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CYTOSOLIC FREE MAGNESIUM, ATP, AND BLEBBING DURING CHEMICAL HYPOXIA IN CULTURED RAT HEPATOCYTES ’ Andrew W. Harman2, Anna-Liisa Nieminen, John J. Lemasters and Brian Herman3 Laboratories for Cell Biology, Department of Cell Biolo & Anatomy, School of Medicine, University of North Carolina at F hapel Hill, CB #7090, Chapel Hill, NC 27599-7090 Received

May 29,


Cytosolic free Mg2+ concentration was determined in l-day cultured rat hepatocytes using Multiparameter Digitized Video Microscopy (MDVM) of the fluorescent probe, magfura-2. Chemical hypoxia with KCN (5 mM) and iodoacetate (1 mM), a model which mi its the ,4TP depletion and reductive stressof hypoxia, caused a rapid increase of free _ 0.2 to 1.6 + 0.2 mM within 4 min. Concurrently, numerous small lasma Mg‘+ from 1.1 + membrane blebs formed and ATP levels dropped from 13.24 to 1.32 nmol/lO E cells. Removal of KCN and iodoacetate resulted in very of ATP to 60-70% of pre-exposure levels, a concomitant decreasein cytosolicfree M + back toward basal levels and rever al of blebbing (bleb resorption . These results indicate that changes of cytosol~c free Mg S+ inversely reflect changesof d. TP in a model of hypoxia and reoxygenation. Bleb formation and resorption were dependent on the fall and rise of ATP. 01990 Academic Press, Inc. Chemical hypoxia has been used to model the cellular events associatedwith hypoxic injury in hepatocytes (1,2). In this model, KCN inhibits mitochondrial respiration and iodoacetate

inhibits gylcolysis. Chemical hypoxia is characterized by reduction of

mitochondrial electron carriers and depletion of ATP leading to cell surface blebbing and, eventually, breakdown of

the plasma membrane permeability barrier with loss of cell

viability. It has been suggestedthat ATP depletion during hypoxic injury results in alterations in the levels of cytosolic ions due to inhibition of ATP-dependent ion transporters. In particular, a rise in cytosolic free Ca+ + soon after the onset of hypoxia may activate Ca++r This work was supported by Grants AGO7218 and DK30874 from the National Institutes of Health, the Gustavas and Louise Pfeiffer Foundation, and Grant J-1433 from the Office of Naval Research. 2 On leave from the Department of Pharmacology, The University of Western Australia, Nedlands 6009, Australia. 3 To whom correspondence should be addressed. Abbreviations used are: KRH, Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 2 mM CaC12,l mM KHzPO4,1.2 mM MgSO4,25 mM NaHEPES buffer, pH 7.4; mag-fura-2, (2-[2-(5-carbo~)oxazole]-5-hydro~-6-aminobenzofuran-N,N,O-triacetic acid; MDVM, Multiparameter Digitized Video Microscopy. ooO6-291x/90



Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.




2, 1990


dependent degradative




enzymes leading to cell death (3). However,




by us

indicates that chemical hypoxia in cultured rat hepatocytes is not associated with an increase of cytosolic free Ca ++, although large increases of free H+ and free Na+ do occur (1,2,4,5). Recently, Raju and colleagues described a new fluorescent of cytosolic free Mg2+

m . suspensions



this procedure


of isolated hepatocytes free M2+

probe for measurement

(6). In the present study, we

in single cultured

rat hepatocytes


MDVM. In hepatocytes, adenine nucloetides magnitude

more than 90% of Mg2+ is bound to cellular constituents,

(7). The binding affinity of Mg 2+ for ATP is more than an order of

greater than for other adenine nucleotides

the decrease

in ATP produced

increase of free Mg2’.

by chemical

Here, using MDVM

single cultured hepatocytes

or inorganic phosphate

hypoxia should produce and mag-fura-2,

(7). Hence,

a corresponding

we monitored

free Mg2+ in

after exposure to KCN plus iodoacetate to determine if changes

of ATP produced by hypoxia are reflected by concomitant MATERIALS


changes of free Mg2+.


