A Flow Injection Renewable Surface Technique for Cell-Based Drug

Anal. Chem. 1999, 71, 1160-1166

A Flow Injection Renewable Surface Technique for Cell-Based Drug Discovery Functional Assays Peter S. Hodder† and Jaromir Ruzicka*,†

Department of Chemistry, Box 351700, University of Washington, Seattle, Washington 98195-1700

A novel flow injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calciumsensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-a` -vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed. In drug discovery research, functional assays are executed to measure the effect a potential drug candidate has on a living cell, tissue, or organism. Since interfacing living biological material to a sensor can be labor intensive, functional assays are usually conducted by repeatedly exposing the material to different concentrations of drug, rather than using fresh material for each dose. In many biological systems, a repeated exposure of drug has the effect of diminishing the response. This can result from a variety of factors, such as desensitization of the cell receptors1 or degradation of the biological matter itself. If the biological material is relatively robust, “rest” intervals between doses are incorporated into the assay, allowing the cells to return to their predose metabolic state.2 Alternatively, mathematical treatment of the collected data can be performed to account for experimental or biological artifact.3 The former remedy comes at a cost of longer assay times; the latter introduces uncertainty and complexity in the interpretation of data. † (e-mail) [email protected]; [email protected]. (1) Maloteaux, J. M.; Gossuin, A.; Pauwels, P. J.; Laduron, P. M. FEBS Lett. 1983, 156, 103-107. (2) McConnell, H. M.; Owicki, J. C.; Parce, J. W.; Miller, D. L.; Baxter, G. T.; Wada, H. G.; Pitchford, S. Science 1992, 257, 1906-1912. (3) Wang, S. S-H.; Thompson, S. H. Cell Calcium 1994, 15, 483-496.

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The principle of the flow injection-renewable surface (FI-RS) technique is the automated fluidic sampling, assay, and disposal of a suspended material. In this way, the suspended material acts as a renewable surface.4 Although FI-RS technology can be used to automate wet chemistry analyses,5,6 one of the principal research endeavors of this laboratory has been its application to biotechnology assays. In many biotechnology assays, samples and reagents are perishable and/or must be quantitatively retained in a form that is suitable for further analysis. FI-RS techniques address these requirements by allowing the suspended material to be either easily discarded or collected after assay. FI-RS techniques have been developed to execute bioligand interaction assays, using both spectrophotometric and fluorometric detection methods.7-9 In each case, the FI-RS assays have yielded data that complement traditional methods of analysis. The application of FI-RS techniques to cell-based assays has been investigated in our laboratory.10 In addition to the ease of sample handling and reproducibility that is inherent to the FI-RS technique, its application to functional assays has two very specific advantages. The first comes from the renewability of the cellular material: target cells can be discarded after measuring the response to a dose of drug, and a fresh batch of cells can be sampled and assayed for the next dose. Therefore, assay data do not contain artifacts resulting from previous drug doses. In this way, shorter assay times and straightforward data interpretation can be realized. FI techniques can also be used to change the shape of the concentration profile of drug that is delivered to the cells. Because many methods of pharmacologic analysis assume the achievement of equilibrium (or steady state) between receptor and ligand, time-dependent concentration gradients are not typically used in the execution of functional assays. However, the manual delivery of drug to biological matter (typical of pipet-based functional assays) lacks reproducibility. Therefore, the use of FI techniques for precise and reproducible control of drug concentration exposed to the cells could be useful in the determination of pharmacologic response kinetics. (4) Ruzicka, J. Anal. Chim. Acta 1995, 308, 14-19. (5) Egerov, O.; Ruzicka, J. Analyst 1995, 120, 1959-1962. (6) Holman, D. A.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1997, 69, 17631765. (7) Ruzicka, J.; Ivaska, A. Anal. Chem. 1997, 69, 5024-5030. (8) Willumsen, B.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1997, 69, 34823489. (9) Ruzicka, J. Analyst 1998, 123, 1617-1623. (10) Ruzicka, J.; Pollema, C. H.; Scudder, K. M. Anal. Chem. 1993, 65, 35663570. 10.1021/ac981102z CCC: $18.00

