The Institute for Enzyne Research and the Department of Biochemistry, The University of Wisconsin, Madison, Wisconsin 53706. Contributed by Henry Lardy, ...
Proc. Nati. Acad. Sci. USA Vol. 75, No. 5, pp. 2234-2238, May 1978
Biochemistry
Norepinephrine, vasopressin, glucagon, and A23187 induce efflux of calcium from an exchangeable pool in isolated rat hepatocytes (gluconeogenesis/a-adreneigic response/intracellular Ca2+/ionophore action) JEN-LING J. CHEN, DONNER F. BABCOCK, AND HENRY A. LARDY The Institute for Enzyne Research and the Department of Biochemistry, The University of Wisconsin, Madison, Wisconsin 53706
Contributed by Henry Lardy, March 1, 1978
Isolated rat hepatocytes do not actively accumulate Ca2+ during prolonged incubation in vitro; however, these cells do exhibit a limited exchange of intracellular with extracellular Ca2+. The exchangeable pool represents about 2 nmol of CaS+ per mg of protein. In medium containing either a low (20 MM) or high (1 mM) concentration of Ca2+, the divalent cation ionophore, A23187 (at concentrations of 0.03-0.1 nmol/ from this exchangeable mg of protein), causes release of of extracellular CaZ+ detectpool but does not allow net influx42Cad+ able by the use of a Ca2+-sensitive electrode. Like A23187, the hormones norepinephrine, vasopressin, and glucagon (at concentrations that stimulate gluconeogenesis) each induces a similar net efflux of Ca2+. Treatment with one hormone decreases the subsequent reponse to the others, whereas treatment with A23187 abolishes the hormonal effects upon both Ca2. release and gluconeogenesis. The action of norepinephrine, but not of glucagon, upon CaO+ efflux is prevented by the a-adrenergic antagonist, phenoxybenzamine. The action of norepinephrine is not prevented by the ,B-adrenergic antagonist, propranolol Together these results indicate that the release of Ca2+ from a common pool of exchangeable Ca2+ is important to the action of a variety of hormones on hepatocytes. This Ca2+ pool in the isolated hepatocyte is characterized as being similar in size and having exchange kinetics that are com arable to those reported for the major intracellular pool of Ca + in the intact liver. The possibility that this pool is intramitochondrial calcium is discussed. The effects of glucagon upon gluconeogenesis in intact, perfused liver are mediated through alterations in cyclic nucleotide levels (1, 2). In contrast, stimulation of hepatic gluconeogenesis by a-adrenergic agonists is accompanied by changes in membrane permeability and resultant alterations in cellular contents of several ionic species (3, 4) but proceeds, under some circumstances at least, without alterations in cyclic nucleotide levels (5-7). Because of its central role in regulation of cellular function, considerable attention has focused upon the possible involvement of changes in cellular Ca2+ in the mechanism of action of the a-adrenergic hormones. In isolated perfused liver, agents that stimulate gluconeogenesis rapidly increase 45Ca2+ efflux and decrease total Ca2+ content (4). Several investigators have reported that gluconeogenic hormones, including catecholamines, glucagon, and vasopressin, enhance the initial rate of both 45Ca2+ influx (8-10) and efflux (10) in isolated hepatocytes. However, isotopic techniques cannot alone establish the direction or the quantity of net changes in cellular calcium after such treatment. The present study reports the application of a sensitive Ca2+-selective electrode and a 45Ca2+-labeling technique for examining the effects of ionophore A23187 and of gluconeogenic hormones on the fluxes and net movements of Ca2+ in hepatocytes. ABSTRACT
