Dynamic Pacing of Cell Metabolism by Intracellular Ca2+ Transients*

THEJOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 44, Issue of November 4, pp. 27310-27314, 1994 Printed in U.S.A.

Dynamic Pacing of Cell Metabolism by Intracellular Ca2+ Transients* (Received for publication, June 22, 1994, and in revised form, July 28, 1994)

William-F. PralongS,Andras Spat#, andClaes B. WollheirnS From the Division of Clinical Biochemistry, Department of Medicine, University of Geneva, Centre Mkdical Universitaire, CH-1211 Geneva 4 , Switzerland a n d the $Department of Physiology, Semmelweis University Medical School, H-1444 Budapest, Hungary

During cell activation, Ca2+, by stimulating the NADH- chondrial metabolism of nutrient stimuli or directly from elecproducing mitochondrial dehydrogenases, triggers the tron donors to the respiratory chain was shown to ATPcontrol generation of reducing equivalents whereby ATP pro- sensitive K' channels (81, whose closure promotes membrane duction is sustained. In cell populations, [Ca2+] changes potential depolarization and Ca2+ influx during insulin secrein the mitochondrial matrix were demonstratedto par- tion from the pancreatic p-cell (10). Another example is the allel rapidly thosein the cytosol ([Ca*+],). There is still no Ca2+-induced mitochondrial NADH formation that supports the indication as to whether metabolic activation follows transhydrogenase-catalyzed NADPHformationrequiredfor oscillatory patterns similar to those of [Ca"Ii. Therefore, steroidogenesis in adrenal cortical cells(7). There is little indichanges in NAD(P)H were monitored in single pancre- cation as to whether regular [Ca2+],transients can pace correatic pcells, adrenal glomerulosa cells, and liver cells sponding metabolic oscillations. In a previous study in single duringoscillatory[Ca2+l,transients.Rapid NAD(P)H primary p-cells (61, we have shownthat NAD(P)H fluorescence and [Ca2+lioscillations with similar frequency andsenincreases beforeCa2' entry in response to glucose or 2-ketoisositive both to changes in glucose concentration and to caproate stimulation,thus demonstrating that glucose or2-keextracellular Ca2+ removal were identified in a subpopulation of pancreatic p-cellsin primary culture. Further- toisocaproate metabolism precedes membrane depolarization more,Ca2+-dependentoscillatory NAD(P)H formation and gating of voltage-sensitive Ca2+ channels.In the present could be evokedby the pulsatile application of depolar- study, to delineate the pattern of metabolic activation caused izing [K'], demonstrating the pacing effect of increased by oscillatory [Ca2+],increases during the sustained phase of p-cell stimulation at intermediate glucose, NAD(P)H fluores[Ca2+],onp-cellmetabolism. In adrenalglomerulosa cells, angiotensin 11, a physiological stimulator of aldo- cence and [Ca2+l,were measured at the single cell level. Furin sterone production, could be shown to elicit the oscilla- thermore, the metabolicpatterns of responseobtained of adrenal glomerulosa cells and tory formation of mitochondrial NAD(P)H through fre- p-cells were compared to those quency modulation of [Ca2+], transients.In contrast to livercells following stimulation with physiologicalagonists the twoformerendocrine cell types, in hepatocytes, that also cause intracellular [Ca2+litransients. [Ar$]vasopressinandepinephrinecausedtheamplitude modulation NAD(P)H of formation. Taken together, MATERIALS AND METHODS these results provide unprecedentedevidence for acellCell Culture and FluorescenceMicroscopy-Dispersed rat islet p-cells transients coor- (6), adrenal glomerulosa cells (7), and hepatocytes (11)were isolated specific pacing of metabolism by [Ca2+], dinated withcell activation and function. and cultured on coverslips as already described. For p-cells,the glucose concentration in the medium wasdecreased from 8.3 to 5 m~ 16 h before the experiments. All cells, with or without prior incubation with fura-2 Many cellular functions suchas hormone secretion and con- acetoxymethyl ester (250 XIM for20min in culture medium),were traction are driven by oscillatory changes in cytosolic calcium mounted in a recording chamber at 37 "C and continuously superfused with a modified Krebs-Ringerbicarbonate solution buffered with Hepes concentrations ([Ca2+li).l Duringcellactivation,increased (10 mM) containing 142 mM Na', 3.6 m~ K', 1.5 mM Ca2+,0.5 mM M e , [Ca2+],is followed within seconds by intramitochondrial CaZ+ 2 m~ HCO,, 145 mM C1-, and 0.5 m~ PO;. Stimuli were delivered as elevation ([Ca2+l,) (1-4). One of the consequences of increased square pulses at final concentrations through microsyringe pumps con[Ca2+], is the stimulationof NADH formation by Ca2+-sensitive nected by polyethylene tubing to two wide-mouthed micropipettes. mitochondrial dehydrogenases (5). In this way, the mitochon- These were positioned close to the cells using side-arm micromanipudrial pyridine nucleotide redox state is adjusted to increased lators (6). Only individual cells not in contact with adjacent cells were analyzed. When the number of [Ca2+],spikes were counted, in order to ATP production by the respiratory chain, thus balancing en- be considered as one oscillation, a transient had to be deflected from ergy demand.In particular, the so-formed NADH can also probasal levels to above 250nM and then returned to basal [Ca2+ljvalues. the The NAD(P)H recordings are expressed as percent of the normalized mote the ATP and/or reducing equivalents essential for metabolic coupling governing specific cellular functions (6-9). initial ratio (1 = 100%) of the signals recorded at 470 nm following excitations at 360 and 390 nm as already described (Ref. 7; see also Fig. Hence, enhanced ATP production resulting either from mito2C). It should be mentioned that both lengthy recordings and detection of oscillatory patterns in the different cell types were possible only when * This work was supported by grants from the Swiss and Hungarian UV excitation was minimized. Indeed, if intense UV illumination was National Science Foundations. The costs of publication of this article applied, NAD(P)H signals decayedexponentially, and onlypoor rewere defrayedin part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 sponses could be obtained. This probably reflects cell injury caused by UV irradiation. U.S.C. Section 1734 solely toindicate this fact. Combined Measurement of NAD(P)H and [Ca2+ljin Single Adrenal $ To whom correspondence and reprint requests should be addressed. Glomerulosa Cells and in Single Hepatocytes-In contrast to p-cells, Tel.: 41-22-70-25-554; Fax: 41-22-702-55-43. The abbreviations used are: [Ca2+li,cytosolic calciumconcentration; NAD(P)H and fura-2 fluorescence can be recorded simultaneously at [Ca2+],, intramitochondrial calcium concentration;MI, angiotensin 11; 470nm in fura-2-loaded glomerulosacells and hepatocytes. Thus, [Ca''], changes can be monitored by exciting the cells at 380 nm (FS8J IP,,inositol 1,4,54risphosphate; AVP, arginine vasopressin.

