Energy metabolism of adult astrocytes in vitro

~ ) Pergamon

0306-4522(95)00480-7

Neuroscience VoI. 71. No. 3. pp. 855-870. 1996 Elsevier Science Ltd Copyright ,(- 1996 IBRO Printed in Great Britain. All rights reserved 0306-4522/96 $15.00 + 0.00

E N E R G Y M E T A B O L I S M OF A D U L T ASTROCYTES IN VITRO S. P E U C H E N , * t M. R. D U C H E N : ~ and J. B. C L A R K t lDepartment of Neurochemistry, Institute of Neurology, Queen Square, London WC1N 3BG, U.K. :[:Department of Physiology, University College London, Gower Street, London, WCIE 6BT, U.K. Abstract--ln this study we established cultures of astrocytes from the forebrain of the adult rat. The homeostatic regulatory mechanisms of the aerobic and anaerobic pathways of energy metabolism in these cells showed that adult astrocytes express many of the regulatory properties previously demonstrated in neonatal astrocytes. Changes in mitochondrial respiration and ATP production were readily evident upon incubation with the relevant substrates. Inhibition of mitochondrial respiration led to a compensatory increase in anaerobic glycolysis as evidenced by an increased release of lactate. We assessed the role of cytosolic calcium in the regulation of the mitochondrial energy metabolism. Increases in cytosolic calcium concentration in response to ATP or stimulation of mechanical receptors were followed by depolarizations of the mitochondrial membrane potential, whose magnitude reflected the amplitude of the cytosolic calcium response. The changes in mitochondrial membrane potential were largely dependent on the presence of external calcium. These results provide the first evidence of a signalling mechanism in astrocytes by which changes in cytosolic calcium mediate changes in respiration, possibly through mitochondrial calcium uptake and subsequent activation of several mitochondrial dehydrogenases. This signalling pathway would thus ensure that energy demands due to changes in cytosolic calcium concentrations are met by increases in energy production through increases in mitochondrial oxidative phosphorylation. Key words: astrocytes, glia, adult, energy metabolism, calcium, mitochondria.

Astrocytes are t h o u g h t to play a n u m b e r o f i m p o r t a n t roles in the overall functioning of the central n e r v o u s system (CNS). 37 M u c h evidence suggests t h a t astrocytes are instrumental in m a i n t a i n i n g the ionic balance a n d micro-homeostasis o f the CNS, m o s t n o t a b l y in terms of p o t a s s i u m a n d p H regulation.~l'8~ T h e expression o f m a n y n e u r o t r a n s m i t t e r receptors o n astrocytes 25"32provides a m e c h a n i s m for c o m m u n i cation between astrocytes a n d neurons, so t h a t astrocyte function can be m a t c h e d to c h a n g i n g neuronal activity. Astrocytes have c o n n e c t i o n s with b o t h vascular endothelial cells a n d with neurons, a n d are therefore t h o u g h t to provide a crucial link in the overall nutritional status a n d energy m e t a b o l i s m *To whom correspondence should be addressed. Abbreviations: BAPTA, 1,2-bis(2-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid; BSA, bovine serum albumin; [Ca2+]~, intracellular calcium concentration; cAMP, adenosine 2":Y-cyclic monophosphate; DIV, days in vitro; EDTA, ethylenediaminetetra-acetate; EGTA, ethylene glycol-bis-(aminoethyl ether); ER, endoplasmic reticulum; FBS, fetal bovine serum; FCCP, p-trifluoromethoxy-phenylhydrazone; FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid; LDH, lactate dehydrogenase; MEM, minimal essential medium; A~bm, mitochondrial membrane potential; NOA, 10-nonyl-acridine orange; PBS, phosphate-buffered saline; TMPD, N,N,N',N'-tetramethylp-phenylenediamine.

within the C N S . 26'37"78 They f u r t h e r m o r e are p a r t o f the barrier between the fully developed C N S a n d the systemic circulation, a n d are thus likely to play a role in the regulation o f entry of metabolic substrates, metabolites, viruses a n d toxins into the CNS. TM M o s t of the k n o w n physiology of astrocytes h a s been derived from in vitro studies o f p r i m a r y c u l t u r e s o f astrocytes derived from a n e o n a t a l animal. H o w ever i m p o r t a n t differences between n e o n a t a l a n d adult astrocytes m a y exist since the adult cell d o e s n o t have to meet the metabolic d e m a n d s specific to development. The possible difference b e t w e e n n e o n a t a l and adult astrocytes is f u r t h e r m o r e illust r a t e d by the o b s e r v a t i o n that adult rats show a m u c h m o r e extensive astrocytic response to brain i n j u r y t h a n the n e o n a t a l rat. 3 T h e r e are only a limited n u m b e r of in vitro studies t h a t have characterized astrocytes derived from a d u l t a n i m a l s 7~'8°'84"88a n d even fewer studies have e x a m i n e d their physiology. 39"72"s7T h e relative paucity of d a t a o n a d u l t astrocytes m a y reflect the technical difficulties in establishing these cultures as well as the low yields o b t a i n e d with existing procedures for isolations f r o m the rat. Cell lines derived from adult astrocytes were therefore established. 63's3 However, with the inevitable modifications i n t r o d u c e d d u r i n g the " i m m o r t a l i z a t i o n " process, these cell lines may n o t necessarily exhibit the same regulatory m e c h a n i s m s as p r i m a r y cultures o f adult astrocytes. 2°

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In this s t u d y we d e s c r i b e the p r e p a r a t i o n o f a s t r o c y t e s f r o m the adult f o r e b r a i n . W e d e s c r i b e in p a r t the r e g u l a t i o n o f e n e r g y m e t a b o l i s m , a n d in p a r t i c u l a r the role o f the c y t o s o l i c free c a l c i u m c o n c e n t r a t i o n in the c o n t r o l o f m i t o c h o n d r i a l e n e r g y p r o d u c t i o n . P r e l i m i n a r y results were p u b l i s h e d e l s e w h e r e ] 8-~954'6°-62 In a s e p a r a t e s t u d y ( s u b m i t t e d as an a c c o m p a n y i n g paper590 we detail t h e r e g u l a t i o n o f intracellular c a l c i u m as p a r t o f o n e o f the signal t r a n s d u c t i o n p a t h w a y s p r e s e n t in the a d u l t a s t r o c y t e . EXPERIMENTAL PROCEDURES Materials

