The elementary mass action rate constants of P-gp transport for a confluent monolayer of MDCKII-hMDR1 cells

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.     

P-Glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites

The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Modeling and optimization of microbial hyaluronic acid production by Streptococcus zooepidemicus using radial basis function neural network coupling quantum‐behaved particle swarm optimization algorithm

Hyaluronic acid (HA) is a natural biopolymer with unique physiochemical and biological properties and finds a wide range of applications in biomedical and cosmetic fields. It is important to increase HA production to meet the increasing HA market demand. This work is aimed to model and optimize the amino acids addition to enhance HA production of Streptococcus zooepidemicus with radial basis function (RBF) neural network coupling quantum‐behaved particle swarm optimization (QPSO) algorithm. In the RBF‐QPSO approach, RBF neural network is used as a bioprocess modeling tool and QPSO algorithm is applied to conduct the optimization with the established RBF neural network black model as the objective function. The predicted maximum HA yield was 6.92 g/L under the following conditions: arginine 0.062 g/L, cysteine 0.036 g/L, and lysine 0.043 g/L. The optimal amino acids addition allowed HA yield increased from 5.0 g/L of the control to 6.7 g/L in the validation experiments. Moreover, the modeling and optimization capacity of the RBF‐QPSO approach was compared with that of response surface methodology (RSM). It was indicated that the RBF‐QPSO approach gave a slightly better modeling and optimization result compared with RSM. The developed RBF‐QPSO approach in this work may be helpful for the modeling and optimization of the other multivariable, nonlinear, time‐variant bioprocesses. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Structural and functional alterations in the surface of vascular endothelial cells associated with the formation of a confluent cell monolayer and with the withdrawal of fibroblast growth factor

Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) devide actively when seeded at low or clonal cell densities and upon reachin confluence adopt a morphologic appearance and differentiated properties similar to those of the vascular endothelium in vovi. In this review, we present some of our recent observations regarding the characteristics (both structural and functional) of these endothelial cells and the role of FGF in controlling their proliferation and normal differentation. At confluence the endothelial cells from a monolayer of closely apposed and nondividing cell that have a nonthrombogenic apical surface and can no longer internalize bound ligands such as low-density lipoprotein (LDL). The adoption of these properties is correlated and possibly causally related to changes in the cell surface such as the appearance of a 60,000 molecular weight protein (CSP-60); the disappearance of fibronectin from the apical cell surface and its concomitant accumulation in the basal lamina; and a restriction of the lateral mobility of various cell surface receptor sites. In contrast, endothelial cells that are maintained in the absence of FGF undergo within three passages alterations that are incompatible with their in vivo morphologic apperarance and physiologic beharior. They grow at confluence on top of each other and hence can no longer adopt both the structural (CSP-60, cell surface polarity) and functional (barrier function, nonthrombogenicity) attributes of differentiated endothelial cell. Since these characteristics can be reacquired in response to readdition of FGF, in addition to being a mitogen FGF may also be involved in controlling the differentitation and phenotypic expression of the vascular endothelium.     

Acute regulation of the facilitative glucose transporter GLUT1 in a hematopoietic cell line

This brief review is focused on the short-term regulation of the facilitative glucose transporter GLUT1 in megakaryocytic cells M07e. The effects of cytokines such as TPO, GM-CSF and SCF and of a low dose of H202 on the transport activity and its kinetic parameters are compared. The possible mechanisms and the signalling pathways involved in the glucose uptake activation are discussed. A role for the cellular redox status in glucose uptake control, possibly related to the status of redox-sensitive enzymes such as tyrosine phosphatases, is suggested.     

Dietary bioactive diindolylmethane enhances the therapeutic efficacy of centchroman in breast cancer cells by regulating ABCB1/P-gp efflux transporter

Overexpression of drug efflux transporters is commonly associated with multidrug-resistance in cancer therapy. Here for the first time, we investigated the ability of diindolylmethane (DIM), a dietary bioactive rich in cruciferous vegetables, in enhancing the efficacy of Centchroman (CC) by modulating the drug efflux transporters in human breast cancer cells. CC is a selective estrogen receptor modulator, having promising therapeutic efficacy against breast cancer. The combination of DIM and CC synergistically inhibited cell proliferation and induced apoptosis in breast cancer cells. This novel combination has also hindered the stemness of human breast cancer cells. Molecular docking analysis revealed that DIM had shown a strong binding affinity with the substrate-binding sites of ABCB1 (P-gp) and ABCC1 (MRP1) drug-efflux transporters. DIM has increased the intracellular accumulation of Hoechst and Calcein, the substrates of P-gp and MRP1, respectively, in breast cancer cells. Further, DIM stimulates P-gp ATPase activity, which indicates that DIM binds at the substrate-binding domain of P-gp, and thereby inhibits its efflux activity. Intriguingly, DIM enhanced the intracellular concentration of CC by inhibiting the P-gp and MRP1 expression as well as activity. The intracellular retaining of CC has increased its efficacy against breast cancer. Overall, DIM, a dietary bioactive, enhances the anticancer efficiency of CC through modulation of drug efflux ABC-transporters in breast cancer cells. Therefore, DIM-based nutraceuticals and functional foods can be developed as adjuvant therapy against human breast cancer.     

