Section of Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616
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ABSTRACT |
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We find that the adrenergic agonist
isoproterenol increases intracellular Ca2+ concentration
([Ca2+]i) in cultured rat brown adipocytes.
At the concentration used (10 µM), isoproterenol-induced
Ca2+ responses were sensitive to block by either
1- or
-adrenergic antagonists, suggesting an
interaction between these receptor subtypes. Despite reliance on
-adrenoceptor activation, the Ca2+ response was not due
solely to increases in cAMP because, administered alone, the selective
3-adrenergic agonist BRL-37344 or forskolin did not
increase [Ca2+]i. However, increased cAMP
elicited vigorous [Ca2+]i increases in the
presence of barely active concentrations of the
-adrenergic agonist
phenylephrine or the P2Y receptor agonist UTP. Consistent with
isoproterenol recruiting only inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, endoplasmic
reticulum store depletion by thapsigargin blocked isoproterenol-induced
Ca2+ increases, but removal of external Ca2+
did not. These results argue that increases in cAMP sensitize the
IP3-mediated Ca2+ release system in brown adipocytes.
adenosine 3',5'-cyclic monophosphate; fura 2 fluorescence imaging; thapsigargin; isoproterenol; caffeine; adipocytes
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INTRODUCTION |
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BROWN ADIPOSE TISSUE (BAT) is a source of nonshivering heat production (thermogenesis) in mammals where it plays a key role, especially in small mammals, in recovery from hibernation and maintenance of body temperature in cold conditions (38). Brown fat, like white fat, is an energy storage site. However, brown fat is also capable, by virtue of its numerous and specialized mitochondria, of rapid conversion of fat stores to heat. This energy-wasting capacity of BAT is also tapped when rats are fed a high-calorie diet (diet-induced thermogenesis) (29), presumably to stave off obesity. This and other observations have encouraged attempts to understand brown fat physiology as both a site of energy wasting to control obesity and a model system for adipocyte physiology in general.
Under sympathetic control, the thermogenic response of BAT is primarily
mediated by 3-adrenergic receptors (43, 44)
via increases in cAMP, release of free fatty acids, and mitochondrial uncoupling through activation of uncoupling protein 1 (UCP1)
(24). Other adrenergic receptor subtypes are also present
on mature adipocytes and include
1-adrenoceptors coupled
to inositol 1,4,5-trisphosphate (IP3) production and
release of Ca2+ from intracellular stores (16, 22,
25, 39, 41). Increased cytosolic Ca2+ affects both
the acute thermogenic response (42) and the long-term capacity of BAT to produce heat (8, 27).
Previous investigations of Ca2+ signaling in brown
adipocytes have noted cytosolic Ca2+ increases in response
to not only -adrenergic stimulation but also the
-adrenergic
agonist isoproterenol (4, 16, 41). Because the
-adrenergic pathway is mediated by changes in cAMP levels, it is not
immediately clear how this signaling pathway also affects cytosolic
Ca2+ levels. Two hypotheses have been put forth to explain
these responses in brown adipocytes.
The first hypothesis points out that significant depolarization of mitochondria likely occurs as a consequence of UCP1 activation (26) and that such depolarization may be sufficient to release mitochondrial Ca2+ stores or to limit mitochondrial Ca2+ uptake of other standing Ca2+ fluxes (41). This is a particularly intriguing hypothesis because its validity would suggest a role for other UCP homologs (28) in the adjustment of mitochondrial Ca2+ levels and/or Ca2+ buffering capacity.
The second hypothesis proposes that isoproterenol has sufficient
activity at 1-adrenoceptors to elicit cytosolic
Ca2+ increases (4, 16). This "dirty drug"
hypothesis is supported by evidence that norepinephrine-induced
Ca2+ increases can be completely blocked by the
-adrenergic antagonist phentolamine (16) and that
isoproterenol-induced Ca2+ increases can be blocked by the
1-adrenergic antagonist prazosin (4).
However, there have been conflicting reports as to whether the
-adrenergic antagonist propranolol blocks (16) or does not block (4) isoproterenol-induced Ca2+
increases. Thus the mechanism(s) by which isoproterenol increases cytosolic Ca2+ in brown adipocytes has remained unclear.
