- Email: [email protected]
[35] Ca2+ fluxes and phosphoinositides in hepatocytes
534
OTHER MAMMALIAN CELLS" INTACT CELLS
[35]
inflammatory processes or during extreme physical exercise. Indeed, under such conditions GSH levels in liver and other organs were significantly decreased. 97,98 Much has to be learned about the detailed mechanisms of the processes. It is known that glutathione conjugates may exert inhibitory effects on some enzyme activities like glutathione transferase 99 and glutathione reductase.100 Efficient biliary disposal of these conjugates is therefore of extreme importance. This becomes especially critical during conditions of oxidative stress accompanied by increased canalicular GSSG transport. GSSG could depress transport of the conjugate and the accumulation of the conjugate could then be amplified by its inhibitory effect on GSSG reductase. Injected leukotriene C4 is rapidly taken up by the liver and metabolized via the mercapturic acid pathway. 101The major product found in the bile depends on the species studied and is in the rat initially LTD4, at a later stage N-acetyl-LTE4 and to-oxidized metabolites predominate. This pathway operates during various types of tissue trauma, including surgical interventions, endotoxin shock, and virus-induced hepatitis.I°1 Acknowledgment Supported by the Deutsche Forschungsgemeinschaft, Grants Ak 8/1-1 and Si 255/8-1. 97 p. C. Bragt and I. L. Bonta, Agents Actions 10, 536 (1980). H. Lew, S. Pyke, and A. Quintanilha, FEBS Lett. 185, 262 0985). 99 I. Jakobson, M. Warholm, and B. Mannervik, J. Biol. Chem. 254, 7985 (1979). 10~M. Bilzer, R. L. Krauth-Siegel, R. H. Schirmer, T. P. M. Akerboom, H. Sies, and G. Schulz, Eur. J. Biochem. 138, 373 (1984). ~0~ M. Huber and D. Keppler, in "Glutathione Conjugation: Its Mechanism and Biological Significance" (H. Sies and B. Ketterer, eds.), 449. Academic Press, London, 1988.
[35] C a 2+ F l u x e s a n d P h o s p h o i n o s i t i d e s in H e p a t o c y t e s
By P. F. BLACKMORE and J. H. EXTON Introduction Vasopressin, angiotensin II, al-adrenergic agonists (epinephrine, norepinephrine), and P2 purinergic agonists (ATP, ADP) exert their effects on liver and certain other tissues by raising the Ca 2+ concentration in the cytosol. 1 On the other hand, glucagon and fl-adrenergic agonists exert i j. H. Exton, Adv. Cyclic Nucleotide Protein Phosphorylation Res. 20, 211 (1986).
METHODS IN ENZYMOLOGY,VOL. 173
Copyright© 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[35]
C a 2+ FLUXES AND PHOSPHOINOSITIDES
535
their effects on liver by raising cAMP, although they also increase cytosolic Ca 2+. The alterations in liver cell Ca 2+ induced by these agents can be monitored by measuring Ca 2+ fluxes across the plasma membrane, changes in the total cellular content of Ca 2+, or changes in cytosolic Ca 2+. Hormones increase cytosolic Ca 2+ in their target cells by mobilizing intracellular Ca 2+ stores and by altering the flux of Ca 2+ across the plasma membrane. There is much evidence that the mobilization of internal Ca 2+ is caused by inositol 1,4,5-trisphosphate generated by the breakdown of phosphatidylinositol 4,5-bisphosphate in the plasma membrane. 1,2 This breakdown occurs very rapidly in liver and other tissues 1,3 and inositol 1,4,5-P3 has been demonstrated to release Ca 2+ from endoplasmic reticulum stores in permeabilized hepatocytes and other cells. 3 Many other inositol phosphates have been identified in liver and other tissues, including inositol 1,3,4-P34.5 and inositol 1,3,4,5-P4.5.6 Their functions are presently unknown. Measurement of myo-Inositol 1,4,5-trisphosphate The most commonly used method for measuring the level of inositol 1,4,5-P3 in liver is to incubate hepatocytes or inject animals with myo-[2-3H]inositol for a period of time (1.5 hr or longer) to label the inositol phospholipids to isotopic equilibrium. Acid extracts of hepatocytes are then subjected to column chromatography on Dowex l-X8. Columns are washed with increasing concentrations of ammonium formate to elute the various inositol phosphates followed by counting the radioactivity in the fractions. Although useful for routine studies, this method does not allow the separation of inositol phosphate isomers such as inositol 1,4,5-P3 and inositol 1,3,4-P3. For this, HPLC methods are used. Both methods are described below. Isolation and Loading of Hepatocytes with myo-[2-3H]Inositol Hepatocytes can be isolated by collagenase digestion 7 from rats injected intraperitoneally with 500/~Ci of myo-[2-3H]inositol as described below. Cell suspensions ( - 4 0 to 50 mg wet wt/ml) are incubated in 2 M. J. Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 3 j. R. Williamson, R. H. Cooper, S. K. Joseph, and A. P. Thomas, Am. J. Physiol. 248, C203 (1985). 4 R. F. Irvine, A. J. Letcher, D. J. Lander, and C. P. Downes, Biochem. J. 223, 237 (1984). 5 C. A. Hansen, S. Mah, and J. R. Williamson, J. Biol. Chem. 261, 8100 (1986). 6 I. R. Batty, S. R. Nahorski, and R. F. Irvine, Biochem. J. 232, 211 (1985). 7 p. F. Blackmore and J. H. Exton, this series, Vol. 109, p. 550.
536
OTHERMAMMALIANCELLS;INTACTCELLS
[35]
Krebs-Henseleit bicarbonate buffer containing 1.5% (w/v) gelatin (Difco) and are continuously gassed with O2/CO2 (19: I). For labeling in vitro, hepatocytes (40-ml cell suspension in 250-ml Erlenmeyer flasks) are incubated for 90 or 120 min with 0.1 mM myo-[2-3H]inositol (25/zCi/ml of cell suspension). The cells are then filtered through nylon mesh to remove any clumps of cells which may form. The cells are then washed once and resuspended in fresh medium without myo-[2-3H]inositol or gelatin for use in experiments. The gelatin is omitted from the buffer at this stage since this protein is difficult to remove with trichloroacetic acid. After incubation for various periods under the experimental conditions desired, l-ml aliquots are pipetted into tubes containing 0.2 ml of 100% (w/v) trichloroacetic acid at 0°. After centrifugation (5000 g for 10 min) the supernatant fluid is extracted six times with 2-ml aliquots of diethyl ether, saturated with water, to remove trichloroacetic acid. The last traces of diethyl ether are removed by blowing a stream of N2 into the solutions which are then stored at - 2 0 ° before analysis. The neutralized extracts are subjected to ion-exchange chromatography or HPLC as follows. Dowex l-X8 Chromatography of Inositol Phosphates One milliliter of 10 mM sodium tetraborate is added to the neutralized extract and then the entire solution is applied to 2 ml of Dowex l-X8 (formate form) ion-exchange resin contained in P-5000 Pipetman disposable pipet tips (Rainin Instrument Co.). The resin is held in place by a small plug of fiberglass wool (Coming Glass Works). A small plug of fiberglass wool is also placed on top of the resin to prevent it from being disturbed by the eluting solutions of ammonium formate. The [3H]inositol phosphates are eluted as follows. Free [3H]inositol is removed from the column by washing with 15 ml of water, glycerophosphoinositol is removed by 2 × 5 ml of 5 mM sodium tetraborate/60 mM ammonium formate, inositol PI is eluted with 2 × 5 ml of 0.2 M ammonium formate/ 0.1 M formic acid, inositol P2 is eluted with 3 × 5 ml of 0.4 M ammonium formate/0.1 M formic acid, inositol P3 is eluted with 5 ml of 0.7 M ammonium forrnate/0.1 M formic acid, and inositol P4 is eluted with 5 ml of 2.0 M ammonium formate/0.1 M formic acid. 8 To each eluate, consisting of 5 ml in 25-ml plastic scintillation vials, is added 15 ml of Beckman ReadySolv EP and the contents are mixed and counted. Typically, inositol P1 contained -9000 cpm/ml cell suspension, inositol P2 -1200 cpm/ml cell suspension, inositol P3 -150 cpm/ml cells suspension, and inositol P4 - 2 0 s M. J. Berridge,R. M. C. Dawson,C. P. Downes,J. P. Heslop,and R. F. Irvine,Biochem. J. 212, 473 (1983).
