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A versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber
ANALYTICALBIOCHEMISTRY
149,301-308(1985)
A Versatile Ca2+ Ion-Sensitive Minielectrode with a Microincubation Chamber’ SIGURDLENZEN~ ANDUWEPANTEN Institute
of Pharmacology
and
Toxicology,
University
of Giittingen,
D-3400
Giittingen,
Federal
Republic
of Germany
Received February 1, 1985 A new, versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber was designed for the direct, continuous monitoring of changes in Ca2’ ion activity in microgram tissue samples. The sample can be stirred in the microincubation chamber and kept at a constant temperature through thermostat&ion. Samples with a protein content ranging from 10 to 40 rg are required for the measurement. This is two to three orders of magnitude lessthan necessary for measurement of Ca2+ ion activity with conventional, commercially available Ca” ion-sensitive electrodes. The device should be useful for a variety of applications in many research areas where sample volumes are small. Some examples are presented in this communication using mitochondria and microsomes from nine different rat tissues. In these experiments it is shown that with mitochondria from all tissues a steady-state ambient free Ca2+ concentration between 0.6 and 0.8 pM is reached, though the Na+ sensitivity of ruthenium red-induced Ca2’ efflux from these mitochondria varies considerably in dependence on the tissue. The additional presence of microsomes resulted in a steadystate Ca2+ concentration between 0.1 and 0.2 jiM. 0 1985 Academic press hc. KEY WORDS: Ca2+ ion-sensitive minielectrode; microincubation chamber; mitochondria; microsomes.
Various intracellular organelles participate in the regulation of intracellular Ca2+ homeostasis. Direct measurement of Ca2+ ion activity with an electrode offers the advantage of direct continuous monitoring of changes in ion activity (l-4). However, when compared with other methods such as spectrophotometric Ca2+ determination using metallochromic indicators or the use of radioactively labeled Ca2+, the Ca2+ ion-sensitive electrodes presently used for potentiometric procedures bear the disadvantage of a lower sensitivity. In contrast to these other methods (5), measurement of Ca2+ fluxes in subcellular organelles with commercially available Ca2+ ion-sensitive
electrodes requires samples with a protein content in the milligram range. This situation prohibits access to the study of ion activities with potentiometric methods in subcellular organelles from many tissues where the amount of tissue available is limited. We now present a new, versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber which overcomes these limitations. With subcellular organelles from nine different rat tissues, we demonstrate the broad spectrum of applicability of this device. MATERIALS
AND
METHODS
Calcium ion-sensitive liquid membrane minielectrode with a thermostatic microincubation chamber. Figure 1 shows a drawing (Fig. 1A) and a schematic diagram (Fig. 1B) of the component parts of the device. All parts are made of Plexiglas except for the membrane
’ Supported by the Deutsche Forschungsgemeinschaft, Bonn and by the Bilttner-Stiftung, Clottingen. 2 To whom correspondence should be addressed at the Institut fur Pharmakologie, Universit& Giiningen, RobertKoch-Str. 40, D-3400 Gottingen, Federal Republic of Germany.
301
0003-2697185 $3.00 Copyrighl 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
302
LENZEN
AND PANTEN
A
FIG. 1. Drawing (A) and schematic diagram showing a cross-section (B) through the component parts of the Ca” ion-sensitive liquid-membrane minielectrode with a microincubation chamber. (1) Coaxial cable; (2) inner lead-off electrode (Ag/AgCI pellet); (3) coaxial cable fixation; (4) electrode head (Plexiglas); (5) filling port; (6) electrolyte solution chamber; (7) male thread; (8) locking ring with female thread for fixation of inset; (9) exchangeable membrane support inset (PVC) (lo), mounted with solvent polymeric membrane; (11) reference electrode (Ag/AgCl pellet); (12) reference electrode holder with coaxial cable; (13) electrode support (Plexiglas); (14) cylinder bore for fixation of electrode support on a thermostatic brass cylinder; (15) microincubation chamber (chamber volume, 45 ~1); (16) magnetic spinbar; (17) filling port (for additions with microliter syringe into microincubation chamber); (18) closure plug for tilling port.
support inset, which is made of PVC3 (polyvinylchloride). An Ag/AgCl pellet electrode (cylinder form; 2 mm diameter X 4 mm long, type E 20 1; IVM, Healdsburg, Calif.) (2), connected to the end of a coaxial cable (l), is fixed in a 2-mm diameter cylinder bore of the electrode head (4) with a two-component adhesive. In order to relieve stress on the coaxial cable, it is fixed with a clamp (3) at the top of the electrode head (4). A solvent polymeric mem3 Abbreviations used: PVC, polyvinylchloride; Hepes, 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid; EGTA, ethylene glycol bis (&aminoethyl ether)-hr,AJ’-tetraacetic acid: Mops, 4-morpholinepropanesulfonic acid; HEDTA, N-hydroxyethylethylenediaminetriacetic acid; NTA, nitrilotriacetic acid; Taps, 3-{ [2-hydroxy- 1, Ibis(hydroxymethyl)ethyl]amino}1-propanesulfonic acid: ETH 1001, WV’-bis[( 1l-ethoxycarbonyl>undecyl]-IVJV’,4, 5-tetramethyL3,6dioxaoctane diamide.
brane based on the calcium ionophore ETH 100 1 is glued on the lower tip of the exchangeable membrane support inset with a PVC adhesive (20 mg PVC dissolved in 1 ml of tetrahydrofuran). The upper tip of the exchangeable membrane support inset (9) is put into the corresponding opening at the lower end of the electrode head (4) and fixed with a locking ring (8). The internal electrolyte solution (1 KM CaC12 solution) is poured into the electrolyte solution chamber (6) through the filling port (5). The filling port is closed with a silicone plug. A grounded hollow brass cylinder (1 mm wall diameter) with an opening on the top for the coaxial cable is used as a shield. This cylinder is put over the electrode head. The electrode support (13) accommodates the microincubation chamber (45 ~1 volume) (15). This chamber has three openings; the first
A
VERSATILE
Ca*+
ION-SENSITIVE
opening for the tip of the exchangeable membrane support inset mounted with the solvent polymeric membrane (5 mm diameter) of the Ca” ion-sensitive electrode (10); the second opening for the tip of the reference electrode (12). An Ag/AgCl pellet electrode (cylinder form, 1.5 mm diameter X 4 mm long (type E 201 M; IVM, Healdsburg, Calif.) (ll), connected to the end of a coaxial cable (l), is fixed in a 1.5-mm-diameter cylinder bore of the reference electrode holder (12) with a two-component adhesive. In order to relieve stress on the coaxial cable, it is fixed with a clamp at the end of the electrode holder (12). If required an intermediate electrolyte chamber separated from the incubation medium in the microincubation chamber by an agar bridge can be interpositioned between reference electrode and microincubation chamber. An alternative is an intermediate electrolyte chamber (containing, e.g., 0.5 M KCl) which connects the reference electrode with the incubation medium in the microincubation chamber. Sample solution is then separated from reference electrolyte solution by a fine-pored ceramic diaphragm (e.g., zirconium dioxide). The third opening is the filling port (17) for additions with a microliter syringe into the microincubation chamber. The filling port can be closed with a closure plug (18). At the bottom the electrode support has a cylinder bore (14) for fixation of the electrode support on a thermostatic brass cylinder. The brass cylinder is inserted into a round, hollow, 20-mm-high PVC thermostatisation chamber which is fixed on the magnetic stirrer and kept at the desired temperature by water which is pumped through this chamber from a thermostated water bath. The microincubation chamber is filled with incubation medium before insertion of the tip of the exchangeable membrane support inset (9) into the opening of the microincubation chamber (15). Superfluous incubation medium can leave the chamber through the filling port (17). This filling method prevents formation of air bubbles. Incubation medium is mixed by a spinbar (1 mm diameter
MINIELECTRODE
303
X 4 mm long) (16) in the microincubation chamber, which is moved through a motordriven magnetic stirrer under the electrode support. Spinbars are made by melting small pieces of iron wire into glass capillaries.
