Introduction

Cell membrane receptors, mainly G-protein-coupled receptors, are currently the largest subgroup (45%) of all drug-target families [1]. Drugs targeting adrenergic receptors (ARs), members of the G-protein-coupled receptor family, are among the most widely used therapeutic agents in clinical medicine. Both α 1 agonists and antagonists have limited success in pharmacological treatment, partly because of undesirable cardiovascular effects of non-selective α 1 -adrenoceptor stimulation. In the last two decades successful cloning of three α 1 -ARs subtypes (α 1A , α 1B , and α 1D -adrenoceptors) has undoubtedly contributed to our better understanding of the functional role of α 1 -ARs subtypes [2]. It is acknowledged that the predominant α 1 -AR subtype in human prostate muscle is α 1A subtype [3]. Because of their less frequent systemic side-effects compared with conventional α 1 -adrenoceptor blockers, several selective α 1A -adrenoceptor antagonists (e.g. tamsulosin and others) have recently been developed for treatment of benign prostatic hyperplasia (BPH) by relaxing smooth muscle in the bladder neck, prostate capsule, and prostatic urethra. These drugs may reduce the urinary obstruction in BPH by reducing prostatic urethral resistance, and selective α 1A -AR agonists have been evaluated for stress incontinence [4, 5]. As an excellent target, the function, distribution, and species discrepancy of the α 1A -adrenoceptor have been studied using a variety of experimental methods for many years. Evaluation of ligand-binding properties and screening of drug candidates for their α 1A -AR binding affinities remain a formidable task, however.

Cell-membrane chromatography (CMC), a novel bio-affinity chromatographic technique introduced by He et al. in 1996, can be used simply and conveniently to observe binding of drug to receptor under dynamic conditions [6, 7]. The cell-membrane stationary phase (CMSP) was prepared by immobilizing cell membrane on the surface of silica, which acted as a carrier, and then used for rapid on-line chromatography. The characteristics of drug-receptor interactions can be determined from chromatographic data. CMC is expected to be a reliable alternative method for initial study of ligand-receptor interactions in which chromatographic separation and receptor-based assays could be performed simultaneously during screening for biologically active substances. Since 1996, our laboratory has been developing an on-line approach for effective screening of components from Chinese traditional natural medicine and herbs using affinity chromatographic techniques and stationary phases containing immobilized trans-membrane receptors [8–13]. Use of homogenized tissue cell membranes in which receptors are present at relatively low densities has, however, previously limited the sensitivity and specificity of CMC [14–16].

The attractiveness of obtaining large amounts of bioinformation while using extremely small volume of sample, and improving sensitivity and specificity, has inspired the extension of cell-membrane chromatography to subtype receptors. We have previously reported on use of cell lines stably overexpressing α1A and α1B-ARs subtypes to prepare subtype receptor CMSP for on-line chromatographic analysis of ligand-receptor interactions [17]. The resulting cell-membrane stationary phase enabled correct determination of the relative affinity rank order of binding affinities of nine test ligands.

The objective of the work discussed in this paper was to determine whether use of cell lines stably overexpressing subtype receptors led to improved sensitivity and specificity of cell-membrane chromatography (CMC), in comparison with homogenized tissue and cells in primary culture. In this work the cell-membrane stationary phases were prepared from homogenized tissue, from cells in primary culture, and from cell lines stably overexpressing subtype receptors. The retention times of nine α1-adrenoceptor ligands and calculated capacity factors, used as a measure of ligand-receptor affinity, were carefully recorded and analyzed. The chromatographic results obtained from the HEK293 α 1A -AR CMSP were compared with those from the CMSP prepared from rabbit hepatocytes α 1A -AR CMSP and rabbit liver homogenate α 1A -AR CMSP. The effects of mobile-phase ionic strength, sample concentration, and the presence of competitive agents on ligand-receptor interactions were also evaluated.