[35]
Ca2+ FLUXESAND PHOSPHOINOSITIDES
537
cpm/ml cell suspension. For more detailed analyses, a linear gradient of ammonium formate (0-2.0 M) in 0.1 M formic acid can be applied to the column. HPLC Analysis of Inositol Phosphates Examination of the [3H]inositol phosphates of labeled hepatocytes and other tissues by HPLC 5'9,1° reveals the presence of inositol bisphosphate isomers, inositol 1,3,4-P3, and inositol 1,3,4,5-P4. Samples (1.0 to 2.0 ml) of extracts from hepatocytes obtained as described above are applied to a Whatman Partisil 10 SAX anion-exchange column together with a RCSS silica Guard-Pak precolumn (Millipore) and the inositol phosphates are eluted as follows. HPLC is performed using a Beckman 421A controller, two 114 M solvent delivery modules, and a 165-variable-wavelength detector. The solvent for pump A is degassed, filtered H20 (Millipore type RA, 1.2 tzm). The solvent for pump B is degassed, Millipore-filtered 2.0 M ammonium formate buffered to pH 3.7 with orthophosphoric acid or 4.0 M ammonium formate buffered to pH 3.2 with formic acid. The column is washed with water at a flow rate of 1.5 ml/min (pump A) for 7 min. Between 7 and 13 rain the eluant is increased linearly from 0 to 50% solvent B, then held at 50% solvent B until 18 min. The eluant is increased linearly from 50 to 100% solvent B between 18 and 28 min, then held at 100% solvent B until 34 min. The gradient is then decreased to 100% solvent A between 34 and 38 min. Fractions are collected at 18-sec intervals using a 2112 Redirac fraction collector (LKB) 2 rain after commencement of the gradient program. The carousel used holds 100 miniscintillation vials, so that scintillant can be added directly and the contents counted in a scintillation counter. When extracts of cells are chromatographed, the eluate is passed through the wavelength detector and the absorbance monitored at 254 nm to monitor the nucleotides. The UV profile serves as a means of determining reproducibility and to verify that the column is not overloaded. If too much salt is present in samples, the UV-absorbing material and most of the 3H-containing material elute very early in the gradient. Figure ! shows a typical HPLC profile. The location of ATP in the fractions coincides with the position of [3H]inositol 1,3,4-P3. The position of [3H]inositol 1,4,5-P3 is verified by chromatography of [3H]inositol 1,4,5P3 obtained from Amersham or New England Nuclear. Inositol 1,3,4,5-P4 9 R. F. Irvine, E. E. Anggard, A. J. Letcher, and C. P. Downes, Biochem. J. 229, 505 (1985). to S. J. Stewart, V. Prpic, F. S. Powers, S. B. Bocckino,F. E. Isaacks, and J. H. Exton, Proc. Natl. Acad. Sci. U.S.A. 83, 6098 (1986).
538
OTHER MAMMALIAN CELLS: INTACT CELLS
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FIG. 1. HPLC analysis of hepatocyte [3H]inositol phosphates formed after 60 sec of 10-7 M vasopressin stimulation. Hepatocytes labeled with myo-[2-3H]inositol (American Radiolabeled Chemicals, Inc.) were incubated for 60 sec with 10-7 M vasopressin. A 1-ml aliquot of cells ( - 4 0 mg wet wt/ml) was deproteinized with trichloroacetic acid, extracted with diethyl ether, and then chromatographed on an HPLC column (Whatman Partisil l0 SAX anionexchange column) as described in the text. A representative elution profile is shown. Peak identification: 1, inositol; 2, glycerophosphorylinositol; 3, inositol monophosphates; 4, inositol 1,4-P2; 5, inositol 3,4-P2 ; 6, inositol 1,3,4-P3 ; 7, inositol 1,4,5-P3 ; and 8, inositol 1,3,4,5P4. The inset is an expanded plot shown to highlight inositol 1,3,4-P3, inositol 1,4,5-P3, and inositol 1,3,4,5-P4.
appears in the column profile between fractions 80 and 90. Its position can be determined by chromatography of [3H]inositol 1,3,4,5-P4 from the above sources. For greater resolution of the inositol Pz and inositol P2 isomers, a linear or nonlinear gradient of ammonium phosphate can be employed with a Whatman Partisil 10 SAX column 11or an Alltech Adsorbosphere column. 12 II N. M. Dean and J. D. Moyer, Biochem. J. 242, 361 (1987). 12 T. Balla, A. J. Baukal, G. Guillemette, R. O. Morgan, and K. J. Catt, Proc. Natl. Acad. Sci. U.S.A. 83, 9323 (1986).
t~o
[35]
Ca2+ FLUXESAND PHOSPHOINOSITIDES
539
Inositol 1,4,5-P3 and other inositol phosphates can also be measured chemically by taking fractions from Dowex l-X8 chromatography or HPLC and desalting and neutralizing them before conversion to myoinositol by alkaline phosphatase. 13The myo-inositol is then quantitated by conversion to scyllo-inosose by myo-inositol dehydrogenase, with NADH generation being measured fluorimetrically after amplification by cycling) TM This method can measure picomole quantities of inositol phosphates) TM When larger amounts of inositol phosphates are present, the liberated myo-inositol can be measured by gas chromatography of its trimethylsilyl derivative) 5 Another method involves separation of inositol 1,4,5-P3 by thin-layer chromatography on polyethyleneimine cellulose, extraction with NH4OH, and acid hydrolysis of the dried extract.~6 The released phosphate is then quantitated using the Malachite Green-phosphomolybdate reaction. ~6 There are also binding assays based on intracellular receptors for inositol 1,4,5-P3. These receptors can be purified from several tissues, but the one most commonly used for assays is from bovine adrenal cortex. 17 An assay kit based on this system which can measure 0.2-25 pmol is commercially available (Amersham). Calcium Fluxes in Hepatocytes Calcium fluxes in hepatocytes can be measured in three basic ways: (1) by monitoring the uptake or efflux of 45Ca2+, (2) by measuring changes in total cell calcium, and (3) by monitoring changes in cytosolic free Ca 2+. Methods (2) and (3) generally yield the most reliable quantitative information and will be described in the greatest detail. M e a s u r e m e n t o f 45Ca2+ Fluxes in H e p a t o c y t e s
Measurements of 45Ca2+ uptake or efflux are frequently the easiest ways to examine hormone effects on Ca 2÷ fluxes in hepatocytes. However, the information derived may be misleading because the cells have several intracellular Ca 2+ pools with which the isotope exchanges at different rates, and because hormones affect not only the fluxes of Ca z+ across the plasma membrane, but also the release and uptake of Ca 2÷ by 13j. A. Shayman,A. R. Morrison, and O. H. Lowry,Anal. Biochem. 162, 562 (1987). 14L. G. MacGregorand F. M. Matschinsky,Anal. Biochem. 141, 382 (1984). 15S. E. Rittenhouseand J. P. Sasson, J. Biol. Chem. 260, 8657 (1985). 16R. H. Underwood,R. Greeley,E. T. Glennon,A. I. Menachery,L. M. Braley,and G. H. Williams, Endocrinology 123, 211 (1988). ~7G. Guillemette,T. Balla, A. J. Baukal, A. Spat, and K. J. Catt, J. Biol. Chem. 262, 1010 (1987).