We have addressed this question by using a mechanistic approach to
determine whether -adrenergic activation increases intracellular Ca2+. We have found that while isoproterenol does have a
modicum of activity at
1-adrenoceptors, additional
cAMP-mediated mechanisms are involved in the Ca2+ response.
In addition, we have found that isoproterenol effects on cytosolic
Ca2+ are largely independent of
-adrenergic effects on
mitochondria. We propose that selective
-adrenergic stimulation of
brown adipocytes potentiates Ca2+ release from
IP3-sensitive stores and that in vivo norepinephrine recruits both
- and
-adrenergic pathways to generate
Ca2+ signals.
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METHODS |
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Cell culture.
Primary cultures of brown adipocytes were obtained by using methods
similar to those previously described (16). Single cells were isolated by collagenase digestion of interscapular fat pads from
neonatal (1-8 days postnatal) Sprague-Dawley rats. Cells from five
to six rat pups were pooled and plated on collagen-coated glass
coverslips within 35-mm culture dishes at a density of
~104 cells/ml. Cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) in an atmosphere of 5%
CO2-95% air at 37°C. DMEM was supplemented with 16 µg/ml insulin, 3.8% (vol/vol) fetal bovine serum (FBS), 1.2% equine
serum, 0.1 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml
amphotericin B. Culture medium was replaced on day 2 following the isolation. Experiments were conducted on days
0 (isolation day) to 7 after isolation. Data were
collected from mature adipocytes as identified visually by their many
fat droplets (see Fig. 1, inset).
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Cytosolic Ca2+ measurements. Cytosolic Ca2+ measurements were made in single cells by using the Ca2+-sensitive dye fura 2 and microfluorometric techniques similar to those previously described (16). Cells cultured on glass coverslips were incubated for 20 min at room temperature in 4 µM fura 2-AM plus 0.02% Pluronic F-127 in Krebs-Ringer bicarbonate (KRB) buffer containing (in mM) 10 D-glucose, 0.5 MgCl2, 4.6 KCl, 120 NaCl, 0.7 sodium phosphate dibasic, 1.3 sodium phosphate monobasic, 24 mM NaHCO3, and 2 CaCl2, pH 7.4. After one wash with KRB buffer, coverslips were mounted in a perfusion chamber fixed to the stage of an inverted microscope where the chamber was continuously perfused with room temperature KRB buffer gassed with 5% CO2-95% O2. Alternating 340- and 380-nm illumination was provided by a xenon arc lamp and rotating filter wheel. Whole field images at ×40 or ×20 were acquired at 510-nm emission via a charge-coupled device camera connected to a personal computer running IonWizard software (IonOptix, Milton, MA). The image acquisition rate was typically 0.1 Hz but was sometimes set to 0.4 Hz during agonist presentation to record rapid changes in fluorescence. Single cells were defined by static user-drawn rectangles within which pixel values were averaged. Ca2+ traces presented were calculated within IonWizard by using ratiometric calculation (11). Ca2+ calibration was conducted post hoc by using glass microcuvettes filled with 100 µM fura 2 (free acid) and varying Ca2+-EGTA mixtures. Free Ca2+ concentrations of these calibrating standards were calculated with WinMAXC (Chris Patton, Stanford University, Stanford, CA). Average traces for multiple cells within a single experiment were generated in Igor Pro (WaveMetrics, Lake Oswego, OR). Quantification of responses for statistical testing was performed in Igor Pro by using a baseline-subtracted averaging technique. Values are given as means ± SE. Statistical tests were performed with StatView 5 (SAS Institute, Cary, NC). Tests noted as chi-squared were continuity-corrected chi-squared tests of 2 × 2 contingency tables.
Materials. Unless otherwise stated, chemicals and solutions were obtained from Sigma (St. Louis, MO). Collagenase was from Worthington Biochemical (Lakewood, NJ). DMEM and insulin were from GIBCO BRL (Grand Island, NY). FBS was from JRH Biosciences (Lenexa, KS). Equine serum was from Hyclone (Logan, UT). Fura 2-AM was from Calbiochem (La Jolla, CA). Pluronic F-127 was from Molecular Probes (Eugene, OR). Bupranolol was a kind gift from Schwarz Pharma/SIFA (Shannon, Ireland). BRL-37344 was a kind gift from SmithKline Beecham Pharmaceuticals.
