Biomedical subjects

L Y Wei

Publications and source records attributed to L Y Wei.

At least 19 recordsLinked to original sources

PubMed2006

The delayed genotoxic effect of N-nitroso N-propoxur insecticide in mammalian cells.

The N-nitroso derivative of an extensively used insecticide, propoxur, consistently induced dose-responsive chromosome aberrations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO-W8) cells. Further investigations indicated that post-treatment incubation with a regular 1.5-cell-cycle period did not offer an unbiased estimation of the genotoxicity of N-nitroso carbamate insecticides. The scale of chromosome aberration induction increased with extension of the post-treatment incubation period. Comparable phenomena were not found in CHO-AGT cells proficient for O(6)-methylguanine-DNA-methyltransferase. In CHO-W8 cells, pulsed-treatment of the insecticide in the 1st replication cycle showed higher SCE induction than in the 2nd cycle. Similar phenomenon was also found in SCE induced by N-nitroso derivatives from other carbamate insecticides including aldicarb, carbofuran and methomyl. Treated cells did not show significantly perturbed cell cycle progression until 12 h after treatment removal. Based on the above observations, the O(6)-methylguanine-DNA adduct is suggested to be the major lesion caused by the delayed genotoxic effect of N-methyl carbamate insecticides as described in this report.

Animals↗

PubMed2006

Reduction of dietary magnesium by only 50% in the rat disrupts bone and mineral metabolism.

INTRODUCTION: The objective of this study was to determine the effect of a moderate reduction of dietary magnesium [50% of nutrient requirement (50% NR)] on bone and mineral metabolism in the rat, and to explore possible mechanisms for the resultant reduced bone mass. METHODS: Female rats were 6 weeks of age at the start of study. Serum magnesium (Mg), calcium (Ca), parathyroid hormone (PTH), 1,25(OH)(2)-vitamin D, alkaline phosphatase, osteocalcin, and pyridinoline were measured during the study at 3- and 6-month time points in control (dietary Mg of 100% NR) and Mg-deficient animals (dietary Mg at 50% NR). Femurs and tibias were also collected for mineral content analyses, micro-computerized tomography, histomorphometry, and immunohistochemical localization of substance P, TNFalpha, and IL-1beta at 3 and 6 months. RESULTS: Although no significant change in serum Mg was observed, Mg deficiency developed, as assessed by the reduction in bone Mg content at the 3- and 6-month time points (0.69+/-0.05 and 0.62+/-0.04% ash, respectively, in the Mg depletion group compared to 0.74+/-0.04 and 0.67+/-0.04% ash, respectively, in the control group; p=0.0009). Hypercalcemia did not develop. Although serum Ca level remained in the normal range, it fell significantly with Mg depletion at 3 and 6 months (10.4+/-0.3 and 9.6+/-0.3 mg/dl, respectively, compared to 10.5+/-0.4 and 10.1+/-0.6 mg/dl, respectively, in the control group; p=0.0076). The fall in serum Ca in the Mg-depleted animals was associated with a fall in serum PTH concentration between 3 and 6 months (603+/-286 and 505+/-302 pg/ml, respectively, although it was still higher than the control). The serum 1,25(OH)(2)-vitamin D level was significantly lower in the Mg depletion group at 6 months (10.6+/-7.1 pg/ml) than in the control (23.5+/- 12.7 pg/ml) (p<0.01 by the t-test). In Mg-deficient animals, no difference was noted in markers of bone turnover. Trabecular bone mineral content gain was less over time in the distal femur with Mg deficiency at 3 and 6 months (0.028+/-0.005 and 0.038+/-0.007 g, respectively, compared to 0.027+/-0.004 and 0.048+/-0.006 g, respectively, in the control group; p<0.005). Histomorphometry at these time points demonstrated decreased trabecular bone volume (15.76+/-1.93 and 14.19+/-1.85%, respectively, compared to 19.24+/-3.10 and 17.30+/-2.59%, respectively, in the control group; p=0.001). Osteoclast number was also significantly increased with Mg depletion (9.07+/-1.21 and 13.84+/-2.06, respectively, compared to 7.02+/-1.89 and 10.47+/-1.33, respectively, in the control group; p=0.0003). Relative to the control, immunohistochemical staining intensity of the neurotransmitter substance P and of the cytokines TNFalpha and IL-1beta was increased in cells of the bone microenvironment in the Mg depletion group, suggesting that inflammatory cytokines may contribute to bone loss. CONCLUSION: These data demonstrate that Mg intake of 50% NR in the rat causes a reduced bone mineral content and reduced volume of the distal femur. These changes may be related to altered PTH and 1,25(OH)(2)-vitamin D formation or action as well as to an increase release of substance P and the inflammatory cytokines TNFalpha and IL-1beta.

