beta -Adrenergic potentiation of endoplasmic reticulum Ca2+ release in brown fat cells

Eric V. Leaver and Pamela A. Pappone

Section of Neurobiology, Physiology, and Behavior, University of California, Davis, California 95616


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1- or beta -adrenergic antagonists, suggesting an interaction between these receptor subtypes. Despite reliance on beta -adrenoceptor activation, the Ca2+ response was not due solely to increases in cAMP because, administered alone, the selective beta 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 alpha -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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 alpha 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 alpha -adrenergic stimulation but also the beta -adrenergic agonist isoproterenol (4, 16, 41). Because the beta -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 alpha 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 alpha -adrenergic antagonist phentolamine (16) and that isoproterenol-induced Ca2+ increases can be blocked by the alpha 1-adrenergic antagonist prazosin (4). However, there have been conflicting reports as to whether the beta -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 beta -adrenergic activation increases intracellular Ca2+. We have found that while isoproterenol does have a modicum of activity at alpha 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 beta -adrenergic effects on mitochondria. We propose that selective beta -adrenergic stimulation of brown adipocytes potentiates Ca2+ release from IP3-sensitive stores and that in vivo norepinephrine recruits both alpha - and beta -adrenergic pathways to generate Ca2+ signals.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Average Ca2+ concentration ([Ca2+]) of 11 cells responding with cytosolic [Ca2+] increases to the first presentation of isoproterenol (gray trace) and [Ca2+] in 2 single cells included in that average (black traces). Agonists were presented for durations indicated by horizontal bars. ISO, 10 µM isoproterenol; PE, 10 µM phenylephrine. The experiment was terminated just before 60 min due to computer memory limitations, so the response to PE washout is not shown. All cells included in the average responded to PE. Another 17 cells from this same experiment responded to PE but not to ISO (not shown). Overall desensitization of the ISO response is clear from the average (gray) trace, which shows the second ISO response to be less than half as large as the naive response. Only 3 of the original 11 cells still responded to ISO by the third presentation, which occurred after ~15 min in agonist-free solution. Two of the three cells increased their responsiveness after ~30 min. The third cell's response did not change (upper black trace at 3rd ISO presentation). A fourth cell regained its ISO response after the ~30-min recovery period (lower black trace at 3rd ISO presentation). Inset: differential interference contrast image of cultured brown adipocytes from a separate experiment. Mature adipocytes in this image can be identified by their many fat droplets.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isoproterenol increases cytosolic Ca2+ in brown adipocytes. Consistent with previously published results (4, 16, 41), we found that the general beta -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 alpha -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).

Responsiveness of cells to isoproterenol was highly variable from one cell isolation to the next and ranged from 0 to 100% of observed cells. The percentage of cells responding to isoproterenol averaged by experiment did not correlate with time spent in culture when expressed as a percentage of phenylephrine-responsive cells (P = 0.37, 0-4 days postisolation) or as a percentage of all cells (P = 0.49, 0-6 days postisolation). Phenylephrine responsiveness was similarly uncorrelated with number of days in culture (P = 0.92, 0-4 days postisolation).

Ca2+ responses to isoproterenol require beta -adrenoceptor activation. Despite its widespread use, isoproterenol is not a completely selective beta -adrenergic agonist. Therefore, an obvious interpretation of these data is that 10 µM isoproterenol is sufficient to appreciably activate alpha 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 alpha 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).

If isoproterenol is acting solely through alpha 1-adrenoceptors, the Ca2+ response should not be affected by the presence of beta -adrenergic antagonists. However, we found that this is not the case (Fig. 2A). The general beta -adrenergic antagonist propranolol (5 µM) significantly attenuated Ca2+ responses to 10 µM isoproterenol compared with subsequent control isoproterenol responses (i.e., in the absence of propranolol) in the same cells (33 ± 7% of control responses; P < 0.0001; paired t-test; n = 28; cells without control responses were excluded). Sensitivity to block by propranolol was variable across the cell population. About half the cells exhibited complete block by propranolol, whereas the balance varied in sensitivity (Fig. 2B). Another general beta -adrenergic antagonist, bupranolol (10 µM), also significantly attenuated Ca2+ responses to isoproterenol (not shown). This finding indicates that Ca2+ responses to isoproterenol require activation of beta -adrenoceptors and cannot be fully explained by isoproterenol activity at alpha 1-adrenoceptors.