Calcium ion-sensitiveliquid membranes. PVC polymeric stock membranes were prepared as described (2,4). PVC ( 140 mg) and 1 mg potassium tetra-(p-chlorophenyl)borate were dissolved in 3 ml tetrahydrofuran. A 6mg sample of the calcium ionophore (neutral carrier ETH 100 I) was dissolved in 200 mg onitrophenyl-n-octyl ether. The combined clear solutions were poured into a glass Petri dish (diameter, 5 cm). After evaporation of the tetrahydrofuran at room temperature a membrane of about 0.2 mm thickness was obtained. Pieces (5 mm diameter) were cut out of this membrane and mounted on the tip of the exchangeable membrane support inset. The parent membranes and the exchangeable membrane support insets mounted with the cutout small pieces of membrane can be stored dry for years. The average lifetime of calcium ion-sensitive membranes in use is at least 1 year. Calcium ion-sensitive membranes were preconditioned overnight before use in a 10 mM CaCIZ solution.
Preparationof mitochondrial and microsomalfractionsfrom rat tissues.Tissue samples were obtained from fasted Wistar rats (200-300 g body wt). Pancreatic islets were isolated from the pancreas following collagenase digestion (6). Liver, parotid gland, pancreatic islets, kidney, adrenal cortex, hypophysis, adrenal medulla, brain, and heart muscle were homogenized in ice-cold homogenization medium (210 mM D-mm&d, 70 mM sucrose, 20 mM Hepes, adjusted to pH 7.0 with KOH) using a Potter-Elvehjem homogenizer with a Teflon pestle and maintained on ice. Medium for preparation of microsomes contained 1 mM EGTA additionally. Sediments obtained after centrifugation for 15 min at 6608 at 4°C were discarded. The mitochondrial pellets were obtained by centrifugation of the supematant for 15 min at 7500g
304
LENZEN
AND PANTEN
at 4°C (7). The microsomal pellets were obtained by centrifugation of the supernatants for another 5 min at 100,OOOg.Mitochondrial and microsomal pellets were resuspended in homogenization medium, recentrifuged, and resuspended in test medium (125 mM KCl, 2 mM KI-12P04, 5 mM succinate, 25 mM Hepes, pH adjusted to 7.0 with KOH). Protein content was determined according to McKnight (8). Measurement of Cazf uptake and calcium e8u.x by subcellular organelles with the calcium ion-sensitive minielectrode. Measurements of medium free Ca2+ concentrations were performed with the Ca2+ ion-sensitive minielectrode in the microincubation chamber containing suspensions with subcellular organelles. The microincubation chamber was filled with a test medium of an ionic composition simulating the composition of the cytosol (9) ( 125 mM KCl, 1 mM MgC12, 2 mM KH2P04, 5 mM succinate, 25 mM Hepes, 3 mM MgATP, adjusted to pH 7.0 with KOH). Measurements were performed at 25°C. The incubation solution was stirred with a spinbar. Samples of subcellular organelles (mitochondria or microsomes) suspended in test medium ( l-2 ~1 containing 1O-40 pg protein) were injected with a microliter syringe through the filling port into the test medium in the microincubation chamber. The same procedure was used for addition of CaC12, NaCl, and ruthenium red dissolved in test medium. Calibration of the Ca2’ ion-sensitive minielectrode was performed with Ca2+ buffer solutions (pCa 5-8, in 0.1 M KCl, pH 7.0), before and after the experiments, which were prepared according to Tsien and Rink (10). The potential of the electrode pair was displayed on a digital millivoltmeter (type pH M 84; Radiometer, Copenhagen, Denmark) and recorded on a chart recorder (type PM 8222; N. V. Philips, Eindhoven, The Netherlands). The response of the Ca2+ ion-sensitive minielectrode was strictly logarithmic with the free Ca2+ concentration down to at least 0.1 PM (27-29 mV per log of Ca2+ concentration) and was linearized graphically. Calcium content of the
incubation medium and of the mitochondrial fractions was measured by atomic absorption spectroscopy with a Massmann cuvette from Beckman Instruments (Munich, Germany). The use of the Massmann cuvette makes this method applicable to the measurement of micromolar Ca2’ concentrations in the microincubation chamber. Chemicals. PVC (high-molecular-weight type), was obtained from Aldrich (Milwaukee, Wise.). The other components for the Ca2+ ion-selective membrane were from Fluka AG (Buchs, Switzerland). Ca*+-ionophore ETH 100 1 potassium tetraphenylborate, 2-nitrophenyl octyl ether, tetrahydrofuran, and succinate (potassium salt) was also from Fluka. The calcium chelating agents EGTA, MOPS, HEDTA, NTA, Taps, for the calcium buffer solutions; and Hepes, MgATP, and ruthenium red were from Sigma (St. Louis, MO.). Collagenase (type IV) was from Worthington (Freehold N. J.). Sucrose was from Serva (Heidelberg, Germany); CaC12 standard solution (Titrisol), Dmannitol, Tris, and all salts and other reagents of analytical grade were from Merck (Darmstadt, Germany). RESULTS
The experiments in Figs. 2-4 demonstrate examples for the use of the Ca2’ ion-sensitive minielectrode with a microincubation chamber (Fig. 1). Mitochondria and microsomes from different rat tissues were incubated in a medium with an ionic composition which simulates that of the cytosol (9) and supplemented with ATP and succinate as the energy sources. Addition of mitochondria from nine different tissues from the rat to this medium produced a rapid decrease in the free Ca2+ (Fig. 2). The total sample protein content in the microincubation chamber necessary for the measurements was in the microgram protein range and varied between 10 and 40 pg of protein (Table 1). A steady-state ambient free Ca2+ level between 0.6 and 0.8 PM was reached with the mitochondria from the various tissues (Fig. 2 and Table 1). Addition of ruthenium red
A VERSATILE
? 3 5.0 .-6 3 E z u0’ & 1.0 2
Mi I
Ca2+ ION-SENSITIVE Mi 1
Liver
305
MINIELECTRODE
Parotid Gland
I
0.7 0.5
Ui
Mi Hypophysls
z 2 so.-s 3 E 8 vs 1.0 l4* 0.7 ”
0.5 -
=-r I
”
0.7
0.7 t
t
Tkme
(mm)
i
0.7 t
--RR
Time
5.0 t
(min)
I
I
‘.-,J
-RR
Time
(mm)
FIG. 2. Regulation ofthe steady-state ambient free Ca” concentration by mitochondria from liver, pancreatic islets, parotid gland, adrenal cortex, kidney, hypophysis, adrenal medulla, brain, and heart muscle from rats and the activation of ruthenium red-induced (RR) (final concentration, 250 nM) Ca2+ efflux from these mitochondria by Na’ (final concentration, I8 mM). Mitochondria from different tissues(I -2.5 ~1, containing IO-40 pg protein as shown in Table I) were added to the test medium (40 ~1 volume: 25°C supplemented with I mM M&l2 and 3 mM MgATP) in the microincubation chamber at min 0 asdescribed under Materials and Methods. At min IO ruthenium red (1 ~1 of a concentration of 10 pM) and at min 12 NaCl (I ~1 of a concentration of 750 mM) were added to the test medium. The lower dashed curve in each experiment represents the control where test medium (I ~1) only was added at min 12. The curves represent typical recordings which were repeated with mitochondria from each tissue four to nine times.