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MATERIALS AND METHODS Cell Isolation and Incubation. Rat liver cells were prepared by a modification* of the method of Zahlten and Stratman (11). Routinely, cells were suspended at 6.5 X 106 cells per ml in Krebs-Henseleit bicarbonate medium containing 1.5% bovine serum albumin, 10 mM Na lactate, and 1 mM Na pyruvate without added calcium (the free calcium in the cell suspension was estimated at 15-30 gM). Cell suspensions (1.0 or 2.5 ml) were incubated in plastic scintillation vials at 370 in a Dubnoff shaker with agitation (110 cycle/min) under 95% 02/5% CO2. 45Ca2+ Exchange Studies. After 40 min of equilibration, 10 gCi of 45Ca2+ was added to each 1.0 ml of cell suspension. Subsequently 20-,ul aliquots were removed for determination of isotopic Ca uptake. Each aliquot was layered on the top of 0.35 ml of 7% bovine serum albumin in 5 mM CaCl2/150 mM NaCl all contained in a 0.45-ml polyethylene centrifuge tube (no. 47/7, Sarstedt Co., Princeton, NJ). The tubes were then centrifuged for 15 sec at 2000 X g. The tips containing the cell pellet were cut off and their contents were flushed into scintillation vials by injecting 0.5 ml of 1% sodium dodecyl sulfate solution. The cells were solubilized with 5 ml of Aquasol and the associated radioactivity was determined. Ca2+ Electrode Studies. Cell suspensions of 2.5 ml were prepared as described above with each vial containing a Ca2+-sensitive and a reference electrode (Radiometer F2112Ca and Calomel K401, respectively, both obtained from Medtron-Chicago, Rosemont, IL). The electrodes were connected to a Radiometer pH meter and a Honeywell visicorder with an intermediate bucking-voltage device similar to that described by Madeira (12). Cell suspensions were routinely incubated for a 30- to 40-min equilibration period before further additions were made. Changes in the external Ca2+ concentration were determined by calibration with aliquots of standard solutions at the end of each experiment. Determination of Net Glucose Production. The concentration of glucose in acid extracts of cell suspensions was determined by a glucose oxidase method (13). Materials. Norepinephrine (NE, L-arterenol bitartrate), arginine vasopressin (VP), grade VI, and bovine serum albumin (fraction V) were obtained from Sigma Chemical Co., St. Louis, MO; glucagon and A23187 from Eli Lilly, Indianapolis, IN; phenoxybenzamine hydrochloride from Smith Kline & French, Philadelphia, PA; and propranolol from Ayerst Laboratories, New York, NY. RESULTS Calcium Electrode Studies on Net Calcium Fluxes Induced by Gluconeogenic Hormones and A23187. Addition of NE, Abbreviations: NE, norepinephrine; VP, vasopressin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. * N. Kneer and H. A. Lardy, unpublished data. 2234
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Chen et al.
2235
NE
ZC VP A23187
1.5
I 0
VP
0 CL 0)
4
,
1.0
0
EC
B
NE 1.3 t
A23187
-~~~~37j
.53
G
C
0
0
0
A23187 (1.0 pM)
I
1.0 nmol Cal' /mg protein
D 1.7 \ NE
)08
16 min
FIG. 1. Effects of hormones and ionophore A23187 on Ca2+ release from isolated hepatocytes. Cell suspensions were prepared and incubated as described in Materials and Methods, except that an equilibration period of 90-110 min preceded the noted additions: NE, 8 ,M; VP, 40 milliunits/ml; glucagon (G), 16 gM; A23187, 10 juM (unless otherwise indicated). The amount of Ca2+ release (nmol of Ca2+ per mg of protein) is noted after each addition.