540
OTHER MAMMALIAN CELLS: INTACT CELLS
[35]
internal organelles. Thus some form of compartmental analysis is necessary to interpret the changes in 45Ca2+ validly and, even then, the conclusions are limited because of the complexity of the system and the difficulty of attaining Ca 2÷ homeostasis prior to 45Ca2+ uptake studies (especially after hormonal perturbation) and of attaining complete isotopic equilibrium prior to 45Ca2÷ efflux studies. In addition, there is usually uncertainty about the anatomical location of the various compartments. The most complete investigations of hormonal effects on hepatocyte Ca 2+ fluxes utilizing 45Ca2+ a r e those of Barritt and associates j8:9 and Borle and associates, z° The methods and analyses employed by these workers will not be described in this chapter, but any reader wishing to use 45Ca2+ to study Ca 2÷ fluxes in any cell is strongly recommended to follow their procedures. As discussed at length elsewhere, simple measurements of hormonally induced changes in 45CaZ+ in cells and/or incubation media are liable to give misleading information about the actual alterations in Ca 2+ fluxes. 21,22
Measurements o f Changes in Total Hepatocyte Ca2+ Using Atomic Absorption Spectroscopy Another approach to analyzing hormone effects on hepatocyte Ca 2÷ fluxes is to measure the changes in total cell Ca 2+ using atomic absorption spectroscopy. 23,z4 Isolated hepatocyte suspensions used for such studies must be equilibrated at 37 ° in Krebs-Henseleit bicarbonate buffer, pH 7.4, with continuous gassing (O2:CO2, 19:1) for at least 10 min after isolation. This allows reaccumulation of Ca 2+ by the cells since the collagenase digestion is carried out with perfusate containing - 5 0 ~ M Ca z+. This equilibration period also allows intracellular K + and Na + to be restored to normal levels. For time-course experiments, 10-20 ml of hepatocyte suspension is incubated with constant shaking (70-90 rpm) in 250-ml polypropylene or polycarbonate Erlenmeyer flasks with continuous gassing with O2:CO2 (19 : 1) at a flow rate of 3-4 liters/min. For dose-response experiments, 2 18 G. J. Barritt, J. C. Parker, and J. C. Wadsworth, J. Physiol. (London) 312, 29 (1981). 19 j, C. Parker, G. J. Barritt, and J. C. Wadsworth, Biochem. J. 216, 51 (1983). 20 R. K. Studer and A. B. Borle, Biochim. Biophys. Acta 762, 302 (1983). 2~ A. B. Bode, this series, Vol. 39, p. 513. 22 j. R. Williamson, R. H. Cooper, and J. B. Hoek, Biochim. Biophys. Acta 639, 243 (1981). 23 F. D. Assimacopoulos-Jeannet, P. F. Blackmore, and J. H. Exton, J. Biol. Chem. 262, 2662 (1977). 24 p. F. Blackmore, F. T. Brumley, J. L. Marks, and J. H. Extort, J. Biol. Chem. 253, 4851 (1978).
[35]
C a 2+ FLUXES AND PHOSPHOINOSITIDES
541
to 3 ml of hepatocyte suspension is incubated in 25-ml polycarbonate or polypropylene Erlenmeyer flasks. Aliquots ranging between 0.5 and 1.0 ml are removed at appropriate times and layered on 10 ml of an ice-cold solution of 150 mM NaCI, 10% (w/v) sucrose, and 0.5 mM EGTA (pH 7.4) contained in 12-ml conical Pyrex test tubes. The tubes are then centrifuged for - 2 0 sec at -3000 rpm to sediment the cells in either an IEC HNSII centrifuge (Damon/International Equipment Division) with a 958 rotor or an IEC size 2 model SBV centrifuge with a 240 rotor and a mechanical foot brake. After centrifugation, the tubes are inverted and allowed to drain for - 5 min. The insides of the tubes are then rinsed with distilled water and allowed to drain for a further 5 min, after which the insides of the tubes are wiped dry with facial tissues. The cell pellets are then dispersed into 0.5 ml of water using a vortex mixer, and 0.5 ml of 0.6 M HC104 containing 0.1% (w/v) LaCI3" 6H20 is added to precipitate protein. The solutions are then centrifuged at 2000 g for 1 min and the Ca z+ in the supernatant fluid is determined by atomic absorption spectroscopy using an atomic absorption spectrophotometer (Perkin-Elmer model 603). Industrial grade acetylene is the fuel and air is used as the oxidant. Calcium standards ranging up to 150/xM are prepared in HCIO4/LaCI3 diluent. Typically, basal Ca 2+ readings are 0.03 to 0.04 absorbance units when the wet weight of the cell suspensions is - 4 0 mg/ml. Contamination of the cells with extracellular Ca 2÷ rarely exceeds 3-5% of cell Ca 2+, thus making correction for extracellular Ca 2+ unnecessary in most experiments. Since the effects of many hormones on net cell Ca 2+ content are relatively small, duplicate samples should be taken and incubations carried out in duplicate or triplicate. For separation of cells and medium 7% (w/v) bovine serum albumin (Pentex) can be used instead of sucrose, and 1% (w/v) LaCI3.6H20 can be used instead of 0.5 mM EGTA to displace extracellular bound Ca 2+. The same results are obtained with either method; however, sucrose is more economical and more effectively minimizes the mixing of the incubation medium with the cell pellet.
Measurement o f Hormone Effects on Free Cytosolic Ca 2+ ([Ca2÷]i) in Hepatocytes An important parameter to measure in hepatocytes when analyzing hormone effects is cytosolic free Ca 2+ ([Ca2+]i). By using fluorescent Ca 2+ chelators (e.g., quin2 and fura2), which can be introduced into cells, changes in [Ca2+]i can be measured. 25,z6When Ca 2+ is bound to quin2 and 25 R. Y. Tsien, T. Pozzan, and T. J. Rink, J. Cell Biol. 94, 325 (1982). 26 G. Grynkiewicz, M. Poenie, and R. Y. Tsien, J. Biol. Chem. 260, 3440 (1985).
