@-Adrenergic Modulation of Ca2' Uptake by Isolated Brown Adipocytes

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Council (to Barbara Cannon and J. N.) and from the Swedish Cancer. Research Council (to J. N.). .... Hettich bench centrifuge with a swing-out rotor. ... take (with 100 p~ arsenazo 111) and membrane potential (with 20 p~ safranine) were ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 263, No. 22, Issue of August 5, pp. 10574-10502,1988 Printed in U.S.A.

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

@-AdrenergicModulation of Ca2' Uptake by Isolated Brown Adipocytes POSSIBLE INVOLVEMENT OF MITOCHONDRIA* (Received for publication, November 10, 1987)

Eamonn ConnollyS and J a n Nedergaard From The Wenner-Gren Institute, University of Stockholm, BiologihusF3, S-10691 Stockholm, Sweden

Rapid, unidirectional Ca2+ influx was examined in al., 1983a; Schimmel et al., 1983) which is probably due, at isolated brown adipocytes by short incubations (30s) least partially, to therestoration of cellular ion gradients that with "Cas+. Ca2+uptake was found to be large in the have been perturbed by the hormone stimulation (Mohell et resting brown adipocyte, but was markedly inhibited al., 1987). when the cells were presented with norepinephrine. It is generally found that stimulation of cells with hormones Specific al-adrenergic stimulation was withouteffect that use Ca2+as an intracellular mediator not only releases on Ca2+ uptake. The effect of norepinephrine (which had an ECao of 140 nM) could be inhibited by &adre- intracellularly stored Ca2' but also leads toan influx of nergic blockade and could be mimicked by forskolin extracellular Ca2+,the latter being necessary for the mainte(an adenylate cyclase activator) and theophylline (a nance of elevated cytosolic Ca2+level and thusof the hormone phosphodiesterase inhibitor). Exogenous free fatty response. The nature of the Ca2+entry mechanism involved acids such as octanoate and palmitate(classical stimu- is under intense study (Reuter, 1983; Irvine and Moore, 1986; lators of respiration in brown adipocytes) were also Putney, 1986; Kuno and Gardner, 1987) and seems to depend able to dramatically inhibit Ca" uptake by the cells. upon the generation of intracellular polyphosphoinositols; The artificial mitochondrial uncoupler carbonyl cya- generation of polyphosphoinositols has also been demonnide p-trifluoromethoxyphenylhydrazone(FCCP) in- strated in al-stimulated brown adipocytes (Nhberg and Putduced a large reduction in cellular Ca" uptake (even ney, 1986; NBnberg and Nedergaard, 1987). The Ca2+entry in the presence of the ATPase inhibitor oligomycin), can be easily followed by radiolabel techniques as performed and in the presence of FCCP the inhibitory effect of in, for example, hepatocytes (Assimacopoulos-Jeannet et al., norepinephrine on Ca" uptake was significantly reduced. The effect of &adrenergic stimulation on Ca2+ 1977; Keppens et al., 1977; Mauger et al., 1984), pancreatic uptake was not directly caused by the large increase acinar cells (Kondo and Schulz, 1976), PC12 cells (Pozzan et in respiration that occurs in response to norepineph- al., 1986), and A431 cells (Sawyer and Cohen, 1981)). We decided to examine unidirectional rates of *Ta2+uptake rine because the respiratory inhibitor rotenone did not affect the Ca2+ response of the cells to the hormone. in the brown adipocyte for two major reasons: firstly, to see The evidence suggests that &adrenergic stimulation of whether al-adrenergic stimulation could lead to an influx of brown adipocyte metabolism leads to a partial inhibi- Ca2+from the extracellular medium, and secondly, to examine tion of Ca2+ uptake into the mitochondrial Ca2+ pool the effect of @-adrenergically mediated brown adipocyte and we discuss the possibility that this represents the thermogenesis on Ca2+metabolism. Interestingly, al-adrenereffect of a reduced membrane potential (and thus re- gic stimulation was unable to affect the cellular Ca2+uptake, duced Ca2+uniport activity)in the partiallyuncoupled while @-stimulationwas found to markedly inhibit Ca2+upmitochondria of the thermogenically active brown adi- take. We conclude that a substantialfraction of the totalCa2+ pocyte. turnover inthe brown adipocyte is mitochondrial, and [email protected] may partially inhibit thismitochondrial turnover, probably as a resultof a decrease in mitochondrial membrane potential. Some of these results have been presented in abCa2+homeostasis in the mammalian cell is paramount for stract form (Connolly and Nedergaard, 1987). the control of intracellular metabolism, as well as for the mediation of the response of the cell to a wide variety of EXPERIMENTALPROCEDURES hormonal stimulations. In brown adipocytes, a1-adrenergic Cell Preparation-Brown adipocytes were prepared by collagenase stimulation induces Ca2+mobilization from intracellular Ca2+ digestion of excised brown fat from adult Syrian hamsters (Mesocristores (Connolly et al., 1984). This leads to an elevation of cetus auratus) in a Krebs-Ringer phosphate buffer exactly as dethe cytosolic Ca2+concentration, as shown by the activation scribed previously (Connolly et al., 1984). Ca2+Uptake Incubations-"'Ca2+ uptake in isolated brown adipoof a Ca2+-dependent K+ channel in the plasma membrane (Nhberg et al., 1984, 1985). Stimulation of the al-receptor cytes was examined using conditions similar to those previously used also leads to an increased respiration by the cells (Mohell et to follow 22Na+accumulation (Connolly et Q L , 1986). The freshly

* Supported by grants from the Swedish Natural Science Research Council (to Barbara Cannonand J. N.) and from the Swedish Cancer Research Council (to J. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ T o whom correspondence should be addressed.

isolated cells were kept (maximum of 1.5 h) in Krebs-Ringer phosphate buffer (containing 4% crude bovine serum albumin) onice until just before the experiment. They were then washed once in a KrebsRinger bicarbonate buffer and resuspended at 2.5-3.5 million cells/ ml. The Krebs-Ringer bicarbonate buffer (pH 7.4) had the following composition (in mM): Na+, 145.5; K', 6.0; M%+, 1.2; Ca2+,2.5; Cl-, 125.1;HCO;,25.5; HPO:-/H2PO;, 1.2; SO:-, 1.2; and contained 10 m M of both glucose and fructose as well as 2% fatty acid-free bovine

