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Pharmacological characterization of the human histamine H 2 receptor stably expressed in Chinese hamster ovary cells
Author(s) -
Leurs Rob,
Smit Martine J.,
Menge Wiro M.B.P.,
Timmerman Hendrik
Publication year - 1994
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1994.tb13157.x
Subject(s) - chinese hamster ovary cell , histamine , receptor , microbiology and biotechnology , histamine h1 receptor , histamine receptor , biology , intracellular , chemistry , antagonist , endocrinology , biochemistry
1 The gene for the human histamine H 2 receptor was stably expressed in Chinese hamster ovary (CHO) cells and characterized by [ 125 I]‐iodoaminopotentidine binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2 After cotransfection of CHO cells with pCMVhumH 2 and pUT626, a phleomycine‐resistant clonal cell line (CHOhumH 2 ) was isolated that expressed 565 ± 35 fmol kg −1 protein binding sites with high affinity (0.21 ± 0.02 n m ) for the H 2 antagonist, [ 125 I]‐iodoaminopotentidine. 3 Displacement studies with a variety of H 2 antagonists indicated that the encoded protein was indistinguishable from the H 2 receptor identified in human brain membranes and guinea‐pig right atrium. The K i ‐values observed in the various preparations correlated very well ( r 2 = 0.996–0.920). 4 Displacement studies with histamine showed that a limited fraction (32 ± 6%) of the binding sites showed a high affinity for histamine (2 ± 1.2 μ m ); the shallow displacement curves were reflected by a Hill‐coefficient significantly different from unity (n H = 0.58 ± 0.09). The addition of 100 μ m Gpp(NH)p resulted in a steepening of the displacement curve (n H = 0.79 ± 0.02) and a loss of high affinity sites for histamine. 5 Displacement studies with other agonists indicated that the recently developed specific H 2 agonists, amthamine and amselamine, showed an approximately 4–5 fold higher affinity for the human H 2 receptor than histamine. 6 Stimulation of CHOhumH 2 cells with histamine resulted in a rapid rise of the intracellular cyclic AMP levels. After 10 min an approximately 10 fold increase in cyclic AMP could be measured. The EC 50 value for this response was 7 ± 1 n m for histamine. This response was effectively blocked by tiotidine and cimetidine, resulting in K i values of 8 ± 1 n m and 0.56 ± 0.24 μ m respectively. 7 Stimulation of CHOhumH 2 cells with histamine neither inhibited the A23187‐induced release of [ 3 H]‐arachidonic acid nor changed the intracellular IP 3 levels. 8 These results show that the cloned human gene encodes a histamine H 2 receptor that is indistinguishable from the H 2 receptor identified in human brain tissue. This receptor is functionally coupled to the adenylate cyclase in CHO cells, but does not influence the inositolphosphate turnover or arachidonic acid release.