br Conclusions br Acknowledgments The authors

Conclusions
Acknowledgments The authors are thankful to Hugo Santos, Olga Martinez and Ricardo Lacerda for the technical assistance provided in the maintenance of the aquaria. This work was supported by national funds, through FCT and co-funded by the European Regional Development Fund (ERDF) through the COMPETE – Operational Competitiveness Programme, under the project PSYCHOBASS (PTDC/AAG MAA/2405/2012 and FCOMP 01 0124 FEDER 027808) and UID/Multi/04423/2013. Virgínia Cunha was supported by FCT with a PhD grant (SFRH/BD/77931/2011).
Introduction The lungs are a site of action for many drugs [1] and a well established route of drug administration [2]. Preclinical data for certain transporters is now recommended for inclusion in new drug applications to licensing authorities [3]. There is therefore interest in the presence and function of transporters in the lung [4,5]. To date transporters in the lung have mainly been studied using gene expression (e.g. polymerase chain reaction [PCR]), immunometric and transporter functional assay methods. For the SLC22A gene family of transporters Horvath et al. [6] have shown OCTN1 and OCTN2 to be present in airway tissues and epithelial cells, from lung donors without preexisting lung disease, using quantitative RT-PCR, immunofluorescence and substrate inhibition assays. The same group has also shown OCT3 to be present in bronchial and vascular smooth muscle INCB3344 using similar methods [7]. Nakamura et al. [8] have also shown OCTN1 and OCTN2 to be present in human bronchial epithelial BEAS–2 B cells using RT-PCR. Data obtained in-house (unpublished work) at GlaxoSmithKline (GSK) indicates significant genomic expression of transporters from this gene family in human lung tissue, with a rank order of OCT3 > OCTN1 > OCTN2, and no significant expression of OCT1 or OCT2. In contrast Bleasby et al. [9] have indicated a rank order of mRNA expression of OCTN1 > OCTN2 > OCT3 > OCT1, with no significant expression of OCT2. For the SLCO gene family of transporters in-house gene expression data (unpublished work) in human lung has indicated noteworthy expression with a rank order of OATP2A1 > 2B1 > 4C1 > 3A1 > 4A1, with OATP1A2 not expressed. Bleasby et al. [9], in contrast, have found the order to be OATP2A1 > 3A1 > 2B1 > 4A1 > 4C1 > 1A2. PEPT2 is the SLC15A2 gene transporter and has been determined by various groups using genetic analysis to be expressed in the lung (GSK [unpublished work]) [9,10]. Takano et al. [11] have shown functional expression of the transporter in the human distal lung epithelial cell line NCI-H441 (H441). Groneberg et al. [12] used immunohistochemical and ex vivo uptake studies to show the presence of PEPT2 in human airway tissue. P-gp (MDR1) (ABCB1 gene transporter), an ATP-binding cassette (ABC) transporter, is found throughout the body [13], including in the lung [4] where its presence has been determined using quantitative RT-PCR [5,13,14]. Lechapt-Zalcman et al. [14], using an immunohistochemical method, found it to localise on the apical surface of airway epithelial cells. BCRP (ABCG2), also an ABC efflux transporter, has also been found to be present in the lung using quantitative RT-PCR [5,13]. Like P-gp, it is widely distributed throughout the body [13]. MRP9 (ABCC12) was observed in the Bleasby et al. [9] study at high levels but was absent in the GSK analysis of lung mRNA levels (data not shown). Langmann et al. [13] reported low expression of MRP9 in many tissues including the lung. Berg et al. [5] also found the transporter to be very low or absent in healthy lung. One comprehensive study employing targeted isotope dilution quantitative proteomics with LC–MS/MS for the quantification of transporters in frozen lung has been reported [15]. Many of the transporters mentioned above were quantified in that study. The same group subsequently used their method to quantify transporters in various immortalized human lung cell lines [16] and in a study also employing OCTN1 and MRP1 activity assays with primary cultured human lung cells [17]. Our study, which uses fresh tissue and a less complex sample digestion preparation method, producing a crude membrane fraction, is complimentary and confirmatory to the above studies and provides an interlaboratory comparison of lung transporter quantification by targeted proteomic isotope dilution LC‐tandem mass spectrometry.