© 1999 American Chemical Society Published on Web 02/13/1999

Figure 1. (A) JR chamber schematic of operation: I, stainless steel tube inlet; W, waste tube outlet; and O, the microscope objective. (1) After the FI apparatus syringe pump aspirates the microbead suspension into a holding coil (not shown), the microbeads are delivered to the JR chamber, where they are captured. (2) The FI apparatus aspirates and dispenses a specified volume of drug to the trapped microcarriers, and the biological response is detected by a fluorescence microscope objective. (3) Lifting the inlet tube and ejecting the microbeads from the JR chamber with wash solution completes the assay cycle. (b) Cell-attached microbeads. Photomicrograph of two Cytodex-2 microbeads with CHO-M1 cells growing on the surface.

EXPERIMENTAL SECTION Materials and Reagents. To evaluate the suitability of mammalian cell microcarriers to the FI-RS format, CultiSpher (Percell, A° storp, Sweden) Biosilon (Nunc, Naperville IL), and Cytodex-2 and Cytodex-3 (Amersham-Pharmacia Biotech, Piscataway, NJ) microcarriers were obtained. Dry microcarriers were sterilized as per manufacturer’s instructions and then resuspended in phosphate-buffered saline to make a 20 g/L stock bead suspension. Chinese hamster ovary (CHO) cells (Cricetulus griseus) transfected with rat M1 muscarinic acetylcholine receptor (CRL-1985, American Type Tissue Collection, Rockville, MD) were cultured in 100-mm cell culture dishes (Falcon, Becton-Dickinson, Franklin Lakes, NJ) using recommended mammalian tissue culture protocols. Cells of passage numbers 50-52 were used for FI-RS functional assays. Two to four days prior to functional assay execution, confluent CHO-M1 cells were released from culture dishes by treatment with a trypsin/EDTA solution (Life Technologies, Gaithersburg, MD). Released cells were seeded at appropriate densities (5 × 105-1 × 105 cells/mL) to 100-mm Petri dishes (Becton-Dickinson) containing ∼100 µL of microbeads. After gentle agitation of the microcarrier and suspended cell mixture, dishes were placed in an incubator, where cell attachment and growth on the beads occurred. After cells had reached confluence on the beads, they were washed and resuspended in a probe loading solution consisting of 12 µM fura-2 acetoxymethyl (AM) ester (Molecular Probes, Eugene, OR) in Ham’s F-12 growth media (Life Technologies) buffered to physiological pH. After cells were loaded with fura-2 AM ester, they were washed and resuspended in a perfusion buffer, also consisting of Ham’s F-12 growth media. The beadcell suspension was then transferred to a small glass vial that interfaced with the FI instrument (see below). Tracer experiments were performed with 1-10 µM solutions of fura-2 free acid (Molecular Probes). All muscarinic receptor drugs were obtained from Sigma (St. Louis, MO). For functional assay measurements, stock solutions (100 mM) of the agonists acetylcholine chloride, carbamylcholine chloride (carbachol), and pilocarpine hydrochloride were made up in perfusion buffer and stored frozen (-20 °C). Fresh standards (0.1, 1, 10, 100, 1000 µM) of each agonist were made prior to each functional assay by serial dilution of the stock solutions. For negative control measurements, the antagonist atropine was dissolved in perfusion buffer to a final concentration of 10 µM.