VP, or glucagon, at concentrations that stimulate gluconeogenesis, caused a net release of approximately 1 nmol of Ca2+ per mg of protein from hepatocytes prepared from starved rats (Fig. 1 A-C). The maximum release induced by the hormones was somewhat less than that induced by a maximally effective level of ionophore A23187 (Fig. ID). When either NE or VP was added sequentially, the second addition was always less effective in promoting release of calcium, suggesting that a common pool of calcium may be involved in the hormonal responses (Fig. 1 A and B). Addition of A23187 alone apparently depletes this pool since subsequent additions of NE did not promote further release (Fig. ID). Data obtained from experiments similar to those shown in Fig. 1 are summarized in Fig. 2 and show that the amount of net Ca2+ efflux induced by A23187 increased linearly with 0.025-0.10 nmol of A23187 per mg of protein. Even when the ionophore concentration was increased to 100 nmol/mg of protein (data not shown), the ionophore produced only net efflux of Ca2+ and not Ca2+ uptake as it does with some other types of cells (14-16). Exchangeable Ca2+ and Its Release Induced by NE, VP, Glucagon, or A2Mi87. The Ca2+ electrode measurements (Fig. 1) indicate that hepatocytes have no detectable net fluxes of Ca2+ in the absence of hormonal stimulation, nor were net movements of Ca2+ detected with the metallochromic indicator dye, Arsenazo III (data not shown). However, hepatocytes did accumulate appreciable quantities of 4QCa2+ during prolonged incubation (Fig. 3), probably as a result of exchange of extracellular with intracellular Ca2+. When NE was added after 60 min of equilibration with isotopically labeled Ca2+, 50-70% of the accumulated 45Ca2+ was rapidly released from the cells (Fig. 3 A and E). The amount of Ca2+ released, as monitored by 45Ca loss, was comparable to the net efflux measured with the Ca2+ electrode (Fig. lA). VP had an effect similar to that of NE (Fig. 3B).
0.2
0.4
0.6
as
A23187, AMM FIG. 2. Effects of A23187 on NE-induced Ca2+ release. Incubations were performed as before except that various amounts of A23187 were added after 60-90 min of preliminary incubation and 15-20 min before addition of 8MuM NE. The Ca2+ electrode was used to determine the amount of Ca2+ released by A23187 (@) and by the subsequent addition of hormone (-).
When 45Ca2+ and NE were added simultaneously to hepatocytes, the amount of isotope accumulated was only 30% of that taken up by cells incubated in the absence of hormones (Fig. 3E). This reduction presumably reflects a net decrease in the size of the exchangeable Ca2+ pool. The simultaneous addition of glucagon and 45Ca2+ diminished the uptake of isotope only slightly (Fig. SF). If cells were equilibrated with 4-Ca2+ in the absence of hormone, the addition of A23187 alone caused the release of more than 90% of the accumulated label (data not shown). When addition of A23187 followed that of NE or VP, the pool of exchangeable calcium was further depleted to less than 10% of its maximal amount (Fig. 3 A and B). When similar experiments were conducted in a medium containing 1 mM CaCl2, the release of exchangeable 45Ca2+ induced by NE or A23187 was less dramatic (26% and 54% of the total, respectively) (Fig. 3 C and D). A slow reuptake of Ca2+ was observed 15-20 min after the efflux that followed addition of each of the three hormones (Fig. 3 A, B, C, and F). No reuptake was observed in the presence of A23187 (Fig. 3 A, B, and D). Net reaccumulation of Ca2+ after hormone-induced efflux was also observed with the calcium electrode in experiments of long duration (data not shown). The nature of this uptake is not understood, but it may represent a refilling of the exchangeable pool after the influence of the hormone is dissipated. Relationship Between Ca2+ Efflux and Gluconeogenesis Induced by Hormones and A23187. Although a strong correlation was found between the levels of NE required to cause release of 45Ca2+ and those required to stimulate gluconeogenesis (Fig. 4A), levels of glucagon that stimulate gluconeogenesis were less effective in promoting Ca2+ release. An increased rate of gluconeogenesis followed exposure to 10-8 M glucagon without detectable release of 45Ca2+ (Fig. 4B), and even high concentrations of glucagon released only 20-25% of the G5Ca2+ present in cells that were equilibrated with the extracellular isotope before addition of the hormone (Figs. 3F and 4B). This relative effectiveness of catecholamines and glucagon in promoting Ca2+ release is comparable to that reported by Assimacopoulos-Jeannet et al. (10). Like the hormones NE and VP (Figs. 1 and 3), the ionophore A23187 (0.2-1.0,uM) also induced net release of Ca2+ (Fig. 2) from the exchangeable pool (Fig. 4C) of the hepatocyte. In
2236
Biochemistry: Chen.et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
.' 4.0, c
A23187
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_
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0
. 50.100
0
50
100
150
I
I
50
100
5
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I 150
Time, min FIG. 3. Hormonal effects on 45Ca2+ uptake and release from isolated hepatocytes. Cell suspensions were prepared and incubated as described in Materials' and Methods, except for the experiments in C and D, in which 1 mM CaCl2 was added after the 40 min of preliminary incubation. In each case 10 zCi of 45CaCl2 was added at time 0. Hormones [NE, 8 gM; VP, 40 milliunits/ml; glucagon (G), 0.3 MM] were added at either time 0 (0) or after equilibration with the isotope was complete (-). Cells were separated from aliquots of the suspension at the indicated times and the associated radioactivity was determined.