It has been proved by RLBA that rabbit liver expresses predominantly the α 1A -AR subtype [18, 19]. The relative levels of α 1 -adrenoceptor subtype mRNA in rabbit liver are α 1a (96.1 ± 62.7), α 1b (<0.1), α 1d (<0.1) taking the level of α 1b mRNA in cerebellum as unity [20]. The mRNA of rabbit α 1A adrenoceptor is much more abundant in liver than in other tissues, which agree with radioligand binding data [21, 22]. These results confirmed that rabbit liver membrane preparations could be used to evaluate the affinity of compounds for α 1A -AR.

Materials and methods

Chemicals and reagents

Prazosin, phentolamine, norepinephrine, phenylephrine, methoxamine, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4,5]decane-7,9-dionedihydrochloride) (BMY7378), trypsin, l-histidinol, and hygromycin were purchased from Sigma (St Louis, MO, USA). N-(2-(2-cyclopropylmethoxy)ethyl)-5-chloro-α-dimethyl-1H-indole-3- thylamine (RS-17053) was provided by Roche Bioscience (Palo Alto, CA, USA). Oxymetazoline and 5-methylurapidil (5-MU) were purchased from Research Biochemical International (Natick, MA, USA). Macroporous spherical silica (7-μm particles, 10-nm pore size) was purchased from the Institute of Chemistry of the Chinese Academy of Sciences (Beijing, P.R. China). Water was of HPLC grade.

Instrumentation

Chromatography was performed with a Waters (Milford, MA, USA) high-performance liquid chromatograph. A Hermel ZK401 high-speed refrigerated centrifuge (Berthold Hermel, Gosheim, Germany), a model TB-85 thermostatted bath (Shimadzu, Kyoto, Japan), and a CS-20 ultrasonic cleaner (Shimadzu) were also used in this study.

Generation of cell lines stably overexpressing subtype receptors

In brief, the pREP8 plasmid with bovine brain α 1a -AR cDNA was transfected into human embryonic kidney 293 (HEK 293) cell lines by the calcium phosphate coprecipitation method. HEK 293 cells stably expressing α 1A , cultured in monolayer in Dubelco’s Modified Eagle Medium (Gibco-BRL, Rockville, MD, USA) with 4.5 g L−1 glucose supplemented with 10% fetal calf serum (FBS), penicillin (50 U mL−1), streptomycin (50 μg mL−1) and propagated for several weeks in the presence of 0.6 mg mL−1 l-histidinol, were seeded into culture flasks (Corning, New York, NY, USA) and grown in a 95% humidified air-5% CO2 incubator at 37 °C. Native HEK 293 cells were cultured in other flasks as a receptor-minus control under identical conditions.

The transfected HEK293 cells were screened and cloned in selection medium and their α 1A -AR was approved. The clonal HEK293 cell lines stably expressing α 1A -AR (643 ± 95 fmol mg−1 protein) was determined by saturation analysis of the binding of the α1-AR antagonist [125I]BE2254 [23].The receptor densities were satisfactory for the requirements of this study.

Tissue sampling and preparation of rabbit liver homogenate

New Zealand rabbits were caged individually with free access to food and water for 1 week before use. Rabbits were sacrificed by exsanguination and the liver was removed immediately. The liver tissue was washed thoroughly, cut into small pieces, and added to ice-cold phosphate-buffered saline (PBS). Rabbits liver pieces were suspended in 50 mmol L−1 Tris-HCl, pH 7.4, and homogenized for 30 s with a Polytron homogenizer. The homogenate was filtered through four layers of cheesecloth and centrifuged at 400 g for 10 min at 4 °C and the cell pellet was removed. The supernatant was collected and centrifuged at 4000 g for 10 min at 4 °C. The pellet was resuspended in ice-cold PBS buffer and the washing procedure was repeated three times. The rabbit liver homogenate and resulting cell pellet were prepared just before experiments.

