and the effect on Ca 2؉ spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca 2؉ signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca 2؉ signal cascade.Antimitotic drugs are used extensively for the treatment of cancer. For example, paclitaxel (Taxol) is used in the treatment of breast and ovarian cancers and for AIDS 1 -related Kaposi's sarcoma, and vinblastine is used in the treatment of Hodgkin's disease (1). The mechanism of action of antimitotic drugs, that leads to cancer cell death, is not clear. It is known that paclitaxel stabilizes microtubule dynamics thereby preventing the proper formation of the mitotic spindle apparatus and arresting cancer cells at the G 2 -M phase of the cell cycle (2, 3). While it is thought that this action of paclitaxel on the cell cycle machinery precedes an apoptotic response of cells (4, 5), some recent work has suggested that paclitaxel-induced apoptosis results from more direct effects of the drug on the mitochondria. In this context paclitaxel has been shown to bind to Bcl-2 (6) and this binding may regulate Bcl-2 effects on the mitochondrial permeability transition pore (PTP) (7,8). Furthermore, deletion of the loop region of Bcl-2 (which prevents Bcl-2 phosphorylation) blocks the apoptotic action of paclitaxel on cancer cells (9). Other proteins may also be involved in the paclitaxel effects on mitochondria, such as APAF-1 (10). In isolated mitochondria paclitaxel acts to release cytochrome c (11). This effect is blocked by cyclosporin A providing further evidence that paclitaxel directly targets mitochondria, independent of actions on microtubules.The effect of antimitotic drugs on microtubule dynamics would confer drug specificity on actively dividing cancer cells. However, the actions of these drugs on Bcl-2 and on the mitochondria might be expected to be non-selective and affect all cells. Indeed paclitaxel treatment is associated with serious side effects, including neuropathy (12) and low white blood cell counts (13). These side effects occur rapidly, appear to be due to drug action on terminally differentiated cells, and are slowly reversible. Thus it is unlikely that these side effects are mediated by either mitotic block or apoptosis. Given that the clinical use of antimitotic drugs is limited by these side effects, understanding the mechanisms by which these drugs act is an important step toward optimizing the therapeutic benefits.In our studies, on terminally differentiated epithelial cells, we now show rapid actions of paclitaxel on the cytosolic Ca 2ϩ signal that can be accounted for by effects of paclitaxel on the PTP of the mitochondria. Given the universality of Ca 2ϩ signaling, it is likely that this action of paclitaxel accounts for some of the side effects of antimitotic drugs. EXPERIMENTAL PROCEDURESCell Preparatio...

Studies have shown that expression of cystic ®brosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) ef¯ux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To de®ne the mechanism underlying CFTR-associated GSH¯ux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as puri®ed and reconstituted CFTR. We show that CFTRexpressing membrane vesicles mediate nucleotide-activated GSH¯ux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that puri®ed CFTR protein alone directly mediates nucleotide-dependent GSH¯ux. Interestingly, although ATP supports GSH ux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These ®ndings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH¯ux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.

The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl− channels in excised inside‐out membrane patches from cells expressing wild‐type human CFTR. Addition of genistein (100 μM) to the intracellular solution caused a small decrease in single‐channel current amplitude (i), but a large reduction in open probability (Po). Single‐channel analysis of channel block suggested that genistein (100 μM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. Genistein inhibition of CFTR Cl− currents was weakly voltage dependent and unaffected by changes in the extracellular Cl− concentration. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide‐binding domains (NBDs) of CFTR to greatly stimulate channel activity. ATP (5 mM) prevented the genistein‐induced decrease in Po, but was without effect on the genistein‐induced decrease in i. The genistein‐induced decrease in i was voltage dependent, whereas the genistein‐induced decrease in Po was voltage independent. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl− permeation.