All materials were obtained from Sigma (Poole, U.K.) unless stated otherwise. All tissue culture plastics and tissue culture media were obtained from Gibco BRL (Paisley, U.K.). All tissue culture media were supplemented (per ml) with 100 units of both penicillin G and Streptomycin, and 250 ng of amphotericin B. All fluorescence indicators were obtained from Molecular Probes (Eugene, OR, U.S.A.) or Calbiochem (Nottingham, U.K.). Isolation and culture o f astrocytes from the fi)rebrain o/" the adult rat

The procedure is essentially that of Booher et al. 5 and Schousboe et al. 7° A sucrose based separation step was added to the procedure to aid in the removal o f cellular debris and dense myelin, thereby promoting cell attachment and growth. 47 Earl's balanced salt solution was used throughout this procedure for incubations and washes. Forebrains were typically taken from five- to eight-week-old female Wistar rats (B&K Universal Ltd, Hull, U.K.). The hemispheres were separated and the meninges and blood vessels removed. The tissue was chopped into small pieces, resuspended, and forced through a 2 9 7 # m metal mesh (tissue pan, Sigma). The filtered suspension was washed by centrifugation at 400 g,v for 5 rain at 5°C (conditions used throughout unless stated otherwise) and the supernatant was discarded. The pellet was resuspended into a solution containing 50,000 units (units established using benzoyl-Larginine ethylester as substrate)/ml trypsin (EC 3.4.21.4, isolated from porcine pancreas), 1.033 units (units establishing using 4-phenylazobenzyl-oxycarbonyl-proline-leucineglycine-proline-D-arginine as substrate)/ml collagenase (EC 3.4.24.3, type XI, isolated from rat pancreatic islet), 336 "Kunitz" units/ml deoxyribonucleate 5"-oligonucleotidohydrolase (EC 3.1.21.1, type IV from bovine pancreas), and 70.67 "Kunitz" units/ml (1.33 mg/ml) ribonuclease A (EC 3.1.27.5, type 1-AS from bovine pancreas). The enzymatic digestion was halted after 15 min at 37°C by the addition o f heat-inactivated fetal bovine serum (FBS, Gibco BRL, of European or Australian origin). The suspension was gravity-filtered through a 7 4 # m metal mesh, centrifuged and washed twice more. The resulting suspension was layered over a 0.4 M sucrose (molecular biology grade, BDH/Merck, Upminster, U.K.) solution and centrifuged at 400 ga~ for 10 min. The resulting pellet was washed several times, followed by one wash in o-valine-based minimum essential medium (MEM) with Earle's salts. The pellet was resuspended in o-valine M E M (which inhibits the growth of fibroblasts ~2) supplemented with 5% FBS, 2 mM glutamine, 1 mM malate, and aliquotted into tissue culture-treated flasks at an approximate initial plating density of 3.2 x 105 cells/cm2. The flasks were placed in an incubator (95% air, 5% CO2, 37°C) for 2 0 - 4 0 h , after which the medium was refreshed. After 6 days in vitro (DIV) the supplemented D-valine M E M was replaced with a supplemented L-valine based MEM. The cells reached confluency at 12-14 DIV, and were harvested and reseeded

on to either a plastic or glass surface appropriate to subsequent analysis. Typical yield for this procedure at 21 DIV was l × 106 viable cells (Trypan Blue stain) per adult forebrain. Analysis was carried out on cells isolated from cultures at nine to 30 days O~ citro. lnlmunocytochenffstry

Cells were incubated with 4% (w/v) paraformaldehyde for 30 min at 5 C . The remaining steps were carried out at room temperature. The cells were washed with Dulbecco's phosphate-buffered saline (PBS) and incubated with 0.1% (v/v) Triton X-100 (Boehringer Mannheim U.K., Lewes, U.K.) for 15 rain. The cells were subsequently incubated with 1% (w/v) bovine serum albumin dissolved in PBS (BSA/PBS) for l h followed by a 3 h incubation with antibodies to the following antigens: anti-glial fibrillary acidic protein (GFAP, rabbit IgG), 1:50 dilution (v/v); anti-galactocerebroside (mouse hybridoma supernate) applied at a 1:50 (v/v) dilution; anti-cellular fibronectin (mouse monoclonal, ascites fluid) applied at a 1:25 (v/v) dilution; 0 4 and Ran-2 antibodies applied at a 1 : 2 dilution (mouse hybridoma supernates) (generous gifts from Mark Noble's laboratory). Control staining was carried out under identical conditions except that the primary antibodies were replaced with PBS, and in some cases with matching preimmune sera. The cells were washed with PBS and incubated with the corresponding fluorescein isothiocyanate (FITC) labelled antibodies: goat anti-rabbit IgG (whole molecule), goat anti-mouse IgM (N-chain specific) or IgG (Fab specific), and rabbit anti-goat lgG (whole molecule). The secondary antibodies were applied at a 1:30 dilution (v/v) with 1% BSA/PBS for 60 min at room temperature. G F A P was detected using similar conditions but employing the biotin/extravidin-FITC detection system. It should be noted that G F A P fluorescence detection with a simple primary/labelled secondary antibody detection system can be used on these cells, but does not result in full spatial resolution of the filaments. Electron microscopy

Cells were prepared for electron microscopy by standard techniques from separate cultures at 14 and 22 DIV. In brief, cells grown on poly-D-lysine-coated plastic were fixed with glutaraldehyde, washed and incubated with osmium tetroxide followed by washes with ascending grades of alcohol. The monolayer of cells was embedded in epoxy resin and sections were prepared on a Vibratome. The sections were examined with a JEOL 1200 EX electron microscope. We examined about 1600 cells derived from two separate cultures. Single cell microfluorimetry, digital imaging and indo-1 calibration