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RESULTS |
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Isoproterenol increases cytosolic
Ca2+ in brown adipocytes.
Consistent with previously published results (4, 16, 41),
we found that the general -adrenergic agonist isoproterenol increased cytosolic Ca2+ in single mature brown adipocytes
(Fig. 1). Ca2+ responses to
isoproterenol were variable between cells, and many cells did not
respond. Of 126 cells later responding to 10 µM phenylephrine (a
general
-adrenergic agonist), 48 (38%) responded to 10 µM
isoproterenol (10 experiments; naive isoproterenol exposure, i.e.,
isoproterenol was the first agonist presented). In aggregate, of all
261 cells exposed to isoproterenol, 92 (35%) responded (15 experiments; naive isoproterenol exposure; 175 cells later tested with phenylephrine).
Ca2+ responses to isoproterenol
require -adrenoceptor activation.
Despite its widespread use, isoproterenol is not a completely selective
-adrenergic agonist. Therefore, an obvious interpretation of these
data is that 10 µM isoproterenol is sufficient to appreciably activate
1-adrenoceptors on brown fat cells and cause
release of stored Ca2+. Indeed, this possibility was first
suggested by our laboratory (16), and the sensitivity of
the isoproterenol response to block by the
1-adrenergic
antagonist prazosin has led others to a similar conclusion
(4). We have since replicated these results with prazosin
(not shown, but see Fig. 8).
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-Adrenergic receptor activation potentiates
-adrenoceptor-mediated Ca2+ increases.
In mature adipocytes,
-adrenergic responses (e.g., to isoproterenol)
are mediated primarily through
3-adrenoceptors
(3). Hence, the involvement of
-adrenoceptors in the
Ca2+ increase induced by isoproterenol is seemingly at odds
with published data indicating no Ca2+ responses to the
3-adrenergic agonist CGP-12177 (4).
Consistent with this observation, the
3-adrenergic
agonist BRL-37344 elicited small responses in only 2 of 118 cells later
responding to 10 µM phenylephrine (5 experiments) (Fig.
3A). However, these results make sense if
3-adrenergic stimulation acts only to
potentiate
1-adrenergic stimulation. Consistent with
this proposal, we found that BRL-37344 consistently elicited
Ca2+ responses if presented along with a low concentration
(100 nM) of phenylephrine (24 of 57 cells, 3 experiments) (Fig.
3B).
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Mechanism of -adrenoceptor stimulation effects on
Ca2+ signaling.
All known effects of
-adrenoceptor stimulation in brown adipocytes
(e.g., lipolysis and thermogenesis) are transduced by the cAMP
second-messenger system through activation of adenylate cyclase.
-Adrenoceptor stimulation can, in many ways, be mimicked by
forskolin, a direct activator of adenylate cyclase. If the Ca2+ responses to
3-adrenergic stimulation
are mediated by increases in cAMP, forskolin application should
elicit Ca2+ responses similarly to BRL. Indeed, we find
this is the case (Fig. 4).
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Minimal 1-adrenergic stimulation promotes
isoproterenol-evoked Ca2+ increases.
During efforts to increase the number of cells responding to
isoproterenol with Ca2+ increases, we experimented with
administering isoproterenol in the presence of a low concentration of
phenylephrine. We found that 100 nM phenylephrine alone produced little
or no Ca2+ response, but cells responded vigorously upon
addition of isoproterenol in the continued presence of 100 nM
phenylephrine. In 6 experiments, 56 of 101 cells (55%) responded to 10 µM isoproterenol in the presence of 100 nM phenylephrine (Fig.
5). This frequency of responses is
significantly higher than the 35% (92 of 261) response frequency seen
in cells responding to isoproterenol without phenylephrine (P < 0.001, chi-squared test). Thus, though
Ca2+ responses to isoproterenol require both
- and
-adrenergic receptor activation, cells are further biased toward
Ca2+ increases if isoproterenol is presented along with
minimal
-adrenergic stimulation.
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External Ca2+ is not required for
isoproterenol-induced Ca2+ responses.