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Ca2+Uptake in Brown Adipocytes serum albumin. The buffer had been bubbled with 5% COz in air before the addition of the albumin. In all the experiments, about 375,000 cells (125 r l ) were preincubated in a 1.5-ml microcentrifuge tube for 5 min a t 37 "C. Then, 125 pl of prewarmed Krebs-Ringer bicarbonate buffer, containing 2 pCi (74 kBq) of r6Caz+and 5 pCi (185 kBq) of [3H]inulin/ml, was added. The tube contents were thoroughly mixed by automatic pipette and the incubation continued as indicated (generally 30 s) before cell recovery. Additions of propranolol, prazosin, or calmodulin antagonists were made to the cells at the start of the 5-min preincubation. Norepinephrine, forskolin (in dimethyl sulfoxide), theophylline, fatty acids, and FCCP' (in ethanol)additions were made in the "Ca2+ buffer, i.e. these agents were mixed into the %a2+ buffer immediately before this was added to thecell suspension, and they were present only for the 30-s incubation. Norepinephrine solutions were always made up in water immediately prior to addition. Low-Na+ incubations were performed in a Krebs-Ringer bicarbonate buffer in which the NaCl and NaHC03 were replaced by N methyl-D-glucamine chloride and choline bicarbonate, respectively (as in Connolly et al. (1986)). The original cell preparation (in isolation buffer) was washed twice with 5 volumes of this buffer, and the '%a2+was also added in this buffer. This procedure led (by dilution) to a calculated final Na+ concentration of less than 1 mM in the incubations. Cell Recouery-The cells were recovered from the incubation medium in a similar way to that described previously (Connolly et al., 1986). After incubation, the cell suspension was transferred to a centrifuge tube containing 800 pl of ice-cold phthalate oil (dibutyl phthalate (density 1.045)/bis(2-ethylhexyl)phthalate(density 0.985), 3:5 (v/v)) overlain by 1ml of warm (37 "C) Krebs-Ringer bicarbonate buffer (referred to as the washing buffer). (Occasionally, as stated under "Results," 5 mM EGTA (Na+-salt)was present in this buffer.) The tube was then immediately centrifuged at 1000 X g for 50 s on a Hettich bench centrifuge with a swing-out rotor. After centrifugation, the cells, which were now separated from the buffer by the oil, were washed from the oil surface by the addition of 2 ml of 154 mM choline chloride solution and immediate recentrifugation of the tubefor 40 s a t 400 X g. The cells were finally recovered from the surface of the choline chloride with an automatic pipette and were counted for 3H and '5Ca on a Beckman LS3801 liquid scintillation counter, in a scintillation mixture (toluene/Triton X-100,2:1(v/v)) containing 5% water and 5.5 g of 2,5-diphenyloxazole/liter. The amount of Ca2+ taken up by the cells was calculated by reference to thespecific activity of the "Ca2+ in the incubation buffer and accounting for the amount of buffer recovered with the cells as measured by the [3H]inulin. Results are expressed either as such or as a percentage of the uptake by control cells. Usually only about 10% of the 46Ca2+recovered with the cells could be ascribed to the extracellular buffer. Mitochondrial Preparation and Zncubntion-Mitochondriawere isolated from the brown adipose tissue of adult hamsters kept at 20'C (as those used for the cell preparations), using the methods previously described (Nedergaard, 1983). Measurements of Ca2+uptake (with 100 p~ arsenazo 111) and membrane potential (with 20 p~ safranine) were performed in an incubation medium consisting of 125 mM purified sucrose, 20 mM Tris, 5 mM glycerol 3-phosphate, 4 p~ rotenone, and 0.08% (11 p ~ fatty ) acid-free bovine serum albumin, as described by Nedergaard (1983). Materiak-"5CaC1z and [G-3H]inulin were purchased from Du Pont-NewEngland Nuclear. N-Methyl-D-glucamine (Meglumine), DL-propranolol hydrochloride, norepinephrine bitartrate, theophylline, forskolin, trifluoperazine, chlorpromazine, sodium palmitate, and oligomycin were all obtained from Sigma. FCCP was obtained from either Pierce Chemical Co. or Aldrich. Octanoic acid was from Fluka and was titrated toneutrality with NaOH. Rotenone was from Penick. Prazosin hydrochloride was a generous gift from Pfizer. RESULTS

In the experiments described here, the brown adipocytes were exposed to 45Ca2+for only very short periods of time to ensure that the 45Ca'+ was far from approaching equilibrium The abbreviations used are: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; EGTA, [ethylene bis(oxyethylenenitrilo)] tetraacetic acid.

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I E. EGTA effect

FIG. 1. Ca2+ uptake by isolated brown adipocytes. Isolated brown adipocytes were exposed for the indicated times to 45Ca2+in a Krebs-Ringer bicarbonate buffer a t 37 "C.After incubation, the cells were recovered from the incubation buffer as described under "Experimental Procedures." For each cell preparation, recoverywas performed with or without 5 mM EGTA as indicated. A , uptake curves; and B , the percentage of the control Ca2+uptake that was removed by the EGTA wash a t each time point.The zero time point was made by addition of cells to the prepared centrifuge tube containing all other components of the incubation. In both A and B , points represent the mean -+ S.E. from 3 different cell preparations (each performed in duplicate).

with the intracellular unlabeled Ca'+. Thus, themovement of 45Ca2+was essentially unidirectional and inward, and the uptake observed in the unstimulated cell was probably in exchange for the unlabeled Ca'+ of the intracellular environment. Time Course of Ca" Uptake