542
OTHER MAMMALIAN CELLS: INTACT CELLS
[35]
fura2 there are 5- and 30-fold increases in fluorescence, respectively. Quin2 and fura2 are introduced into cells as the membrane-permeant tetraacetoxymethyl and pentaacetoxymethyl esters, respectively (quin2/ AM and fura2/AM). Intracellular esterases hydrolyze quin2/AM and fura2/AM to yield the membrane-impermeant free acids quin2 and fura2, which accumulate in the cytosol and thus permit the continuous measurement of [ C a 2 + ] i . Hepatocytes loaded with quin2 and fura2 can be used to measure hormone effects o n [Ca2+]i as follows. Hepatocytes are isolated as previously described by collagenase digestion of the liver. Cells ( - 5 0 mg wet wt/ml) are preincubated (5 ml in 25-ml polypropylene Erlenmeyer flasks) for 5-10 min at 37° before quin2/AM or fura2/AM is added. The stock solutions of quin2/AM and fura2/AM are 50 and 1 mM, respectively, in dimethylsulfoxide (Me2SO). Quin2/AM (10/xl) or fura2/AM (20 /zl) stock solution is added to 5 ml of cell suspension and incubated for 15 min with continuous gassing (02 : CO2, 19 : 1). Control cells are incubated with an equivalent amount of Me2SO. After 15 min of incubation, the cells are sedimented (50 g for 1 min) and resuspended in 7 ml of fresh KrebsHenseleit bicarbonate buffer containing 5 mg/ml gelatin and 0.5 or 2.5 mM Ca 2÷, depending on the experimental protocol. Following a 5- to 15-min incubation with continuous gassing, 3 ml of cell suspension is added to a 12 × 50 mm borosilicate glass test tube containing a 0.9-cm-long, 0.2-cmdiameter stirring bar passing through the middle of a triangular shaped piece of polypropylene (each side - 1 cm long). Although many spectrofluorimeters specifically designed to measure certain intracellular ion changes are now commercially available (see below), a Varian model SF330 spectrofluorometer fitted with a magnetic stirrer (Rank Brothers, Bottisham, England) and a thermostatically controlled cell housing can be employed. The cells are stirred and oxygenated by infusing O2:CO2 (19 : 1) through a polyethylene tube (Intramedic PE50, Clay Adams). Hormones, drugs, and agents are injected through another piece of PE50 tubing ( - 4 0 cm long) into the hepatocyte suspension, avoiding the need to open the cell housing. Routinely, 130/zl of the agent to be added is drawn up into a No. 1750 Hamilton syringe. Following injection, 30/xl of agent is added to the cells, with 100/xl still remaining in the syringe needle and PE50 tubing (dead space). For the quin2-1oaded hepatocytes, the excitation and emission wavelengths are 340 and 500 nm, respectively, with a slit width of 10 nm. At the emission wavelength of 500 nm there is a substantial contribution of pyridine nucleotide reduction when hormones are added. 27 By increasing the 27 R. Charest, P. F. Blackmore, B. Berthon, and J. H. Exton, J. Biol. Chem. 258, 8769 (1983).
[35]
543
C a 2+ FLUXES AND PHOSPHOINOSITIDES
emission wavelength to 520 nm, the contribution to the pyridine nucleotide fluorescence can be eliminated. However, this results in a slight loss in sensitivity. The spectrofluorometer output (millivolts) can be recorded directly on a strip chart recorder, but if averaging of several experiments is required it is better to channel the signal into an analog/digital converter and store the data in a computer. For our experiments we use a Hewlett Packard 18652A A/D converter and the computer is a Hewlett Packard 3356 laboratory automation system. The data are usually collected for 30 sec to 1 min before hormone injection and then for various times up to 30 min after hormone addition, with data in millivolts being sampled twice each second. Another advantage of using a computer is that changes in
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TIME (mln) FIG. 2. Effect of vasopressin (vaso), angiotensin II (angio), and epidermal growth factor (EGF) on hepatocyte [Ca2+]~ using fura2. Hepatocytes were loaded with fura2 as described in the text. Agonists were added at 0.5 rain and the changes in fluorescence measured at excitation wavelengths of 340 and 380 nm on two separate aliquots of cells. The data are expressed as the fluorescence values at 340 nm divided by the values at 380 nm. The traces shown are representative (in duplicate) of a single batch of hepatocytes, Autofluorescence 26,27was subtracted in these experiments. The resting [Ca2÷]i calculated using Eq. (1) was 146 nM and the maximum value obtained with vasopressin was 270 nM. In another experiment (not shown) the resting level was 92 nM while the stimulated level was 293 nM. The Ka, Rmi,, Rm~,, and (St'2/Sb2) values used were those given in Ref. 26. However, as detailed in this reference, these values should be determined for each fluorometer due to instrumental variations in sensitivity.
544
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OTHER MAMMALIAN CELLS: INTACT CELLS
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FIG. 3. Dose response of vasopressin to increase [Ca2+]i in quin2-1oaded bepatocytes. Hepatocytes were loaded with quin2 as described in the text. Agonists were added at 0.5 rain. Each curve represents the mean of three separate traces. Each trace was normalized such that the time of hormone addition was zero fluorescence. For this to be done, a base point mean value was obtained over the 5 sec preceding the hormone injection, and this value then subtracted from all other values in the data file. Calculating the base point mean over 5 sec tends to reduce the noise level, since 10 values are averaged (slice width normally is 2/sec). For this experiment the resting [Ca2+]i was 210 nM and the maximum [Ca2+]i obtained with 10 nM vasopressin was 550 nM using Eq. (2). The data illustrate that the rate, magnitude, and lag (time taken before [Ca2+]~ increases) are dose dependent. The concentration of vasopressin for each curve is given in nanomolar units. (P. F. Blackmore, unpublished observations, 1986.)
control fluorescence (non-quin2-1oaded cells) can be subtracted from the fluorescence in quin2-1oaded cells. There are now many spectrofluorometers which have been specifically designed to measure [Ca2+]i in either suspensions of cells or in single cells. The software packages that come with these instruments are designed to be very user friendly. Some of the more widely used spectrofluorometers are manufactured by SPEX Industries, Inc. (Edison, NJ), Photon Tech-
[35]
C a 2+ FLUXES AND PHOSPHOINOSITIDES
545
nology International, Inc. (Princeton, N J), and Tracor, Inc. (Austin, TX), although there are other excellent fluorometers available. For the fura2-1oaded hepatocytes, the two excitation wavelengths used are 340 and 380 nm, while the emission wavelength is 500 nm. If the instrument used is not capable of alternating excitation wavelengths rapidly and calculating the ratio of fluorescence intensities, then two separate runs need to be performed. To this end, the data at both wavelengths are stored in the computer which generates the ratio between the two raw data files (340 nm/380 nm). The [Ca2+]i is then calculated using Eq. (1): [Ca2+]i =
Kd[(R
-
Rmin)/(Rmax
-
R)](Sf2/Sb2)
(I)
The apparent Kd value used in the equation is 224 nM, R is the observed ratio (340 nm/380 nm), while Rmi, and Rmax are the minimum and maximum ratios when the [Ca 2+] is zero and saturating, respectively. Rmaxcan be determined in hepatocytes by adding 50/zM ionomycin to cells incubated in the presence of external Ca z+. This will cause a large net uptake of Ca z+ into the cells, which will then saturate the dye. Rmin c a n then be measured by adding 5 mM EGTA to the same cells treated with ionomycin. This will cause a slow release of intracellular Ca 2+ and a decrease in fura2 fluorescence. The value (Sr2/Sb2) is the fluorescence of the free dye at 380 nm (St2) divided by the fluorescence of the CaZ+-bound dye at 380 n m ( S b 2 ) • A full explanation of each of these terms is given in Grynkiewicz et a l . 26 Figure 2 shows the data from a representative experiment using fura2-1oaded hepatocytes stimulated with several hormones. To quantitate the fluorescence measurements in quin2-1oaded cells, the cells are lysed with Triton X-100 (10 mg/ml) after the fluorescence measurements are made and the fluorescence with saturating Ca z+ (at least 1.0 mM Ca 2+, Fmax) and low Ca 2+ (1.0 mM EGTA in excess of Ca z+ or 1.0 mM Mn 2+, Fmi.) are measured. The [Ca2+]i is calculated using Eq. (2): [Ca2+]i
= Kd[(F -
Fmin)/Frnax -
F)]
(2)
The apparent Kd value used in the equation is 115 nM, which was determined under ionic conditions which are similar to cytosol, namely high K + and low Na +.z5 Figure 3 shows the effects of several concentrations of vasopressin on [Ca2+]~ in quin2-1oaded hepatocytes; similar data are obtained with angiotensin II and epinephrine as agonists. 27
OTHER MAMMALIAN CELLS" INTACT CELLS
[35]
inflammatory processes or during extreme physical exercise. Indeed, under such conditions GSH levels in liver and other organs were significantly decreased. 97,98 Much has to be learned about the detailed mechanisms of the processes. It is known that glutathione conjugates may exert inhibitory effects on some enzyme activities like glutathione transferase 99 and glutathione reductase.100 Efficient biliary disposal of these conjugates is therefore of extreme importance. This becomes especially critical during conditions of oxidative stress accompanied by increased canalicular GSSG transport. GSSG could depress transport of the conjugate and the accumulation of the conjugate could then be amplified by its inhibitory effect on GSSG reductase. Injected leukotriene C4 is rapidly taken up by the liver and metabolized via the mercapturic acid pathway. 101The major product found in the bile depends on the species studied and is in the rat initially LTD4, at a later stage N-acetyl-LTE4 and to-oxidized metabolites predominate. This pathway operates during various types of tissue trauma, including surgical interventions, endotoxin shock, and virus-induced hepatitis.I°1 Acknowledgment Supported by the Deutsche Forschungsgemeinschaft, Grants Ak 8/1-1 and Si 255/8-1. 97 p. C. Bragt and I. L. Bonta, Agents Actions 10, 536 (1980). H. Lew, S. Pyke, and A. Quintanilha, FEBS Lett. 185, 262 0985). 99 I. Jakobson, M. Warholm, and B. Mannervik, J. Biol. Chem. 254, 7985 (1979). 10~M. Bilzer, R. L. Krauth-Siegel, R. H. Schirmer, T. P. M. Akerboom, H. Sies, and G. Schulz, Eur. J. Biochem. 138, 373 (1984). ~0~ M. Huber and D. Keppler, in "Glutathione Conjugation: Its Mechanism and Biological Significance" (H. Sies and B. Ketterer, eds.), 449. Academic Press, London, 1988.
