THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 263, No. 22, Issue of August 5, pp. 10574-10502,1988

Printed in U. S. A.

@-Adrenergic Modulation of Ca2' Uptake by Isolated Brown Adipocytes POSSIBLE INVOLVEMENT OF MITOCHONDRIA*

(Received for publication, November 10, 1987)

Eamonn ConnollyS and Jan Nedergaard From The Wenner-Gren Institute, University of Stockholm, Biologihus F3, S-106 91 Stockholm, Sweden

Rapid, unidirectional Ca2+ influx was examined in isolated brown adipocytes by short incubations (30 s) with "Cas+. Ca2+ uptake was found to be large in the resting brown adipocyte, but was markedly inhibited when the cells were presented with norepinephrine. Specific al-adrenergic stimulation was without effect on Ca2+ uptake. The effect of norepinephrine (which had an ECao of 140 nM) could be inhibited by &adre- nergic blockade and could be mimicked by forskolin (an adenylate cyclase activator) and theophylline (a phosphodiesterase inhibitor). Exogenous free fatty acids such as octanoate and palmitate (classical stimu- lators of respiration in brown adipocytes) were also able to dramatically inhibit Ca" uptake by the cells. The artificial mitochondrial uncoupler carbonyl cya- nide p-trifluoromethoxyphenylhydrazone (FCCP) in- duced a large reduction in cellular Ca" uptake (even in the presence of the ATPase inhibitor oligomycin), and in the presence of FCCP the inhibitory effect of norepinephrine on Ca" uptake was significantly re- duced. The effect of &adrenergic stimulation on Ca2+ uptake was not directly caused by the large increase in respiration that occurs in response to norepineph- rine because the respiratory inhibitor rotenone did not affect the Ca2+ response of the cells to the hormone. The evidence suggests that &adrenergic stimulation of brown adipocyte metabolism leads to a partial inhibi- tion of Ca2+ uptake into the mitochondrial Ca2+ pool and we discuss the possibility that this represents the effect of a reduced membrane potential (and thus re- duced Ca2+ uniport activity) in the partially uncoupled mitochondria of the thermogenically active brown adi- pocyte.

Ca2+ homeostasis in the mammalian cell is paramount for the control of intracellular metabolism, as well as for the mediation of the response of the cell to a wide variety of hormonal stimulations. In brown adipocytes, a1-adrenergic stimulation induces Ca2+ mobilization from intracellular Ca2+ stores (Connolly et al., 1984). This leads to an elevation of the cytosolic Ca2+ concentration, as shown by the activation of a Ca2+-dependent K+ channel in the plasma membrane (Nhberg et al., 1984, 1985). Stimulation of the al-receptor also leads to an increased respiration by the cells (Mohell et

* Supported by grants from the Swedish Natural Science Research Council (to Barbara Cannon and 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 solely to indicate this fact.

$ To whom correspondence should be addressed.

al., 1983a; Schimmel et al., 1983) which is probably due, at least partially, to the restoration of cellular ion gradients that have been perturbed by the hormone stimulation (Mohell et al., 1987).

It is generally found that stimulation of cells with hormones that use Ca2+ as an intracellular mediator not only releases intracellularly stored Ca2' but also leads to an influx of extracellular Ca2+, the latter being necessary for the mainte- nance of elevated cytosolic Ca2+ level and thus of the hormone response. The nature of the Ca2+ entry mechanism involved is under intense study (Reuter, 1983; Irvine and Moore, 1986; Putney, 1986; Kuno and Gardner, 1987) and seems to depend upon the generation of intracellular polyphosphoinositols; generation of polyphosphoinositols has also been demon- strated in al-stimulated brown adipocytes (Nhberg and Put- ney, 1986; NBnberg and Nedergaard, 1987). The Ca2+ entry can be easily followed by radiolabel techniques as performed in, for example, hepatocytes (Assimacopoulos-Jeannet et al., 1977; Keppens et al., 1977; Mauger et al., 1984), pancreatic acinar cells (Kondo and Schulz, 1976), PC12 cells (Pozzan et al., 1986), and A431 cells (Sawyer and Cohen, 1981)).