Apparatus. All experiments were conducted using a flow injection instrument equipped with a 1-mL syringe, six-port sampling valve, and a peristaltic pump (FIAlab 3000, Alitea USA, Medina, WA). All fittings and tubing were obtained from Upchurch Scientific (Oak Harbor, WA). The glass vial containing the cellattached microcarriers was mounted on a 12 VDC motor with a spur gearhead (Maxon Precision Motors, Burlingame, CA) and connected to the multiport valve via 0.8-mm-i.d. stainless steel tubing. When the motor was turned on, the rotating vial served as a miniature microbead suspension device, from which aliquots were aspirated into a 500-µL holding coil consisting of 0.8-mmi.d. Teflon tubing. Agonist solution vials, wash buffer containers, and detector flow cell were all connected to the multiport valve via 0.8-mm-i.d. stainless steel tubing. A jet ring chamber (JR chamber) was used to both capture and release microcarriers delivered by the FIAlab apparatus and allow interrogation of the captured microcarriers by a fluorescence microscope objective (Figure 1A). The JR chamber consisted of a 24 VDC solenoid (Guardian Electric, Woodstock, IL) mounted on top of a LabTek tissue culture chamber (Nunc), equipped with a cover glass bottom designed for use with a high-magnification microscope objective. The core of the solenoid was drilled out, and the stainless steel microcarrier delivery tube from the FIAlab multiport valve was fastened to it. By turning the solenoid off, the tube was brought in close proximity (∼100 µm) to the cover glass bottom of the chamber, creating a gap suitable for trapping the beads. Actuation of the solenoid during fluid delivery created a larger gap between the tube and the cover glass bottom, allowing ejection of the trapped beads. Located ∼2 mm from the cover glass bottom was a piece of PEEK 1.0-mm-i.d. tubing that was connected to the peristaltic pump of the FIAlab apparatus, maintaining a constant level of fluid in the JR chamber. All components of the JR chamber were firmly fastened to the microscope stage, which prevented wandering of the microcarrier delivery tube from the objective field of view during repeated solenoid actuation. A dual-monochromator photometry system (Ratiomaster, Photon Technology International, South Brunswick, NJ) and inverted epiluminescence microscope (Axiovert 100, Carl Zeiss, Oberkochen, Germany) were used for fluorescence measurements of intracellular calcium elevation. Excitation wavelengths for the fura-2 fluorescent calcium probe were set to 345 and 380 nm, at a 4-nm Analytical Chemistry, Vol. 71, No. 6, March 15, 1999

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Table 1. FI-RS Functional Assay Procedure FI-RS functional assay step microbead delivery 1 2 3 microbead perfusion 1 2 microbead ejection 1 2 3

Figure 2. Results of an FI-RS functional assay. The delivery of the muscarinic receptor agonist carbachol to trapped CHO-M1 cells on microbeads results in an intracellular calcium elevation. This increase is reflected in the ratio of fluorescence spectra of the intracellular calcium probe fura-2. Also shown is the biological response resulting from delivery of a control dose of the muscarinic receptor antagonist atropine (see text).

band-pass. Light was delivered to the microscope via a bifurcated fiber optic cable. A 20×, 0.75 NA microscope objective (Olympus, Tokyo, Japan) was used to center and focus the trapped microcarriers into the field of view of the photometry system’s photomultiplier tube. All fluorescence spectra data were analyzed with the software utility included with the photometry system. Dose-response curves were constructed with spreadsheet software. Standard error of the mean calculations were performed to assess the reproducibility of dose-response data. FI-RS Functional Assay Summary. A typical calcium response measured by the FI-RS functional assay is illustrated in Figure 2. Prior to data collection, a well-defined volume of microbead suspension was aspirated from the rotating vial. Data collection began when delivery of the suspension to the JR chamber was initiated. The JR chamber quantitatively trapped all of the delivered microbeads (∼300) in the internal diameter of the stainless steel tube, forming a small column of ∼2 mm in height. The syringe pump and multiport valve were then used to aspirate and deliver a dose of agonist to the trapped cells. As the agonist reached the JR chamber, it bound to M1 receptors on the surface of CHO-M1 cells, resulting in an elevation of the cytosolic calcium concentration. This intracellular calcium response was measured as a rise in the ratio of the emitted fluorescence from the selected excitation wavelengths of the fura-2 probe.11 To end the assay, the syringe was loaded with perfusion buffer, the solenoid was turned on, and the beads were flushed from the photomultiplier tube’s field of view. The assay was repeated for the different concentrations of each agonist. The individual steps of the FIAlab program used in the FI-RS functional assay are shown in Table 1. RESULTS AND DISCUSSION Choice of Functional Assay. Central to the success of the FI-RS functional assay was the choice of a biological material to be used as a renewable surface. With the evolution of sophisticated recombinant DNA technologies and sensitive assay techniques, (11) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 34403450.

