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23Na NMR study of intracellular sodium ions in Dictyostelium discoideum amoeba
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 254, No. 2, May 1, pp. 559-567,1987
23Na NMR Study of Intracellular in Dictyostelium discoideum JEAN-BAPTISTE
MARTIN,*
GERARD
Sodium Ions Amoeba
KLEIN,?
AND
MICHEL
SATREt”
*DRF, SPh, R&mance Magn&que en Biologic et en h&?&x&e, and TDRF, Labratoire de Biochimie, UA 1130 du CNRS, Centre d’Etude.s Nuclhires, 85x, $8041 Grenoble C&, France Received
August
18,1986,
and in revised
form
November
26,1986
The intracellular
sodium concentration in the amoebae from the slime mold Dictyohas been studied using 23Na NMR. The 23Na resonances from intracellular and extracellular compartments could be observed separately in the presence of the anionic shift reagent Dy(PPPi)z- which does not enter into the amoebae and thus selectively affects Na+ in the extracellular space. 31P NMR was used to control the absence of cellular toxicity of the shift reagent. The intracellular Na+ content was calculated by comparison of the intensities of the two distinct peaks arising from the intra- and extracellular spaces. It remained low (0.6 to 3 mM) in the presence of external Na+ (20 to 70 mM), and a large Na+ gradient (20- to 40-fold) was maintained. A rapid reloading of cells previously depleted of Na+ was readily measured by %Na NMR. Nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular Na+ concentration. The time dependence of the %Na and 31P NMR spectra showed a rapid degradation of Dy(PPPi)z- which may be catalyzed by an acid phosphatase. stelium discoideum
0 1987 Academic Press. Inc.
The control of the concentrations of intracellular cations, such as H+, Na+, K+, Mg2+, and Ca2’, is of crucial importance for cells. In the social amoeba Dictyostelium disco&urn, it has been shown that the intracellular pH is highly regulated. Both cytoplasmic and mitochondrial pHs are kept constant when the external pH is varied over a large range (l-4). Na+ content was measured in submerged monolayer cultures and evidence for a moderate transmembrane gradient was presented (5). Na+ transport has not been studied in D. disco&urn, although an amiloride sensitive Na+ carrier was postulated to be present on the basis of intracellular pH recovery after an acid load (6). ?Na NMR has been used as a noninvasive technique to study Na+ in a variety of isolated cells (7-15) and tissues (16-19). The NMR signals from intra- and extracellular i To whom
correspondence
should
Na+ normally occur at the same frequency but the development of anionic paramagnetic shift reagents which do not penetrate the internal space of cells has made it possible to achieve a satisfactory separation between intra- and extracellular %a+ (2022). In the current report we describe the use of =Na NMR to study intracellular Na+ ions in D. discoideum amoebae. MATERIALS
AND
METHODS
ShQ? reagents. Dysprosium chloride, dysprosium oxide, sodium, and potassium tripolyphosphate were purchased from Alfa-Ventron. Triethylenetetramine hexaacetic acid (I-QITHA)2 was purchased from Fluka. NaTDy(PPP&, 3 NaCl was formed by titrating DyCla with Na,(PPPJ until the disappearance of the
’ Abbreviations used: PPPiq tripolyphosphate; I&TTHA, triethylenetetramine hexaacetic acid; Mes: 4-morpholineethanesulfonic acid; NTP, nueleotide triphosphate; NDP, nucleotide diphosphate; CCCP, carbonyl cyanide phenylhydrazone.
be addressed. 559
0003-9861/W $3.00 Copyright 8 1987 by Academic Press. Inc. All rights of reproduction in any form resewed.
560
MARTIN,
KLEIN,
white precipitate observed when molar ratios are lower than 1:2 (20). Stock solutions (50 mM in dysprosium, pH 7.5) were stored at 4°C and never kept more than 1 week. In the complex solution, the Na+ concentration measured by flame photometry was 525 + 29 mM (n = 4) and the phosphate content, measured calorimetrically after hydrolysis by 10 N HeS04, was found to be 308 + 14 mM (n = 5), in accordance with the expected concentrations. Stock solutions of TrisaDy(TTHA) (100 mM in dysprosium, pH 6.5) were prepared by the heterogenous reaction between stoichiometric amounts of DyaOa, HGTTHA, and Tris base (22). Cells. D discoideum strain AX2 (ATCC 24397) was grown at 22 + 0.5”C in axenic medium (23). Amoebae in their exponential phase of growth were collected at 2-4°C by eentrifugation at 800~ for 4 min in a Jouan GR4.11 centrifuge. The cell pellet was washed three times with ice-cold ‘20 mM Mes-Na buffer, pH 6.3, or with 20 mM Mes-10 mM Tris, pH 6.2. The Na+ concentration measured by flame-photometry was 10.4 mM in Mes-Na buffer. The washed cells were kept at 0°C as packed pellets, always less than 2 h, until used for the NMR measurements. IntraceUulur Na+ concentration Cell volume was determined with =Na NMR by comparing directly, at a given concentration of N~Dy(PPPi)Z, the resonance intensities of extracellular Na+ in the cell suspension and in the cell free supernatant obtained from the same suspension. Intracellular sodium concentration (Nai,) was calculated according to
(Nad = (N%,t) X (lin/Iout) X [(I- V,)/ Xl, where (Na& was the extracellular Na concentration, lin and Iout were respectively the resonance intensities of the intracellular and extracellular Na+ signals and V, was the fractional cellular volume in ml/ml suspension (20). NMR spectroscopy. NMR spectra were collected in the pulsed Fourier transform mode and at frequencies of 52.94 MHz for wa and 81.01 MHz for ‘iP in a Bruker WM200 spectrometer using a lo-mm NMR tube (%a) or a 25-mm NMR tube (*iP). For “Na experiments, interpulse intervals were 0.2 s and the flip-angle was 90’. For alP NMR experiments, conditions were as described previously (3,4). To perform wa or 31P NMR measurements, washed amoebae were suspended in the required buffer and supplemented with Da0 to give a final concentration of 6% (v/v). The total volume was 3 ml in a lo-mm NMR tube or 25 ml in a 25-mm tube and the cell concentrations were from 2 to 5 X 10Ecells/ml. When the small diameter tube was used, 1~1 antifoam A (Sigma) was added per each milliliter of medium to control frothing. The NMR spectrometer was field-frequency locked on the deuterium resonance from DsO present in the samples. The NMR tube was fitted with a poly(tetrafluoroethylene) insert through which the cell
AND
SATRE
