changed, and cells were maintained in fresh DMEM containing 20 m~ glucose ..... Nishino, H., Christopher, C. W., Schiller, R., Gammon, M. T., Ullrey, D., and.
Vol. 269,No.51,Issue of December 23. pp. 32098-32103.
THEJ o w u OF BIOLGGICM. CHEMISIRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed
1994
in U S A .
Glucose Deprivation and Acute Cycloheximide TreatmentStimulate System L Amino AcidTransport in Cultured Vascular Smooth Muscle Cells* (Received for publication,May 11, 1994, and in revised form, September 9, 1994)
Boon Chuan Low, Ian K. Ross, and Murray R. GrigorS From the Department of Biochemistry, University of Otago, Dunedin, New Zealand 9001
The effect of glucose deprivation on the uptake of complications that are also associated with diabetes mellitus leucine has been examinedin cultured vascular smooth (Alipui et al., 1993; Yorek and Winegrad, 1989; Brownlee and muscle cellsisolated from rat aortae. Equimolar substi- Cerami, 1984). Although prolonged hyperglycemia in diabetes complications tution of sucrose or fructose for glucose in the culture has been suggested tocontributetothese by which mechanism medium enhanced the uptake of leucine in a time- and (Ruderman etal., 19921, it is still unclear concentration-dependent manner. The effect was first it alters the metabolism in vascular cells and their functions. It has been recently shown that elevated glucose levels indetectable after 12 h and reached the maximum, 2-fold, after 48 h with an apparent half-maximal effectat 1mM crease the growth of porcine and rat VSMC by increasing total glucose and could be reversed after 48 h of glucose cellular protein and DNA synthesis (Natarajan et al., 1992; refeeding. Theenhanced leucine uptake was completely Koibuchi et al., 1992), the mRNA levels for both transforming inhibited by 2-amino-2-norbornane-carboxylic acid, a growth factora and basic fibroblast growth factorin rat VSMC specific substrate for SystemL, but not by a-(methylami-(McClain et al., 19921, and the expression and activity of 12no)isobutyric acid or lysine. Kinetic analysesindicated lipoxygenase in porcine VSMC (Natarajan et al., 1993). Howthat this stimulation was mediated via a homogenous ever, no studies have so far addressed the directeffect of glusystem with a 1.7-fold increase in the V,, without any cose availability on the transport of amino acids, which is a Prolonged treatments with potential regulatory point controlling vascular hypertrophyby change in theK,,, (0.15 m). cycloheximide (10 pg/ml) or actinomycin D (10 pg/ml) modulating the availability of precursors for protein synthesis. blocked this glucose deprivation effect and its reversal. Previously, we have characterized System L and System y+ However, cycloheximide also very rapidly stimulated amino acid transport activity in culturedrat VSMC (Low et al., leucine uptake, reaching the maximum, 2.5-fold over the 1993). System L mediates the uptake of nonpolar, branched basal at 1h. This effectoccurred at concentrations that chain andaromatic amino acidsand is also largely responsible matched its inhibition on protein synthesis (half-maxi- for the transportof nutritionally essential neutral amino acids, mal at 0.1 pg/ml) and could be reproduced with puromy-whereas System y+ mediates the flux of cationic amino acids. cin as well as actinomycin D. The stimulatory effect of Both of these systemscatalyze the exchange of substrate amino cycloheximide was also accompanied by an increase in acids across the cell membrane, and the apparent uptake rate the V,, but not in theK,,,, being sensitiveto 2-amino-2can be stimulated by the presence of substrate amino acids on norbornane-carboxylicacid inhibition only, and apthe opposite side of the membrane (trans-activation) (Lowet al., peared to occur in anadditive manner to thatof glucose 1993). Although these two systems are ubiquitously expressed deprivation. Although the uptake of leucine was stimuin many cell types (Christensen, 1990; Cheeseman, 19911, little lated by glucose deprivation and brief exposure to cycloheximide, these treatments had no effect on the ef- is known about theirregulation at a molecular level. Here, we flux of the substrate. These results are all consistent report studies