CHO CELLS EXPRESSING HUMAN ENDOTHELIN RECEPTORS

Abstract: DNA encoding human endothelin receptor subtype A (ETA) and human endothelin receptor subtype B (ETB) are identified, cloned, sequenced and expressed in CHO cells. Transformed CHO cells expressing endothelin receptors are useful pharmacological tools.

Description: TΓΓLE OF THE INVENTION CHO CELLS EXPRESSING HUMAN ENDOTHELIN RECEPTORS BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a partial restriction map of the expression construct of the human ETA and human ETβ receptors. Figure 2 shows the DNA and amino acid sequences of human ETA receptor. BACKGROUND OF THE INVENTION This is a continuation-in-part of U. S. S. N. 07/961,555 filed October 15, 1992, now pending and U. S. S. N. 07/961,568 filed October 15, 1992, now pending. Endothelin is a substance endogenous to mammals that induces a wide variety of physiological responses. Excess endothelin is associated with pathogeneses such as hypertension, bronchial asthma, myocardial infarction and arteriosclerosis. Endothelin controls the release of physiological substances such as renin, atrial naturetic peptide and angiotensin. Endothelin causes contraction of smooth muscle cells of the cardiovascular system, lungs, and uterus. In vitro studies have shown that endothelin promotes the growth of vascular smooth muscle cells and fibroblasts, suggesting that endothelin may be linked to arterial hypertrophy. Endothelin is a neuropeptide that acts on the posterior pituitary gland, thus modulating the release of hormones such as vasoprssin and oxytocin. Endothelin also affects the endocrine system, particularly the adrenal glands, and increases the level of adrenaline in plasma. Release of endotoxin promotes the release of endothelin, suggesting that endothelin may be an important mediator of endotoxin- induced disease. Endothelin may also play a role in the pathogenesis of cyclosporin-induced disease. Experiments have shown that the addition of cyclosporin to renal cell cultures promotes the release of endothelin. Other experiments have shown that the administration of cyclosporin to rats was followed by a decrease in the rate of glomerular filtration, an increase in blood pressure and an increase in the level of circulating endothelin. The administration of endothelin antibody suppressed these changes. Endothelin receptors are present in high concentrations in peripheral tissues and the central nervous system. Substances that specifically inhibit the binding of endothelin to its receptors are believed to antagonize the effects of endothelin. The administration of such an inhibitor would be a useful method of treatment for a person in need of treatment for endothelin-related pathogeneses. Identification of compounds that inhibit endothelin may be accomplished by screening. Prior to the recombinant cells of the present invention, screens for endothelin inhibitors employed cell membranes prepared from tissues as reagents. The use of the tissues limited the specificity, accuracy and efficiency of the screens. The specificity of the screens is affected by the presence of a variety of cell types in the tissue and a variety of receptors on the cells. The accuracy of the screen is affect by variations in the source and condition of the tissue. The efficiency of the screen is limited by the relative scarcity of acceptable human tissue. The present invention provides reagents that improve the specificity, accuracy and efficiency of screens for substances that act as inhibitors of endothelin. The present invention encompasses a Chinese Hamster Ovary (CHO) cells that is transformed with DNA encoding human endothelin receptor subtype A and CHO cells that are transformed with DNA encoding human endothelin receptor subtype B. SUMMARY OF THE INVENTION DNA encoding human endothelin receptor subtype A (ETA) and human endothelin receptor subtype B (ETβ) are identified, cloned, sequenced and expressed in CHO cells. Transformed CHO cells expressing endothelin receptors are useful pharmacological tools. DETAILED DESCRIPTION OF THE INVENTION The endothelins (ET) comprise a class of 21 amino acid peptides that have potent vasoactive properties. ET were first isolated ° from mammalian endothelin cells, but are also produced by other cell types. The three major endothelins are designated ET-1, ET-2 and ET-3. ET-1 and ET-2 differ by two amino acids while ET-1 and ET-3 differ by six amino acids. Endothelins are closely related to sarafotoxins, potent 5 peptides isolated from the middle eastern burrowing asp. ET and sarafotoxins have a high degree of homology to each other and bind to the same cell receptors. ET have a number of potent effects on a variety of cells and exert