Proc. Nat. Acad. Sci. USA
Vol. 72, No. 9, pp. 3561-3564, September 1975 Cell Biology
Stimulation of epinephrine-sensitive fat cell adenylate cyclase by cytosol: Effect of cholera toxin (catecholamine effect)
UMA GANGULY AND WILLIAM B. GREENOUGH III* Department of Medicine, Infectious Diseases Division, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland 21205
Communicated by Victor A. McKusick, June 17,1975
ABSTRACT Cytosol prepared from rat epididymal fat cells by centrifugation at 100,00d X g for 1 hr was found to enhance the basal and epinephrine-sensitive adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] of fat cell ghosts. Cholera toxin also stimulated adenylate cyclase and increased the response to epinephrine in fat cells. A possible relationship between the adenylate cyclase modifying activities of cytosol and the effects of cholera toxin was sought. Cytosol from freshly prepared fat cells added to ghosts prepared from cells that had been exposed to toxin for varying periods showed a progressive loss of responsiveness to cytosol epinephrine-enhancing activity. The effect appeared within 15 min after toxin exposure, a full 30 min before any direct effect of toxin on adenylate cyclase was seen. Since exposure to toxin decreased membrane response to cytosol epinephrine-enhancing activity, the possibility that epinephrine-enhancing activity in cytosol might be altered by toxin was explored. Cytosol from cells exposed to toxin for varying periods lost epinephrine-enhancing activity to an a preciable degree within 15 min. Examination of these ear y events after exposure to toxin should clarify the way in which this bacterial substance affects mammalian cells. The cytosol epinephrine-enhancing activity was destroyed by boiling for 3 min and was partially inactivated by trypsin. It was nondialyzable and stable at -70°.
The effect of the enterotoxin of Vibrio cholerae on cells is due to its stimulation of the cell membrane-associated enzyme adenylate cyclase [EC 4.6.1.1; ATP pyrophosphatelyase (cyclizing)] (2). Recent studies from this laboratory (3) have shown that adenylate cyclase in ghosts prepared from fat cells that had been exposed to cholera toxin for 3 hr were more responsive to stimulation by epinephrine than those that were not..A similar phenomenon has recently been described in turkey erythrocytes (4) and solubilized rat liver preparations (5). There is a delay in the onset of action of this toxin (2) which has not been decreased by using detergent or by changing the incubation temperature (6). Because this delay in the onset of action has not been well explained and since there has been difficulty in demonstrating effects in broken cell systems, we felt it was important to study whether intracellular factors affected by toxin might be present which were involved in modulating the response of adenylate cyclase. In this paper we describe the activation of membrane adenylate cyclase and sensitization of response to epinephrine by cytosol derived from fat cells (1) and relate these properties to the effects of cholera toxin. MATERIALS AND METHODS Adenosine 5'-triphosphate (ATP), adenosine 3':5'-cyclic mo*
Reprint requests: Dr. William B. Greenough III, Department of Medicine, The Johns Hopkins Hospital, Baltimore, Md. 21205.
3561
nophosphate (cAMP), phosphoenolpyruvate, pyruvate kinase, and myokinase were obtained from Sigma Chemicals. [a-32P]ATP and [3H]cAMP were purchased from New England Nuclear. Dowex AG 50 W-X2, 200-400 mesh, hydrogen form, was supplied by Bio-Rad Laboratories. Collagenase was obtained from Worthington Biochemical Corp. Bovine serum albumin Fraction V was purchased from Armour Pharmaceutical Co. Highly purified cholera toxin was produced by Dr. R. A. Finkelstein (University of Texas Southwestern Medical School, Dallas, Texas) (7) and supplied by The National Institutes of Health. Preparation of Fat Cell Ghosts and Cytosol. Fat cells isolated (8) from the epididymal fat pads of male SpragueDawley rats were incubated with collagenase (10 mg/g of fat pad) in Krebs-Ringer phosphate (pH 7.2) buffer with albumin (3 g/100 ml) for 1 hr at 370 with gentle shaking. They were filtered through a silk screen, washed three times, and resuspended in the same buffer (1 g of cells per 5 ml of buffer). Glucose was omitted from incubation mixture. Toxin was added at time zero at a concentration of 1 Ag/iml of fat cell suspension, and the mixture was incubated for the indicated times at 370 while shaking at 60 cycles per min. Cells were removed at desired intervals and centrifuged for 1 min at 1500 rpm, and the infranatant buffer was removed. The whole cells were washed once with cold saline, then lysed by exposure to chilled water at 40 with mixing twice in a Vortex mixer and centrifuged at 12,000 X g for 10 min at 40. The pellet was washed once with 5 mM Tris-HCl, pH 8, and resuspended in cold 150 mM Tris-HCl, pH 8, 25 mM MgCl2. The volume was adjusted to make a protein concentration of 2.0-3.0 mg/ml