on the regulation of one of these systems, System we present strongly suggest with the System L amino acids transport activity in cul- L. The biochemical and kinetic data L transport activity in cultured rat VSMC is under that System tured rat vascular smooth muscle cells being under the control of at least two non-hormonal regulatory mecha-the control of at least two non-hormonal regulatory mechanisms, one that is likely to involve a labile repressor nisms, one that is likely to involve a labile repressor molecule molecule and the other involving de nouo protein syn- and the otherinvolving CEe novo protein synthesisas a result of chronic glucose deprivation. The latter result contrasts with the thesis as a result of chronic glucose deprivation. The growth and proliferation of vascular smooth muscle cells (VSMC)l is a key feature in the development of essential hypertension (Connell, 1986; Olivetti et al., 1982; Owens and Schwartz, 1982) and atherosclerosis (Ross, 19861, two vascular *This work was supported in part by grants from theHealth Research Council of New Zealand and the University of Otago. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom all correspondenceshouldbeaddressed:Dept. of Biochemistry,University of Otago, P. 0. Box 56, Dunedin,New Zealand 9001. Tel.: 64-3-479-7840; Fax: 64-3-479-7866. The abbreviations used are: VSMC, vascular smooth muscle cells; BCH, 2-amino-2-norbornane-carboxylicacid; DMEM, Dulbecco's modified Eagle's medium; methyl-AIB,a-(methy1amino)isobutyricacid.
observations relating glucose availability and growth in these cells and shows that amino acid uptake and growth aredifferentially regulated insmooth muscle. EXPERIMENTAL.PROCEDURES aortae of 17-18-week-old male Wistar-derivedrats by enzymatic digestion and passaged weekly as previously described (Low et al., 1992). Briefly, replicatesof VSMC subcultures from passages 6-10 were plated at approximately 2 x 10' celldm1 andgrown to confluence ina humidified CO, incubator at 37 "C in 24-well plasticculture dishes containing Dulbecco's modified Eagle's medium (DMEM) with10%fetal calf serum. The cells were then made quiescent for2-3 days in serum-freeDMEM supplemented with0.05% (w/v) fatty acid-freebovineserumalbumin.Themediumwas then changed, and cells were maintainedin fresh DMEM containing 20 m~ glucose, fructose, or sucrose for times indicated in each legend before uptake of substrates was measured. Dunsport Assays-VSMC were washed twice with 0.5 ml of prewarmed sugar-free HEPES buffer (140 m~ NaC1, 0.5 rn KCl, 0.9 m~ Cell Culture-Cells wereisolatedfrom
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System L Amino Acid Dansport in Smooth Muscle Cells CaCI,, 1m MgCl,, 25 m HEPES, pH 7.4) and incubated in thisbuffer containing glucose, fructose, or sucrose for 3 h in the absence or presence of cycloheximide orother metabolic inhibitors. Previously, we have found that incubation of cells in amino acid-free buffer for3 h isSUEcient to reduce the intracellular concentrations of System L substrates to undetectable levels.2To measure leucine uptake, cells wereincubated in 0.25 ml of HEPES buffer containing 0.1 mM r3H1leucine(2 pCi/ml) for 1 min unless otherwise stated. Uptake was terminated by two rapid washes with ice-cold HEPES buffer, and cells were lysedin 0.5 ml of 5% (w/v) trichloroacetic acid.Total radioactivity in trichloroacetic acidsoluble fraction was counted in 6 ml of scintillant containing 4.5 g/liter diphenyloxazolein toluene/Tkiton X-100(3:l by volume). Similar assays were used to measure the uptake of ['Hllysine (4 min), ['Hlalanine (5 min), or r3H]histidine(5 min), each at 50 p (2 pCi/ml), or 2-deoxyglucose (10 min at 0.1 mM, 1 pCVm1). The times for these measurements were chosento be within the linear portions of the uptakecurves (data not shown). To correct for nonspecific uptake and binding, cells were incubated in parallel wells with buffer containing 100 mM of the appropriate substrates; the fraction of the radioactivity associated with the cells was determined, and this fraction was then subtracted from each data point. Eflm Studies-VSMC were incubated in HEPES buffer containing 50 1.1~r3H]leucine(2 pCi/ml) at 37 "C for 7 min and washed once very quickly (