Animals↗

PubMed2004

Alterations in growth plate and articular cartilage morphology are associated with reduced SOX9 localization in the magnesium-deficient rat.

Insufficient dietary magnesium (Mg) intake has been associated with low bone mass in humans,and recent basic science studies have indicated that this bone loss may be secondary to increased release of substance P and TNFc Much less is known about the effects of low Mg intake on cartilage. We have evaluated growth plate and articular cartilage in rats following a 6 month dietary Mg restriction. Histomorphometry demonstrated significantly decreased distal femur articular cartilage chondrocyte density and decreased tibial growth plate width in experimental animals compared to controls. Growth plates of Mg-restricted animals showed reduced chondrocyte column formation. Extracellular matrix of both articular cartilage and growth plates in experimental animals contained reduced amounts of proteoglycans. Immunolocalization of Sox9 was decreased in both articular and growth plate cartilage in experimental animals compared to controls, suggesting that reduced Mg intake causes cartilage changes that may be secondary to reduced levels of the SOX9 transcription factor.

Animal Feed↗

PubMed2002

Magnesium deficiency: effect on bone and mineral metabolism in the mouse.

Insufficient dietary magnesium (Mg) intake has been associated in humans with low bone mass. Mg deficiency in the rat has suggested bone loss is due to increased bone resorption and/or inadequate bone formation during remodeling. The purpose of this study was to assess the effect of a low Mg diet on bone and mineral metabolism in the young and mature BALB/c mouse and explore the hypothesis that inflammatory cytokines may contribute to Mg deficiency-induced osteoporosis. Using an artificial diet, we induced targeted Mg depletion (0.002% Mg) with all other nutrients maintained at the normal level. In all Mg-depleted mice, hypomagnesemia developed and skeletal Mg content fell significantly. The serum Ca in Mg-deficient mice was higher than in control mice; however, serum PTH levels were not significantly different. Osteoprotegerin (OPG) in dosages that inhibit osteoclastic bone resorption did not prevent hypercalcemia in Mg-deficient animals. No significant difference in serum Ca was observed between groups when dietary Ca was reduced by 50%, suggesting that a compensatory increase in intestinal absorption might account for the hypercalcemia. Growth plate width decreased 33% in young Mg-deficient animals and chondrocyte columns decreased in number and length, suggesting that Mg deficiency reduced bone growth. Trabecular bone volume in the metaphysis of the tibia in these animals was decreased and osteoclast number was increased by 135%. Osteoblast number was significantly reduced. Immunohistochemistry revealed that substance P increased 230% and 200% in megakaryocytes and lymphocytes, respectively, after 1 day of Mg depletion. IL-1 increased by 140% in osteoclasts by day 3 and TNF alpha increased in osteoclasts by 120% and 500% in megakaryocytes on day 12. This study demonstrates a profound effect of Mg depletion on bone characterized by impaired bone growth, decreased osteoblast number, increased osteoclast number in young animals, and loss of trabecular bone with stimulation of cytokine activity in bone.

Animals↗

PubMed2002

Separation of pure elemental and oxygen influenced signal in ELNES.

The energy loss near edge structure (ELNES) of many elements is strongly influenced by the presence of oxygen or other elements at surfaces, grain boundaries, or in the bulk material. The presented investigation deals mainly with the influence of oxygen at the surface. A method for the separation of both, the pure bulk signal and the oxidized surface signal, was evaluated and tested on Al, Cu, Mg, and Si. A comparison of experimental data with ab initio bandstructure calculations and other proofs of the accuracy of ELNES separation are presented. Influences of error propagations were tested and are exemplarily given for Al and Si.

Journal Article↗

PubMed2001

Modulation of intraocular pressure by unilateral and forced unilateral nostril breathing in young healthy human subjects.