contrast to the effects of the hormones, however, treatment with
ionophore resulted in an inhibition of gluconeogenesis (Fig. 4C and ref. 10). It may be that A23187 causes redistribution of Ca2+ that differs in some respect from that induced by the hormones or, alternatively, the ionophore may have other effects that secondarily inhibit glucose production. Fig. 4C also shows that an intermediate concentration of the ionophore (0.2 ,gM) prevented a detectable incremental release of 45Ca2+ in response to NE, but still permitted increased gluconeogenesis after hormonal stimuli. This observation may simply reflect the choice of sampling times used rather than demonstrate a true dissociation of the gluconeogenic response from the release of exchangeable Ca2+. Alternatively, these data may indicate that only a small portion of the exchangeable pool is obligatorily involved in hormone action. Effects of a- and j3-Adrenergic Antagonists on NE-Induced Calcium Release. The a-adrenergic antagonist, phenoxybenzamine, at 20 MiM, blocked 50% of the net calcium efflux (Fig. 5 A and C) and completely suppressed the response to NE when present at 40MM (Fig. SD). In contrast, the f3-adrenergic blocker, propranolol, at concentrations up to 40 MM, had no effect on the release of Ca2+ induced by NE (Fig. 5B). The stimulatory effect of glucagon upon gluconeogenesis in hepatocytes is resistant to a-adrenergic antagonists (5). It is therefore not surprising that the release of Ca2+ induced by glucagon was also resistant to phenoxybenzaihine (Fig. 5D). The effects of
and ,B-adrenergic antagonists were duplicated in experiments examining 45a2+ fluxes (data not shown). Other Factors that Affect Release of Ca2+ Ibduced by NE. The release of Ca2+ induced by NE was influenced by the feeding regimen of the rats from which the hepatocytes were a-
prepared. Hormones consistently caused a larger releaseof Ca2+ from hepatocytes that were prepared from fed animals (data not shown). The condition of the cell preparations, as influenced by the isolation procedure, incubation time, and storage time, and by the substrates present, also apparently influences the magnitude of the hormonal response. In addition, the release of Ca2+ by NE also depends upon the incubation temperature (Fig. 6). When hepatocytes were incubated for 25 min at 370, NE released 1.1 nmol of Ca2+ per mg of protein. When another sample of hepatocytes was transferred from 370 to 250 and then incubated for 13 min, an identical dosage of NE released 1.0 nmol of Ca2+ per mg of protein. After an additional 54 min of incubation at 25°, the same quantity of NE released only 0.25 nmol of Ca2+ per mg of protein. Thus, incubation at 25° resulted in the slow loss of the ability of NE to cause Ca2+ efflux. If hepatocytes were incubated for 46 min at 370 after 67 min of incubation at 250, NE again released 1.0 nmol of Ca2+ per mg of protein. The effect of low temperature upon the ability of NE to cause release of Ca2+ is thus both slow and reversible. Effect of Inhibitors of Mitochondrial Function on Calcium
Biochemistry: Chen et at.