(final concentration, 250 nM) to the incubation medium induced Ca2+ efflux from mitochondria from all tissues (Fig. 2 and Table 1). Addition of Na+ ( 18 IrIM) to the incubation medium activated ruthenium red-induced Ca2+
efflux from mitochondria from all nine tissues (Fig. 2 and Table 1). However, sensitivity toward Na+ varied considerably depending on the source of the mitochondria. Activation of ruthenium red-induced Ca2+ efflux by Na+ was
306
LENZEN
AND PANTEN
weak in mitochondria from liver, parotid gland, and pancreatic islets; the effect of Na+ was greater in mitochondria from kidney, adrenal cortex, and hypophysis; in mitochondria from adrenal medulla, brain, and heart ruthenium red-induced Ca2+ efflux was strongly activated by Na+ (Fig. 2 and Table 1). Ca2+ content of the medium in the microincubation chamber as measured by atomic absorption spectroscopy was reduced after a lo-min incubation period with mitochondria from liver, kidney, heart muscle, or brain from rats by that amount of calcium, which had been taken up by the mitochondria during this period of lowering of the ambient free Ca2+ concentration to values between 0.6 and 0.8 PM (Fig, 3). Upon addition of a pulse of Ca2+ (5.4 + 0.9 pmol/pg protein; N = 15) to the medium in the microincubation chamber there was an
immediate rapid rise in the free Ca2+ concentration followed by a fast return to steady-state Ca2+ concentrations (Fig. 3). Figure 4 demonstrates that the addition of microsomes to mitochondria suspended in medium in the microincubation chamber decreased the ambient free Ca2+ concentration maintained by liver mitochondria at 0.57 t 0.02 pM (N = 9) to 0.13 f 0.0 1 pM (N = 9) after addition of liver microsomes. The ambient free Ca2+ concentration maintained by brain mitochondria at 0.75 f 0.04 PM (N = 5) was reduced to 0.23 + 0.04 PM (N = 5) after addition of brain microsomes (Fig. 4). DISCUSSION
The new, small, Ca2+ ion-sensitive minielectrode with a microincubation chamber
TABLE REGULATION
OF STEADY-STATE
AMBIENT
FREE Ca”
1
CONCENTRATION
BY MITOCHONDRIA
FROM LIVER,
PAROTID
GLAND, PANCREATIC ISLETS, KIDNEY, ADRENAL CORTEX, HY~OPHYSIS, ADRENAL MEDULLA, BRAIN, AND HEART MUSCLE FROM RATS AND THE ACTIVATION OF RUTHENIUM RED-INDUCED (FINAL CONCENTRATION,250 nM) Ca*+ EFFLUX FROM THESE MITOCHONDRIA BY Na+ (FINAL CONCENTRATION, 18 mM) Free Ca” concentration (FM)
N
Sample protein content (Mcp)
A
B
C
Liver
4
20.5 + 3.1
0.61 + 0.06
1.93 + 0.19
2.83 + 0.19
0.90
Parotid gland
3
34.1 + 4.9
0.74 z!z0.07
1.82 k 0.15
3.32 f 0.48
1.50
Pancreatic islets
I
10.8 f 3.9
0.66 + 0.06
1.62 + 0.30
2.60 f 0.28
0.90
Kidney
6
29.5 f 3.8
0.14 If- 0.04
3.00 f 0.59
5.62 f 0.58
2.62
Adrenal cortex
5
20.6 f 1.6
0.69 f 0.03
2.03 + 0.16
4.84 f 0.48
2.81
Hypophysis
I
16.2 + 6.1
0.66 f 0.05
1.51 * 0.22
5.68 + 0.63
4.17
Adrenal medulla
9
40.8 + 6.4
0.93 + 0.05
2.38 +- 0.54
7.79 + 0.96
5.54
Brain
5
38.8 + 5.8
0.73 * 0.03
4.85 f 0.39
14.50 f 1.47
9.65
Heart muscle
5
37.9 + 2.9
0.78 -t 0.10
2.90 f 0.11
22.30 -c 1.23
19.40
Source of mitochondria
D K-B)
Note. Experiments were performed as described in Fig. 1. This table presents the number of experiments (N), the sample protein content (pg) in the microincubation chamber, and the steady-state ambient free Ca” concentration (PM) maintained by the mitochondria from each of the various tissues: the lowest Ca*+ concentration recorded in the presence of the mitochondria from the various tissues after uptake of Ca*’ during a lo-min incubation period (A); the Ca2’ concentration recorded 10 min after addition of ruthenium red to the incubated mitochondria (B); the Ca*’ concentration recorded 10 min after addition of ruthenium red to the incubated mitochondria during the last 8 min of which Na+ was present additionally in incubation medium (C). Column D shows the difference between column C and column B, which represents the potentiatory effect of Na+ on ruthenium red-induced Ca2’ efflux from mitochondria.