their action by interacting with specific receptors on cell 0 membranes. There are at least two subtypes of ET receptors (ETA and ETβ), which are classified by their selectivity in binding various ET. The ETA receptor subtype binds ET-1 more potently than ET-3, while the ETβ receptor binds all of the ET equally. ETA and ETβ receptor subtypes have been cloned from rat and cow. Oligonucleotide probes 5 from the rat and cow sequences have been used to clone human ET receptors. ET receptors may be distinguished by binding characteristics. Sarafotoxin S6c binds potently to ETβ receptors and weakly to ETA receptors. The cyclic peptides BQ-153 and BQ-123 bind ° strongly to ETA receptors and weakly to ETβ receptors, and may be used to distinguished between receptor subtypes. Identification of compounds that inhibit endothelin may be accomplished by screening. Prior to the recombinant cells of the present invention, screens for endothelin inhibitors employed cell membranes prepared from tissues as reagents. The use of the tissues limited the specificity, accuracy and efficiency of the screens. The specificity of the screens is affected by the presence of a variety of cell types in the tissue and a variety of receptors on the cells. The accuracy of the screen is affect by variations in the source and condition of the tissue. The efficiency of the screen is limited by the relative scarcity of acceptable human tissue. Recombinant DNA technology may be used to provide an alternative source of endothelin receptors. Using methods that are well- known in the art, CHO cells may be transfected with DNA encoding human ETA receptor or human ETβ receptor to produce a stably- transformed CHO cell that expresses either human ETA receptor or human ETβ receptor. Such transformed cells provide a consistent source of reagent useful in screening assays. A variety of methods may be used to clone human ET receptor. The methods include, but are not limited to direct functional expression of human ET receptor following construction of a human ET receptor-containing cDNA library and screening a commercially- available library for a human ET receptor. Examples of such methods are outlined in the text Molecular Cloning (Maniatis et al., CSH Laboratory Press, Cold Spring Harbor, NY, 1982). DNA encoding ETA receptor or ETβ receptor may be cloned into a variety of expression vectors. The vector may then be transferred into a host cell to produce a transformed host cell. Host cells may be prokaryotic or eukaryotic. Mammalian cell lines which may be suitable for transformation and expression of ET receptor include cell lines such as CHO cells. The expression vector-containing cells are clonally isolated, propagated and analyzed to determined whether they express ET receptor. The following examples illustrate the present invention without, however, limiting the same thereto. EXAMPLE 1 Cloning of Human ET Receptor Subtype A A 1.1 kb BstXI fragment from a bovine ETA receptor cDNA clone was labeled by random priming and was used to screen plaques of a human lung cDNA library (Clontech). Two positive recombinant bacteriophage were identified, purified and subcloned into the EcoRI site of pSP73 (Promega). The two clones were digested with EcoRV and ligated together to place a 790 bp human ETA receptor coding sequence on one piece of DNA. The complete human ETA receptor was obtained by generating two additional DNA fragments representing the amino and carboxy terminal ends of the cDNA. The resulting expression vector is shown in Figure 1, and the DNA and amino acid sequences of human ETA receptors are shown in Figure 2. EXAMPLE 2 Expression of recombinant human ET Areceptor in CHO cells The ETA receptor of Example 1 was transfected into CHO cells, grown in Hams F12 medium containing 10% fetal bovine serum (FBS), glutamine, penicillin, and streptomycin essentially acccording to the calcium phosphate procedure of Graham et al. (Virology 52:456, 1973). After 48 hr the cells were selected for stable integration of human ETA by adding genticin (400 ug/ml) to the medium and incubating for two weeks. Clones growing in the selective medium were assayed for endothelin receptor expression by measuring the relative binding of radiolabeled ET-1 by wild-type and transformed cells. EXAMPLE 3 Characterization of CHO cells expressing human ET A receptor The transfected cells of Example 2 were cultivated in Hams F-12 medium supplemented with 10% FBS, glutamine, penicillin, streptomycin and genticin; harvested; and concentrated by centrifugation and 250 x g. Cell membranes were prepared by homogenizing the cell pellets in an ice cold solution (250 mM sucrose, 50 mM Tris-HCl, pepstatin and leupeptin) using a Brinkman