The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S -conjugate S -(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1 -transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance. ( J. Clin. Invest. 1996.

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Eukaryotic cells contain genes termed proto-oncogenes (c-onc) which have the potential to transform cells in culture and induce tumours in vivo. Most of these genes have been identified by their occasional incorporation into retroviral genomes which can act as natural transducing vectors for these and perhaps other cellular genes. Cell-derived oncogenes of retroviruses (v-onc) are associated mostly with the induction of mesenchymal tumours whereas carcinoma induction is rare. One of these rare carcinoma-inducing viruses is the acutely transforming avian retrovirus MH2 (refs 3-5). Recently we and others have shown that this virus carries a novel putative oncogene, v- mil , in addition to the known oncogene v-myc. While the transforming ability of v- mil has not been directly established, we have recently discovered by hybridization analysis that v- mil is homologous to v-raf (ref. 9), the transforming gene of the murine retrovirus 3611 MSV (ref. 10). Both viruses express the mil /raf oncogene product as a gag-fusion polyprotein, while the myc oncogene of MH2 is expressed via a subgenomic mRNA. Here we report the complete nucleotide sequence of v- mil and compare it with that of v-raf. The 80% homology between the nucleotide sequences and the 94% homology between the predicted amino acid sequences of the two viral genes clearly indicate that these are the avian and murine forms of the same gene. Comparison of the two sequences with that of the human cellular homologue (T. I. Bonner et al., manuscript in preparation) indicates that v-raf has more 3' untranslated sequences while v- mil has additional sequences from two 5' exons of the cellular homologue. Although the mil /raf amino acid sequences reveal partial homology to that of the v-src product, no tyrosine-specific protein kinase activity is detected for the gag- mil and the gag-raf hybrid proteins.

Molecularly cloned proviral DNA of avian replication‐defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5′ terminus, and a 1.3‐kb segment of the v‐myc transforming gene near the 3′ terminus. These v‐myc sequences are closely related to the v‐myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c‐myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v‐mil, which are unrelated to v‐myc, or to other oncogenes or structural genes of the avian leukemia‐sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v‐mil, termed c‐mil. Analyses of chicken c‐mil clones isolated from a recombinant DNA library of the chicken genome reveal that c‐mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v‐mil is expressed via genome‐sized mRNA as a gag‐related hybrid protein, p100gag‐mil, while v‐myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell‐derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.

Hypercapnia as well as lowered intracellular pH (pHi) increase the bioelectric activity of CO2/H+-sensitive neurones (VLNcs) of the ventrolateral medulla oblongata. Here we describe that immunoreactive Na+/H+ exchanger (NHE3) is present in ventrolateral neurones from medullary organotypic cultures (obex level). To test whether VLNcs can be acidified and thereby activated by inhibition of NHE3, we used the novel high-affinity NHE3-inhibitors S1611 and S3226. Both drugs raised the firing rates of VLNcs to at least 150% of the control values, and depolarized membrane potential by up to 15 mV at concentrations (0.5-1 micromol/l) suitable for selective inhibition of NHE3. The changes in bioelectric activity strongly resembled the responses to hypercapnia (PCO2: 60-100 mmHg). In BCECF-AM-loaded cultures a subfraction of ventrolateral VLNcs was found to be intracellularly acidified by 0.05-0.1 pH units following treatment with S1611; the time course of this acidification was similar to that evoked by hypercapnia. All drug effects were sustained and readily reversible upon washing. Non-CO2/H+-responsive medullary neurones as well as hippocampal CA3 neurones were unaffected by up to 20 micromol/l S1611. It is concluded that the selective inhibition of NHE3 acidifies and activates CO2/H+-sensitive neurones within the ventrolateral medulla oblongata.