Isolation and in-vitro primary culture of rabbit hepatocytes

Hepatocytes were isolated from male New Zealand white rabbits (2–3 kg) by use of the two-step collagenase digestion method as described elsewhere [24]. Briefly, the portal vein of the rabbit was cannulated and rabbit livers were first perfused with calcium-free buffer containing 5.5 mmol L−1 glucose and 0.5 mmol L−1 EGTA equilibrated with 95% O2/5% CO2 for 10 min at a rate of 30 mL min−1 and then with buffer containing 1.5 mmol L−1 calcium and collagenase type-IV (0.7 U mL−1) for 25 to 30 min at a flow rate of 30 to 35 mL min−1. The liver was removed from the rabbit and immersed in ice-cold medium (DMEM containing 5% FBS, 50 U mL−1 penicillin, 50 μg mL−1 streptomycin, 4 mg L−1 insulin, and 1 μmol L−1 dexamethasone). The hepatocytes were released from the liver (by gently tearing the capsule surrounding the liver) and the suspension obtained was filtered through a 100-μm pore size Nylon mesh and then centrifuged at 50 g for 2 min at 4 °C. The resulting cell pellet was resuspended in fresh supplemented DMEM and the washing procedure was repeated three times. The culture medium was supplemented with 10% FBS during the first 4 h after plating to improve the attachment of cells. Hepatocytes (1.0×105) were added to 5 mL supplemented DMEM in a 25-mL plastic disposable tissue-culture flask (Corning, New York, NY, USA). The medium was then replaced every 24 h. Cultures were maintained at 37 °C in a humid atmosphere of air and 5% carbon dioxide. The viability of the freshly isolated rabbit hepatocytes, as assessed by the trypan blue exclusion test, was 90% or greater.

Membrane preparation

In the same way as for HEK293 α 1A -AR cell and rabbit hepatocytes in primary culture, digestion with trypsin is necessary to dislodge the cells from the flask bottom before the experiment. Cells at confluence were washed twice with PBS solution and detached from the flasks by incubation for 10 min at 37 °C with 0.25% trypsin, with gentle agitation to dislodge adhering cells. The cell suspension was then centrifuged at 4000 g for 10 min. Pellets identical in quality from homogenized tissue, hepatocytes in primary culture, and cell lines were then resuspended in hypotonic distilled water and vibrated for 20 min in an ultrasonic cleaner. The membrane suspension obtained was recentrifuged at 12000 g for 10 min. The concentration of the membrane protein in the suspensions, determined by the Lowry method [25], should be more than 1 mg mL−1. All procedures were performed at 4 °C. Cell-membrane suspension was usually prepared from 8×107 cells or 3–5 g tissue. The CMSP for one column was prepared from 1 mL cell-membrane suspension (2 mg membrane protein per 1 mL suspension).

Preparation of CMSP and rapid on-line chromatography

Cell-membrane stationary phase (CMSP) was prepared by immobilizing cell membrane on the surface of silica (7-μm particles, 10-nm pore size), as carrier, and used for the rapid on-line chromatography. The preparation procedure was similar to that described previously [6, 7, 17]. In brief, silica activated at 120 °C, then the suspension of the cell membranes, were placed in a 10-mL reaction tube. All three CMSPs were prepared by use of 1 mL cell-membrane suspension with an identical amount of membrane protein. Adsorption of the cell membranes on the activated silica surface was conducted at 4 °C until equilibrium was reached. The whole adsorption process was performed under vacuum with ultrasonication. The reaction mixture was then diluted with an equal volume of deionized water. The phospholipids of the cell membranes were able to fuse spontaneously with each other (self-fusion) on the silica surface in the aqueous solution until a resealed cell membrane layer was obtained. The residual free cell membranes were washed out with Tris-HCl buffer (pH 7.4). The prepared CMSP particles in the buffer were then packed into a chromatography column (50 mm×2 mm, i.d.) under low pressure by the slurry method and equilibrated with at least 100 mL 5 mmol L−1 phosphate-buffered saline solution buffer (pH 7.4) for 3–4 h at 37 °C before different ligand runs.

