L-Lysine uptake in giant vesicles from cardiac ventricular sarcolemma: two components of cationic amino acid transport

Abstract

Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[(14)C]lysine uptake experiments were conducted within a 10(3)-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies. Two Na(+)-independent, N-ethylmaleimide-sensitive uptake components were found, one with high apparent affinity (K(m)=222+/-71 microM) and low transport capacity (V(max)=121+/-36 pmol/min per mg of vesicle protein) and the other with low apparent affinity (K(m)=16+/-4 mM) and high capacity (V(max)=4.0+/-0.4 nmol/min per mg of vesicle protein). L-Lysine uptake mediated by both components was stimulated by the presence of intravesicular L-lysine as well as by valinomycin-induced membrane hyperpolarization. Altogether, this behaviour is consistent with the functional properties of the CAT-1 and CAT-2A members of the system y(+) family of cationic amino acid transporters. Furthermore, mRNA transcripts for these two carrier proteins were identified in freshly isolated rat cardiac myocytes, the amount of CAT-1 mRNA, relative to beta-actin, being 33-fold larger than that of CAT-2A. These two transporters appear to function simultaneously as a homoeostatic device that supplies cardiac-muscle cells with cationic amino acids under a variety of metabolic conditions. Analysis of two carriers acting in parallel with such an array of kinetic parameters shows significant activity of the low-affinity component even at amino acid plasma levels far below its K(m).

Nitric oxide can acutely modulate its biosynthesis through a negative feedback mechanism on l-arginine transport in cardiac myocytes

“…These plasma membrane-bound carriers transport the L-enantiomers of arginine, lysine (L-Lys), and ornithine (L-Orn) with similar efficiency and in a Na ϩ -independent manner (8). We have recently found two L-Lys uptake components in cardiac sarcolemmal vesicles, with functional properties that are consistent with the activity of the high-affinity, low-capacity CAT-1, and the low-affinity, high-capacity CAT-2A members of system y ϩ (17). These transporters are likely to play a key role in the various metabolic pathways that involve L-Arg and L-Lys (18).…”

“…The uptake reaction was started by adding 100 l of a solution containing twice the final concentrations of L-[ 14 C]Lys and unlabeled L-Lys, in KCl-MOPS. Uptake was stopped at the desired times and vesicles were washed, radioactivity counted, and the amount of vesicle protein determined as previously described (17).…”

“…The rapid onset of inhibition suggests either a direct interaction between NO and protein residues involved in L-Arg binding/transport or transporter phosphorylation via NO-sensitive cGMP-dependent protein kinases (13,34). To distinguish between these possibilities, the effect of NO donors on cationic amino acid uptake was studied in giant sarcolemmal vesicles prepared from rat heart ventricles (16,17). The rate of NEM-sensitive L-[ 14 C]Lys uptake was measured in vesicles incubated with 0.05-50 mM L-Lys in the presence of 0, 10, 20, and 100 M SNAP.…”

“…The rate of NEM-sensitive L-[ 14 C]Lys uptake was measured in vesicles incubated with 0.05-50 mM L-Lys in the presence of 0, 10, 20, and 100 M SNAP. Radiolabeled L-Lys instead of L-Arg was chosen for these experiments as previously discussed (17). Giant sarcolemmal vesicles, loaded with and suspended in 140 mM KCl and 20 mM MOPS-KOH, pH ϭ 7.4, were incubated in the absence or presence of NEM for at least 10 min.…”

“…Instead, these cells must import L-Arg from the circulation to ensure adequate intracellular levels of this amino acid. Cationic amino acids appear to enter cardiomyocytes through the activity of a family of transporters known as system y ϩ (17,27,31). These plasma membrane-bound carriers transport the L-enantiomers of arginine, lysine (L-Lys), and ornithine (L-Orn) with similar efficiency and in a Na ϩ -independent manner (8).…”