Microfluorimetric analysis was carried out as previously described. 16 Three or more days before analysis, astrocytes were plated on to glass coverslips with an initial plating density of approximately 7000 cells/cm2. Ceils were washed with a physiological saline containing 156mM NaC1, 1.25mM KH2PO 4, 3 m M KC1, 2 m M MgSO4, 7.5mM HEPES, 2 mM CaCI 2 and I0 mM glucose at pH 7.3. Experiments were typically carried out at room temperature, in the above saline. Rhodamine 123 fluorescence. Cells were loaded with 10#g/ml rhodamine 123 for 10min at room temperature and washed thoroughly. Rhodamine 123 was excited at 490 nm and emitted fluorescence was measured at 530 nm. Indo-1 fluorescence. Cells were loaded with 5 p M of the acetoxymethyl ester of indo-I for 3 0 - 4 0 m i n at room temperature. Indo-I was excited at 350nm, and emitted fluorescence was monitored simultaneously by two photomultiplier tubes at 410 and 490 nm (using 10 nm bandpass filters). For experiments involving digitonin, indo-1 fluor-

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Energy metabolism of adult astrocytes escence was measured using a wide-band interference filter centred at 450 nm ( __+40 nm). Fura-2 fluorescence. Cells were loaded with 5 tt M of the acetoxymethyl ester of fura-2 for 30-40 min at room temperature. Fura-2 was excited sequentially at 340, 360 and 380 nm using a spinning filter wheel (Cairn Research Ltd, U.K.) and emitted fluorescence was measured between 510 and 540 nm using a combination of long and short pass filters. For experiments involving digitonin, fura-2 was excited at 365 nm and emission was monitored between 410 and 540 nm using a combination of long and short pass filters. Calibration of indo-1 ratios. The intracellular calcium concentration [Ca2+]~ as reflected by the ratio of indo-1 fluorescence at 410 and 490nm (excitation at 350nm), was determined by in situ calibration. The Ca2+/Mn 2+ ionophore 4-Bromo-A23187 was applied at 200/~M by a pressurized micropipette to individual cells to determine the ratio at saturating [Ca2+]~ (Rmax).13 The ratio at minimal [Ca2+]i (Rmin) was determined from cells loaded with 50/~M BAPTA-acetoxymethyl (the acetoxymethyl ester form of the calcium chelating buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) incubated in a calcium free physiological saline (supplemented with I mM EGTA) for 45 min. High concentrations of intracellular BAPTA have been shown to be as effective in chelating calcium as microinjection o f EGTA. 5~ Neither de-esterified BAPTA or BAPTA-acetoxymethyl interfered with indo-I fluorescence, lntracellular calcium concentrations were determined from the equation established by Grynkyewicz et al.27: [Ca2+]i = K~([R - Rmin]/[Rmax - R ])( Sf2/ Sb2) using the quoted K a of indo-I of 250 nM, the measured Rmin o f 0.93 + 0.018 (n = 8), the measured RmaX of 6.121 + 0.109 (n = t2), the measured S n (at 490nm) o f 3.299 + 0.216 (n = 8), and the measured Sb2 (at 490 nm) of 0.924 +__0.082 (n = 12). Digital imaging. Digital imaging techniques were applied to identify the distribution of fluorescence within the cells. In this case, the image was projected onto the face of a cooled CCD camera (Digital Pixel, U.K.). Images were acquired and analysed under the control o f software from Kinetic Imaging (Liverpool, U.K.). To gather images of rhodamine 123 and 10-nonyl-acridine orange (NOA; 10min incubation with 0.1 # M ) fluorescence, the cells were excited at 490 nm, and the fluorescence distribution was collected using a long pass filter with a cut-off at 510 nm in front of the camera. To image the distribution of indo-1 fluorescence, indo-I was excited at 350 nm and fluorescence was collected at wavelengths longer than 410 nm. Respiration media. Experiments with digitonin (monitoring fura-2, indo-I or rhodamine 123 fluorescence) were carried out in a medium containing 0.25M sucrose, 2raM HEPES, 2.5mM o f both malate and pyruvate, 100#M A D P and 1 mM EGTA at pH 7.2. Experiments with N O A were carried out in a medium containing 100 m M NaCI, 75mM mannitol, 2 5 m M sucrose, 10mM phosphate-Tris, 10 mM Tris-HC1, 50/~M EDTA, 5.5 mM glucose, pH 7.3. 6 Lactate experiments and determination

Astrocytes were plated into plastic wells at a plating density of about 3000 cells/cm2 (16,000 cells per well) and incubated for 48 h in I ml o f the supplemented L-valinebased M E M with and without the addition o f several concentrations of antimycin. The initial glucose concentration of the medium was 5.56 mM. After 48 h the medium was removed and deproteinized with 0.8 M perchloric acid. Lactate concentrations were determined by the method of Guttmann and Wahlefeld 29 adapted as a fluorimetric assay for the Cobas Fara (Roche, U.K.) centrifugal analyser. Experimental measurements were corrected for lactate endogenous to the medium (0.8 mM).

