The effects of Na+ and Cl- on the binding of [3H]imipramine and the cocaine analog [125I]-beta-carbomethoxy-3 beta-(4-iodophenyl)tropane([125I]-beta-CIT) to the human platelet serotonin transporter have been measured. The ion dependence of beta-CIT binding is consistent with binding beta-CIT together with one Na+ ion, but not in an ordered sequence. Imipramine affinity, like beta-CIT affinity, is increased by Na+, but imipramine binding involves at least two Na+ ions. This conclusion is based on the observation that both imipramine association rate constants and equilibrium affinity constants show a sigmoidal Na+ dependence. As with beta-CIT, the imipramine and Na+ binding sequence is not strictly ordered. Cl- increases imipramine affinity, apparently by slowing dissociation. beta-CIT binding occurs even in the absence of Na+ and Cl-. This provided a means to measure substrate and inhibitor affinity in both the presence and absence of cotransported ions. Nontransported inhibitors, such as imipramine and citalopram, as well as the transport substrates serotonin and 3,4-(methylenedioxy)methamphetamine all displaced beta-CIT binding in the absence of NaCl. In the absence of Cl-, Na+ increased the affinity of nontransported inhibitors but not of substrates. The results suggest that Na+ and Cl- induce independent changes in the transporter binding site and that binding of substrates and inhibitors is affected differently by these changes.

The biochemical basis for bleomycin-induced pulmonary toxicity was studied in vitro and in vivo with an improved HPLC system. The in vitro metabolism of bleomycin A2 to desamido-bleomycin A2 was measured in tissue homogenates from a species sensitive (mice) and relatively resistant (rabbits) to the pulmonary fibrogenic properties of bleomycin. Lung tissue from mice lacked detectable bleomycin hydrolase activity, whereas rabbit lung tissue homogenates had high levels of the enzyme activity, equaling that seen in rabbit kidneys and spleen. Injection of radiolabeled bleomycin A2 into mice demonstrated that only a small percentage of the total dose was taken up by any organ and that extensive metabolism of this drug occurred within 1 hr in liver, kidneys, and spleen but not in lungs in vivo. In addition, metabolites other than desamido-bleomycin A2 were prominent, and their relative amounts increased with time. Mice injected subcutaneously with bleomycin A2 developed pulmonary fibrosis, while animals treated with equivalent doses of desamido-bleomycin A2 did not, indicating that this metabolite is not as toxic to the lungs as is the parent compound. These results provide direct evidence that metabolism plays a major role in determining the toxic potential of bleomycin to the lungs.Bleomycin (BLM) is a glycoprotein that is extensively used in combination with other anticancer agents because of its relative lack of hematological and gastrointestinal toxicity. However, pulmonary toxicity is common with BLM and limits its therapeutic utility (1). Life-threatening, irreversible pulmonary fibrosis is associated with high cumulative doses of BLM; thus, it is important to understand the biochemical basis of its lung toxicity. Furthermore, BLM may be a useful prototype for studying the general mechanism by which other substances, including anticancer drugs, cause pulmonary toxicity.Cellular toxicity from BLM exposure is believed to result from interactions between the drug and DNA (2 Previous experiments (4-7) indicated that repeated injections of mice with 5-10 mg of BLM per kg of body weight for 4 weeks or more produced pulmonary fibrosis as detected by an increase in lung hydroxyproline content and by histological analyses, whereas a similar injection protocol failed to yield fibrosis in rabbits (8). The experiments reported here examined the relative BLM hydrolase activities in various organs of these two species to evaluate the hypothesis of Umezawa et al. (3). A modification of our recently described sensitive HPLC system for detection of BLM B2 and desamido-BLM B2 (9) was used. In addition, the metabolism of radiolabeled BLM A2 in organs of mice in vivo and the pulmonary toxicity of the only previously described metabolite of BLM A2, desamido-BLM A2 (BLM dA2), were determined.MATERIALS AND METHODS Drugs. BLM (Blenoxane) and Cu-free BLM A2 that were used as HPLC standards were obtained from W. T. Bradner (Bristol Laboratories, Syracuse, NY). The Cu-free metabolite of BLM A2, BLM dA2 (lot TN-A-029), that was us...