of buffer. Adenylate cyclase activity was measured on this suspension. For the extraction of cytosol, fat cells were dispersed in a minimum volume of hypotonic (10 mM) sodium phosphate buffer, pH 7, (1 g of cells per 1 ml of buffer). The cells were held for one minute on ice, then stirred twice for 10 sec on a Vortex mixer. This mixture was centrifuged at 12,000 X g for 10 min, which resulted in three layers-a topmost layer of fat, an intermediate turbid aqueous layer, and a small pellet. The turbid aqueous phase was withdrawn into a Pasteur pipette and centrifuged again at 100,000 X g for 1 hr. A volume of 20 Ml of this 100,000 X g supernatant fraction (containing a total of 30-40 jig of protein) was used in the assay mixture for adenylate cyclase. All steps were accomplished at 40. An equal volume of boiled cytosol was used for control experiments. The cytosol itself possessed no adenylate cyclase activity in either control or toxin-treated cells. Adenylate Cyclase Assay. Fat cell ghosts were incubated for 10 min at 370 in 0.05 ml of medium containing 30 mM Tris-HCl (pH 8.0), 10 mM MgC12, 10 mM theophylline, 1.5 mM ATP, 1 MCi of [at-32P]ATP, and an ATP-regenerating
Cell Biology: Ganguly and Greenough
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Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. Stimulation of adenylate cyclase by cytosol in rat fat cell ghosts
Table 2. Effect of cytosol on basal and toxin-stimulated adenylate cyclase activity of fat cell ghosts
Adenylate cyclase, pmol/mg of protein per min (± SEM) Addition of cytosol +
Basal
Epinephrine
63.5 ± 2.8 127 ± 4.6 n = 20
373± 13
238 ± 7.5
Fat cells ghosts and cytosol were prepared as described in Materials and Methods. Cytosol (20 Ml) used in this experiment was obtained from fresh cells; 5.5 uM epinephrine was used in this experiment.
system consisting of 5 mM phosphoenolpyruvate, 50 Mg/ml of pyruvate kinase, and 20,Mg/ml of myokinase. Reactions were stopped by adding 0.5 ml of a recovery mixture (50 gg of cAMP, 100 ,g of ATP, and approximately 2000 cpm of [3H]cAMP) and placing the tubes in a boiling waterbath for 3 min. The radioactive cAMP formed from ATP was separated as described by Krishna et al. (9). The products in the supernate from the barium hydroxide zinc sulfate precipitation step were identified by thin-layer chromatography on polyethyleneimine (PEI) impregnated cellulose MN 300. Nonradioactive AMP and ATP were added, and the chromatogram was developed with 0.6 M LiCl adjusted to pH 7.3 with 1.0 M formic acid as the solvent (10). Nucleotides were localized under ultraviolet light. The radioactive 32p present in the AMP and ATP spots from samples with cytosol present did not differ from those of the control and reaction blank, and radioactivity in the fraction containing cyclic AMP was restricted to this compound. Furthermore, we also found increased adenylate cyclase activity due to cytosol by the more specific method described by Salomon et al. (11). Boiled enzyme samples were used as controls. Protein was measured by the method of Lowry et al. (12). Synthesis of cyclic AMP was linear with time for at least 10 min and proportional to the enzyme concentration up to 1 mg/ml. Fat cell ghosts were prepared fresh each day for each experiment. RESULTS The supernatant fluid fractions of fat cells prepared in water or 10 mM sodium phosphate buffer stimulated basal and epinephrine-sensitive adenylate cyclase when incubated with freshly prepared fat cell ghosts (Table 1). Stimulation by cytosol plus epinephrine produced a 5-fold increase over basal activity. By comparison, stimulation by epinephrine alone was 3.5-fold (Table 1). Since cholera toxin is known to increase the fat cell adenylate cyclase, the activity of cytosol was also tested in toxintreated cells. Accordingly, cytosol from freshly prepared cells was added to fat cell ghosts prepared from cells that had been incubated for 3 hr with or without toxin. Although cytosol stimulated basal adenylate cyclase activity of fat cell ghosts prepared from both control and toxin-treated cells (Table 2), the enzyme response to cytosol was significantly increased in cells that had been exposed to toxin as compared to controls. In addition to a direct stimulation of adenylate cyclase, the cytosol fraction potentiated the effect of epinephrine in ghosts prepared from control cells. However, ghosts from cells exposed to toxin did not show such a synergistic effect of epinephrine and cytosol. The potentiating ef-
Adenylate cyclase, pmol/ mg of protein per min (± SEM) -
Basal Cytosol Epinephrine Epinephrine + cytosol
Toxin
60 95 126 228 n =
± ± ±
±
8.2 5.5 7.5 15.0
+ Toxin
175 305 397 477
±
12 22
±
15
±
22
±
5
Fat cells were incubated without and with cholera toxin (1 Mg/ml of cell suspension) for three hours. Ghosts were prepared from these cells as described in Materials and Methods. Cytosol (20 il) used in this experiment was obtained from fresh cells; 5.5 uM epinephrine was used in this experiment.