A VERSATILE
Ca2’ ION-SENSITIVE
10.0 5.0 -
1.0 0.5
I 3
ii
t
0.7 0.5 -
o-o ';"I
(9’
5.0 I
I
I Heart 1.muscle
0.5 I
o-
10.0
Time
10 lmm)
20
0.5 I-
0
10
20
0
10
20
Time (mm)
FIG. 3. Regulation of steady-state ambient free Ca2+ concentration by mitochondria from liver, kidney, brain, and heart muscle from rats. Liver mitochondria (1 pl, containing 22.8 + 3.0 pg protein and 12.1 + 4.3 pmol Ca2’/pg protein; N = 3) kidney mitochondria (1 ~1, containing 25.4 + 1.8 rg protein and 11.4 f 2.4 pmol Ca*‘/ fig protein; N = 4) heart muscle mitochondria (I ql, containing 19.5 f 3.0 rg protein and 16.1 + 1.8 pmol Ca2+/ rg protein; N = 3), or brain mitochondria (1 ~1,containing 12.6 + 1.6 pg protein and 24.1 ? 4.2 pmol Ca2’/wg protein; N = 5) were added to the test medium (40 ~1 volume; 25°C; supplemented with 1 mM MgCl, and 3 mM MgATP) in the microincubation chamber as described under Materials and Methods. Mitochondria took up Ca2+ from the medium. After 10 min of incubation, the Ca2’ content was in liver mitochondria 17.4 + 4.5 pmol Ca2+/pg protein, in kidney mitochondria 17.1 + 2.5 pmol Ca2+/rg protein, in heart muscle mitochondria 22.9 f 2.3 pmol Ca2+/rg protein, and in brain mitochondria 32.5 + 5.8 pmol Ca2+/ rg protein. At min 10 1 rl of a CaC12solution was added to the mitochondria in the test medium. In liver mitochondria addition of Ca2+ (5.1 + 1.O pmol/Fg protein) raised the Ca2+ content to 23.3 f 5.6 pmol/rg protein. In kidney mitochondria addition of Ca*+ (4.0 + 0.4 pmol/ pg protein) raised the Ca2+ content to 20.6 f 2.0 pmol/ r.igprotein. In heart muscle mitochondria addition ofCa2+ (6.3 ? 1.6 pmol/rg protein) raised the Ca2+ content to 29.3 f 2.6 pmol/pg protein. In brain mitochondria addition of Ca*+ (6.3 + 0.7 pmol/pg protein) raised to Ca2+ content to 38.6 + 7.9 pmol/rg protein. The curves represent typical recordings which were repeated with mitochondria from each tissue three to five times.
(Fig. 1) is a versatile device for direct, continuous monitoring of Ca2+ ion activity in microgram tissue samples. The present investi-
307
MINIELECTRODE
gation shows examples for the general applicability of the new device for measurement of Ca2+ uptake and Ca2+ efflux in mitochondria and microsomes from different organs (Figs. 2-4). Samples of mitochondria or microsomes required for measurement of Ca2+ ion activity with this device ranged from 10 to 40 pg of protein (Table 1). Thus, the sensitivity of this new device is greater by two to three orders of magnitude than that of conventional commercially available Ca2+ ion-sensitive electrodes, which require milligram amounts of mitochondrial or microsomal protein for measurement. Thus this new device enables the measurement of the ambient free Ca2+ concentrations in suspensions of organelles from tissues such as pancreatic islets, hypophysis, adrenal cortex, and adrenal medulla from small laboratory animals such as the rat (Fig. 2) which has not been possible so far due to the availability of limited amounts of tissue from these organs. The device also permits the
5.0
MI 1
itver
5.0 -
‘;‘I
Brain
1 g
1.0
b5 0.7
1.0 MC
I
s” 0.5 50-L
MC I
0.7 0.5 :’
Time 1min)
Time
I mln)
FIG. 4. Regulation of steady-state ambient free Ca2+ concentration by mitochondria and microsomes from rat liver and rat brain. Liver mitochondria (1-2 ~1, containing 34.8 f 1 I.1 pg protein) or brain mitochondria (l-2 ~1. containing 28.0 f 1.7 rg protein) were added to the test medium (40 ~1 volume; 25°C; supplemented with 1 mM MgC& and 3 mM MgATP) in the microincubation chamber at min 0; liver microsomes (2 ~1,containing 52.8 + 1I .O pg protein) or brain microsomes (2 ~1, containing 49.6 + 13.6 rg protein) were added to the test medium at min 5 as described under Materials and Methods. The curves represent typical recordings which were repeated nine and five times, respectively.
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LENZEN
AND PANTEN
investigation of Ca*+ fluxes in subcellular organelles from certain localized areas of many organs and may provide an approach to the study of the different functional characteristics of subpopulations of cell organelles such as of mitochondria from liver. Another good example for the versatile applicability of the new device is the possibility to compare functional characteristics of the same organelle from different tissues as demonstrated in the present investigation for the Na+ sensitivity of Ca*+ efflux from mitochondria from nine different rat tissues (Fig. 2) (11). ACKNOWLEDGMENTS The authors thank Prof. Dr. B. Lindemann, Institut fiir Physiologie II, Universitat Homburg/&at’; Prof. Dr. G. Schwedt, lnstitut fur Lebensmittelchemie, Universitat Stuttgart; and Dr. H.-G. Wetzstein, Institut fur Mikrobiologie, Universitlt Gottingen, for valuable advice. The skillful technical assistance of Mrs. B. Jlnsch-Paulsen is gratefully acknowledged. The authors thank Mr. R. Wegener for help with the construction of the device, and Mrs. G. Degenhardt for help with the illustrations.
REFERENCES 1. Simon, W., Ammann, D., Oehme, M., and Morf, W. F. (1978) Ann. N. Y. Acad. Sci. 307, 52-70. 2. Affolter, H., and Sigel, E. (1979) Anal. Biochem. 97, 315-319. 3. Walker, J. L. (1979) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 56, pp. 359-368, Academic Press, New York. 4. Ammann, P., Morf, W. E., Anker, P., Meier, P. C., Pretsch, E., and Simon, W. (1983) Ion-Select. Electr. Rev. 5, 3-92. 5. Lenzen, S., and Panten, U. (1983) Anal. Biochem. 134,56-59. 6. Lacy, P. E., and Kostianowsky, U. (1967) Diabetes 16, 35-39. 7. Pedersen, P. L., Greenawalt, J. W., Reynafarje, B., Hullihen, J., Decker, G. L., Soper, J. W., and Bustamente, E. (1978) Methods Cell Biol. 20, 41 l481. 8. McKnight, G. S. (1977) Anal. Biochem. 78, 86-92. 9. Becker, G. L., Fiskum, G., and Lehninger, A. L. (1980) J. Biol. Chem. 255,9009-9012. 10. Tsien, R. Y., and Rink, T. J. (1980) B&him. Biophys. Acta. 599, 623-638. 11. Akerman, K. E. O., and Nicholls, D. G. (1983) Rev. Physiol. Biochem. Pharmacol. 95, 149-20 I.
