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G G Guidotti

Publications and source records attributed to G G Guidotti.

At least 19 recordsLinked to original sources
PubMed1991

Energization of amino acid uptake by system A in cultured human fibroblasts.

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.

Amino Acids↗
PubMed1990

Phorbol esters stimulate the transport of anionic amino acids in cultured human fibroblasts.

The effect of phorbol esters on the transport of amino acids has been evaluated in cultured human fibroblasts. The activity of the Na(+)-dependent system XAG- for anionic amino acids is selectively and markedly stimulated by phorbol esters. The effect is maximal within 15 min; it is attributable to an increase in transport maximum (Vmax) and not prevented by inhibitors of protein synthesis. The half-maximal stimulation is observed at concentrations of phorbol 12,13-dibutyrate lower than 100 nM. Prolonged incubations in the presence of 1 microM phorbol 12,13-dibutyrate lower the binding of the ligand to its receptor with a loss of the stimulatory effect on transport. The results presented indicate that the stimulation of amino acid transport through system XAG- by phorbol esters requires the activation of protein kinase C.

Amino Acids↗
PubMed1990

The transport of L-glutamine into cultured human fibroblasts.

The transport of L-glutamine has been studied in diploid human fibroblasts in culture. Mathematical discrimination by nonlinear regression, competition analysis, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of this amino acid. The adopted criteria showed that L-glutamine enters the fibroblast by the Na(+)-dependent systems ASC and A and by a Na(+)-independent route identified as system L. The relative contribution of these agencies to the total saturable uptake of glutamine varied with the concentration of the amino acid and with the nutritional state of the cell. At amino acid concentrations approaching those encountered in human plasma: (1) system ASC represented the primary mediation for entry of L-glutamine in human fibroblasts; (2) the contribution of system A was lower, though significant, in unstarved repressed cells and became predominant in starved derepressed cells; (3) the Na(+)-dependent system L accounted for less than one-fifth of glutamine uptake in either nutritional condition. The changes in the relative contribution of the various systems to the uptake of glutamine as a function of its concentration may have implications in pathophysiology under conditions associated with enhanced glutamine concentrations in the extracellular fluids.

Amino Acids↗
PubMed1989

Influx of L-arginine is an indicator of membrane potential in human fibroblasts.

The net influx of L-arginine (JARG) was employed as an indicator of the membrane potential in human fibroblasts. Cell depolarization, obtained by increasing [K+]out, decreased both JARG and the net influx of the lipid soluble cation tetraphenylphosphonium (JTPP), a probe of membrane potential. JTPP, but not JARG, was influenced by the mitochondrial potential and exhibited a component dependent on intracellular and/or extracellular binding. JARG was sensitive to changes in the membrane potential induced by Na+-dependent transport of L-proline or by the activity of Na+-K+-ATPase. In the presence of 50 microM valinomycin, JARG was markedly influenced by the distribution ratio of K+ in a range of [K+]out from 1.5 to 100 mM. In this range of [K+]out, membrane potential (Em) varied from -90 to -23 mV, and calibration of JARG vs. the membrane potential yielded a linear relationship. These results indicate the following: 1) that the net influx of TPP+ is not a reliable indicator of membrane potential in cultured human fibroblasts; 2) that in the same cells the net influx of L-arginine can be employed as an index of membrane potential; 3) that in a range of Em from -23 to -90 mV the activity of system y+ (the membrane agency devoted to L-arginine transport in cultured human fibroblasts) exhibits no saturation of potential-dependent activation of transport.

Arginine↗
PubMed1987

Dependence of L-arginine accumulation on membrane potential in cultured human fibroblasts.

The cell-to-medium distribution ratios at steady state of L-arginine (RArg) and of the lipid-soluble cation tetraphenylphosphonium (RTPP) were studied as a function of the membrane potential (Em) in adult human fibroblasts. The relationship between RArg and Em was qualitatively similar to that of RTPP and Em. Quantitatively, RArg and RTPP differed in that 1) RTPP was much greater than RArg when Em was near zero, indicating a significant binding component in the uptake of TPP+ but not of L-arginine, and 2) after a correction for binding when Em is near zero, RTPP was still greater than RArg so that RT/F . ln RTPP exceeded RT/F . ln RArg by 10-25 mV. The pattern of the redistribution of accumulated TPP+ and arginine after an alteration of Em was identical. In null-point experiments, the external [K+] for which there were no changes in cellular TPP+ or L-arginine in the presence of high valinomycin (the null points) were very similar for the two probes. Em calculated from the null-point measurements (-70(-)-80 mV) was also very similar to RT/F . ln RArg and thus smaller than RT/F.ln RTPP. It was concluded that 1) there was an additional TPP+ binding as cellular [TPP] rose in response to more negative membrane potentials, 2) the transport system for L-arginine in these cells (system y+) operates as a facilitated diffusion system driven by the membrane potential, and 3) in some circumstances, L-arginine could be employed as a probe of Em.

Arginine↗
PubMed1986

Perturbation of Na+ and K+ gradients in human fibroblasts incubated in unsupplemented saline solutions.