fect of cytosol could not be accurately judged because of the nearly maximal stimulation that was achieved by either cytosol or epinephrine alone (Table 2) in ghosts prepared from toxin-treated cells that already had a 3-fold increase in adenylate cyclase. The degree of adenylate cyclase stimulation by cytosol in ghosts prepared from cells that had been exposed to toxin was tested during the early phase of incubation at a time when the direct stimulating effect of toxin on this enzyme was not yet apparent. The adenylate cyclase activity in the presence and absence of epinephrine and cytosol in toxintreated fat cell ghosts is shown (Fig. 1). Ghosts prepared from cells exposed to toxin for 30 min showed no increase of adenylate cyclase activity. At 60 min an increase of 50 pmol/ mg of protein per min over the basal value had occurred. Cytosol added to fresh fat cell ghosts stimulated basal activity 2-fold and increased the epinephrine response. Thirty minutes after exposure to toxin a progressive increase in the responses both to cytosol and epinephrine occurred. In fresh cells, when cytosol was added with epinephrine a greater than additive response occurred (indicated by "I" adjacent to histogram). This additional response to epinephrine is progressively lost beginning 15 min after exposure to toxin (P < 0.02). Since exposure to toxin for 30 min appreciably enhanced the response of fat cell ghosts to cytosol and diminished the capacity of cytosol to enhance the epinephrine response, it seemed reasonable to determine the effect of toxin on cytosol itself. Accordingly, cytosol from cells incubated with and without cholera toxin for varying periods of time was tested in fresh fat cell ghosts. As shown in Fig. 2, cytosol from both groups of cells stimulated basal adenylate cyclase to the same degree; however, cytosol from cells exposed to toxin showed a reduced ability to enhance the response of ghosts to epinephrine within 15 min after exposure to toxin (P < 0.01). This alteration preceded detectable direct stimulation of adenylate cyclase by the toxin, which occurred at approximately 60 min. This loss of the epinephrine-enhancing effect from the cytosol of toxin-treated cells correlated with the increase in the epinephrine response of toxin-treated ghosts (Fig. 1). This enhanced epinephrine response has been described (3). It occurred as a significant effect within 30 min after exposure of fat cells to the toxin. No activity of adenylate cyclase was detected directly in cytosol from either control or toxin-treated cells. The catalytic activity of adenylate cyclase was increased in a nonlinear manner with increasing amounts of cytosol tested from 5
Cell Biology:
Proc. Nat. Acad. Sci. USA 72 (1975)
Ganguly and Greenough
E C
c v 0
o-Basoal
Dc"-Cy+Cytosol 600 *g600
sum of ind lividual stimuli (epi + cytosol)
500
I
0.
0,
400
E
300 00
60 min(+)toxin f resh(-)toxi n 15 min(+) toxi n 30 min(+)toxin FIG. 1. Effect of cytosol prepared from fresh cells on basal and epinephrine-stimulated adenylate cyclase activity of toxin-treated fat cell ghosts. Fat cells were incubated in the presence of cholera toxin (1 tig/ml of cell suspension) at 370 for various times. Epinephrine was present at a final concentration of 5.5 MAM. Cytosol (20 Al) was added to a final reaction volume of 50 Ail. The loss of synergistic effect of epinephrine response produced by cytosol in toxin-treated fat cells is indicated by an "I" adjacent to the histogram, at 0, 15, 30, and 66 min. Results are means of four separate experiments.
to 50 /d. With the maximum amount of cytosol used (50 Ml), the enzyme was stimulated approximately 6-fold. The cytosol activity was lost after boiling for 3 min. It was partly inactivated (40-50%) by trypsin (1 mg/ml of cytosol per 30 min at 370). It was stable at -700, but lost activity after repeated freezing and thawing. Separation of the adenylate cyclase stimulating activity was done initially by adding cold saturated (NH4)2SO4 solution to the cytosol to bring the salt concentration to 60% saturation. After 30 min of standing on ice, the precipitated protein was collected by centrifugation (27,000 X g for 30 min at -4°) and was dissolved in 10 mM Na-phosphate buffer, pH 7.0, containing 2 mM dithioerythritol. Both the supernatant and precipitate were dialyzed against 10 mM Na-phosphate buffer, 2 mM dithioerythritol overnight at 40. The basal activity was not increased by either fraction. The epinephrine-enhancing activity was present only in the supernatant fraction.
DISCUSSION The present study indicates that cytosol obtained from fresh fat cells exerted two effects on fat cell adenylate cyclase: (i) it increased the basal activity 2-fold and (ii) it enhanced the * Cytosol obtained from control cells (So) o Cytosol obtained from toxin- treated cells (ST)
.EE a.
._
Epi + So
Epi+ST
CL ED 0
0
Epinephrine
E
XA-'0
15
60 30 TIME (minutes)
So and ST 180
FIG. 2. Cytosol from cells exposed to toxin for varying periods of time was tested against ghosts from fresh fat cells. Basal values of adenylate cyclase were 62 pmol/mg of protein per min. A 12,000 x g supernatant was used in these experiments.