suspension was oxygenated and simultaneously stirred with a steady stream of small oxygen bubbles (flow rate of about 20 ml/min). In the absence of bubbling, a rapid sedimentation of the cells would occur. In anaerobic experiments, nitrogen instead of oxygen was bubbled through the cell suspensions. All NMR measurements were conducted at a temperature of 21 * 2°C. All spectra shown were plotted with an artificial line broadening of 15 Hz for “P or 10 Hz for %Na. RESULTS
Cell Membrane Perrxeabilit~ Shift Reagents
to the
Separation of intra- and extracellular 23Na NMR signals required the addition of a shift reagent to the medium bathing the cells. Two different dysprosium complexes were investigated in this study. Dy(PPPi)$-, introduced by Gupta and Gupta (20), induced an upfield shift, while DY(TTHA)~(8, 22) caused a downfield shift. The reagents should not cross the plasma membrane or adversely affect the cell. This was examined in D. disco&urn amoeba by studying the 31P NMR signals of intracellular phosphorylated compounds. These metabolites, identified as described previously (l-4), were not qualitatively or quantitatively modified (Fig. 1A). For example, considering the /3P-NTP resonance, its peak width (100 Hz) was only slightly enlarged as compared to the value (80 Hz) in a control spectrum without shift reagent, and its intensity was not significantly reduced. 31P NMR spectra were followed as a function of time in the presence of the shift reagent. The cells remained impermeable to Dy(PPPi)i-, as assessed by the constancy of the /3P-NTP signal, but a progressive increase of a signal downfield of the intracellular Pi peaks at 2 to 4 ppm was observed (Figs. 1A and 1C). The compound responsible for this resonance remained mostly extracellular as shown directly by the respective spectra of the supernatant and of the cells separated by centrifugation (Fig. 1B). It was identified as extracellular Pi on the basis of the observation that the presence of N%Dy(PPPi)z caused a large
%Na
NMR
OF
Dictyostelium
dkcoideum
561
AMOEBAE
0
-20
-10
wm
0
-10
pm pm
-20
0
IO
20
30
TIME
~~INUTESI
40
50
60
FIG. 1. *‘P NMR spectra of 0. discoidearn amoebae in the presence of NqDy(PPPi)r. (A) Spectra were obtained every 10 min from oxygenated cell suspensions (4.0 X 10’ cells/ml) in 20 mM MesNa, pH 6.3. A control spectrum is shown in (a). The signals were identified as described previously (3,4) as peak 1, monophosphoesters; peak 2, Pi; peak 3, -rP-NTP and BP-NDP; peak 4, (YP-NTP and (YP-NDP; and peak 5, BP-NTP. Spectra obtained after addition of 2.8 mM N%Dy(PPPi)zp 8.4 mM NaCl are shown in (b) O-10 min, (c) 20-30 min, and (d) 50-60 min. Peak (6) corresponded to small amounts of long-chain polyphosphate contaminants brought by the shift reagent itself. The methylene diphosphonate signal (MDP) at 16.4 ppm arose from a sealed capillary tube serving as an external field standard. (B) After 60 min, cells were separated from their incubation medium by centrifugation at SOOg for 4 min and resuspended directly in 20 mM Mes-Na, pH 6.3, at the original cell concentration. Spectra of cells and incubation medium are presented in (e) and (f), respectively. (C) The evolution of the intensity of the Pi peak at 2 to 4 ppm was plotted as a function of time after addition of 2.8 mM NaTDy(PPP&, 8.4 mM NaCL Spectra were collected every 5 min, otherwise conditions were as described above.
downfield shift of Pi resonance. The increase in Pi was caused by a direct degradation of the shift reagent, Na,Dy(PPPi)z, by D. dticoideum amoebae. 31P NMR spectra of D. discoideum amoebae were also recorded in the presence of 10 mM DY(TTHA)~-, a concentration needed to obtain a Na+ shift of sufficient magnitude (8). A significant broadening of
intracellular resonance peaks of Pi and nucleotides was observed; for example, the peak width of the /LIP-NTP signal was 1’70 Hz, a twofold enlargement (Fig. 2). Either the D. discoideum plasma membrane did not remain fully impermeable to DY(TTHA)~or it was affected in another way. Despite the broadening of the intracellular signals, the intracellular NTP level
562
MARTIN,
al
KLEIN,
0
-10
SATRE
responded to extracellular Na+ (Na,,J whereas the unshifted signal was intracellular Na+ (Nai,). The upfield shift of the Naout peak was dependent on the shift reagent concentration but the value of the shift between NaOUt and N%,, was not stable and decreased as a function of time. Na,Dy(PpP& was degraded in the presence of D. discoideum cells, as was previously observed by 31P NMR (Fig. 1). The position of the Nain signal remained fixed, as opposed to the Naout peak which slowly shifted back to the Nai,, value. This led to a complex set of %Na NMR responses, as illustrated in Figure 4A. Representative decay curves for different concentrations of shift reagent are presented in Fig. 4B. The maximal separation between Naout and Nain extrapolated back to the time of addition of the shift reagent to the cells is presented as a function of Na,Dy(PPP& concentration (Fig. 4C).
MDP
10
AND
-20
pm
FIG. 2. ‘iP NMR spectra of D. disco&Gum amoebae in the presence of Tris,Dy(TTHA). Spectra were recorded from aerobic amoebae (3.7 X 10’ cells/ml) suspended in 20 mM Mes-10 mM Tris, pH 6.2, and containing either 0 (spectrum a) or 10 mM TrisJly(‘I”l’HA) (spectrum b). Data were originally collected every 5 min and found to be stable as a function of time. The traces presented were sums of individual spectra and corresponded to total accumulation times of 15 and 50 min, respectively. Mitochondrial, cytosolic, and external Pi were assigned as described in (1,3,4). For other peak identifications see Fig. 1.
Na: out
was similar to that of a control without shift reagent, indicating that the energetic machinery of the amoebae was not inhibited by DY(TTHA)~-. In contrast to what was observed with the other shift reagent, Na,Dy(PPP&, spectra were stable as a function of time. 40
20
-20
I
-40
P&
BNa NMR Detection
of Intracellular
Na
Figure 3 shows representative =Na spectra of an aerobic D. discoideum amoebae suspension at 22°C before and after addition of 3 InM NqDy(PPPi)z. In the presence of shift reagent two well-resolved peaks were observed. The upfield peak cor-
FIG. 3. =Na
NMR spectra of D. discoideum amoebae in the presence of NeDy(PPPi)ze Spectra were obtained from oxygenated cells (4.6 X 10’ cells/ml) suspended in 20 mM Mes-10 rnM Tris, pH 6.2, and containing 0 (a) or 2.8 rnM NRDy(PPP&, 8.4 m?d NaCl (b). Each spectrum corresponded to 6.7 min accumulation time. The scale expansion is given along the trace.
%a
,
r
I.
1
10
0
NMR
I
.
OF
Dictyostelium
.