The CMSPs prepared from rabbit liver homogenate, rabbit hepatocytes in primary culture, and cell lines stably overexpressing subtype receptors were used for rapid on-line chromatographic determination of the binding affinities of nine α 1 -adrenoceptor ligands for the subtype receptor. At the same time, the receptor minus control CMSP was also prepared by using the same method. All the drugs were dissolved in thrice-distilled water. The mobile phase was pumped through the column at a flow rate of 0.5 mL min−1 at 37 °C and the ligands were detected on-line by ultraviolet absorbance at 280 nm. On the basis of results from pre-experiment trial runs, 5 μL sample was injected into the sample loop, except for the rabbit liver homogenate α 1A -AR chromatographic column, for which sample size was 10 μL. The nine ligands, concentration of 3×10−3 mol L−1, were drawn by 10-μL syringes with blunt-tip needles and loaded into the sample loop. All solvents were filtered through a 0.45 μm membrane filter.

Mobile phases of different ionic strength were obtained by changing the concentration of phosphate buffer from 1 mmol L−1 to 200 mmol L−1 while maintaining the pH at 7.4. RS17053, a highly subtype-selective antagonist of the A subtype of the α 1 -adrenoceptor, was dissolved in thrice-distilled water and serial dilution was used to prepare samples of concentration 3×10−4, 1×10−3, 3×10−3, 6×10−3, and 9×10−3 mol L−1. Elution profiles were obtained by pumping these dilute samples through the HEK293 α 1A CMSP. The effect of the absence or presence of competitive antagonist on ligand-receptor interaction also was taken into account. When 0.01 mmol L−1 prazosin was added to the phosphate buffer, the chromatographic column was equilibrated again with the previous mobile phase. After equilibration with mobile phase containing prazosin for 3–4 h at 37 °C the retention of RS17053 is determined after setting the elution profile to near zero baseline. Standard deviations were obtained by using retention volumes from three or four runs.

Data analysis

The retention times of the nine α 1 -adrenoceptor ligands and the calculated capacity factors, as chromatographic data, were recorded carefully. The relationships between the three CMSP-based capacity factors were analyzed by correlation analysis. Correlation constants (r) were calculated using SPSS software (8.0 for Windows; SPSS; Chicago, IL, USA). All data were expressed as means±SEM and values of P < 0.01 (two tailed) were regarded as indicative of statistically significance. A graphical representative profile for CMSP-based capacity factors was generated using SigmaPlot software (SPSS). The resulting data were smoothened by use of Microsoft (Redmond, WA, USA) Excel software. The mean and standard deviations of retention times were obtained from three or four runs.

Results

Capacity factors were calculated using the formula k′=(t R−t 0)/t 0, where t R is the retention time of the ligands and t 0 is the retention time of the solvent (n = 3 or 4). The capacity factor k′ is specific for a given substance and reflects the ratio of the mole fractions in the stationary and mobile phases, at equilibrium. It depends on the stationary phase, the mobile phase, the temperature, quality of the packing, etc. In CMC experiments, because the silica surface was completely coated by cell membrane in CMSP [6, 7, 17], capacity factors were used to describe ligand-receptor affinity, i.e. the larger the capacity factor the stronger the affinity. The immobilized subtype selective CMSP was stable and reproducible.

Effects of the ionic strength of the mobile phase, the concentration of the sample, and the presence of competitive agent on ligand-receptor interaction

The effects of mobile-phase ionic strength, sample concentration, and the presence of competitive agent on the binding affinities of RS-17053 were determined with an HEK293 α 1A cell-membrane chromatographic column. The retention time of RS-17053 was >32 min at low ionic strength (1 mmol L−1 phosphate buffer) and decreased as the ionic strength of the mobile phase was increased (Fig. 1). The retention time of RS-17053 (3×10−4 mol L−1) was 33.38 ± 0.44 min and decreased to 17.55 ± 0.35 min when the RS-17053 concentration was increased to 9×10−3 mol L−1 (Fig. 2). The binding of RS-17053 to the α 1A -AR involved in the HEK293 α 1A cell-membrane stationary phase could be reduced in competitive displacement experiments by using a known competitive agent in mobile phase. The retention time of RS-17053 was also reduced from 22.00 min to 16.14 min when a selective α 1 -adrenoceptor antagonist, prazosin (0.01 mmol L−1), was added to the mobile phase, and fell further when the prazosin concentration was increased (Fig. 3).