149,301-308(1985)
A Versatile Ca2+ Ion-Sensitive Minielectrode with a Microincubation Chamber’ SIGURDLENZEN~ ANDUWEPANTEN Institute
of Pharmacology
and
Toxicology,
University
of Giittingen,
D-3400
Giittingen,
Federal
Republic
of Germany
Received February 1, 1985 A new, versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber was designed for the direct, continuous monitoring of changes in Ca2’ ion activity in microgram tissue samples. The sample can be stirred in the microincubation chamber and kept at a constant temperature through thermostat&ion. Samples with a protein content ranging from 10 to 40 rg are required for the measurement. This is two to three orders of magnitude lessthan necessary for measurement of Ca2+ ion activity with conventional, commercially available Ca” ion-sensitive electrodes. The device should be useful for a variety of applications in many research areas where sample volumes are small. Some examples are presented in this communication using mitochondria and microsomes from nine different rat tissues. In these experiments it is shown that with mitochondria from all tissues a steady-state ambient free Ca2+ concentration between 0.6 and 0.8 pM is reached, though the Na+ sensitivity of ruthenium red-induced Ca2’ efflux from these mitochondria varies considerably in dependence on the tissue. The additional presence of microsomes resulted in a steadystate Ca2+ concentration between 0.1 and 0.2 jiM. 0 1985 Academic press hc. KEY WORDS: Ca2+ ion-sensitive minielectrode; microincubation chamber; mitochondria; microsomes.
Various intracellular organelles participate in the regulation of intracellular Ca2+ homeostasis. Direct measurement of Ca2+ ion activity with an electrode offers the advantage of direct continuous monitoring of changes in ion activity (l-4). However, when compared with other methods such as spectrophotometric Ca2+ determination using metallochromic indicators or the use of radioactively labeled Ca2+, the Ca2+ ion-sensitive electrodes presently used for potentiometric procedures bear the disadvantage of a lower sensitivity. In contrast to these other methods (5), measurement of Ca2+ fluxes in subcellular organelles with commercially available Ca2+ ion-sensitive
electrodes requires samples with a protein content in the milligram range. This situation prohibits access to the study of ion activities with potentiometric methods in subcellular organelles from many tissues where the amount of tissue available is limited. We now present a new, versatile Ca2+ ion-sensitive minielectrode with a microincubation chamber which overcomes these limitations. With subcellular organelles from nine different rat tissues, we demonstrate the broad spectrum of applicability of this device. MATERIALS
AND
METHODS
Calcium ion-sensitive liquid membrane minielectrode with a thermostatic microincubation chamber. Figure 1 shows a drawing (Fig. 1A) and a schematic diagram (Fig. 1B) of the component parts of the device. All parts are made of Plexiglas except for the membrane
’ Supported by the Deutsche Forschungsgemeinschaft, Bonn and by the Bilttner-Stiftung, Clottingen. 2 To whom correspondence should be addressed at the Institut fur Pharmakologie, Universit& Giiningen, RobertKoch-Str. 40, D-3400 Gottingen, Federal Republic of Germany.
301
0003-2697185 $3.00 Copyrighl 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
302
LENZEN
AND PANTEN
A
FIG. 1. Drawing (A) and schematic diagram showing a cross-section (B) through the component parts of the Ca” ion-sensitive liquid-membrane minielectrode with a microincubation chamber. (1) Coaxial cable; (2) inner lead-off electrode (Ag/AgCI pellet); (3) coaxial cable fixation; (4) electrode head (Plexiglas); (5) filling port; (6) electrolyte solution chamber; (7) male thread; (8) locking ring with female thread for fixation of inset; (9) exchangeable membrane support inset (PVC) (lo), mounted with solvent polymeric membrane; (11) reference electrode (Ag/AgCl pellet); (12) reference electrode holder with coaxial cable; (13) electrode support (Plexiglas); (14) cylinder bore for fixation of electrode support on a thermostatic brass cylinder; (15) microincubation chamber (chamber volume, 45 ~1); (16) magnetic spinbar; (17) filling port (for additions with microliter syringe into microincubation chamber); (18) closure plug for tilling port.
support inset, which is made of PVC3 (polyvinylchloride). An Ag/AgCl pellet electrode (cylinder form; 2 mm diameter X 4 mm long, type E 20 1; IVM, Healdsburg, Calif.) (2), connected to the end of a coaxial cable (l), is fixed in a 2-mm diameter cylinder bore of the electrode head (4) with a two-component adhesive. In order to relieve stress on the coaxial cable, it is fixed with a clamp (3) at the top of the electrode head (4). A solvent polymeric mem3 Abbreviations used: PVC, polyvinylchloride; Hepes, 4-(2-hydroxyethyl)-I-piperazineethanesulfonic acid; EGTA, ethylene glycol bis (&aminoethyl ether)-hr,AJ’-tetraacetic acid: Mops, 4-morpholinepropanesulfonic acid; HEDTA, N-hydroxyethylethylenediaminetriacetic acid; NTA, nitrilotriacetic acid; Taps, 3-{ [2-hydroxy- 1, Ibis(hydroxymethyl)ethyl]amino}1-propanesulfonic acid: ETH 1001, WV’-bis[( 1l-ethoxycarbonyl>undecyl]-IVJV’,4, 5-tetramethyL3,6dioxaoctane diamide.
brane based on the calcium ionophore ETH 100 1 is glued on the lower tip of the exchangeable membrane support inset with a PVC adhesive (20 mg PVC dissolved in 1 ml of tetrahydrofuran). The upper tip of the exchangeable membrane support inset (9) is put into the corresponding opening at the lower end of the electrode head (4) and fixed with a locking ring (8). The internal electrolyte solution (1 KM CaC12 solution) is poured into the electrolyte solution chamber (6) through the filling port (5). The filling port is closed with a silicone plug. A grounded hollow brass cylinder (1 mm wall diameter) with an opening on the top for the coaxial cable is used as a shield. This cylinder is put over the electrode head. The electrode support (13) accommodates the microincubation chamber (45 ~1 volume) (15). This chamber has three openings; the first
A
VERSATILE
Ca*+
ION-SENSITIVE
opening for the tip of the exchangeable membrane support inset mounted with the solvent polymeric membrane (5 mm diameter) of the Ca” ion-sensitive electrode (10); the second opening for the tip of the reference electrode (12). An Ag/AgCl pellet electrode (cylinder form, 1.5 mm diameter X 4 mm long (type E 201 M; IVM, Healdsburg, Calif.) (ll), connected to the end of a coaxial cable (l), is fixed in a 1.5-mm-diameter cylinder bore of the reference electrode holder (12) with a two-component adhesive. In order to relieve stress on the coaxial cable, it is fixed with a clamp at the end of the electrode holder (12). If required an intermediate electrolyte chamber separated from the incubation medium in the microincubation chamber by an agar bridge can be interpositioned between reference electrode and microincubation chamber. An alternative is an intermediate electrolyte chamber (containing, e.g., 0.5 M KCl) which connects the reference electrode with the incubation medium in the microincubation chamber. Sample solution is then separated from reference electrolyte solution by a fine-pored ceramic diaphragm (e.g., zirconium dioxide). The third opening is the filling port (17) for additions with a microliter syringe into the microincubation chamber. The filling port can be closed with a closure plug (18). At the bottom the electrode support has a cylinder bore (14) for fixation of the electrode support on a thermostatic brass cylinder. The brass cylinder is inserted into a round, hollow, 20-mm-high PVC thermostatisation chamber which is fixed on the magnetic stirrer and kept at the desired temperature by water which is pumped through this chamber from a thermostated water bath. The microincubation chamber is filled with incubation medium before insertion of the tip of the exchangeable membrane support inset (9) into the opening of the microincubation chamber (15). Superfluous incubation medium can leave the chamber through the filling port (17). This filling method prevents formation of air bubbles. Incubation medium is mixed by a spinbar (1 mm diameter
MINIELECTRODE
303
X 4 mm long) (16) in the microincubation chamber, which is moved through a motordriven magnetic stirrer under the electrode support. Spinbars are made by melting small pieces of iron wire into glass capillaries.
