The MDR1 (ABCB1) gene is located on chromosome 7q21.12 and encodes for P-gp. P-gp plays a role in cell defence against environmental attacks such as generated by xenobiotics. It transports many hydrophobic substrates and anti-cancer drugs including etoposide, doxorubicin and vinblastine (for review, see 11) and is mainly apically expressed in organs involved in excretion such as liver and intestine. In the lung, P-gp is expressed at the apical side of ciliated epithelial cells or ciliated collecting ducts, and on apical and lateral surfaces of serous cells of bronchial glands but not in mucus-secreting goblet cells (Figure 1 and 2) (Table 1) 12. Epithelial cells of the trachea and major bronchi stain strongly while staining of the smaller bronchi is patchy or absent. In addition, P-gp is present in the lateral membranes of normal nasal respiratory mucosa 13. In human and rat type I alveolar epithelium, P-gp is located at the lumenal side whereas freshly isolated type II cells lack P-gp 14. In another study, pneumocytes did not stain for P-gp 15. Some antibodies visualised P-gp in endothelial cells of blood vessels (Figure 1) 121516. In addition, alveolar and peripheral blood monocyte-derived macrophages stained positive but variably for P-gp 41617. The observed apical epithelial expression may signify that P-gp plays a role in transport of compounds from the interstitium into the lumen. However, the precise function of P-gp in the lung is as yet unknown. Interestingly, P-gp was found to play a role in the regulation of cell volume activated chloride channels and to possess channel activities 1819. However, Cl^- and K⁺ conductances were not affected by the level of P-gp expression and the physiological role of P-gp in volume-activated chloride currents is still unclear 202122. Many lipophilic amines, such as fentanyl, highly accumulate in the lung. It was suggested that P-gp plays a role in the disposition of these pulmonary amine drugs 23. Cellular uptake in lung microvascular endothelial cells of fentanyl was inhibited by the P-gp substrate verapamil, but not by the P-gp blocking antibody UIC2. The authors suggested that this results from an inhibition of fentanyl uptake. This is however unlikely, since P-gp is primarily responsible for removal of drugs out of cells.

P-gp mRNA expression was not increased in smokers (n = 11) compared to ex-and non-smokers (n = 7) 12. Whether P-gp expression levels may play a defensive role towards tobacco-derived agents remains to be investigated.

High P-gp expression can imply chemotherapeutic resistance due to increased chemotherapeutic drug efflux. In cancer therapy, many attempts have been made to reverse MDR mechanisms. However, in a randomised double-blind trial in 130 SCLC patients no positive effects were seen with the P-gp modulator megestrol acetate in addition to chemotherapeutic drugs, suggesting that levels of P-gp expression in lung tumours were not relevant or that modulation of P-gp activity was not complete in this treatment 24. Some studies show higher P-gp expression at the invasion front of lung tumours and it was suggested that P-gp expression augments invasion properties of tumour cells 25. Only two out of 22 NSCLC samples (both adenocarcinomas) stained positive with three P-gp antibodies 15 and no P-gp was detected on pulmonary carcinoids. Other studies revealed a relation between P-gp and glutathione S-transferase-pi (GST-pi) expression in NSCLC that were exposed in vitro to doxorubicin 26, suggesting that these two factors play a role in doxorubicin resistance. There was also a correlation between current smoking and doxorubicin resistance of NSCLC. Forty-two out of 72 NSCLC smokers expressed P-gp, whereas only two out of 22 tumours of non-smokers were P-gp positive 27.