We decided to examine unidirectional rates of *Ta2+ uptake in the brown adipocyte for two major reasons: firstly, to see whether al-adrenergic stimulation could lead to an influx of Ca2+ from the extracellular medium, and secondly, to examine the effect of @-adrenergically mediated brown adipocyte thermogenesis on Ca2+ metabolism. Interestingly, al-adrener- gic stimulation was unable to affect the cellular Ca2+ uptake, while @-stimulation was found to markedly inhibit Ca2+ up- take. We conclude that a substantial fraction of the total Ca2+ turnover in the brown adipocyte is mitochondrial, and that @- stimulation may partially inhibit this mitochondrial turnover, probably as a result of a decrease in mitochondrial membrane potential. Some of these results have been presented in ab- stract form (Connolly and Nedergaard, 1987).

EXPERIMENTAL PROCEDURES

Cell Preparation-Brown adipocytes were prepared by collagenase digestion of excised brown fat from adult Syrian hamsters (Mesocri- cetus auratus) in a Krebs-Ringer phosphate buffer exactly as de- scribed previously (Connolly et al., 1984).

Ca2+ Uptake Incubations-"'Ca2+ uptake in isolated brown adipo- cytes was examined using conditions similar to those previously used to follow 22Na+ accumulation (Connolly et Q L , 1986). The freshly isolated cells were kept (maximum of 1.5 h) in Krebs-Ringer phos- phate buffer (containing 4% crude bovine serum albumin) on ice until just before the experiment. They were then washed once in a Krebs- Ringer 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

10574

Ca2+ Uptake in Brown Adipocytes 10575

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 preincu- bated in a 1.5-ml microcentrifuge tube for 5 min at 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. Norepi- nephrine, 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 the cell 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 bicarbon- ate 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 me- dium 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 1 ml 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 tube for 40 s at 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 the specific 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-Mitochondria were 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+ up- take (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-New England Nuclear. N-Methyl-D-glucamine (Meglumine), DL-propranolol hydrochloride, norepinephrine bitartrate, theophyl- line, 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 to neutrality 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-trifluoro- methoxyphenylhydrazone; EGTA, [ethylene bis(oxyethylenenitrilo)] tetraacetic acid.

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 "Ex- perimental Procedures." For each cell preparation, recovery was 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 at each time point. The zero time point was made by addition of cells to the prepared centrifuge tube con- taining 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, the movement 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 environ- ment.

Time Course of Ca" Uptake

When exposed to 45Ca2+, the brown adipocytes showed a steady increase in 45Ca2+ accumulation which was approxi- mately linear over the first 2 min (Fig. lA, control). The uptake of 45Ca2+ corresponds to a movement of approximately 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+ concentration in the order of 100-200 nM (although this has not yet been 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 et al., 1984), equilibration of 45Ca'+ with the cytosolic pooI of Ca2+ would require an apparent entry of only about 1 pmol 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 a constant absolute amount of the observed cell- associated Ca'+. However, when uptake studies were per- formed with cells recovered either in the presence or absence of EGTA in the washing buffer (see "Experimental Proce- dures") (Fig. lA), it was clear that the EGTA 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.

10576 ea2+ Uptake in Brown Adipocytes

These observations must mean that the intracellular CaZ+ was being markedly depleted by the EGTA washing procedure and that the extracellular binding of Caz+ must be very low compared to the amount 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 1 h 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 main- tained 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 prein- cubation (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 preincu- bation at 37 "C for 5 min, did not show a significantly de- creased Ca2+ uptake, compared to cells from the same prepa- ration 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 the presence 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-The biological nature of the Ca2+ uptake was confirmed by incubation of the cells at tempera- tures 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 extra- cellular environments. From the above experiments, it would seem that the apparent Ca" uptake was not due to extracel- lular 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 intracellular pools.

The Response to Adrenergic 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 incuba- tion 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 60 s (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 condi- tions where the cyI- and 8-adrenergic effects of norepinephrine on brown adipocyte respiration can 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 "C incubations, 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 concentra- tions of norepinephrine. The control cell Ca2+ uptake (mean: 6.0 & 1.2 nmol of Caz+/million cells) was set to 100% for each cell prepa- ration, 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. from four different cell preparations (each performed in duplicate). Statistical analysis: *** and ** indicate a significant dif- ference from control ( p < 0.001 and 0.01, respectively; Student's t test).

adrenergic blockade (with a 10-fold excess of the al-adrenergic antagonist prazosin) did not inhibit the effect of norepineph- rine (Fig. a), and selective al-adrenergic stimulation (with