I
-10
-
I
-20
dticoideum
AMOEBAE
563
-
-30
km
0 si” 2II . LT. 21 4
0
2
Na,Dy(PPPd2
4
6
(mm)
FIG. 4. Chemical shift difference between the intraand extracellular =Na resonances in D. diswideurn as a function of time and of N%Dy(PPPi)z concentration. (A) Aerobic amoebae suspended in 20 mM Mes-10 mM Tris, pH 6.2, at 4.0 X lo* cells/ml were supplemented at t = 0 min with 5.4 mM N%Dy(PPPi)a, 16.4 mM NaCl and 2-min spectra were recorded and stored. The trace displayed was the sum of eight individual spectra whose data collection was started at t = 0, 10,20,30,40,50,60, and 70 min. The first spectrum corresponded to the rightmost peak and the last spectrum to the leftmost peak in the constructed substructure of the Nav,,,t peak. Note the stability of the Nq. resonance position. (B) Data were obtained from spectra collected as a function of time and similar to that shown in A. The concentrations of NqDy(PPPi)c (mM) and of amoebae (cells/ml) were, respectively, (A) 1.9 and 3.8 X 108; (A) 2.8 and 4.6 X 108; (0) 3.7 and 3.8 X 10’; and (0) 5.4 and 3.8 X 10s. (C) The initial chemical shift differences between the intra- and extracellular %a resonances in LI. diswideum were derived from above data by extrapolation back to t = 0 and plotted as a function of N~$Y(PPP~)~ concentrations.
We were unable to detect a Nqn peak in D. discoideum in the presence of both externally added Na+ (up to 40 InM) and 10 IIIM TrissDy(TTHA). Since this reagent gave shifts of lower magnitude than Na,Dy(PPP&, this was likely to result from the low intensity of the Nai,, peak buried in the base of the large NaUt peak. A successful detection (Fig. 5) was achieved in the presence of the polyene antibiotic nystatin (see below), which increased the intracellular Na+ content. IntraceUular
Na+ Concentration
A knowledge of the cell volume was needed to calculate the Nain concentration.
From %a NMR and with the shift reagent, Dy(PPPi)i-, to define the extracellular space, the cellular volume was found to be 550 f 70 pm3 (n = 3). This value is identical to the one measured previously (24). From the integrated intensities of the Naout and Nain resonances and using the above determined cellular volume, the Nai,, concentrations were calculated for different Naout concentrations according to the formula given under Materials and Methods and are reported in Table I. When Naout was varied from 25 to 70 RIM, the Nai,, concentration was found to vary from 0.6 to about 2.5 mM. A marked Na+ gradient was thus present between the cell and the external medium. In the absence of added Na+ in the in-
564
MARTIN,
KLEIN,
,Na+in
x32
J1/ Na* in
(b)
(a)
/\
1 60
40
20
0 Ppm
-20
-40
-60
FIG. 5. %a NMR spectra of D. disco&urn amoebae in the presence of TrisaDy(TTHA). Aerobic amoebae (4.2 X lo* cells/ml) were suspended in 20 mM MesNa, pH 6.2, and spectrum (a) was recorded. They were then supplemented with 10 mM TrisxDy(TTHA) and 0.4 mg nystatin/ml. After 15 min of incubation, spectrum (b) was obtained. Accumulation times for spectra were 6.7 min.
cubation medium, and using cells previously washed in a buffer without Na+, the intracellular Na+ level was below the limits of detection (Fig. 6, spectrum b). Amoebae depleted of their intracellular Na+ were rapidly reloaded (half-time < 3 min) by addition of NaCl (Fig. 6). Anaerobiosis or the presence of 20 PM carbonyl cyanide phenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, have been shown to deplete cell ATP and to collapse the intracellular pH gradients (1, 3, 4). As shown in Fig. ‘7, they had no drastic effect on the intensity of the Nain signal, except for a moderate increase (about 30%). Perturbation of Intracellular Ngstutin: NlKR Visibility Polyene antibiotics, such tin B or nystatin, bind to
Na+ by of Na+
as amphoteristerols in the
AND
SATRE
membranes and cause modification of their permeability characteristics. An enhancement of Nain with amphotericin B or nystatin has been demonstrated in renal tubule preparations (18, 19). D. discoideum amoebae have been shown to be sensitive to nystatin, and sterols can be detected in plasma and endosomal membranes (25,26). Accordingly, the N%,, peak increased about ZO-fold in the presence of nystatin (0.7 mg/ ml), as compared to a control without antibiotic as shown in Fig. 7. The increase in the Nai,, signal reflected a tendency toward equilibration of the Na+ gradient. NMR visibility of Na+ in D. discoideum was determined by comparing the changes in the areas of the Nai,, and Naout resonances following Na+ entry (7,lZ). The relative visibility of the two Na+ pools was equal to the ratio of the increase in Nain to the decrease in NaoUt. From the data shown in Fig. 7, most of the intracellular Na+ (more than 75%) was found to be NMR visible. DISCUSSION
Nondestructive =Na NMR spectroscopy was used successfully to measure intracellular Naf in D, diswideum amoebae. The major experimental problem encountered during these determinations is linked to stability of the shift reagent, Dy(PPPi)P-, itself. During a limited period after the
TABLE INTRACELLULAR
D. disctideum EXTERNAL
Na+
CONCENTRATION
(Nai,)
AMOEBAE AS A FUNCTION Na+ CONCENTRATION (Na,,J
Na,,t
Nain
MM)
(n-f)
0”
I
25
0.05 0.6
39 48 63 70
LO-l.6 1.3 1.4 1.7-3.2
IN OF
Gradient (out/in) 42 24-39 37 45 22-41
a This experiment was conducted with cells washed and suspended in 20 mM Mes-Tris, pH 6.2, and in the presence of 2.8 my KIDy(PPPi)a, 8.4 mM KCl.
%Na
NMR
Dictyostelium
OF
dticoideum
565
AMOEBAE
I
cNa+
i-
out
T
30
5 TIME
20
10
10 ( MINUTES)
-10
-20
15
-30
20
FIG. 6. Kinetics of Na+ entry in D. discoi&eum amoebae. Cells (6.2 X 10’ cells/ml) in 20 mM Mes10 mM Tris, pH 6.2, were bubbled with oxygen and spectrum (a) was recorded before addition of 2.8 mM K,Dy(PPP&, 8.4 mM KC1 (spectrum b). At time t = 0 min, 40 mM NaCl was added and Na+ entry was followed as a function of time. Spectrum (c), shown as an example, was recorded at t = 13.3 min after the addition of NaCl. All NMR spectra were recorded during periods of 3.3 min. Intracellular Na+ (0) calculated as described under Materials and Methods is represented as a function of time.
addition of the shift reagent (up to about 1 h), the quantitative determination of intracellular Na+ is easily achieved. A rapid hydrolysis of the PPPi ligand precludes extended measurements. D. discoideum amoebae are known to possessintracellular phosphatases and enzymes of polyphosphate metabolism and also to secrete actively lysosomal enzymes in a process linked to endocytosis and membrane recycling (2’7-30). The degradation of Na,Dy(PPP& by living cells was also shown in Saccharcmzyces cerevisiae (7), Escherichia coli (12), brain or muscle tissue (31).