Ox)'gen consumption measurements

The measurement of oxygen consumption rates was performed at 30:C using a 250ttl capacity incubation chamber with a water jacket, a Clark electrode (Yellow Springs Instrument Co., OH, U.S.A.) fitted into the top o f the chamber, a biological oxygen monitor (Yellow Springs Instrument Company, OH, U.S.A.), and a chart recorder. The electrode (oxygen sensor) was calibrated using air-saturated water, assuming an oxygen concentration of 440 ng atoms oxygen per ml at 30°C. Astrocytes were trypsinized and resuspended in a 100 mM NaC1 based respiration medium (see above) and kept on ice until analysis. One to two million cells were used per 250/~1 incubation medium. The incubation mixture was constantly stirred. BSA was added to the incubation medium at the start of the experiment to a final concentration of 0.5mg/ml. Various mitochondrial substrates and inhibitors (see Results section) were sequentially added to the incubation medium. Postanalysis cell viability was greater than 80%. hTtracellular adenine nucleotide analysis by high-performance liquid chromatography

Cells were incubated under conditions similar to those described for the oxygen consumption measurements. Sodium succinate was added to the respiration medium to a final concentration o f 1 mM. Incubations were stopped after 10rain by precipitation with 9.5 M perchloric acid, followed by neutralization with 1 M K2HPO 4. The supernatants were frozen at - 7 0 ° C until analysis. Nucleotides were separated using an isocratic ion pair reversed-phase method modified from Perret et al. 57 A Beckman System Gold was used and the separation was performed at room temperature with an Anachem $50D2 column (250 × 4.6 mm) and UV detection at 254 nm. The mobile phase consisted of 85 mM triethylamine, pH 6.0 (neutralized with H3PO 4 and 2% (v/v) methanol), the flow rate was 1 ml/min. Assignment of adenine nucleotide (ATP, A D P and AMP) peaks and calculation of absolute concentrations (by integration of peak areas) were obtained by comparison with known concentrations o f external standards. The limit of detection using this method is 10 pmol. RESULTS Characterization of adult astrocytes

A t 14 D I V , all cells were fully differentiated a n d exhibited both protoplasmic, fibrous and intermediate (including b i p o l a r ) m o r p h o l o g i e s (see Figs 1, 2). 79 T h e r e was little cell division at this time o r t h e r e a f t e r since only o n e o f 1600 cells e x a m i n e d by e l e c t r o n m i c r o s c o p y at 14 D I V w a s f o u n d to c o n t a i n c h r o m a t i n in a m i t o t i c p h a s e . E l e c t r o n m i c r o s c o p y f u r t h e r m o r e revealed (Fig. 1) nuclei c o n t a i n i n g h o m o g e n e o u s c h r o m a t i n a n d a d e n s e rim a r o u n d t h e nuclear membrane. The cytoplasm and astrocytic f o o t p r o c e s s e s c o n t a i n e d easily identifiable s t r a n d s o f glial filaments, with b u n d l e s o f f i l a m e n t s m o s t l y f o u n d n e a r the cell m e m b r a n e . T h e a b o v e f e a t u r e s were p r e s e n t in m o r e t h a n 9 5 % o f the cells e x a m i n e d a n d are c h a r a c t e r i s t i c o f a d u l t a s t r o c y t e s in vitro a n d in vivo. ~4 M i t o c h o n d r i a a n d e n d o p l a s m i c r e t i c u l u m ( E R ) were f o u n d t h r o u g h o u t the c y t o p l a s m . T h e spatial r e l a t i o n s h i p s a n d relative a b u n d a n c e o f E R a n d m i t o c h o n d r i a varied c o n s i d e r ably f r o m cell to cell. M o r e t h a n 9 5 % o f t h e cells s h o w e d d e t e c t a b l e levels o f G F A P i m m u n o f l u o r e s cence with o c c a s i o n a l cells ( < 5 % ) s h o w i n g s t r o n g

Fig. 1. 858

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Fig. 2. GFAP immunofluorescence in adult astrocyte cultures at 22 DIV (magnification: x 25). Glial filaments are readily identified throughout the cytoplasm. Note that the antibody appears to cross react with material in the nucleus. immunofluorescence (Fig. 2). There was no immunoreactivity towards the 0 4 and Ran-2 antigens, markers for immature astrocytes. 9 At the same time there was no evidence for any immunoreactivity to fibronectin, a marker for fibroblasts and galactocerebroside, a marker for oligodendrocytes. Removal of serum from the growth medium or increasing the intracellular c A M P (adenosine 2':3'-cyclic monophosphate) concentration by incubating the cells with adenosine (100/~M), 8-Br-cAMP (1 m M ) or forskolin (7/~M) for 2 h at 37°C resulted in the conversion of polygonal astrocytes from flat to more stellate forms (results not shown). The latter observation has been described by Schwartz and Wilson 71 in adult astrocytes and by numerous groups in neonatal astrocytes.34"36.79

Control of respiration by mitochondrial substrates and inhibitors Cell metabolism and its modulation are central to the cellular economy. Chances of cell survival during

acute or chronic changes in its environment will be dictated largely by the available metabolic machinery. We therefore first examined the status of aerobic metabolism using single cells loaded with rhodamine 123, a fluorescent dye that localizes to mitochondria in response to mitochondrial membrane potential (AqJm), and once accumulated its fluorescence reflects the relative mitochondrial membrane potential. 1°'15'23'33 Digital imaging o f rhodamine 123, showed a very discrete distribution of the dye, clearly staining rod-shaped structures (Fig. 3). Fluorescence intensities in the nucleus were minimal. This discrete distribution was present in all cells examined (n > 100, representing experiments carried out on at least 10 separate cultures). We subsequently examined the control of mitochondrial respiration by assessing changes in A~bm in response to various modulators applied to single cells loaded with rhodamine 123. Since the accumulation of rhodamine 123 within mitochondria leads to

Fig. 1. Morphology of three individual (A, B, C) adult astrocytes at 22 DIV. (A) Predominance of bundles of glial filaments near the cell membrane (magnification: x 10,000). (B) Predominance of single strands of glial filaments in the cytoplasm (magnification: x 10,000) and abundance of ribosomes (including the ER; evident as black dots) and mitochondria (bottom left). (C) Rim of dense chromatin around the nucleus, but homogeneous within. Mitochondria, ribosomes and bundles of filaments within the cytoplasm (magnification: x 7500).