[35] C a 2+ F l u x e s a n d P h o s p h o i n o s i t i d e s in H e p a t o c y t e s
By P. F. BLACKMORE and J. H. EXTON Introduction Vasopressin, angiotensin II, al-adrenergic agonists (epinephrine, norepinephrine), and P2 purinergic agonists (ATP, ADP) exert their effects on liver and certain other tissues by raising the Ca 2+ concentration in the cytosol. 1 On the other hand, glucagon and fl-adrenergic agonists exert i j. H. Exton, Adv. Cyclic Nucleotide Protein Phosphorylation Res. 20, 211 (1986).
METHODS IN ENZYMOLOGY,VOL. 173
Copyright© 1989by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[35]
C a 2+ FLUXES AND PHOSPHOINOSITIDES
535
their effects on liver by raising cAMP, although they also increase cytosolic Ca 2+. The alterations in liver cell Ca 2+ induced by these agents can be monitored by measuring Ca 2+ fluxes across the plasma membrane, changes in the total cellular content of Ca 2+, or changes in cytosolic Ca 2+. Hormones increase cytosolic Ca 2+ in their target cells by mobilizing intracellular Ca 2+ stores and by altering the flux of Ca 2+ across the plasma membrane. There is much evidence that the mobilization of internal Ca 2+ is caused by inositol 1,4,5-trisphosphate generated by the breakdown of phosphatidylinositol 4,5-bisphosphate in the plasma membrane. 1,2 This breakdown occurs very rapidly in liver and other tissues 1,3 and inositol 1,4,5-P3 has been demonstrated to release Ca 2+ from endoplasmic reticulum stores in permeabilized hepatocytes and other cells. 3 Many other inositol phosphates have been identified in liver and other tissues, including inositol 1,3,4-P34.5 and inositol 1,3,4,5-P4.5.6 Their functions are presently unknown. Measurement of myo-Inositol 1,4,5-trisphosphate The most commonly used method for measuring the level of inositol 1,4,5-P3 in liver is to incubate hepatocytes or inject animals with myo-[2-3H]inositol for a period of time (1.5 hr or longer) to label the inositol phospholipids to isotopic equilibrium. Acid extracts of hepatocytes are then subjected to column chromatography on Dowex l-X8. Columns are washed with increasing concentrations of ammonium formate to elute the various inositol phosphates followed by counting the radioactivity in the fractions. Although useful for routine studies, this method does not allow the separation of inositol phosphate isomers such as inositol 1,4,5-P3 and inositol 1,3,4-P3. For this, HPLC methods are used. Both methods are described below. Isolation and Loading of Hepatocytes with myo-[2-3H]Inositol Hepatocytes can be isolated by collagenase digestion 7 from rats injected intraperitoneally with 500/~Ci of myo-[2-3H]inositol as described below. Cell suspensions ( - 4 0 to 50 mg wet wt/ml) are incubated in 2 M. J. Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 3 j. R. Williamson, R. H. Cooper, S. K. Joseph, and A. P. Thomas, Am. J. Physiol. 248, C203 (1985). 4 R. F. Irvine, A. J. Letcher, D. J. Lander, and C. P. Downes, Biochem. J. 223, 237 (1984). 5 C. A. Hansen, S. Mah, and J. R. Williamson, J. Biol. Chem. 261, 8100 (1986). 6 I. R. Batty, S. R. Nahorski, and R. F. Irvine, Biochem. J. 232, 211 (1985). 7 p. F. Blackmore and J. H. Exton, this series, Vol. 109, p. 550.
536
OTHERMAMMALIANCELLS;INTACTCELLS
[35]
Krebs-Henseleit bicarbonate buffer containing 1.5% (w/v) gelatin (Difco) and are continuously gassed with O2/CO2 (19: I). For labeling in vitro, hepatocytes (40-ml cell suspension in 250-ml Erlenmeyer flasks) are incubated for 90 or 120 min with 0.1 mM myo-[2-3H]inositol (25/zCi/ml of cell suspension). The cells are then filtered through nylon mesh to remove any clumps of cells which may form. The cells are then washed once and resuspended in fresh medium without myo-[2-3H]inositol or gelatin for use in experiments. The gelatin is omitted from the buffer at this stage since this protein is difficult to remove with trichloroacetic acid. After incubation for various periods under the experimental conditions desired, l-ml aliquots are pipetted into tubes containing 0.2 ml of 100% (w/v) trichloroacetic acid at 0°. After centrifugation (5000 g for 10 min) the supernatant fluid is extracted six times with 2-ml aliquots of diethyl ether, saturated with water, to remove trichloroacetic acid. The last traces of diethyl ether are removed by blowing a stream of N2 into the solutions which are then stored at - 2 0 ° before analysis. The neutralized extracts are subjected to ion-exchange chromatography or HPLC as follows. Dowex l-X8 Chromatography of Inositol Phosphates One milliliter of 10 mM sodium tetraborate is added to the neutralized extract and then the entire solution is applied to 2 ml of Dowex l-X8 (formate form) ion-exchange resin contained in P-5000 Pipetman disposable pipet tips (Rainin Instrument Co.). The resin is held in place by a small plug of fiberglass wool (Coming Glass Works). A small plug of fiberglass wool is also placed on top of the resin to prevent it from being disturbed by the eluting solutions of ammonium formate. The [3H]inositol phosphates are eluted as follows. Free [3H]inositol is removed from the column by washing with 15 ml of water, glycerophosphoinositol is removed by 2 × 5 ml of 5 mM sodium tetraborate/60 mM ammonium formate, inositol PI is eluted with 2 × 5 ml of 0.2 M ammonium formate/ 0.1 M formic acid, inositol P2 is eluted with 3 × 5 ml of 0.4 M ammonium formate/0.1 M formic acid, inositol P3 is eluted with 5 ml of 0.7 M ammonium forrnate/0.1 M formic acid, and inositol P4 is eluted with 5 ml of 2.0 M ammonium formate/0.1 M formic acid. 8 To each eluate, consisting of 5 ml in 25-ml plastic scintillation vials, is added 15 ml of Beckman ReadySolv EP and the contents are mixed and counted. Typically, inositol P1 contained -9000 cpm/ml cell suspension, inositol P2 -1200 cpm/ml cell suspension, inositol P3 -150 cpm/ml cells suspension, and inositol P4 - 2 0 s M. J. Berridge,R. M. C. Dawson,C. P. Downes,J. P. Heslop,and R. F. Irvine,Biochem. J. 212, 473 (1983).
