Force Measurements For μ-opiate Receptor And Ligand With AFM On The Living Cells In Physical Solutions

After immobilizing the β-endorphine on the AFM tip and observing the living μ-66 cells, on which μ-opiate receptors were highly expressed, we measured the forces between μ-opiate receptor and β-endorphine and the changes in these forces after the receptor was persistently stimulated by morphine. The aim of the present study is to provide methodological bases for the application of AFM force measurements in receptor pharmacology and reveal some information about whether there is any changes in the property of μ-receptor-ligand interaction.(1) Establishment of the technology to observe the living cells in physical solution with AFMThe basic methods to image living cells in physical solutions with atomic force microscope (AFM) have been established and various factors that affect the quality of the image have been studied. The factors that influence image quality include the non-specific interactions between the AFM tip and the surface of the cells, the elastic constant of the cantilever, the softness of the cells and so on. Several ways have been proposed to solve the problems often encountered in AFM imaging of the living cells in physical solutions, which provided basis for the studies on the other surfacial properties of the living cells. The result mainly include following aspects:(i) The influence of the non-specific interactions between the AFM tip and the surface of the cellsCarrying out experiments in solutions with suitable ionic strength, such as pH 7.4PBS, that shield charges on the surfaces being brought into contact.(ii) The influence of the spring constant of the cantileverThe results of the experiment showed that the living cells would be deformed seriously or detached from the substrate if the spring constant was too large. But the lateral resolution would decrease if the spring constant was too small. So the probes spring constant ranged in 0.02-0.06N/m are recommended for use.(iii) The influence of the softness of the cellsBecause the cell surface is soft, the too large force will make the living cell deform. So during the observing, the force excerted to the tip should be as small as possible. Another method is to increase the hardness by fixation in glutaraldehyde. It is a good choice for some experiments in which higher resolution rather than the activity of cells is needed.(iv) The influence of the cell adhensionThe cells is easy to detach from the substrate during the observing if the cell adhension is poor, and that will effect the observation. To increase the adhension, we used the polylysine and increased the days of cell culture.(v) The influence of the contamination tipIn experiments, we found tips can easily become contaminated during thescanning process. This can result in double tip images or reduction of the lateral resolution. So cleaning is very important if we need to reuse the tip. For some highly strict experiments, had better new tips be used.(vi) The influence of the radius of curvature of the tipThe radius of curvature of the tip, or tip sharpness, determines the lateral resolution. Most of the time, optimal resolution on biological samples requires the possible minimum tip radius. Nevertheless it is preferable to use dull tips rather than sharp tips, when imaging cells for example, because the pressure exerted on the sample by the tips with larger curvature radius is less than those with smaller ones. This makes it possible to prevent the damage of the cell.With the method reported in this paper, the membrane surface of the living cells can be clearly distincted from that of the fixed cells. The membrane of the living cells is intact and smooth while that of the fixed cells is rough and incomplete in their margin.(2) The establishment of the method to measure the force between the opiate receptor and the ligand on the living cell in physical solution with AFMThe method to measure the force between the opiate receptor and the ligand on the living cells in physical solution with AFM include following key aspects:(i) The U.-66 cell cultivation and fixation on the AFM substrateThe cell was maintained at 37 °C and under 5 % CO2 in DMEM-F12 medium supplemented with 10% fetal bovine serum, G418(200/ig/ml), penicillin(50 U/ml) and streptomycin(50 U/ml). The best grown cell was picked for the experiment. Before use, the culture medium was discarded, the cell was washed for 3 times with PBS (PH 7.4) quickly, and the culture dish was fixed on the substrate of the AFM liquid cell after it was manicured into lxl cm pieces.(ii) Cantilever functionalizationThe ligand must be immobilized on the AFM tip to acquire direct measurement of ligand-receptor pairs. 6-endorphine was immobilized to the tip via an extended linker in our experiment. The linker between the tip and the ligand lends greater mobility and access to receptors on the surface being probed. This method can be adopted widely.(iii) Observation of the living fi-66 cellBefore observation, we changed the medium culture in the liquid cell by PBS, adjusted the atmosphere chamber to make the surrounding temperature same to the growing temperature of the cell( 37 °C) and adjusted the parameters. Finishing all of that, we can observe the cell in touch mode.(iv) The record of the force curveA clear image of these cells could be obtained under AFM in touch mode. A single cell was chosen as an the scanning centre and we changed the touch mode to force scanning mode. Every time the tip approaching and leaving the base, a force-distance graph was recorded.(v) The confirmation of the specific receptor-ligand adhensive peaks in the force curvesThe recognition of the specific adhesive peaks in force curves was the key in this experiment. To confirm the specificity, excess naloxone was added to the solution and the adhesive force peaks were eliminated.(vi) Measurement of the force curveBecause more than one B-endorphine were immobilized on the tip, when the tip was approaching the cell surface , one or several receptor-ligand bonds possibly formed. When the tip retracted, the retract trace of the force measurement may exhibit several transitions in force due to the sequential unbinding of multiple receptor-ligand complexes. In our analysis, we considered only the last step of the unbinding as the adhesive force (Fad), because it is conceivable that other steps are a nonlinear convolution of multiple unbinding processes. But it maybe the result of multiple bonds broke simultaneously, so we called it as "comprehensive force". A histogram from a plenty of these comprehensive forces shows three peaks. The first peak and the periodic intervals reflected the individual interaction between the modified tip and an ^-receptor.(3) Unbinding force of/t-receptor and B-endorphine (i) Unbinding force before morphine stimulationThe statistic result of the individual unbinding force is 232.5+29.3 pN.(ii) Unbinding force after persistent morphine stimulation...