Proc. Nati. Acad. Sci. USAVol. 86, pp. 453-457, January 1989Biochemistry

ATP stimulates Ca2+ uptake and increases the free Ca2+concentration in isolated rat liver nuclei

(Ca2+ transport/calmodulin/fura-2)

PIERLUIGI NICOTERA, DAVID J. MCCONKEY, DEAN P. JONES*, AND STEN ORRENIUStDepartment of Toxicology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden

Communicated by Bengt Samuelsson, October 10, 1988 (received for review August 19, 1988)

ABSTRACT Addition of ATP to a highly purified fractionof rat liver nuclei incubated with submicromolar concentra-tions of Ca2l and trace amounts of 45Ca2+ resulted in the rapidaccumulation of 45Ca2+ in the nuclei. This was associated withan increase in intranuclear free Ca21 concentration as mea-sured with the fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphe-noxy)ethane-N,N,N',N'-tetraacetic acid (fura-2). Inhibitors ofmicrosomal and mitochondrial Ca2+ translocases had no effecton nuclear Ca21 sequestration, indicating that it was distinctfrom previously known intracellular Ca2+-transporting sys-tems. Ca2+ uptake and the associated increase in intranuclearfree Ca2+ concentration were prevented by calmidazolium, apotent calmodulin antagonist. Partial characterization of theATP-stimulated nuclear Ca2+ uptake showed that maximalrates of Ca21 uptake and increase in intranuclear free Ca2+level occurred at concentrations of Ca2+ normally present inthe cytosol of mammalian cells. Together, these results showthat a distinct, ATP- and calmodulin-dependent Ca2+ uptakesystem exists in liver nuclei. This system may play an importantrole in the regulation of intranuclear Ca2'-dependent pro-cesses.

Changes in the cytosolic free Ca2l concentration control avariety of cell functions, including metabolism, growth, anddifferentiation (1, 2). Transient increases in cytosolic Ca2+level can also affect the behavior ofthe nucleus during the cellcycle (3, 4), activate nuclear processes, such as DNAfragmentation and repair (5, 6), and modulate gene transcrip-tion (7). Although it is well established that alterations incytosolic Ca2+ level can affect the activity of nuclear en-zymes (5), or cause the modification of nuclear structure (4),it is not yet known how this occurs and to what extentfluctuations in cytosolic Ca2+ level are reflected in changesin intranuclear free Ca2+ concentration.The presence of pores in the nuclear membrane has been

taken as evidence that free diffusion of ions and smallmolecules can occur in and out of the nucleus (8, 9).However, Ca2+ concentration gradients have recently beenshown to exist between the nucleus and the cytoplasm (10,11), suggesting that there are mechanisms that modulate thetransport of Ca2+ across the nuclear membrane and regulateintranuclear Ca2+ concentration. The assumption that theintranuclear Ca2+ concentration is regulated by the activity ofspecific transport systems is also supported by the presenceof Ca2+-stimulated ATPase activity in skeletal muscle nuclei(12). In addition, the intranuclear Ca2+ concentration mayalso be modulated by Ca2+ binding to proteins-e.g., cal-modulin-that have been identified in the nuclei of differentcell types and have been associated with some of theintranuclear effects of Ca2+ (13, 14).

Although early work had shown that Ca2+-dependentnuclear enzymes required millimolar Ca2+ concentrations (5,15), in recent studies we found that a constitutive endonu-clease in intact, isolated liver nuclei was activated at submi-cromolar concentrations of Ca2+ when ATP was included inthe incubation medium (16). Activation of Ca2+-dependentDNA fragmentation was also strictly dependent on calmod-ulin, since the presence of a calmodulin inhibitor, calmida-zolium (17), resulted in the complete inhibition of DNAcleavage. The results of this study suggested that ATP andcalmodulin affected the availability of intranuclear Ca2`required for activation ofthe endonuclease. Thus, the presentstudy was designed to investigate whether the Ca2+ concen-tration in isolated nuclei could be actively regulated by ATPand/or calmodulin. Here we report the existence of anATP-stimulated Ca2+ sequestration system in rat liver nucleithat requires calmodulin and generates a net increase innuclear matrix free Ca2+ concentration.