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A

B

Fig. 3. Digital imaging of single cell rhodamine 123 fluorescence (A) and corresponding phase microscopy view (B) (magnification: x 60).

autoquenching of fluorescence, the redistribution that accompanies depolarization of A~bmis associated with an increase in the fluorescence signal. 7'16 A period of anoxia (Fig. 4A; n = 5; or sodium cyanide, not shown), or application of the uncoupler p-trifluoromethoxy-phenylhydrazone (FCCP); (Fig. 4B) resulted in predictable changes in the ml]/m(result shown is representative of experiments on five separate cultures (n i> 30)), consistent with the chemiosmotic theory. Figure 5 illustrates the imaging correlate of the microfluorimetric data. Figure 5A shows the localized discrete distribution of the rhodamine 123 fluorescence in the resting cell. Following application of FCCP (Fig. 5B), the fluorescence redistributed to fill the cell diffusely, and the total rhodamine 123 fluorescence increased by 68.4%. Results shown are representative of two cultures (n = 8). Control of mitochondrial respiration was also assessed by monitoring oxygen consumption in suspensions of intact cells in response to a range of substrates and inhibitors of each complex of the mitochondrial respiratory chain. Table 1 shows the results of these experiments. The basal rate of oxygen consumption was established at the start of each experiment. Malate combined with either pyruvate or glutamate did not cause a significant rise in oxygen consumption, which suggests that the basal rate consists in part of endogenous consumption of these substrates. Moreover, malate and L-glutamine were both present in the growth medium at millimolar concentrations, and may thus have already been present at saturating intracellular concentrations at the start of the experiment. Rotenone, an inhibitor of

mitochondrial Complex I reduced the basal oxygen consumption to zero, further confirming that the initial basal rate of oxygen reflects consumption of nicotinamide adenine dinucleotide-linked endogenous substrates. In contrast, succinate and N , N , N ' , N'-tetramethyl-p-phenylenediamine (TMPD) and ascorbate caused a near trebling of oxygen consumption compared with the initial basal oxygen consumption rate. As expected, antimycin, an inhibitor of Complex III, and cyanide, an inhibitor of Complex IV, reduced the existing respective oxygen consumption rates to near zero. Since succinate caused a constant oxygen consumption rate over at least 10 min we used similar conditions for examining the changes in total cellular adenine nucleotide concentrations in response to the addition of 1 mM succinate (final concentration) to the incubation medium. Addition of 1 mM succinate resulted in an ATP production of 255 _+ 74 pmol/10 min per 1 × 106 ceils (n = 3). The calculated energy charge after 10 rain was 0.66 + 0.03 (n = 3). We further assessed the contribution of mitochondrial metabolism to the overall metabolism of the cell by inhibiting mitochondrial respiration with antimycin and monitoring the lactate released from the cell into the culture medium. The optimum concentration of antimycin (greatest increase in lactate release without cell death) was 3/tM (10 and 1/~M were also tested). In the absence of antimycin, basal lactate release into the culture medium was 92.0 _+ 5.3 ~tmol/48 h per mg protein (n = 18) or 1.92 + 0.11/~mol/h per mg protein. This correlates well with published measurements of lactate release in the neonatal astrocyte of 1.42 and 2.0 pmol/h per mg protein by Pauwels et al. s6 and Walz and Mukerji, s2 respectively. The addition of antimycin resulted in a lactate release of 157.8 + 7.4/tmol/48 h per mg protein (n = 8 ) or 3.3+0.16/~mol/h per mg protein comprising a 171% increase over basal lactate release, which compares reasonably well with the results of Pauwels et al. 56 in neonatal astrocytes, who reported a 260% increase [but used 10% (v/v) fetal bovine serum based Dulbecco's modified Eagle's medium]. Role o f cytosolic calcium in the control o f mitochondrial respiration

In several cell types a signalling pathway has been established by which changes in cytosolic calcium lead to changes in mitochondrial calcium, with subsequent activation of the three key rate limiting enzymes of the Krebs cycle, leading to an increase in respirationJ 5"2s'3°'46'65'66 We therefore wished to determine whether such mechanisms operate in (adult) astrocytes. Changes in calcium are easily followed with calcium indicators, and changes in respiration can be followed by changes in the mitochondrial membrane potential which reflects mitochondrial respiration, as it forms part of the proton-motive force generated by the electron

Energy metabolism of adult astrocytes transport chain. However, correct interpretation of the experimental data are contingent upon verification that the [Ca2+]~ measured with fluorescent calcium indicators (indo-1 or fura-2) actually represents the free cytosolic calcium concentration, and is not unduly contaminated by signals from other cellular compartments which sequester the dye. 44'49'67 Significant accumulation of the dye into the ER would result in large systematic errors in presumed cytosolic calcium measurements as significant amounts of calcium are released from the ER into the cytosol when various signalling pathways are activated (see also accompanying paper). Substantial accumulation of

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calcium indicator into the mitochondria would prevent correct interpretation of the temporal changes of the parameters to be examined (see below). Compartmental distribution of indo-1 and fura-2 Roe et al. 67 have shown that low concentrations of digitonin (~< 20/2M) permeabilize the plasma membrane, whereas high concentrations (of the order of 100 # M) permeabilize all cell compartments except the mitochondria. Selective use of this detergent thus allows determination of the compartmental distribution of a particular dye. We therefore monitored indo-1 and fura-2 fluorescence by microfluorimetry

11

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Anoxia 60

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Fig. 4. Modulators of the AOm.Representative microfluorimetric results are shown. (A) A short period of anoxia (indicated by black bar) leads to a reversible increase in rhodamine 123 fluorescence.(B) FCCP application (5/~M; indicated by open bar) leads to a reversible increase in rhodamine 123 fluorescence. Rhodamine 123 fluorescence shown was.monitored in single and separate cells.

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A

B

C

Fig. 5. Effect of FCCP on distribution of rhodamine 123 fluorescence. (A) Digital image of single cell rhodamine 123 fluorescence before FCCP application. (B) Digital image of rhodamine 123 fluorescence (of the same cell) recorded 12 s after a 2 s application of FCCP (5 pM). Exposure times for A and B were identical. (C) Corresponding phase microscopic view (magnification: x 40). before and after application of 2 0 p M and 1 0 0 p M digitonin, respectively. The cellular compartmentation of indo-I and fura-2, respectively, was as follows ( m e a n + S.E.M.): cytosol, 89.1 + 1.1% (n = 4), 80.4 + 2.3% (n = 6); mitochondria,

et al.

