Tracer membrane transport

After release from storage sites in presynaptic monoamine transporter vesides, DA is bound by postsynaptic DA receptors, and unbound DA is broken down by enzymes, or taken back into presynaptic neurons, called reuptake into presynaptic neurons by spedfic presynaptic protein transporters. The uptake of neurotransmitters by presynaptic neurons is a major mechanism for stopping neurotransmission. The recyded neurotransmitter is repackaged in vesicles in presynaptic neurons, and then rdeased again in response to stimulation by presynaptic electrical action potentials. Tracers have been developed to assess the vesicular membranes, as well as the specific reuptake protein transporters. The selectivity with which monoaminergic neurons store DA, 5-HT, NER, or histamine in presynaptic vesides depends on the spedficity of the membrane transporters. 

The results of the tracer studies including the elucidation of the stereochemistry involved, provided a firm basis for a biochemical approach to PA biosynthesis, i. e., characterization of the enzymes that catalyze biosynthetic key steps and the specific mechanisms involved in translocation,subcellular accumulation, and metabolism of PAs. Early tracer work was carried out with intact plants to which tracers were applied for days or weeks. Meanwhile, in vitro plant systems, such as cell cultures and root-organ cultures of PA-producing plants are available. Root cultures were found to be excellent systems for biochemical and enzymatic studies of PA biosynthesis [20-22]. Dedifferentiated cell cultures do not synthesize PAs, but retain the ability to accumulate PAs. They are excellent systems to study the membrane transport of PAs and to identify the subcellular storage sites. 

Reporter genes can also encode for extracellular receptors such as dopamine D2 (200) and SST type-2 receptors (201), or membrane transporters such as the sodium/iodide symporter (202). These human genes have been suggested as candidate reporter genes because they exhibit limited expression in the body. At the same time, radiolabeled tracers with high affinity for these extracellular gene-products have been extensively studied and are approved for human use [e.g., [ F]fluoroethylspiperone for D2 receptors (203), " in-pentetreotide for SST receptors (121), and 123/124/I3ij... 

Several factors might account for this apparent plateau elfect. Competition for phosphorylation between [ F]-FHBG and cellular thymidine is one possibility, as recently noted by Min et al. (66). Another possible cause is that tracer availability to the viral transgene was restricted in some way. For instance, [1 F]-FHBG was recently shown to accumulate much more slowly in mammalian cells expressing the wild type HSVl-tA in vitro than other radiolabeled pyrimidine nucleoside derivatives (13), and it was hypothesized that [ Fj-FHBG uptake might be limited by cell membrane transport (13). 

Powerful solvents such as dimethyl sulfoxide (common laser dye solvent) and solubilizing substituents (K" and R " = sulfoalkyl in stmcture 32) may enhance the transport of dyes in solution through skin and other membranes. Reference 88 (on laser dye solutions and toxicity) is recommended to researchers working with dye solutions. Other dyes, such as Indocyanine Green, attain useful properties (blood tracer dye) as a result of having solubilizing substituents in their stmcture. 

The permselectivity of the corneal and conjunctival paracellular routes was investigated by Huang et al. [159] in an attempt to show that nutrients can be extracted from the blood by the conjunctiva. Neither the blood vessels supplying the conjunctiva nor its basement membrane are rate-limiting to the transport of horseradish peroxidase. This 40 kDa tracer is restricted underneath the conjuncti-... 

Meares and his collaborators are especially interested in transport processes across biological membranes. They wish to distinguish experimentally between the active and the passive transport of a solute. For that purpose they determined the fluxes of the sodium ions in each direction through the membrane, using the technique of radio-tracers. The ratio of these experimental fluxes was compared with the theoretical ratios. The same is done with regard to the chlorine ions. 

Zelsmann and co-workers [88] have reported tracer diffusion coefficients for water in Nafion membranes exposed to water vapor of controlled activity. These were determined by various techniques, including isotopic exchange across the membrane. They reported apparent self-diffiision coefficients of water much lower than those determined by Zawodzinski et al. [64], with a weaker dependence on water content, varying from 0.5 x 10 cm to 3 x 10 cm /s as the relative humidity is varied from 20 to 100%. It is likely that a different measurement method generates these large differences. In the experiments of Zelsmaim et al., water must permeate into and through the membrane from vapor phase on one side to vapor phase on the other. Since the membrane surface in contact with water vapor is extremely hydrophobic (see Table 7), there is apparently a surface barrier to water uptake from the vapor which dominates the overall rate of water transport in this type of experiment. 

A laboratory membrane brine electrolysis cell, designed for automated operation, was constructed ( 1,2). This system enables the measurement of the sodium ion transport number of a membrane under specific sets of conditions using a radiotracer method. In such an experiment, the sodium chloride anolyte solution is doped with 22Na radio-tracer, a timed electrolysis is performed, and the fraction of current carried by sodium ion through the membrane is determined by the amount of radioactivity that has transferred to the sodium hydroxide catholyte solution. The voltage drop across the membrane during electrolysis is simultaneously measured, so that the overall performance of the material can be evaluated. 