[35]
Ca2+ FLUXESAND PHOSPHOINOSITIDES
537
cpm/ml cell suspension. For more detailed analyses, a linear gradient of ammonium formate (0-2.0 M) in 0.1 M formic acid can be applied to the column. HPLC Analysis of Inositol Phosphates Examination of the [3H]inositol phosphates of labeled hepatocytes and other tissues by HPLC 5'9,1° reveals the presence of inositol bisphosphate isomers, inositol 1,3,4-P3, and inositol 1,3,4,5-P4. Samples (1.0 to 2.0 ml) of extracts from hepatocytes obtained as described above are applied to a Whatman Partisil 10 SAX anion-exchange column together with a RCSS silica Guard-Pak precolumn (Millipore) and the inositol phosphates are eluted as follows. HPLC is performed using a Beckman 421A controller, two 114 M solvent delivery modules, and a 165-variable-wavelength detector. The solvent for pump A is degassed, filtered H20 (Millipore type RA, 1.2 tzm). The solvent for pump B is degassed, Millipore-filtered 2.0 M ammonium formate buffered to pH 3.7 with orthophosphoric acid or 4.0 M ammonium formate buffered to pH 3.2 with formic acid. The column is washed with water at a flow rate of 1.5 ml/min (pump A) for 7 min. Between 7 and 13 rain the eluant is increased linearly from 0 to 50% solvent B, then held at 50% solvent B until 18 min. The eluant is increased linearly from 50 to 100% solvent B between 18 and 28 min, then held at 100% solvent B until 34 min. The gradient is then decreased to 100% solvent A between 34 and 38 min. Fractions are collected at 18-sec intervals using a 2112 Redirac fraction collector (LKB) 2 rain after commencement of the gradient program. The carousel used holds 100 miniscintillation vials, so that scintillant can be added directly and the contents counted in a scintillation counter. When extracts of cells are chromatographed, the eluate is passed through the wavelength detector and the absorbance monitored at 254 nm to monitor the nucleotides. The UV profile serves as a means of determining reproducibility and to verify that the column is not overloaded. If too much salt is present in samples, the UV-absorbing material and most of the 3H-containing material elute very early in the gradient. Figure ! shows a typical HPLC profile. The location of ATP in the fractions coincides with the position of [3H]inositol 1,3,4-P3. The position of [3H]inositol 1,4,5-P3 is verified by chromatography of [3H]inositol 1,4,5P3 obtained from Amersham or New England Nuclear. Inositol 1,3,4,5-P4 9 R. F. Irvine, E. E. Anggard, A. J. Letcher, and C. P. Downes, Biochem. J. 229, 505 (1985). to S. J. Stewart, V. Prpic, F. S. Powers, S. B. Bocckino,F. E. Isaacks, and J. H. Exton, Proc. Natl. Acad. Sci. U.S.A. 83, 6098 (1986).
538
OTHER MAMMALIAN CELLS: INTACT CELLS
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FIG. 1. HPLC analysis of hepatocyte [3H]inositol phosphates formed after 60 sec of 10-7 M vasopressin stimulation. Hepatocytes labeled with myo-[2-3H]inositol (American Radiolabeled Chemicals, Inc.) were incubated for 60 sec with 10-7 M vasopressin. A 1-ml aliquot of cells ( - 4 0 mg wet wt/ml) was deproteinized with trichloroacetic acid, extracted with diethyl ether, and then chromatographed on an HPLC column (Whatman Partisil l0 SAX anionexchange column) as described in the text. A representative elution profile is shown. Peak identification: 1, inositol; 2, glycerophosphorylinositol; 3, inositol monophosphates; 4, inositol 1,4-P2; 5, inositol 3,4-P2 ; 6, inositol 1,3,4-P3 ; 7, inositol 1,4,5-P3 ; and 8, inositol 1,3,4,5P4. The inset is an expanded plot shown to highlight inositol 1,3,4-P3, inositol 1,4,5-P3, and inositol 1,3,4,5-P4.
appears in the column profile between fractions 80 and 90. Its position can be determined by chromatography of [3H]inositol 1,3,4,5-P4 from the above sources. For greater resolution of the inositol Pz and inositol P2 isomers, a linear or nonlinear gradient of ammonium phosphate can be employed with a Whatman Partisil 10 SAX column 11or an Alltech Adsorbosphere column. 12 II N. M. Dean and J. D. Moyer, Biochem. J. 242, 361 (1987). 12 T. Balla, A. J. Baukal, G. Guillemette, R. O. Morgan, and K. J. Catt, Proc. Natl. Acad. Sci. U.S.A. 83, 9323 (1986).
t~o
[35]
Ca2+ FLUXESAND PHOSPHOINOSITIDES
539
Inositol 1,4,5-P3 and other inositol phosphates can also be measured chemically by taking fractions from Dowex l-X8 chromatography or HPLC and desalting and neutralizing them before conversion to myoinositol by alkaline phosphatase. 13The myo-inositol is then quantitated by conversion to scyllo-inosose by myo-inositol dehydrogenase, with NADH generation being measured fluorimetrically after amplification by cycling) TM This method can measure picomole quantities of inositol phosphates) TM When larger amounts of inositol phosphates are present, the liberated myo-inositol can be measured by gas chromatography of its trimethylsilyl derivative) 5 Another method involves separation of inositol 1,4,5-P3 by thin-layer chromatography on polyethyleneimine cellulose, extraction with NH4OH, and acid hydrolysis of the dried extract.~6 The released phosphate is then quantitated using the Malachite Green-phosphomolybdate reaction. ~6 There are also binding assays based on intracellular receptors for inositol 1,4,5-P3. These receptors can be purified from several tissues, but the one most commonly used for assays is from bovine adrenal cortex. 17 An assay kit based on this system which can measure 0.2-25 pmol is commercially available (Amersham). Calcium Fluxes in Hepatocytes Calcium fluxes in hepatocytes can be measured in three basic ways: (1) by monitoring the uptake or efflux of 45Ca2+, (2) by measuring changes in total cell calcium, and (3) by monitoring changes in cytosolic free Ca 2+. Methods (2) and (3) generally yield the most reliable quantitative information and will be described in the greatest detail. M e a s u r e m e n t o f 45Ca2+ Fluxes in H e p a t o c y t e s
Measurements of 45Ca2+ uptake or efflux are frequently the easiest ways to examine hormone effects on Ca 2÷ fluxes in hepatocytes. However, the information derived may be misleading because the cells have several intracellular Ca 2+ pools with which the isotope exchanges at different rates, and because hormones affect not only the fluxes of Ca z+ across the plasma membrane, but also the release and uptake of Ca 2÷ by 13j. A. Shayman,A. R. Morrison, and O. H. Lowry,Anal. Biochem. 162, 562 (1987). 14L. G. MacGregorand F. M. Matschinsky,Anal. Biochem. 141, 382 (1984). 15S. E. Rittenhouseand J. P. Sasson, J. Biol. Chem. 260, 8657 (1985). 16R. H. Underwood,R. Greeley,E. T. Glennon,A. I. Menachery,L. M. Braley,and G. H. Williams, Endocrinology 123, 211 (1988). ~7G. Guillemette,T. Balla, A. J. Baukal, A. Spat, and K. J. Catt, J. Biol. Chem. 262, 1010 (1987).
