Biochimica et Biophysica Acta, 1092 (1991) 79-84 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001238


BBAMCR 12883

Protein synthesis is required for cholera toxin-induced stimulation of arachidonic acid metabolism Johnny W. Peterson 1, James C. Reitmeyer 1, Christopher A. Jackson 1 and G.A.S. Ansari 2 ! Department of Microbiology, University of Texas Medical Branch, Galveston, TX (U.S.A.) and z Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX (U.S.A.)

(Received 1 August 1990) (Revised manuscript received 11 October 1990)

Key words: Cholera toxin; Arachidonic acid metabolism; cylcic AMP synthesis; Prostaglandin synthesis; Protein synthesis; (Mouse Chinese hamster ovary cell)

The molecular events in the mechanism of action of cholera toxin were analyzed using Chinese hamster ovary (CHO) cells. Cholera toxin stimulated both 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and arachidonic acid metabolism in these cells. The turnover of phospholipid by cholera toxin-induced stimulation of phospholipase activity evoked the synthesis of PGE z and other prostagland|ns. Cholera toxin-induced release of both [3H]arachidonic acid and PGE z was blocked by addition of either cycloheximide or actinomycin D. In contrast, accumulation of cAMP in cholera toxin-treated CHO cells was unaffected by adding these drugs. Further, dibutyryl cAMP or forskolin caused [3H]arachidonic acid release, which also was blocked by cycloheximide and actinomycin D. We concluded that the sequence of molecular events in cholera toxin-treated CHO cells first involved activation of adenylate cyc|ase, which caused an increase in cAMP. In turn, cAMP promoted transcription of mRNA that encoded either a specific phospholipase or a phospholipase-activating protein. The emerging arac~hadonic acid metabolites (e.g., PGEz and PGF2a) might be important mediators of cholera toxin's stimulatory effects on vascular permeability and smooth muscle contraction in the intestine during cholera. Introduction

Cholera toxin (CT) stimulated both adenylate cyclase and a phospholipase in eukaryotic cells; these events culminated in the increased synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid metabolites, including various prostaglandins [1-3]. The precise sequence of molecular events in the pathogenic mechanism was not clear, but there was some evidence for the involvement of a protein intermediate. Earlier investigators, for example, observed that cycloheximide, puromycin and actinomycin D markedly blocked cholera toxin-induced edema activity in the mouse or rat foot edema models (4,5]. Cycloheximide also blocked

Abbreviations: CHO, Chinese hamster ovary cells; cAMP, 3',5'-cyclic adenosine monophosphate; DHKPGF2,,, 13,14-dihydro-15-ketoprostaglandin F:a; CT, cholera toxin. Correspondence: J.W. Peterson, Department of Microbiology, University of Texas Medical Branch, Galveston, Texas 77550, U.S.A.

intestinal fluid accumulation in rabbit intestinal loops challenged with cholera toxin [6,7]. Furthermore, Modtz et al. [8] observed that cycloheximide inhibited cholera toxin-stimulated chloride ion transport in isolated small intestinal mucosa in vitro. These reports suggested th~.t a newly synthesized protein might be important in mediating the secretory response to cholera toxin; however, a protein mediator was not identified. Recently, Peterson et al. [6] observed that rabbit intestinal epithelial cells and Chinese hamster ovary (CHO) cells synthesized increased amounts of a membrane-associated protein(s) during the first few hours of incubation with cholera toxin. The identity and the importance of the protein in the pathogenic mechanism of cholera remained unclear. The purpose of this report was to clarify the relationship between cholera tox/n-induced synthesis of cAMP, prostaglandins and protein in CHO cells. We observed th:,t cholera toxin caused the rapid release of arachidonic acid and subsequent synthesis of prostaglandins [9,10]. Both cycloheximide and actinomycin D blocked CT..induced release of arachidonic acid and PGE 2, but cAMP


accumulation was not affected. This new information suggested that a protein(s) might be important in the mechanism of action of cholera toxin in altering arachidonic acid metabolism.