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4

explanation of step

vol (µL)

flow rate (µL/s)

aspirate perfusion buffer aspirate microbead suspension deliver microbeads to JR chamber

700 250 950

50 10 5

aspirate agonist deliver agonist to JR chamber

300 300

75 1

aspirate perfusion buffer turn JR chamber solenoid on deliver perfusion buffer to JR chamber turn JR chamber solenoid off

1000

50

1000

150

the use of eucaryotic cell lines as the biological material for functional assays has become a popular.12 For the FI-RS functional assay research, CHO cells transfected with the rat muscarinic type 1 (M1) acetylcholine receptor served as the biological material. Muscarinic receptors are pharmacologically well-characterized targets for the discovery of neurological and gastrointestinal pharmaceuticals,13 and different types of functional assays have been conducted with the CHO-M1 cell line. In this way, the CHOM1 cells served as a model system for which the performance of the FI-RS system could be evaluated. Microbead Selection. To employ the cells as a renewable surface, they were cultured on a variety of different commercially available microcarriers (see Experimental Section). Excellent cell growth was observed with all microcarrier brands. Cytodex-2 microbeads were determined most useful for the FI-RS functional assay, since their spherical shape and compressibility prevented clogging of the FIAlab instrument. In addition, the transparency of the Cytodex-2 facilitated observation of the cell growth kinetics with a brightfield microscope. When cells reached confluence on Cytodex-2, many (50-100) cells were visible on a single (∼200-µm diameter) microbead (Figure 1B). Therefore, the microbeads preconcentrated cells in a well-defined area, suitable for sensitive measurements in the FI-RS functional assay. In addition, the microbeads packed closely in the JR chamber, further increasing the amount of cells visible to the detector. To maximize the signal-to-noise ratio (S/N) and improve assay reproducibility, assays were conducted only after cells had reached confluence on the microbeads. The S/N of the FI-RS functional assay was found to be significantly improved over an identical functional assay conducted in our laboratory that used cultured cell monolayers on cover slips as a renewable surface. FI-RS Functional Assay Design and Optimization. In FI techniques, the size, shape, and height of the resulting FI peak measured depends on the internal diameter of the tubing, the volume of sample injected, and the flow rate of the carrier stream.14 For FI-RS assays, the volume of renewable surface trapped in the JR chamber could also have an effect on peak characteristics.7 (12) Schwarz, R. D.; Davis, R. E.; Jaen, J. C.; Spencer, C. J.; Tecle, H.; Thomas, A. J. Life Sci. 1993, 52, 465-472. (13) Katzung, B. G. Basic Clinical Pharmacology, 5th ed.; Appleton and Lange: Norwalk, CT, 1992. (14) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; Wiley: New York, 1988.

Figure 3. Effect of flow rate on concentration gradient of agonist. Fluorescence ratio spectra resulting from the injection of 300 µL of 5 µM fura-2 free acid dye at different flow rates to CHO-M1 cells trapped inside the JR chamber. Increasing the flow rate from 2 to 6 µL/s results in a more rapid attainment of steady-state fluorophore concentration in the JR chamber.

Figure 4. Effect of captured microbead volume on calcium response. Calcium responses are measured after the delivery of decreasing volumes of CHO-M1 cell-attached microbead suspension to the JR chamber. Calcium responses obtained by delivering 300 µL of 10 µM carbachol at a flow rate of 1 µL/s.

Each of these parameters was investigated and optimized. To prevent clogging, 0.8-mm-i.d. tubing was used, and a sample volume of 300 µL was chosen, since at the FI peak maximum it yielded a final concentration of agonist in the JR chamber equal the concentration of agonist before the dispersion process. The effect of flow rate on the dispersion of the injected agonist volume was investigated by constructing “tracer curves” that mimicked the agonist concentration profile (Figure 3). As expected from classical FI principles, increase in the flow rate of injected agonist varied inversely with the residence time of the injected tracer volume and, therefore, shortened the arrival time of the tracer peak maximum. This also had a concomitant relationship to the “rise time” of the injected tracer curve: a higher flow rate resulted in a faster attainment of the peak maximum. At the selected flow rates, all the tracer peaks reached a steady-state signal, showing that an increase in flow rate increased the amount of injected agonist per unit time delivered to the cells, but not the final concentration of agonist. To investigate the effect that the amount of trapped cells in the JR chamber would have on the measured calcium response, varying volumes of bead suspension were injected and then perfused with 10 µM carbachol (Figure 4). A slight decrease in S/N with decreasing injection volume was attributed to the smaller amounts of microcarriers that were trapped in the JR chamber and therefore the volume of beads probed by the microscope objective. This resulted in lower, noisier fluorescence intensities