MATERIALS AND METHODS

Isolation of Nuclei. Male Sprague-Dawley rats (200-300 g,fed ad libitum) were anesthetized with sodium pentobarbital.Livers were cannulated and perfused in situ with =50 ml ofice-cold TKM solution (50 mM Tris-HCI, pH 7.5/25 mMKCI/5 mM MgCl2) to remove blood. Livers were thenremoved, cut into small pieces, and homogenized with aglass/Teflon homogenizer (nine strokes) in 40 ml of the samesolution supplemented with 0.25 mM sucrose. The homoge-nate was filtered through three layers of cheesecloth. Thenuclei were pelleted by centrifugation at 700 x g for 10 min.The pellets were homogenized (five strokes) in 40 ml of theTKM/sucrose solution and centrifuged again at 700 x g for10 min. The resulting pellet was resuspended in 24 ml of thesame solution by homogenization (three to five strokes).Six-milliliter samples of this suspension were added to eachoffour tubes containing 12 ml ofTKM solution supplementedwith 2.3 M sucrose. The tubes were gently mixed and a 6-mlcushion (TKM solution containing 2.3 M sucrose) wascarefully added to the bottom of each tube prior to centrif-ugation at 37,000 x g for 30 min. The resulting pellet (nuclei)was resuspended in TKM solution and sedimented at 1000 Xg for 5 min. The highly purified pellet of nuclei was gentlyresuspended in incubation medium (125 mM KCI/2 mMK2HPO4/25 mM Hepes/4mM MgCl2/2 mM EGTA, pH 7.0).All steps were performed at 0-4°C. Contamination of thenuclear fraction by plasma membrane fragments was <5%,by microsomes was <1%, and by mitochondria was <3%, asmeasured by assaying the activities of 5'-nucleotidase, glu-

Abbreviations: fura-2, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofu-ran-5-oxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,NN ',N'-tetraacetic acid; fura-2 AM, tetraacetoxymethyl ester of fura-2.*Present address: Department of Biochemistry, Emory UniversitySchool of Medicine, Atlanta, GA 30322.tTo whom reprint requests should be addressed.

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454 Biochemistry: Nicotera et al.

cose-6-phosphatase, and succinate-cytochrome c reductase,respectively.

Buffering of Ca2" Concentration. The concentrations ofEGTA and Ca2+ required to achieve free Ca2` concentrationsranging from 1 nM to 1 gM were determined according toBartfai (18). Free Ca2` concentrations were verified by using1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5 '-methylphenoxy)ethane-N,N,N' ,N'-tetra-acetic acid (fura-2) free acid in a solution containing 100 mMKCI, 2 mM K2HPO4, 25 mM Hepes, 4 mM MgCl2, and 2 mMEGTA with 10 ,uM fura-2 (pH 7.0) at 20'C as described (19).

"5Ca2+ Uptake. Isolated nuclei (150-200 jig ofDNA per ml)were resuspended in the Ca2+-buffered medium describedabove supplemented with trace amounts (0.1 ,Ci/ml; 1 Ci =37 GBq) of 45Ca2 . Samples were then incubated in thepresence or absence of ATP or other additions at 370C asindicated in figure and table legends. At appropriate times,0.1-ml samples were collected and placed on 0.22-num nitro-cellulose filters (Sartorius) and washed four times withice-cold buffer containing 50 mM Tris HCl and 150 mM KCl(pH 7.0). Filters were placed in 5 ml of scintillation cocktail(Beckman "Ready Safe") prior to the determination of45Ca2+ radioactivity in a Beckman LS 1801 liquid scintillationcounter.

Fluorescence Microscopy. Fluorescent images of fura-2-loaded nuclei were obtained using a Nikon Diaphot micro-scope equipped for fluorescence microscopy with excitationfilter block at 340-380 nm and emission barrier at 435 nm.Nuclei were photographed using a Nikon Fx35Va cameraloaded with Kodak T-max 400 ASA film rated at 800 ASA.