4.7_+0.6% ( n = 6 ) , 11.6+0.6% (n=ll). From the difference between the residual cytosolic and mitochondrial fluorescence the extramitochondrial/non-cytosolic compartmental uptake was calculated to represent 6.2 and 8.0%, respectively. Some variation was observed in compartmental uptake between batches of cells, but the total non-cytosolic compartmentation never exceeded 20%. Digital imaging of total indo-1 fluorescence showed an initial fluorescence distribution that was relatively uniform across the cell, appearing brightest in the centre of the cell where the cell has the greatest volume (Fig. 6A). Application of digitonin (100/~M) resulted in a 90% loss of total fluorescence (Fig. 6B), with the remaining dye locally and discretely distributed within the cytosol, but excluded from the nucleus, resembling the rhodamine 123 fluorescence distributions shown in Figs 3A and 5A. Results shown are representative of four separate experiments. The above observations suggest that a uniform appearance of fluorescence across the cell is not sufficient to conclude that the distribution of the particular dye is purely cytosolic. We independently verified that digitonin did not permeabilize the mitochondria by incubating cells with 0.1/~ M N O A (fluorescence spectrum identical to rhodamine 123) for 10 min. N O A has been shown to be taken up into mitochondria independent of the AI//m .43'59 N O A fluorescence was largely unaffected by brief applications of digitonin up to 1 m M [concentrations tested: 1 m M (n = 3), 100 ktM (n = 3) and 20/~ M (n = 3)]. Application of Triton X- 100 (0.1%, v/v), which fully permeabilizes the mitochondria, resulted in a full loss of N O A fluorescence (n = 4; results not shown). In summary, we have shown that more than 80% of indo-1 and fura-2 fluorescence is localized to the cytosol, roughly 10% localizes to the mitochondria, and another ! 0% localizes to the non-cytosolic/extramitochondrial sites such as the E R and the lysosomes. We can therefore reasonably accept that the [Ca:+]i measured with these dyes in these cells primarily reflects the cytosolic free calcium concentration.

Table 1. Oxygen consumption of adult astrocytes in response to various mitochondrial substrates and inhibitors Additions None Pyruvate (5 mM) and malate (2.5 mM) Glutamate (10 mM) and malate (2.5 mM) Rotenone (5 #M) and succinate (10mM) Antimycin (10/~M), TMPD (200tiM) and ascorbate (1 mM)

02 consumption (ng atoms/min/1 × 106cells) 0.63 + 0.09 (n = 7) 0.92+0.19 ( n = 4 ) 0.76+0.17 ( n = 3 ) 1.99 + 0.07* (n = 5) 1.92 __+0.21" (n = 4)

Cells were incubated sequentially with pyruvate and malate or glutamate and malate, followed by rotenone, succinate, antimycin then followed by TMPD and ascorbate. Typical recording time was 25 mins. Time between additions was 2-3 min. Data were derived from three separately developed and analysed preparations of adult astrocytes. *Statistically significantly different (P < 0.001) compared with no additions.

Energy metabolism of adult astrocytes

863

A

B

Fig. 6. Digital imaging of indo-I fluorescence before (A) and after (B) digitonin application (100/~M; magnification: × 40). Exposure times for A and B were identical.

Changes in [Ca2+]i in response to known modulators

The study of the relationship between [Ca2+]i and control of mitochondrial respiration furthermore requires a tool which reversibly and reliably modulates [Ca2+]i. Changes in [Ca2+]i in response to potassium, glutamate, noradrenaline, isoproterenol, ATP and mechanical stimulation (touching of the cell membrane with a micropipette) have all been documented in cultured neonatal astrocytes. 32 We thus tested these modulators on adult astrocytes. In brief, calcium changes in response to K + were highly variable between cells and between preparations (38 out of 63 cells tested responded, representing eight cultures). Most cells responded to 1 mM L-glutamate with changes in [Ca2+]~ (25 out of 34 cells tested responded, representing six cultures), but responses were less frequently seen when agonist concentration was decreased to 100pM (19 out of 32 cells tested responded, representing five separate cultures). Noradrenaline (100/t M) caused only small ( < 500 nM) rises in [Ca2+]i in some cells (19 out of 34 cells tested responded, representing four cultures),

whereas isoproterenol (100/~M) caused large rises (up to l p M ) in [Ca2+]i in most cells (eight of 10 tested, representing two cultures; results not shown). Both ATP (10-500 #M; see accompanying paper for dose-response relationship) and touch resulted in predictable and large increases in [Ca2+]i in all cells (45 cultures tested, n > 100) and were thus used to raise [Ca2+]i in subsequent experiments (see below). Relationship respiration

between

[Ca2+]i and

mitochondrial

The next step in this study was to assess whether changes in [Ca2+]~ produced by ATP or touch are sufficient to modulate mitochondrial respiration. Initially, preliminary experiments were carried out by simply loading cells with rhodamine 123 and applying ATP. Comparison of Fig. 7A and B, shows that A T P induced changes in AqJmappear to parallel changes in [Ca2+]i in shape and magnitude (albeit in an identical experiment on a separate cell). Of 33 cells tested, 14 responded with a significant change in rhodamine 123 fluorescence in response to ATP (in experiments on three separate cultures).