Ca2' Uptake in Brown Adipocytes 10577

A. Prazosin

6. Prop! 'ar l0 lO l -

FIG. 4. Adrenergic blockade of the norepinephrine re- sponse. Brown adipocytes were incubated for 30 s with '%a2+ and with the following additions as indicated NE, 500 nM norepinephrine; PRA, 5 p~ prazosin; EtOH, 0.4% ethanol (the prazosin carrier); PRO, 20 p M DL-propranolol. The adrenergic antagonists were added to the cells at the start of the 5-min preincubation before the addition of '%a2+ and norepinephrine. The mean control (con) cell Ca2+ uptake was 5.8 * 0.6 nmol of Ca2+/million cells. Bars represent the mean + S.E. from three different cell preparations (each performed in tripli- cate). Statistical analysis: *(*) and ** indicate significant differences from the control cell Ca2+ uptake ( p < 0.02 and 0.01, respectively); +(+) indicates a significant effect of norepinephrine compared to controls with antagonists alone ( p < 0.02; Student's t test).

a 40-fold excess of the fl-antagonist DL-propranolol) was unable in itself to affect the Ca2+ uptake of the cells (Fig. 4B). This is in marked contrast to observations in other cell types stimulated via al-adrenergic pathways where a stimulated Ca2+ uptake is generally seen under similar experimental conditions (see ''Discussion").

From the same experiments, it may thus be concluded that complete @-adrenergic blockade abolished the effect of nor- epinephrine on Ca" uptake (Fig. 4B), whereas selective @- adrenergic stimulation (Fig. 4A) was equivalent to norepi- nephrine in inducing the effect.

The cyclic-AMP elevating agents forskolin (an adenylate cyclase activator) and theophylline (a phosphodiesterase in- hibitor) are able to induce @-adrenergic-like respiratory re- sponses in isolated brown fat cells under incubation condi- tions similar to those described here (Connolly et al., 1986). Both forskolin and theophylline also reduced Ca" uptake by the cells (Fig. 5), indicating that a CAMP-dependent process was involved in the response.

It may be mentioned that the response of the cells to both norepinephrine and forskolin, i.e. the percent inhibition of Ca2+ uptake, was similar whether an EGTA wash was used or not (with EGTA in the recovery wash: controls loo%, + norepinephrine 77% -+ 3 (9), + forskolin 74% k 2 (11); mean k S.E. from the number of cell preparations in parentheses, each performed in triplicate), which, assuming norepineph- rine only affects intracellular Ca2', further confirms that the

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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 the mean + S.E. from the number of cell preparations shown in parentheses (each performed in triplicate). Statistical analysis: *** indicates a significant effect of the agent 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

mediated exclusively through the @-adrenergic receptor. The effect of norepinephrine and the CAMP-elevating agents on Ca2+ uptake is in marked contrast to the stimulatory effects of these agents, under almost identical conditions, on Na+ influx into brown adipocytes (Connolly et al., 1986), thus excluding the possibility that the effect of adrenergic stimu- lation observed on 4SCa2+ uptake is due, for example, to a decreased cell recovery in the experimental procedure.

The Influence of CAMP-stimulated Processes on Ca" Entry

The substrate for the respiratory response of the brown adipocyte to norepinephrine (the @-response) is the free fatty acids released by CAMP-stimulated lipolysis (for review, see Nedergaard and Lindberg (1982)). Externally applied free fatty acids are potent stimulators of brown adipocyte thermo- genesis, inducing high rates of respiration similar to those seen with norepinephrine (Prusiner et aL, 1968a, 1968b; Reed and Fain, 1968 Bukowiecki et al., 1981). If the inhibition of Ca2+ entry was a consequence of the CAMP-mediated lipolysis induced by P-adrenergic stimulation of the brown adipocytes, it would be expected that exogenous free fatty acids could also induce such an inhibition.