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Dynamic Pacingof Cell Metabolism and NAD(P)H with an excitation at 360 nm (F,,,), taking advantage of the isosbestic point of fura-2, which is not sensitive to [Ca"], (12). However, the F36a signal also reflects the kinetics of leakage and of photobleaching of the Ca2+indicator. Therefore, in long recordings, which resulted in the almost complete lossof the indicator (see Fig. 21, the contaminating fura-2 component was estimated in order to be subtracted. A good fit of the latter was obtained with a double exponential equation using the F380signal as reference: Y = (a, A e-k1t + a, A + B , where t is time, A corresponds to F380,0- B, and B = the estimatedF,,, after complete lossof fura-2; a, = 0.13 and 0.128, a, = 0.87 and 0.872, k , = 0.005 and 0.008,and k, = 0.001 and 0.0008 werethe best estimates for the double exponentials, indicated by asterisks in Fig. 2 (A and B , respectively). Thus, the fura-2 contamination could be removed from the F3MI signal by dividing directly F360by Y , giving NAD(P)H in arbitrary units (see Figs. 2 and 3). The traces indicated as [Ca2+li(Figs. 2 and 3) correspond to the YIF,,,, ratio. As a control, only solutions reasonably close to the truekinetics of fura-2 disappearance permit, at the same time, normalization of NAD(P)H and [Ca2+libase lines. When calculated, data are expressed as means *. S.E.