Calcium ion-sensitiveliquid membranes. PVC polymeric stock membranes were prepared as described (2,4). PVC ( 140 mg) and 1 mg potassium tetra-(p-chlorophenyl)borate were dissolved in 3 ml tetrahydrofuran. A 6mg sample of the calcium ionophore (neutral carrier ETH 100 I) was dissolved in 200 mg onitrophenyl-n-octyl ether. The combined clear solutions were poured into a glass Petri dish (diameter, 5 cm). After evaporation of the tetrahydrofuran at room temperature a membrane of about 0.2 mm thickness was obtained. Pieces (5 mm diameter) were cut out of this membrane and mounted on the tip of the exchangeable membrane support inset. The parent membranes and the exchangeable membrane support insets mounted with the cutout small pieces of membrane can be stored dry for years. The average lifetime of calcium ion-sensitive membranes in use is at least 1 year. Calcium ion-sensitive membranes were preconditioned overnight before use in a 10 mM CaCIZ solution.
Preparationof mitochondrial and microsomalfractionsfrom rat tissues.Tissue samples were obtained from fasted Wistar rats (200-300 g body wt). Pancreatic islets were isolated from the pancreas following collagenase digestion (6). Liver, parotid gland, pancreatic islets, kidney, adrenal cortex, hypophysis, adrenal medulla, brain, and heart muscle were homogenized in ice-cold homogenization medium (210 mM D-mm&d, 70 mM sucrose, 20 mM Hepes, adjusted to pH 7.0 with KOH) using a Potter-Elvehjem homogenizer with a Teflon pestle and maintained on ice. Medium for preparation of microsomes contained 1 mM EGTA additionally. Sediments obtained after centrifugation for 15 min at 6608 at 4°C were discarded. The mitochondrial pellets were obtained by centrifugation of the supematant for 15 min at 7500g
304
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AND PANTEN
at 4°C (7). The microsomal pellets were obtained by centrifugation of the supernatants for another 5 min at 100,OOOg.Mitochondrial and microsomal pellets were resuspended in homogenization medium, recentrifuged, and resuspended in test medium (125 mM KCl, 2 mM KI-12P04, 5 mM succinate, 25 mM Hepes, pH adjusted to 7.0 with KOH). Protein content was determined according to McKnight (8). Measurement of Cazf uptake and calcium e8u.x by subcellular organelles with the calcium ion-sensitive minielectrode. Measurements of medium free Ca2+ concentrations were performed with the Ca2+ ion-sensitive minielectrode in the microincubation chamber containing suspensions with subcellular organelles. The microincubation chamber was filled with a test medium of an ionic composition simulating the composition of the cytosol (9) ( 125 mM KCl, 1 mM MgC12, 2 mM KH2P04, 5 mM succinate, 25 mM Hepes, 3 mM MgATP, adjusted to pH 7.0 with KOH). Measurements were performed at 25°C. The incubation solution was stirred with a spinbar. Samples of subcellular organelles (mitochondria or microsomes) suspended in test medium ( l-2 ~1 containing 1O-40 pg protein) were injected with a microliter syringe through the filling port into the test medium in the microincubation chamber. The same procedure was used for addition of CaC12, NaCl, and ruthenium red dissolved in test medium. Calibration of the Ca2’ ion-sensitive minielectrode was performed with Ca2+ buffer solutions (pCa 5-8, in 0.1 M KCl, pH 7.0), before and after the experiments, which were prepared according to Tsien and Rink (10). The potential of the electrode pair was displayed on a digital millivoltmeter (type pH M 84; Radiometer, Copenhagen, Denmark) and recorded on a chart recorder (type PM 8222; N. V. Philips, Eindhoven, The Netherlands). The response of the Ca2+ ion-sensitive minielectrode was strictly logarithmic with the free Ca2+ concentration down to at least 0.1 PM (27-29 mV per log of Ca2+ concentration) and was linearized graphically. Calcium content of the
incubation medium and of the mitochondrial fractions was measured by atomic absorption spectroscopy with a Massmann cuvette from Beckman Instruments (Munich, Germany). The use of the Massmann cuvette makes this method applicable to the measurement of micromolar Ca2’ concentrations in the microincubation chamber. Chemicals. PVC (high-molecular-weight type), was obtained from Aldrich (Milwaukee, Wise.). The other components for the Ca2+ ion-selective membrane were from Fluka AG (Buchs, Switzerland). Ca*+-ionophore ETH 100 1 potassium tetraphenylborate, 2-nitrophenyl octyl ether, tetrahydrofuran, and succinate (potassium salt) was also from Fluka. The calcium chelating agents EGTA, MOPS, HEDTA, NTA, Taps, for the calcium buffer solutions; and Hepes, MgATP, and ruthenium red were from Sigma (St. Louis, MO.). Collagenase (type IV) was from Worthington (Freehold N. J.). Sucrose was from Serva (Heidelberg, Germany); CaC12 standard solution (Titrisol), Dmannitol, Tris, and all salts and other reagents of analytical grade were from Merck (Darmstadt, Germany). RESULTS
The experiments in Figs. 2-4 demonstrate examples for the use of the Ca2’ ion-sensitive minielectrode with a microincubation chamber (Fig. 1). Mitochondria and microsomes from different rat tissues were incubated in a medium with an ionic composition which simulates that of the cytosol (9) and supplemented with ATP and succinate as the energy sources. Addition of mitochondria from nine different tissues from the rat to this medium produced a rapid decrease in the free Ca2+ (Fig. 2). The total sample protein content in the microincubation chamber necessary for the measurements was in the microgram protein range and varied between 10 and 40 pg of protein (Table 1). A steady-state ambient free Ca2+ level between 0.6 and 0.8 PM was reached with the mitochondria from the various tissues (Fig. 2 and Table 1). Addition of ruthenium red
A VERSATILE
? 3 5.0 .-6 3 E z u0’ & 1.0 2
Mi I
Ca2+ ION-SENSITIVE Mi 1
Liver
305
MINIELECTRODE
Parotid Gland
I
0.7 0.5
Ui
Mi Hypophysls
z 2 so.-s 3 E 8 vs 1.0 l4* 0.7 ”
0.5 -
=-r I
”
0.7
0.7 t
t
Tkme
(mm)
i
0.7 t
--RR
Time
5.0 t
(min)
I
I
‘.-,J
-RR
Time
(mm)
FIG. 2. Regulation ofthe steady-state ambient free Ca” concentration by mitochondria from liver, pancreatic islets, parotid gland, adrenal cortex, kidney, hypophysis, adrenal medulla, brain, and heart muscle from rats and the activation of ruthenium red-induced (RR) (final concentration, 250 nM) Ca2+ efflux from these mitochondria by Na’ (final concentration, I8 mM). Mitochondria from different tissues(I -2.5 ~1, containing IO-40 pg protein as shown in Table I) were added to the test medium (40 ~1 volume: 25°C supplemented with I mM M&l2 and 3 mM MgATP) in the microincubation chamber at min 0 asdescribed under Materials and Methods. At min IO ruthenium red (1 ~1 of a concentration of 10 pM) and at min 12 NaCl (I ~1 of a concentration of 750 mM) were added to the test medium. The lower dashed curve in each experiment represents the control where test medium (I ~1) only was added at min 12. The curves represent typical recordings which were repeated with mitochondria from each tissue four to nine times.