PURPOSE: To determine the effects of unilateral right/left nostril breathing (URNB/ULNB) and forced unilateral right/left nostril breathing (FURNB/FULNB) on intraocular pressure (IOP) and to examine the differences in the IOP during the various phases of nasal cycle. METHODS: Young healthy volunteers of either sex aged between 19-24 years, participated in the sessions using URNB/ULNB (n = 52) and FURNB/FULNB (n = 28). The nostril dominance was calculated from signals recorded on the PowerLab equipment, representing pressure changes at the end of the nostrils during respiration. The IOP was measured with Tono-Pen. The subjects were divided into 4 groups viz. right nostril dominant (RND), left nostril dominant (LND), transitional right nostril dominant (TRND) and transitional left nostril dominant (TLND) groups. The IOP data 'before and after' URNB/ULNB or FURNB/FULNB were compared by using paired t-test. The baseline data of IOP between the groups were analysed by using independent samples t-test. RESULTS: The URNB decreased the IOP in the LND and TLND (p < 0.01) and also in the RND (p < 0.05) groups but not significantly in the TRND group. The ULNB decreased the IOP in the RND group (p < 0.01) only. The FURNB significantly reduced the IOP (p < 0.05) only in the LND and RND groups. The FULNB decreased the IOP but not significantly. The baseline IOP did not differ significantly between the LND, RND, TLND and TRND groups. CONCLUSION: The URNB/FURNB reduced the IOP, while ULNB/FULNB failed to increase the IOP significantly. It is suggested that the lowering of IOP by URNB indicated sympathetic stimulation.

Adult↗

PubMed1999

Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo.

Two organotin pesticides, triphenyltin acetate (TPTA) and triphenyltin hydroxide (TPTH), were evaluated for their ability to induce micronuclei (MN) and sister chromatid exchange (SCE) in vitro using cultured Chinese hamster ovary (CHO) cells and in vivo BALB/c mouse erythrocytes. Both pesticides induced a dose-dependent increase but only TPTH induced a significant increase in MN at the highest dose (150 ng/ml) tested in CHO cells. With adding S9 microsomal fractions, both pesticides induced a meaningful MN induction at 150 ng/ml and a dose-dependent significant increase in SCE. In vivo MN induction in erythrocytes was conducted by treating BALB/c mice orally or intraperitoneally with these pesticides either in a single or triple treatments. Oral gavage (p.o.) of TPTA resulted in a dose-related significant increase of MN induction in peripheral blood and of TPTH induced a significant increase in micronucleated reticulocyte (MNRETs) only in a single treatment. Intraperitoneal administration of TPTA or TPTH, however, resulted in meaningless random increases in MN though these increases might be attributable to toxic effects. The MNRETs levels in the treatment with both pesticides were independent to the sampling time. This study demonstrated that TPTA and TPTH was potential chromosome mutagens.

Animals↗

PubMed1999

Preparation for TEM of layered samples with fragile microstructure and weak layer interface.

The objective of this work was to prepare for transmission electron microscopy (TEM) a layered structure of materials with fragile microstructure. The samples consisted of two layers of different materials, silicon nitride and borosilicate glass, loosely bonded together. The low strength of the sample resulted in fragmentation during more conventional preparation. However, it was possible to prepare the fragments by mounting them in a titanium specimen carrier with aluminium strips as support. After grinding and polishing, a technique of low-angle ion milling was used to obtain electron beam transparent areas at the nitride/glass interface.

Microscopy, Electron↗

PubMed1997

Ultramicrotomy of powder material for TEM/STEM study.

This paper summarizes methods conventionally used to prepare thin foil samples of powder materials for transmission electron microscopy (TEM) and introduces another variant, ultramicrotomy, for the preparation of TEM samples of industrial dust powder. The choice of ultramicrotoming in the present work was based on two features of this technique: (1) it can produce thin-sectioned specimens with a uniform thickness; (2) it can retain the original elemental distribution in phases of the sample during sectioning. Dust powder preparation and the sectioning procedure are described in this paper. The results of the method are illustrated by examples of TEM/STEM micrographs of industrial dust.

Microscopy, Electron↗

PubMed1997

Altered pHi regulation in 3T3/CFTR clones and their chemotherapeutic drug-selected derivatives.