864

S. Peuchen

Since we determined the uptake of calcium indicators into the mitochondria to be minimal (see above), we were able to accurately examine the kinetics of the changes in calcium and mitochondrial potential by loading single cells with both fura-2 and rhodamine 123. Figure 8A shows that application of ATP results in reversible changes in [Ca2+]~, that are immediately followed by reversible changes in the A0m. Although 12 of 12 cells showed substantial rises of [Ca2+]i in response to ATP, only two of the 12 cells showed a subsequent change in the rhodamine 123 signal. The small proportion of cells showing concurrent changes in rhodamine 123 in these experiments may reflect buffering of the cytosolic calcium by fura-2. :4 Any calcium buffering would therefore have

3

m

et al.

to be overcome before substantial mitochondrial uptake would occur. In contrast, when cells were mechanically stimulated (Fig. 8B), all cells (three separate experiments, n = 12) responded with a reversible change in [Ca2+]i, which was immediately followed by a reversible change in A~bm. The latter observation may be explained by the observation that touch promotes calcium influx, and consistently raises the [Ca2+]i to micromolar levels, thus saturating the calcium buffering capacity of fura-2, leaving excess cytosolic calcium available for mitochondrial uptake. So far we have demonstrated a direct association of changes in calcium with changes in A~bm, at least in a proportion of cells. The question arises whether the

..

m

A

O

E o O

E

2

8 O

i

I

240

I

480 Time (seconds)

72O

B

180

140

g 100

60

m

m

m

20 0

240

480 Time (seconds)

720

Fig. 7. Modulation of [Ca2+]i and A~bm by consecutive applications of ATP. (A) Changes in [Ca2+]i in response to consecutive applications of ATP (100 #M; indicated by black bars). (B) Changes in A~bm in response to consecutive applications of ATP (100 #M; indicated by black bars). It is important to note that the representative results shown were recorded from single and separate cells by microfluorimetry, using indo-1 and rhodamine 123, respectively. Note the gradual decline in baseline which may reflect bleaching of fluorescence, and/or loss of dye from the cell.

Energy metabolism of adult astrocytes

865

Rh 123 fluorescence (%)

Fura-2 ratio

A

4

.19o

~ -2

Rh 123

-170 -150 -130 -110 -90

!~!!!!!!!!iiiii~ii~

ATP

70 6o

Time (seconds)

Fura-2 ratio

Rh 123 fluorescence (%)

B

195

Fura-2

.145

,

i

0

,

i

30

,

60

i

,

90

I

J

9 5

120

"lqme (seconds)

Rh 123 fluorescence (%)

Fura-2 ratio

-

' 3

C ~

120

110

2 1

100 t

0 . 0

130

i 4-Br-A23"I87

9O 3O

60

9O

120

150

Time (seconds) Fig, 8. Concurrent monitoring of [Ca2+]i and A ~ , in response to various stimuli. (A) Application of ATP (10pM; indicated by hatched bar). (B) Mechanical stimulation (indicated by vertical black bar). (C) Application of 4-Br-A23187 (50/~M; indicated by open bar). The representative results shown were recorded from single and separate cells by microfluorimetry, using fura-2 and rhodamine 123.

866

S. Peuchen et al.

rise in cytosolic calcium modulates z~bm, or whether it plays a secondary role to some other signal (such as sodium). ATP application in zero calcium (replaced by equimolar concentrations of magnesium, and supplemented with 1 mM EGTA) in rhodamine 123-loaded cells resulted in a further reduction of both the relative magnitude of changes seen as well as the number of responding cells (three out of 14 cells responded), suggesting that calcium plays a direct role (results not shown). The effect of touch on the potential was clearly mediated by calcium since touch failed to elicit any change in either [Ca2+]i or rhodamine 123 fluorescence following the removal of calcium from the medium (n = 4). The mechanism of touch-induced calcium influx is likely to be explained by the presence of stretch activated nonselective cation channels on the outer cell membrane of astrocytes. 55The direct involvement of calcium was furthermore demonstrated by the effect of the calcium ionophore 4-Br-A23187 ( n = 5 ) , which caused changes similar to that of touch, as shown in Fig. 8C. It is important to empirically establish the correct concentration of this ionophore for the latter response to occur, as increasing concentrations lead to a fully calcium permeant cell (such as used in this study for the calibration of indo-1 fluorescence) followed by a continuous depolarization of the AOm, whereas at even higher concentrations the protonophoric properties of 4-Br-A23187 cause the loss of dye from the mitochondria and also from the cell. 48

DISCUSSION Characterization o f cultured adult astroo'tes

The astrocytic nature of the isolated and examined cultures can be derived from the following observations: (1) expression of glial filaments (shown by electron microscopy and immunofluorescence). The relatively weak expression of GFAP (compared with neonatal astrocytes) by immunocytochemistry has been observed in many in vitro studies of adult rat and human astrocytes, and correlates well with numerous in vivo studies. Strong expression of G F A P in vivo is typically only seen in selected areas of the brain (striatum, glial limitans) or upon injury; 2"3"2°'31A2"sS"69"7a'89(2) morphological features of the nuclei of the cells; and (3) metabolic (high LDH levels) and physiological/pharmacological evidence (see also accompanying paper). It is likely that some of the cells in our cultures were mature adult mitotic astrocytes in vivo but a lack of specific markers for the identification of this population of cells precludes any further comment on this matter. Contamination of these cultures with adult progenitor cells s3`Ss'86 at the time of analysis is unlikely since, (1) there was little evidence for any cell division, a defining characteristic of any stem cell population, as evidenced by visible light and electron microscopy, (2) the cultures stained negative for antibodies against the 0 4 and Ran-2

antigens,4° antigens that are present on the adult progenitor cells derived from the optic nerve, (3) light and electron microscopy showed fully differentiated cells, with clearly identifiable structures, and the absence of any large uncharacterized dense bodies or a euchromatic nucleus, I'~'a°79 and finally (4) the cells showed metabolic and physiological properties consistent with mature cells. Control o f rnitochondrial respiration by substrates and inhibitors