D. disco&urn amoebae can be depleted of their intracellular Na+ by incubation in a Nat-free medium, and rapidly reloaded by addition of Na+. Intracellular Na+ content was found to respond in a predictable manner to perturbation of plasma membrane permeability by a polyene antibiotic. In the presence of external Na+, D. discoideum amoebae have a low intracellular sodium content which results in a large inwardly directed Na+ gradient. Under anoxic conditions, or in the presence of an uncoupler of oxidative phosphorylation, there was no significant change in the level of intracellular Na+ and thus cells main-
566
MARTIN,
10
0 mm
-10
KLEIN,
-20
AND
0
SATRE
10 TIME
20 30 (MINUTES)
40
FIG. 7. Effect of anaerobiosis, CCCP, and nystatin on intracellular Na+ in D. discoideum amoebae. Amoebae (3.8 X lO* cells/ml) were suspended in 20 mM Mes-Na, 3 mrd NarDy(PPPi)e, 9 mM NaCI, pH 6.3. The total Na* concentration was 40 mM. As indicated by the arrow, one sample was supplemented with nystatin (0.7 mg/ml) (0) and another with 20 pM CCCP (0). In the third, anaerobic conditions were ensured by nitrogen bubbling instead of oxygen (Cl). Spectra were collected and intracellular Na+ concentrations were calculated. (A) Spectrum shown in (a) was a control spectrum before addition of nystatin. Spectra (b) and (c) were obtained after 6.7 and 27 min of incubation with nystatin, respectively. (B) Intracellular Na+ is represented as a function of time.
tain a 20- to IO-fold gradient under conditions where intracellular ATP content is negligible. A similar situation was reported in yeast during a OS/N2 cycle (7) and is possibly linked to a very restricted passive permeability of the cell plasma membrane toward Na+. A complication to an in-depth interpretation of the =Na NMR results arises because of the existence of different intracellular compartments such as endocytic vacuoles or mitochondria. The NMR method gives a measure of the total intracellular amount of Na+ but does not show whether Na+ gradients exist within the cell and are affected in various ways. The presence in mitochondria of a rapid (Na+-H+) antiport has been documented and its function was proposed to be linked to the regulation of the pH difference between the mitochondrial matrix and the cytosol (32,33). In conclusion, 23NaNMR app ears to provide a convenient quantitative method to determine intracellular Na+ in intact D. disco&urn amoebae. Dy(PPPi)z- has no apparent toxic effects on the cells and continuous measurements can be made with reasonably short-time resolution.
ACKNOWLEDGMENTS We thank Professor P. V. Vignais for his generous support and Dr. Joel Lunardi for his help with the flame-photometry measurements. This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS/UA 1130), and from the Institut National de la Sante et de la Recherche Medicale. REFERENCES 1. JENTOFT, J. E., AND TOWN, C. D. (1985) J. CeUBiol. 101,778-784. 2. KAY, R. R., GADIAN, D. G., AND WILLIAMS, S. R. (1986) J. CeU Sci 83,165179. 3. SATRE, M., AND MARTIN, J.-B. (1985) B&hem Bio-phys. Res. Cornmun 132,140-146. 4. SATRE, M., KLEIN, G., AND MARTIN, J.-B. (1986) Biochimie 68,1253-1261. 5. MARIN, F. T., AND ROTHMAN, F. G. (1980) J. Cell Biol
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6. JAMIESON, G. A., JR., FRAZIER, W. A., AND SCHLESINGER, P. H. (1984) .I. CeU BioL 99,1883-1887. 7. OGINO, T., DEN HOLLANDER, J. A., AND SHULMAN, R. G. (1983) Proc Nati Ad .‘% USA SO, 51855189. 8. PIKE, M. M., FOSSEL, E. T., SMITH, T. W., AND SPRINGER, C. S., JR. (1984) Amer. J. Physiol. 246 (CeU PhysioL 15), C528-C536. 9. BOWLANGER, Y., VINAY, O., AND DESROCHES, M. (1985) Biophys. J. 47,553-561.
BNa
NMR
OF
Dictyostelium
10. PETTEGREW, J. W., WOESSNER, D. E., MINSHEW, N. J., AND GLONEK, T. (1984) J. Magn Res. 57, 185-196. 11. WITTENBERG, B. A., AND GUPTA, R. K. (1985) J. Biol Chem 260,2031-2034. 12. CASTLE, A. M., MACNAB, R. M., AND SHIJLMAN, R. G. (1986) J. BioL Chem. 261,3288-3294. 13. CASTLE, A. M., MACNAB, R. M., AND SHULMAN, R. G. (1986) J. BioL Chem. 261,7797-‘7806. 14. GUPTA, R. K., KOSTELLOW, A. B., AND MORRILL, G. A. (1985) J. Biol Chem 260,9203-9208. 15. MORRILL, G. A., WEINSTEIN, S. P., KOSTELLOW, A. B., AND GUPTA, R. K. (1985) Biochim. Biophys. Acta 844,377-392. 16. CIVAN, M. M., DEGANI, H., MARGALIT, Y., AND SHPORER, M. (1983) Amer. J. Physiol. 245 (Cell Physiol la), C213-C219. 17. FOSSARELLO, M., ORZALESI, N., CORONGIU, F. P., BIAGINI, S., CASU, M., AND LAI, A. (1985) FEBS Lett. 184,245-248. 18. GLUANS, S. R., AVISON, M. J., OGINO, T., GIEBISCH, G., AND SHIJLMAN, R. G. (1985) Amer. J. Physiol. 249 (Renal, Fluid, Electrolyt. PhysioL 18), F160F168. 19. RAYSON, B. M., AND GUPTA, R. K. (1985) J. Biol. Chem. 260,7276-7280. 20. GUPTA, R. K., AND GUPTA, P. (1982) J. Mag% Reson 47,344-350.
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21. GUPTA, R. K., AND GUPTA, P. (1984) Annu. Rev. Biophys Bioeng. 13,221-246. 22. CHU, S. C., PIKE, M. M., FOSSEL, E. T., SMITH, T. W., BALSCHI, J. A., AND SPRINGER, C. S., JR. (1984) J. Magn Reson. 56,33-47. 23. WATTS, D. J., AND ASHWORTH, J. M. (1970) Biochem J. 119,171-174. 24. KLEIN, G., AND SATRE, M. (1986) Biochem Biophys. Res. Commun. 138,1146-1152. 25. BARRAND, P., PEREIRA DA SILVA, L., AND BRACHET, P. (1974) Bull Sot. Fr. Mycol Med 3,29-32. 26. FAVARD-SERENO, C., LUDOVSKY, M.-A., AND RYTER, A. (1981) J. Cell Sci. 51,63-84. 27. WIENER, E., AND ASHWORTH, J. M. (1970) Biochem J 118,505-512. 28. GEZELIUS, K. (1974) Arch. MicrobioL 98,311-329. 29. DIMOND, R. L., BURNS, R. A., AND JORDAN, K. B. (1981) J. Biol. Chem. 256,6565-6572. 30. LOOMIS, W. F., AND KUSPA, A. (1984) Dev. BioL 102,498-503. 31. MATWIYOFF, N. A., GASPAROVIC, C., WENK, R., WICKS, J. D., AND RATH, A. (1986) Magn. Reson Med. 3,164-168. 32. MITCHELL, P., AND MOYLE, J. (1967) Biochem. J. 105,1147-1162. 33. TYLER, D. D., AND SUITON, C. M. (1984) in Membrane Structure and Function (Bittar, E. E., Ed.), pp. 181-270, Wiley, New York.