540
OTHER MAMMALIAN CELLS: INTACT CELLS
[35]
internal organelles. Thus some form of compartmental analysis is necessary to interpret the changes in 45Ca2+ validly and, even then, the conclusions are limited because of the complexity of the system and the difficulty of attaining Ca 2÷ homeostasis prior to 45Ca2+ uptake studies (especially after hormonal perturbation) and of attaining complete isotopic equilibrium prior to 45Ca2÷ efflux studies. In addition, there is usually uncertainty about the anatomical location of the various compartments. The most complete investigations of hormonal effects on hepatocyte Ca 2+ fluxes utilizing 45Ca2+ a r e those of Barritt and associates j8:9 and Borle and associates, z° The methods and analyses employed by these workers will not be described in this chapter, but any reader wishing to use 45Ca2+ to study Ca 2÷ fluxes in any cell is strongly recommended to follow their procedures. As discussed at length elsewhere, simple measurements of hormonally induced changes in 45CaZ+ in cells and/or incubation media are liable to give misleading information about the actual alterations in Ca 2+ fluxes. 21,22
Measurements o f Changes in Total Hepatocyte Ca2+ Using Atomic Absorption Spectroscopy Another approach to analyzing hormone effects on hepatocyte Ca 2÷ fluxes is to measure the changes in total cell Ca 2+ using atomic absorption spectroscopy. 23,z4 Isolated hepatocyte suspensions used for such studies must be equilibrated at 37 ° in Krebs-Henseleit bicarbonate buffer, pH 7.4, with continuous gassing (O2:CO2, 19:1) for at least 10 min after isolation. This allows reaccumulation of Ca 2+ by the cells since the collagenase digestion is carried out with perfusate containing - 5 0 ~ M Ca z+. This equilibration period also allows intracellular K + and Na + to be restored to normal levels. For time-course experiments, 10-20 ml of hepatocyte suspension is incubated with constant shaking (70-90 rpm) in 250-ml polypropylene or polycarbonate Erlenmeyer flasks with continuous gassing with O2:CO2 (19 : 1) at a flow rate of 3-4 liters/min. For dose-response experiments, 2 18 G. J. Barritt, J. C. Parker, and J. C. Wadsworth, J. Physiol. (London) 312, 29 (1981). 19 j, C. Parker, G. J. Barritt, and J. C. Wadsworth, Biochem. J. 216, 51 (1983). 20 R. K. Studer and A. B. Borle, Biochim. Biophys. Acta 762, 302 (1983). 2~ A. B. Bode, this series, Vol. 39, p. 513. 22 j. R. Williamson, R. H. Cooper, and J. B. Hoek, Biochim. Biophys. Acta 639, 243 (1981). 23 F. D. Assimacopoulos-Jeannet, P. F. Blackmore, and J. H. Exton, J. Biol. Chem. 262, 2662 (1977). 24 p. F. Blackmore, F. T. Brumley, J. L. Marks, and J. H. Extort, J. Biol. Chem. 253, 4851 (1978).
[35]
C a 2+ FLUXES AND PHOSPHOINOSITIDES
541
to 3 ml of hepatocyte suspension is incubated in 25-ml polycarbonate or polypropylene Erlenmeyer flasks. Aliquots ranging between 0.5 and 1.0 ml are removed at appropriate times and layered on 10 ml of an ice-cold solution of 150 mM NaCI, 10% (w/v) sucrose, and 0.5 mM EGTA (pH 7.4) contained in 12-ml conical Pyrex test tubes. The tubes are then centrifuged for - 2 0 sec at -3000 rpm to sediment the cells in either an IEC HNSII centrifuge (Damon/International Equipment Division) with a 958 rotor or an IEC size 2 model SBV centrifuge with a 240 rotor and a mechanical foot brake. After centrifugation, the tubes are inverted and allowed to drain for - 5 min. The insides of the tubes are then rinsed with distilled water and allowed to drain for a further 5 min, after which the insides of the tubes are wiped dry with facial tissues. The cell pellets are then dispersed into 0.5 ml of water using a vortex mixer, and 0.5 ml of 0.6 M HC104 containing 0.1% (w/v) LaCI3" 6H20 is added to precipitate protein. The solutions are then centrifuged at 2000 g for 1 min and the Ca z+ in the supernatant fluid is determined by atomic absorption spectroscopy using an atomic absorption spectrophotometer (Perkin-Elmer model 603). Industrial grade acetylene is the fuel and air is used as the oxidant. Calcium standards ranging up to 150/xM are prepared in HCIO4/LaCI3 diluent. Typically, basal Ca 2+ readings are 0.03 to 0.04 absorbance units when the wet weight of the cell suspensions is - 4 0 mg/ml. Contamination of the cells with extracellular Ca 2÷ rarely exceeds 3-5% of cell Ca 2+, thus making correction for extracellular Ca 2+ unnecessary in most experiments. Since the effects of many hormones on net cell Ca 2+ content are relatively small, duplicate samples should be taken and incubations carried out in duplicate or triplicate. For separation of cells and medium 7% (w/v) bovine serum albumin (Pentex) can be used instead of sucrose, and 1% (w/v) LaCI3.6H20 can be used instead of 0.5 mM EGTA to displace extracellular bound Ca 2+. The same results are obtained with either method; however, sucrose is more economical and more effectively minimizes the mixing of the incubation medium with the cell pellet.
Measurement o f Hormone Effects on Free Cytosolic Ca 2+ ([Ca2÷]i) in Hepatocytes An important parameter to measure in hepatocytes when analyzing hormone effects is cytosolic free Ca 2+ ([Ca2+]i). By using fluorescent Ca 2+ chelators (e.g., quin2 and fura2), which can be introduced into cells, changes in [Ca2+]i can be measured. 25,z6When Ca 2+ is bound to quin2 and 25 R. Y. Tsien, T. Pozzan, and T. J. Rink, J. Cell Biol. 94, 325 (1982). 26 G. Grynkiewicz, M. Poenie, and R. Y. Tsien, J. Biol. Chem. 260, 3440 (1985).
