Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.     

Specificity of intestinal brush-border proline transport: cyanine dye studies

The ability of rabbit jejunal brush borders to transport inhibitors of the imino carrier was investigated in membrane vesicles by measuring their ability to depolarize the membrane potential. Membrane potentials were monitored using a voltage-sensitive cyanine dye. Piperidine and pyrrolidine carboxylic acids, which are potent inhibitors of Na+-dependent proline transport (Ki less than 0.5 mM) depolarize the potential in a Na+-dependent, saturable manner indicating transport. On the other hand, N-methylated amino acids, which are fair inhibitors (Ki 2-10 mM), do not depolarize the membrane to any significant extent, but they competitively inhibit the L-proline transport signal. This indicates that these analogs are nontransported inhibitors of the imino carrier. The poor inhibitors niacin and pipolinic acid (Ki greater than 60 mM) depolarize the membrane about twice as much as proline and with low Kf values. This suggests separate carriers for these substrates.     

The use of membrane vesicles in transport studies

Transport-competent plasma membrane vesicles isolated from mammalian cells provide a system to investigate mechanisms and regulation of nutrient and ion transport systems. The characteristics of membrane vesicle systems to study transport in erythrocytes, renal and epithelial membranes, Ehrlich ascites cells, and mouse fibroblasts are discussed. Studies of Na+-stimulated and Na+-independent amino acid and glucose transport in these systems are evaluated, with emphasis on experimental verification of concepts stated in the Na+ gradient hypothesis. Nucleoside, phosphate, and calcium transport systems in plasma membrane vesicles from mouse fibroblast cultures are discussed. Also, current biochemical approaches to investigate mechanisms of regulation of nutrient transport systems by hormones or cellular proliferative state are described.     

The transport of chemicals in semen

Three mechanisms have been proposed for exposure of the conceptus to chemicals in semen: access of chemicals to the maternal circulation after absorption from the vagina, direct chemical exposure of the conceptus following transport from the vagina to the uterine cavity, and delivery to the egg and subsequent conceptus of chemical bound to the sperm cell. We review published data for each of these three mechanisms. Human seminal fluid chemical concentrations are typically similar to or lower than blood concentrations, although some antimicrobial agents achieve higher concentrations in semen than in blood. Vaginal absorption of medications has been shown to occur, although the vehicles in which these medications are delivered to the vagina may maintain contact with the vaginal epithelium to a greater extent than does semen. Assuming total absorption of a seminal dose of a chemical with a high semen:blood concentration ratio, distribution within the recipient woman would result in a blood concentration at least three orders of magnitude lower than that in the man. Direct delivery of seminal chemicals into the uterine cavity of humans has not been shown to occur, although it may occur in species such as the rat in which seminal fluid has access to the uterine cavity. Chemicals in or on human sperm cells have been demonstrated with respect to tetracycline and cocaine in vitro and aluminum, lead, and cadmium in vivo. The in vitro cocaine study offers sufficiently quantitative data with which to predict that oocyte concentrations would be five orders of magnitude lower than blood concentrations associated with cocaine abuse, assuming a maximally cocaine-bound sperm were capable of fertilizing. Thus, even using liberal assumptions about transmission of chemicals in semen or sperm, predicted exposure levels of a pregnant woman or of the conceptus are three or more orders of magnitude lower than blood concentrations in the man whose semen is the putative vehicle for chemical transport.     

Meiotic studies in human semen

Summary Meiotic studies can be carried out in the spermatogenic cells present in ejaculate. Using this technique, we identified one man with a reciprocal translocation and six oligochiasmatic males among 180 patients studied. The technique is easy and reliable; good-quality figures can be obtained, and meiotic studies can be carried out as often as needed.     