Proc. Natl. Acad. Sci. USA 75 (1978)
2237
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0)
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0) +1 Cu
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l-" 1-9 Glucagon, M
Norepinephrine, M
0.2
0
10-5
0.4 0.6 A23187, MM
0.8
1.0
FIG. 4. Effect of NE, glucagon, and A23187 on gluconeogenesis and 45Ca2+ content of hepatocytes. Cell suspensions (1.0 ml each) were prepared. (A and B) After 40 min of equilibration, 10 MiCi of 45Ca2+ was added to each vial to give a final concentration of 0.3 mM. After a further incubation of 30 min, various concentrations of the hormones, 10 mM Na lactate, and 1 mM Na pyruvate were added. Fifteen minutes after substrates were added, duplicate 20-Al aliquots were removed for determination of intracellular 45Ca. After an additional 25 min, the remainder of each suspension was treated with perchloric acid and net glucose production was determined. (C) After 60 min of equilibration, CaCl2 was added to give a final concentration of 0.5 mM containing 10 MCi of '45Ca; 45 min later lactate and pyruvate were added as above together with the indicated amounts of A23187. NE was added to a final concentration of 10MuM in half of the vials (open symbols). Twenty minutes and 40 min later samples were removed for 45Ca (-, A) and glucose (0, 0) determinations, respectively.
Content and Hormone-Induced Efflux of Ca2+. Inhibitors of the electron transport chain (CN-) and agents that uncouple oxidative phosphorylation [carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) and dinitrophenol] each caused 25-50% release of the exchangeable Ca2+ pool of the hepatocyte. After treatment with these agents subsequent release of Ca2+ induced by NE was inhibited by 50-90% (Table 1). These preliminary experiments suggest that mitochondrial function is required to maintain the exchangeable Ca2+ pool and its responsiveness to hormonal stimulation. DISCUSSION Claret-Berthon et al. (17) recently examined Ca2+ pools in NE
PN PN NE
B
1.1
1.0
intact, perfused rat liver. By application of compartmental analysis, two extracellular pools (1.3 and 3.3 nmol of Ca2+ per mg of protein) and an intracellular pool (1.9 nmol of Ca2+ per mg of protein) were identified. The exchange of 45Ca2+ found here for isolated hepatocytes resembles that into the intracellular pool of intact liver in both its kinetics (t1/2 -15 min) and its extent (-.2 nmol of Ca2+ per mg of protein). The net release of exchangeable Ca2+ that was demonstrated here after addition of hormones to hepatocytes is likewise consistent with the release of exchangeable Ca2+ found during perfusion with hormones that stimulate glycogenolysis and gluconeogenesis in rat (4), rabbit, and guinea pig (18) liver. In contrast to the results of Kleineke and Stratman (19), who used lesser quantities of collagenase in the preparation technique, we did not detect a net increase in Ca2+ content of hepatocytes incubated in the absence of hormones. This is consistent with reports (20, 21) that improved procedures produce cell preparations with decreased membrane permeability. 1.25f
7..1
cam
Cc1 00 PB NE
I
C0.35
I
ME
PB PB NE
G
m
G
^ 0.75 Fan
J C 0 50 D
0.58
0.48
1-
.cc
Lu
z
025
0.5 nmol/mg I
0
I 8
I
T
16 min
FIG. 5. Effects of a- and fl-adrenergic antagonists on hormoneinduced release of Ca2+. Conditions were as in Fig. 1. Additions of 50 nmol each of propranolol (PN) or phenoxybenwvmine (PB) were made as indicated. Glucagon (G) and NE were added to final concentrations of 0.08MgM and 2 MM, respectively.
:--------_
25
370 -:-~13
2505
I
54 250 25 46 Incubation conditions
I
I
j
337min
FIG. 6. Effect of temperature on release of Ca2+ induced by NE. Cell suspensions were prepared and incubated as described in Materials and Methods except for the indicated alterations of incubation temperature. The release of Ca2+ induced by the addition of 8MM NE was measured by the Ca2+ electrode method.