in the individual excitation spectra, which propagated to the ratio spectra. However, these changes did not significantly effect the arrival time of the calcium response or its shape, showing that the detection method was insensitive to the volume of beads captured in the JR chamber. A final concern in design of the FI-RS functional assay was the elimination of artifacts that would give false positive biological responses. Hydrodynamic flow produces shear stresses that can result in intracellular calcium responses unrelated to drugreceptor interactions.15 Therefore, the effect that the flow rate of injected agonist had on calcium response was investigated. This effect could be measured on the FI-RS system by exposing cells trapped inside the JR chamber to a “blank” dose of perfusion buffer. In all flow rates investigated (1-6 µL/s), no shear stress responses from the cells were observed. Another artifact could result from interaction between the agonist and other types of receptors located on the cell surface; this would elicit calcium responses unrelated to the binding of the muscarinic receptor. Since the muscarinic receptor is not normally expressed in the CHO cells, this type of artifact was assumed to be of no consequence. A control experiment where the muscarinic receptor antagonist atropine was delivered to trapped cells proved the correctness of this assumption (Figure 2). Evaluation of the FI-RS Functional Assay as an Analytical Method. The success of the FI-RS functional assay in producing useful pharmacological data was evaluated by assaying different concentrations of carbachol, a muscarinic receptor agonist (Figure 5A). Interestingly, not only did the calcium response peak maximum (the maximum amount of calcium fluxed) correlate with the concentration of injected agonist but also a correlation was seen with the peak area (the total amount of calcium fluxed). Also apparent in the response spectra were other important pieces of information: reproducible, dose-dependent differences in the arrival time and rate of onset and decay of the calcium response, signifying the presence of biological kinetic information. To visualize the relationship between agonist concentration present in the JR chamber and the calcium response measured, a tracer curve of 5 µM fura-2 free acid solution was injected in place of the agonist. As can be seen in the figure, the concentration profile of the injected agonist approached its maximum concentration in a gradual, linear fashion. Within a given batch (passage number) of cell suspension, assay results were highly reproducible. To demonstrate batchto-batch reproducibility, the functional assay was repeated for three different batches of CHO-M1 cells (spectra not shown). The peak maximum and peak area of the calcium responses were quantified and used to construct dose-response curves. For each batch of cells, the baseline signal (ratio or peak area) was subtracted from each response and then all responses were normalized as a percent of the maximum response. The percent of maximal response was then graphed versus the log of the agonist concentration, as typical for dose-response curve construction.16 Averages of the three different dose-response curves are shown in Figure 5B. Since the three batches of CHO cells were of successive passage numbers, i.e., three successive generations of cells, differences in receptor expression and cell senescence (15) Yang, X. C.; Sachs, F. EXS 1993, 66, 79-92. (16) Tallarida, R. J.; Jacob, L. S. The Dose Response Relation in Pharmacology; Springer-Verlag: New York, 1979.

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Figure 6. Ranking of different muscarinic receptor agonists with the FI-RS functional assay. Dose-response curves constructed by assaying three different muscarinic agonists, acetylcholine (ach), carbachol (cch), and pilocarpine (pilo) with CHO-M1 cells. See discussion in text and also Figure 6.