Dynamic transport and reaction model for azo dye removal in a UAFB reactor

An Upflow Anaerobic Fixed Bed (UAFB) reactor packed with activated carbon was used to remove the azo dye Reactive red 272. The biomass grown on the activated carbon surface was composed of an adapted consortium of microorganisms. Residence time distribution test indicated that the reactor was a plug flow behavior. A dynamic mathematical model is presented for dye flux along the reactor and within the bioparticles composed of two regions: activated carbon core and biofilm. The model considers that the reaction is performed in the biofilm and in the liquid phase and includes dye transport by dispersion and diffusion. The concentration profile within the bioparticles changes with reactor height and time as the equilibrium is achieved. Changes in dye concentrations affect the concentration profile in the reactor and reduce the removal efficiency. The effectiveness factor depends on the reactor height and on the dye concentration at the inlet.     

Comparison of three containers used for the transport of cooled stallion semen

Three containers commonly used to transport cooled equine semen (Equitainer, ExpectaFoal and a Swedish-designed semen-transport container, previously called the Salsbro Box and now called Equine Express) were compared, using four ejaculates from each of three stallions. Each ejaculate was diluted to a spermatozoal concentration of 25 x 10(6)/ml with a nonfat dry milk-glucose extender containing amikacin sulfate (1 mg/ml) and potassium penicillin G (1000 units/ml). Extended semen was divided into three 40-ml aliquots for placement in each of the three semen-transport containers. The extended semen was stored in the containers for 24 h prior to analysis. Stored semen was warmed for 15 min at 37 degrees C, then video records of sperm motility were obtained for evaluation using a Hamilton-Thorne motility analyzer equipped with a stage warmer set at 37 degrees C. The temperature of 40-ml aliquots of semen extender stored in each container was also measured for 60 h using a copper-constantan thermocouple placed in the center of the stored samples. Intervals from onset of storage until sample temperature exceeded 10 degrees C during the warming phase were 27.5, 33.5 and 53 h, for the Expecta-Foal, Equine Express and Equitainer, respectively. Semen extender stored in the Equitainer compared most favorably to ideal cooling rates and storage temperatures published previously. Following a 24-h storage period, the mean percentages of motile, progressively motile, and rapidly motile spermatozoa, as well as the mean spermatozoal curvilinear velocity were similar (P > 0.05) among the three containers.     

Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception

The present study was based on data obtained from artificial inseminations (AIs) performed with cryopreserved semen from elite bulls used in the Norwegian breeding program. Semen was diluted to standardize the number of spermatozoa to 18 million per AI dose. The aim of the study was to investigate whether the net sperm concentration at semen collection and the storage period in liquid nitrogen have any effect on probability of conception in dairy cattle. We demonstrated that the natural range of sperm concentration at semen collection within some of the bulls was associated with the probability of conception. However, no primary trend among bulls was found on the effect of sperm concentration at semen collection. This appears to be due to differences among bulls in their response to the dilution ratio of seminal plasma to extender. The effect of storage time was investigated in semen that had been stored between 1000 days and 2400 days in AI straws in liquid nitrogen at the AI center. Our findings showed that use of semen with the longest storage period, i.e. 1951-2400 days, resulted in a more than one percentage point lower probability of conception than semen with a shorter storage period. In conclusion, the net sperm concentration at semen collection, which affects the dilution ratio of seminal plasma to extender, should be considered individually among bulls to achieve optimal reproductive performance. Furthermore, this study gives support to the idea that a measurable degree of damage to the spermatozoa could occur during the preservation time in liquid nitrogen.     

Comparative studies of pollen and fluorescent dye transport by bumble bees visiting Erythronium grandiflorum

Summary In the Colorado Rocky Mountains the glacier lily Erythronium grandiflorum exhibits a striking dimorphism in pollen color and is commonly pollinated by the bumble bee Bombus occidentalis. We induced bees to visit sequences of flowers in a flight cage, and compared dispersal of distinctively-colored pollen and fluorescent pigment (dye) that the bee had picked up at a single donor flower. Nonparametric and parametric analyses showed that dispersal properties of pollen and dye differed; consistently less pollen was deposited and it was carried consistently shorter distances than dye. Dye thus does not provide an accurate means of assessing exacty where or how far pollen travels in this plant-pollinator system. On the other hand, both pollen and dye responded similarly to several experimental manipulations of donor and recipient flowers. Hence dye may well be of value for a qualitative investigation of how floral traits influence pollen dispersal.     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97-98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO2 decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