When exposed to 45Ca2+,the brown adipocytes showed a steady increase in 45Ca2+accumulation which was approximately linear over the first 2 min (Fig. lA, control). The corresponds to a movement of approximately uptake of 45Ca2+ 5-10 nmol extracellular Ca'+ into 1 million cells/min. It is likely that, in common with many other cell types, resting brown adipocytes have a cytosolic free Ca2+ concentrationin yet been the order of 100-200 nM (although this has not directly shown due to difficulties in using the Quin-2 probe in these cells.)' If one assumes a cytosolic volume of 3 pl/ million cells (Nibberg e t al., 1984), equilibration of 45Ca'+ with the cytosolic pooIof Ca2+ would require an apparent entry of only about 1pmol of Ca'+/million cells. The observed Ca'+ uptake was thus large, and this prompted us to examine whether it was artificially elevated in some way. Extracellular ea2+ and the Effect of EGTA-Since plasma membrane proteins provide extracellular binding sites for Ca'+, the specific uptake of 45Ca2+by the cells could be overestimated. To remove such extracellular binding, the Ca" chelator EGTA was added in some experiments to wash the cells before estimation of the intracellularly trapped 45Ca'+. Assuming that some 45Ca2+was rapidly bound to a constant number of extracellular binding sites, an EGTA wash should remove aconstant absolute amount of the observed cellassociated Ca'+. However,when uptakestudies were performed with cells recovered either in the presence or absence of EGTA in the washing buffer (see "Experimental Procedures") (Fig. lA),it was clear that theEGTA always removed approximately 40% of the cell-associated 45Ca2+,irrespective of how much 45Ca'+had been trapped by the cells (Fig. 1B). E. Connolly and J. Nedergaard, unpublished observations.

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ea2+Uptake in Brown Adipocytes

These observations must mean that theintracellular CaZ+ was being markedly depleted by the EGTA washing procedure and that the extracellular binding of Caz+ must be very low compared to theamount trapped within the cells. Possible Artifactual Ca2+Depletion of the Cells-Before the 5-min preincubation period at 37 "C, the cells were normally stored for 1h or more on ice (see "Experimental Procedures") during which time the intracellular Ca2+stores may have been depleted, because Ca2+gradients within the cells are maintained by energy-requiring processes. Thus, the high rate of Ca2+uptake may have resulted from the restoration of these gradients on return of the cells to 37 "C. This was shown not to be the case by the following observations. Prolonged preincubation (15 min) a t 37 "C only slightly reduced the Ca2+ uptake, probably due to cell breakage (from 7.0 f 0.3 to 5.3 f 0.4 nmol of Ca2+/million cells after 30 s). Also, cells isolated as usual, but stored at room temperature before the preincubation at 37 "C for 5 min, did not show a significantly decreased Ca2+uptake, compared to cells from the same preparation that were stored on ice prior to preincubation (6.5 f 0.2 and 7.0 f 0.3 nmol of Ca2+/million cells after 30 s, respectively). Effect of Extracellular Phosphate-Elevated rates of Ca2+ entry have been observed in white adipocytes in thepresence of slightly higher than normal extracellular phosphate levels (Martin et al., 1975). Although the phosphate concentration in our incubation buffer is normal, there may have been a slight carry-over of phosphate from the isolation buffer which could have increased the phosphate level to about 3 mM. However, cells that were washed thoroughly to remove this contamination showed a Ca2+uptake (4.2 f 0.9 nmol of Ca2+/ million cells after 30 s) which was unchanged by the addition of 5 mM phosphate to the cells (4.1 f 0.3 nmol of Ca"). Temperature Effects-Thebiological nature of the Ca2+ uptake was confirmed by incubation of the cells at temperatures below 37 "C which led to a significant decrease in the rate of cellular uptake of Ca2+(Fig. 2). Thus, under steady-state conditions, brown adipocytes had a rapid rate of Ca2+exchange between the intra- and extracellular environments. From the above experiments, it would seem that the apparentCa" uptake was not due to extracellular binding, was not due to restoration of ion gradients after isolation, and was not induced by high extracellular phosphate levels, but represented a true uptake into intracellularpools. The Response toAdrenergic Stimulation Norepinephrine Stimulation-Brown adipocyte respiration is stimulated 10-20-fold by norepinephrine, leading to very high rates of oxygen consumption by the cells (for a review, see Nedergaard and Lindberg, 1982). As a result of oxygen electrode studies we have concluded that, under the incubation conditions used here, the oxygen supply is adequate well beyond 30 s of incubation with cells which are respiring maximally, but may be depleted by 60s (Connolly et al., 1986). Thus, a short term uptake (30 s) was considered suitable for the following experiments. When norepinephrine was presented to the cells together with the 46Ca2+for the 30-9 uptake period, the apparent Ca2+ uptake by the cells was reduced in a dose-dependent manner, with an value of around 140 nM (Fig. 3), which is within the expected range for a true physiological effect of the hormone. Pharmacological Characterization-Experimental conditions where the cyI- and 8-adrenergic effects of norepinephrine on brown adipocyte respirationcan be distinguished have recently been described (Mohell et al., 1987). Complete al-

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FIG. 2. Temperature dependence of Ca" uptake by brown adipocytes. Brown adipocytes were preincubated for 5 min and then incubated for the indicated times with "Ca2+ at 37, 20, and 0 'C. Recovery was performed as under "Experimental Procedures" except that for both the 20 and 0 "Cincubations, the recovery wash buffer was at room temperature. Points represent mean + S.E. from three different cell preparations.

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FIG. 3. Dose response to norepinephrine. Brown adipocytes were incubated for 30 s with %a2+ and with the indicated concentrations of norepinephrine. The control cell Ca2+uptake (mean: 6.0 & 1.2 nmol of Caz+/million cells) was set to 100% for each cell preparation, and the percent reduction in Ca2+ uptake induced by the norepinebhrine addition was calculated. In the figure the control Ca*+ uptake is represented as 0%reduction. The points represent the mean f S.E. fromfour different cell preparations (each performed in duplicate). Statistical analysis: *** and ** indicate a significant difference from control ( p < 0.001 and 0.01, respectively; Student's t test).

adrenergic blockade (with a 10-foldexcess of the al-adrenergic antagonist prazosin) did not inhibit the effect of norepinephrine (Fig. a), and selective al-adrenergic stimulation (with

Ca2' Uptake in Brown Adipocytes 120

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FIG. 5. Cyclic-AMP-mediated inhibition of Ca" uptake in brown adipwytes. Brown adipocytes were incubated for 30 s at 37 "C with ''Ca2+ and with the following additions as indicated NE, 1 p M norepinephrine; MezSO, 5 pl of dimethyl sulfoxide (forskolin carrier); FORSK, 50 p~ forskolin; THEO, 1 mM theophylline. The mean control (con) cell Ca2+uptake was 5.6 f 0.4 nmol of Ca2+/ million cells. Bars represent themean + S.E. from the number of cell preparations shown in parentheses (each performed in triplicate). Statistical analysis: *** indicates a significanteffect of theagent compared to the control cells( p < 0.001; Student's t test).