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Fig. 2.   ISO-evoked Ca2+ increases are blocked by the beta -adrenergic antagonist propranolol. A: traces are averages of cells grouped according to sensitivity of the ISO response to antagonist block (gray trace, completely blocked; black trace, less sensitive to block). Cells were exposed first to propranolol (5 µM) to block beta -adrenoceptors and then to ISO (10 µM) in the continued presence of propranolol (5 µM). After >30 min of wash (to alleviate any desensitization), cells were exposed to ISO (10 µM) in the absence of propranolol. Bath solution was then exchanged for one containing the alpha -adrenergic agonist PE (10 µM). In the absence of propranolol, 22 cells produced Ca2+ responses to ISO in this experiment, and each cell is included in 1 of the 2 averages. B: sensitivity to block by propranolol was variable among cells. Histogram shows single-cell Ca2+ responses to 10 µM ISO in the presence of 5 µM propranolol, expressed as a percentage of the subsequent 10 µM ISO response alone (control response). Data are from 28 cells with control Ca2+ responses and include the 22 cells from the experiment shown in A.

beta -Adrenergic receptor activation potentiates alpha -adrenoceptor-mediated Ca2+ increases. In mature adipocytes, beta -adrenergic responses (e.g., to isoproterenol) are mediated primarily through beta 3-adrenoceptors (3). Hence, the involvement of beta -adrenoceptors in the Ca2+ increase induced by isoproterenol is seemingly at odds with published data indicating no Ca2+ responses to the beta 3-adrenergic agonist CGP-12177 (4). Consistent with this observation, the beta 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 beta 3-adrenergic stimulation acts only to potentiate alpha 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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Fig. 3.   Selective beta 3-adrenergic stimulation does not increase cytosolic [Ca2+] on its own but potentiates Ca2+ increases elicited by alpha -adrenergic stimulation. A: the beta 3-adrenergic agonist BRL-37344 (BRL; 1 µM) had essentially no effect on cytosolic [Ca2+] compared with control responses evoked by 10 µM PE. Data sampling frequency was increased (see METHODS) during the beginning of BRL presentation to ensure capture of any fast Ca2+ dynamics. All cells responding to 10 µM PE in this experiment (16 cells) are included in the average. B: in a separate experiment on cells from the same isolation and day in culture, a low PE concentration (100 nM) produced only small increases in cytosolic [Ca2+]. Addition of BRL (1 µM) in the continued presence of 100 nM PE evoked a robust Ca2+ response. All cells responding to BRL/PE in this experiment (15 cells) are included in the average.

Of the 118 cells tested with BRL, 66 were also tested with isoproterenol soon after BRL-37344 treatment (3 experiments, not shown). Only 9 of these 66 cells (14%) responded to isoproterenol, which is a significantly lower response frequency than the 35% (92 of 261) isoproterenol-responsive cells seen without prior BRL-37344 treatment (P = 0.001, chi-squared test). This finding indicates that prior BRL-37344 exposure prevents subsequent isoproterenol-induced Ca2+ increases, presumably due to BRL-induced desensitization of beta 3-adrenergic receptors (40).

Mechanism of beta -adrenoceptor stimulation effects on Ca2+ signaling. All known effects of beta -adrenoceptor stimulation in brown adipocytes (e.g., lipolysis and thermogenesis) are transduced by the cAMP second-messenger system through activation of adenylate cyclase. beta -Adrenoceptor stimulation can, in many ways, be mimicked by forskolin, a direct activator of adenylate cyclase. If the Ca2+ responses to beta 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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Fig. 4.   Direct stimulation of adenylate cyclase by forskolin produces Ca2+ responses similar to those elicited by beta 3-adrenergic stimulation. A: application of 10 µM forskolin to brown adipocytes evoked a small shift in baseline [Ca2+] that appeared significant in 3 cells. Average trace is of 16 cells subsequently responding to 10 µM PE in this experiment. B: separate cells from those shown in A, but from the same isolation and day in culture. Treatment of brown adipocytes with 100 nM PE elicited small but significant changes in cytosolic [Ca2+] in most cells (12 of 15) that relaxed back toward baseline levels. Addition of 10 µM forskolin in the continued presence of PE evoked additional clear responses in 14 of 15 cells. Average (gray) trace represents the 14 cells responding to 10 µM forskolin-100 nM PE. Responses are understated in average due to predominantly oscillatory responses in these cells that tended to average out (18). Black trace represents a single typical cell included in the average.

Like BRL, forskolin had little effect by itself on cytosolic Ca2+ in brown adipocytes (37). Of 24 cells later responding to 10 µM phenylephrine, 18 responded with small shifts in baseline Ca2+ and the remaining 6 had either small sustained increases in baseline Ca2+ or transient Ca2+ responses (Fig. 4A). These minimal Ca2+ changes resemble the responses to BRL-37344 (compare to Fig. 3A).