Hepatocyte Isolation - Hepatocytes were isolated form male S rague-Dawley rats (200-250 g) by collagenase perfusion as described previously (8). C!ells (106/ml) were cultured overnight on glass coverslips in 35 mm culture dishes in Waymouths Medium MB752/2 containing 5% fetal calf serum, 100 ng/nl streptomycin and 100 III/ml penicillin. The cells were then loaded with 2 PM mag-furaacetorqmethyl ester (Molecular Probes, Eugene, OR) for 10 min at 37” C and rinsed twice with 2 ml of KRH. MDVM - Following labeling, coverslips were mounted on the stage of the MDVM system. Temperature was maintained at 37O C using an air-curtain incubator. Fluorescence images were obtained using 340 nm and 380 nm excitation light, a 395 nm dichroic mirror and a 440 nm long pass barrier filter. After background subtraction, images at 340 nm were divided by images at 380 nm on a pixel by pixel basis. Details of the imaging system have been published elsewhere (1,8,9). Standard curves relating intensity to free M$’ concentration were obtained using mag-furafree acid (a gift from Dr. Elizabeth Murphy, HS, Research Triangle Park, NC) and EDTA buffers containing known amounts of + (10). The free acid was found to have the same fluorescence spectral properties as the de-esterified product formed by incubating mag-furaacstoxymethyl ester with hepatocytes. The excitation maximum of mag-fura was 370 nm. Mg + shifted the excitation maximum of to 3$8+nm with an isoasbestic point of 348 nm. There was a linear relationship between free Mg concentration and the fluorescence ratio at 340 and 380 nm (data not shown). The dissociation constant for mag-fura(KJJ) was estimated from a Hill plot to be 1.0 mM. ATP, AD and AMP Determination - ATP, ADP and AMP content of cultured hepatocytes (1 J cells/dish was determined by HPLC after perchloric acid extraction as previously described (11). I’n some experiments, ATP was determined by a coupled enzyme assay as previously described (12). RESULTS Probe Localization hepatocytes

and Basal Free i@’

in 2 PM mag-fura-

acetoxymethyl 478

in Hepatoqtes

- Incubation

of cultured rat

ester for 10 min resulted in diffuse cellular




2, 1990


labeling. Free Mg2+ in individual (kS.E.M.) mM

of 1.1 + 0.13 mM (n=14).


for hepatocyte





cells ranged form 0.5 mM to 2.1 mM with This value is somewhat


digitonin, which selectively permeabilizes 99% of cellular


(6). Treatment

higher than the value of 0.57 of hepatocytes

the plasma membrane

This indicated


a mean


20 PM

(5), released more than


was localized


exclusively to the cytosolic compartment. Relationship Between Free M?’

and ATP During Chemical Hypoxia - Free Mg2+


cultured hepatocytes was essentially unchanged during incubations for up to 30 min in KRH (Fig. 1A). However, than doubled

after addition of 5 mM KCN and 1 mM iodoacetate, free Mg2+ more

over 30 min (Fig. 1A). Most

minutes. In Mg2+-free shown)



of the increase


over the first ten

cytosolic free Mg2+ increased to a similar degree (data not

that the rise in cytosolic free Mg2+ was not dependent

on extracellular

Mg2+. ATP content of control hepatocytes was 13.24 nmol/106 cells. After addition of KCN and iodoacetate,


decreased by >84% within 2 min (Fig. 2B). This decrease of ATP

coincided with the increase in free M$+

(Fig. 2A). Concomitant

with this decrease in ATP,

ADP and AMP content increased from 0.84 and .39 nmol/106 cells, respectively, 11.48 nmol/l06

cells (Fig. 2B). These findings are

binding ligand for M2’


to 3.63 and

with ATP being the major

in the cytosol(7).








Figure 1. Effect of Chemical Hypoxia on Free M2’ - Cultured rat hepatocytes were l(3aded with mag-fura- as described in the Materials and Methods and incubated ‘“+ KRH buffer. KCN 15 mM> and iodoacetate 1mM) were added (arrow) and free M I2 was determined ik single cells k)y MD Al . Data represent mean f S.E.M. from 5 determinations. 479




2, 1990


75 5

” “0r








B 15.