Fura-2 Loading of Nuclei and Fluorescence Measurements ofFree Ca2+ Concentration. Isolated nuclei (=150 ,ug of DNAper ml) were incubated at 4°C for 45 min with 7 ,uM fura-2AM(the tetraacetoxymethyl ester of fura-2) in a buffer containing125 mM KCl, 2 mM K2HPO4, 25 mM Hepes, 4 mM MgCl2,and 2 mM EGTA (pH 7.0). After 45 min the nuclear sus-pension was diluted 1:10 and washed free of extranuclearfura-2 AM. Nuclei (100 ,g ofDNA per ml) were resuspendedin the same buffer supplemented with Ca2+-EGTA to achievethe range of free Ca2+ concentrations described above andtransferred to a dual-wavelength Sigma ZWII fluorometerwhere measurements of Ca2+ were performed at 37C using

z0

cn

Ex

a

.00

Ec

7

z

C-)Ex

N

C

L 5 10 5 1 0 15

TIME (min)

the ratio of excitation wavelengths 350/380 nm with emissioncutoff at 500 nm. Free Ca2+ concentration was calculatedaccording to ref. 19. Since the fluorescence of fura-2 free acidcan be quenched by Mn2 , whereas the fluorescence of fura-2AM is insensitive to Mn2 , the appearance of Ca2+-depen-dent, Mn2+-sensitive fluorescence can be used to measure thehydrolysis of fura-2 AM (19). Addition of 2.5 mM MnC12, or5 mM EGTA in the presence of 1 AM free Ca2 , to nuclei thathad been loaded with fura-2 AM for 45 min, or to the supernateobtained by washing loaded nuclei, had no effect on thefluorescence signal, indicating that no fura-2 free acid waspresent in the extranuclear medium. In contrast, addition ofdetergent (Nonidet P-40; 0.05% final concentration) to fura-2-loaded nuclei resulted in the appearance of Mn2+- andEGTA-sensitive fluorescence, indicating that fura-2 AM washydrolyzed and the fura-2 free acid was trapped by the nuclei.Following the loading procedure, nuclei contained about 24pmol of fura-2 free acid per ,g of DNA as determined bycomparing the intensity of Ca2 -dependent and Mn2 -sensitive fluorescence (19) with the fluorescence intensity ofknown amounts offura-2 free acid in the presence ofdetergent-treated, unloaded nuclei in the same incubation medium.Nuclear autofluorescence was determined in fura-2-loaded

nuclei after detergent treatment in the presence of 2.5 mMMnCl2 and was accounted for in calculations of Ca2+ con-centration. Leakage of fura-2 from the nuclei was minimal(<2% after 15 min at 37°C), as measured by the inclusion of2.5 mM MnCl2 at the end of each experiment.

Materials. 45Ca2+ (10 mCi/mg) was purchased from Am-ersham. Fura-2 and fura-2 AM were obtained from Calbio-chem. ATP, ADP, and AMP came from Boehringer Man-nheim. Calmidazolium was from Sigma. All other reagentswere of the highest grade of purity available and wereobtained from local commercial sources.

RESULTSATP-Stimulated 45Ca2+ Uptake by the Nuclear Fraction.

Addition of 1 mM ATP to liver nuclei incubated in a mediumcontaining 400 nM Ca2+ and a trace amount of45Ca2+ resultedin rapid nuclear Ca2+ uptake (Fig. 1A). Ca2' sequestrationwas dependent on the Ca2+ concentration in the medium,

40r B 200 r C

Ca02+ (nM) TIME (min)

FIG. 1. ATP-stimulated Ca2+ uptake in rat liver nuclei. (A) Time course ofCa2' accumulation. Isolated nuclei were incubated with or withoutATP in the presence of 400 nM Ca2' and trace amounts of 45Ca2+ for the times indicated prior to filtration. o, Control; *, 1 mM ATP. Valuesare mean ± SEM (where applicable) of three separate experiments. (B) Ca2+ concentration dependence of Ca2+ accumulation. Isolated nucleiwere incubated with or without ATP in the presence of the indicated concentrations of Ca2+ and trace amounts of 45Ca2+ for 5 min at 37°C priorto filtration. o, Control; *, 1 mM ATP. Values are mean of four separate experiments. (C) Maintenance of intranuclear Ca22+ accumulationrequires ATP. Isolated nuclei were incubated in the standard 45Ca2+ incubation medium in the presence of 1 mM ATP and 400 nM Ca24 for5 min at 37°C. Nuclei were then pelleted by centrifugation for 30 sec at 1500 x g in a Microfuge. Samples were resuspended in an equal volumeof the standard 45Ca24. incubation medium with or without 1 mM ATP. Aliquots of each incubation were collected and filtered at the timesindicated. The arrow indicates time of centrifugation. A, Without ATP in the resuspension medium; A, 1 mM ATP in the resuspension medium.Values are results of one experiment, which is typical of three.