extracellular Ca2+binding was very low. Thus, the effect of norepinephrine on Ca2+ uptake was FIG. 4. Adrenergic blockade of the norepinephrine re- mediated exclusively through the @-adrenergicreceptor. The sponse. Brown adipocytes were incubated for 30 s with '%a2+and effect of norepinephrine and the CAMP-elevating agents on with the followingadditionsas indicated NE, 500 nM norepinephrine; PRA, 5 p~ prazosin; EtOH, 0.4% ethanol (the prazosin carrier); PRO, Ca2+uptake is in marked contrast to the stimulatory effects 20 p M DL-propranolol. The adrenergic antagonists were added to theof these agents, under almost identical conditions, on Na+ cells at the start of the 5-min preincubation before the addition of influx into brown adipocytes (Connolly et al., 1986), thus '%a2+and norepinephrine. The mean control (con) cell Ca2+ uptake excluding the possibility that the effect of adrenergic stimuwas 5.8 0.6 nmol of Ca2+/million cells. Bars represent the mean + lation observed on 4SCa2+uptake is due, for example, to a S.E. from three different cell preparations (each performed in triplidecreased cell recovery in theexperimental procedure. cate). Statistical analysis: *(*) and ** indicate significant differences from the control cell Ca2+ uptake ( p < 0.02 and 0.01, respectively); The Influence of CAMP-stimulated Processeson Ca" Entry +(+) indicates a significant effect of norepinephrine compared to controls with antagonists alone( p < 0.02; Student's t test). The substrate for the respiratory response of the brown adipocyte to norepinephrine (the @-response)is the free fatty a 40-fold excess of the fl-antagonist DL-propranolol) was acids released by CAMP-stimulated lipolysis (for review, see unable in itself to affect the Ca2+uptake of the cells (Fig. 4B). Nedergaard and Lindberg (1982)). Externally applied free This is in marked contrast to observations in othercell types fatty acids are potent stimulatorsof brown adipocyte thermostimulated via al-adrenergic pathways where a stimulated genesis, inducing high rates of respiration similar to those Ca2+ uptake is generally seen under similar experimental seen with norepinephrine (Prusiner et aL,1968a, 1968b; Reed conditions (see ''Discussion"). and Fain, 1968 Bukowiecki et al., 1981). If the inhibition of From the same experiments, it may thus be concluded that Ca2+ entry was a consequence of the CAMP-mediatedlipolysis complete @-adrenergicblockade abolished the effect of nor- induced by P-adrenergic stimulation of the brown adipocytes, epinephrine on Ca" uptake (Fig. 4B), whereas selective @- it would beexpected that exogenous free fatty acids could also adrenergic stimulation (Fig. 4A) was equivalent to norepi- induce such an inhibition. nephrine in inducing the effect. Octanoate, as well as palmitate (a more physiological subThe cyclic-AMP elevating agents forskolin (an adenylate strate for brown adipocytes), could induce a reduction in the cyclase activator) and theophylline (a phosphodiesterase in- apparent Ca2+uptake of the cells, similar to that seen with hibitor) are able to induce @-adrenergic-likerespiratory re- norepinephrine (Fig. 6). Norepinephrine added together with sponses in isolated brown fat cells under incubation condi- a fattyacid led to a greater inhibitionof Caz+entry thaneach tions similar to those described here (Connolly et al., 1986). agent alone (Fig. 6), but theeffects were found not to be fully Both forskolin and theophylline also reduced Ca" uptake by additive. Thus, in contrast to the case of Na+ ion influx the cells (Fig. 5), indicating that a CAMP-dependent process (Connolly et al., 1986), the effect of p-stimulation on Caz+ion was involved in theresponse. influx could be mimicked by the addition of free fatty acids It may be mentioned that the response of the cells to both and was thus probably primarily a consequence of CAMPnorepinephrine and forskolin, i.e. the percent inhibition of mediated lipolysis. In control experiments performed under Ca2+ uptake,was similar whetheran EGTA wash was used or identical conditions (not shown), palmitate was found to be not(with EGTA in the recovery wash: controls loo%, + effective in stimulating cellular respiration, inducing rates norepinephrine 77% -+ 3 (9), forskolin 74% k 2 (11); mean which were identical to those with norepinephrine; the stimk S.E. from the number of cell preparations in parentheses, ulation with octanoate was slightly lower. When added toeach performed in triplicate), which, assuming norepineph- gether with norepinephrine, neither palmitate nor octanoate rine only affects intracellular Ca2', further confirms that the could further stimulaterespiration. These effects of fatty acids

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FIG. 6. Effect of exogenous free fattyacids on Ca2+uptake by brown adipocytes. Brown adipocytes were incubated for 30 s with "Ca" and with the following additions as indicated N E , 1 PM norepinephrine; OCT, 2 mM sodium octanoate; PAL, 2.4 mM sodium palmitate. Note that the incubation buffer contained 2% (0.3 mM) fatty acid-free bovine serum albumin. The mean control (con) cell Ca2+uptake was 5.6 0.7 ( A ) and 7.8 2.4 ( B )nmol of Ca2+/million cells. Bars represent the mean + S.E. from four different cell preparations (each performed in triplicate). Statistical analysis: *(*) and *** indicate significant differences from control cell Ca2+uptake ( p < 0.02 and 0.001, respectively).

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FIG. 7. Titration of the effectof FCCP on brown adipocyte Ca" uptake. Brown adipocytes were incubated for 30 s with "Ca2+ together withthe indicated concentrationsof FCCP. Points are means with the range (wheregreater than the size of the symbol) of duplicate determinations inone cell preparation.

