/ . Biochem., 78, 811-815 (1975)

Correlation between Adenosine 3', 5'-Cyclic Monophosphate Levels, Adenylate Cyclase Activity, and Adenosine 3', 5'-Cyclic Monophosphate Phosphodiesterase Activity in Tissue Culture Cells Stimulated by Serum1 Takeshi MATSUMOTO and Takahiro UCHIDA Cancer Research Institute, Sapporo Medical College, Chuo-ku, Sapporo, Hokkaido 060 Received for publication, March 14, 1975

Changes in the activity of adenylate cyclase [EC 4.6.1.1] of serum-stimulated hamster BHK cells in culture were investigated in relation to changes in the intracellular level of adenosine 3', 5'-cyclic monophosphate (cyclic AMP). Addition of calf serum to quiescent cultures decreases the activity of adenylate cyclase, followed by cellular DNA synthesis. Cyclic AMP levels drop in parallel with the decrease in adenylate cyclase activity. Kinetic analysis of cyclic AMP phosphodiesterase [EC 3.1.4.17] revealed that BHK cells contain two forms of the enzyme, one with a Km of 0.77 i"M and the other with a Km of 17.0 fiM. The activity of these enzymes is not affected by the addition of serum to tissue culture cells. The findings indicate that the decrease in adenylate cyclase activity is directly responsible for the decrease in cyclic AMP levels that is observed immediately after serum addition. Low levels of cyclic AMP continue for several hours during serum treatment, followed by a transient increase in cyclic AMP in the early stages of cellular DNA synthesis, at which time the activity of cyclic AMP phosphodiesterase with a low Km shows a slight decrease.

Several lines of evidence suggest that the proliferation of tissue culture cells is regulated by adenosine 3', 5'-cyclic monophosphate (cyclic AMP) {1-9). Cyclic AMP levels are higher in contact-inhibited cells than in logarithmically growing cells (3, 7), and there is an inverse relationship between growth rate and intracellular cyclic AMP content (4,8). Treat1

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education of Japan. Vol. 78, No. 4, 1975

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ment of quiescent cells with substances (fresh serum, insulin, and trypsin) that stimulate cell growth leads to a decrease in the intracellular concentration of cyclic AMP (7, 8, 10-12). The cyclic AMP metabolism is controlled mainly by its rate of synthesis and hydrolysis, which are catalyzed by adenylate cyclase [EC 4.6.1.1] and cyclic AMP phosphodiesterase [EC 3.1.4.17], respectively. A direct correlation between adenylate cyclase activity and cyclic AMP levels has been reported using cells transformed by a temperature-sensitive

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T. MATSUMOTO and T. UCHIDA

Rous sarcoma virus (13). On the other hand, it has been shown that no change takes place in cyclic AMP levels when no significant alteration occurs in the ratio between adenylate cyclase and cyclic AMP phosphodiesterase activities {14). In our study of hamster BHK cells and their virally-transformed derivatives, the adenylate cyclase activity and cyclic AMP level were found to rise in parallel without any decrease in phosphodiesterase activity when the cells were cultured at a low serum concentration (15). In this paper we describe a relationship between cyclic AMP levels and the activities of two enzymes involved in cyclic AMP metabolism in serum-stimulated BHK cells. MATERIALS AND METHODS

Cell Culture—The BHK 21 hamster cell line (C13 clone) was used. The cells were maintained in Dulbecco's modification of Eagle's medium (GIBCO), supplemented with 10% calf serum (Flow Laboratories, Inc.), 500 units/ml of penicillin, and 100 ftg/ml of streptomycin in a Petri dish at 37° in a 5% CO2 atmosphere. Quiescent BHK cells were prepared by seeding 3-5x10° cells per dish (9 cm) with 10 ml of medium containing 0.5% calf serum, and incubating for 2 days. DNA Synthesis Assay—Cellular DNA synthesis was measured as follows. Quiescent BHK cells grown on cover slips (15 mm in diameter) in a Petri dish (5 cm) were treated with serum as described in the text. At intervals, the cells were pulse-labeled with 2 j«Ci/ml of [3H]thymidine (45.3 Ci/mmole) for 1 hr. Cover slips were rinsed in Tris-buffered saline (pH 7.4), washed with 5% trichloroacetic acid, and counted with Triton-toluene scintillation solution in a Horiba scintillation spectrometer. Cyclic AMP Assay—The culture medium was removed and 5 ml of cold 5% trichloroacetic acid was added to the dish. To estimate the recovery, 0.25 pmole of cyclic PHIAMP (30 Ci/mmole) was added to the dish. The cells were scraped from the dish with a rubber policeman and the suspension was centrifuged to remove the acid precipitate. The supernatant fluid was extracted 5 times with 3

volumes of water-saturated ether after the addition of 0.1 ml of I N HC1. The aqueous phase was separated and lyophilized. The residue was then dissolved in a small volume of water, and cyclic AMP was isolated by column chromatography (Dowex 50W-X4) according to the method of Krishna and Birnbaumer (16). The fraction containing cyclic AMP was lyophilized and dissolved in a small volume of 40 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl2. The recovery of radioactivity was about 80%. The amounts of isolated cyclic AMP were measured by the protein-binding method of Gilman (17). All values were corrected in each assay by subtracting the values obtained on samples that had been treated with cyclic AMP phosphodiesterase for 4 hr at 37°. Adenylate Cyclase Assay—Cell sheets were rinsed twice with Tris-buffered saline (pH 7.4), and scraped from dishes with a rubber policeman. The cells were collected by centrifugation, suspended in 40 mM Tris-HCl buffer (pH 7.5) containing 3 mM MgSO

Correlation between adenosine 3',5'-cyclic monosphosphate levels, adenylate cyclase activity, and adenosine 3',5'-cyclic monophosphate phosphodiesterase activity in tissue culture cells stimulated by serum.

/ . Biochem., 78, 811-815 (1975) Correlation between Adenosine 3', 5'-Cyclic Monophosphate Levels, Adenylate Cyclase Activity, and Adenosine 3', 5'-C...
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