Alveolar Macrophages from Normal Subjects Lack the NOS-Related System y⁺for Arginine Transport

Abstract

Systems y+ and y+L represent the main routes for arginine transport in mammalian cells. While system y+ activity is needed for the stimulated NO production in rodent alveolar macrophages (AM), no information is yet available about arginine transport in human AM. We study here arginine influx and genes for arginine transporters in AM from bronchoalveolar lavage of normal subjects. These cells express the y+ -related genes SLC7A1/CAT1 and SLC7A2/CAT2B, as well as the y+L genes SLC7A7/y+LAT1 and SLC7A6/y+LAT2. However, compared with human endothelial cells, AM express much less SLC7A2 mRNA and higher levels of SLC7A7 mRNA. Granulocyte macrophage colony-stimulating factor or IFN-gamma do not change the expression of any transporter gene, while lipopolysaccharide induces SLC7A2/CAT2B. Under all the conditions tested, leucine inhibits most of the arginine transport in the presence of Na+ and N-ethylmaleimide, an inhibitor of system y+, is completely ineffective, indicating that system y+L operates most of the arginine influx. Comparable results are obtained in AM from patients with interstitial lung disease, such as Nonspecific Interstitial Pneumonia (NSIP), although these cells have a higher SLC7A1 and a lower SLC7A7 expression than AM from normal subjects. It is concluded that AM from normal subjects or patients with NSIP lack a functional transport system y+, a situation that may limit arginine availability for NO synthesis. Moreover, since mutations of SLC7A7/y+LAT1 cause Lysinuric Protein Intolerance, a disease often associated with AM impairment and alveolar proteinosis, the high SLC7A7 expression observed in human AM suggests that y+LAT1 activity is important for the function of these cells.

Arginine transport in human monocytic leukemia THP-1 cells during macrophage differentiation

“…9). These results are in agreement with previous data from our group, demonstrating that y ϩ LAT1 is the transporter mainly active in cells of monocytic lineage [17,18] and with a recent report documenting that in differentiated human blood cells, SLC7A7 expression is restricted to monocytes [36].…”

“…For expression studies, total RNA from 1 ϫ 10 6 cells was isolated with the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich, Milano, Italy) and reverse-transcribed as described previously [18]. cDNA (30 ng) from each sample was amplified in a total volume of 25 l with Mesa Green qPCR 2ϫ Master Mix Plus (Explera s.r.l., Jesi, Ancona, Italy), along with the transporter genes and GAPDH or RPL15 primers described previously [18,20].…”

Inhaled Sargramostim Induces Resolution of Pulmonary Alveolar Proteinosis in Lysinuric Protein Intolerance

“…Toll-like receptor signalling, especially a response to viral DNA, has been shown to be impaired, and nitric oxide (NO) production decreased in LPI monocyte-derived macrophages reinforcing the role of macrophages in the development of secondary complications of LPI (Kurko et al 2015). We hypothesized that, in contrast to the earlier suggestions by other groups (Sebastio et al 2011;Ogier de Baulny et al 2012), arginine reservoirs and the subsequent NO levels may actually be diminished in LPI macrophages as a result of reduced arginine influx by the defective y + LAT1 transporter, known to be the most important transporter of arginine in macrophages, including those of alveolar origin (Barilli et al 2011;Rotoli et al 2007). In addition, the defective CAA transport also results in impaired phagocytic activity of LPI macrophages (Barilli et al 2012).…”

Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles

“…The pellet was then washed with urea and extracted with 200μl of 5% sodium deoxycholate in 1 N NaOH. After 1 h, 100μl of the cell extracts were used for radioactivity determination with a Wallac Trilux 2 liquid scintillation spectrometer (Perkin Elmer, Wellesley, MA, USA), while the remaining aliquot was used for protein determination with a modified Lowry procedure (see [27] for details). A modified EBSS, in which N-methyl-D-glucamine chloride substituted sodium chloride [28], was used for sodium-free determinations.…”