response to epinephrine. Both effects were observed, but at a reduced level, in fat cell ghosts prepared from cells that were preincubated at 370 for 3 hr without any additives, although the actual increase of epinephrine response due to cytosol remained the same. Fat cell ghosts prepared after exposure to toxin for three hours demonstrated a 2-fold increase in their response to cytosol, The reason for the diminished effect of cytosol on basal activity after 3 hr of incubation and its restoration by cholera toxin is not known. Experiments during the early phase of incubation with toxin (Fig. 1) indicate a change occurring in toxin-treated ghosts that rendered them more sensitive to stimulation both by epinephrine and cytosol. A simultaneous loss of epinephrine-enhancing activity from the cytosol of cells exposed to toxin (Fig. 2) may indicate that toxin mediates a reaction between the membrane and cytosol which results in changes in the catalytic activity of adenylate cyclase. The lack of change in the basal adenylate cyclase stimulating activity of cytosol from cells that have been exposed to toxin suggests that this activity is distinct from cytosol epinephrine-enhancing activity. This conclusion is supported by the separation of the epinephrine-enhancing activity from the basal stimulating activity by ammonium sulfate fractionation. It is likely that the basal stimulating activity may be a complex of many factors, such as ions or nucleotides (13, 14). Since cholera toxin alters the responsiveness of fat cells to epinephrine nea'rly 30 min before the activation of adenylate cyclase can be detected, and before this occurs disappearance of an epinephrine-enhancing activity from the cytosol can be measured, it is likely that intracellular events occur well before activation of the membrane-associated adenylate cyclase. These events are likely to be even more specific to the mechanisms of action of the enterotoxin of V. cholerae than the stimulation of adenylate cyclase which occurs at a later time. In addition, specific components of the cytosol are probably important in the regulation of this enzyme. It has been shown that the response to toxin is more rapid in disrupted cells (15-17), and in our own laboratory this is also true in broken fat cells. In all systems, cytosol factors are necessary for toxin to act, although it is apparent that the sequence of events is compressed and may be different from that in intact cells.
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Cell Biology: Ganguly and Greenough
We are grateful to Mr. Edward Moore for expert assistance in this work. We are also grateful to the Gerontology Research Center of The National Institute of Child Health and Human Development, for the use of facilities provided under its Guest Scientist Program. This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by The National Institute of Allergy and Infectious Diseases of The National Institutes of Health, Bethesda, Maryland, Grant nos. AI 08209 and Al 07625. 1. Ganguly, U. & Greenough, W. B., III (1974) "A cytoplasmic factor enhancing adenylate cyclase response to epinephrine: Effect of cholera toxin," Clin. Res. 22, 710A. 2. Pierce, N. F., Greenough, W. B., III & Carpenter, C. C. J., Jr. (1971) "Vibrio cholerae enterotoxin and its mode of action," Bacteriol. Rev. 35, 1-13. 3. Hewlett, E. L., Guerrant, R. L., Evans, D. J., Jr. & Greenough, W. B., III (1974) "Toxins of Vibrio cholerae and Escherichia coli stimulate adenyl cyclase in rat fat cells," Nature 249, 371-373. 4. Field, M. (1974) "Mode of action of cholera toxin stabilization of catecholamine-sensitive adenylate cyclase in turkey erythrocytes, " Proc. Nat. Acad. Sci. USA 71, 3299-3303. 5. Beckman, B., Flores, J., Witkum, A. P. & Sharp, W. G. G. (1974) "Studies on the mode of action of cholera toxin effects on solubilized adenylate cyclase," J. Clin. Invest. 53, 12021205. 6. Cuatrecasas, P. (1973) "Cholera toxin-fat cell interaction and the mechanism of action of the lipolytic response," Biochemistry 12, 3567-3577.
Proc. Nat. Acad. Sci. USA 72 (1975) 7. Finkelstein, R. A. & LoSpalluto, J. J. (1970) "Production, purification and assay of cholera toxin," J. Infect. Dis. 121, S63S72. 8. Rodbell, M. (1964) "Metabolism of isolated fat cells," J. Biol. Chem. 239,375-380. 9. Krishna, G., Weiss, B. & Brodie, B. B. (1968) "A simple sensitive method for assay of adenyl cyclase," J. Pharm. Exp. Ther. 163,379-385. 10. Randerath, K. & Randerath, E. (1969) "Ion exchange chromatography of nucleotides on poly-(ethylene-imine)-cellulose thin layer," J. Chromatogr. 16, 111-125. 11. Salomon, Y., Londos, C. & Rodbell, M. (1974) "A highly sensitive adenylate cyclase assay," Anal. Biochem. 58,541-548. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) "Protein measurement with Folin phenol reagent," J. Biol. Chem. 193,265-275. 13. Birnbaumer, L. & Yang, P. C. (1974) "Studies on receptor mediated activities of adenylyl cyclase," J. Biol. Chem. 249, 7867-7873. 14. Kalish, M. I., Pineyno, A. M., Cooper, B. & Gregerman, R. I. (1974) "Adenylyl cyclase activation by halide anions other than fluoride," Biochem. Biophys. Res. Commun. 61, 731737. 15. Zieve, P. D., Pierce, N. F. & Greenough, W. B., III (1971) "Stimulation of- glycogenolysis by purified cholera enterotoxin in disrupted cells," Johns Hopkins Med. J. 129, 300-303. 16. Gill, M. D. (1975) Proc. Nat. Acad. Sci. USA 72,2064-2068. 17. vanHeyningen, S. & King, C. A. (1975) "Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes," Biochem. J. 146, 269-271.