23Na NMR Study of Intracellular in Dictyostelium discoideum JEAN-BAPTISTE
MARTIN,*
GERARD
Sodium Ions Amoeba
KLEIN,?
AND
MICHEL
SATREt”
*DRF, SPh, R&mance Magn&que en Biologic et en h&?&x&e, and TDRF, Labratoire de Biochimie, UA 1130 du CNRS, Centre d’Etude.s Nuclhires, 85x, $8041 Grenoble C&, France Received
August
18,1986,
and in revised
form
November
26,1986
The intracellular
sodium concentration in the amoebae from the slime mold Dictyohas been studied using 23Na NMR. The 23Na resonances from intracellular and extracellular compartments could be observed separately in the presence of the anionic shift reagent Dy(PPPi)z- which does not enter into the amoebae and thus selectively affects Na+ in the extracellular space. 31P NMR was used to control the absence of cellular toxicity of the shift reagent. The intracellular Na+ content was calculated by comparison of the intensities of the two distinct peaks arising from the intra- and extracellular spaces. It remained low (0.6 to 3 mM) in the presence of external Na+ (20 to 70 mM), and a large Na+ gradient (20- to 40-fold) was maintained. A rapid reloading of cells previously depleted of Na+ was readily measured by %Na NMR. Nystatin, an antibiotic known to perturb the ion permeability of membranes, increased the intracellular Na+ concentration. The time dependence of the %Na and 31P NMR spectra showed a rapid degradation of Dy(PPPi)z- which may be catalyzed by an acid phosphatase. stelium discoideum
0 1987 Academic Press. Inc.
The control of the concentrations of intracellular cations, such as H+, Na+, K+, Mg2+, and Ca2’, is of crucial importance for cells. In the social amoeba Dictyostelium disco&urn, it has been shown that the intracellular pH is highly regulated. Both cytoplasmic and mitochondrial pHs are kept constant when the external pH is varied over a large range (l-4). Na+ content was measured in submerged monolayer cultures and evidence for a moderate transmembrane gradient was presented (5). Na+ transport has not been studied in D. disco&urn, although an amiloride sensitive Na+ carrier was postulated to be present on the basis of intracellular pH recovery after an acid load (6). ?Na NMR has been used as a noninvasive technique to study Na+ in a variety of isolated cells (7-15) and tissues (16-19). The NMR signals from intra- and extracellular i To whom
correspondence
should
Na+ normally occur at the same frequency but the development of anionic paramagnetic shift reagents which do not penetrate the internal space of cells has made it possible to achieve a satisfactory separation between intra- and extracellular %a+ (2022). In the current report we describe the use of =Na NMR to study intracellular Na+ ions in D. discoideum amoebae. MATERIALS
AND
METHODS
ShQ? reagents. Dysprosium chloride, dysprosium oxide, sodium, and potassium tripolyphosphate were purchased from Alfa-Ventron. Triethylenetetramine hexaacetic acid (I-QITHA)2 was purchased from Fluka. NaTDy(PPP&, 3 NaCl was formed by titrating DyCla with Na,(PPPJ until the disappearance of the
’ Abbreviations used: PPPiq tripolyphosphate; I&TTHA, triethylenetetramine hexaacetic acid; Mes: 4-morpholineethanesulfonic acid; NTP, nueleotide triphosphate; NDP, nucleotide diphosphate; CCCP, carbonyl cyanide phenylhydrazone.
be addressed. 559
0003-9861/W $3.00 Copyright 8 1987 by Academic Press. Inc. All rights of reproduction in any form resewed.
560
MARTIN,
KLEIN,
white precipitate observed when molar ratios are lower than 1:2 (20). Stock solutions (50 mM in dysprosium, pH 7.5) were stored at 4°C and never kept more than 1 week. In the complex solution, the Na+ concentration measured by flame photometry was 525 + 29 mM (n = 4) and the phosphate content, measured calorimetrically after hydrolysis by 10 N HeS04, was found to be 308 + 14 mM (n = 5), in accordance with the expected concentrations. Stock solutions of TrisaDy(TTHA) (100 mM in dysprosium, pH 6.5) were prepared by the heterogenous reaction between stoichiometric amounts of DyaOa, HGTTHA, and Tris base (22). Cells. D discoideum strain AX2 (ATCC 24397) was grown at 22 + 0.5”C in axenic medium (23). Amoebae in their exponential phase of growth were collected at 2-4°C by eentrifugation at 800~ for 4 min in a Jouan GR4.11 centrifuge. The cell pellet was washed three times with ice-cold ‘20 mM Mes-Na buffer, pH 6.3, or with 20 mM Mes-10 mM Tris, pH 6.2. The Na+ concentration measured by flame-photometry was 10.4 mM in Mes-Na buffer. The washed cells were kept at 0°C as packed pellets, always less than 2 h, until used for the NMR measurements. IntraceUulur Na+ concentration Cell volume was determined with =Na NMR by comparing directly, at a given concentration of N~Dy(PPPi)Z, the resonance intensities of extracellular Na+ in the cell suspension and in the cell free supernatant obtained from the same suspension. Intracellular sodium concentration (Nai,) was calculated according to
(Nad = (N%,t) X (lin/Iout) X [(I- V,)/ Xl, where (Na& was the extracellular Na concentration, lin and Iout were respectively the resonance intensities of the intracellular and extracellular Na+ signals and V, was the fractional cellular volume in ml/ml suspension (20). NMR spectroscopy. NMR spectra were collected in the pulsed Fourier transform mode and at frequencies of 52.94 MHz for wa and 81.01 MHz for ‘iP in a Bruker WM200 spectrometer using a lo-mm NMR tube (%a) or a 25-mm NMR tube (*iP). For “Na experiments, interpulse intervals were 0.2 s and the flip-angle was 90’. For alP NMR experiments, conditions were as described previously (3,4). To perform wa or 31P NMR measurements, washed amoebae were suspended in the required buffer and supplemented with Da0 to give a final concentration of 6% (v/v). The total volume was 3 ml in a lo-mm NMR tube or 25 ml in a 25-mm tube and the cell concentrations were from 2 to 5 X 10Ecells/ml. When the small diameter tube was used, 1~1 antifoam A (Sigma) was added per each milliliter of medium to control frothing. The NMR spectrometer was field-frequency locked on the deuterium resonance from DsO present in the samples. The NMR tube was fitted with a poly(tetrafluoroethylene) insert through which the cell
AND
SATRE