Octanoate, as well as palmitate (a more physiological sub- strate for brown adipocytes), could induce a reduction in the apparent Ca2+ uptake of the cells, similar to that seen with norepinephrine (Fig. 6). Norepinephrine added together with a fatty acid led to a greater inhibition of Caz+ entry than each agent alone (Fig. 6), but the effects were found not to be fully additive. Thus, in contrast to the case of Na+ ion influx (Connolly et al., 1986), the effect of p-stimulation on Caz+ ion influx could be mimicked by the addition of free fatty acids and was thus probably primarily a consequence of CAMP- mediated lipolysis. In control experiments performed under identical conditions (not shown), palmitate was found to be effective in stimulating cellular respiration, inducing rates which were identical to those with norepinephrine; the stim- ulation with octanoate was slightly lower. When added to- gether with norepinephrine, neither palmitate nor octanoate could further stimulate respiration. These effects of fatty acids

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FIG. 6. Effect of exogenous free fatty acids on Ca2+ uptake by brown adipocytes. Brown adipocytes were incubated for 30 s with "Ca" and with the following additions as indicated NE, 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 prepa- rations (each performed in triplicate). Statistical analysis: *(*) and *** indicate significant differences from control cell Ca2+ uptake ( p < 0.02 and 0.001, respectively).

FIG. 8. Influence of mitochondrial uncoupling on Cas' up- take 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+/million cells. 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).

are in agreement with classical observations (Prusiner et al., 1968a, 1968b).

Effect of Mitochondrial Uncoupling One of the consequences of the @-adrenergically stimulated

release of intracellular free fatty acids in the brown adipocyte is believed to be a partial uncoupling of the mitochondria, leading to a large stimulation of cellular respiration (thermo- genesis). This is due to the presence of the uncoupling protein thermogenin in the brown fat mitochondrial membrane (for review, see Nedergaard and Lindberg, 1982). We have there- fore examined the effects of artificial mitochondrial uncou- pling and of stimulated respiration on Ca2+ uptake.

To test the possibility that the @-mediated effect was due to mitochondrial uncoupling, we mimicked this by adding the mitochondrial uncoupler FCCP. The uncoupling effect of FCCP can be seen even in mitochondria within intact brown adipocytes, as clearly shown by respiration studies (e.g. Moh- ell et al. (1987)).

Titration of different concentrations of FCCP led to an inhibition of Ca2+ entry into the brown adipocytes with full effect being observed at 100 pM or higher (Fig. 7). This shows

that some of the Ca2+ taken up is moving into an uncoupler- sensitive intracellular Ca2+ pool which is probably the mito- chondria. An alternative explanation would be that FCCP was acting via a decrease in cellular ATP levels (due to a reversal of the mitochondrial F1-ATPase), thereby inhibiting ATP-dependent Ca2+ uptake into, for example, the endo- plasmic reticulum. However, when the cells were exposed to the F1-ATPase inhibitor oligomycin (which is effective in these cells (Mohell et al., 1987)) for the 5-min preincubation period, the full effect of FCCP was still observable (cells preincubated with 5 pg/ml oligomycin showed a Ca2+ uptake of 6.4 +_ 0.4 nmol of Ca"+/million cells after 30 s loading; oligomycin-treated cells loaded in the presence of 100 PM FCCP had an uptake of 3.9 f 0.2 nmol of Ca2+; FCCP thus inhibited Ca2+ uptake by 40%). This clearly shows that the effect of FCCP cannot be mediated via a lowering of cellular ATP levels. Neither can this be the case for norepinephrine since the hormone was also able to inhibit Ca2+ uptake in the cells even in the presence of oligomycin (not shown).

The addition of the maximally effective dose of FCCP for the duration of the 45Ca2+ uptake period led in an inhibition of Ca2+ entry that was larger than that seen with norepineph-

Ca2+ Uptake in Brown Adipocytes 10579

rine (Fig. 8). Furthermore, FCCP was able to further decrease Ca2+ uptake in cells stimulated with norepinephrine (Fig. 8). These results are in agreement with the idea that p-stimula- tion, via fatty acid release, leads to only a partial mitochon- drial uncoupling (i.e. a limited decrease in the mitochondrial membrane potential) within the brown adipocyte. They also demonstrate that inhibition of Caz+ entry into an intracellular Ca2+ pool will be reflected 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 norepineph- rine, reducing Ca2+ uptake below the FCCP level, must be due to an effect 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+ uptake even in the presence of free fatty acids (Fig. 6).

The Influence of Respiratory Stimulation Norepinephrine, forskolin, theophylline, and free fatty

acids can all induce high rates of respiration in brown adipo- cytes under these incubation conditions (Connolly et al., 1986). To examine whether the decreased Ca2+ uptake was a consequence of the stimulation of respiration in itself, rote- none 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 effect of norepinephrine on T a 2 + influx was unaltered (Table I).