MDR1 polymorphisms were first described by Hoffmeyer et al. 28 who found a correlation between lower intestinal expression of P-gp and a polymorphism in exon 26. Many single nucleotide polymorphisms (SNPs) have been recognised in the MDR1 gene (see reference 29 for recent review about clinical aspects). The impact of these polymorphisms on lung diseases is still speculative. It was proposed that polymorphisms in the MDR1 gene may have clinical consequences in patients with cystic fibrosis, since MDR1 plays a role in CFTR regulation. Rodents contain two Mdr1 genes, denoted as Mdr1a and Mdr1b. It was shown that Mdr1b mRNA expression in lung parenchyma of outbred rats is very variable and this may also be the case in humans 30. The possible effects of MDR1 polymorphisms was studied in tobacco-related lung cancer 31. No clear association was found between the T/T genotype of the C3435T polymorphism and susceptibility to lung cancer in a group of 268 Caucasian men who were current smokers. No relation was found between SNP C3435T in MDR1 and survival in 62 docetaxel-cisplatin-treated NSCLC patients 32. Immunosuppressive agents such as cyclosporin A and tacrolimus (both calcineurin antagonists) are P-gp substrates. No relation was found of MDR1 G2677T and C3435T genotypes with tacrolimus blood levels in 83 lung transplant patients treated with tacrolimus 33. Altogether, these data implicate that there is still no clear association between MDR1 polymorphisms and effects on outcome of treatment of lung cancer or lung transplant patients.

Scheffer et al. detected high P-gp levels in lungs of mice 4. In rats, Mdr1a and Mdr1b mRNA expression were highest in the ileum 34. The Mdr1a expression level in rat lung was 2% of the expression in ileum and expression of Mdr1b was 47% of that in ileum. In mice orally treated with dexamethasone for 24 hours, Mdr1b mRNA expression in lungs was decreased, from which the authors deduce that dexamethasone treatment of lung tumours may reverse MDR 35. To study the in vivo distribution of P-gp, nude rats were injected with a P-gp overexpressing SCLC cell line (GLC4/Pgp) and with a P-gp negative cell line (GLC4) 36. P-gp function was visualised with radiolabeled P-gp substrate [¹¹C]verapamil by positron emission tomography (PET) with or without P-gp modulator cyclosporin A. The accumulation of [¹¹C]verapamil was significantly increased by cyclosporin A in brains and GLC4/Pgp tumours in these rats. In all other investigated organs including lungs, the accumulation after cyclosporin A treatment was unaltered. In intact rabbit lung, vascular P-gp kinetics were measured in vivo using the lipophilic amine dye rhodamine 6G (R6G) by measuring R6G in the perfusate during circulation 37. Inhibition of P-gp function with verapamil or GF120918 resulted in higher accumulation of R6G in lung. It was proposed that the opposite would happen when epithelial P-gp was inhibited because R6G would then be retained in the airspace. We propose another possibility that inhibition of epithelial P-gp will also result in higher R6G accumulation. In that case, R6G transport to the lumen is inhibited and as a compensation mechanism it may be transported back to the interstitial side where it either may be retained in the tissue or subsequently be transported into the circulation. This model could be useful in testing a large variety of pulmonary therapeutic agents, such as corticosteroids and sympathicomimetics that may be substrates for transporters in the lung or modulate their activity. Similar studies were also carried out in perfused rat liver to assess the effect of P-gp modulators on the hepatobiliary system, supporting the usefulness of this approach 38.

Mdr1a/1b (-/-) mice lack both genes encoding for P-gp and these mice seem physiologically normal. The penetration of [³H]digoxin (a P-gp substrate) was higher in brain, ovary and adrenal gland. In the lung tissue of these (-/-) mice 39, the level of [³H]digoxin was rather low compared to other organs. The level was 2.6 times higher in (-/-) mice than in (+/+) mice, but this was not significant. The pharmacokinetics of the central nervous system (CNS) drug amitryptiline (a P-gp substrate) and its metabolites were examined in Mdr1a/1b (-/-) mice by high performance liquid chromatography (HPLC) in several organs. Higher concentrations of these metabolites were measured in the brain of these mice, but not in other examined organs including the lung 40. Thus, P-gp likely plays a more active role in exporting CNS drugs out of the brain than out of the lung. Another possibility is that other transporters in the lung were capable to efflux these drugs as a compensation mechanism.