Figure 2. Free M2’ and Adenine Nucleotides During Chemical Hypoxia - Culturedrat hepatocytesloadedwith mag-fura- were incubatedin KRH, and KCN and iodoacetate wereadded(exposure).Aft r 4 min,cellswererinsed5 timesin KRH andfurther incubated (recovery). In A, free M!J + of singlecells was determinedby MDW. In B, adenine nucleotides(ATP, ADP and AMP) of extracts of whole cultures were determined by HPLC. Error barsare 2 S.E.M.


is a major factor determining cytosolic free Mg’,

recover, free Mg*+

then as ATP levels

should fall. To test this hypothesis, we exposed hepatocytes to KCN

plus iodoacetate for 4 minutes and then washed out the metabolic inhibitors. Under these conditions, ATP levels quickly recovered from 1.32 nmol/106 cells (10% of control values) to 7.6 nmol/106 cells (43% of control levels) by 5 min after washing out the inhibitors (Fig. 2B). In parallel MDVM

studies of mag-fura- loaded single cells, washout of KCN and

iodoacetate caused the free Mg’

concentration to return to pre-exposure levels within 4

min (Fig 2A). Relationship






Free ikf2’

- Cell surface bleb

formation is an early event in hypoxic cell injury to hepatocytes (1,8,13). Bleb formation has been described to occur in three stages(8). Stage I is the formation of small surface blebs 480




2, 1990






Figure 3. Bleb Resorption During Recovery From Chemical Hpoxia - A he atocytes was imaged by phase microscopy between measurements of free Mg + by MD vhf . Prior to addition of metabolic inhibitors, the cell surface was unblebbed (Panel A), and free Mg2+ was 1.7 mM. After 6 min of exposu e to KCN and iodoacetate, numerous small surface blebs had formed (Panel B)Z+and Mg 1 + had increased to 2.7 mM. After 12 min., blebs were larger (Panel C), and Mg was 4.0 mM. Subsequently, the metabolic inhibitors were removed and the cell was incubated another 30 min (Panel D). The surface blebs were resorbed and the free M2’ had decreased to 2.0 mM.

(Fig. 3B) which coalesce into fewer larger blebs (Stage II), one of which eventually ruptures resulting in breakdown of the plasma membrane permeability barrier and onset of cell death (Stage III). Figure 3C illustrates a hepatocyte in Stage I of bleb formation after 12 min of exposure to KCN and iodoacetate. Free Mg*+ increased during the exposure from 1.7 mM to 4.0 mM, whereas ATP was lessthan 2% of control levels based on measurementsin whole cultures (data not shown). Removal of KCN and iodoacetate caused free Mg*+ to decrease, and subsequently blebs resorbed such that the cell surface regained its spherical shape after 30 minutes (Fig. 3D). These findings indicate that bleb formation is preceded by a decrease of ATP

(increase of free Mg*+), and that bleb resorption follows


resynthesis(decrease in free M$‘). DISCUSSION It is generally accepted that ATP depletion is the primary factor leading to irreversible hypoxic injury. Since ATP is a major binding site for Mg*+, a consequence of ATP depletion is an increase in free MS’. for the first time that digital video

The experiments described here demonstrate

fluorescence microscopy using mag-fura-

may be

employed to measure cytosolic free MS+ and indirectly cytosolic ATP in single hepatocytes. 481




Free Mg2’

2, 1990




rose and fell during the onset and recovery

with a decrease and increase in ATP. However,



from chemical hypoxia in parallel

free M$+

continued to increase after ATP

depletion (Figure 1) and returned to baseline levels before complete ATP recovery (Figure 2). Since we measured ATP in acid extracts of whole cells, descrepancies content and free Mg 2+ levels may be a consequence

of ATP compartmentalization

the cells. The loss and recovery of ATP in the cytosol (where other cellular compartments