Fig. 1
figure 1

Effect of mobile-phase ionic strength on the retention time of RS-17053 on HEK293 α1A-AR CMSP in cell-membrane chromatography. The error-limit bars indicate maximum differences between retention times in different runs (n = 3 or 4) for mobile phase of a given ionic strength. Mobile phases of different ionic strengths were obtained by changing the concentration of phosphate buffer (1, 5, 20, 50, 100, and 200 mmol L−1) while maintaining the pH at 7.4

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Fig. 2
figure 2

Effect of sample concentration on the retention time of RS-17053 on HEK293 α1A-AR CMSP in cell-membrane chromatography. RS-17053 was dissolved in thrice-distilled water and samples of 3×10−4, 1×10−3, 3×10−3, 6×10−3, and 9×10−3 mol L−1 were prepared by serial dilution. The error-limits bars indicate maximum differences between retention time in different runs (n = 3 or 4) for sample of a given concentration

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Fig. 3
figure 3

Elution profiles of RS-17053 (3×10−3 mol L−1) in HEK293 α1A-AR CMSP (37 °C) in the absence (profile A) and presence of prazosin (0.01 mmol L−1, profile B, and 0.05 mmol L−1, profile C) in the mobile phase (PBS solution, 5 mmol L−1, pH 7.4). The flow rate was 0.5 mL min−1 and the detection wavelength 280 nm

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Determination of capacity factors on CMSPs by use of CMC

The capacity factors determined by rapid on-line chromatography were in the order prazosin (307 ± 14)>RS-17053 (271.2 ± 7.6)>5-methylurapidil (232.4 ± 3.8)>phentolamine (209 ± 27)>oxymetazoline (184 ± 13)>norepinephrine (110 ± 17)>BMY7378 (61.4 ± 2.3)>phenylephrine (34.7 ± 3.6)>methoxamine (14.164 ± 0.075). These calculated capacity factors for the HEK293 α 1A -AR CMSP (k′ HEK293 α 1A ) correlate positively with published pK i values obtained from radioligand binding assays using homogenous membrane and were in the same order as published pK i values. No specific binding was detected in the receptor-minus control CMSP. The resulting cell-membrane stationary phase correctly determined the order of relative affinity of the binding affinities of the nine test ligands reported elsewhere [17]. Other capacity factors calculated using corresponding CMSPs are listed in Table 1.

Table 1 Comparative analysis of capacity factors on the three CMSP in CMC (x±s)
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Comparative analysis of three CMSP-based capacity factors

It is apparent from Tables 1 and 2 and from Fig. 4 that there were strong positive correlations between the capacity factors obtained on the three CMSP by use of CMC (r = 0.969, 0.961, 0.886; P < 0.01). The capacity factors were in the same order, albeit with minor variations. In these experiments, selective α 1 -adrenoceptor antagonist prazosin, which has the strongest affinity, ranked first in the list. RS-17053 and 5-methylurapidil, highly subtype-selective antagonists of α 1A -adrenoceptor, always ranked second and third.

Table 2 Results from correlation analysis of three CMSP-based capacity factors
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Fig. 4
figure 4

Results from comparative analysis of capacity factors on three CMSP in CMC (from high to low). Abbreviations used in figure: Praz, prazosin; RS, RS-17053; 5-MU, 5-methylurapidil; Phentol, phentolamine; Oxy, oxymetazoline; Nor, norepinephrine; BMY, BMY7378; Phenyl, phenylephrine; Metho, methoxamine; k′ HEK293 , k′ Hepatocytes , and k′ Homogenate are explained in Table 1

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With all other conditions identical to those given above, retention of the analyte was obvious and visible when 10 μL ligand samples were loaded on to the rabbit liver homogenate α 1A -AR chromatographic column; 5-μL samples were sufficient for experiments with the HEK293 α 1A -AR CMSP and rabbit hepatocytes α 1A -AR CMSP. In general, all nine test ligands had greater affinity toward the HEK293 α 1A CMSP than the other CMSPs. The solutes were retained on the HEK293 α 1A -AR CMSP for the longest time with the largest capacity factors, and retained in the rabbit liver homogenate CMSP for the shortest time with the lowest capacity factors.