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FIG. 8. Influence of mitochondrial uncoupling on Cas' uptake in brown adipocytes. Brown adipocytes were incubated for 30 s with &Ca2+and with the following additions as indicated NE, 1 PM norepinephrine; &OH, 1.8%ethanol (the FCCP carrier); FCCP, 100 p M FCCP. The mean control (con)cell Caz+uptake was 6.4 & 0.8 nmol of Ca2+/millioncells. Bars represent the mean + S.E. from five different cell preparations (each performed in triplicate). Statistical analysis: *** indicates significant difference from control cell Ca2+ uptake ( p < 0.001; Student's paired t test).

that some of the Ca2+taken up ismoving into an uncouplersensitive intracellular Ca2+ pool which is probably the mitochondria. An alternative explanation would be that FCCP Effect of Mitochondrial Uncoupling was acting via a decrease in cellular ATP levels (due to a One of the consequences of the @-adrenergicallystimulated reversal of the mitochondrial F1-ATPase), thereby inhibiting release of intracellular free fatty acids in the brown adipocyte ATP-dependent Ca2+ uptakeinto, forexample, theendois believed to be a partial uncoupling of the mitochondria, plasmic reticulum. However, when the cells were exposed t o leading to a large stimulation of cellular respiration (thermo- theF1-ATPaseinhibitor oligomycin (which is effective in of the uncoupling protein these cells (Mohell et al., 1987)) for the 5-min preincubation genesis). This is due to the presence thermogenin in the brown fat mitochondrial membrane (for period, the fulleffect of FCCP was still observable(cells review, see Nedergaard and Lindberg, 1982). We have there- preincubated with 5 pg/ml oligomycin showed a Ca2+ uptake fore examined the effects of artificial mitochondrial uncou- of 6.4 +_ 0.4 nmol of Ca"+/million cells after 30 s loading; oligomycin-treated cellsloaded in the presence of 100 PM pling andof stimulated respiration onCa2+uptake. To test the possibility that the @-mediatedeffect was due FCCP had an uptake of 3.9 f 0.2 nmol of Ca2+;FCCP thus to mitochondrialuncoupling, we mimicked this by adding the inhibited Ca2+ uptake by 40%). This clearly shows that the mitochondrialuncouplerFCCP. The uncouplingeffect of effect of FCCP cannot be mediatedvia a lowering of cellular FCCP can be seen even in mitochondria within intact brown ATP levels. Neither can this be thecase for norepinephrine Ca2+uptake in the adipocytes, as clearly shown by respiration studies (e.g. Moh- since the hormonewas also able to inhibit cells even in the presenceof oligomycin (not shown). ell et al. (1987)). T h e addition of the maximally effective dose of FCCP for Titration of different concentrations of FCCP led to an inhibition of Ca2+ entry into thebrown adipocytes with full the duration of the 45Ca2+uptake period led in an inhibition was larger than that seen with norepinepheffect being observedat 100 pM or higher (Fig. 7). This shows of Ca2+ entry that

Ca2+ Uptake in Brown Adipocytes rine (Fig. 8). Furthermore, FCCP was able to furtherdecrease Ca2+ uptake incells stimulated with norepinephrine (Fig. 8). These results are in agreement with the idea that p-stimulation, via fatty acid release, leads to only a partial mitochondrial uncoupling (i.e. a limited decrease in the mitochondrial membrane potential) within the brown adipocyte. They also demonstrate that inhibition of Caz+entry intoan intracellular Ca2+pool will bereflected in a reduction of the total cell Caz+ uptake. Even in the presence of FCCP, norepinephrine was able to further reduce the Caz+ uptake (Fig. 8), but this effect of norepinephrine was much smaller here, in agreement with the idea that part of the effect of norepinephrine was to inhibit entry of Ca2+into the mitochondria. As it must be assumed that FCCP fully uncouples the mitochondria within the intact, unstimulated cell, then this additional effect of norepinephrine, reducing Ca2+uptake below the FCCP level, must be due to aneffect of adrenergic stimulation on some Caz+flux which is uncoupler-insensitive. This is also in accordance with the effect of norepinephrine in reducing the Ca2+ uptakeeven in the presence of free fattyacids (Fig. 6).

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1 A. Ca2+ uptake

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i

5

The Influence of Respiratory Stimulation Norepinephrine, forskolin, theophylline, and free fatty -C m acids can all induce high rates of respiration in brown adipo0 cytes underthese incubation conditions (Connolly et al., cm 1986). To examine whether the decreased Ca2+uptake was a consequence of the stimulation of respiration in itself, rotenone was added 40 s prior to the 46Ca2+uptake period. This is sufficient to completely abolish the respiratory response to stimulation in these cells (Connolly et al., 1986). Despite the complete respiratory inhibition in presence of rotenone, the UM effect of norepinephrine on T a 2 +influx was unaltered (Table FIG. 9. Changes in Cas+ uptake rate and membrane potenI). tial induced by palmitate and its derivatives in isolated brown fat mitochondria. Ca2+ uptake(with arsenazo) and membrane potential (with safranine) were examined exactly as described previously (Nedergaard, 1983). Palmitoyl-CoA (Palm-CoA), palmitate (Palm), and palmitoyl-carnitine (Palm-Cn) were added at the conMitochondria centrations shown. Note that the medium contained 0.08% (11PM) Since fatty acids and FCCP had similar effects on cellular fatty acid-free bovine serum albumin. The data for Ca2+uptake ( A ) “Ca2+ uptake and since both partially mimicked the effect of are expressed as a percentage of the control initial rateof Ca2+uptake after addition of 10 p M Ca2+ (mean: 19 f 2 nmol of Ca2+/min/mg norepinephrine, a unifying theory wouldbe that all these agents acted by decreasing the mitochondrial membrane po- protein). The dataon membrane potential ( B ) are shown both as the percent change in safranine absorbance and with reference to the tential and thereby reducing the flux through the mitochon- millivolt scale calculated by Nedergaard (1983).Due to thelimitations drial Ca2+uniport. However, an alternative possibility could of the method used, the absolute values in millivolts are less accurate be that the fattyacids, or their cellular derivatives acyl-CoA than the magnitude of the changes in potential (Nedergaard, 1983). The corresponding curves on Ca2+uptake and membrane potential and acyl-carnitine, had a direct inhibitory effect on the Ca” uniport of the mitochondria. Using isolated mitochondria, we for each agent were performed on the same mitochondrial preparation therefore examined whether the effects of fatty acids (or their under identical conditions except for the dye used.

The Effect of Fatty Acids and Their Derivatives on Ca2+ Uptake and Membrane Potential inIsolated Brown Fat

esters) on Ca2+uptake could be fully accounted for as being secondary to their effects on the mitochondrial membrane potential.