Vol. 72, No. 9, pp. 3561-3564, September 1975 Cell Biology
Stimulation of epinephrine-sensitive fat cell adenylate cyclase by cytosol: Effect of cholera toxin (catecholamine effect)
UMA GANGULY AND WILLIAM B. GREENOUGH III* Department of Medicine, Infectious Diseases Division, The Johns Hopkins University School of Medicine and Hospital, Baltimore, Maryland 21205
Communicated by Victor A. McKusick, June 17,1975
ABSTRACT Cytosol prepared from rat epididymal fat cells by centrifugation at 100,00d X g for 1 hr was found to enhance the basal and epinephrine-sensitive adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] of fat cell ghosts. Cholera toxin also stimulated adenylate cyclase and increased the response to epinephrine in fat cells. A possible relationship between the adenylate cyclase modifying activities of cytosol and the effects of cholera toxin was sought. Cytosol from freshly prepared fat cells added to ghosts prepared from cells that had been exposed to toxin for varying periods showed a progressive loss of responsiveness to cytosol epinephrine-enhancing activity. The effect appeared within 15 min after toxin exposure, a full 30 min before any direct effect of toxin on adenylate cyclase was seen. Since exposure to toxin decreased membrane response to cytosol epinephrine-enhancing activity, the possibility that epinephrine-enhancing activity in cytosol might be altered by toxin was explored. Cytosol from cells exposed to toxin for varying periods lost epinephrine-enhancing activity to an a preciable degree within 15 min. Examination of these ear y events after exposure to toxin should clarify the way in which this bacterial substance affects mammalian cells. The cytosol epinephrine-enhancing activity was destroyed by boiling for 3 min and was partially inactivated by trypsin. It was nondialyzable and stable at -70°.
The effect of the enterotoxin of Vibrio cholerae on cells is due to its stimulation of the cell membrane-associated enzyme adenylate cyclase [EC 4.6.1.1; ATP pyrophosphatelyase (cyclizing)] (2). Recent studies from this laboratory (3) have shown that adenylate cyclase in ghosts prepared from fat cells that had been exposed to cholera toxin for 3 hr were more responsive to stimulation by epinephrine than those that were not..A similar phenomenon has recently been described in turkey erythrocytes (4) and solubilized rat liver preparations (5). There is a delay in the onset of action of this toxin (2) which has not been decreased by using detergent or by changing the incubation temperature (6). Because this delay in the onset of action has not been well explained and since there has been difficulty in demonstrating effects in broken cell systems, we felt it was important to study whether intracellular factors affected by toxin might be present which were involved in modulating the response of adenylate cyclase. In this paper we describe the activation of membrane adenylate cyclase and sensitization of response to epinephrine by cytosol derived from fat cells (1) and relate these properties to the effects of cholera toxin. MATERIALS AND METHODS Adenosine 5'-triphosphate (ATP), adenosine 3':5'-cyclic mo*
Reprint requests: Dr. William B. Greenough III, Department of Medicine, The Johns Hopkins Hospital, Baltimore, Md. 21205.
3561
nophosphate (cAMP), phosphoenolpyruvate, pyruvate kinase, and myokinase were obtained from Sigma Chemicals. [a-32P]ATP and [3H]cAMP were purchased from New England Nuclear. Dowex AG 50 W-X2, 200-400 mesh, hydrogen form, was supplied by Bio-Rad Laboratories. Collagenase was obtained from Worthington Biochemical Corp. Bovine serum albumin Fraction V was purchased from Armour Pharmaceutical Co. Highly purified cholera toxin was produced by Dr. R. A. Finkelstein (University of Texas Southwestern Medical School, Dallas, Texas) (7) and supplied by The National Institutes of Health. Preparation of Fat Cell Ghosts and Cytosol. Fat cells isolated (8) from the epididymal fat pads of male SpragueDawley rats were incubated with collagenase (10 mg/g of fat pad) in Krebs-Ringer phosphate (pH 7.2) buffer with albumin (3 g/100 ml) for 1 hr at 370 with gentle shaking. They were filtered through a silk screen, washed three times, and resuspended in the same buffer (1 g of cells per 5 ml of buffer). Glucose was omitted from incubation mixture. Toxin was added at time zero at a concentration of 1 Ag/iml of fat cell suspension, and the mixture was incubated for the indicated times at 370 while shaking at 60 cycles per min. Cells were removed at desired intervals and centrifuged for 1 min at 1500 rpm, and the infranatant buffer was removed. The whole cells were washed once with cold saline, then lysed by exposure to chilled water at 40 with mixing twice in a Vortex mixer and centrifuged at 12,000 X g for 10 min at 40. The pellet was washed once with 5 mM Tris-HCl, pH 8, and resuspended in cold 150 mM Tris-HCl, pH 8, 25 mM MgCl2. The volume was adjusted to make a protein concentration