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 455

being half-maximal at about 75 nM Ca2l and maximal atabout 200 nM Ca2+ (Fig. 1B). ATP-independent Ca2+ accu-mulation was directly related to the level of free Ca2+ in theincubation medium and was minimal (<16% of the ATP-stimulated uptake) at Ca2+ concentrations up to 400 nM Ca2+.Maintenance of the nuclear Ca2+ gradient depended on thepresence of ATP, since removal ofATP from the incubationmedium resulted in rapid release of Caa2+ from loaded nuclei,whereas readdition ofATP resulted in rapid reuptake ofCa2+(Fig. 1C).Comparison of the effectiveness of ATP, ADP, and AMP

to support nuclear Ca2+ accumulation revealed a preferentialdependency on ATP. ATP-stimulated Ca2' uptake wastemperature-dependent, being almost absent when nucleiwere incubated at 40C. Moreover, vanadate, an inhibitor ofATP-dependent ion pumps (20), partially inhibited nuclearCa2+ uptake (Table 1). To determine whether Ca2+ uptakewas associated with ATP hydrolysis, Ca2+-dependent Pirelease was measured in nuclear suspensions incubated with0.1-1 AtM Ca2' and 1 mM ATP. Ca2+ was found to stimulatenuclear ATP hydrolysis in a concentration-dependent man-ner, half-maximal Pi release occurring at 200-300 nM Ca2+.Assays of various marker enzymes indicated minimal

contamination of the nuclear fraction with other organelles(<5%, see Materials and Methods). However, to rule out thepossibility that contaminating organelles contributed to theobserved Ca2+ uptake, the effects ofinhibitors ofmicrosomal[2,5-di(tert-butyl)-1,4-benzohydroquinoneJ (21) and mito-chondrial (ruthenium red) (22) Ca2+ sequestration on nuclearCa2+ uptake were determined. Neither agent was found toinhibit nuclear Ca2+ accumulation. In contrast, incubation ofisolated nuclei with calmidazolium, a specific inhibitor ofcalmodulin (17), resulted in marked inhibition of Ca2+ uptake(Table 1). Since calmodulin is not involved in the activationof the hepatic plasma membrane Ca2+ pump (23), this findingnot only provides strong evidence for the involvement ofcalmodulin in nuclear Ca2+ uptake but also excludes thepossibility that the observed Ca2+ uptake was due to con-tamination with inverted plasma membrane vesicles.To investigate whether the ATP-stimulated Ca2+ uptake

was due to a nonspecific increase in the permeability of thenuclear membrane caused by ATP, we measured the nuclearuptake of deoxy[3H]glucose in the absence or presence ofATP. The results show that uptake of deoxy[3H]glucose bynuclei was minimal and unaffected by the presence of ATPover the course of a. 15-min incubation (not shown). To-gether, these results show that an ATP-stimulated Ca2+transport system exists in liver nuclei that requires calmod-

Table 1. Effects of various agents on Ca2+ uptake by ratliver nuclei

Ca2+ uptake,*nmol/min per

Addition mg of DNA Activity, %1 mM ATP 29.4 ± 1.8 1001 mM ADP 19.4 ± 5.2 661 mM AMP 9.3 ± 1.5 381 mM ATP, 4°C 7.4 ± 0.52 321 mM ATP + 10,M 2,5- di(tert-butyl)-1,4-benzohydroquinone 30.7 ± 4.1 104

1 mM ATP + 5,M ruthenium red 37.5 ± 4.2 1281 mM ATP + 100,M vanadate 18.3 ± 0.8 621 mM ATP + 10,Mcalmidazolium 9.7 ± 3.9 33

Incubations contained nuclei (100 ,ug ofDNA per ml) and 400 nMCa2+, with a trace amount of 45Ca2+. Ca2+ uptake is presented as

ulin and is distinct from the translocases found in theendoplasmic reticulum, mitochondria, and plasma mem-brane.ATP-Stimulated Increase in Intranuclear Free Ca22. To

investigate the relationship between the observed uptake of45Ca2+ and changes in the intranuclear free Ca2+ concentra-tion, isolated nuclei were loaded with the Ca2+ indicatorfura-2 AM. Following the loading procedure, fura-2 AM was

hydrolyzed and fura-2 free acid was trapped within the nuclei(see Materials and Methods). Fluorescence microscopy offura-2-loaded nuclei revealed that localization of the dye was

diffuse, consistent with a homogeneous distribution of fura-2in the nuclear matrix (Fig. 2). Fura-2 exclusion from the