Several lung cell culture models have been described for determining P-gp expression and functionality. The immortalised human bronchial epithelial cell line 16HBE14o^- resembles primary epithelium and was reported to be suitable for drug metabolism studies 41. In 16HBE14o^- cells, P-gp was expressed at the apical side. Its functional activity was measured with P-gp substrate rhodamine 123 and its transport was inhibited by verapamil 42. Two lung cell lines, Calu-3 and A549 cells, were compared for P-gp expression and functionality 43. The bronchiolar adenocarcinoma cell line Calu-3 is suitable for drug transport studies because these cells form tight junctions in culture, which is not the case for the type II alveolar carcinoma cell line A549. P-gp expression was higher in A549 cells than in Calu-3 cells. However, efflux of rhodamine 123 was higher in Calu-3 cells. This may be explained by additional MRP1 activity in these cells because rhodamine 123 is also an MRP1 substrate 44. In primary rat alveolar type II cells, Mdr1b mRNA levels increased in a time dependent manner in cultures at day 1, 2 and 3 compared to freshly isolated cells. Mdr1b mRNA was present at low levels and increased after oxygen radical induction with paraquat 45. P-gp expression was below the detection limit in these cells at time of isolation. In freshly isolated primary human bronchial epithelial cells, P-gp was present and increased after 24 hours paraquat exposure. Rhodamine 123 efflux could be measured in these cells, confirming functional activity of P-gp 45. These results demonstrate that P-gp is upregulated during stress, both from radical production and from ex vivo culturing or differentiation.

The MRP1 (ABCC1) gene is located on chromosome 16p13.12. Cole et al. discovered in 1992 a non-Pgp mediated MDR mechanism in the human lung cancer cell line H69AR 46. MRP1 was overexpressed in these cells and it was found that glutathione-, glucuronide-, and sulfate-conjugated organic anions are substrates for MRP1 4748. MRP1 confers resistance to several chemotherapeutic agents including vincristine, daunorubicin and methotrexate 495051. Physiological substrates for MRP1 are e.g. leukotriene C₄ (LTC₄) and glutathione disulfide (GSSG) 52. Interestingly, these substrates play an important role in lung physiology with respect to inflammation and oxidative stress. MRP1 is highly expressed mainly at the basolateral side of human bronchial epithelial cells (Figure 1 and 2) 53. Ciliated and basal cells have been collected from brushes of main or lobar bronchi. Basal cells stained strongly on the entire circumference of the plasma membrane. Ciliated epithelial cells and mucous cells stained positive at the basolateral membrane but not at the apical membrane. No intracytoplasmic staining was observed. However, strong apical cytoplasmic staining has been detected below the cilia in respiratory columnar epithelial cells in paraffin sections with antibody MRPr1 41354. The discrepancy between these findings may be due to different fixation procedures. Basal cells of seromucinous glands of the lungs also stain positive for MRP1 with a higher intensity in the serous area than mucinous cells. Alveolar macrophages are MRP1 positive with variable staining between individual lung samples (Figure 2) 4. Kool et al. investigated MRP1, 2, 3, 4, and 5 with an RNase protection assay in total lung RNA and found high MRP1, absence of MRP2, low MRP3 and MRP4 and moderate MRP5 gene transcripts 55. Total RNA was obtained from lung tissue collected during surgery or autopsy. Therefore, the original cell types can not be distinguished in these samples.

Possibly, the high MRP1 expression at the basolateral side of lung epithelium may assist in the clearance of toxins coming from the luminal or interstitial side (back) into the interstitial fluid 56. Function of MRP1 expression in the lung epithelium, glands and alveolar macrophages may include extrusion of toxic intracellular substances, antioxidant defence or production of LTC₄ as an inflammatory response. Given the fact that MRP1 expression is higher in the lung compared to other solid organs, decreased or increased functional MRP1 expression may have a high impact on development and/or progression of lung diseases and protection against air pollution and inhaled toxic substances such as present in cigarette smoke 10.

