]i and mitochondrial membrane depolarization. A: Effects of a Ca2+ free, Mg2+
free, EGTA solution on the FCCP-induced changes in the ratio of Magfura-2 fluorescence. The ratio of fluorescence at 340
nm and 380 nm was taken. FCCP (2 mM) was applied for 3 min as shown by a black bar, while a Ca2+ free, Mg2+ free, EGTA
solution was applied for the period indicated by a gray bar. Magfura-2 was loaded and ratio-imaging was made. B: Effects ofoligomycin on FCCP-induced rises in [Ca2+]i and mitochondrial membrane depolarization. FCCP (2 mM) was applied for 4 min
as shown by a black horizontal bar, while a solution containing oligomycin (5 mg/ml) was applied for the period indicated by agray bar. The lower trace (F480) represents changes in mitochondrial membrane potential. Reproduced from Kuba et al. (14) bypermission of Am. J. physiol.

Possible roles of mitochondrion-ER-plasmalemma couplings in thermogenesis
The central questions are how the couplings among the mitochondrion, ER and plasmalemma operate
under the physiological conditions and how they affect or regulate heat production in brown adipocytes. Clues
to answer these questions should lie in the modes of the b-action of noradrenaline involved in thermogenesis,
and must be obtained by observing changes in heat production under the blockade of each of these couplings. In
addition, it is highly possible that the a-action of noradrenaline plays a crucial role in thermogenesis, although
it is overlooked for a long time.

50

Mitochondrion-ER-plasmalemma couplings in adipocytes

Fig. 9. Effects of FCCP on [Ca2+]i during the sustained rise in [Ca2+]i induced by thapsigargin. A: Reduction of [Ca2+]i by FCCP
(2 mM) during the thapsigargin-induced rise in [Ca2+]i. The record also shows that the second spiky component of rises remains
during the FCCP-induced reduction of [Ca2+]i, but is blocked in Ca2+ free, EGTA solution. FCCP and Thapsigargin was applied
during the period indicated by a black or dark gray bar, respectively. A Ca2+ free, EGTA solution was superfused to the bath
during the period indicated by a light gray bar. B: Diphasic rises induced by FCCP during the sustained rise in [Ca2+]i caused bythapsigargin. The record also shows that the reduction of [Ca2+]i following the initial transient rise underlies the second spikyrise. FCCP and thapsigargin were applied during the period indicated by a black and dark gray bar, respectively. CPA (10 mM)
was applied during the period indicated by a light gray bar simply to examine the potency of thapsigargin to deplete Ca2+ in the
ER. Reproduced from Kuba et al. (14) by permission of Am. J. physiol.

Under the physiological conditions, the actions of FCCP shown above is reproduced by the activationof uncoupling proteins by the b-action of noradrenaline released from sympathetic nerves in response to cold
exposure (see the previous section for references). On the other hand, the action of CPA and thapsigargin are
reproduced by the a-action of noradrenaline (see the previous section for references). Thus, the two modes of
the actions of noradrenaline can produce those induced by FCCP, CPA and thapsigargin.
Isoprenaline, b-agonist, elicits Ca2+ release from mitochondria and then activates metabolite-dependent
Ca2+ entry, Ca2+ release via the mitochondrion-ER coupling and subsequently SOC (unpublished observations).
Moreover, under the physiological conditions, another mode of b-actions occurs that enhances lipolysis and so
the driving force for heat production. The notable characteristic of the b-actions is that it lasts for more thanseveral tens of minutes. Thus, the two modes of the b-actions, increasing the mH and shunting it, must be in51

Fig. 10. New mechanisms of Ca2+ signaling and possible roles in thermogenesis in brown adipocytes. a1 is a1 adrenergic
receptor. b3 is b3 adrenergic receptor. AC is adenylate cyclase. UCP is the type-1 uncoupling protein. Explanations for othersymbols are the same as those in Fig. 2. The dark gray lines show newly found the Ca2+ couplings between mitochondria and the
ER and those between mitochondria and the plasma membrane. See text.

the steady state during heat genesis. Imagine the situation for a bathtub, whose stopcocks for hot water andsewage are both opened, dumping potential energy and heat without achieving work. To maintain the steadystate for energy accumulation and dissipation in mitochondria, a constant production of NADH and FADH2
and their constant oxidation of NADH and FADH2 for pumping H+ across the inner membrane are needed.
The former is achieved by continuous activation of lipolysis and the latter is maintained by the constant highlevel of [Ca2+]i that supply Ca2+ through Ca2+ uniporters for the activation of Ca2+-dpendent dehydrogenesesin mitochondria. This high level of [Ca2+]i can be maintained by continuous Ca2+ entry via the activation of
metabolite-dependent Ca2+ entry and SOC, which are activated by both the a- and b-actions of noradrenaline
via mitochondrion-ER-plasmalemma couplings (Fig. 10).

Acknowledgements
This work was supported by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture
to K.K. (No. 18590211)

52Mitochondrion-ER-plasmalemma couplings in adipocytes

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