No abstract

Amino acid transport in Madin-Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na+-independent leucine or niethionine uptake at pH 7.4. The activity of the A system was measured as Na+-dependent a(methy1amino)isobutyric acid (mAl €3) uptake at p H 7.4, the activity of the ASC system was measured as Na+-dependent alanine uptake in the presence of 0.1 m M mAlB at pH 6.0, and the activity of system N was observed by measuring Na+-dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na+-dependent amino acid transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE,, and was elevated 3-7 fold in the transformed cell line, MDCK-T,. The activity of the ASC system wa5 slightly stimulated by insulin and by PGE,, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.

Glycolysis in normal resting rat kidney cells (NRK-49F) was stimulated by a 2-hr exposure to transforming growth factors prior to assay. Transforming growth factor (3 (TGF-3) was effective when added alone, and further addition of epidermal growth factor (EGF) had little effect. The stimulation by TGF-. was abolished when cycloheximide was present during the incubation, suggesting that protein synthesis Is required for the effect. Incubation of the cells with 25 mM methionine abolished the stimulation of glycolysis by TGF-(3. The uptake of methylaminoisobutyrate via system A was stimulated by either TGF-1 or EGF. The >3-fold stimulation of uptake by 1 ng of pure TGF-( per ml was usually somewhat enhanced on addition of 0.5 ng of EGF per ml.Moreover, an antiserum against EGF receptor partially depressed the response to TGF-fi, suggesting some overlapping interactions of EGF and TGF-13. We have observed recently that glycolysis in a variety of normal cell lines was markedly stimulated in the presence of a crude preparation of TGF-13 from human placenta during a 60-min assay (10). In this paper we report that both partly purified and pure preparations of TGF-P from human platelets stimulate glycolysis as well as the uptake of MeAIB into normal rat kidney cells (NRK-49F) after an incubation period of 24 hr. In an accompanying paper, an independent study is reported on the stimulation of glucose uptake into 3T3 cells by epidermal growth factor (EGF) and Transport assays were performed basically as described (6).[3H]Alanine uptake was measured after 2 min at pH 6.0 in the presence of 1 mM MeAIB to ensure that alanine uptake occurred via system ASC and not via system A.[3H]Methionine uptake was measured after 1 min with N-methyl-D-glucosamine substituted for NaCl to ensure uptake via system L (13). After washing, the cells were dissolved in 2 ml of 0.2 M NaOH/1% sodium dodecyl sulfate and assayed for radioactivity in 10 ml of Liquiscint (National Diagnostics, Somerville, NJ). RESULTS 1350The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Adaptive regulatory control of System A activity was investigated using MDCK cells and a chemically induced, oncogenic transformant of MDCK cells, MDCK-T1. Within 7 hours after transfer to an amino-acid-deficient medium, A activity of subconfluent MDCK cells had maximally derepressed, but this activity in confluent MDCK cells and in subconfluent transformed cells showed little capacity for derepression. Amino-acid-starved, subconfluent MDCK cells were used to study trans-inhibition and repression of A activity by individual amino acids. Trans-inhibition and repression were defined as the cycloheximide-insensitive and cycloheximide-sensitive components, respectively, of the total inhibition. Trans-inhibition correlated well with substrate affinity, but repression did not. Trans-inhibition and repression were further characterized using alpha-(methylamino) isobutyric acid (mAIB), a trans-inhibitor, and glutamate, an effective repressor. The apparent initial T 1/2 for inhibition by mAIB in the presence of cycloheximide was 0.5 hours, while that for repression by glutamate was 4.7 hours. Half-maximal inhibition by mAIB and repression by glutamate occurred at approximately 0.02 mM and 0.07 mM, respectively. Reversal of trans-inhibition by methionine occurred in the presence of cycloheximide within 1-4 hours after removal of methionine. The A system of the transformed MDCK-T1 cells showed elevated activity, little capacity for derepression, resistance to repression by amino acids, but retention of sensitivity to trans-inhibition. Kinetic analysis of mAIB uptake indicated that the A system of MDCK-T1 cells has become kinetically more complex in a manner which resembled amino-acid-starved rather than amino-acid-fed MDCK cells. These results suggest that the A system of MDCK-T1 cells has become resistant to adaptive regulatory control.

The synthesis and processing of the 15S and 21S rRNAs have been studied in isolated yeast mitochondria. When mitochondrial transcripts were labeled with [alpha-32p]UTP in an incubation mixture containing 50 microM ATP, the transcripts from the genes for the large and small ribosomal RNAs accumulated in the form of putative precursor molecules. The labeled pre-21S rRNA was converted to mature 21S rRNA during a chase period in the presence of 1 mM ATP. Thus, the maturation of 21S rRNA, a process which includes trimming at the 3' end and, in omega+ strains, the excision of a 1.1 kb intervening sequence, can occur in isolated mitochondria and appears to be dependent on ATP. In contrast, the maturation of 15S rRNA by the removal of approximately 80 nucleotides from the 5' end of a 15.5S transcript is severely restricted in isolated mitochondria, even in the presence of 2.5 mM ATP.