Increasing concentrations of palmitate and its derivatives palmitoyl-CoA and palmitoyl-carnitine were found to have an inhibitory effect on mitochondrial Ca2+uptake (Fig. 9A), in the order of potency palmitoyl-Cob > palmitate > palmitoylTABLE I Effect of inhibitionof respiration on Ca2+ uptake in norepinephrine- carnitine. The agents also had a depolarizing effect on the mitochondrial membrane potential (Fig. 9B). The similarity stirnuluted brown adipocytes Rotenone (10 p M ) was added to thecells 40 s prior to theaddition between the relative effects of the palmitate derivatives on of the “Ca2+containing buffer (see “Experimental Procedures”). NE, both parameters suggests that the inhibition of Caz+uptake 1 p~ norepinephrine. The mean control cell Ca” uptake was 3.7 & into the isolated mitochondria canbe fully ascribed to a 0.6 nmol of Ca2‘/million cella. The values represent the mean S.E. reduction of the membrane potential. The fact that the Ca2+ from three different cell preparations each performed in triplicate. uptake is more sensitive to these agentsthan is the membrane % of control potential is in accordance with our earlier demonstration that Conditions Ca*+ uptake only a small reduction in membrane potential has a large effect on Ca2+ uptake (Nedergaard, 1983). Thus, it is clear Control 100 NE 76 k 3” that fatty acids and their derivatives, the levels of which are Rotenone 99 4 expected to increase in the thermogenically active brown 74 4” Rotenone + NE adipocyte, could have the ability to reduce the mitochondrial A significant effect of norepinephrine (p < 0.01;Student’s t test). membrane potential and thereby inhibitCa2+uptake.

* *

Ca2+Uptake Adipocytes in Brown

10580

TABLEI1 The effect of inhibition of Ca2+exit pathways O R the @stimulated reduction of Ca2+uptake Low-Na+ buffer incubations wereperformed as describedunder "Experimental Procedures." NE, 1 PM norepinephrine. Trifluoperazine (50 pM) and chlorpromazine (50 /A" were present during the 5min preincubation of the cells prior to the addition of ''Ca''. In each case the control CaZ+ uptake (mean: low-Na+buffer, 3.2 & 0.8; normal buffer, 5.7 f 0.9) was set to 100%.Trifluoperazine and chlorpromazine had no effect on Ca2+ uptake when added alone. The values represent mean & S.E. from threecell preparations each performed in triplicate. 5% of control Conditions Cas+uptake

Low-Na+ buffer Control Forskolin (50p M ) Normal buffer Control

+

72 f 5"

NE 62 f 3b Trifluoperazine 100 Trifluoperazine NE 77 1b 100 Chlorpromazine Chlorpromazine + NE 64 & lb "Different from control cell Ca'+ uptake (p < 0.01, Student's t test). bDifferent from control cell Ca*+uptake (p c 0.001,Student's t test).

+

*

Possible Non-mitochondrial Effects of Norepinephrine While it is evident from the above experiments that the major effect of norepinephrine on brown adipocyte Ca2+uptake couldbe ascribed to changes in mitochondrial Ca2+ uptake resulting from the effects of norepinephrine on the mitochondrial membrane potential, it was also clear that even under conditions where the mitochondrial uptake was fully abolished, norepinephrine could still influence the Ca2+uptake of the adipocytes. Thus, asecond effect of norepinephrine on Ca2+uptake, of non-mitochondrial origin, was implied and we attempted to identify this other mechanism. Although the experimental conditions used essentially allow only unidirectional '%a2+ influx, an apparent inhibition of Caz+ entry could theoretically result from a CAMP-mediated stimutationof Ca2+exit from the cells (if this Ca2+exit mechanism could successfully compete with the intracellular pools for cytosolic "Ca2+). To examine this possibility we attempted to block the two major mechanisms for Ca2+expulsion that exist in cells: the Na+/Ca2+exchanger and the Ca2-ATPase. Na+/Ca2+ Exchange-Because the Ca2+ response occurs within the same time scale as the earlier demonstrated Na+ influx (Connolly et al., 1986) and is also CAMP-mediated, it was reasonable to suspect that thetwo ionic movements were linked, i.e. that elevated CAMPlevels might stimulate a Na+/ Ca2+exchange in the plasma membrane of these cells. The Na+/Ca2+exchanger would be driven by the Na' gradient across the plasma membrane, and removal of this gradient would therefore lead to a loss of the exchanger activity. However, under such conditions, i.e. in a low-Na+buffer, the effect of forskolin was identical to that seen in the presence of normal Na+ levels (Table 11). The same was true in alowNa+ buffer in which choline was the only ion replacing Na+ (results not shown), a condition which completely inhibited Na+/Ca2+exchange in renal cells (Hanai et al., 1986). (Forskolin was used to avoid possible problems with the binding of norepinephrine in the low-Na+ medium?) Thus, the Na+ and Ca2+ion movements occurring after @-stimulationseem L. Unelius, N. Mohell, and J. Nedergaard, unpublished observations.

to be unrelated, and this distinction is confirmed by the fact that the Na+ influx event is independent of other known CAMP-mediated intracellular events in the brown adipocyte, such as triglyceride breakdown, mitochondrial uncoupling, and elevated respiration (Connolly et al., 1986), while the Ca2+ influx inhibition was not (see above). Ca2-ATPase-The plasma membrane Ca2-ATPasehas been shown, at least in parotid basolateral membranes, to be activated by CAMP-dependent phosphorylation (Helman et al., 1986), making it a possible mechanism for CAMP-stimulated Ca2+efflux. In most cells the binding of calmodulin may also activate the enzyme (see Carafoli, 1984). Thus, we attempted to block CaZ-ATPaseactivity by exposing the cells during the 5-min preincubation period to the calmodulin antagonists trifluoperazine and chlorpromazine. The response to norepinephrine was, however, unaffected by the calmodulin antagonist treatment (Table11). The Plasma Membrane-Another possibility would bethat there was an additional, direct effect of norepinephrine on the uptake of Ca" across the plasma membrane. If this uptake should be regulated, it should be associated with a transport mechanism or channelfor Ca2+.However, the Ca2+ antagonist diltiazem (which blocks Ca2+entry into other tissues(Fleckenstein-Griin et al., 1984)) was without effect on Ca2+uptake into brown adipocytes, when tested in the range of 100 PM to 1 mM (not shown). Although diltiazem-insensitive Ca2+transporters may, of course, exist, it would seem most likely that the Ca2+uptake over the plasma membrane occurs through leak mechanisms which are inherently unregulated. As the driving force for the Ca2+entry into thecell consists of both the Ca2+gradient and theplasma membrane potential, it could be envisaged that theadditional effect of norepinephrine on Ca2+uptake could be due to the plasma membrane depolarization observed after norepinephrine stimulation of these cells (Girardier and Schneider-Picard, 1983; Horwitz and Hamilton, 1984). However, when the cells were experimentally depolarized by elevation of the extracellular K' concentration (upto 46 mM, corresponding to a depolarization of about 55 mV), the rate of Ca2+uptake was unaffected (not shown). This indicated that a change in plasma membrane potential could not explain the additional effect of norepinephrine. Thus, we have not found evidence for an effect of norepinephrine stimulation at the plasma membrane level, neither stimulatory nor inhibitory, and we consider it most likely that the effect of norepinephrine in excess of that which can be mimicked by FCCP is associated with an effect on the uptake of Ca2+into an intracellular, non-mitochondrial pool, e.g. the endoplasmic reticulum. DISCUSSION