Membrane Diffusion in Dilute Solution Environments. The measurement of ionic diffusion coefficients provides useful information about the nature of transport processes in polymer membranes. Using a radioactive tracer, diffusion of an ionic species can be measured while the membrane is in equilibrium with the external solution. This enables the determination of a selfdiffusion coefficient for a polymer phase of uniform composition with no gradients in ion or water sorption. In addition, selfdiffusion coefficients are more straightforward in their interpretation compared to those of electrolyte flux experiments, where cation and anion transport rates are coupled. 

Burkhardt, S.F., Radioactive tracer measurement of sodium transport efficiency in membrane cell, presented at Electrochem. Soc. Meeting, Atlanta Georgia U.S.A. 

Blaedel and Haupert (28) demonstrated the feasibility of using this phenomenon as a preconcentration technique using isotope tracer studies on the ions Na4", Cs4", Zn24", and later Blaedel and Christensen (29) extended the work to include the anions 1 and HP01 . They found anion transport to be much slower than the previously reported cation transport. Coion transport in the anion exchange membranes was much higher and apparently dependent on the anionic charge of the bulk electrolyte. Further studies with more recently available membranes (1) seem to be needed. 

When an ion exchange membrane divides solutions of the same concentration and composition, the flux of solute and solvent is zero. However, ion, solute and solvent do transport across the membrane, a process known as self-diffusion. The self-diffusion flux is proportional to the self-diffusion coefficient and the concentration of species in the membrane and inversely proportional to the membrane thickness. The self-diffusion coefficient is evaluated by diffusion of a radiotracer (isotopic or tracer diffusion).16... 

A simple classification of the main macroscopic techniques is shown in Table 1, and this provides a useful framework for our review. Macroscopic measurements generally yield transport diffusivities, although variants of the techniques, using isotopically tagged tracers, can be devised to measure self-diffusivities. The large majority of the macroscopic techniques involve transient measurements. Steady-state or quasi-steady-state methods, notably membrane permeation and catalyst effectiveness measurements, have been demonstrated, but their application has been limited to a few systems. 

Of course, there are more bulk properties of interest than the above parameters related to transport of the fast ions and electrons. Metal cation transport is minor, but still a most crucial parameter, because it eventually leads to membrane walkout, demixing, or decomposition in chemical gradients. Methods used for investigating metal cation diffusion comprise reactivity studies, interdiffusion couples, and tracer studies, using analytical SEM, EPMA, SIMS or radioactivity for the diffusion profile analyses. 

Probably the most systematic and complete study on the influence of temperature on water transfer has been performed on mammalian red cells [10,20,28]. The dependence on temperature of both the tracer diffusional permeability coefficient (cotho) 3 nd the hydraulic conductivity (Lp) of water in human and dog red-cell membranes have been studied. The apparent activation energies calculated from these results for both processes are given in Table 2. The values for the apparent activation energies for water self-diffusion and for water transport in a lipid bilayer are also included in the table. For dog red cells, the value of 4.9 kcal/mol is not significantly different from that of 4.6-4.8 kcal/mol for the apparent activation energy of the water diffusion coefficient ( >,) in free solution. Furthermore, it can be shown that the product L — THOV )rt, where is the partial molar volume of water and the viscosity of water remains virtually independent of temperature for dog, hut not for the human red-cell membrane [20]. The similarity of the transmembrane diffusion with bulk water diffusion and the invariance of the... 

Carrier-mediated transport across membranes adds additional complexity to the system and, thus, to the model. For even the simplest transporter, the concentration of the transporter and its affinity for the substrate must be known before it can be modeled. Also, active transport is inherently a saturable process. Thus, to analyze the dynamics of tracer-labeled substrate, the model must account for both labeled and unlabeled substrate as the transport dynamics will depend on total substrate concentration. 

Carrier-mediated transport is a saturable process. The carrier has a finite concentration in the membrane. When the concentration of the substrate is high enough, all the carrier will be bound to the substrate and adding additional substrate will not result in increased transport. When a tracer is used, the carrier will exhibit competitive inhibition. Labeled substrate must compete with unlabeled substrate for available carrier. Thus, increased concentrations of unlabeled substrate may result in decreased transport of tracer. To account for this, the model must account for both tracer and mother (nontracer) substances. 

FIG. 7. Outflow concentration (a) for plasma (circles) and intestine (squares) and average membrane conduaance (b) for the intestine-mucosal cell (circles) and mucosal cell-isf (squares) exchange following constant infusion of substrate and tracer into the inflow to the intestinal lumen at a total substrate concentration of 10 mAf. See the legends to Figs. S and 7 for the values of the parameters for flows and volumes and of the transporter. 

However, although cellular uptake of [ " TcjMIBI is driven by the plasma and mitochondrial membrane potentials, the overall accumulation of the tracer is inversely proportional to the expression levels of Pgp in tumor cells. The earlier observations that intracellular accumulation of f Tc]MIBI varied widely among tumor cell lines [60] prompted several groups to explore the role of Pgp in transport kinetics of [ Tc]MIBI in tumor cells. Several studies performed in tumor cell lines exhibiting the MDR phenotype, either by selection with cytotoxic agents or by transfection with the MDR recombinant transporters, generally showed a reduced net uptake of [ Tc]MIBI, a feature that is closely related with the outward transport activity of Pgp. 

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