Polytron (setting 10). Crude particulate matter was pelleted by centrifugation at 750 x g. The membrane-containing supernatant was centrifuged at 48,000 x g to pellet the membranes. The membranes were resuspended and stored at -70°C until use. Control membranes were prepared from frozen nonpregnant human uterine tissue, essentially according to the methods described above. Binding assays were performed in Skatron tube strips (Lier, Norway). Various concentrations of unlabelled competitor compounds were placed in the strips along with radiolabeled ET-1. The binding reaction was initiated by the addition of either whole cells or membranes and then incubated at 37°C for 60 min. The reaction was terminated by filtration through a filtermat. The filtermats were washed and them counted in a gamma counter. Nonspecific binding, defined as cpm bound in the presence of 100 nM ET-1, was subtracted from all samples. Phosphatidyl inositol (PI) turnover in membranes was measured by loading cells with tritiated myoinositol for about 24 hr, harvesting and washing the cells with 10 mM LiCl. Hydrolysis reactions were initiated by the addition of agonist compound and terminated by the addition of tricloroacetic acid (TCA). Competition binding experiments showed that the Ki value for the cloned ETA receptor was 0.77 nM while the Ki value of the uterine-derived ETA receptor was 0.63 nM. EXAMPLE 4 Cloning of human ETβ receptor The human ETβ receptor was cloned in two pieces by reverse transciption polymerase chain reaction (RT-PCR) and assembled into the expression vector of Figure 1. RT-PCR was based on published DNA and amino acid sequences of human ETβ receptor (Figure 3). EXAMPLE 5 Expression of recombinant human ETft_receptor in CHO cells The ETβ receptor of Example 4 was transfected into CHO cells, grown in Hams F12 medium containing 10% fetal bovine serum (FBS), glutamine, penicillin, and streptomycin essentially acccording to the calcium phosphate procedure of Graham et al. (Virology 52:456, 1973). After 48 hr the cells were selected for stable integration of human ETβ by adding genticin (400 ug/ml) to the medium and incubating for two weeks. Clones growing in the selective medium were assayed for endothelin receptor expression by measuring the relative binding of radiolabeled ET-1 by wild-type and transformed cells. EXAMPLE 6 Characterization of CHO cells expressing human ETR receptor The transfected cells of Example 5 were characterized essentially according to the methods outlined in Example 3. In competition binding experiments with radiolabeled ET-1, the Ki value for unlabelled ET-1 with the cloned human ETβ receptor was 0.14 nM. EXAMPLE 7 Detection of Human ETA ^ and ETR Receptor Antagonists The binding assays of Example 3 are used to evaluate the potency of interaction of substances with endothelin receptors. To determine whether these substances are endothelin antagonists, the ability of the substance to inhibit ET-stimulated PI hydrolysis is determined. Control values (100%) are values in the presence of endothelin minus basal values. Test samples are values in the presence of endothelin and test sample values minus basal values. Inhibitor concentration is the concentration of the test compound required to give a sample activity of 50% of control value.

Claims: WHAT IS CLAIMED IS: 1. A transformed CHO cell expressing human endothelin receptor. 2. The transformed CHO cell of Claim 1 wherein the human endothelin receptor is selected from the group consisting of human endothelin receptor subtype A and human endothelin receptor subtype b. 3. The transformed CHO cell of Claim 2 wherein the human endothelin receptor subtype A has the DNA sequence shown in Figure 2. 4. The transformed CHO cell of Claim 2 wherein the human endothelin receptor subtype B has the DNA sequence shown in Figure 3. 5. A method of detecting substances that interfere with the binding of endothelin to human endothelin receptor which comprises: (a) incubating the transformed CHO cells of Claim 1 in a solution containing competitor compounds and endothelin; (b) filtering the solution of step (a) through a filter; and (c) comparing the filter of step (b) to a control filter.

Inventors: ALVES KENNETH, CHAN CHRISTINE PUI-CHE, HOLLIS GREGORY F, JONES KATHRYN, TUNG JWU-SHENG, WILLIAMS DAVID L JR, ALVES, KENNETH, CHAN, CHRISTINE, PUI-CHE, HOLLIS, GREGORY, F., JONES, KATHRYN, TUNG, JWU-SHENG, WILLIAMS, DAVID, L., JR.
Applicants: ALVES KENNETH, CHAN CHRISTINE PUI CHE, HOLLIS GREGORY F, JONES KATHRYN, MERCK & CO INC, TUNG JWU SHENG, WILLIAMS DAVID L JR, MERCK & CO., INC.
Application year: 1993
Main classification: C12N5
IPC codes: A61K39, A61K45, A61P9, A61P11, A61P43, C07K14, C12N5, C12N15, G01N33