Discussion

This study shows that use of cell lines stably overexpressing subtype receptors results in clearly improved sensitivity and specificity in CMC. The data show that α1A-AR was immobilized on the silica surface with retention of binding activity. Under binding conditions, CMSP will bind molecules according to its specificity only whereas all other sample components will pass through the chromatography medium unabsorbed. The resulting CMSP could be used for rapid on-line chromatographic determination of potential subtype-selective α 1 -adrenoceptor ligands with binding affinity for α 1 -adrenoceptor subtypes.

The liver is one of the largest glands. Hepatic tissue consists of hepatocytes, hepatic stellate cells (HSC), Kupffer’s cells, endothelial cells, and pit cells, etc. The parenchymal cells of the liver are hepatocytes. Hepatocytes are responsible for the synthesis, degradation, and storage of a vast number of substances. Tissue distribution of mRNA of total α1A-AR isoforms in rabbit detected by competitive RT-PCR have made it clear that α1A-AR was expressed most abundantly in the liver, vas deferens, and brain stem whereas rabbit heart does not express α1A-AR [20–22]. According to results from comparative analysis of the capacity factors of three CMSP, rabbit liver homogenate α1A-AR CMSP results in a higher background that severely hampers the sensitivity of the CMC system compared with use of HEK293 α 1A -AR CMSP and rabbit hepatocytes α 1A -AR CMSP. Use of the last two CMSPs eliminates the negative effect of liver non-parenchymal cells on ligand-receptor interaction, and results in improved sensitivity and specificity of CMC.

The effects of mobile-phase ionic strength and the concentration of sample have also been evaluated. At low ionic strengths all components will be tightly adsorbed in CMSP and elution takes a relatively long time. When the ionic strength of the mobile phase is increased by adding phosphate, the drug is partially desorbed and movement down the column begins to accelerate. Increasing the ionic strength even further causes more of the sample drug to be desorbed, and the speed of movement down the column will increase. The phenomenon is partly because the salt ions increase the solubility of the analyte in the mobile phase. The stronger binding affinity of RS-17053 at low ionic strength and the reduction in binding with increasing ionic strength of the mobile phase is indicative of an electrostatic-based retention mechanism. These results are consistent with the pharmacophore models of the α1A-AR binding site. In the model, a positive charge in the middle of the system, a hydrogen bond acceptor group, and an aromatic ring system were included [26, 27]. The retardation of the retention time accompanying increased sample concentration was primarily because of the specific binding to the receptors. The reduced retention time of RS-17053 suggest a dose-dependent, saturable effect. Similarly, the dependence of the retention time on RS-17053 or displacer concentration reflected the specific binding activity of α1A-AR. The reason for this effect might be their occupation or saturation of the receptor sites on the CMSP. With increasing RS-17053 or displacer concentration, receptors were occupied or saturated; non-binding ligands were eluted and passed through in the flow through during washing with buffer, resulting in reduction of the retention time.

It is well known to biologists and chemists that the fundamental processes of drug action, absorption, distribution, excretion, and receptor activation are dynamic in nature and have much in common with the basic mechanisms involved in chromatographic distribution. Indeed, the same basic intermolecular interactions, whether hydrophobic, electrostatic, or hydrogen-bonding, determine the behavior of chemical compounds in both biological and chromatographic environments. Thus, chromatographic techniques can be used for drug-affinity studies by involving active biological components in chromatographic systems [28].