Juurlink and Hertz 35 concluded from their studies that rhodamine 123 was a reliable marker of the viability of neonatal astrocytes. They established an inverse relationship between rhodamine 123 accumulation and lactate dehydrogenase release from the cell. The authors found that full dissipation of the mitochondrial membrane potential was required for propidium iodide entry to occur. Our present results confirm their study by showing that all adult astrocytes analysed stained with rhodamine 123, thus exhibiting a mitochondrial membrane potential, consistent with an active aerobic metabolism. We extended their observations by showing that the potential can be predictably manipulated, in accordance with the basic principles of the chemiosmotic theory. Edmond et al., 21"2"- have described the developing neonatal astrocyte as being metabolically "multifunctional", since it can adapt to changes in its environment by using alternative substrates for aerobic metabolism. In our study we have identified succinate as a metabolic substrate (TMPD as a non-metabolic substrate), that stimulates respiration, leading to a transient increase in ATP production. The immediate uptake of succinate (a negatively charged molecule) into the cell suggests that there must be an active carrier for this substrate. One group of investigators explored the differences in substrate (glucose, glutamine and ketone bodies) utilization between adult and developing brain cells and homogenates, 68"77 but were unable to reach firm conclusions about the nature of these differences, which may be explained by the complexity of the preparations used. However, since the glial cell is the most common cell type in the CNS, one might assume that the results of their study largely reflect substrate oxidation by glial cells. The above authors did, however, confirm the general notion that the metabolism of the adult brain cell can be supported by many substrates other than glucose. The second aspect of "multifunctionality" relates to the ability of the astrocyte to switch to an increased anaerobic glycolysis if the mitochondrial respiratory chain activity is limited. This mechanism appears to also operate in the adult astrocyte, since compromising mitochondrial respiration (with antimycin) led to an increased anaerobic metabolism by the cell in order to maintain its energy demands. The pathophysiological importance of this compensatory

Energy metabolism of adult astrocytes mechanism is illustrated in a study of neonatal astrocytes, where nitric oxide was shown to mediate inhibition of the mitochondrial respiratory chain, leading to an enhancement of anaerobic metabolism,4 in an attempt to maintain existing energy levels. It appears paradoxical that astrocytes (both adult and neonatal), with functional mitochondria, whose respiratory chain activity can be easily upregulated for increased energy demands, keep using their anaerobic pathways (as indicated by the high basal lactate release levels) for ATP production to a large extent. Several observations may explain this discrepancy. The unusually high basal lactate release in astrocytes 41's6'82is reflected by the high endogenous activities of LDH reported in astrocytes and to some degree in neurons.38'41'52 Recent studies have shown that both astrocytes and neurons are able to use lactate as a metabolic fuel. The continuous production of lactate would therefore ensure a readily available source of energy upon re-oxygenation after a period of anoxia or ischaemia, when glucose supplies may not yet be replenished) 4'75'76 Role o f cytosolic calcium in mitochondrial metabolism

In this study we have shown that ATP and touch raise [Ca2+]i consistently and reliably, and for the first time show that in astrocytes these manoeuvres may also lead to a depolarization of the mitochondrial membrane potential that closely follows the change in [Ca2+]~ and whose magnitude seems to reflect the amplitude of the change in [Ca2+]~. Our data are consistent with the proposed signalling pathway, where changes in cytosolic [Ca2+]i, and thus changes in energy demands, are met by appropriate changes in energy production. The above mechanisms could thus serve as part of the maintenance of the overall energy metabolism of the astrocyte during normal physiological conditions) 8'3°'45'46'65,66 However, a discrepancy in our data remains, since in some cells exogenous ATP-mediated changes in calcium did not result in any change in the mitochondrial membrane potential, whilst touching (or applying 4-Br-A23187) the same cell did result in changes in potential. There are several possible explanations for the above discrepancy. In a separate study (see accompanying paper) we have detailed the mechanisms by which calcium is released into the cytosol in response to exogenous nucleotides such as ATP. The latter study suggests that nucleotide receptor activation leads to the release of calcium from the ER, which is followed by re-uptake of the calcium into the ER. There was no evidence for subsequent influx of

867

extracellular calcium. Several elegant studies by Rizzutto et al. 65"66 have suggested the existence of "microdomains" of high calcium, consisting of local areas of high calcium between the ER and mitochondria upon receptor stimulation. The lack of mitochondrial changes seen in a subpopulation of cells in response to ATP may therefore be due to a heterogeneity of structure and/or function of these microdomains in a particular population of these cells, and is consistent with the observations made by electron microscopy. The mostly uniform changes seen in transfected cells (used in part by Rizzutto et al.) may therefore not necessarily apply to cells in primary culture. Secondly, the discrepancy described above may be due in part to differences in the activities of the ER-Ca2+ATPase. A certain population of cells may have pumps that are more easily saturated, allowing excess calcium to enter neighbouring mitochondria, whereas in other populations of cells large quantities of calcium may be rapidly pumped back into the ER, minimizing any substantial mitochondrial uptake. 3°'5° In the case of external calcium influx, such as occurs with touch, the activity of the ER-Ca2+ATPase pumps should be minimal as the ER stores remain filled. The accumulated calcium is therefore readily available for uptake into the mitochondria and stimulation of respiration.

CONCLUSION

In summary, we have provided evidence that adult astrocytes have functional aerobic energy pathways stimulated by glucose, various metabolites as well as free cytosolic calcium, coupled with a highly active anaerobic pathway. The multifunctional adult astrocyte would therefore appear to be uniquely versatile, capable of dealing with acute and dramatic changes in the energy metabolism of the brain. Acknowledgements--We thank Mark Noble (Ludwig Insti-

tute for Cancer Research, London) for the helpful suggestions regarding the characterization of our cultures and the generous gift of several antibodies, Dr J. P. Bolafios (Department of Neurochemistry) for the oxygen electrode studies, Mr B. Young and Prof. D. N. Landon (Institute of Neurology) for the processing and interpretation of the electron microscopic data, Dr S. Davies (Department of Neurochemistry) for the adenine nucleotide analysis, and Dr T. Briddon (Department of Chemical Pathology) for the lactate determinations. This research was funded in part by the Medical Research Council (U.K.) and the Brain Research Trust (U.K.).

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