Whereas forskolin had little effect by itself, forskolin accompanied by minimal alpha -adrenergic stimulation elicited clear Ca2+ responses in 24 of 29 other cells in 2 experiments (Fig. 4B). The Ca2+ responses to forskolin are underreported in the averaged data (Fig. 4B) because of predominantly oscillatory responses in the experiment shown (18). Cells displayed a variety of Ca2+ responses to forskolin: single spikes, sustained elevation relaxing with forskolin washout, sustained activation persisting until 100 nM phenylephrine washout, initiation of oscillations that subsided during forskolin exposure, and, in some cases, reinitiation of oscillations upon forskolin washout. Despite this variability across single cells, these experiments provide strong evidence that beta -adrenergic stimulation potentiates alpha -adrenoceptor-mediated Ca2+ release through a cAMP-mediated mechanism.

Minimal alpha 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 alpha - and beta -adrenergic receptor activation, cells are further biased toward Ca2+ increases if isoproterenol is presented along with minimal alpha -adrenergic stimulation.


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Fig. 5.   PE increases the frequency of brown fat cell Ca2+ responses to ISO. Responses to 100 nM PE + 10 µM ISO are defined as Ca2+ increases beyond the response, if any, to 100 nM PE just before the 2 agonists were applied together. Responses to ISO were significantly more frequent when 10 µM ISO was presented with 100 nM PE than when 10 µM ISO was presented alone (P < 0.001, chi-squared test). Response frequency to a high dose of PE (10 µM), which was often used as a positive control, is also shown for comparison.

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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Fig. 6.   Isoproterenol-induced Ca2+ responses do not require Ca2+ influx. Average [Ca2+] is shown for 6 cells bathed in Krebs-Ringer bicarbonate buffer with no added Ca2+ (0 Calcium KRBB) during the time indicated. All 6 cells responded to both 10 µM PE in 0 Ca2+ KRBB and 5 µM ISO in 0 Ca2+ KRBB. The increase in cytosolic [Ca2+] upon return to normal (2 mM) Ca2+ KRBB is presumably due to store-operated influx triggered by decreases in endoplasmic reticulum (ER) Ca2+ stores during PE-induced Ca2+ release (2, 16).

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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Fig. 7.   Ca2+ responses to ISO require ER release and are not due to Ca2+ release from mitochondria. ER Ca2+ stores were depleted with thapsigargin (100 nM) during the time indicated. Thapsigargin elicited a large increase in cytosolic [Ca2+] that relaxed toward baseline levels upon removal of external Ca2+. In the absence of external Ca2+, and with empty ER stores, ISO (10 µM) application did not increase cytosolic [Ca2+]. However, 5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) produced a tremendous increase in cytosolic [Ca2+], indicating that mitochondrial Ca2+ stores were well loaded by the previous period of high cytosolic [Ca2+] (13). No cells responded to ISO in this experiment. An average trace of 11 cells with responses to FCCP is shown.

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 beta -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 alpha 1-adrenoceptor that increases IP3 levels. To test whether another IP3-producing agent could substitute for the alpha 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 alpha 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 beta -adrenoceptor stimulation potentiates IP3-based Ca2+ increases that are not dependent on alpha 1-adrenergic stimulation.


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Fig. 8.   P2Y receptor stimulation can substitute for the alpha 1-adrenergic component of isoproterenol. Prazosin (5 µM) was present to block alpha 1-adrenoceptors. No cells responded to 100 nM PE and 10 µM ISO in the presence of prazosin. Washout of prazosin in this experiment was initially accomplished with KRBB that had not yet been fully gassed with CO2, and the apparent Ca2+ rebound is most likely due to changes in external pH, [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>], and/or PCO2 (15, 21). A long wash period was used to relieve beta -adrenergic desensitization. Subsequent exposure to prazosin and a low concentration of UTP (200 nM) evoked small responses in some cells (1 oscillated). Addition of 10 µM ISO to the mix evoked strong responses (the oscillating cell increased its oscillation frequency). The trace represents an average of the 9 cells responding to this later ISO presentation. All 9 cells later responded to 10 µM UTP. In this experiment, 8 other cells responded to 10 µM UTP but to neither of the ISO presentations (not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have found that beta -adrenoceptor activation amplifies but does not trigger ER Ca2+ release in brown fat cells. Consistent with previous observations (4), we have found that selective beta 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 alpha -adrenergic stimulation. Furthermore, selective beta -adrenergic stimulation potentiates Ca2+ release elicited by P2Y receptor stimulation, demonstrating that the sensitization of Ca2+ release is not confined to alpha -adrenergic pathways.

Effects of cAMP on IP3-mediated Ca2+ release in other cell types. Although Ca2+ increases in response to the beta -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 beta -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 beta -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).

Not all cells responding to 10 µM phenylephrine with Ca2+ increases also responded to isoproterenol with Ca2+ increases. An interesting possible explanation for this observation is that beta -adrenergic stimulation potentiates Ca2+ release in the some brown fat cells but inhibits Ca2+ release in other brown fat cells. Although our data cannot completely rule out this possibility, it is difficult to reconcile this idea with the fact that low concentrations of phenylephrine tended to increase the probability that a given cell would respond to isoproterenol with a Ca2+ increase. It seems more likely that the lack of responsiveness in some cells is due to low expression and/or sensitivity of beta - and/or alpha 1-adrenoceptors.