of 2.0-3.0 mg/ml of buffer. Adenylate cyclase activity was measured on this suspension. For the extraction of cytosol, fat cells were dispersed in a minimum volume of hypotonic (10 mM) sodium phosphate buffer, pH 7, (1 g of cells per 1 ml of buffer). The cells were held for one minute on ice, then stirred twice for 10 sec on a Vortex mixer. This mixture was centrifuged at 12,000 X g for 10 min, which resulted in three layers-a topmost layer of fat, an intermediate turbid aqueous layer, and a small pellet. The turbid aqueous phase was withdrawn into a Pasteur pipette and centrifuged again at 100,000 X g for 1 hr. A volume of 20 Ml of this 100,000 X g supernatant fraction (containing a total of 30-40 jig of protein) was used in the assay mixture for adenylate cyclase. All steps were accomplished at 40. An equal volume of boiled cytosol was used for control experiments. The cytosol itself possessed no adenylate cyclase activity in either control or toxin-treated cells. Adenylate Cyclase Assay. Fat cell ghosts were incubated for 10 min at 370 in 0.05 ml of medium containing 30 mM Tris-HCl (pH 8.0), 10 mM MgC12, 10 mM theophylline, 1.5 mM ATP, 1 MCi of [at-32P]ATP, and an ATP-regenerating
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3562
Proc. Nat. Acad. Sci. USA 72 (1975)
Table 1. Stimulation of adenylate cyclase by cytosol in rat fat cell ghosts
Table 2. Effect of cytosol on basal and toxin-stimulated adenylate cyclase activity of fat cell ghosts
Adenylate cyclase, pmol/mg of protein per min (± SEM) Addition of cytosol +
Basal
Epinephrine
63.5 ± 2.8 127 ± 4.6 n = 20
373± 13
238 ± 7.5
Fat cells ghosts and cytosol were prepared as described in Materials and Methods. Cytosol (20 Ml) used in this experiment was obtained from fresh cells; 5.5 uM epinephrine was used in this experiment.
system consisting of 5 mM phosphoenolpyruvate, 50 Mg/ml of pyruvate kinase, and 20,Mg/ml of myokinase. Reactions were stopped by adding 0.5 ml of a recovery mixture (50 gg of cAMP, 100 ,g of ATP, and approximately 2000 cpm of [3H]cAMP) and placing the tubes in a boiling waterbath for 3 min. The radioactive cAMP formed from ATP was separated as described by Krishna et al. (9). The products in the supernate from the barium hydroxide zinc sulfate precipitation step were identified by thin-layer chromatography on polyethyleneimine (PEI) impregnated cellulose MN 300. Nonradioactive AMP and ATP were added, and the chromatogram was developed with 0.6 M LiCl adjusted to pH 7.3 with 1.0 M formic acid as the solvent (10). Nucleotides were localized under ultraviolet light. The radioactive 32p present in the AMP and ATP spots from samples with cytosol present did not differ from those of the control and reaction blank, and radioactivity in the fraction containing cyclic AMP was restricted to this compound. Furthermore, we also found increased adenylate cyclase activity due to cytosol by the more specific method described by Salomon et al. (11). Boiled enzyme samples were used as controls. Protein was measured by the method of Lowry et al. (12). Synthesis of cyclic AMP was linear with time for at least 10 min and proportional to the enzyme concentration up to 1 mg/ml. Fat cell ghosts were prepared fresh each day for each experiment. RESULTS The supernatant fluid fractions of fat cells prepared in water or 10 mM sodium phosphate buffer stimulated basal and epinephrine-sensitive adenylate cyclase when incubated with freshly prepared fat cell ghosts (Table 1). Stimulation by cytosol plus epinephrine produced a 5-fold increase over basal activity. By comparison, stimulation by epinephrine alone was 3.5-fold (Table 1). Since cholera toxin is known to increase the fat cell adenylate cyclase, the activity of cytosol was also tested in toxintreated cells. Accordingly, cytosol from freshly prepared cells was added to fat cell ghosts prepared from cells that had been incubated for 3 hr with or without toxin. Although cytosol stimulated basal adenylate cyclase activity of fat cell ghosts prepared from both control and toxin-treated cells (Table 2), the enzyme response to cytosol was significantly increased in cells that had been exposed to toxin as compared to controls. In addition to a direct stimulation of adenylate cyclase, the cytosol fraction potentiated the effect of epinephrine in ghosts prepared from control cells. However, ghosts from cells exposed to toxin did not show such a synergistic effect of epinephrine and cytosol. The potentiating ef-
Adenylate cyclase, pmol/ mg of protein per min (± SEM) -
Basal Cytosol Epinephrine Epinephrine + cytosol
Toxin
60 95 126 228 n =
± ± ±
±
8.2 5.5 7.5 15.0
+ Toxin
175 305 397 477
±
12 22
±
15
±
22
±
5
Fat cells were incubated without and with cholera toxin (1 Mg/ml of cell suspension) for three hours. Ghosts were prepared from these cells as described in Materials and Methods. Cytosol (20 il) used in this experiment was obtained from fresh cells; 5.5 uM epinephrine was used in this experiment.