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

Mitochondria Since fatty acids and FCCP had similar effects on cellular

“Ca2+ uptake and since both partially mimicked the effect of norepinephrine, a unifying theory would be that all these agents acted by decreasing the mitochondrial membrane po- tential and thereby reducing the flux through the mitochon- drial Ca2+ uniport. However, an alternative possibility could be that the fatty acids, or their cellular derivatives acyl-CoA and acyl-carnitine, had a direct inhibitory effect on the Ca” uniport of the mitochondria. Using isolated mitochondria, we therefore examined whether the effects of fatty acids (or their esters) on Ca2+ uptake could be fully accounted for as being secondary to their effects on the mitochondrial membrane potential.

TABLE I Effect of inhibition of respiration on Ca2+ uptake in norepinephrine-

stirnuluted brown adipocytes Rotenone (10 pM) was added to the cells 40 s prior to the addition

of the “Ca2+ containing buffer (see “Experimental Procedures”). NE, 1 p~ norepinephrine. The mean control cell Ca” uptake was 3.7 & 0.6 nmol of Ca2‘/million cella. The values represent the mean S.E. from three different cell preparations each performed in triplicate.

Conditions % of control Ca*+ uptake

Control NE Rotenone Rotenone + NE

100 76 k 3” 99 * 4 74 * 4”

A significant effect of norepinephrine (p < 0.01; Student’s t test).

1 A. Ca2+ uptake

loo]

* ‘“1 B. Membrane potential mV

5 i

C - m 0

m c

UM

FIG. 9. Changes in Cas+ uptake rate and membrane poten- 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 previ- ously (Nedergaard, 1983). Palmitoyl-CoA (Palm-CoA), palmitate (Palm), and palmitoyl-carnitine (Palm-Cn) were added at the con- centrations shown. Note that the medium contained 0.08% (11 PM) fatty acid-free bovine serum albumin. The data for Ca2+ uptake ( A ) are expressed as a percentage of the control initial rate of Ca2+ uptake after addition of 10 pM Ca2+ (mean: 19 f 2 nmol of Ca2+/min/mg protein). The data on membrane potential ( B ) are shown both as the percent change in safranine absorbance and with reference to the millivolt scale calculated by Nedergaard (1983). Due to the limitations of the method used, the absolute values in millivolts are less accurate than the magnitude of the changes in potential (Nedergaard, 1983). The corresponding curves on Ca2+ uptake and membrane potential for each agent were performed on the same mitochondrial preparation under identical conditions except for the dye used.

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 > palmitoyl- carnitine. The agents also had a depolarizing effect on the mitochondrial membrane potential (Fig. 9B). The similarity between the relative effects of the palmitate derivatives on both parameters suggests that the inhibition of Caz+ uptake into the isolated mitochondria can be fully ascribed to a reduction of the membrane potential. The fact that the Ca2+ uptake is more sensitive to these agents than is the membrane potential is in accordance with our earlier demonstration that only a small reduction in membrane potential has a large effect on Ca2+ uptake (Nedergaard, 1983). Thus, it is clear that fatty acids and their derivatives, the levels of which are expected to increase in the thermogenically active brown adipocyte, could have the ability to reduce the mitochondrial membrane potential and thereby inhibit Ca2+ uptake.

10580 Ca2+ Uptake in Brown Adipocytes TABLE I1

The effect of inhibition of Ca2+ exit pathways OR the @stimulated reduction of Ca2+ uptake

Low-Na+ buffer incubations were performed as described under "Experimental Procedures." NE, 1 PM norepinephrine. Trifluopera- zine (50 pM) and chlorpromazine (50 /A" were present during the 5- min 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 three cell preparations each performed in triplicate.

Conditions 5% of control Cas+ uptake

Low-Na+ buffer Control 100 + Forskolin (50 pM) 72 f 5"

Control 100 NE 62 f 3b Trifluoperazine 100 Trifluoperazine + NE 77 * 1b Chlorpromazine 100 Chlorpromazine + NE 64 & lb

Normal buffer

"Different from control cell Ca'+ uptake (p < 0.01, Student's t

bDifferent from control cell Ca*+ uptake (p c 0.001, Student's t test).