Mechanism. The experiments presented here do not address the mechanism by which beta -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.

It is unlikely that beta -adrenergic stimulation affects cytosolic Ca2+ through non-IP3-based mechanisms such as NAADP (10) or cADP-ribose (14) production. NAADP-sensitive stores are resistant to Ca2+ store depletion by thapsigargin, but we have found that Ca2+ responses to isoproterenol in brown adipocytes are, in fact, sensitive to thapsigargin treatment (Fig. 7). Ca2+ responses to cADP-ribose appear to occur through recruitment of ryanodine receptors. On the basis of the lack of Ca2+ responses to caffeine treatment, we have concluded that brown adipocytes do not express functional ryanodine receptors and, by extension, do not use cADP-ribose to produce Ca2+ signals.

Receptors involved in isoproterenol-induced Ca2+ release. We propose that the isoproterenol-induced Ca2+ response is comprised of a small alpha 1-adrenergic component that is strongly amplified by a large beta -adrenergic component. In contrast, more specific beta -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., alpha 1-adrenoceptors) even though IP3-based Ca2+ release machinery is highly sensitized by the agonist's activity at beta -adrenergic receptors.

By using combined drug treatment, we were able to elicit isoproterenol-like Ca2+ responses by using agonists more selective than isoproterenol. The beta 3-adrenergic agonist BRL-37344 evoked Ca2+ responses when presented along with a low concentration of the alpha -adrenergic agonist phenylephrine. Thus a combination of weak alpha -adrenergic stimulation and strong beta 3-adrenergic stimulation is sufficient to evoke isoproterenol-like Ca2+ responses in brown fat. We also have shown that even when the alpha 1-adrenergic component of isoproterenol is blocked with prazosin, Ca2+ responses to isoproterenol can be rescued by inclusion of weak P2Y receptor stimulation. Thus the beta -adrenergic component of isoproterenol is a real and important factor in eliciting increases in cytosolic Ca2+.

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 beta 1- and/or beta 3-adrenergic receptors, both of which are known to desensitize in brown adipocytes (40).

Also supporting the proposal that desensitization of isoproterenol Ca2+ responses occur due to beta -adrenoceptor desensitization is that the beta 3-adrenergic agonist BRL-37344 diminished responses to isoproterenol for a short time after washout (see RESULTS). We have drawn two conclusions from this result. First, because BRL-37344 did not appreciably increase cytosolic Ca2+, Ca2+ increases are not required for desensitization of isoproterenol-induced Ca2+ responses. Second, beta 3-adrenoceptors may be the important subtype involved in desensitization of isoproterenol-induced Ca2+ increases, either because isoproterenol acts mainly through beta 3-adrenoceptors or because beta 3-adrenoceptor activation leads to desensitization of all beta -adrenoceptors in brown adipocytes.

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 alpha - and beta -adrenergic receptors. This added level of complexity in adipocyte Ca2+ signaling invites us to speculate why beta -adrenergic receptors would be (albeit indirectly) coupled to Ca2+ release when alpha 1-adrenoceptors are perfectly capable of eliciting Ca2+ release on their own.

One possible function is related to the fact that beta -adrenoceptors, and their consequent Ca2+ signals, desensitize quickly compared with alpha 1-adrenoceptors (16, 25). This feature likely sensitizes Ca2+ release mechanisms to particular types of stimulation from innervating fibers. For example, norepinephrine released onto brown fat cells after a long period of inactivity, as might be brought about by quickly moving an animal to a cooler environment, would likely produce more robust Ca2+ increases than the same rate of norepinephrine release would elicit after several minutes of stimulation. Given the positive influence of Ca2+ on long-term thermogenic capacity, it seems perfectly reasonable to suggest that novel cold stimuli are more capable of generating Ca2+ increases than chronic stimuli. Novel stimuli, by definition, would be a surprise to the system and would likely require some system modification to enable appropriate and efficient responses.

Another possible function of beta -adrenoceptor effects on Ca2+ signaling is in the integration of adrenergic and nonadrenergic signals. We have shown that beta -adrenergic stimulation can potentiate Ca2+ release not only by alpha 1-adrenergic stimulation but also by P2Y receptor stimulation (Fig. 8). This result suggests that cAMP increases may potentiate all IP3-based signals regardless of their origin. This could provide a means by which brown adipocyte responses to nonadrenergic, Ca2+-elevating stimuli such as purine nucleotides (17, 25), prostaglandins (23), and cell adhesion molecules (33; unpublished observations) are modified when those stimuli are received simultaneously with heat production requests from the animal.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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