Using mRNA in situ hybridisation Thomas et al. 57 detected high MRP1 expression in normal epithelium whereas the major component of the tumour epithelium showed a negative hybridisation signal. However, MRP1 transcript expression at the invasive front of the lung tumours was consistently stronger and particularly strong in areas with lymphatic or blood vessels. In addition, endothelial cells and lymphocytic infiltrates stained strongly positive for MRP1. These results may indicate a role of MRP1 in invasion or in mitotic activity. In a recent study, all 102 NSCLC tumours were MRP1 positive. In addition, the level of expression was 3-fold higher in DNA-aneuploid cells compared to normal bronchial and carcinomatous DNA-diploid cells 58. This was associated with more frequent gain of chromosome 16 where the MRP1 gene is located. Overexpression of MRP1 may therefore be an important factor of intrinsic resistance to chemotherapy in NSCLC. High MRP1 expression can also result in increased apoptosis as shown by experiments with verapamil in MRP1 transfected baby hamster kidney-21 (BHK-21) cells 59. Verapamil acts as an apoptogen in these cells when compared to MRP1 negative cells or mutant MRP1 transfected control cells. This was accompanied by depletion of intracellular GSH due to transport of GSH by MRP1. Indeed, addition of extracellular GSH prevented cell death. This mechanism may be valuable for treatment of MRP1-positive tumours.

Cigarette smoking is the principle risk factor for the development of COPD. Our preliminary results indicate that MRP1 expression is diminished in bronchial epithelium of COPD patients (ex-smokers) and that lower expression is related to worse lung function 10. In addition, bronchial MRP1 expression was higher during smoking than after one year of smoking cessation (Van der Deen et al., submitted). The expression of MRP1 measured with RT-PCR was similar in lungs of smokers and a combined group of ex- and non-smokers, suggesting that current smoking does not affect MRP1 gene expression 53. This was semi-quantitatively analysed and the power of this study was rather low (smokers n = 13 and ex- or non-smokers n = 8). Cigarette smoke extract is a complex mixture of many substances. One of these is nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic nitrosamine. NNK is converted intracellularly in NNAL-O-glucuronide which is transported by MRP1 in the presence of glutathione (GSH) 60. Thus, functional activity of MRP1 in the lung may play an important role in the antioxidant defence against toxic compounds generated by cigarette smoke.

Polymorphisms of the MRP1 gene or in regulatory genes may influence function of MRP1 in the lung. MRP1 has been screened for genetic variations and several mutations have been found in the MRP1 gene in the human population 6162. Ethnic differences for MRP1 expression were observed between Caucasian and Japanese subjects but the clinical consequences have to be determined to date 6364. In preclinical models, a single mutation in the MRP1 gene can result in loss of transport of some, but not all, MRP1 substrates 65 which implies that potentially minor differences in the MRP1 gene may result in aberrant functional capacities of the MRP1 protein. A low frequency (<1%) naturally occurring mutation in MRP1 was related to functional differences in organic anion transport and drug resistance 66. Therefore, some individuals could be more susceptible to a set of xenobiotics than others.