fect of cytosol could not be accurately judged because of the nearly maximal stimulation that was achieved by either cytosol or epinephrine alone (Table 2) in ghosts prepared from toxin-treated cells that already had a 3-fold increase in adenylate cyclase. The degree of adenylate cyclase stimulation by cytosol in ghosts prepared from cells that had been exposed to toxin was tested during the early phase of incubation at a time when the direct stimulating effect of toxin on this enzyme was not yet apparent. The adenylate cyclase activity in the presence and absence of epinephrine and cytosol in toxintreated fat cell ghosts is shown (Fig. 1). Ghosts prepared from cells exposed to toxin for 30 min showed no increase of adenylate cyclase activity. At 60 min an increase of 50 pmol/ mg of protein per min over the basal value had occurred. Cytosol added to fresh fat cell ghosts stimulated basal activity 2-fold and increased the epinephrine response. Thirty minutes after exposure to toxin a progressive increase in the responses both to cytosol and epinephrine occurred. In fresh cells, when cytosol was added with epinephrine a greater than additive response occurred (indicated by "I" adjacent to histogram). This additional response to epinephrine is progressively lost beginning 15 min after exposure to toxin (P < 0.02). Since exposure to toxin for 30 min appreciably enhanced the response of fat cell ghosts to cytosol and diminished the capacity of cytosol to enhance the epinephrine response, it seemed reasonable to determine the effect of toxin on cytosol itself. Accordingly, cytosol from cells incubated with and without cholera toxin for varying periods of time was tested in fresh fat cell ghosts. As shown in Fig. 2, cytosol from both groups of cells stimulated basal adenylate cyclase to the same degree; however, cytosol from cells exposed to toxin showed a reduced ability to enhance the response of ghosts to epinephrine within 15 min after exposure to toxin (P < 0.01). This alteration preceded detectable direct stimulation of adenylate cyclase by the toxin, which occurred at approximately 60 min. This loss of the epinephrine-enhancing effect from the cytosol of toxin-treated cells correlated with the increase in the epinephrine response of toxin-treated ghosts (Fig. 1). This enhanced epinephrine response has been described (3). It occurred as a significant effect within 30 min after exposure of fat cells to the toxin. No activity of adenylate cyclase was detected directly in cytosol from either control or toxin-treated cells. The catalytic activity of adenylate cyclase was increased in a nonlinear manner with increasing amounts of cytosol tested from 5
Cell Biology:
Proc. Nat. Acad. Sci. USA 72 (1975)
Ganguly and Greenough
E C
c v 0
o-Basoal
Dc"-Cy+Cytosol 600 *g600
sum of ind lividual stimuli (epi + cytosol)
500
I
0.
0,
400
E
300 00
60 min(+)toxin f resh(-)toxi n 15 min(+) toxi n 30 min(+)toxin FIG. 1. Effect of cytosol prepared from fresh cells on basal and epinephrine-stimulated adenylate cyclase activity of toxin-treated fat cell ghosts. Fat cells were incubated in the presence of cholera toxin (1 tig/ml of cell suspension) at 370 for various times. Epinephrine was present at a final concentration of 5.5 MAM. Cytosol (20 Al) was added to a final reaction volume of 50 Ail. The loss of synergistic effect of epinephrine response produced by cytosol in toxin-treated fat cells is indicated by an "I" adjacent to the histogram, at 0, 15, 30, and 66 min. Results are means of four separate experiments.
to 50 /d. With the maximum amount of cytosol used (50 Ml), the enzyme was stimulated approximately 6-fold. The cytosol activity was lost after boiling for 3 min. It was partly inactivated (40-50%) by trypsin (1 mg/ml of cytosol per 30 min at 370). It was stable at -700, but lost activity after repeated freezing and thawing. Separation of the adenylate cyclase stimulating activity was done initially by adding cold saturated (NH4)2SO4 solution to the cytosol to bring the salt concentration to 60% saturation. After 30 min of standing on ice, the precipitated protein was collected by centrifugation (27,000 X g for 30 min at -4°) and was dissolved in 10 mM Na-phosphate buffer, pH 7.0, containing 2 mM dithioerythritol. Both the supernatant and precipitate were dialyzed against 10 mM Na-phosphate buffer, 2 mM dithioerythritol overnight at 40. The basal activity was not increased by either fraction. The epinephrine-enhancing activity was present only in the supernatant fraction.
DISCUSSION The present study indicates that cytosol obtained from fresh fat cells exerted two effects on fat cell adenylate cyclase: (i) it increased the basal activity 2-fold and (ii) it enhanced the * Cytosol obtained from control cells (So) o Cytosol obtained from toxin- treated cells (ST)
.EE a.
._
Epi + So
Epi+ST
CL ED 0
0
Epinephrine
E
XA-'0
15
60 30 TIME (minutes)
So and ST 180
FIG. 2. Cytosol from cells exposed to toxin for varying periods of time was tested against ghosts from fresh fat cells. Basal values of adenylate cyclase were 62 pmol/mg of protein per min. A 12,000 x g supernatant was used in these experiments.
