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Transport processes form part of the body's defense mechanism, and they determine the intracellular levels of many endogenous and exogenous compounds. The multidrug resistance-related protein MRP1 and the lung resistance-related protein LRP are associated with drug resistance against chemotherapeutics; they protect cells against toxic compounds. There is much experimental evidence to suggest that both of these transporter proteins serve important physiological functions. The expression of LRP and MRP1 was studied in normal human bronchial epithelial cells (NHBEC) and peripheral lung cells (PLC) obtained from explant cultures from morphologically-normal human lung tissue taken from patients with lung cancer. LRP (mRNA and protein) was detected in the cells of the bronchi as well as the peripheral lung with low (a factor of 2.6) inter-individual variation in the first generation. No significant alterations were noted for LRP within three-to-four generations in the same patient. LRP expression was not substantially different between cultures from different topographic regions of the human lung. MRP1 protein and MRP1 mRNA could also be detected in all of the NHBEC and PLC cultures studied, but with substantially higher (a factor of 7.7) intra-individual variation in the first generation than for LRP. MRP expression was the same for bronchial cells and PLC when the material was obtained from both sites. The level of mRNA for MRP1 was, in general, less stable than that for LRP. In multigeneration explant cultures, the levels of LRP mRNA and protein and MRP1 protein did not fluctuate greatly, but the level of MRP1 mRNA dropped to about 25% of the reference value within four generations (after about 8-10 weeks of culture). In one case, NHBEC subpassages were followed over a period of 20 weeks. In this system MRP mRNA levels increased by more than threefold, while levels of MRP1 protein and LRP mRNA and protein were expressed at almost constant rates.

Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2'-7'-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells.

A novel approach to triethyl 5-methoxy-1H-pyrrolo[2,3-fjquinoline-2,7,9-tricarboxylate (6), a close precursor of the redox cofactor F'QQ of bacterial dehydrogenases, is described. Diethyl 2-oxopent-3-ynedioate (5) was used as building block for the annelation of the quinoline unit. The annelation proceeds in two steps via the diastereoselecfive formation of diethyl(2)-4-{ [2-(ethoxycarbonyl)-5-methoxyindol-6-yl]amino}-2-oxopent-3-enedioate ((2)-7) followed by :acid-catalyzed cyclocondensation.1. Einleitung. -Die Trisaure PQQ (= Pyrrolo Quinoline Quinone, 4,5-Dihydro-4,5-dioxo-1 H-pyrrolo[2,~l-flchinolin-2,7,9-tricarbonsaure) [ Den Schlusselschritt der effizientesten bisher bekanniten PQQ-Synthese [14] stellt die diastereoselektive Doebner-van-Miller-Reaktion vork 6-Amino-2-methoxyindol-2-carbonsaure-ethylester (1) mit (E)-2-Oxoglutaconsaure-dimethylester (2) dar, die zu cis -6,7,8,9-Tetrahydro-9-hydroxy-5-methoxy-l H-pyrrolo[ 2,3-flchinolin-2,7,9-tricarbonsaure-2-ethyl-7,9-dimlethylester (3) fuhrt (Schema 1). Der in CH,Cl, geloste Ester 3 konnte dann durch Dehydratisierung und Oxidation in Gegenwart von Cu(OAc),, HC1-Gas und Luft-0, Zuni PQQ-Triester 4 aromatisiert werclen, aus dem PQQ uber zwei Stufen zuganglich ist.Aufgrund der interessanten Eigenschaften von PQQ sind PQQ-Triester als dessen Vorlaufer daher auch weiterhin ein lohnendes Ziel der Nafurstoffsynthese.In der vorliegenden Arbeit wird gezeigt, dass ein Triester der 5-Methoxy-lH-pyrrolo-[2,3-flchinolin-2,7,9-tricarbonsaure durch eine alternative Annelierung zuganglich ist, die vom kurzlich durch uns beschriebenen Baustein 2-Oxopent-3-indisaure-diethylester (5) [ 171 Gebrauch macht.

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