542
OTHER MAMMALIAN CELLS: INTACT CELLS
[35]
fura2 there are 5- and 30-fold increases in fluorescence, respectively. Quin2 and fura2 are introduced into cells as the membrane-permeant tetraacetoxymethyl and pentaacetoxymethyl esters, respectively (quin2/ AM and fura2/AM). Intracellular esterases hydrolyze quin2/AM and fura2/AM to yield the membrane-impermeant free acids quin2 and fura2, which accumulate in the cytosol and thus permit the continuous measurement of [ C a 2 + ] i . Hepatocytes loaded with quin2 and fura2 can be used to measure hormone effects o n [Ca2+]i as follows. Hepatocytes are isolated as previously described by collagenase digestion of the liver. Cells ( - 5 0 mg wet wt/ml) are preincubated (5 ml in 25-ml polypropylene Erlenmeyer flasks) for 5-10 min at 37° before quin2/AM or fura2/AM is added. The stock solutions of quin2/AM and fura2/AM are 50 and 1 mM, respectively, in dimethylsulfoxide (Me2SO). Quin2/AM (10/xl) or fura2/AM (20 /zl) stock solution is added to 5 ml of cell suspension and incubated for 15 min with continuous gassing (02 : CO2, 19 : 1). Control cells are incubated with an equivalent amount of Me2SO. After 15 min of incubation, the cells are sedimented (50 g for 1 min) and resuspended in 7 ml of fresh KrebsHenseleit bicarbonate buffer containing 5 mg/ml gelatin and 0.5 or 2.5 mM Ca 2÷, depending on the experimental protocol. Following a 5- to 15-min incubation with continuous gassing, 3 ml of cell suspension is added to a 12 × 50 mm borosilicate glass test tube containing a 0.9-cm-long, 0.2-cmdiameter stirring bar passing through the middle of a triangular shaped piece of polypropylene (each side - 1 cm long). Although many spectrofluorimeters specifically designed to measure certain intracellular ion changes are now commercially available (see below), a Varian model SF330 spectrofluorometer fitted with a magnetic stirrer (Rank Brothers, Bottisham, England) and a thermostatically controlled cell housing can be employed. The cells are stirred and oxygenated by infusing O2:CO2 (19 : 1) through a polyethylene tube (Intramedic PE50, Clay Adams). Hormones, drugs, and agents are injected through another piece of PE50 tubing ( - 4 0 cm long) into the hepatocyte suspension, avoiding the need to open the cell housing. Routinely, 130/zl of the agent to be added is drawn up into a No. 1750 Hamilton syringe. Following injection, 30/xl of agent is added to the cells, with 100/xl still remaining in the syringe needle and PE50 tubing (dead space). For the quin2-1oaded hepatocytes, the excitation and emission wavelengths are 340 and 500 nm, respectively, with a slit width of 10 nm. At the emission wavelength of 500 nm there is a substantial contribution of pyridine nucleotide reduction when hormones are added. 27 By increasing the 27 R. Charest, P. F. Blackmore, B. Berthon, and J. H. Exton, J. Biol. Chem. 258, 8769 (1983).
[35]
543
C a 2+ FLUXES AND PHOSPHOINOSITIDES
emission wavelength to 520 nm, the contribution to the pyridine nucleotide fluorescence can be eliminated. However, this results in a slight loss in sensitivity. The spectrofluorometer output (millivolts) can be recorded directly on a strip chart recorder, but if averaging of several experiments is required it is better to channel the signal into an analog/digital converter and store the data in a computer. For our experiments we use a Hewlett Packard 18652A A/D converter and the computer is a Hewlett Packard 3356 laboratory automation system. The data are usually collected for 30 sec to 1 min before hormone injection and then for various times up to 30 min after hormone addition, with data in millivolts being sampled twice each second. Another advantage of using a computer is that changes in
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TIME (mln) FIG. 2. Effect of vasopressin (vaso), angiotensin II (angio), and epidermal growth factor (EGF) on hepatocyte [Ca2+]~ using fura2. Hepatocytes were loaded with fura2 as described in the text. Agonists were added at 0.5 rain and the changes in fluorescence measured at excitation wavelengths of 340 and 380 nm on two separate aliquots of cells. The data are expressed as the fluorescence values at 340 nm divided by the values at 380 nm. The traces shown are representative (in duplicate) of a single batch of hepatocytes, Autofluorescence 26,27was subtracted in these experiments. The resting [Ca2÷]i calculated using Eq. (1) was 146 nM and the maximum value obtained with vasopressin was 270 nM. In another experiment (not shown) the resting level was 92 nM while the stimulated level was 293 nM. The Ka, Rmi,, Rm~,, and (St'2/Sb2) values used were those given in Ref. 26. However, as detailed in this reference, these values should be determined for each fluorometer due to instrumental variations in sensitivity.
544
[35]
OTHER MAMMALIAN CELLS: INTACT CELLS
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FIG. 3. Dose response of vasopressin to increase [Ca2+]i in quin2-1oaded bepatocytes. Hepatocytes were loaded with quin2 as described in the text. Agonists were added at 0.5 rain. Each curve represents the mean of three separate traces. Each trace was normalized such that the time of hormone addition was zero fluorescence. For this to be done, a base point mean value was obtained over the 5 sec preceding the hormone injection, and this value then subtracted from all other values in the data file. Calculating the base point mean over 5 sec tends to reduce the noise level, since 10 values are averaged (slice width normally is 2/sec). For this experiment the resting [Ca2+]i was 210 nM and the maximum [Ca2+]i obtained with 10 nM vasopressin was 550 nM using Eq. (2). The data illustrate that the rate, magnitude, and lag (time taken before [Ca2+]~ increases) are dose dependent. The concentration of vasopressin for each curve is given in nanomolar units. (P. F. Blackmore, unpublished observations, 1986.)
control fluorescence (non-quin2-1oaded cells) can be subtracted from the fluorescence in quin2-1oaded cells. There are now many spectrofluorometers which have been specifically designed to measure [Ca2+]i in either suspensions of cells or in single cells. The software packages that come with these instruments are designed to be very user friendly. Some of the more widely used spectrofluorometers are manufactured by SPEX Industries, Inc. (Edison, NJ), Photon Tech-
[35]
C a 2+ FLUXES AND PHOSPHOINOSITIDES
545
nology International, Inc. (Princeton, N J), and Tracor, Inc. (Austin, TX), although there are other excellent fluorometers available. For the fura2-1oaded hepatocytes, the two excitation wavelengths used are 340 and 380 nm, while the emission wavelength is 500 nm. If the instrument used is not capable of alternating excitation wavelengths rapidly and calculating the ratio of fluorescence intensities, then two separate runs need to be performed. To this end, the data at both wavelengths are stored in the computer which generates the ratio between the two raw data files (340 nm/380 nm). The [Ca2+]i is then calculated using Eq. (1): [Ca2+]i =
Kd[(R
-
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R)](Sf2/Sb2)
(I)
The apparent Kd value used in the equation is 224 nM, R is the observed ratio (340 nm/380 nm), while Rmi, and Rmax are the minimum and maximum ratios when the [Ca 2+] is zero and saturating, respectively. Rmaxcan be determined in hepatocytes by adding 50/zM ionomycin to cells incubated in the presence of external Ca z+. This will cause a large net uptake of Ca z+ into the cells, which will then saturate the dye. Rmin c a n then be measured by adding 5 mM EGTA to the same cells treated with ionomycin. This will cause a slow release of intracellular Ca 2+ and a decrease in fura2 fluorescence. The value (Sr2/Sb2) is the fluorescence of the free dye at 380 nm (St2) divided by the fluorescence of the CaZ+-bound dye at 380 n m ( S b 2 ) • A full explanation of each of these terms is given in Grynkiewicz et a l . 26 Figure 2 shows the data from a representative experiment using fura2-1oaded hepatocytes stimulated with several hormones. To quantitate the fluorescence measurements in quin2-1oaded cells, the cells are lysed with Triton X-100 (10 mg/ml) after the fluorescence measurements are made and the fluorescence with saturating Ca z+ (at least 1.0 mM Ca 2+, Fmax) and low Ca 2+ (1.0 mM EGTA in excess of Ca z+ or 1.0 mM Mn 2+, Fmi.) are measured. The [Ca2+]i is calculated using Eq. (2): [Ca2+]i
= Kd[(F -
Fmin)/Frnax -
F)]
(2)
The apparent Kd value used in the equation is 115 nM, which was determined under ionic conditions which are similar to cytosol, namely high K + and low Na +.z5 Figure 3 shows the effects of several concentrations of vasopressin on [Ca2+]~ in quin2-1oaded hepatocytes; similar data are obtained with angiotensin II and epinephrine as agonists. 27