Recently (L. Y. Wei, M. J. Stutts, M. M. Hoffman, and P. D. Roepe. Biophys. J. 69: 883-895, 1996), 3T3 cells overexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) were found to exhibit chemotherapeutic drug resistance and other traits of multidrug resistant (MDR) cells. In the present work, NIH 3T3/CFTR clones were selected with either doxorubicin or vincristine in incremental fashion to generate series of stable MDR cell lines that exhibit increasing levels of drug resistance. Thus C3D6 (grown in the presence of 600 nM doxorubicin) was selected from C3D4 (grown in the presence of 400 nM doxorubicin), which was selected from C3D1 (grown in the presence of 100 nM doxorubicin), which was in turn selected from the original 3T3/CFTR clone C3 (M. J. Stutts, S. E. Gabriel, J. C. Olsen, J. T. Gatzy, T. L. O'Connell, E. M. Price, and R. C. Boucher. J. Biol. Chem. 268: 20653-20658, 1993), which was not grown in the presence of chemotherapeutic drug. A similar series was generated via selection with vincristine. In both series, as well as series derived from a different CFTR clone, initial low-level drug selection increases CFTR expression without promoting MDR 1 or multidrug resistance-associated protein expression. On continued selection at higher drug concentrations, CFTR mRNA levels decrease while MDR 1 mRNA levels concomitantly increase. At each incremental step of selection, intracellular pH (pHi) increases (e.g., pHi of C3D6 > C3D4 > C3D1 > C3). Cl-/HCO3- exchange activity is significantly reduced in the drug-selected derivatives overexpressing MDR 1 but not the parental CFTR clones. The apparent set point of Na+/H+ exchange activity is significantly lower for the non-drug-selected 3T3/CFTR clones, relative to controls, but it increases on initial selection with chemotherapeutic drug. Overexpression of MDR 1 in the higher-level selectants does not appear to further perturb apparent Na+/H+ exchange. These data further describe how CFTR and MDR proteins may affect pHi regulation.

3T3 Cells↗

PubMed1997

Assessment of the ability of propoxur, methomyl, and aldicarb, three carbamate insecticides, to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice.

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, on optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment.

Aldicarb↗

PubMed1996

Altered drug translocation mediated by the MDR protein: direct, indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ItpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential (delta psi) and/or intracellular pH (pHi) and thereby indirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pHi or delta psi-dependent manner. A third model proposes that the protein alternates between drug pump and Cl- channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pHi, delta psi, and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Are altered pHi and membrane potential in hu MDR 1 transfectants sufficient to cause MDR protein-mediated multidrug resistance?

Multidrug resistance (MDR) mediated by overexpression of the MDR protein (P-glycoprotein) has been associated with intracellular alkalinization, membrane depolarization, and other cellular alterations. However, virtually all MDR cell lines studied in detail have been created via protocols that involve growth on chemotherapeutic drugs, which can alter cells in many ways. Thus it is not clear which phenotypic alterations are explicitly due to MDR protein overexpression alone. To more precisely define the MDR phenotype mediated by hu MDR 1 protein, we co-transfected hu MDR 1 cDNA and a neomycin resistance marker into LR73 Chinese hamster ovary fibroblasts and selected stable G418 (geneticin) resistant transfectants. Several clones expressing different levels of hu MDR 1 protein were isolated. Unlike previous work with hu MDR 1 transfectants, the clones were not further selected with, or maintained on, chemotherapeutic drugs. These clones were analyzed for chemotherapeutic drug resistance, intracellular pH (pHi), membrane electrical potential (Vm), and stability of MDR 1 protein overexpression. LR73/hu MDR 1 clones exhibit elevated pHi and are depolarized, consistent with previous work with LR73/mu MDR 1 transfectants (Luz, J.G. L.Y. Wei, S. Basu, and P.D. Roepe. 1994. Biochemistry. 33:7239-7249). The extent of these perturbations is related to the level of hu MDR 1 protein that is expressed. Cytotoxicity experiments with untransfected LR73 cells with elevated pHi due to manipulating percent CO2 show that the pHi perturbations in the MDR 1 clones can account for much of the measured drug resistance. Membrane depolarization in the absence of MDR protein expression is also found to confer mild drug resistance, and we find that the pHi and Vm changes can conceivably account for the altered drug accumulation measured for representative clones. These data indicate that the MDR phenotype unequivocally mediated by MDR 1 protein overexpression alone can be fully explained by the perturbations in Vm and pHi that accompany this overexpression. In addition, MDR mediated by MDR protein overexpression alone differs significantly from that observed for MDR cell lines expressing similar levels of MDR protein but also exposed to chemotherapeutic drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype.