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+ up- take could be 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+ up- take of the adipocytes. Thus, a second 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 al- low only unidirectional '%a2+ influx, an apparent inhibition of Caz+ entry could theoretically result from a CAMP-me- diated stimutation of 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+ ex- pulsion 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 the two ionic movements were linked, i.e. that elevated CAMP levels 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 a low- Na+ 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). (For- skolin 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 @-stimulation seem

L. Unelius, N. Mohell, and J. Nedergaard, unpublished observa- tions.

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-A TPase-The plasma membrane Ca2-ATPase has been shown, at least in parotid basolateral membranes, to be acti- vated 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-ATPase activity by exposing the cells during the 5-min preincubation period to the calmodulin antagonists trifluoperazine and chlorpromazine. The response to norepi- nephrine was, however, unaffected by the calmodulin antag- onist treatment (Table 11).

The Plasma Membrane-Another possibility would be that 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 channel for Ca2+. However, the Ca2+ antagonist diltiazem (which blocks Ca2+ entry into other tissues (Fleck- enstein-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+ trans- porters 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 the cell consists of both the Ca2+ gradient and the plasma membrane potential, it could be envisaged that the additional effect of norepineph- rine 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 experi- mentally depolarized by elevation of the extracellular K' concentration (up to 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 norepi- nephrine.

Thus, we have not found evidence for an effect of norepi- nephrine 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 data we present here provide evidence for the entry of Ca2+ into two intracellular Ca2+ pools in the brown adipocyte: one which is FCCP-sensitive and another which is FCCP- insensitive.

The FCCP-sensitive intracellular Ca2+ pool, which is prob- ably 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 (Neder- gaard et al., 1977), this corresponds to about 2 nmol of Ca"/ min/mg mitochondrial protein, a value which is in remarkably good agreement with the Ca2+ turnover observed in isolated brown fat mitochondria (measured as basal, steady-state re- lease) (Nedergaard, 1981, 1983). We have made a series of attempts at rapid isolation of mitochondria from digitonin-

Ca2+ Uptake in Brown Adipocytes 10581

treated brown adipocytes but, even at high concentrations of digitonin, it was not possible to release sufficient amounts of the intact organelles 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 par- tially inhibited by norepinephrine stimulation of the cells. As we have shown that the norepinephrine effect on Ca2+ uptake occurred via the @adrenergic receptor, involved CAMP pro- duction and could be mimicked by the addition of both exog- enous 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 ther- mogenically active brown adipocyte. Any alternative theory suggesting that norepinephrine was working on a non-mito- chondrial compartment, for example the endoplasmic reticu- lum, would involve postulating that, although both fatty acid release and partial uncoupling occur within the /%stimulated brown adipocyte, these events do not then lead to the effects seen on Ca2+ uptake when these processes are mimicked by the addition of free fatty acids or FCCP to the cells.

Thus, although the present experiments do not finally prove that norepinephrine works on mitochondrial Caz+ uptake through the sequence of events suggested, alternative hy- potheses seem much less plausible.

The other intracellular Ca2+ pool is somewhat larger, is insensitive to uncoupler, and probably corresponds to (a frac- tion of) the endoplasmic reticulum. Norepinephrine is appar- ently 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" concentration during stimulation with hormones that use Ca2+ as the intracellular 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 the al-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 media- tion 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 CAMP levels 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,

some of these studies were performed under conditions which closely resemble those used in our study of brown adipocytes.

