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Effect of Glucagon on Amino Acid Transport and Cyclic Adenosine 3':5'-Monophosphate Production in Rat Hepatoma Cell Line McA-RH 8994 in Culture¹

McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

The rates of -aminoisobutyrate (AIB) transport and cyclic adenosine 3':5'-monophosphate (cAMP) production in response to glucagon (2 x 10^–7 M) have been determined in monolayer cultures of hepatoma cell line 8994 (refers to hepatoma cell line McA-RH 8994) under three different calcium treatments. The calcium treatments were: (a) control, cultures maintained in calcium-containing medium without prior calcium depletion; (b) calcium depleted, cultures maintained in calciumfree medium after treatment with ionophore A23187 and ethylene glycol bis (ß-aminoethyl ether)-N,N'-tetraacetic acid; (c) calcium repleted, cultures maintained in calcium-containing medium after treatment with A23187 and ethylene glycol bis (ß-aminoethyl ether)-N,N'-tetraacetic acid. In the control cultures, glucagon in the absence of methylisobutylxanthine (MIX) has no effect on the rate of AIB transport. AIB transport is approximately doubled when glucagon is added in the presence of MIX. Calcium depletion and calcium repletion enhance the rate of AIB transport by about 50 and 100%, respectively. Glucagon and N⁶,O^2'-dibutyryl cyclic adenosine 3':5'-monophosphate (1 x 10^–4 M) further increase the rate of AIB transport by 10% in calcium-depleted cultures and by 50% in calcium-repleted cultures. The stimulation of AIB transport by glucagon as well as by the ionophore can be blocked by the simultaneous addition of cycloheximide (0.1 mM) or actinomycin D (3.2 µM). In the absence of MIX, there is about a 2-fold increase in the level of cAMP in response to glucagon. In the presence of MIX, glucagon causes more than 100-fold increase in the level of cAMP. The presence or absence of calcium does not affect the basal or glucagon-stimulated production of cAMP in these cultures. We have reported previously that, in monolayer cultures of rat hepatocytes, calcium repletion enhanced the glucagon stimulation of AIB transport while it markedly inhibited the production of cAMP in response to glucagon. It is suggested that calcium rather than cAMP has a direct effect on the amino acid transport system.

¹ Financial support was provided in part by Grants CA-07175, CA-09020, CA-224484, and CA-17334 from the National Cancer Institute and by Grant 1N-27B from the American Cancer Society.

² Present address: West Virginia University, School of Medicine, Department of Biochemistry, Morgantown, W. Va. 26506.

Received 12/23/81. Accepted 4/ 8/82.