Because of the similarities between the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) proteins, recent observations of decreased plasma membrane electrical potential (delta psi) in cells overexpressing either MDR protein or the CFTR, and the effects of delta psi on passive diffusion of chemotherapeutic drugs, we have analyzed chemotherapeutic drug resistance for NIH 3T3 cells overexpressing different levels of functional CFTR. Three separate clones not previously exposed to chemotherapeutic drugs exhibit resistance to doxorubicin, vincristine, and colchicine that is similar to MDR transfectants not previously exposed to chemotherapeutic drugs. Two other clones expressing lower levels of CFTR are less resistant. As shown previously these clones exhibit decreased plasma membrane delta psi similar to MDR transfectants, but four of five exhibit mildly acidified intracellular pH in contrast to MDR transfectants, which are in general alkaline. Thus the MDR protein and CFTR-mediated MDR phenotypes are distinctly different. Selection of two separate CFTR clones on either doxorubicin or vincristine substantially increases the observed MDR and leads to increased CFTR (but not measurable MDR or MRP) mRNA expression. CFTR overexpressors also exhibit a decreased rate of 3H -vinblastine uptake. These data reveal a new and previously unrecognized consequence of CFTR expression, and are consistent with the hypothesis that membrane depolarization is an important determinant of tumor cell MDR.

3T3 Cells↗

PubMed1994

Novel Cl(-)-dependent intracellular pH regulation in murine MDR 1 transfectants and potential implications.

Previously [Luz et al. (1994) Biochemistry 33, 7239-7249], we determined that Cl(-)- and -HCO3-dependent pHi homeostasis was perturbed in multidrug resistant (MDR) cells created by transfecting LR73 Chinese hamster ovary fibroblasts with wild-type mu (murine) MDR 1 (Gros et al., 1991). Via single-cell photometry experiments performed under various conditions, we are now able to separate Na(+)-dependent and Na(+)-independent components of Cl-/-HCO3 exchange in the MDR transfectants and the parental LR73 cells. Cl(-)-dependent, Na(+)-independent reacidification of pHi, mediated by the anion exchanger 2 isoform in LR73 cells, is dramatically inhibited by mild overexpression of MDR protein. Analysis of H+ flux at different pHi shows that Cl(-)-dependent reacidification approaches 0.2 mM H+/s for LR73 cells at pHi = 8.0 but is at least 10-fold slower for MDR 1 transfectants that were never exposed to chemotherapeutics (EX4N7 cells). MDR 1 transfectants selected on the chemotherapeutic vinblastine (1-1 cells), which express approximately 10-fold more MDR protein relative to EX4N7 cells, exhibit similar behavior; however, alterations in Cl(-)-dependent pHi regulation are more severe. Hypotonic conditions, which have been shown to increase anomalous Cl- conductance in some cells overexpressing MDR protein (Valverde et al., 1992), are found to amplify the altered pHi homeostasis features in the primary transfectants that express lower levels of MDR protein such that they then mimic the behavior of the drug-selected cells that express substantially more MDR protein. Verapamil reverses the anomalous behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Low external pH and osmotic shock increase the expression of human MDR protein.

We have studied the effects of extracellular pH (pHo) and osmotic strength on the expression of the human multidrug resistance (MDR) protein. Both lowered pHo and hypertonic shock increase the level of hu MDR protein 5-10-fold in membranes isolated from the human colon carcinoma cell lines SW620 and HCT15 and the human kidney carcinoma line SKRC-39. Increased protein expression is dependent on the duration of acid or osmotic shock and is reversed within several days when normal growth conditions are restored. Quantitative northern blot analysis with a hu MDR 1 specific probe reveals increased MDR mRNA in the acid and hypertonically shocked cells. Interestingly, we find a greater increase in mRNA levels for hypotonically shocked colon cells, without an apparent increase in protein levels. Overexpressing cells are found to retain less [3H]vinblastine relative to cells cultured under normal conditions, and they are resistant to the cytotoxic effects of doxorubicin, vinblastine, and colchicine, but not methotrexate. This resistance appears to be reversed by treatment with verapamil. In contrast, SW620 cells previously induced to overexpress MDR protein via the administration of differentiation agents [Mickley et al. (1989) J. Biol. Chem. 264, 18031-18040] did not exhibit decreased retention of [3H]vinblastine; thus low-pHo-induced overexpression of MDR protein in these cells may provide additional factors that promote the full expression of the MDR phenotype. These data may help to explain why many solid tumors (e.g., of colon and kidney origin) develop MDR prior to chemotherapy, since they usually grow under similar acidic conditions. These data also support the contention that MDR protein may play a role in intracellular pH and volume homeostasis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Transfection of mu MDR 1 inhibits Na(+)-independent Cl-/-HCO3 exchange in Chinese hamster ovary cells.