Scheffer et al. detected, besides P-gp, high levels of Mrp1 in lungs of mice 4. Mrp1 (-/-) mice develop normally 67. Viability, fertility, and a range of histological, hematological, and serum-chemical parameters were similar in Mrp (+/+) and Mrp (-/-) mice. However, Mrp1 (-/-) mice are hypersensitive to exposure to several drugs e.g. etoposide, resulting in loss of body weight and death 566768. In Mrp1 deficient mice there were no gross abnormalities in lungs and other tissues after treatment with the chemotherapeutic drug etoposide-phosphate, but abnormal mucous production was seen around the mouth. Further examination of tissues showed that these mice suffered from oropharyngeal toxicity 56. In Mrp1 (-/-) mice, GSH levels were elevated in e.g. lung, kidney, heart, testes and skeletal muscle. In organs that express little MRP1, such as the liver and small intestine, GSH levels were unchanged 6769. The increase in GSH was not related to increased levels of gamma-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme for production of the tripeptide GSH. This suggests that MRP1 plays a role in GSH metabolism because MRP1 expression determines cellular GSH levels which is γ-GCS independent. In liver tissue, Mrp2 and Mrp5 mRNA levels were increased in Mrp1(-/-) mice compared to wild-type mice, probably due to a compensation mechanism. Mdr1a and Mrp3 levels were unchanged in these animals. Unfortunately, lung tissue was not examined 69. Mrp1 (-/-) mice showed impaired inflammatory responses accompanied by a decreased LTC₄ excretion by leukotriene producing cells 6768. The outgrowth of Mycobacterium tuberculosis was enhanced in Mrp1 (-/-) mice, but there was no difference in survival compared to wild-type mice 70. Strikingly, Schultz et al. 71 observed that the survival of Mrp1 (-/-) mice inoculated with Streptococcus pneumoniae was better than of wild-type mice. This was accompanied by a lower LTC₄ concentration but a higher LTB₄ level in bronchoalveolar lavage fluid (BALF). Treatment with an LTB₄ antagonist abolished the positive effect on survival rate. Altering MRP1 function may therefore be a target for further studies on treatment of pneumonia. Interestingly, LTB₄ levels are also elevated in sputum and exhaled breath condensate of COPD patients 7273. This is thought to result from a higher amount of neutrophils present, yet also lower functional MRP1 expression may play a role, according to the observations in Mrp1 (-/-) mice 1071. The pulmonary and hepatic carcinogen aflatoxin B₁ (a mycotoxin) and its GSH conjugate are MRP1 substrates in vitro 74. MRP1 may therefore play an important role in detoxification of aflatoxin B₁ in the lung. No difference in occurrence of lung tumours (and liver tumours) 12 months after an 8 week exposure to aflatoxin B₁ was observed in Mrp1 (-/-) and wild-type mice 75. This observation may be explained by differences in exposure time compared to humans (who may be chronically exposed for many years) or by redundancy of other ABC transporters in knock-out mice to export aflatoxin B₁ and its conjugates.

NRF2 (nuclear factor-E2 p45-related factor), a transcription factor for many genes that play a role in antioxidant defence and detoxification processes, was recently identified as a transcription factor for MRP1 76. Interestingly, the onset of cigarette smoke-induced emphysema was earlier and the extent of emphysema was more severe in Nrf2 (-/-) mice as compared to wild-type mice 77. The number of inflammatory cells, mainly consisting of macrophages, in bronchoalveolar lavage fluid and lung tissue was higher in Nrf2 (-/-) mice, accompanied by more extensive apoptosis of endothelial cells and alveolar type II cells. Forty-five Nrf2-dependent protective genes were induced by smoke exposure in wild-type mice compared to knock-out mice as measured with microarray analysis. Regretfully, Mrp1 was not present on this gene expression array. Similar smoke exposure experiments with Mrp1 (-/-) mice would be interesting to investigate if these mice are also more susceptible to develop emphysema. Three polymorphisms were identified in the promotor region of NRF2 in healthy individuals (n = 81) and in patients with COPD (n = 87) and sytemic lupus erythematosis(n = 51) (all Japanese individuals) 78. There was no relation between these polymorphisms and risk of these diseases, but the power of this study was rather low.