From the beginning, many years of hard work have been devoted to ensuring the CMC model is a success [6–17]. Its correctness and feasibility in the study of ligand-receptor interactions have been proved for many years. He demonstrated that the cell membrane was tightly immobilized on the surface of silica in CMC systems; this has been proved by results from scanning electron microscopy and surface energy spectrometry. The mechanism of retention of drugs on cell-membrane stationary phases is totally different from that on unmodified silica. No specific binding as a result of non-specific interaction between the compounds and the membranes or the column matrix was detected in receptor-minus control CMSP [17]. More important, what we must emphasize, because CMSP has the dual effect of biologically membrane activity and chromatographic separation, is that both chromatographic separation and receptor-based assays in screening for biologically active substances could be performed simultaneously. CMC is, therefore, a superior method for screening for active ingredients in plant extracts and natural product-like libraries. This process, commonly called dereplication, is important to prevent the unnecessary use of resources for isolation, from extracts used in the screening process, of compounds for development with little or no value. Resources can then be focused on samples containing the most promising leads. This method can therefore save money and time dramatically and increase throughput.

Recently publications have reported several chromatographic methods that can be used for direct analysis of the binding of ligand-proteins and quantification of protein-protein interactions. As most representative of this period, Lundahl et al. developed a method for immobilization of liposomes or liposome-containing membrane proteins (human red cell glucose transporter) on chromatographic stationary phases [29]. Human red cell membrane vesicles stripped of peripheral proteins and proteoliposomes with reconstituted red cell glucose transporter (Glut1) were sterically immobilized in gel beads by freeze thawing. Hage et al. combined use of immobilized serum albumin and high-performance affinity chromatography as tools for study of serum protein binding processes [30]. Most of his work focused on human serum albumin (HSA), bovine serum albumin (BSA), and α 1 -acid glycoprotein (AGP). Wainer et al. described immobilization of membrane receptors (nAChR, nicotinic acetylcholine receptors) in the phospholipid monolayer of immobilized artificial membrane (IAM) liquid chromatography stationary phase [28]. The techniques also can be used to immobilize transporter proteins, for example the d-glucose transporter and the P-glycoprotein transporter, carrier proteins such as human serum albumin, and receptor proteins such as nicotinic acetylcholine receptors and estrogen receptor [31]. The nicotinic receptor was immobilized by hydrophobic insertion into the interstitial spaces of an immobilized artificial membrane (IAM) stationary phase; the estrogen receptor was tethered to a hydrophilic stationary phase; and membranes containing the P-gp transporter were coated on the surface of the IAM stationary phase. Liquid chromatographic stationary phases containing either the opioid receptors or beta 2 adrenergic receptor also have been developed to study the binding between the receptors and their ligands [32, 33]. The results from these studies suggest that the immobilized receptors retained their ability to undergo ligand-induced conformational changes and could be used to screen for ligand-binding interactions in both the resting and active states of the receptor. All of these receptor and transporter proteins must be solubilized with detergent solution before immobilization, however, a process which might, to some extent, change the natural state of protein. In contrast with the IAM LC stationary phase, use of cell-membrane stationary phases (CMSP) prepared by immobilizing, on the silica surface, cell membranes expressing receptors could simulate the fundamental processes of drug action more exactly and precisely.

CMC, a reliable tool, has been successfully used to isolation and analysis of active lead compounds or effective components from Chinese traditional medicine extracts, for example Angelica sinensis [8], Herba eplmedii [9], Cladonia alpestris [10], Leontice robustum [11], Libanoits buethorimensis [12], and Ligusticum chuanxiong [13]. Cell lines stably overexpressing receptor subtypes in our experiment clearly result in improved sensitivity and specificity in CMC. This technique provides useful procedures and cell-membrane stationary phases that may facilitate exploration of ligand-receptor interaction, determination of ligand-receptor binding affinity in the process of initial screening, and separation of lead compounds or effective components from Chinese traditional natural medicine and herbs. We have convincingly shown that CMC is an accurate viable alternative to other assays used to study ligand-receptor interactions in initial screening in which chromatographic separation and receptor-based assays for biologically active substances are performed simultaneously. The non-radioactive receptor-ligand binding technology might eventually be an important contribution and addition to our collection of methods.