Isoproterenol activates a large cation-selective conductance in brown
fat cells (20) that could mediate the
isoproterenol-induced Ca2+ increase. However, cells
produced vigorous Ca2+ responses to 10 µM isoproterenol
in the presence of nominally Ca2+-free media [0
Ca2+ KRB (KRB buffer with no added Ca2+)] that
were comparable to maximal (10 µM) phenylephrine responses under the
same 0 Ca2+ conditions (Fig.
6). This finding indicates that
isoproterenol evokes substantial release from internal Ca2+
stores and rules out activation of a Ca2+ influx pathway as
being the sole effect of isoproterenol on Ca2+ signaling.
In 4 experiments similar to that presented in Fig. 6, 19 of 32 cells
(59%) responding to 10 µM PE also responded to 10 µM isoproterenol
in Ca2+-free media.
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Isoproterenol releases Ca2+ from ER
stores.
We tested the possibility that isoproterenol releases Ca2+
from ER stores. Emptying ER stores with thapsigargin, a potent
inhibitor of the smooth ER Ca2+-ATPase (36),
increased cytosolic Ca2+ dramatically within 5 min (Fig.
7) (25). The increased
Ca2+ level was sensitive to removal of external
Ca2+ and is presumably due to the same store-operated
influx mechanism (2) previously shown to activate during
ER Ca2+ release in this cell type (4, 16, 17, 25,
41). After thapsigargin treatment, isoproterenol application
failed to elicit Ca2+ increases in 145 of 145 cells in 7 experiments. These experiments included 27 cells in 4 experiments in
which application of carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (a mitochondrial
uncoupler) after isoproterenol washout increased cytosolic
Ca2+ (Fig. 7), indicating that mitochondria were well
loaded with Ca2+ (13). Thus isoproterenol
primarily taps thapsigargin-sensitive (e.g., ER) Ca2+
stores, and contributions from thapsigargin-insensitive stores (e.g.,
mitochondria) are small or negligible under these conditions.
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Ryanodine receptor presence is unlikely in brown adipocytes. Ca2+ release from the ER could potentially be through either ryanodine receptors (e.g., via cyclic ADP-ribose production; Ref. 32) or IP3 receptors. To our knowledge, the presence of ryanodine receptors has not been investigated in brown adipocytes, so we tested this possibility by applying 10 mM caffeine to isolated cells. This treatment had little or no effect on cytosolic Ca2+ in 63 of 63 cells in 2 experiments despite later responses to isoproterenol in 19 of 24 cells. The lack of Ca2+ increases in response to caffeine treatment argues against the presence of functional ryanodine receptors in brown adipocytes and suggests that the Ca2+ response elicited by isoproterenol is through an IP3 receptor-based system.
Synergy of -adrenergic and P2Y receptor stimulation.
The results presented indicate that increasing cAMP is not itself
sufficient to substantially increase cytosolic Ca2+ in
brown fat cells. Presumably isoproterenol can elicit Ca2+
responses because it has some activity at the
1-adrenoceptor that increases IP3 levels. To
test whether another IP3-producing agent could substitute
for the
1-adrenergic component of isoproterenol stimulation, we administered a low concentration of UTP to activate P2Y
receptors (6, 25). Prazosin (5 µM) was present to block
1-adrenoceptors. Under these conditions we found that
isoproterenol could elicit robust Ca2+ responses (Fig.
8). Of 102 cells ultimately responding to
10 µM UTP, 51 (50%) also responded to a 200 nM UTP-10 µM
isoproterenol mixture in the presence of 5 µM prazosin (10 experiments). Of these 51 responding cells, 13 were also previously
exposed to 100 nM phenylephrine-10 µM isoproterenol in the presence
of 5 µM prazosin (4 experiments) (Fig. 8). Only 1 of these 13 cells responded with Ca2+ increases. Isoproterenol was also found
to similarly evoke Ca2+ increases when 400 nM ATP (another
P2Y agonist) was used instead of UTP (not shown). These data indicate
that
-adrenoceptor stimulation potentiates IP3-based
Ca2+ increases that are not dependent on
1-adrenergic stimulation.