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As previously observed (13) when testing the response to changes in glucose concentrations in single isolated rat p-cells, fewer than 10% of the cells examined (Fig. lA)produce oscillatory [Ca2+],patterns similar to those reported in intactmouse islets (10, 14). Correlated oscillatory patterns in NAD(P)H fluorescence could also be observed in such a subpopulation of cells under similar stimulation conditions (Fig. 1B). Thus, at 8.3 mM glucose, the numbers of eventdminute were 1.4 2 0.4 ( n = 5 ) and 1.3 2 0.1 ( n = 4) for the [Ca2+liand NAD(P)H transients, respectively. To test for the implication of Ca2+entry in thegeneration of an oscillatory metabolicpattern, chelation of extracellular Ca2+with EGTA was used (Fig. 1, C and D). This caused the abolition of oscillatory patterns in both [Ca*+], and NAD(P)H and lowered the pyridine nucleotide redox state (Fig. 1D). To increase the number of observations of NAD(P)H formation during oscillatory [Ca2+l,increases, pulses of depolarizing K+ concentrations (24 mM for 40 s) were applied. This maneuver resulted, at 4 m glucose, in Ca2+-dependentand [Ca2+l,-correlated NAD(P)H oscillations (Fig. 1,E-H). Since the amplitude of [Ca2+l,and the patterns of NAD(P)H varied from cell to cell, two traces indicative of the obtained responses are shown. Notethat some cellsdisplayed both a clear drift in their basal [Ca"], and reached higher levels during depolarization (Fig. lG, thin truce). Therefore, as previouslyobserved (7), [Ca"], overload may also negatively influence mitochondrial NAD(P)H fluorescence. This probably underlies the inverted NAD(P)H signals displayed by some cells after repeated depolarization (Fig. 3 u , thin trace).Moreover, the mean reoxidation rate constant of the NAD(P)H transients evaluated after K+ stimulation was generally slower (assuming first-order kinetics, T = 101 2 49 s, n = 6) and displayed greater heterogeneity from cellto cell than thatestimated inthe NAD(P)Hoscillating subpopulation at 8.3 mM glucose (T = 27 2 4 s, n = 4). To investigate whether such metabolic oscillations also occur in other cell types, we extended our observations to adrenal glomerulosa cells (Fig. 2) and liver cells (Fig. 3). These cells represent appropriate experimental models forthe study of the relationship between [Ca2+],and NAD(P)H formation as, in contrast with p-cells, NAD(P)H fluorescenceis sufficiently intense for its simultaneous recording with [Ca2+],transients. In adrenal glomerulosa cells, AI1 causes [Ca2+],oscillations through both the mobilization of intracellular Ca2+via inositol 1,4,5-trisphosphate (IP,) and Ca2+influx from the extracellular space (15). AI1 application 'promoted, in these fura-2-loaded cells, the rapid appearance of [Ca2+l,transients preceding (Fig. 2 4 ) or immediately associated with (Fig. 2 B ) oscillations in NAD(P)H fluorescence. At later time points (Fig. M ) , a remarkable correlation between the two parameters was ob-

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FIG.1. Effects of glucose and depolarizingpotassium(KCl, 24 mrd on [Ca2+li and NAD(P)H fluorescence in single isolated of extracellularCa2+ ratp-cellsinthepresenceorabsence (EGTA,3 m). NAD(P)H fluorescence (percent of basal values) and [Ca"], (nanomolar) measurements using the Ca2+indicator fura-2 (12) were recorded in parallel experiments by dual excitation microspectrofluorometry (see "Materials and Methods"); when otherwise not indicated, the basal glucose ( g l c )concentration was 4 mM. In G and H , the two traces (thick and thin) represent typical patterns obtained during pulsatile K+stimulations.

served. To ascertain the mitochondrial origin of the NAD(P)H oscillations, Amytal was applied to block site I of the respiratory chain. This caused the selective and reversible cessation of the NAD(P)H oscillations (Fig. 2 A ) . The specificity of the signals was further demonstrated by the time-dependent disappearance of the fura-2 signal (due to the loss of the indicator), which contrasted with the preservation ofAII-evoked NAD(P)H oscillations. Frequency modulation of the mitochondrial pyridine nucleotide redox state could also be demonstrated when two increasing AI1 concentrations were tested consecutively (Fig. 2B). MI-evoked NAD(P)H oscillations were blocked by removal of extracellular Ca2+(data not shown) and were also generated in cells not loaded with fura-2 (Fig. 2C). Single fura-2-loaded hepatocytes (Fig. 3 A ) were stimulated first with arginine vasopressin (AVP; 0.5 m),which generates IP,-dependent [Ca2+l,oscillations (16). In contrast to the adre-