We have used single-cell photometry to measure intracellular pH (pHi) for several MDR cell lines constructed by stably transfecting LR73 chinese hamster ovary fibroblasts with mutant and wild type murine MDR 1 genes. In addition, plasma membrane electrical potential (delta psi) has been measured for the same cells by the K+/valinomycin null point titration method using the ratiometric styryl probe di-4-ANEPPS. Both the untransfected, parental cell line and a cell line expressing substantial mutant MDR 1 protein (K432R/K1074R) that is unable to confer the MDR phenotype are found to have delta psi > or = -40 (+/- 5) mV and pHi < or = 7.16 (+/- 0.03) units. In contrast, MDR cell lines constructed by transfecting wild type mu MDR 1 cDNA are found to exhibit delta psi from 15 to 19 mV lower and pHi from 0.13 to 0.34 units higher. A cell line that overexpresses crippled MDR protein (S941F) that is not resistant to colchicine or doxorubicin, but which is resistant to vinblastine [Gros, P., Dhir, R., Croop, J., & Talbot, F. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7289-7293], exhibits elevated pHi and slightly elevated delta psi, relative to LR73. Northern and western blot analyses confirm the substantial overexpression of the mu MDR genes and proteins in these lines, as well as the mild overexpression of endogenous hamster p-GP mRNA in some lines. In general agreement with previous studies that examined myeloma cells overexpressing hu MDR 1 protein [Roepe, P.D., Wei, L.-Y., Cruz, J., & Carlson, D. (1993) Biochemistry 32, 11042-11056] we find that overexpression of wild type mu MDR 1 protein inhibits Cl(-)- and -HCO3-dependent pHi homeostasis. Via single-cell photometry studies we now conclude that this is due to inhibition of Na(+)-independent Cl-/-HCO3 exchange (strict anion exchange or AE). As concluded previously for other MDR cells, decreased AE activity is not due to decreased expression of the exchanger; in fact, again similar to previous work [Roepe et al. (1993) Biochemistry 32, 11042-11056], we find increased levels of AE mRNA in some MDR cell lines. Models that may explain these data that are also consistent with the known physiology of cells that endogenously express MDR protein are suggested. These data are consistent with a model for MDR protein function wherein overexpression of the protein decreases delta psi and/or elevates pHi via Cl(-)- and -HCO3-dependent mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Lower electrical membrane potential and altered pHi homeostasis in multidrug-resistant (MDR) cells: further characterization of a series of MDR cell lines expressing different levels of P-glycoprotein.

Recently [Roepe, P.D. (1992) Biochemistry 31, 12555-12564], increased steady-state levels of chemotherapeutic drug efflux from multidrug-resistant (MDR) myeloma cells were correlated with intracellular alkalinization. To better understand elevated pHi in MDR cells, Na(+)- and Cl-dependent recovery of pHi upon intracellular acid or alkaline shock has been examined for this same series of MDR cell lines. In agreement with another recent report [Boscoboinik, D., Gupta, R.S., & Epand, R.M. (1990) Br. J. Cancer 61, 568-572], we find that the rate of Na(+)-induced alkalinization after an intracellular acid shock is increased in the MDR cells, relative to the drug-sensitive parent. Interestingly, we also now find that mRNA encoding the human Na+/H+ exchanger (NHE) is overexpressed in these MDR cells, but the level of overexpression does not correlate with the relative drug resistance or steady-state pHi. It is also found that the efficiency of Cl(-)dependent reacidification of pHi, after an intracellular alkaline shock is reduced in the MDR cells. This effect appears to correlate with the relative expression of MDR protein, but not the relative expression of Cl-/HCO3- exchanger (AE), which we now find is also altered in the series of cells. Since elevated pHi will increase delta pH across the plasma membrane, we have also measured the electrical potential for these cells using three different methods. Most interestingly, the magnitude of the plasma membrane electrical potential (delta psi) decreases concomitant with increased expression of the MDR protein. Energy provided by increased delta pH compensates for the lowered delta psi, such that the total electrochemical membrane potential (delta mu H+) remains similar among the cells in this series (delta mu H+ = delta psi - Z delta pH). These data, along with other recent experiments that associated an increased Cl- conductance with the expression of MDR protein [Valverde, M., Diaz, M., Sepúlveda, F.V., Gill, D.R., Hyde, S.C., & Higgins, C.F. (1992) Nature 355, 830-833], are consistent with a model for MDR protein-mediated multidrug resistance that does not entail direct active transport of lipophilic drugs by the MDR protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