Figure 5. Results of the FI-RS functional assay: Carbachol. (A) The calcium responses resulting from injecting different concentrations (0-1000 µM) of muscarinic receptor agonist carbachol (cch) to CHOM1 cells trapped inside the JR chamber. A fresh set of cell-attached microbeads was used for each dose of cch. A tracer curve of 5 µM fura-2 free acid is also displayed to show the concentration of cch inside the JR chamber during an assay. (B) Dose-response curves resulting from conducting the FI-RS functional assay on successive generations of CHO-M1 cells. Calcium responses were normalized to the maximum response.

would affect the reproducibility of the assay results. With both methods of data analysis, the effective concentration of agonist that gave half the maximal response (EC50) was determined to be ∼1 µM. This value compares favorably to those found in the literature (2-5 µΜ), which were quantitated with a variety of different assay techniques.2,17,18 Efficacy Ranking of Muscarinic Receptor Agonists. By comparing the EC50s of different agonists, drug candidates can be ranked in terms of efficacy. To evaluate the FI-RS technique’s ability to rank the EC50s of different drugs, three different muscarinic receptor agonists (acetylcholine, carbachol, pilocarpine) were assayed. The resulting dose-response curves are shown in Figure 6. Although EC50 values have not been published for acetylcholine using this CHO-M1 biological system, the EC50s calculated from the FI-RS functional assay follow expected trends. Acetylcholine and carbachol are both considered full agonists, capable of producing a maximal response. Also, acetylcholine is considered more efficacious; i.e., a lower concentration of acetylcholine is necessary to produce a half-maximal response.13 This is reflected in the dose-response curves: the acetylcholine and carbachol both elicited the maximal response from the CHO-M1 cells, and acetylcholine’s EC50 of ∼0.3 µM was lower than (17) Buck, M. A.; Fraser, C. M. Biochem. Biophys. Res. Commun. 1990, 173, 666-672. (18) Baxter, G. T.; Young, M. L.; Miller, D. L. Owicki, J. C. Life Sci. 1994, 55, 573-583.

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carbachol’s EC50. Also, the FI-RS functional assay results distinguished pilocarpine as a partial agonist with low efficacy for the M1 receptor: it could only achieve 90% of the maximal response measured for the full agonists and had the highest EC50 (∼5 µM) of all agonists assayed. Although pilocarpine has not been previously assayed with CHO-M1 cells, research with transfected human muscarinic receptors has defined pilocarpine as a partial agonist.19 Kinetic Discrimination of Agonists. Because the FI-RS technique allows precise control of the agonist concentration with respect to time, it is useful for studying the kinetics of biological processes. For example, highly reproducible time differences in the time delay of the onset of calcium responses (i.e., latency) were apparent both in the experimental data for a single agonist at different concentrations (Figure 5A) and for different muscarinic receptor agonists at the same concentration (Figure 7). This latency increased with decreasing agonist concentration and also decreasing agonist efficacy. Other researchers have related this latency to the kinetics of the calcium response signal transduction.20,21 Although latency of response has been observed in these pipet-based assays, the desensitization of response and imprecision in fluidic control complicates the fitting of experimental data to mathematical models. In addition to latency, other interesting kinetic phenomena related to the delivery of agonist to the JR chamber were observed in the FI-RS functional assay. In all assays, a linear gradient of agonist concentration was delivered to the JR chamber, reaching its maximum concentration at ∼500 s (Figure 5A). However, calcium responses appeared prior to the arrival of the maximum agonist concentration. This reproducible difference in time between the onset of the calcium response and the arrival of the maximum concentration of agonist was more pronounced at higher concentrations of agonist. Also observed was that all calcium responses at a given concentration of agonist appeared at roughly the same time, regardless of agonist type, perhaps signifying that a threshold concentration of agonist was necessary to elicit a response. This kinetic discrimination cannot be measured using pipet-based assay techniques, due to poor fluidic (19) Baumgold, J.; Dyer, K., Falcone, J. F.; Bymaster, F. P. Cell. Signalling 1995, 7, 39-43. (20) Mahama, P. A.; Linderman, J. J. Ann. Biomed. Eng. 1995, 23, 299-307 (21) Wang, S. S-H.; Alousi, A. A.; Thompson, S. H. J. Gen. Physiol. 1995, 105, 149-171.