A

B

FIG. 2. Phase-contrast and fluorescence microscopy of fura-2-loaded rat liver nuclei. (A) Phase-contrast image of isolated nucleishowing granular structure (chromatin) and the nucleoli. (B) Fura-2fluorescent image of the same field following treatment with 1 mMATP, showing diffuse distribution of the dye within the nuclei withapparent exclusion from the nucleoli. Nuclei were loaded with 7 ,uMfura-2 AM for 45 min and washed free of extranuclear fura-2 AM;10-/Ll samples were then taken for microscopy. (x290.)

mean -+- SEM of three separate experiments.*Rate of active Ca2+ uptake (passive Ca2+ uptake was subtractedfrom total).

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456 Biochemistry: Nicotera et al.

nucleoli was apparent. The addition of 1 mM ATP tofura-2-loaded nuclei incubated with 200 nM Ca2+ resulted ina marked increase in fluorescence intensity, suggestive of anATP-stimulated increase in intranuclear free Ca2' concen-tration.The effect of ATP on the intranuclear free Ca2+ concen-

tration was measured in fura-2-loaded nuclei using a dual-wavelength fluorometer. As shown in Fig. 3, trace A, addi-tion of ATP to the nuclear suspension in the presence of 100nM Ca2+ resulted in a rapid increase in intranuclear Ca2+concentration, from a basal value of 4100 nM Ca2+ to about540 nM Ca2+. Addition of 1 mM ADP increased the intranu-clear Ca2+ concentration to a lesser extent, whereas AMP,adenosine, and pyrophosphate had no effect on intranuclearCa2+ concentration (Fig. 3), confirming that the process waspreferentially dependent on ATP.

In view of the previous observation that calmodulin wasrequired for the ATP-dependent nuclear 45Ca2+ uptake, wetested the effect of calmidazolium on the ATP-stimulatedincrease in intranuclear free Ca2+ concentration. As shown inFig. 3, pretreatment of nuclei with calmidazolium abolishedthe ATP-dependent increase in free Ca2+ concentration(trace B), whereas the addition of calmidazolium to nucleipreloaded with Ca2+ in the presence of ATP caused about70% release of Ca2+ (trace A). Addition of detergent tocalmidazolium-treated, Ca2+-loaded nuclei resulted in a re-turn to the basal Ca2' level. Total release ofCa2+ from nucleiwas also observed when the detergent was added alone afterATP-dependent Ca2+ uptake was complete (not shown).Calmidazolium had little or no effect on fluorescence inten-sity (Fig. 3), demonstrating that its effect was not due toquenching of Ca2+-dependent fura-2 fluorescence.The relationship between the changes in the intranuclear

free Ca2+ level and the Ca2+ concentration in the incubationmedium was determined for fura-2-loaded nuclei, using arange ofCa2+ concentrations from 1 nM to 1 ,uM (Fig. 4). Theincrease in the intranuclear free Ca2+ level was half-maximalat a Ca2+ concentration of 61 ± 10 nM (mean ± SD of fourdifferent experiments), whereas maximal increase in iptra-

[Ca21] (nM)

600_

300.

200_

100_

ATP

cmz

50 sec

APNP-40

cmz i/ AMP

ATP

FIG. 3. ATP-stimulated increase in intranuclear free Ca2l con-centration. Nuclei (100 Mg of DNA per ml), loaded with fura-2 AM,were incubated in the presence of 100 nM Ca2' at 37°C. In trace A,1 mM ATP was added to the nuclei and the fluorescence changeswere monitored. After the increase in intranuclear Ca2+ level hadreached its maximum, 25 MM calmidazolium (cmz) and 0.05%

(vol/vol) Nonidet P-40 (NP-40) were added sequentially, causing a

rapid decrease in intranuclear free Ca2+ concentration. In trace B, 25MM calmidazolium (cmz) was added prior to the addition of 1 mMATP, preventing the ATP-stimulated increase in intranuclear Ca2+concentration. In trace C, the effects of 1 mM ADP, 1 mM AMP, or

1 mM pyrophosphate (PP) on intranuclear Ca2W concentration areshown.

c 800 -

0O 600 -

o400z

Y 200z

0 200 400 600 800 1000

EXTRANUCLEAR Ca2+ (nM)