Mdr1a/1b(-/-)/Mrp1(-/-) triple knockout (TKO) mice are indistinguishable from wild-type mice under normal physiological conditions 7980. Intravenous injection of [³H]etoposide in TKO mice resulted after 4 hours in a higher accumulation in brown adipose tissue, colon, urogenital tract, salivery gland and heart when compared to Mdr1a/1b (-/-) mice. The accumulation of [³H]etoposide in lung did not increase 79. Accumulation of [³H]vincristine was 26-fold higher in lungs of weaning TKO mice compared to adult TKO mice, 4 hours after a single dose intraperitoneal injection 81. In TKO compared to wild-type mice, [³H]vincristine was 82.7 and 5.8-fold increased in respectively weaning and adult mice after 4 hours in this experimental setting. Eight hours post-injection, these ratios were 33 and 51.1. Thus, accumulation of vincristine is both time and age-related. In another study, toxicity of the chemotherapeutic drugs vincristine and etoposide was elevated in TKO mice (respectively 128-fold and 3–5 fold) compared to wild-type mice. Lung tissue was not further examined 80.

MRP1 overexpression was first described in the human lung cancer cell line H69AR 46. The human SCLC cell line GLC4/ADR, which displays multiple copies of the MRP1 gene, is often used to study MRP1 function 47. The lung cell line Calu-3 (from bronchiolar adenocarcinoma/glandular origin) has been reported to express MRP1 basolaterally and has functional MRP1 activity. It may serve as a good model for in vitro analysis of transport activities 44. Besides P-gp, MRP1 protein was present in freshly obtained primary human bronchial epithelial cells. Efflux of MRP1 substrate carboxy-dichlorofluorescein could be measured in these cells and was inhibited by MRP modulator MK571 45. In contrast to upregulation of P-gp, epithelial cells did not respond with MRP1 upregulation to paraquat and MRP1 levels were stable in time until 12 weeks, but highly increased after 18 weeks culturing. An interesting observation was made by Bandi et al. 82 who incubated the airway epithelial cell line Calu-1 with budesonide, an anti-asthma drug. MRP1 expression decreased after 7 and 14 days incubation with budesonide (10 μM) but not at day 1 and 4. MRP1 function was also diminished after 14 days incubation with increasing amounts of budesonide (1, 10 and 100 μM). It was therefore proposed that budesonide could be used as a chemosensitiser in lung tumours. The immortalised bronchial epithelial cell line 16HBE14o^- expresses high MRP1 protein levels and activity 83. MRP1 activity, but not P-gp, was blocked by incubation with cigarette smoke extract. Thus, the expression of MRP1 in human lungs may contribute to defence mechanisms of toxic inhaled substances present in cigarette smoke to protect against pulmonary diseases such as lung cancer or COPD. It was shown that benzo [a]pyrene (B [a]P), a polycyclic aromatic hydrocarbon that is a.o. present in cigarette smoke and car exhaust fuels, increased the efflux of monochlorobimane GSH conjugate (mBCl-SG, which is an MRP substrate) out of primary rabbit alveolar type II cells 84. This was explained by an increase in GSH levels. MK571 reduced the efflux of mBCL-SG, suggesting that the transport was MRP-mediated. Surprisingly, the transport direction of this substrate was higher to the apical than to the basolateral compartment. This may imply that MRP1 (or other MRPs) is apically located in alveolar type II cells in contrast to the basolateral location that is usually reported for MRP1, or that another transporter is responsible for this unexpected result. MRP1-5 expression was studied in primary human lung cell cultures and in A549 cells 85. MRP1 and MRP3 expression were membrane-associated whereas MRP2, 4, and 5 were located in intracellular structures in primary cells (both bronchial and peripheral epithelial cells). In A549 cells, all transporters were expressed in the cellular membrane. The fluorescent microscopic pictures, however, did not precisely show the exact cellular localisation and in several cases, the Golgi apparatus also seems to stain beside the cellular membrane.

MRP2 (ABCC2) is located on chromosome 10q24.2 and is also named the canalicular multispecific organic anion transporter (cMOAT) in the liver 86. MRP2 and MRP1 share very similar substrate specificities. High affinity endogenous substrates for MRP2 include amphiphilic anions, such as LTC₄ 8788. and bilirubin glucuronosides 8990. Data on the presence of MRP2 protein in the lung are conflicting. Only one out of three antibodies stained positive at the apical side in bronchial epithelium (Figure 1) 490. If MRP2 is expressed apically, it could be involved in transport of noxious compounds into the lumen of the lung.