suspension was oxygenated and simultaneously stirred with a steady stream of small oxygen bubbles (flow rate of about 20 ml/min). In the absence of bubbling, a rapid sedimentation of the cells would occur. In anaerobic experiments, nitrogen instead of oxygen was bubbled through the cell suspensions. All NMR measurements were conducted at a temperature of 21 * 2°C. All spectra shown were plotted with an artificial line broadening of 15 Hz for “P or 10 Hz for %Na. RESULTS
Cell Membrane Perrxeabilit~ Shift Reagents
to the
Separation of intra- and extracellular 23Na NMR signals required the addition of a shift reagent to the medium bathing the cells. Two different dysprosium complexes were investigated in this study. Dy(PPPi)$-, introduced by Gupta and Gupta (20), induced an upfield shift, while DY(TTHA)~(8, 22) caused a downfield shift. The reagents should not cross the plasma membrane or adversely affect the cell. This was examined in D. disco&urn amoeba by studying the 31P NMR signals of intracellular phosphorylated compounds. These metabolites, identified as described previously (l-4), were not qualitatively or quantitatively modified (Fig. 1A). For example, considering the /3P-NTP resonance, its peak width (100 Hz) was only slightly enlarged as compared to the value (80 Hz) in a control spectrum without shift reagent, and its intensity was not significantly reduced. 31P NMR spectra were followed as a function of time in the presence of the shift reagent. The cells remained impermeable to Dy(PPPi)i-, as assessed by the constancy of the /3P-NTP signal, but a progressive increase of a signal downfield of the intracellular Pi peaks at 2 to 4 ppm was observed (Figs. 1A and 1C). The compound responsible for this resonance remained mostly extracellular as shown directly by the respective spectra of the supernatant and of the cells separated by centrifugation (Fig. 1B). It was identified as extracellular Pi on the basis of the observation that the presence of N%Dy(PPPi)z caused a large
%Na
NMR
OF
Dictyostelium
dkcoideum
561
AMOEBAE
0
-20
-10
wm
0
-10
pm pm
-20
0
IO
20
30
TIME
~~INUTESI
40
50
60
FIG. 1. *‘P NMR spectra of 0. discoidearn amoebae in the presence of NqDy(PPPi)r. (A) Spectra were obtained every 10 min from oxygenated cell suspensions (4.0 X 10’ cells/ml) in 20 mM MesNa, pH 6.3. A control spectrum is shown in (a). The signals were identified as described previously (3,4) as peak 1, monophosphoesters; peak 2, Pi; peak 3, -rP-NTP and BP-NDP; peak 4, (YP-NTP and (YP-NDP; and peak 5, BP-NTP. Spectra obtained after addition of 2.8 mM N%Dy(PPPi)zp 8.4 mM NaCl are shown in (b) O-10 min, (c) 20-30 min, and (d) 50-60 min. Peak (6) corresponded to small amounts of long-chain polyphosphate contaminants brought by the shift reagent itself. The methylene diphosphonate signal (MDP) at 16.4 ppm arose from a sealed capillary tube serving as an external field standard. (B) After 60 min, cells were separated from their incubation medium by centrifugation at SOOg for 4 min and resuspended directly in 20 mM Mes-Na, pH 6.3, at the original cell concentration. Spectra of cells and incubation medium are presented in (e) and (f), respectively. (C) The evolution of the intensity of the Pi peak at 2 to 4 ppm was plotted as a function of time after addition of 2.8 mM NaTDy(PPP&, 8.4 mM NaCL Spectra were collected every 5 min, otherwise conditions were as described above.
downfield shift of Pi resonance. The increase in Pi was caused by a direct degradation of the shift reagent, Na,Dy(PPPi)z, by D. dticoideum amoebae. 31P NMR spectra of D. discoideum amoebae were also recorded in the presence of 10 mM DY(TTHA)~-, a concentration needed to obtain a Na+ shift of sufficient magnitude (8). A significant broadening of
intracellular resonance peaks of Pi and nucleotides was observed; for example, the peak width of the /LIP-NTP signal was 1’70 Hz, a twofold enlargement (Fig. 2). Either the D. discoideum plasma membrane did not remain fully impermeable to DY(TTHA)~or it was affected in another way. Despite the broadening of the intracellular signals, the intracellular NTP level
562
MARTIN,
al
KLEIN,
0
-10
SATRE
responded to extracellular Na+ (Na,,J whereas the unshifted signal was intracellular Na+ (Nai,). The upfield shift of the Naout peak was dependent on the shift reagent concentration but the value of the shift between NaOUt and N%,, was not stable and decreased as a function of time. Na,Dy(PpP& was degraded in the presence of D. discoideum cells, as was previously observed by 31P NMR (Fig. 1). The position of the Nain signal remained fixed, as opposed to the Naout peak which slowly shifted back to the Nai,, value. This led to a complex set of %Na NMR responses, as illustrated in Figure 4A. Representative decay curves for different concentrations of shift reagent are presented in Fig. 4B. The maximal separation between Naout and Nain extrapolated back to the time of addition of the shift reagent to the cells is presented as a function of Na,Dy(PPP& concentration (Fig. 4C).
MDP
10
AND
-20
pm
FIG. 2. ‘iP NMR spectra of D. disco&Gum amoebae in the presence of Tris,Dy(TTHA). Spectra were recorded from aerobic amoebae (3.7 X 10’ cells/ml) suspended in 20 mM Mes-10 mM Tris, pH 6.2, and containing either 0 (spectrum a) or 10 mM TrisJly(‘I”l’HA) (spectrum b). Data were originally collected every 5 min and found to be stable as a function of time. The traces presented were sums of individual spectra and corresponded to total accumulation times of 15 and 50 min, respectively. Mitochondrial, cytosolic, and external Pi were assigned as described in (1,3,4). For other peak identifications see Fig. 1.
Na: out
was similar to that of a control without shift reagent, indicating that the energetic machinery of the amoebae was not inhibited by DY(TTHA)~-. In contrast to what was observed with the other shift reagent, Na,Dy(PPP&, spectra were stable as a function of time. 40
20
-20
I
-40
P&
BNa NMR Detection
of Intracellular
Na
Figure 3 shows representative =Na spectra of an aerobic D. discoideum amoebae suspension at 22°C before and after addition of 3 InM NqDy(PPPi)z. In the presence of shift reagent two well-resolved peaks were observed. The upfield peak cor-
FIG. 3. =Na
NMR spectra of D. discoideum amoebae in the presence of NeDy(PPPi)ze Spectra were obtained from oxygenated cells (4.6 X 10’ cells/ml) suspended in 20 mM Mes-10 rnM Tris, pH 6.2, and containing 0 (a) or 2.8 rnM NRDy(PPP&, 8.4 m?d NaCl (b). Each spectrum corresponded to 6.7 min accumulation time. The scale expansion is given along the trace.
%a
,
r
I.
1
10
0
NMR
I
.
OF
Dictyostelium
.