Figure 7. Kinetic discrimination of different muscarinic receptor agonists by the FI-RS functional assay. Each graph shows the calcium responses resulting from delivering a specified concentration of the different agonists acetylcholine (ach), carbachol (cch), and pilocarpine (pilo) to CHOM1 cells trapped inside the JR chamber. Different muscarinic receptor agonists can be distinguished on the basis of a variety of kinetic parameters. See discussion in text.

control. It is interesting to note that this control of the drug concentration profile delivered to the JR chamber mimics physiological dosing mechanisms in living organisms, where gradual release and subsequent dilution of a drug into the bloodstream occurs prior to drug-receptor interaction. Comparison of the tracer and calcium response curves of the FI-RS functional assay provides some insight into the relationship between the binding event of the receptor by the agonist and the subsequent eliciting of response. The tracer curve shows that only a fraction of the maximum concentration of agonist is present in the JR chamber when a calcium response is evoked. In terms of classical pharmacological theory, this observation could signify that only a small fraction of all receptors on the cell surface need to be bound by agonist in order to elicit a maximal calcium response. This is further supported by comparison of the calcium responses of different agonists at the same concentration: the more efficacious the agonist, the quicker the onset of the response; i.e., a lower amount of agonist was necessary to elicit a response. Since a fresh, random sample of CHO-M1 cells was aspirated from the cell suspension device for each dose of agonist, and the calcium response was measured from a large population of cells, it can be assumed that the quantity of receptors and the biological signal transduction mechanism did not change during the assay. The differences in the onset of calcium responses could be related to the one or more of the following: (1) a “spareness” of M1 receptors (i.e., the receptors were not the limiting reagent in the ensuing biological response mechanism), (2) different affinities of the agonists for the M1 receptor, and/or (3) different efficacies of the agonists in eliciting the response. Another interesting observation was the dose-dependent shape of the calcium response curves. At lower concentrations of dosed agonist, very gradual increases in calcium occurred, while higher concentration doses resulted in sharper, more pronounced increases. Also, there was a difference in the decay rates of the calcium response. Since the rate of increase or decrease in the calcium response is proportional to rate of intracellular calcium

production or destruction, yet another kinetic parameter can be quantified in the response spectra of the FI-RS functional assay. CONCLUSIONS This paper introduces a novel methodology for performing cellbased functional assays. The FI-RS functional assay provides automated and highly reproducible fluidic manipulation of the cellular material and drug stimulant and sensitive, real-time collection of data that is free of biological artifacts. Further, the automated perfusion provided by the FI instrument allows precise control of an agonist’s concentration profile, which has not been previously possible with other automated perfusion-based functional assays. The use of microbeads as a disposable and renewable surface circumvents the problems of conventional mammalian cell functional assays that involve repetitive stimulations of the same group of cells. The results obtained from this instrument compare well with those of conventional functional assay techniques. In addition to producing results comparable to those published in the literature, the FI-RS technique yields new pharmacological information, since it can reproducibly measure the initial kinetics of the biological response. Therefore, data from these assays can provide a wealth of information: the response curves can be analyzed not only for traditional functional assay parameters (e.g., calcium response height) but also in terms of how long it takes the response to develop, what concentration of agonist is necessary to trigger a response, and the shape of the response (i.e., the rate of onset, sustainment, and decay of intracellular calcium concentration) at different drug doses. This complex behavior can be expected from the myriad of interconnected biochemical processes that exist in biological systems; mathematical analysis of these response curves will reveal the underlying kinetics of cellular response. In addition, analysis of kinetic parameters such as initial rate of the calcium response can be used as a rapid analytical method for discrimination of different drugs. Analytical Chemistry, Vol. 71, No. 6, March 15, 1999

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ACKNOWLEDGMENT The authors thank Profs. Craig Beeson, Gary Christian, and Dr. Louis Scampavia (Department of Chemistry, University of Washington) and Pat McKernan (ZymoGenetics, Seattle, WA) for thoughtful discussions. The authors are indebted to ZymoGenetics for use of its tissue culture facilities. This research was funded

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by National Institutes of Health General Medical Sciences Grant RO1GM45260. Received for review October 6, 1998. Accepted January 7, 1999. AC981102Z