There are several rat strains that have a mutation in Mrp2/cMOAT and the phenotypes resemble that of the human Dubin-Johnson syndrome, a disease in which MRP2 is mutated. Examples are the GY/TR^- rat 91 and the Eisai hyperbilirubinemic rat (EHBR) 92. Subjects with the Dubin-Johnson syndrome and also Mrp2 deficient rats display jaundice and have impaired organic anion transport. Recently, it was shown that deficient Mrp2 function in EHBR rats was compensated by upregulation of Mrp3 in liver and kidney 93. Mrp2 (-/-) mice have not been described. There are no reports in the literature of pulmonary malfunction in rats or humans with defects in the MRP2 gene.

CFTR (ABCC7) is located on chromosome 7q31.31. CFTR is the only member of the ABC superfamily which is not an active transporter. It functions as a chloride channel and in normal human airway tissue CFTR is highly expressed at the luminal side in serous cells of the submucosal glands. In addition, it is restricted to the apical membrane domain of well-differentiated epithelial cells such as ciliated cells, and probably also non-ciliated Clara cells and alveolar type I cells (Figure 1) 117118119120. CFTR is also expressed in normal nasal respiratory mucosa 13.

Mutations in the CFTR gene can cause cystic fibrosis 7 and are associated with abnormal Cl^- and Na⁺ ion transport in several tissues including the lungs, pancreas, gastrointestinal tract, liver, sweat glands and male reproductive organs. Although the normal expression of CFTR in the lung is lower compared to tissues such as the intestine and pancreas, its function in the lung is of major importance. The most frequent mutation in this gene is the delta F508 mutation which leads to cystic fibrosis. Defective CFTR function can cause viscous secretions in the lungs which leads to chronic inflammation with acute exacerbations by impaired mucociliary clearance. There are major risks for colonisation with Pseudonomas aeruginosa which leads to pneumonia and respiratory insufficiency 121. Besides abnormal CFTR localisation and expression in cystic fibrosis, also in non-cystic fibrosis airway tissue CFTR can be abnormally expressed in remodelled or dedifferentiated epithelium 122123. whereas in delta F508 CFTR epithelial cells there may be a normally processed CFTR 120124. The regulation and transport function of CFTR are dependent of the state of differentiation and polarisation of epithelial cell cultures 125126. Dedifferentiation with hyperplasia or metaplasia was associated with an intracellular localisation or absence of CFTR protein 122. Hurbain et al. 127 analysed MRP1-5 transcript levels in nasal respiratory cells from cystic fibrosis patients with homozygous delta F508 mutation. Surprisingly, low MRP1 levels were associated with more severe disease and in addition, MRP1 levels were related to cAMP-independent chloride transport suggesting that MRP1 regulates another chloride channel in the apical membrane. CFTR function is cAMP regulated 121, and therefore, MRP4 or MRP5 activity may play a role in regulation of CFTR, since these proteins transport cAMP. Another study showed that CFTR function is blocked by two MRP substrates (taurolithocholate-3-sulphate and beta-estradiol) 128. This implies that these two substances are also substrates for CFTR, or that CFTR and MRP proteins possess similar anion binding sites. Bebok et al. showed that nitric oxide (NO) and reactive oxygen nitrogen species (RONS) decrease wild-type CFTR protein levels in airway epithelial cell monolayers 129. Natural sources of NO and RONS are activated in alveolar and interstitial macrophages 130131, neutrophils 132, alveolar type II cells 133134. and airway epithelial cells 135. In this view, CFTR function may be important in pulmonary infections and in the effect of oxidative stress generated by e.g. cigarette smoke.