Materials and Methods

Cell culture and labeling. Phospholipids were labeled by adding 0.3/~Ci of [5,6,8,9,11,12,14,15-3H]arachidonic acid (American Radiolabled Chemicals, St. Louis, MO) to a suspension of 2. 105/ml CliO cells (American Type Culture Collection) suspended in Ham's F-12 medium (Gibco, Gaithersburg, MD) containing 10% fetal calf serum [9]. CHO cell monolayers were established in 12-well tissue culture plates (Costar) and were incubated overnight at 37"C with 5% CO2. Antimicrobial agents were added to the medium as follows: penicillin (100 units/ml), streptomycin (100 /~g/ml), gentamicin (50/~g/ml) and nystatin (50 units/ml). The monolayers were washed three times with phosphatebuffered saline (composition in g/l: NaCI, 8.0; KCL, 0.2; Na2HPO4, 1.15; KH2PO4, 0.2; CaCl 2 • 6H20, 1.0 (pH 7.4)). Cholera toxin (List Laboratories, Campbell, CA) was diluted in Ham's F-12 medium with 10% fetal calf serum and antimicrobial agents to a final concentration of 100 ng/ml. Triplicate wells were used for each test or control in each experiment. The experiments always were perfoL'med at least twice. The data summarized in Table III were derived from six to seven experiments. Cycloheximide and actinomycin D (Sigma, St. Louis, MO) were dissolved to a final concentration of 10 /tg/ml in Ham's F-12 medium containing fetal calf serum and antimicrobial agents. Dibutyryl cAMP and forskolin (Sigma) similarly were prepared to a concentration of 2 mM and 10/~M, respectively. All plates were incubated at 37"C in 5% CO2 with or without cholera toxin (100 ng/ml) for 3 h unless indicated otherwise. Aliquots (200 tal) of culture medium from each well were pipetted into 5 rnl scintillation cocktail (Hydrofluor, National Diagnostics) and counted in a liquid scintillation counter (Table I). The remaining medium from each well was discarded, the monolayer was washed once with PBS, and fresh medium was added (1 ml/well). Tli,e plates were incubated again for 3 h. Then, 200 lal was removed, added to scintillation cocktail, and counted (Table I). Assay for PGE2. Tissue culture procedures were as described above except that [3H]arachidonie acid was omitted, and monolayers were established in 35-ram dishes containing 2 ml CHO cells (2.10S/m!) suspended in medium. Conditions for exposure to cholera toxin and drugs were as described, and the cells were incubated for 4 h. The culture medium was removed,

and prostaglandins were extracted before quantifying PGE 2 by radioimmunoassay using procedures recommended by the manufacturer (Advanced Magnetics, Cambridge, MA). Percentages of PGE2 recoveries were estimated after sample extraction. Assay for cyclic AMP. After removing the medium from the cell monolayer for the prostaglandin assay above, I ml of 7.5% trichloroacetic acid (TCA) was placed on the cell monolayers and allowed to remain for 30 min at room temperature. The supernatants were removed for the cAMP assay [11] and the precipitates were solubilized with 1 ml aliquots of 0.5 M KOH and assayed for total protein by the method of Bradford [12]. The percentage of cAMP recovery was estimated by adding 0.3 laCi of radiolabeled cAMP (Amersham) to samples before extraction. The TCA supernatant was extracted five times (with twice the volume of ice-cold anhydrous ether) to remove the TCA, and the aqueous phase was evaporated to dryness in a vacuum centrifuge (Savant). The concentrated dried material was resuspended in 200/~1 of 0.1 M sodium acetate buffer (pH 4.0) for determination of cAMP, using a modification by Brostrom and Kon [13] of the Gilman method [14]. The accuracy of this radiometric cAMP assay was confirmed by comparing initial results with those achieved with a commercial cAMP RIA kit (Advanced Magnetics), which had a several-fold greater sensitivity. The results of the CT dose response experiments were comparable by both assays. Detection of prostaglandins by HPLC CHO cell monolayers (containing approx. 1.107 cells) were established in 75-cm2 tissue culture flasks (10 ml). Some flasks were exposed to cholera toxin (2 /~g/ml), while others also contained indomethacin (10 #g/ml). Culture supernatants were frozen and iyophilized. After reconstituting the dried samples with 2 ml H20, proteins were precipitated with 6 ml cold acetone, and neutral lipids were extracted with 4 ml petroleum ether. The lower phase was acidified to pH 3-4 with acetic acid, and extracted with 6 ml ethyl acetate. The samples were centrifuged, and the upper ethyl acetate phase was evaporated in a vacuum centrifuge (Savant). The dried extracts were resuspended in 500/~1 of 30% acetonitrile in water containing 0.1% trifluoroacetic acid. The entire sample was chromatographed through a 10 #m column (4.5 x 250 mm) of C18 (Serva) equilibrated with the same buffer at a flow rate of 1.5 ml/min. Prostaglandins were identified by comparison of retention time with standards (Sigma, St. Louis, MO). Analysis of data. Variation in the means of each experimental group (cholera toxin control, drugs and drugs plus cholera toxin) were compared with that of the untreated control using an Analysis of Variance. The moderately conservative Tukey test was used to determine any significant difference between the experimental groups and the controls.

