Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells

Abstract

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.

ALL blasts drive primary mesenchymal stromal cells to increase asparagine availability during asparaginase treatment

“…The repertoire of amino acid transporters in human MSCs has been only recently evaluated in stromal cells from healthy donors. 40 Comparing the expression of several transporters for Asn and Gln in HD- and ALL-MSCs, we found that SLC38A5 , the gene that encodes for the transporter SNAT5, is significantly more expressed in mesenchymal cells from ALL patients. Consistently, Asn efflux is significantly faster in ALL-MSCs than in HD-MSCs, suggesting that SNAT5 is the carrier exploited for Asn secretion by stromal cells.…”

“…However, the pro-survival intercellular amino acid flux implies the activity of amino acid transporters. The repertoire of amino acid transporters in human MSCs has been only recently evaluated in stromal cells from healthy donors 40 . Comparing the expression of several transporters for Asn and Gln in HD-and ALL-MSCs, we found that SLC38A5, the gene that encodes for the transporter SNAT5, is significantly more expressed in mesenchymal cells from ALL patients.…”

Sodium-Dependent Neutral Amino Acid Transporter 2 Can Serve as a Tertiary Carrier for l-Type Amino Acid Transporter 1-Utilizing Prodrugs

“…Preincubation was done with 500 μL of prewarmed HBSS at 37 °C for 10 min before adding substrates (250 μL in HBSS) for the uptake experiments. The functionality of SNATs in MCF-7 cells was studied with a known SNAT substrate; the cells were incubated either with [ 3 H]- l -proline [1 μM (PerkinElmer, Waltham, MA, USA) in uptake buffer HBSS, 250 μL] at 37 °C for 1–60 min or with the uptake buffer (250 μL) consisting of 1–600 μM l -proline (containing 2 μCi [ 3 H]- l -proline) at 37 °C for 60 min . After incubation, the uptake was stopped by adding 500 μL of ice-cold HBSS, and the cells were washed two times with ice-cold HBSS (2 × 500 μL).…”

“…The functionality of SNATs in MCF-7 cells was studied with a known SNAT substrate; the cells were incubated either with [ 3 H]- l -proline [1 μM (PerkinElmer, Waltham, MA, USA) in uptake buffer HBSS, 250 μL] at 37 °C for 1–60 min or with the uptake buffer (250 μL) consisting of 1–600 μM l -proline (containing 2 μCi [ 3 H]- l -proline) at 37 °C for 60 min. 38 After incubation, the uptake was stopped by adding 500 μL of ice-cold HBSS, and the cells were washed two times with ice-cold HBSS (2 × 500 μL). The cells were then lysed with 500 μL of 0.1 M NaOH (60 min), and the lysate was mixed with 3.5 mL of Emulsifier safe cocktail (Ultima Gold, PerkinElmer, Waltham, MA, USA).…”

The Role of Amino Acids in the Crosstalk Between Mesenchymal Stromal Cells and Neoplastic Cells in the Hematopoietic Niche

“…Through glutamate, Gln sustains the synthesis of GSH (also fueled by cysteine/Glu exchange, see above) and several non–essential amino acids (NEAAs). Moreover, Gln is a precursor for nucleotides and glucosamine and behaves as a compatible osmolyte to maintain cell volume under hypertonic conditions ( Chiu et al, 2020a ). In addition, through its capability to interact with several active and exchange transporters, it is accumulated at high levels into the cell and can fuel by exchange the uptake of many NEAA and essential amino acids (EAAs).…”