Sensitivity to values of the rate constants in a neurochemical metabolic model

Equations were derived for the instantaneous relative sensitivities of reaction rates (controllability indices) and metabolite concentrations (response indices) to perturbations in the values of rate constants and were used to analyze the behavior of a model of in vivo glutamate metabolism in rat brain. Controllabilities of reversible reactions were found to increase as the values of the corresponding rate constants (i.e., the rate of approach to equilibrium) increased. Response indices generally declined with the metabolic distance between the metabolite and the rate constant, but they were unexpectedly high for reversible reactions with high controllabilities. The transient response of a given metabolite is most sensitive to reactions involving metabolites which are changing most rapidly relative to their respective pool sizes. Rapidly reversible reactions are most important for communication between metabolite pools.     

Optimization of culture conditions for the production of Pleuromutilin from Pleurotus Mutilus using a hybrid method based on central composite design, neural network, and particle swarm optimization

This study aims at optimizing the culture conditions (agitation speed, temperature and pH) of the Pleuromutilin production by Pleurotus mutilus. A hybrid methodology including a central composite design (CCD), an artificial neural network (ANN), and a particle swarm optimization algorithm (PSO) was used. Specifically, the CCD and ANN were used for conducting experiments and modeling the non-linear process, respectively. The PSO was used for two purposes: Replacing the standard back propagation in training the ANN (PSONN) and optimizing the process. In comparison to the response surface methodology (RSM) and to the Bayesian regularization neural network (BRNN), PSONN model has shown the highest modeling ability. Under this hybrid approach (PSONN-PSO), the optimum levels of culture conditions were: 242 rpm agitation speed; temperature 26.88 and pH 6.06. A production of 10,074 ± 500 ??g/g, which was in very good agreement with the prediction (10,149 ??g/g), was observed in verification experiment. The hybrid PSONN-PSO gave a yield of 27.5% greater than that obtained by the hybrid BRNN-PSO. This work shows that the combination of PSONN with the generic PSO algorithm has a good predictability and a good accuracy for bio-process optimization. This hybrid approach is sufficiently general and thus can be helpful for modeling and optimization of other industrial bio-processes.     

Hormonal regulation of oligopeptide transporter Pept-1 in a human intestinal cell line

The intestinal oligopeptide transporter (cloned as Pept-1) hasmajor roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. Inthis experiment, we investigated whether insulin has such a role. Weused a human intestinal cell monolayer (Caco-2) as the in vitro modelof human small intestine and glycylglutamine (Gly-Gln) as the modelsubstrate for Pept-1. Results showed that addition of insulin at aphysiological concentration (5 nM) to incubation medium greatlystimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blockedwhen genistein, an inhibitor of tyrosine kinase, was added toincubation medium. Studies of the mechanism of insulin stimulationshowed the following. 1) Stimulationoccurred promptly (30-60 min) after exposure to insulin.2) There was no significant changein the Michaelis-Menten constant of Gly-Gln transport, but there was anearly twofold increase in its maximal velocity.3) Insulin effect persisted evenwhen Golgi apparatus, which is involved in trafficking of newlysynthesized Pept-1, was dismantled.4) However, there was completeelimination of insulin effect by disruption of microtubules involved intrafficking of preformed Pept-1. 5)Finally, with insulin treatment, there was no change in Pept-1 geneexpression, but the amount of Pept-1 protein in the apical membrane wasincreased. In conclusion, the results show that insulin, when it bindsto its receptor, stimulates Gly-Gln uptake by Caco-2 cells byincreasing the membrane population of Pept-1. The mechanism appears tobe increased translocation of this transporter from a preformedcytoplasmic pool.     

Direct determination of intracellular daunorubicin in intact confluent monolayers of AT1 prostate carcinoma cells using a multiwell-multilabel counter

The cytostatic drug daunorubicin exerts its toxic action by intercalating into the DNA. The efficacy of daunorubicin depends on the intracellular amount in the tumor cell. Here we have evaluated the use of a multiwell-multilabel reader for the direct determination of the fluorescent cytostatic drug daunorubicin in a prostate carcinoma cell line (AT1 R-3327 Dunning prostate carcinoma cells) grown on 24-well plates. We present evidence that this simple fluorescent parameter is a good measure for the toxicologically relevant amount of the drug intercalated into the DNA and, therefore, is a good predictor for the drug’s cytotoxicity. The amount of cationic cytostatics in a tumor cell is primarily a function of the efflux pump protein p-gycoprotein (pGP). Therefore, it is of great value that the assay is also suitable for the estimation of the multidrug resistance efflux pump (pGP) activity.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

Function-altering SNPs in the human multidrug transporter gene ABCB1 identified using a Saccharomyces-based assay

The human ABCB1 (MDR1)-encoded multidrug transporter P-glycoprotein (P-gp) plays a major role in disposition and efficacy of a broad range of drugs including anticancer agents. ABCB1 polymorphisms could therefore determine interindividual variability in resistance to these drugs. To test this hypothesis we developed a Saccharomyces-based assay for evaluating the functional significance of ABCB1 polymorphisms. The P-gp reference and nine variants carrying amino-acid–altering single nucleotide polymorphisms (SNPs) were tested on medium containing daunorubicin, doxorubicin, valinomycin, or actinomycin D, revealing SNPs that increased (M89T, L662R, R669C, and S1141T) or decreased (W1108R) drug resistance. The R669C allele's highly elevated resistance was compromised when in combination with W1108R. Protein level or subcellular location of each variant did not account for the observed phenotypes. The relative resistance profile of the variants differed with drug substrates. This study established a robust new methodology for identification of function-altering polymorphisms in human multidrug transporter genes, identified polymorphisms affecting P-gp function, and provided a step toward genotype-determined dosing of chemotherapeutics.     

Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.     

Evaluation of growth rate in adhering cell cultures using a simple colorimetric method

In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures. This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue. To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method. In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord. Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data. As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.     

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.