FIG. 4. Relationship between extra- and intranuclear free Ca2"concentrations. Nuclei were loaded with fura-2 AM and incubated inthe presence (e) or absence (o) of 1 mM ATP with Ca2+ concen-trations in the incubation medium ranging from 1 nM to 1000 nM.Intranuclear free Ca2+ concentrations were then measured.

nuclear Ca2+ concentration was achieved in the presence of200 ± 22 nM Ca2+ (mean ± SD offour separate experiments).Passive Ca2+ influx into nuclei was minimal and directlyrelated to the Ca2+ concentration in the medium (Fig. 4), aswas observed in the experiments with 45Ca2+.The requirement for ATP was studied in fura-2-loaded

nuclei incubated at ATP concentrations ranging from 1 AM to1 mM, in the presence of 100 nM Ca2+. Kinetic analysisrevealed a Km for ATP of 75 ± 9 uM (mean ± SD of threeseparate experiments) and saturation at about 500 AM ATP,suggesting that ATP-dependent Ca2` transport into the nu-cleus is fully active at physiological ATP concentrations.

DISCUSSIONThe results of the present work provide evidence for theexistence of a Ca2+ uptake system in rat liver nuclei. Thissystem operates at Ca2+ concentrations normally present inthe cytosol of mammalian cells (24), and it is distinct fromother known Ca2+-transporting systems in the hepatocyte.Several lines of evidence support this contention: (i) theisolation procedure employed yields a highly purified nuclearfraction, containing <5% contamination by other organelles;(it) specific inhibitors of microsomal (21) and mitochondrial(22) Ca2` sequestration did not affect ATP-stimulated Ca2+uptake in the nuclear fraction; (iii) calmidazolium inhibitednuclear Ca2+ uptake and the subsequent increase in intranu-clear free Ca2+ concentration, suggesting that calmodulin iscritically involved in the control of this transport system andexcluding the possibility that inverted plasma membranevesicles were involved; (iv) ATP- and calmodulin-dependentCa2+ uptake was confirmed in studies with fura-2-loadednuclei, which indicated that the ATP-dependent Ca2+ uptakeresulted in an increase in intranuclear free Ca2+ concentra-tion. The fact that two independent methods for monitoringCa2+ uptake yielded identical results further strengthens thiscontention.The characteristics of the ATP-dependent Ca2+ uptake

system suggest that nuclear Ca2+ sequestration is mediatedby a calmodulin-dependent Ca2+ pump. The generation of aCa2+ concentration gradient across the nuclear membraneimplies the existence of an active process. Vanadate, whichis a potent inhibitor of various ATPases coupled with iontransport (20), partially blocked nuclear Ca2+ sequestration,suggesting that ATP hydrolysis is involved. Furthermore, thesaturation of the process observed at submicromolar Ca2+levels implies that Ca2l uptake is not. the result of diffusionand that it involves a finite number of translocation sites. In

Proc. Natl. Acad. Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 457

addition, agents that have previously been shown to affectthe activity of other Ca2"-transporting systems, including thethiol alkylating agents, N-ethylmaleimide (25 AsM) and p-chloromercuribenzoic acid (10 KM), were found to preventthe ATP-stimulated Ca2' increase. Finally, the K+ iono-phore, valinomycin (15 ,M) did not affect the ATP-inducedincrease in intranuclear free Ca2+ concentration, indicatingthat under these experimental conditions the Ca2+ uptakewas not dependent on the nuclear membrane potential.Though it remains to be established whether the nuclear Ca2+uptake system is located in the outer membrane or at thenuclear pore complex, it is clear that its stimulation results inan increased Ca2' concentration in the nuclear matrix. Thisis evident from the distribution pattern of fura-2 fluorescencein nuclei exposed to ATP (Fig. 3) and from the observationthat ATP-stimulated Ca2+ uptake results in the activation ofan endonuclease present in the nuclear matrix (16).The ability to load isolated nuclei with fura-2 AM allows for

the continuous monitoring of intranuclear Ca2+ fluctuations.Intranuclear localization of fura-2 has previously been ob-served in situ (3, 4). Our results show that fura-2 AM canenter the nuclear matrix and be hydrolyzed and that the freeacid can be trapped there (only a minimal amount of fura-2was found to be associated with the nuclei, when fura-2 AMwas substituted with fura-2 free acid in the loading proce-dure). This suggests that when cells are loaded with fura-2AM, incomplete hydrolysis of the dye in the cytoplasm (25)may result in nuclear loading, which should be taken intoaccount when fura-2 is used to measure the cytosolic Ca2+concentration in intact cells.Although it seems unlikely that the mere availability of