The datawe present here provide evidence for the entry of Ca2+into two intracellular Ca2+pools in thebrown adipocyte: one which is FCCP-sensitive and another which is FCCPinsensitive. The FCCP-sensitive intracellular Ca2+pool, which is probably localized in the mitochondria, is large and accounts for some 40% of the Ca2+turnover in the resting cell, ie. about 2 nmol of Ca2+/min/million cells. Based on a mitochondrial protein content of approximately 1 mg/million cells (Nedergaard et al., 1977), this corresponds to about 2 nmol of Ca"/ min/mg mitochondrial protein, avalue which is in remarkably good agreement with the Ca2+turnover observed in isolated brown fat mitochondria (measured as basal, steady-state release) (Nedergaard, 1981, 1983). We have made a series of attempts at rapid isolation of mitochondria from digitonin-

Ca2+ Uptake in Brown Adipocytes treated brown adipocytes but, even a t high concentrations of digitonin, it was not possible to release sufficient amounts of the intactorganelles from the cell suspensions. Thus, we could not, using this approach, provide more direct evidence for the mitochondrial localization of this Ca2+pool within the cells. Calcium uptake into this mitochondrial pool could be partially inhibited by norepinephrine stimulation of the cells. As we have shown that thenorepinephrine effect on Ca2+uptake occurred via the @adrenergic receptor, involved CAMP production and could be mimicked by the addition of both exogenous free fatty acids and artificial uncoupling agents, it is logical to imagine that the effect of norepinephrine on Ca2+ uptake occurs via the well known events of CAMP-dependent lipolysis and partial mitochondrial uncoupling in the thermogenically active brown adipocyte. Any alternative theory suggesting that norepinephrine was working on a non-mitochondrial compartment, for example the endoplasmic reticulum, would involve postulating that, although both fattyacid release and partial uncoupling occur within the /%stimulated brown adipocyte, these events do not then lead to theeffects seen on Ca2+uptake when these processes are mimicked by the addition of free fattyacids or FCCP to thecells. Thus, althoughthe present experimentsdo not finally prove that norepinephrine works on mitochondrial Caz+ uptake through the sequence of events suggested, alternative hypotheses seem much less plausible. The other intracellular Ca2+ pool is somewhat larger, is insensitive to uncoupler, and probably corresponds to (a fraction of) the endoplasmic reticulum. Norepinephrine is apparently also able to inhibit the uptake into this pool, although to a rather small extent. No al-adrenergically induced Ca2+ entry into the brown adipocyte could be demonstrated. In many other cell types, Ca2+entry from the external medium has been demonstrated as being necessary to maintain the elevated cytosolic Ca" concentrationduringstimulation with hormones that use Ca2+as theintracellular mediator of the response (Kondo and Schulz, 1976 Assimacopoulus-Jeannet et al., 1977; Keppens et al., 1977; Sawyer and Cohen 1981; Mauger et al., 1984; Hesketh et al., 1985; Joseph et al., 1985; Pozzan et al., 1986). In many of these studies, the time scale and conditions of measurement of the Ca2+ uptake were very similar to those we describe here for brown adipocytes. Thus, the lack of an al-adrenergically mediated Ca2+entry was very conspicuous, as there is good evidence for a series of al-adrenergic effects in brown adipocytes (Mohell et al,, 1983a, 1983b, 1984, 1987; Mohell, 1984; Silva and Larsen, 1983; Nhnberg et al., 1984; Ninberg and Putney, 1986), some of which have been shown to be Ca2+-mediated (Schimmel et al., 1983; Connolly et al., 1984; NBnberg et al., 1985; Ninberg and Nedergaard, 1987). Thus, it would seem that the elevated cytosolic Caz+ levels required to elicit and maintain theal-adrenergic response of these cells can be fully derived from pools of intracellularly stored Ca2+. This unusual property of this cell makes it a useful candidate for studying Ca2+involvement in the mediation of hormone responses in general. The CAMP-mediated inhibition of cellular Ca2+uptake in brown adipocytes is also unusual. In other cell types, agents that elevate CAMPlevels have been shown to either stimulate Ca2+entry (for example in pancreatic P-cells (Henquin and Meissner, 1983; Prentki et al., 1987), hepatocytes (Keppens et al., 1977; Mauger et al., 1985; Poggioli et al., 1986), Leydig cells (Sullivan and Cooke, 1986), and a mouse pituitary cell line (Luini et al., 1985)) or to have no effect (for example in submandibular acini (McPherson and Dormer, 1984) and in pancreatic exocrine cells (Kondo and Schulz, 1976)). Again,