(e.g. the mitochondrial

not be the same. Alternatively,



ATP inside

is localized)


matrix which excludes mag-fura-2),


other factors (such as cell swelling) may influence levels of

free. Mg2’ during hypoxia. Iodoacetate

is an alkylating agent, and it has been suggested that cell blebbing during

chemical hypoxia is the consequence of irreversible depletion

(14). However,

This finding eliminates covalent binding

blebbing reversed the possibility

of iodoacetate

appear to be the consequence The consequences

when KCN and iodoacetate

were removed.

that blebbing during chemical hypoxia is caused by

to protein.


bleb formation

and bleb resorption

of ATP depletion and repletion.

of abrupt changes of free Mg2+ on cell function are unknown.

is critical in DNA transcription, M2’

protein thiol alkylation rather than ATP


protein synthesis (15), and many membrane functions (16).

is also essential in a number of enzymatic reactions (17,18). In addition, alterations


the levels of free Mg2+ have been observed to alter the organization

of the microtrabecular

lattice and cell morphology

changes of free Mg2+

(18). It is therefore

conceivable that

during hypoxia may have adverse effects on cell function that could contribute

to lethal cell


REFERENCES 1. Lemasters, J.J., DiGuiseppi, J., Nieminen, A.-L., and Herman, B., (1987) Nature 325,78-81. 2. Nieminen, A.-L., Gores. G.J., Wray, B.E., Tanaka, Y., Herman, B., and Lemasters, J.J. (1988) Cell Calcium 9,237-246. 3. Jewell, S.A., Bellomo, G., Thor, H., Orrenius, S., and Smith, M.T. (1982) Science 214: 1257-1259. 4. Herman, B., Gores, G.J., Nieminen, A.-L., Kawanishi, T., Harman, A., and Lemasters, J.J. (1990) CRC Critical Reviews in Toxicology in press. 5. Gores, G-J., Nieminen, A.-L., Wray, B.E., Herman, B., and Lemasters, J.J. 1989) J. Clin. Invest, 83: 386-396. 6. k aju, B., Murphy, E., Levy, L.A., Hall, R.D., London, R.E. (1989) Am. J. Physiol. 256, C540-C548. 7. Velesco, D., Guyann, R.W., Oskarsson, M., Veech, R.L. (1973) J. Biol. Chem. 248, 4811-4819. 482




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8. Herman, B., Nieminen, A.-L., Gores, G.J., Lemasters, J.J. (1988) FASEB J. 2, 146-151. 9. DiGuiseppi, J. Inman, R., Ishihara, A., Jacobson, K., Herman, B. (1985) Biotech. 3,394-4Q3. 10. Tsien, R., (1980) Biochemistry 19,2396-2402. 11. Nieminen, A.-L., Gores, G.J., Dawson, T.L., Herman, B., and Lemasters, J.J. 1990) J. Biol. Chem. 265: 2399-2408. 12. a illiamson, D.H. and Mellanby, J. (1974) in Methods of Enzvmatic Analvsis Ed., Bergmeyer, H.U., pp 1836-1839, Verla Chemie/Academic Press NY. 13. Lemasters, J.J., Ji, S., and Thurman, R.G. t 1981) Sceince 231: 661-663. 14. Thomas, C.E. and Reed, D.J. (1989) Hepatol. 10: 375-384. 15. Rubin, H. (1976) J. Cell Physiol. 89,613-626. 16. Aikawa, J.K. (1981) In Magnesium: Its BioloPical Significance, pp. 21-29, CRC Press, Boca Raton, FL. 17. Ebel, H., Gunther, T. (1980) J. Clin. Chem. Clin. Biochem. 18,257-270. 18. Garfinkel, L., Garfinkel, D. (1985) Magnesium, 4,60-72. 19. Porter, K.R. 1988) In The Liver: Biologv and Pathobiolonv 2nd ed., Eds, Arias, I.M., Jacoby, L .B., Popper, H., Schacter, D., and Shafritz, D.A. pp. 41, Raven Press, NY, NY.


Cytosolic free magnesium, ATP and blebbing during chemical hypoxia in cultured rat hepatocytes.

Cytosolic free Mg2+ concentration was determined in 1-day cultured rat hepatocytes using Multiparameter Digitized Video Microscopy (MDVM) of the fluor...
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