I
-10
-
I
-20
dticoideum
AMOEBAE
563
-
-30
km
0 si” 2II . LT. 21 4
0
2
Na,Dy(PPPd2
4
6
(mm)
FIG. 4. Chemical shift difference between the intraand extracellular =Na resonances in D. diswideurn as a function of time and of N%Dy(PPPi)z concentration. (A) Aerobic amoebae suspended in 20 mM Mes-10 mM Tris, pH 6.2, at 4.0 X lo* cells/ml were supplemented at t = 0 min with 5.4 mM N%Dy(PPPi)a, 16.4 mM NaCl and 2-min spectra were recorded and stored. The trace displayed was the sum of eight individual spectra whose data collection was started at t = 0, 10,20,30,40,50,60, and 70 min. The first spectrum corresponded to the rightmost peak and the last spectrum to the leftmost peak in the constructed substructure of the Nav,,,t peak. Note the stability of the Nq. resonance position. (B) Data were obtained from spectra collected as a function of time and similar to that shown in A. The concentrations of NqDy(PPPi)c (mM) and of amoebae (cells/ml) were, respectively, (A) 1.9 and 3.8 X 108; (A) 2.8 and 4.6 X 108; (0) 3.7 and 3.8 X 10’; and (0) 5.4 and 3.8 X 10s. (C) The initial chemical shift differences between the intra- and extracellular %a resonances in LI. diswideum were derived from above data by extrapolation back to t = 0 and plotted as a function of N~$Y(PPP~)~ concentrations.
We were unable to detect a Nqn peak in D. discoideum in the presence of both externally added Na+ (up to 40 InM) and 10 IIIM TrissDy(TTHA). Since this reagent gave shifts of lower magnitude than Na,Dy(PPP&, this was likely to result from the low intensity of the Nai,, peak buried in the base of the large NaUt peak. A successful detection (Fig. 5) was achieved in the presence of the polyene antibiotic nystatin (see below), which increased the intracellular Na+ content. IntraceUular
Na+ Concentration
A knowledge of the cell volume was needed to calculate the Nain concentration.
From %a NMR and with the shift reagent, Dy(PPPi)i-, to define the extracellular space, the cellular volume was found to be 550 f 70 pm3 (n = 3). This value is identical to the one measured previously (24). From the integrated intensities of the Naout and Nain resonances and using the above determined cellular volume, the Nai,, concentrations were calculated for different Naout concentrations according to the formula given under Materials and Methods and are reported in Table I. When Naout was varied from 25 to 70 RIM, the Nai,, concentration was found to vary from 0.6 to about 2.5 mM. A marked Na+ gradient was thus present between the cell and the external medium. In the absence of added Na+ in the in-
564
MARTIN,
KLEIN,
,Na+in
x32
J1/ Na* in
(b)
(a)
/\
1 60
40
20
0 Ppm
-20
-40
-60
FIG. 5. %a NMR spectra of D. disco&urn amoebae in the presence of TrisaDy(TTHA). Aerobic amoebae (4.2 X lo* cells/ml) were suspended in 20 mM MesNa, pH 6.2, and spectrum (a) was recorded. They were then supplemented with 10 mM TrisxDy(TTHA) and 0.4 mg nystatin/ml. After 15 min of incubation, spectrum (b) was obtained. Accumulation times for spectra were 6.7 min.
cubation medium, and using cells previously washed in a buffer without Na+, the intracellular Na+ level was below the limits of detection (Fig. 6, spectrum b). Amoebae depleted of their intracellular Na+ were rapidly reloaded (half-time < 3 min) by addition of NaCl (Fig. 6). Anaerobiosis or the presence of 20 PM carbonyl cyanide phenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, have been shown to deplete cell ATP and to collapse the intracellular pH gradients (1, 3, 4). As shown in Fig. ‘7, they had no drastic effect on the intensity of the Nain signal, except for a moderate increase (about 30%). Perturbation of Intracellular Ngstutin: NlKR Visibility Polyene antibiotics, such tin B or nystatin, bind to
Na+ by of Na+
as amphoteristerols in the
AND
SATRE
membranes and cause modification of their permeability characteristics. An enhancement of Nain with amphotericin B or nystatin has been demonstrated in renal tubule preparations (18, 19). D. discoideum amoebae have been shown to be sensitive to nystatin, and sterols can be detected in plasma and endosomal membranes (25,26). Accordingly, the N%,, peak increased about ZO-fold in the presence of nystatin (0.7 mg/ ml), as compared to a control without antibiotic as shown in Fig. 7. The increase in the Nai,, signal reflected a tendency toward equilibration of the Na+ gradient. NMR visibility of Na+ in D. discoideum was determined by comparing the changes in the areas of the Nai,, and Naout resonances following Na+ entry (7,lZ). The relative visibility of the two Na+ pools was equal to the ratio of the increase in Nain to the decrease in NaoUt. From the data shown in Fig. 7, most of the intracellular Na+ (more than 75%) was found to be NMR visible. DISCUSSION
Nondestructive =Na NMR spectroscopy was used successfully to measure intracellular Naf in D, diswideum amoebae. The major experimental problem encountered during these determinations is linked to stability of the shift reagent, Dy(PPPi)P-, itself. During a limited period after the
TABLE INTRACELLULAR
D. disctideum EXTERNAL
Na+
CONCENTRATION
(Nai,)
AMOEBAE AS A FUNCTION Na+ CONCENTRATION (Na,,J
Na,,t
Nain
MM)
(n-f)
0”
I
25
0.05 0.6
39 48 63 70
LO-l.6 1.3 1.4 1.7-3.2
IN OF
Gradient (out/in) 42 24-39 37 45 22-41
a This experiment was conducted with cells washed and suspended in 20 mM Mes-Tris, pH 6.2, and in the presence of 2.8 my KIDy(PPPi)a, 8.4 mM KCl.
%Na
NMR
Dictyostelium
OF
dticoideum
565
AMOEBAE
I
cNa+
i-
out
T
30
5 TIME
20
10
10 ( MINUTES)
-10
-20
15
-30
20
FIG. 6. Kinetics of Na+ entry in D. discoi&eum amoebae. Cells (6.2 X 10’ cells/ml) in 20 mM Mes10 mM Tris, pH 6.2, were bubbled with oxygen and spectrum (a) was recorded before addition of 2.8 mM K,Dy(PPP&, 8.4 mM KC1 (spectrum b). At time t = 0 min, 40 mM NaCl was added and Na+ entry was followed as a function of time. Spectrum (c), shown as an example, was recorded at t = 13.3 min after the addition of NaCl. All NMR spectra were recorded during periods of 3.3 min. Intracellular Na+ (0) calculated as described under Materials and Methods is represented as a function of time.
addition of the shift reagent (up to about 1 h), the quantitative determination of intracellular Na+ is easily achieved. A rapid hydrolysis of the PPPi ligand precludes extended measurements. D. discoideum amoebae are known to possessintracellular phosphatases and enzymes of polyphosphate metabolism and also to secrete actively lysosomal enzymes in a process linked to endocytosis and membrane recycling (2’7-30). The degradation of Na,Dy(PPP& by living cells was also shown in Saccharcmzyces cerevisiae (7), Escherichia coli (12), brain or muscle tissue (31).