10581

some of these studieswere performed under conditions which closely resemble those used in our study of brown adipocytes. Thus, thebasis for the inhibitory effect of B-stimulation on Ca" uptake inthe brown adipocyte may be related to a special property of these cells. Brown adipocytes contain avery large amount of mitochondria and apparently a very low amount of endoplasmic reticulum (Lindberg et al., 19671, and it is probably this dominance of the mitochondria that allows US to reveal the mitochondrial Ca" metabolism even in studies with isolated cells. The large Ca" turnover of the brown adipocyte observed here may also be related to the avid Ca" metabolism of the tissue implied by Lilien et al. (19%). The Ca2+ uniport, the major entry mechanism for mitochondrial Ca", is driven by the mitochondrial membrane potential (Nicholls and Akerman, 1982). Thus, the decrease in Ca2+uptake into the mitochondrial pool occurring after norepinephrine stimulation could be explained as being due to a decreased membrane potential. Our experiments with FCCP are inagreement with this tenet;however, as theeffect of FCCP is larger than that of norepinephrine, it may be concluded that the decrease in the mitochondrial membrane potential with norepinephrine is less than with FCCP. The decrease in mitochondrial membrane potential could either be due to a "coupled" process, i.e. to anincreased ATP synthesis, or to an uncoupling, ie. a decrease in membrane potential occurring in theabsence of ATP synthesis. Control experiments showed that the norepinephrine effect was also visible in the presence of the ATP synthetase inhibitor oligomycin (not shown). Thus,the norepinephrine-mediated decrease in membrane potential (which here is reflected as a decrease of entry of Ca2+into themitochondria), must be due to a partial uncoupling of the mitochondria within the cells. Our ability to mimic the effect of norepinephrine by the addition of free fatty acids to the cell makes it highly likely that the uncoupling occurs due to the release of free fatty acids within the cell and their subsequent interaction with the mitochondria. Although it cannot be ascertained from the present experiments, the mitochondrial uncoupling is probably mediated by the uncoupling protein thermogenin. Thermogenin is exclusiveto brown adipocytes (Cannon et al., 1982) and thus, only in these cells can the mitochondria be uncoupled in vivo to give high rates of respiration (heatproduction). This thenprovides the basis of the highly unusual observation that &stimulation, via CAMP, inhibits mitochondrial Ca2+ entry; thus cellular Ca" uptake is inhibited by B-stimulation, which is not seen in other mammalian cells (see above). The difference in the Ca2+ metabolism of the brown adipocyte compared to many other cells probably arises as aconsequence of the thermogenic function of this cell. From our earlier experiments(Nedergaard, 1983), values of the decrease in mitochondrial membrane potential necessary to account for the norepinephrine-mediated decrease in mitochondrial ca'+ can be estimated. A reduction in the rateof mitochondrial Ca2+uptake of approximately 50%, as observed here, corresponds to a decrease in themembrane potential of only 25 mV or less. This decrease is fully sufficient to maximally stimulate respiration (thermogenesis), but it should be noted that theextramitochondrial Ca'+ steady state level was only marginally affected by such a decrease in membrane potential (Nedergaard, 1983). By the use of cationic dyes, Rafael and Nicholls (1984) and LaNoue et al. (1986) have attempted to establish the degree of mitochondrial membrane depolarization after norepinephrine stimulation of isolated brown adipocytes. The estimates were different (15 and 60mV, respectively), andneither allowed for the changes in the plasma membrane potential

10582

Ca2+Uptake in Brown Adipocytes

known to occur after norepinephrine Stimulation (Girardier and Schneider-Picard, 1983; Horwitz and Hamilton, 1984); also long incubation times with the dyes and with norepinephrine were used. Thus, our results may be the first to demonstrate a rapid functional decrease in mitochondrial membrane potential as a consequence of uncoupling of the mitochondria within the intact,thermogenically active brown adipocyte. It might be thought that @-adrenergicstimulation would therefore lead to an increase in cytosolic Caz+ within the brown adipocyte. These cells possess a plasma membrane CaZ+-dependent K+ channel which conveniently actsas a marker for functionally elevated cytosolic Ca2+levels (NBnberg et al., 1985). The K+ efflux through this channel seems to be exclusively stimulated by al-adrenergicstimulation, implying that the partial inhibition of mitochondrial Caz+ uptake reported here is not in itself sufficient to increase the cytosolic Ca2+ level to an extent required to activate those processes which are regulated by cytosolic Ca2+. However, current work with several different cell types suggests that Ca2+ mobilizedfrom the endoplasmic reticulum of the stimulated cell elevates the cytosolic Caz+concentration, which in turn leads to Ca2+uptake into mitochondrial the pool (Crompton et al., 1983; Shears and Kirk,1984; Akin and Bygrave, 1986; Biden et al., 1986). Thus, the mitochondria tend to counteract the elevation of cytosolic Ca2+, and a redistribution of intracellular CaZ+ occurs after stimulation (Denton and McCormack, 1985). The evidence we present could suggest that the brown adipocyte does not follow this pattern of events. The physiological mediator of stimulation of brown adipose tissue, norepinephrine, acts simultaneously on both al- and @-adrenergicreceptors on the cell surface (Mohell et al., 1983a,1983b, 1987). Because al-stimulation leads to intracellular Ca2+mobilization (Connolly et al., 1984; NBnberg et al., 1985) and @-stimulation apparently leads to an inhibition of Ca2+uptake into the mitochondria, then the @-adrenergicstimulus would be expected to potentiate the a1induced rise in cytosolic CaZ+ (perhaps removing the need for, and explaining the absence of, an al-mediated stimulation of influx of extracellular Ca2+).This may be an explanation at the subcellular level for some of the observations of cooperativity between al-and @-stimulationsin brown adipose tissue that have recently been observed (Ma and Foster, 1984; Jacobsson et al., 1986a, 1986b;Cunningham and Nicholls, 1987). Acknowledgments-We would like to thank Nina Mohell and Barbara Cannon for valuable discussions and Barbro Svensson for performing the mitochondrial studies. REFERENCES Akin, J. G. & By ave, F. L. (1986)Biochem. J. 238,653-661 Assimacopoulos-xannet,F. D., Blackmore, P. F. & Exton, J. H.(1977)J. Biol. Chem. 252,2662-2669 Biden, T. J., Wollheim, C. B. & Schlegel, W. (1986)J.Biol. Chem. 261,72237229 Bukowiecki, L. J., Follha, N., Lupien, J. & Paradis, A. (1981)J. Biol. Chem. 256,12840-12848 Cannon, B., Hedin, A. & Nedergaard, J. (1982)FEBS Lett. 150,129-132 Carafoli, E. (1984)Fed. Pmc. 43,3005-3010

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