Effective use of renal cortical slices in transport and metabolic studies

The uptake and metabolism of two water-soluble vitamins were measured in rat renal cortical slices, isolated tubules, and vesicles of the brush-border and basolateral cell membranes to determine (a) whether it is possible to produce slices that have open tubules and, (b) whether slices and tubules metabolize vitamins similarly. Transport of ascorbic acid is sodium-dependent in slices and in brush-border vesicles but is sodium-independent in basolateral vesicles, suggesting that the brush-border membrane of slices is accessible to components of the bathing solution. Nicotinic acid was metabolized similarly (97–98%) in both slices and isolated tubules. Oxygen consumption by slices maintained in a closed chamber was constant as pO₂ decreased from 88% to 58%. Slices are concluded to be a suitable model for transport and metabolic studies providing that care is taken in their preparation and use.     

Glucose transport by mixed ruminal bacteria from a cow.

The glucose transport of mixed ruminal bacteria harvested from a holstein cow fed 5.0 kg of Italian ryegrass and 1.5 kg of flaked corn a day was investigated. The Eadie-Hofstee plot characterized two transport systems: a high-affinity, low-velocity system and a low-affinity, high-velocity system. The former system (K(m) = 16 microM; Vmax = 2.2 nmol/min/mg of protein) is considered dominant under this feeding condition based on the glucose concentration in the rumen (< 1 mM). In light of the facts that the protonophore SF6847 and the lipophilic triphenylmethyl phosphonium ion had no effect on the high-affinity system and an artificially generated proton gradient and electrical potential across the cell membrane did not increase glucose transport, a proton motive force is not be involved in the system. On the other hand, from the facts that chlorhexidine inhibited about 90% of the high-affinity system while iodoacetate showed no significant effect, and a high phosphoenolpyruvate-dependent phosphorylation of glucose was actually shown, the phosphoenolpyruvate-dependent phosphotransferase system is considered the main system in the high-affinity system. Moreover, as shown by the facts that harmaline inhibited about 30% of the high-affinity system and the artificially generated sodium gradient across the cell membrane significantly stimulated glucose transport, this system also includes sodium symport to some degree. The high-affinity system was sensitive to a decrease in pH (< 6.5) and was inhibited by the presence of sucrose, mannose, and fructose.     

The calibration and use of a calcium ion-specific electrode for kinetic studies of mitochondrial calcium transport.

A calcium ion-specific electrode has been used to study calcium transport by isolated,hepatic mitochondria. The methodology used requires only a sensitive pH meter operated in the millivolt mode with the electrode. Free calcium ion concentrations may be followed continuously. Using incubation conditions which cause release of intramitochondrial calcium, the calcium electrode system may also be used to determine total. intramitochondrial calcium. Techniques for the calibration of the electrode response are discussed. Free calcium ion concentrations have been calculated from total calcium concentrations and the association constants for the binding species present in the assay medium. The observation that the electrode response is linear to submicromolar concentrations allows calculation of a linear least-squares fit of millivolt reading to computed free calcium ion concentration. A computer program written in BASIC for these computations is included in Appendix material. The half-maximal rate constant for mitochondrial calcium uptake has been found to occur at a free calcium ion concentration of 6.5 μm. The interaction or Hill coefficient for the process is 2.3, indicating positive cooperativity.     

A modified method for the production of 36ClO3- for use in plant nitrate transport studies

A modified method for the production and purification of 36ClO3- in high yield is described. This procedure, involving the electrolytic oxidation of 36Cl- in a cell with simple electrode design and purification of the electrolysis products (36Cl-, 36ClO3-, and 36ClO4-) by aqueous column chromatography, allows for the recovery of about 80% of the initial radiolabel as 36ClO3-. The method is rapid and suitable for the production of this radiolabeled anion for use as a tracer analog for nitrate in plant membrane transport experiments.