Cftr knock-out mice show reduced viability in contrast to most other ABC transporter knock-outs which are viable and fertile. Trezise et al. developed Cftr knock-out mice that have a severe cystic fibrosis phenotype accompanied by a lack of Cftr-related chloride conductance in e.g. tracheal epithelium 136137. They found that the P-gp mRNA level was four-fold increased in intestines of neonatal and 3- to 4-week-old Cftr knock-out mice compared to littermates of the same age. However, in 10 weeks-old mice P-gp levels were three-fold decreased. Apparently, a reduction or loss of Cftr function influences P-gp expression.

The Calu-3 cell line, which has properties from serous cells of the pulmonary submucosal glands, is often used to study function and expression of CFTR 129138. It was demonstrated in Calu-3 cells that CFTR mRNA expression is downregulated after ouabain incubation whereas P-gp expression was upregulated 139. The bronchial epithelial cell line 16HBE14o^- is also invaluable in CFTR research 129140. In wild-type and mutant CFTR expressing Sf9 insect cells, it was demonstrated that besides chloride transport, another function of CFTR is transport of GSH in a nucleotide-dependent manner 141. This observation suggests that CFTR plays a role in the control of oxidative stress. Indeed, several studies in patients, mice and cell lines have shown that GSH levels are lower in case of defective CFTR function.

BCRP (ABCG2) is the breast cancer resistance protein (BCRP), located on chromosome 4q22. BCRP is a half-transporter that probably acts as a homo- or heterodimer 142 and is involved in resistance against toxins and several chemotherapeutic agents (e.g. mitoxantrone and topoisomerase 1 inhibitors) 143144. Protein levels of BCRP in the lung are lower than P-gp and MRP1 but distinct in the epithelial cell layer and in seromucinous glands (Figure 1) 4. BCRP was absent in alveolar macrophages, suggesting that BCRP does not play a major role in innate inflammatory responses in the lung. Small endothelial capillaries also stain positive for BCRP, thus BCRP may protect the lungs against noxious compounds that enter systemically.

In a study of untreated solid lung tumours, i.e. squamous cell carcinoma (n = 5), adenocarcinoma (n = 2) and SCLC (n = 3), most cases expressed moderate or strong BCRP 145. In addition, higher BCRP expression in blood vessels was observed than in vessels of the surrounding tumour, indicating that BCRP may play a role in tumour angiogenesis. In 72 cases of advanced NSCLC, BCRP expression, but not P-gp, MRP1, MRP2 and MRP3, predicted poor clinical outcome 146.

Bcrp knock-out mice do not display any abnormalities compared to wild-type mice under normal conditions, except for the colour of their bile which is red instead of yellow on certain diets 147. However, these mice appear to be extremely sensitive to the phototoxin pheophorbide A, showing that BCRP is important to protect against toxic food components. Abnormalities of lungs in Bcrp (-/-) mice have not been reported thus far. Recently, Bcrp1 (Bcrp in the mouse is also called Bcrp1) localisation in the lung of 4- to 8-week old C57Bl/6J mice was studied to identify lung stem cells. These cells possess high activity of efflux of Hoechst dye that is transported by Bcrp1 148. In peripheral blood and bone marrow, cells with side population activity represent BCRP positive (haematopoietic) stem cells and this might also be the case for side population cells in the lungs. Indeed, Bcrp1 (-/-) mice did not display side population cells as shown by Hoechst staining of lung digests. In paraffin-embedded tissue sections, Bcrp1 was restricted to smooth muscle cells (of both arteries and airways) and a subpopulation of unidentified round cells in the alveolar space but not detectable in endothelial or epithelial cells. BAL cells of mice were Bcrp1 positive, but did not display side population (SP) activity 148. Clara cells, a nonciliated bronchiolar epithelial cell type, were also phenotypically described to possess stem cell characteristics 149. Further investigation is required to identify the stem cell pool in the lung in relation to expression of ABC transporters. Repairing and replacing lost lung tissue is a research area of promising therapeutic possibilities 150.