Thus, the basis for the inhibitory effect of B-stimulation on Ca" uptake in the brown adipocyte may be related to a special property of these cells. Brown adipocytes contain a very 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 mito- chondrial 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 in agreement with this tenet; however, as the effect 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 an increased ATP synthesis, or to an uncoupling, ie . a decrease in membrane potential occurring in the absence of ATP synthesis. Control experiments showed that the norepinephrine effect was also visible in the presence of the ATP synthetase inhibitor oli- gomycin (not shown). Thus, the norepinephrine-mediated decrease in membrane potential (which here is reflected as a decrease of entry of Ca2+ into the mitochondria), 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 prob- ably mediated by the uncoupling protein thermogenin. Ther- mogenin is exclusive to brown adipocytes (Cannon et al., 1982) and thus, only in these cells can the mitochondria be uncou- pled in vivo to give high rates of respiration (heat production). This then provides 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 a consequence 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 mi- tochondrial ca'+ can be estimated. A reduction in the rate of mitochondrial Ca2+ uptake of approximately 50%, as observed here, corresponds to a decrease in the membrane potential of only 25 mV or less. This decrease is fully sufficient to maxi- mally stimulate respiration (thermogenesis), but it should be noted that the extramitochondrial 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 norepineph- rine stimulation of isolated brown adipocytes. The estimates were different (15 and 60 mV, respectively), and neither 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 norepi- nephrine 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 @-adrenergic stimulation 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 acts as a marker for functionally elevated cytosolic Ca2+ levels (NBn- berg et al., 1985). The K+ efflux through this channel seems to be exclusively stimulated by al-adrenergic stimulation, 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+ mobilized from the endoplasmic reticulum of the stimulated cell elevates the cytosolic Caz+ concentra- tion, which in turn leads to Ca2+uptake into the mitochondrial 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 @-adrenergic receptors 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 @-adrenergic stimulus would be expected to potentiate the a1-

induced 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 coopera- tivity between al- and @-stimulations in brown adipose tissue that have recently been observed (Ma and Foster, 1984; Ja- cobsson et al., 1986a, 1986b; Cunningham and Nicholls, 1987).

Acknowledgments-We would like to thank Nina Mohell and Bar- bara Cannon for valuable discussions and Barbro Svensson for per- forming 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.

Biden, T. J., Wollheim, C. B. & Schlegel, W. (1986) J. Biol. Chem. 261,7223-

Bukowiecki, L. J., Follha, N., Lupien, J. & Paradis, A. (1981) J. Biol. Chem.

Cannon, B., Hedin, A. & Nedergaard, J. (1982) FEBS Lett. 150,129-132 Carafoli, E. (1984) Fed. Pmc. 43,3005-3010

Chem. 252,2662-2669

7229

256,12840-12848

Connolly, E., Nhberg, E. & Nedergaard, J. (1984) Eur. J . Biochem. 141,187- 193

Connolly, E., Ninberg, E. & Nedergaard, J. (1986) J. Bwl. Chem. 261, 14377- 14385

Connolly, E. & Nedergaard, J. (1987) Ninth European Society for Comparative Physiology and Biochemisty Conference, August 16-21, 1987, Abstr. A26,

Crompton, M., Kessar, P. & Al-Nasser, I. (1983) Biochem. J. 216,333-342 Copenhagen, in press

Cunningham, S. A. & Nicholls, D. G. (1987) Biochem. J . 246,485491 Denton, R. M. & McCormack, J. G. (1985) Am. J. Physiol. 249, E543-E554 Fleckenstein-Grun, G., Frey, M. & Fleckenstein, A. (1984) Trends Phmmacol.

Girardier, L. & Schneider-Picard, G. (1983) J. Physiol. 336, 629-641 Hanal, H., Ishida, M., Liang, C. T. & Sacktor, B. (1986) J. Biol. Chem. 261,

Helman, J., Kuyatt, B. L., Takuma, T., Seligmann, B. & Baum, B. J. (1986) J.

Henquin, J.-C. & Meissner, H. P. (1983) Biochem. Biophys. Res. Commun. 112,

Hesketh, T. R., Moore, J. P., Morris, J. D. H., Taylor M. V., Rogers, J., Smith,

Horwitz, B. A. & Hamilton J. (1984) Comp. Biochem. Physiol. 78C, 99-104 Imine, R. F. & Moor, R. M: (1986) Biochem. J. 240,917-920 Jacobsson, A., Nedergaard, J. & Cannon, B. (1981%) Biosci. Rep. 6,621-631 Jacobsson, A., Nedergaard, J. & Cannon, B. (1986b) EBEC Rep. 4,386 Joseph, S. K., Coll, K. E., Thomas, A. P., Rubin, R. &Williamson, J. R. (1985)

Keppens, S., Vandenheede, J. R. & De Wulf, H. (1977) Biochim. Bwphys. Acta

Kondo, S. & Schulz, I. (1976) Biochim. Biophys. Acta 419,76-92 Kuno, M. & Gardner, P. (1987) Nature 326,301-304 LaNoue, K. F., Stnelecki, T., Stnelecki, D. & Koch, C. (1986) J. Biol. Chern.