2238
Biochemistry:
Chen et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 1. Effects of uncouplers and inhibitors on NE-induced Ca2+ efflux from hepatocytes
Inhibitor 800 AM KCN 8 AM FCCP
375MM dinitrophenol 375MM dinitrophenol + 25 MM oligomycin
Net efflux* induced by % NE inhibition Inhibitor NE after of NE alone alone inhibitor response 0.88 0.54 0.43
1.40 2.10 2.21
0.69 0.19
50 91
1.20
45
1.07
0.71
0.14
80
Except for the last experiment (using "5Ca2+ measurements), all incubations were performed as in the legend for Fig. 1 and net release of Ca2+ was measured by the Ca2+ electrode. * nmol of Ca2+ per mg of protein.
Work by Assimacopoulos-Jeannet et al. (10) and by Keppens et al. (8) indicates that glycogenolytic hormones or ionophore A23187 each increases 45Ca2+ influx into hepatocytes. Our experiments using cells equilibrated with 45Ca2+ prior to treatment with ionophore or hormone indicate that, indeed, membrane permeability increases in response to these stimuli, but more importantly, each treatment promotes a net release of Ca2+ from the exchangeable pool of hepatocytes. Comparable results were obtained in medium containing either 20,gM or 1 mM Ca2+. Thus, although hormones or ionophore each increases the rate of entry of 45Ca2+ into an exchangeable pool present in isolated hepatocytes (8, 10, 22), efflux is apparently increased to an even greater extent so that the size of the pool decreases rapidly and dramatically after exposure to these agents. The importance of efflux from this pool to the mechanism of hormone action is suggested by the inhibition of NE-induced efflux by the aadrenergic antagonist, phenoxybenzamine, and is supported by the preliminary observation that the size of the Ca2+ pool in hepatocytes prepared from starved and fed rats correlates with the hormonal state of the animals. In addition, the action of NE upon Ca2+ content (shown here) and upon gluconeogenesis (23, 24) is reversibly inhibited by low temperature. Depletion of the exchangeable pool by treatment with A23187 prevents both further release of Ca2+ and the stimulation of gluconeogenesis that normally result from exposure to NE. Although the efflux of exchangeable Ca2+ induced by low concentrations of A23187 alone is comparable to that induced by NE, the ionophore does not stimulate gluconeogenesis, and at higher concentrations it inhibits. We conclude, therefore, either that low concentrations of A23187 have secondary, inhibitory effects upon gluconeogenesis or that efflux of Ca2+ from the exchangeable pool per se does not induce the gluconeogenic response. Treatment with hormones (8, 10) or inhibitors of mitochondrial function (25) results in activation of liver phosphorylase, phosphorylase kinase, or both. Because these events are presumably mediated by elevation of intracellular Ca2+ concentrations (26, 27), it is probable that efflux of Ca2+ from the exchangeable pool results in the transient elevation of the levels of Ca2+ in other cellular pools before its expulsion from the cell. This explanation accounts for the ability of hormones to stimulate glycogenolysis (8, 10, 22) in the absence of extracellular Ca2 . However, it is also possible that lowering of available Ca2+ in an exchangeable pool, such as mitochondria, may activate enzymes that participate in gluconeogenesis (28, 29). Ionophore A23187 causes release of Ca2+ from intracellular stores in several other cells (14, 30, 31) with concomitant activation of Ca2+-dependent processes. In one cell type, at least, the Ca2+ pool that is susceptible to the action of A23187 has
