Herein we employ an adapted targeted quantitative proteomic

Herein we employ an adapted targeted quantitative proteomic isotope dilution method, developed in our laboratory [[18], [19], [20], [21]], to quantify membrane transporters in fresh lung from five donors, in four immortalized human lung cell lines and in three control cell lines. We quantify twelve transporters, namely OCT2 (SLC22A2), OCT3 (SLC22A3), OCTN1 (SLC22A4) and OCTN2 (SLC22A5) from the SLC22A cationic uptake transporter family, OATPs (SLCOs) 1A2, 2A1, 2B1 and 4C1 from the SLCO organic anion transporting polypeptide family, the SLC15A oligopeptide transporter PEPT2 (SLC15A2) and three ABC transporters P-gp (ABCB1), BCRP (ABCG2) and MRP9 (ABCC12). The transporters were selected based on the gene expression and immunohistochemical information presented above and on other observations which included 1) in vitro or preclinical data showing involvement in the transport of inhaled drug molecules, 2) known involvement in clinically relevant drug transport and drug–drug interactions in other parts of the body and 3) potential target transporters for respiratory molecules yet to be developed or investigated. MRP9 was included primarily as a negative control.
Materials and methods
Results and discussion We have successfully evaluated transporter protein expression in five fresh human lung samples and four human immortalized lung epithelial cell lines. Our method involves first isolating pexidartinib from tissue samples by homogenization, filtration and centrifugation and from cell line cultures by trypsinization and centrifugation. Membrane fractions of cells are then prepared by lysis, homogenization and differential centrifugation and analyzed by a modified targeted quantitative proteomic isotope dilution nanoLC–MS/MS method previously developed in our lab [[18], [19], [20], [21]]. Representative total and extracted ion chromatograms for a ‘Lung 3′ membrane sample digestion (1st of five replicates) are shown in Fig. 1. The extracted chromatogram shows a low amount of the OATP4C1·LGSLFEDTCVTTR quantified.
Conclusion We have quantified transporters in five fresh human lung membrane fraction samples and four immortalized human lung epithelial cell membrane fraction samples using isotope dilution nanoLC–MS/MS targeted quantitative proteomics. Our results can be compared to those obtained by another group [[15], [16], [17]], who used frozen lung samples, and to semiquantitative gene expression data in the literature. We found the OATP transporters to be the most highly expressed in the lung, followed by PEPT2 and the ABC transporters BCRP and P-gp. SLC22A uptake transporters could also be detected. OATP2A1 was the transporter of highest concentration with measurements markedly lower in two donors that were at least occasional smokers. This is perhaps associated with prostaglandins and the inflammatory response (OATP2A1 is also known as the prostaglandin transporter [22,23]). The highest OATP2A1 concentration determined was 69 pmol/mg protein, approximately 8.4 times higher than the next highest OATP2A1 concentration, which itself was higher than the concentration of any other transporter. The results inform the understanding of xenobiotic disposition in the lung and show that the lung has a distinct profile of transporters compared to other tissues such as liver, with considerable variability across individuals.
Notes
Acknowledgements Funded by GlaxoSmithKline and in part by NIH instrumentation grant, S10, RR024595.
Introduction Maintaining a physiological level of extracellular glutamate, a major excitatory amino acid neurotransmitter in the spinal dorsal horn, is key to preventing excitotoxicity and neurotoxicity produced by excessive activation of its receptors, which can occur under many pathological conditions (Danbolt, 2001). Regulation of the glutamate concentration in the synaptic cleft is carried out primarily by an efficient, high-affinity glutamate transporter localized in the cell membranes of glia and neurons (Mao, 2007). Thus, spinal glutamate transporter inhibition can lead to decreased uptake of and then excessive accumulation of glutamate in the synaptic cleft, which may, in turn, result in over-activation of glutamate receptors, producing a pronociceptive effect under normal conditions (Liaw et al., 2005).