response to epinephrine. Both effects were observed, but at a reduced level, in fat cell ghosts prepared from cells that were preincubated at 370 for 3 hr without any additives, although the actual increase of epinephrine response due to cytosol remained the same. Fat cell ghosts prepared after exposure to toxin for three hours demonstrated a 2-fold increase in their response to cytosol, The reason for the diminished effect of cytosol on basal activity after 3 hr of incubation and its restoration by cholera toxin is not known. Experiments during the early phase of incubation with toxin (Fig. 1) indicate a change occurring in toxin-treated ghosts that rendered them more sensitive to stimulation both by epinephrine and cytosol. A simultaneous loss of epinephrine-enhancing activity from the cytosol of cells exposed to toxin (Fig. 2) may indicate that toxin mediates a reaction between the membrane and cytosol which results in changes in the catalytic activity of adenylate cyclase. The lack of change in the basal adenylate cyclase stimulating activity of cytosol from cells that have been exposed to toxin suggests that this activity is distinct from cytosol epinephrine-enhancing activity. This conclusion is supported by the separation of the epinephrine-enhancing activity from the basal stimulating activity by ammonium sulfate fractionation. It is likely that the basal stimulating activity may be a complex of many factors, such as ions or nucleotides (13, 14). Since cholera toxin alters the responsiveness of fat cells to epinephrine nea'rly 30 min before the activation of adenylate cyclase can be detected, and before this occurs disappearance of an epinephrine-enhancing activity from the cytosol can be measured, it is likely that intracellular events occur well before activation of the membrane-associated adenylate cyclase. These events are likely to be even more specific to the mechanisms of action of the enterotoxin of V. cholerae than the stimulation of adenylate cyclase which occurs at a later time. In addition, specific components of the cytosol are probably important in the regulation of this enzyme. It has been shown that the response to toxin is more rapid in disrupted cells (15-17), and in our own laboratory this is also true in broken fat cells. In all systems, cytosol factors are necessary for toxin to act, although it is apparent that the sequence of events is compressed and may be different from that in intact cells.
3564
Cell Biology: Ganguly and Greenough
We are grateful to Mr. Edward Moore for expert assistance in this work. We are also grateful to the Gerontology Research Center of The National Institute of Child Health and Human Development, for the use of facilities provided under its Guest Scientist Program. This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by The National Institute of Allergy and Infectious Diseases of The National Institutes of Health, Bethesda, Maryland, Grant nos. AI 08209 and Al 07625. 1. Ganguly, U. & Greenough, W. B., III (1974) "A cytoplasmic factor enhancing adenylate cyclase response to epinephrine: Effect of cholera toxin," Clin. Res. 22, 710A. 2. Pierce, N. F., Greenough, W. B., III & Carpenter, C. C. J., Jr. (1971) "Vibrio cholerae enterotoxin and its mode of action," Bacteriol. Rev. 35, 1-13. 3. Hewlett, E. L., Guerrant, R. L., Evans, D. J., Jr. & Greenough, W. B., III (1974) "Toxins of Vibrio cholerae and Escherichia coli stimulate adenyl cyclase in rat fat cells," Nature 249, 371-373. 4. Field, M. (1974) "Mode of action of cholera toxin stabilization of catecholamine-sensitive adenylate cyclase in turkey erythrocytes, " Proc. Nat. Acad. Sci. USA 71, 3299-3303. 5. Beckman, B., Flores, J., Witkum, A. P. & Sharp, W. G. G. (1974) "Studies on the mode of action of cholera toxin effects on solubilized adenylate cyclase," J. Clin. Invest. 53, 12021205. 6. Cuatrecasas, P. (1973) "Cholera toxin-fat cell interaction and the mechanism of action of the lipolytic response," Biochemistry 12, 3567-3577.
Proc. Nat. Acad. Sci. USA 72 (1975) 7. Finkelstein, R. A. & LoSpalluto, J. J. (1970) "Production, purification and assay of cholera toxin," J. Infect. Dis. 121, S63S72. 8. Rodbell, M. (1964) "Metabolism of isolated fat cells," J. Biol. Chem. 239,375-380. 9. Krishna, G., Weiss, B. & Brodie, B. B. (1968) "A simple sensitive method for assay of adenyl cyclase," J. Pharm. Exp. Ther. 163,379-385. 10. Randerath, K. & Randerath, E. (1969) "Ion exchange chromatography of nucleotides on poly-(ethylene-imine)-cellulose thin layer," J. Chromatogr. 16, 111-125. 11. Salomon, Y., Londos, C. & Rodbell, M. (1974) "A highly sensitive adenylate cyclase assay," Anal. Biochem. 58,541-548. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) "Protein measurement with Folin phenol reagent," J. Biol. Chem. 193,265-275. 13. Birnbaumer, L. & Yang, P. C. (1974) "Studies on receptor mediated activities of adenylyl cyclase," J. Biol. Chem. 249, 7867-7873. 14. Kalish, M. I., Pineyno, A. M., Cooper, B. & Gregerman, R. I. (1974) "Adenylyl cyclase activation by halide anions other than fluoride," Biochem. Biophys. Res. Commun. 61, 731737. 15. Zieve, P. D., Pierce, N. F. & Greenough, W. B., III (1971) "Stimulation of- glycogenolysis by purified cholera enterotoxin in disrupted cells," Johns Hopkins Med. J. 129, 300-303. 16. Gill, M. D. (1975) Proc. Nat. Acad. Sci. USA 72,2064-2068. 17. vanHeyningen, S. & King, C. A. (1975) "Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes," Biochem. J. 146, 269-271.