ATP in the cytosol directly regulates fluctuations in intranu-clear Ca2' concentration, the presence of an ATP-dependentCa2+ uptake system provides a basis for the potential"fine-tuning" of the intranuclear free Ca2+ level by otherfactors. The involvement of second messengers, such asinositol trisphosphate, in the regulation of cytosolic Ca2+concentration is well established (1), and it seems possiblethat the intranuclear free Ca2+ concentration may be modu-lated by a similar regulatory system. Control of intranuclearCa2+ concentration by second messengers could potentiallymediate the effects of hormones and other agents known toaffect Ca2+-dependent events within the nucleus. Furtherstudies of the regulation of the intranuclear free Ca2+ con-centration should provide important insight into the com-plexity of genomic modulation.

This study was supported by grants from the Swedish MedicalResearch Council (03X-2471), the Karolinska Institute, and Fondazi-one Clinica del Lavoro Istituto di Ricerca e Cura a CarattereScientifico, UniversitA di Pavia, Italy.

1. Berridge, M. J. (1984) Biochem. J. 220, 345-360.2. Charest, R., Blackmore, P. F., Berthon, B. & Exton, J. H.

(1983) J. Biol. Chem. 258, 8769-8773.3. Poenie, M., Alderton, J., Tsien, R. Y. & Steinhardt, R.A.

(1985) Nature (London) 315, 147-149.4. Twigg, J., Patel, R. & Whitaker, M. (1988) Nature (London)

332, 366-369.5. Vanderbilt, J. N., Bloom, K. S. & Anderson, J. N. (1982) J.

Biol. Chem. 257, 13009-13017.6. Chafouleas, J. C., Bolton, W. E. and Means, A. R. (1984)

Science 224, 1346-1348.7. White, B. A. (1985) J. Biol. Chem. 260, 9341-9344.8. Bonner, W. M. (1987) in The Cell Nucleus, ed. Bush, H.

(Academic, New York), Vol. 6, pp. 97-148.9. Paine, P. L. & Horowitz, S. B. (1980) in Cell Biology: A

Comprehensive Treatise, eds. Prescot, D. M. & Goldstein, L.(Academic, New York), Vol. 4, pp. 299-338.

10. Williams, D. A., Fogarty, K. E., Tsien, R. Y. & Fay, F. S.(1985) Nature (London) 318, 558-560.

11. Williams, D. A., Becker, P. L. & Fay, F. S. (1988) Science 235,1644-1648.

12. Kulikova, 0. G., Savostianov, G. A., Beliavsteva, L. M. &Razumovskaia, N. I. (1982) Biochimica 47, 1216-1221.

13. Harper, J. F. (1980) Proc. Natl. Acad. Sci. USA 77, 366-370.14. Bachs, 0. & Carafoli, E. (1988) J. Biol. Chem 262, 10786-

10790.15. Osheroff, N. & Zechiedrich, E. L. (1987) Biochemistry 26,

4303-4309.16. Jones, D. P., McConkey, D. J., Nicotera, P. & Orrenius, S.

(1989) J. Biol. Chem., in press.17. Gietzen, K., Wuthrich, A. & Bader, H. (1981) Biochem.

Biophys. Res. Commun. 101, 418-425.18. Bartfai, T. (1979) in Advances in Cyclic Nucleotide Research,

eds. Brooker, G., Greengard, P. & Robinson, G. A. (Raven,New York), Vol. 10, pp. 219-242.

19. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol.Chem. 260, 3440-3450.

20. Delfert, D. M. & McDonald, J. M. (1985) Arch. Biochem.Biophys. 241, 665-672.

21. Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien,P. J. & Orrenius S. (1987) FEBS Lett. 224, 331-336.

22. Moore, C. (1971) Biochem. Biophys. Res. Commun. 42, 298-305.

23. Lotersztajn, S., Hanoune, J. & Pecker, F. (1981) J. Biol. Chem.256, 11209-11215.

24. Malgaroli, A., Milani, D., Meldolesi, J. & Pozzan, T. (1987) J.Cell. Biol. 105, 2145-2155.

25. Oakes, S. G., Martin, W. M., II, Lisek, C. A. & Powis, G.(1988) Anal. Biochem. 169, 159-166.

Biochemistry: Nicotera et al.