Dynamic Pacing of Cell Metabolism

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FIG.2. Simultaneous NAD(P)H and [Ca2+], measurements(arTime (s) bitrary units) in single rat adrenal glomerulosa cells following AI1 stimulation. In C , NAD(P)H fluorescence was measured by dual FIG.3. Simultaneous NAD(P)H and [Ca2+li measurementsin excitation (6). The traces in A and B are representative of 10 independ- single fura-2-loaded rathepatocytes following stimulation with ent recordings. The biexponential traces (*) and the corrected NAD(P)H AVP (A) and epinephrine ( B ) .When not indicated, the basal glucose and [Ca2'li traces were calculated as explained under "Materials and ( g k ) concentration was 4 mM. The traces indicated as NAD(P)H and Methods." Long lasting oscillations (>30min) following AI1 removal [Ca2+li(arbitrary units)in the three panels result from a mathematical were observed in 8 out of 26 responsive cells; increased NAD(P)H flu- procedure identical to that shown in Fig. 2. Similar oscillations were orescence is detected as anupward deflection of the signal recorded at observed with AVP in 10 individual hepatocytes out of 18 tested and 470 nm followingexcitation at the isosbestic pointof fura-2 (12), 360 nm with epinephrine in 9 out of 16 cells tested. ( F 360). Changes in [Ca2+Iiare reflected by the downward deflectionof the fluorescence signal excited at 380 nm ( F 380)(12). evated pyridine nucleotide reduction. AVP receptors were still

nal cell, the [Ca2+],transients resulted in hepatocytes in the cumulative reduction of NAD(P). Such a situation is certainly explained by the slow apparent reoxidation rate observed in hepatocytes (see Fig. 3 A , end of stimulation), which prevents the expression of an oscillatory metabolicsignal. Thus, inthese cells, [Ca2+], transients are translated into theamplitude modulation of the metabolic signal. In most cells, a progressive inhibition of the [Ca2+],spikes was found coincidental with el-

functional as two additions of maximal AVP concentration (1 I"; Fig. 3A) each produced a correlated single transient, followed by a plateau both in [Ca2+Ji and in NAD(P)H. The latter persisted until the withdrawal of the agonist, although only a small [Ca2+],signal could be detected. Epinephrine, which acts both on &-adrenoreceptors (generating CAMP)and qadrenoreceptors (generating IP3), promoted more rapid [Ca2+], oscillations than AVP (16). Here, the first two [Ca2+l,transients stepwise increased the level of NAD(P)H to a largely non-oscillatory