D. disco&urn amoebae can be depleted of their intracellular Na+ by incubation in a Nat-free medium, and rapidly reloaded by addition of Na+. Intracellular Na+ content was found to respond in a predictable manner to perturbation of plasma membrane permeability by a polyene antibiotic. In the presence of external Na+, D. discoideum amoebae have a low intracellular sodium content which results in a large inwardly directed Na+ gradient. Under anoxic conditions, or in the presence of an uncoupler of oxidative phosphorylation, there was no significant change in the level of intracellular Na+ and thus cells main-
566
MARTIN,
10
0 mm
-10
KLEIN,
-20
AND
0
SATRE
10 TIME
20 30 (MINUTES)
40
FIG. 7. Effect of anaerobiosis, CCCP, and nystatin on intracellular Na+ in D. discoideum amoebae. Amoebae (3.8 X lO* cells/ml) were suspended in 20 mM Mes-Na, 3 mrd NarDy(PPPi)e, 9 mM NaCI, pH 6.3. The total Na* concentration was 40 mM. As indicated by the arrow, one sample was supplemented with nystatin (0.7 mg/ml) (0) and another with 20 pM CCCP (0). In the third, anaerobic conditions were ensured by nitrogen bubbling instead of oxygen (Cl). Spectra were collected and intracellular Na+ concentrations were calculated. (A) Spectrum shown in (a) was a control spectrum before addition of nystatin. Spectra (b) and (c) were obtained after 6.7 and 27 min of incubation with nystatin, respectively. (B) Intracellular Na+ is represented as a function of time.
tain a 20- to IO-fold gradient under conditions where intracellular ATP content is negligible. A similar situation was reported in yeast during a OS/N2 cycle (7) and is possibly linked to a very restricted passive permeability of the cell plasma membrane toward Na+. A complication to an in-depth interpretation of the =Na NMR results arises because of the existence of different intracellular compartments such as endocytic vacuoles or mitochondria. The NMR method gives a measure of the total intracellular amount of Na+ but does not show whether Na+ gradients exist within the cell and are affected in various ways. The presence in mitochondria of a rapid (Na+-H+) antiport has been documented and its function was proposed to be linked to the regulation of the pH difference between the mitochondrial matrix and the cytosol (32,33). In conclusion, 23NaNMR app ears to provide a convenient quantitative method to determine intracellular Na+ in intact D. disco&urn amoebae. Dy(PPPi)z- has no apparent toxic effects on the cells and continuous measurements can be made with reasonably short-time resolution.
ACKNOWLEDGMENTS We thank Professor P. V. Vignais for his generous support and Dr. Joel Lunardi for his help with the flame-photometry measurements. This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS/UA 1130), and from the Institut National de la Sante et de la Recherche Medicale. REFERENCES 1. JENTOFT, J. E., AND TOWN, C. D. (1985) J. CeUBiol. 101,778-784. 2. KAY, R. R., GADIAN, D. G., AND WILLIAMS, S. R. (1986) J. CeU Sci 83,165179. 3. SATRE, M., AND MARTIN, J.-B. (1985) B&hem Bio-phys. Res. Cornmun 132,140-146. 4. SATRE, M., KLEIN, G., AND MARTIN, J.-B. (1986) Biochimie 68,1253-1261. 5. MARIN, F. T., AND ROTHMAN, F. G. (1980) J. Cell Biol
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6. JAMIESON, G. A., JR., FRAZIER, W. A., AND SCHLESINGER, P. H. (1984) .I. CeU BioL 99,1883-1887. 7. OGINO, T., DEN HOLLANDER, J. A., AND SHULMAN, R. G. (1983) Proc Nati Ad .‘% USA SO, 51855189. 8. PIKE, M. M., FOSSEL, E. T., SMITH, T. W., AND SPRINGER, C. S., JR. (1984) Amer. J. Physiol. 246 (CeU PhysioL 15), C528-C536. 9. BOWLANGER, Y., VINAY, O., AND DESROCHES, M. (1985) Biophys. J. 47,553-561.
BNa
NMR
OF
Dictyostelium
10. PETTEGREW, J. W., WOESSNER, D. E., MINSHEW, N. J., AND GLONEK, T. (1984) J. Magn Res. 57, 185-196. 11. WITTENBERG, B. A., AND GUPTA, R. K. (1985) J. Biol Chem 260,2031-2034. 12. CASTLE, A. M., MACNAB, R. M., AND SHIJLMAN, R. G. (1986) J. BioL Chem. 261,3288-3294. 13. CASTLE, A. M., MACNAB, R. M., AND SHULMAN, R. G. (1986) J. BioL Chem. 261,7797-‘7806. 14. GUPTA, R. K., KOSTELLOW, A. B., AND MORRILL, G. A. (1985) J. Biol Chem 260,9203-9208. 15. MORRILL, G. A., WEINSTEIN, S. P., KOSTELLOW, A. B., AND GUPTA, R. K. (1985) Biochim. Biophys. Acta 844,377-392. 16. CIVAN, M. M., DEGANI, H., MARGALIT, Y., AND SHPORER, M. (1983) Amer. J. Physiol. 245 (Cell Physiol la), C213-C219. 17. FOSSARELLO, M., ORZALESI, N., CORONGIU, F. P., BIAGINI, S., CASU, M., AND LAI, A. (1985) FEBS Lett. 184,245-248. 18. GLUANS, S. R., AVISON, M. J., OGINO, T., GIEBISCH, G., AND SHIJLMAN, R. G. (1985) Amer. J. Physiol. 249 (Renal, Fluid, Electrolyt. PhysioL 18), F160F168. 19. RAYSON, B. M., AND GUPTA, R. K. (1985) J. Biol. Chem. 260,7276-7280. 20. GUPTA, R. K., AND GUPTA, P. (1982) J. Mag% Reson 47,344-350.
discoideum
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21. GUPTA, R. K., AND GUPTA, P. (1984) Annu. Rev. Biophys Bioeng. 13,221-246. 22. CHU, S. C., PIKE, M. M., FOSSEL, E. T., SMITH, T. W., BALSCHI, J. A., AND SPRINGER, C. S., JR. (1984) J. Magn Reson. 56,33-47. 23. WATTS, D. J., AND ASHWORTH, J. M. (1970) Biochem J. 119,171-174. 24. KLEIN, G., AND SATRE, M. (1986) Biochem Biophys. Res. Commun. 138,1146-1152. 25. BARRAND, P., PEREIRA DA SILVA, L., AND BRACHET, P. (1974) Bull Sot. Fr. Mycol Med 3,29-32. 26. FAVARD-SERENO, C., LUDOVSKY, M.-A., AND RYTER, A. (1981) J. Cell Sci. 51,63-84. 27. WIENER, E., AND ASHWORTH, J. M. (1970) Biochem J 118,505-512. 28. GEZELIUS, K. (1974) Arch. MicrobioL 98,311-329. 29. DIMOND, R. L., BURNS, R. A., AND JORDAN, K. B. (1981) J. Biol. Chem. 256,6565-6572. 30. LOOMIS, W. F., AND KUSPA, A. (1984) Dev. BioL 102,498-503. 31. MATWIYOFF, N. A., GASPAROVIC, C., WENK, R., WICKS, J. D., AND RATH, A. (1986) Magn. Reson Med. 3,164-168. 32. MITCHELL, P., AND MOYLE, J. (1967) Biochem. J. 105,1147-1162. 33. TYLER, D. D., AND SUITON, C. M. (1984) in Membrane Structure and Function (Bittar, E. E., Ed.), pp. 181-270, Wiley, New York.