Changes in the intracellular concentrations of Na+ and K+ of fetal human fibroblasts have been followed after replacement of serum-containing growth media with unsupplemented and serum-supplemented saline solution (Earle's balanced salt solution). Incubation in unsupplemented salt solution was followed by a progressive increase of the internal Na+ counterbalanced by a decrease of internal K+, without major alterations of the internal osmolarity. After 3 h incubation the intracellular Na+ and K+ concentrations were 120 mM and 50 mM, respectively. These intracellular ion derangements were not associated with a failure of the (Na+ + K+)-ATPase pump, whose activity actually increased with enhanced intracellular Na+ concentration. Ion changes did not take place when serum (in excess of 0.5%, final concentration) was present in the saline solution and a complete restoration to normal of the Na+ and K+ gradients occurred upon addition of serum to cells previously incubated in plain saline solution. The effects of serum were mimicked by furosemide, thus suggesting that channels sensitive to this diuretic are involved in the movement of Na+ and K+ following fibroblast incubation in unsupplemented saline solution.

Amiloride↗
PubMed1986

Effect of extracellular potassium on amino acid transport and membrane potential in fetal human fibroblasts.

The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K+. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), X-AG (for anionic amino acids), and y+ (for cationic amino acids). High doses (100 microM) of the K+-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y+. Transport systems L (for neutral amino acids) and x-C (for anionic amino acids) are insensitive to changes in extracellular K+ or to valinomycin. System X-AG is inhibited by the addition of 100 microM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x-C is potential-independent and (b) the activity of systems A, ASC, X-AG and y+ is sensitive to alterations of external [K+] associated to changes in membrane potential.

Amino Acids↗
PubMed1985

Effect of insulin on the activity of amino acid transport systems in cultured human fibroblasts.

The regulation of amino acid transport by insulin has been studied in cultured human fibroblasts. Among the six amino acid transport systems operating in cultured human fibroblasts, two systems (A and X-C) are strongly stimulated by insulin and four (ASC, X-AG, y+ and L) are essentially not sensitive to the presence of the hormone in the incubation medium. The hormonal stimulation of system A and system X-C became significant after 3 h of incubation and increased up to 12 h. The stimulatory effect was related to insulin concentration, with a half-maximal stimulation at 10(-9) M hormone concentration. Insulin enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in Km values.

Amino Acids↗
PubMed1984

Post-translational control by carrier availability of amino acid transport in fetal human fibroblasts.

The action of serum on the expression of the starvation-enhanced amino acid transport by System A (as a part of the adaptive regulation mechanism) has been studied in cultured fetal human fibroblasts. Serum enhanced L-proline uptake of cells starved in serum-free medium. This effect was rapid, proportional to the amount of pre-existing transporters, insensitive to cycloheximide and kinetically characterized by an increase of transport Vmax. These results can be interpreted to indicate that serum is essential for a vectorial post-translational event leading to insertion of transport proteins into the cell membrane.

Amino Acids↗
PubMed1983

Pathways of L-glutamic acid transport in cultured human fibroblasts.

The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax.

Aminoisobutyric Acids↗
PubMed1982

The transport of alanine, serine, and cysteine in cultured human fibroblasts.

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.

Alanine↗
PubMed1982

Effect of cell density on growth rate and amino acid transport in simian virus 40-transformed 3T3 cells.

Rate of proliferation and amino acid transport were assessed in simian virus 40 (SV40)-transformed 3T3 cells by measurements of growth rate quotient and L-serine uptake via System ASC, respectively. Growth rate and cell density of the cultures were varied by modifying: (a) the number of cells initially plated; and (b) the period spent by the cells in culture. The growth rate quotient of SV40 3T3 cells was not correlated with cell density. Sparse cultures exhibited marked fluctuations in their growth rate as a function of time, whereas, under comparable conditions, crowded cultures retained some form of growth control by density. Transport activity by System ASC decreased as a function of increased cell density following a complex trend described by a double-exponential equation. The density-dependent decrease in amino acid transport was not accompanied by a parallel change in the rate of cell proliferation. These results indicate that alterations in amino acid transport are not linked with cell growth and suggest that an increase in transport activity is not a prerequisite for an optimal rate of proliferation in SV40-transformed 3T3 cells.

Animals↗
PubMed1981

Adaptive regulation of amino acid transport in cultured human fibroblasts. Sites and mechanism of action.

The regulation of the transport of neutral amino acids across the cell membrane by adaptive mechanisms has been studied in cultured human fibroblasts. Among the three transport systems (A, ASC, and L) individually discriminated, only the Na+-dependent System A was subject to adaptive regulation, showing enhancement of its activity when the cells were incubated under conditions of amino acid shortage (derepression phase) and decrease of its activity when the cells were exposed to a medium supplied with Site A-reactive amino acids (repression phase). Starvation-induced derepression of transport activity and its reversal by amino acid refeeding required active RNA and protein synthesis. Derepression involved an early mRNA synthesis which started within 30 min from the abrupt change in extracellular amino acid concentration and apparently lasted 90 min. The transcribed mRNA was rather stable and translatable for a few hours (presumably into transport proteins) in cells maintained in the absence of amino acids. Repression by amino acid refeeding also involved an early mRNA synthesis, the product of its translation being presumably a protein capable of causing degradation or inactivation of transport proteins. The rate of decay in transport activity of previously derepressed cells was somewhat faster in the presence of added Site A-reactive amino acids than in their absence. A model is proposed in which the concentration of Site A-reactive amino acids affects transport activity of System A by modulating transcription of mRNA species coding for transport proteins and their putative inactivators and by regulating the efficiency of transport protein inactivation at the cell membrane.

Amino Acids↗