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The Na+ and Cl- dependence of imipramine binding and dissociation were determined in platelet plasma membrane vesicles. Equilibrium imipramine binding affinity depends on Na+ binding to two non-interacting, low-affinity sites. Binding of a single Cl- ion also enhances imipramine affinity. Imipramine dissociation is inhibited by Na+ and Cl-, indicating that both ions can bind after imipramine. Of the two Na+ ions required for imipramine binding, only one is involved in slowing imipramine dissociation, indicating that imipramine binding makes the two Na+ ions non-equivalent. The initial rate of imipramine association is strongly Na(+)-dependent, suggesting that Na+ binds prior to imipramine. Cl-, however, affects imipramine dissociation but not association. Thus, while Na+ and Cl- can bind either before or after imipramine, kinetic considerations impose a most likely binding order of first Na+, then imipramine and finally Cl-. We have confirmed and extended these conclusions using serotonin exchange and efflux measurements. Efflux of radioactivity from vesicles preloaded with [3H]serotonin is stimulated by both external K+ and external unlabelled serotonin. K+ acts to accelerate a step that is rate-limiting for net efflux but that does not involve Na+, Cl- or serotonin translocation. Unlabelled serotonin accelerates radioactivity efflux by exchanging with intravesicular label. This serotonin exchange requires external Cl-, but not external Na+. These results suggest that first Na+, then serotonin and finally Cl- bind from the external medium. Although serotonin exchange requires external Cl-, internal Cl- is not required. These results suggest that translocation does not disturb the spatial order of bound substrates, which dissociate internally in a first-in-first-out order.

Several platinum(II) complexes of 3',5'-diamino-3',5'-dideoxythymidine (compound 1), 5'-amino-5'-deoxythymidine (compound 2), and 3'-amino-3'-deoxythymidine (compound 3) and the respective 2'-deoxyuridine amino nucleoside complexes, 4-6, have been synthesized. Whereas compounds 1, 2, and 4-6 had no inhibitory effect on the replication of murine L1210 cells in cell culture, compound 3 [(3'-AdThd)2PtCl2] inhibited these cells with an ED50 of 0.8 microM. Incubation of L1210 cells with 10-20 microM compound 3 for 2 h produced less than 18% inhibition of RNA, DNA, or protein synthesis, which is of questionable significance. However a 16-h incubation resulted in an increased uptake of labeled thymidine into DNA (77%), labeled uridine into RNA (17%), and labeled amino acids into protein (100%). These unexpected results indicate that inhibition of macromolecules may not be involved in the inhibition of the replication of L1210 cells. The increased incorporation of labeled metabolites into macromolecules may be related to the increase in cell volume after a 2-h incubation of L1210 cells with compound 3 plus a marked increase after 2 h in the proportion of cells in their S phase. Compound 3 appears to delay the progression of cells through their cell cycle. A marked inhibitory effect on the transport of methionine or aminoisobutyric acid into L1210 cells was found with compound 3, which was slightly greater than that produced with cisplatin. Compound 3 had a dose-dependent effect on the survival of mice bearing the L1210 ascites neoplasm, with a T/C X 100 of 175 at a dose of 320 mg/kg. Investigation of the kinetics of decomposition in aqueous systems demonstrated that the primary UV-absorbing decomposition product is 3'-amino-3'-deoxythymidine and that only a limited amount of the compound is formed (less than 8%). Although 3'-amino-3'-deoxythymidine could account for a part of the inhibition of the replication of L1210 cells in culture, it cannot account for the inhibition of amino acid transport by compound 3, the platinum complex of 3'-amino-3'-deoxythymidine. Compound 3 has been shown to limit part of the amino acid uptake into L1210 cells in a similar manner to cisplatin.