(final concentration, 250 nM) to the incubation medium induced Ca2+ efflux from mitochondria from all tissues (Fig. 2 and Table 1). Addition of Na+ ( 18 IrIM) to the incubation medium activated ruthenium red-induced Ca2+
efflux from mitochondria from all nine tissues (Fig. 2 and Table 1). However, sensitivity toward Na+ varied considerably depending on the source of the mitochondria. Activation of ruthenium red-induced Ca2+ efflux by Na+ was
306
LENZEN
AND PANTEN
weak in mitochondria from liver, parotid gland, and pancreatic islets; the effect of Na+ was greater in mitochondria from kidney, adrenal cortex, and hypophysis; in mitochondria from adrenal medulla, brain, and heart ruthenium red-induced Ca2+ efflux was strongly activated by Na+ (Fig. 2 and Table 1). Ca2+ content of the medium in the microincubation chamber as measured by atomic absorption spectroscopy was reduced after a lo-min incubation period with mitochondria from liver, kidney, heart muscle, or brain from rats by that amount of calcium, which had been taken up by the mitochondria during this period of lowering of the ambient free Ca2+ concentration to values between 0.6 and 0.8 PM (Fig, 3). Upon addition of a pulse of Ca2+ (5.4 + 0.9 pmol/pg protein; N = 15) to the medium in the microincubation chamber there was an
immediate rapid rise in the free Ca2+ concentration followed by a fast return to steady-state Ca2+ concentrations (Fig. 3). Figure 4 demonstrates that the addition of microsomes to mitochondria suspended in medium in the microincubation chamber decreased the ambient free Ca2+ concentration maintained by liver mitochondria at 0.57 t 0.02 pM (N = 9) to 0.13 f 0.0 1 pM (N = 9) after addition of liver microsomes. The ambient free Ca2+ concentration maintained by brain mitochondria at 0.75 f 0.04 PM (N = 5) was reduced to 0.23 + 0.04 PM (N = 5) after addition of brain microsomes (Fig. 4). DISCUSSION
The new, small, Ca2+ ion-sensitive minielectrode with a microincubation chamber
TABLE REGULATION
OF STEADY-STATE
AMBIENT
FREE Ca”
1
CONCENTRATION
BY MITOCHONDRIA
FROM LIVER,
PAROTID
GLAND, PANCREATIC ISLETS, KIDNEY, ADRENAL CORTEX, HY~OPHYSIS, ADRENAL MEDULLA, BRAIN, AND HEART MUSCLE FROM RATS AND THE ACTIVATION OF RUTHENIUM RED-INDUCED (FINAL CONCENTRATION,250 nM) Ca*+ EFFLUX FROM THESE MITOCHONDRIA BY Na+ (FINAL CONCENTRATION, 18 mM) Free Ca” concentration (FM)
N
Sample protein content (Mcp)
A
B
C
Liver
4
20.5 + 3.1
0.61 + 0.06
1.93 + 0.19
2.83 + 0.19
0.90
Parotid gland
3
34.1 + 4.9
0.74 z!z0.07
1.82 k 0.15
3.32 f 0.48
1.50
Pancreatic islets
I
10.8 f 3.9
0.66 + 0.06
1.62 + 0.30
2.60 f 0.28
0.90
Kidney
6
29.5 f 3.8
0.14 If- 0.04
3.00 f 0.59
5.62 f 0.58
2.62
Adrenal cortex
5
20.6 f 1.6
0.69 f 0.03
2.03 + 0.16
4.84 f 0.48
2.81
Hypophysis
I
16.2 + 6.1
0.66 f 0.05
1.51 * 0.22
5.68 + 0.63
4.17
Adrenal medulla
9
40.8 + 6.4
0.93 + 0.05
2.38 +- 0.54
7.79 + 0.96
5.54
Brain
5
38.8 + 5.8
0.73 * 0.03
4.85 f 0.39
14.50 f 1.47
9.65
Heart muscle
5
37.9 + 2.9
0.78 -t 0.10
2.90 f 0.11
22.30 -c 1.23
19.40
Source of mitochondria
D K-B)
Note. Experiments were performed as described in Fig. 1. This table presents the number of experiments (N), the sample protein content (pg) in the microincubation chamber, and the steady-state ambient free Ca” concentration (PM) maintained by the mitochondria from each of the various tissues: the lowest Ca*+ concentration recorded in the presence of the mitochondria from the various tissues after uptake of Ca*’ during a lo-min incubation period (A); the Ca2’ concentration recorded 10 min after addition of ruthenium red to the incubated mitochondria (B); the Ca*’ concentration recorded 10 min after addition of ruthenium red to the incubated mitochondria during the last 8 min of which Na+ was present additionally in incubation medium (C). Column D shows the difference between column C and column B, which represents the potentiatory effect of Na+ on ruthenium red-induced Ca2’ efflux from mitochondria.