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DISCUSSION |
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We have found that -adrenoceptor activation amplifies but does
not trigger ER Ca2+ release in brown fat cells. Consistent
with previous observations (4), we have found that
selective
3-adrenergic stimulation and forskolin-induced
increases in cAMP do not appreciably increase cytosolic
Ca2+ by themselves. However, either treatment sensitizes
cells to Ca2+ release elicited by
-adrenergic
stimulation. Furthermore, selective
-adrenergic stimulation
potentiates Ca2+ release elicited by P2Y receptor
stimulation, demonstrating that the sensitization of Ca2+
release is not confined to
-adrenergic pathways.
Effects of cAMP on IP3-mediated
Ca2+ release in other cell types.
Although Ca2+ increases in response to the -adrenergic
agonist isoproterenol have been documented in brown adipocytes over the
past decade (4, 16, 41), we have demonstrated for the first time that this occurs primarily through
-adrenergic
potentiation of IP3-mediated Ca2+ release.
Sensitization of IP3-mediated Ca2+ release by
increases in cAMP is not unique to brown fat and has been reported in
other cell types, including hepatocytes (5, 12, 31, 34),
pancreatic
-cells (19), articular chondrocytes (9), and parotid acinar cells (30). The
effect of cAMP increases in brown fat on IP3-mediated
Ca2+ release contrasts with other cell types, such as
megakaryocytes, pancreatic acinar cells, and nonvascular smooth muscle,
in which cAMP inhibits IP3-mediated Ca2+
release (1, 7, 35).
Mechanism.
The experiments presented here do not address the mechanism by which
-adrenergic stimulation potentiates IP3-mediated
Ca2+ release in brown fat cells. In hepatocytes, for
example, cAMP increases lead to both an increase in IP3
receptor sensitivity to IP3-stimulated channel opening and
a recruitment of Ca2+ stores previously insensitive to
IP3 (12). These mechanisms may also be at work
in brown adipocytes, but other mechanisms are also possible including
increased IP3 production, decreased IP3
breakdown, increased filling of Ca2+ stores, modification
of IP3 receptor Ca2+ sensitivity, changes in
local Ca2+ buffering near IP3 receptors, or
modification of membrane surface receptors coupled to IP3 generation.
Receptors involved in isoproterenol-induced
Ca2+ release.
We propose that the isoproterenol-induced Ca2+ response is
comprised of a small 1-adrenergic component that is
strongly amplified by a large
-adrenergic component. In contrast,
more specific
-adrenergic agonists (such as BRL-37344, used here)
are incapable on their own of eliciting Ca2+ responses
presumably because they lack sufficient activity at receptors coupled
to IP3 production (e.g.,
1-adrenoceptors)
even though IP3-based Ca2+ release machinery is
highly sensitized by the agonist's activity at
-adrenergic receptors.
Desensitization of Ca2+ responses.
Ca2+ responses to isoproterenol in brown adipocytes
appeared to desensitize quickly. While a 30-min wait period between
applications was sufficient in some cells to restore sensitivity to
isoproterenol, in other cells it was not (see Fig. 1). This observation
is consistent with the activation of either 1- and/or
3-adrenergic receptors, both of which are known to
desensitize in brown adipocytes (40).
Physiological significance.
Ca2+ signals in brown adipocytes affect a wide range of
cellular responses ranging from acute stimulation of thermogenesis
(42) to long-term modifications of thermogenic capacity
(8). On the basis of the data presented in this study, it
appears clear that, in vivo, the endogenous ligand norepinephrine
generates Ca2+ signals by recruiting both - and
-adrenergic receptors. This added level of complexity in adipocyte
Ca2+ signaling invites us to speculate why
-adrenergic
receptors would be (albeit indirectly) coupled to Ca2+
release when
1-adrenoceptors are perfectly capable of
eliciting Ca2+ release on their own.
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ACKNOWLEDGEMENTS |
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We thank Christina Stroup and Xiaochun Liu for valuable technical assistance, Robert Berman for use of laboratory space and equipment, and Sherwin Lee and Joel Keizer for insightful discussions.
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FOOTNOTES |
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This work was supported by National Science Foundation Training Grant 9602226 "Nonlinear Dynamics in Biology."
Address for reprint requests and other correspondence: P. A. Pappone, Neurobiology, Physiology, and Behavior, Univ. of California, Davis, CA 95616 (E-mail: papappone{at}ucdavis.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 2, 2002;10.1152/ajpcell.00204.2001
Received 3 May 2001; accepted in final form 20 December 2001.
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