Influx assay

Note with filter assays glass fiber filters with adsorbed or Ca must be incubated with scintillator for at least 18 h, with occasional shaking. Only then the cpm/vial will have stabilized. Preincubation of the filters with a solution of 10 mM CaCl2 should inhibit the nonspecific binding of to the filters. 

For the prolis to survive a filtration without losing their content, the temperature is lowered below the phase change point of the phospholipids being used. The filtration procedure resembles those for membrane binding assays (see Section 2.2.3). However, they are different insofar as you need to keep an eye on the stability and tightness of the compartments (and perhaps on ion gradients between compartment and media). The compartments must not be subjected to osmotic stress. 

In indirect methods of influx measurement, a color reaction, fluorescence reaction, enzyme reaction, or density change of the compartments indicate the translocation of the substrate. The indicator molecules are generally inside the compartments, but not in the outer medium. If the substrate enters the vesicles, it reacts with the indicator molecule, the indicator molecule changes its properties, and this change is measured. The reaction with the indicator generally happens quickly, so that with adsorption or fluorescence measurements the substrate intake or loss can be traced continuously. 

Proiis without Na channei do not form a diffusion potentiai 

Of course, the properties of the indicator molecule may only change with the substrate. Thus, membrane-permeating ligands of the translocator— whose effect on the translocator should be ascertained—can (for example) quench the fluorescence of the indicator and thus create the appearance of a change in the substrate translocation. 

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In a second example, researchers at Hoffmann-LaRoche in Basel applied a program they developed called CATS (Chemically Advanced Template Search) to mibefradil, a known 1.7 pM calcium channel blocker. In-house libraries were virtually screened using this pharmacophore search and the highest-ranking molecules went into a real, cellular Ca influx assay. The best of... 

In one example (Lawrence and Casida 1984, Abalis et al. 1985) rat brain microsacs were used to test the action of cyclodiene insecticides such as dieldrin and endrin on the GABA receptors contained therein. The influx of radiolabeled CL into the microsacs via the pore channel of the receptor was inhibited by these chemicals. A similar assay was developed using microsacs from cockroach nerve. Assays with this preparation showed again the inhibitory effect of a cyclodiene (this time heptachlor epoxide) on CL influx. Also, that microsacs from cyclodiene resistant cockroaches were insensitive to the inhibitory effect of picrotoxinin, which binds to the same site on the GABA receptor (Kadous et al. 1983). 

Na Influx Studies. Na influx was monitored according to the procedure of Owen and Villereal (34), with some modifications. Cells were seeded onto 60-mm culture dishes, grown, and serum starved as described for the assays above. The cells were washed with incubation media and incubated in 3 ml of the appropriate agent at 37 C. After incubation the cells were rapidly washed in ice cold 0.1 mM MgCL and extracted with 5% TCA/0.5% KNO3 for sodium determination or 0.2% SDS for protein determination. Sodium concentration was measured using a Varian Model 275 Atomic Absorption Spectrophotometer. Protein was determined fluorimetrically. 

Assay of Na and Ca Influxes. Experiments to determine Na and Ca influxes into PC12 cells were done as reported previously (72). 

Biochemical studies with purified preparations incorporated into liposomes have also been performed [32,33,96-98]. Reconstituted receptors from skeletal muscle bound DHPs, PAAs and diltiazem with high affinity and in a 1 1 1 stoichiometry [97], In general, the reconstituted proteins exhibit the characteristic pharmacological properties expected for these channels. In recent studies, our laboratory has reconstituted partially purified channels into liposomes containing the Ca -sensitive fluorescent dye, fluo-3 [33,96]. These channels exhibit Ca influx that is sensitive to activation by Ca channel activators and inhibitors with affinities similar to those observed in intact cells, and the Ca influx is dependent on the establishment of a gradient in the presence of valinomycin [132]. This assay provides a convenient and rapid approach to obtaining a macroscopic picture of the activity of the channels under different conditions, while the more complex studies in lipid bilayers provide a more complete analysis of the single channel behavior. 

Recent reports have emerged of several TRPV1 antagonists possessing a biaryl amide (14-16), urea (17), or urea isostere (18-20) scaffolds. Bicyclic derivatives 14-16 block capsaicin- or pH-stimulated calcium influx in FLIPR-based assays... 

Figure 4. The transport of by Chlorella under Os stress A. Control efflux from pre-loaded cells. A culture of 38°C-grown C. sorokiniana was preloaded with for 24 hrs. Cells were concentrated by centrifugation, washed, and resuspended as described in Figure 1, and assayed for Rfo loss by millipore filtration. The cells were dried, bleached, and counted by liquid scintillation as described by Fredrick and Heath (24). B. Influx into cells. A culture of exponentially growing C. sorokiniana was centrifuged, washed, resuspended in the Tris-Cl solution (see Figure 1) plus 100 fjM KCl, and placed in a 38°C water bath. At time intervals after RB addition, samples of cells were assayed as described in A.

One of the main in vitro permeability assays used in the pharmaceutical industry has been for many years the Caco-2 monolayer. Therefore, most of the in silica models developed to predict permeability were based on Caco-2 data. Hou and Johnson produced a couple of reviews that comprehensibly summarizes the recent efforts using Caco-2 permeability data [92, 94]. All those models are designed to predict the influx or apparent permeability of drugs in the same direction as intestinal absorption occurs, that is, from the apical to the basal side of the cell line, regardless of the extent of active transport involved in the permeation process. 

A medium throughput approach to evaluating sodium channel activity is the measurement of sodium flux across cell membranes [103]. In these experiments, a tracer that permeates the channel and is easily quantifiable is used to analyze sodium influx. Traditionally, radioactive tracers such as 22Na+ or [14C]guanidinium have been used. Alternatively, Li+ can be used as a tracer and analyzed by atomic absorption spectrometry. Sodium flux assays can be used to test approximately 105 compounds per year. They offer a robust readout of channel activity, but lack voltage control and temporal resolution. To examine sodium channel blockade by measuring sodium flux,... 

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