A VERSATILE
Ca2’ ION-SENSITIVE
10.0 5.0 -
1.0 0.5
I 3
ii
t
0.7 0.5 -
o-o ';"I
(9’
5.0 I
I
I Heart 1.muscle
0.5 I
o-
10.0
Time
10 lmm)
20
0.5 I-
0
10
20
0
10
20
Time (mm)
FIG. 3. Regulation of steady-state ambient free Ca2+ concentration by mitochondria from liver, kidney, brain, and heart muscle from rats. Liver mitochondria (1 pl, containing 22.8 + 3.0 pg protein and 12.1 + 4.3 pmol Ca2’/pg protein; N = 3) kidney mitochondria (1 ~1, containing 25.4 + 1.8 rg protein and 11.4 f 2.4 pmol Ca*‘/ fig protein; N = 4) heart muscle mitochondria (I ql, containing 19.5 f 3.0 rg protein and 16.1 + 1.8 pmol Ca2+/ rg protein; N = 3), or brain mitochondria (1 ~1,containing 12.6 + 1.6 pg protein and 24.1 ? 4.2 pmol Ca2’/wg protein; N = 5) were added to the test medium (40 ~1 volume; 25°C; supplemented with 1 mM MgCl, and 3 mM MgATP) in the microincubation chamber as described under Materials and Methods. Mitochondria took up Ca2+ from the medium. After 10 min of incubation, the Ca2’ content was in liver mitochondria 17.4 + 4.5 pmol Ca2+/pg protein, in kidney mitochondria 17.1 + 2.5 pmol Ca2+/rg protein, in heart muscle mitochondria 22.9 f 2.3 pmol Ca2+/rg protein, and in brain mitochondria 32.5 + 5.8 pmol Ca2+/ rg protein. At min 10 1 rl of a CaC12solution was added to the mitochondria in the test medium. In liver mitochondria addition of Ca2+ (5.1 + 1.O pmol/Fg protein) raised the Ca2+ content to 23.3 f 5.6 pmol/rg protein. In kidney mitochondria addition of Ca*+ (4.0 + 0.4 pmol/ pg protein) raised the Ca2+ content to 20.6 f 2.0 pmol/ r.igprotein. In heart muscle mitochondria addition ofCa2+ (6.3 ? 1.6 pmol/rg protein) raised the Ca2+ content to 29.3 f 2.6 pmol/pg protein. In brain mitochondria addition of Ca*+ (6.3 + 0.7 pmol/pg protein) raised to Ca2+ content to 38.6 + 7.9 pmol/rg protein. The curves represent typical recordings which were repeated with mitochondria from each tissue three to five times.
(Fig. 1) is a versatile device for direct, continuous monitoring of Ca2+ ion activity in microgram tissue samples. The present investi-
307
MINIELECTRODE
gation shows examples for the general applicability of the new device for measurement of Ca2+ uptake and Ca2+ efflux in mitochondria and microsomes from different organs (Figs. 2-4). Samples of mitochondria or microsomes required for measurement of Ca2+ ion activity with this device ranged from 10 to 40 pg of protein (Table 1). Thus, the sensitivity of this new device is greater by two to three orders of magnitude than that of conventional commercially available Ca2+ ion-sensitive electrodes, which require milligram amounts of mitochondrial or microsomal protein for measurement. Thus this new device enables the measurement of the ambient free Ca2+ concentrations in suspensions of organelles from tissues such as pancreatic islets, hypophysis, adrenal cortex, and adrenal medulla from small laboratory animals such as the rat (Fig. 2) which has not been possible so far due to the availability of limited amounts of tissue from these organs. The device also permits the
5.0
MI 1
itver
5.0 -
‘;‘I
Brain
1 g
1.0
b5 0.7
1.0 MC
I
s” 0.5 50-L
MC I
0.7 0.5 :’
Time 1min)
Time
I mln)
FIG. 4. Regulation of steady-state ambient free Ca2+ concentration by mitochondria and microsomes from rat liver and rat brain. Liver mitochondria (1-2 ~1, containing 34.8 f 1 I.1 pg protein) or brain mitochondria (l-2 ~1. containing 28.0 f 1.7 rg protein) were added to the test medium (40 ~1 volume; 25°C; supplemented with 1 mM MgC& and 3 mM MgATP) in the microincubation chamber at min 0; liver microsomes (2 ~1,containing 52.8 + 1I .O pg protein) or brain microsomes (2 ~1, containing 49.6 + 13.6 rg protein) were added to the test medium at min 5 as described under Materials and Methods. The curves represent typical recordings which were repeated nine and five times, respectively.
308
LENZEN
AND PANTEN
investigation of Ca*+ fluxes in subcellular organelles from certain localized areas of many organs and may provide an approach to the study of the different functional characteristics of subpopulations of cell organelles such as of mitochondria from liver. Another good example for the versatile applicability of the new device is the possibility to compare functional characteristics of the same organelle from different tissues as demonstrated in the present investigation for the Na+ sensitivity of Ca*+ efflux from mitochondria from nine different rat tissues (Fig. 2) (11). ACKNOWLEDGMENTS The authors thank Prof. Dr. B. Lindemann, Institut fiir Physiologie II, Universitat Homburg/&at’; Prof. Dr. G. Schwedt, lnstitut fur Lebensmittelchemie, Universitat Stuttgart; and Dr. H.-G. Wetzstein, Institut fur Mikrobiologie, Universitlt Gottingen, for valuable advice. The skillful technical assistance of Mrs. B. Jlnsch-Paulsen is gratefully acknowledged. The authors thank Mr. R. Wegener for help with the construction of the device, and Mrs. G. Degenhardt for help with the illustrations.
REFERENCES 1. Simon, W., Ammann, D., Oehme, M., and Morf, W. F. (1978) Ann. N. Y. Acad. Sci. 307, 52-70. 2. Affolter, H., and Sigel, E. (1979) Anal. Biochem. 97, 315-319. 3. Walker, J. L. (1979) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 56, pp. 359-368, Academic Press, New York. 4. Ammann, P., Morf, W. E., Anker, P., Meier, P. C., Pretsch, E., and Simon, W. (1983) Ion-Select. Electr. Rev. 5, 3-92. 5. Lenzen, S., and Panten, U. (1983) Anal. Biochem. 134,56-59. 6. Lacy, P. E., and Kostianowsky, U. (1967) Diabetes 16, 35-39. 7. Pedersen, P. L., Greenawalt, J. W., Reynafarje, B., Hullihen, J., Decker, G. L., Soper, J. W., and Bustamente, E. (1978) Methods Cell Biol. 20, 41 l481. 8. McKnight, G. S. (1977) Anal. Biochem. 78, 86-92. 9. Becker, G. L., Fiskum, G., and Lehninger, A. L. (1980) J. Biol. Chem. 255,9009-9012. 10. Tsien, R. Y., and Rink, T. J. (1980) B&him. Biophys. Acta. 599, 623-638. 11. Akerman, K. E. O., and Nicholls, D. G. (1983) Rev. Physiol. Biochem. Pharmacol. 95, 149-20 I.