81 Results


Effects o[ CT on arachidonic acid metabolism

Effect of cycloheximide and actinomycin D on the synthesis of prostaglandin E 2 and cAMP by CHO cells exposed to cholera toxin

Cholera toxin caused an increase in the release of [3H]arachidonic acid from CHO cells that was blocked by either cycloheximide or actinomycin D during the initial 3 h incubation period (Table I). The CHO cells treated with cholera toxin averaged 26.970 more release of [3H]arachidonic acid than did the untreated control cells (P < 0.05). Cycloheximide, when added with cholera toxin, blocked completely the release of [3H]arachidonic acid (-5.270), yielding slightly negative values comparable to that of the cycloheximide control (-7.870). Similarly, actinomycin D restricted cholera toxin-induced [3H]arachidonic acid release ( - 10.670) to levels comparable to that of the actinomycin D control (-10.170). Table I also shows the amount of [aH]arachidonic acid released from the same cells during a subsequent 3 h incubation period after the drugs were washed away and fresh medium was added. Cells that had been exposed previously to CT alone produced only a 13.270 increase in [aH]arachidonic acid release during the second incubation period, compared to 26.970 earlier. It appeared that the initial cholera toxin response reduced the amount of [3H]arachidonic acid released during the subsequent incubation period. The wells that had contained cycloheximide alone had an increase of 18.370 above the control during the second incubation. Neither of these effects were considered significantly different from the cell control (P > 0.05). In contrast, [3H]arachidonic acid release from cells that earlier were protected from cholera toxin by incubation with cycloheximide, increased significantly (P < 0.05) above the cycloheximide control and the cell control (33.670 and 58.070, respectively) when the drug was removed. Cells that had been exposed to actinomycin D, alone or with cholera toxin added, remained negative


Prostaglandin E 2 (pg/mg cell protein)

Cyclic AMP (pmol/mg cell protein)

Cell control Cholera toxin Cycloheximide Cholera toxin + cycloheximide Actinomycin D Cholera toxin + actinomycin D

648 + 174 1585 + 142 a 508 + 43

23 + 5 183 + 62 a 23 + 5

667 + 182 736+ 160

158 =l:24 b 20+ 4

857 + 98

159 + 34 c

a Significantly greater than the cell control ( P < 0.05, Tukey). b Significantly greater than the cycloheximide control ( P < 0.05, Tukey). c Significantly greater than the actinomycin D control ( P < 0.05, Tukey).

relative to the cell control (-16.97o and -21.170, respectively) irregardless of whether the drug was present. Effects of cholera toxin on PGE 2 and cAMP levels Cholera toxin caused a significant (P < 0.05) increase in the release of PGE 2 from CHO cells (Table If). Both cycloheximide and actinomycin D blocked the stimulatory effects of cholera toxin on PGE2 release, and PGE 2 levels were maintained near the respective drug control levels (P > 0.05). Cholera toxin also evoked a significant (P < 0.05) increase in cAMP in CHO cells (Table If). Interestingly, neither cycloheximide nor actinomycin D affected intraceUular concentrations of cAMP in CHO cells exposed to cholera toxin (P < 0.05). Thus, the stimulatory effects of cholera toxin on adenylate cyclase and phospholipase activity was distinguished with either of these drugs. Effects of cAMP on [ 3H]arachidonic acid release The stimulatory effect of cholera toxin on [3H]arachidonic acid release in CHO cells was dupficated by



inhibiticm of cholera toxin.induced [3H]arachidonic acid release from ClIO cells by cycloheximide and actinomycin D




Cell control Cholera toxin Cycloheximide


first 3 h incubation (with drugs)

second 3 h incubation (without drugs)

.>_ 400

1682 4-109 2135 4-118 1551 4- 84

1706+ 51 1930 + 177 2 O18 4- 248

1549 4- l 11 1504 4-120

2696 + 168 b 1417+139

1512 4-121

1346 :t=234

a Significantly greater than the cell con'~rol ( P < 0.05, Tukey). b Significantly greater than the cycloheximide control ( P

Protein synthesis is required for cholera toxin-induced stimulation of arachidonic acid metabolism.

The molecular events in the mechanism of action of cholera toxin were analyzed using Chinese hamster ovary (CHO) cells. Cholera toxin stimulated both ...
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