been identified with the mitochondria (14). As shown here, A23187 and the hormones NE, VP, and glucagon all apparently act upon the major pool of exchangeable Ca2+ present in hepatocytes. Some evidence suggests that this pool is also of mitochondrial origin. For example, the similar kinetics of 45Ca2+ exchange into the intracellular pool of intact liver and into mitochondria in situ (17) suggest that the mitochondria are a major repository of exchangeable Ca2+ in liver cells. Carafoli (32) and Ash and Bygrave (33) reached a similar conclusion based upon studies of the binding of Ca2+ to isolated cell fractions. This suggestion is consistent with our demonstration that agents that inhibit mitochondrial function both deplete the exchangeable pool and inhibit the response to catecholamines. This work was supported by Grants AM20678 and AM10334 from the National Institutes of Health. 1. Robison, A., Butcher, R. W. & Sutherland, E. W. (1971) Cyclic AMP (Academic, New York), pp. 232-240. 2. Exton, J. H. (1972) Metabolism 21, 945-990. 3. Friedman, N. (1972) Biochim. Biophys. Acta 274,214-225. 4. Friedman, N. & Park, C. R. (1968) Proc. Nati. Acad. Sci. USA 61,504-508. 5. Kneer, N. M., Bosch, A. L., Clark, M. G. & Lardy, H. A. (1974) Proc. Natl. Acad. Sci. USA 71, 4523-4527. 6. Tolbert, M. E. M., Butcher, F. R. & Fain, J. N. (1973) J. Biol. Chem. 248, 5686-5692. 7. Exton, J. H. & Harper, S. C. (1975) Adv. Cyclic Nucleotide Res. 5,519-532. 8. Keppens, S., Vandenheede, J. R. & DeWulf, H. (1977) Btochtm. Biophys. Acta 496,448-457. 9. Pilkis, S. J., Claus, T. H., Johnson, R. A. & Park, C. R. (1975) J. Biol. Chem. 250,6328-66. 10. Assimacopoulos-Jeannet, F. D., Blackmore, P. F. & Exton, J. H. (1977) J. Biol. Chem. 252,2662-2669. 11. Zahlten, R. N. & Stratman, F. W. (1974) Arch. Biochem. Biophys. 163,600-608. 12. Madeira, V. M. C. (1975) Biochem. Biophys. Res. Commun. 64, 870-876. 13. Zahlten, R. N., Stratman, F. W. & Lardy, H. A. (1973) Proc. Natl. Acad. Sci. USA 70,3213-3218. 14. Babcock, D. F., First, N. L. & Lardy, H. A. (1976) J. Biol. Chem. 251,3881-3886. 15. Jensen, P. & Rasmussen, H. (1977) Biochim. Blophys. Acta 468, 146-156. 16. Ferreira, H. G. & Lew, V. L. (1976) Nature 259,47-49. 17. Claret-Berthon, B., Claret, M. & Mazet, J. L. (1977) J. Physiol. 18. 19. 20. 21.
(London) 272,529-552. Haylett, D. G. (1976) Br. J. Pharmacol. 57, 158-160. Kleineke, J. & Stratman, F. W. (1974) FEBS Lett. 43,75-80. Mapes, J. P. & Harris, R. A. (1975) FEBS Lett. 51, 80-3. Dubinsky, W. P. & Cockrell, R. S. (1975) FEBS Lett. 59,39-
43. 22. Pointer, R. H., Butcher, F. R. & Fain, J. N. (1976) J. Biol. Chem.
251,2987-2992. 23. Bosch, A. L. (1976) Ph.D. Dissertation, University of Wisconsin, pp. 82-89. 24. Friedman, N. & Rasmussen, H. (1970) Biochim. Biophys. Acta
222,41-52. 25. Fain, J. N. & Shepherd, R. E. (1977) J. Biol. Chem. 252,80668070. 26. Khoo, J. C. & Steinberg, D. (1975) FEBS Lett. 57,68-72. 27. Shimazu, T. & Amakawa, A. (1975) Biochim. Biophys. Acta 385, 242-256. 28. Gevers, W. & Krebs, H. A. (1966) Biochem. J. 98,720-735. 29. Foldes, M. & Barrit, G. J. (1977) J. Biol. Chem. 252, 53725380. 30. Hainaut, K. & Desmedt, J. E. (1974) Nature 252,407-408. 31. Steinhardt, R. A. & Epel, D. (1974) Proc. NatI. Acad. Sd. USA 71, 1915-1919. 32. Carafoli, E. (1967) J. Gen. Physiol. 50, 1849-1864. 33. Ash, G. R. & Bygrave, F. L. (1977) FEBS Lett. 78,166-168.