Lilien, 0. M., Krauss, D. J., Manke, C. G., Euser, B. A, & Subramanian, G. M.

Lindberg, 0.. de Pierre, J., Rylander, E. & Afzelius, B. A. (1967) J. Cell Biol.

Luini, A., Lewis, D., Guild, S., Corda, D. & Axelrod, J. (1985) Pmc. Natl. Acad.

Ma, S. W. Y. & Foster, D. 0. (1984) Can. J. Physiol. Pharmol . 62,943-948 Martin, B. R Clausen, T. & Gliemann, J. (1975) Biuchem. J. 162,121-129 Mauger, J.-P: Poggioli, J., Guesdon, F. & Claret, M. (1984) Biochem. J. 221,

Mauger, J.-P., Poggioli, J. & Claret, M. (1985) J. Biol. Chem. 260, 11635-

McPherson, M. A. & Dormer, R. L. (1984) Biochem. J. 224,473-481 Mohell, N., Connolly, E. & Nedergaard, J. (1987) Am. J. Physiol. 253. C301-

Mohell, N., Nedergaard, J. & Cannon, B. 6983a) Eur.2. Phurmacol. 93, 183- Mohell, N. (1984) Acta Physiol. S c a d . (su pl ) 530, 1 64

Mohell, N., Svartengren, J. & Cannon, B. (1983b) Eur. J. Phurmacol. 92, 15-

Mohell, N., Wallace, M. & Fain, J. N. (1984) Mol. Phurmaeol. 26,64-69 Ninberg, E. & Nedergaard, J. (1987) Biochim. Biophys. Acta 930,438-445 Ninberg, E. & Putney, J., Jr. (1986) FEBS Lett. 196,319-322 Ninberg, E., Nedergaard, J. & Cannon, B. (1984) Biochim. Bwphys. Acta 804,

Ninberg, E., Connolly, E. & Nedergaard, J. (1985) Biochim. Biophys. Acta 844,

Nedergaard, J. (1981) Eur. J. Biochem. 114,159-167 Nedergaard, J. (1983) Eur. J . Biochem. 133,185-191 Nedergaard, J. & Lindberg, 0. (1982) Int. Rev. Cytol. 74, 187-286 Nedergaard, J., C non, B. & Lindberg, 0. (1977) Nature 267,518-520 Nicholls, D. G. &ykerman, K. (1982) Biochim. Biophys. Acta 683,57-88 Poggioli, J., Mauger, J.-P. & Claret, M. (1986) Biochem. J. 235,663-669 Pozzan, T., Di Virgilio, F., Vicentini, L. M. & Meldolesi, J. (1986) Biochem. J.

Prentki, M., Glennon, M. C., Geschwind, J.-F., Matschinsky, F. M. & Corkey,

Prusiner, S. B., Cannon, B., Ching, T. M. & Lindberg, 0. (1968a) Eur. J.

Prusiner, S. B., Cannon, B. & Lindberg, 0. (1968b) Eur. J. Biochem. 6, 15-22 Putney J. W. (1986) Cell Calcium 7,l-12 Rafae1,’J. & Nicholls, D. G. (1984) FEBS Lett. 170, 181-185 Reed, N. & Fain, J. N. (1968) J. Biol. Chem. 243,2843-2848 Reuter, H. (1983) Nature 301,569-574

Schimmel, R. J., McCarthy, L. & McMahon, K. K. (1983) Am. J. Physiol. 244, Sawyer, S. T. & Cohen, S. (1981) Biochemistry 20,6280-6286

Shears, S. B. & Kirk, C. J. (1984) Biochem. J. 220,417-421 Silva, J. E. & Larsen, P. R. (1983) Nature 306,712-713 Sullivan, M. H. F. & Cooke, B. A. (1986) Biochem. J. 236,45-51

Sci. 6, 283-286

5419-5425

Biol. Chem. 261,8919-8923

614-620

G. A. & Metcalfe, J. C. (1985) Nature 313,481-4h

J. Biol. Chm. 260,12508-12515

496,448-457

26 1,298-305

(1978) J. UmL 119,170-174

34,293-310

Sci. U. S. A. 82,8034-8038

121-127

11642

C308

193

25

291-300

42-49

234,547-553

B. E. (1987) FEBS Lett. 220,103-107

Biochem. 7,51-57

C362-C368