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A contrasting metabolic pattern was revealed in thehepatocyte, where [Ca'+], transients resulted in the amplitude modulation of reduced pyridine nucleotides. It is noteworthy that the [Ca2+],oscillations were similar (even slower) in frequency to those in both p-cells and adrenal glomerulosa cells. Dueto the slow apparent reoxidation rate of reduced pyridine nucleotides, [Ca2+],spikes of low frequency should therefore be sufficient to sustain NAD(P)H formation in liver cells. Simultaneous stimulation by Ca" of both Ca'+-sensitive mitochondrial dehydrogenases (5)and glycogenolysisin thecytosol (28,291(the latteralso being stimulated by CAMPin thecase of epinephrine) probably underlies the apparent preponderance of reductive over oxidative fluxes. The slow reoxidation of the reduced pyridine nucleotides may also be the consequence of the highly stabilized mitochondrial power supply reported in this cell even during oscillatory [Ca"], transients (30).Another finding that also points to the presence of functional microdomains in the hepaDISCUSSION tocyte is illustrated by the sustained NAD(P)H signal despite a The demonstration that physiological agonists may induce small [Ca"], plateau phase recorded under maximal AVP conCa'+-dependent NAD(P)Hoscillations provides new insight in centration (Fig. 3 A ) . It can be speculated that these Ca'' the kinetics of metabolic activation during cell stimulation. changes could in fact reflect only [Ca2+l,increases close t o miFurthermore, the most significant implication of this observa- tochondria, as suggested by the sustained effect on the tion is that Ca",by triggering metabolic pathways (51,may NAD(P)H signal. lead to the oscillatory generation of metabolism-derived intraFurthermore, as previously reported (171, our results suggest cellular signals (9, 18). Such metabolic signals are relevant to that interactions exist between redox potential and IP,-sensithe control of hormonal secretion both in the pancreatic p-cell tive Ca2+stores. On the one hand, increased reduction of pyri(6, 9, 10, 18, 19) and in the adrenal glomerulosa cell (7). The dine nucleotide may provide a direct means for delaying unobservation that (i)only a limited number of rat p-cells display necessary [Ca"], transients in hepatocytes through a decrease glucose-modulated oscillations either in [Ca2+lior in NAD(P)H in IP, sensitivity, as conversely observed when permeabilized and (ii) K+-induced[Ca"], transients stimulate NAD(P)H for- hepatocytes were incubated under oxidizing conditions (17). mation in the presence of glucose, but not in its absence (81, Similarly, it is noteworthy that hypoxia, which is known to may provide new information on the mechanisms whereby os- increase dramatically the reduction of pyridine nucleotides, is cillatory metabolic signals (18) are generated in theislet micro- able t o block immediately the generation of [Ca"], oscillations organ during pulsatile insulin secretion (20-22). It isnotewor- in hepatocytes (31). On the other hand, by analogy t o the effect thy that, even if only a small number of P-cells display of oxidizing agents indifferent cell types (17,32,33),increased oscillatory patterns, gap junctions functionally couple islet free radical formation in microdomains of adrenal glomerulosa p-cells (23, 24).Therefore, such a subpopulation of cells could cells during cholesterol hydroxylations (34) may underlie the often spontaneous [Ca2+l,and NAD(P)H oscillations persisting electrically pace oscillatory Ca'' influx in islet territories in response to glucose. This would favor the Ca2+-inducedsynthe- after removal of AI1 (Fig. 2C). sis of mitochondrion-derived coupling factors that permit exoThe main conclusion emerging from this study is that, by cytosis (6, 8-10, 19) in metabolically heterogeneous subpopu- controlling the activity of key metabolic enzymes(5),Ca2+may lations of p-cells (6, 25, 26). The metabolic heterogeneity ensure the cell-specific transition of cellular metabolism froma existing between p-cell subpopulations may explain why close to equilibrium state under resting conditions to a dynamic NAD(P)H oscillations cannot be observed in single islets (19) in oscillating state directly coordinated with cell activation and contrast to the electrically coupled [Ca"], transients (14,231. It function. Furthermore, the stimulation in the p-cell of mitois nevertheless possible that thesmall number of metabolically chondrial metabolism by [Ca2+l,transients (4) may represent a oscillating p-cells observed in this study represents an under- central mechanism for the oscillatory generation of metabolic estimation of such a population in vivo. Indeed, cellular coupling factors underlying pulsatile insulin secretion from the changes due to isolation procedures and t o primary culture may islet micro-organ in response to glucose. occur, consequently altering the functional expression of these Acknowledgments-We thank D. Harry for skilled technical assistcells in vitro. ance and Drs. M. Prentki and G. Rutter for useful discussions. In the adrenal glomerulosa cell,the frequency modulation of mitochondrial metabolism by the AII-evoked [Ca2+],transients REFERENCES was observed. The striking correlation between the changes in 1. McCormack, J. G., Browne, H. M., and Dawes, N. J. (1989) Biochim. Biophys. Acta 973, 420-427 [Ca"], in the cytosol and those observed in the redox state of 2. Miyata, H., Silverman, H. S., Sollot, S. J., Lakatta, E. G., Stem, M. D., and mitochondrial pyridine nucleotide strongly suggests the presHansford, R. G . (1991) Am. J. Physiol. 261, H1123-H1134 ence of [Ca2+l,oscillations in this cell type. 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steady-state during the subsequent [Ca'+], oscillations (Fig. 3, B and C ) .As with AVP (Fig. 3A), progressive inhibition of the [Ca2+],spikes was also noticed paralleling the augmented pyridine nucleotide reduction (Fig. 3B). Such inhibition may represent themirror phenomenon of the spontaneous or enhanced IP,-evoked intracellular Ca2+transients that occur in hepatocytes under oxidizing conditions(17). To probe fora correlation between elevated pyridine nucleotide redox state and inhibition of the generation of[Ca"], spikes, a high extracellular glucose concentration, aimed at further increasing the stateof reduction of pyridine nucleotides, was applied t o hepatocytes displaying regular oscillations (Fig. 3C). Indeed, the additional rise in NAD(P)H evoked by glucose was associated with the immediate decrease in frequency and eventual inhibition of the [Ca'+], spikes (Fig. 3 0 .

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