Glucocorticoids increase bradykinin B2 receptor gene transcription in cultured guinea-pig tracheal smooth muscle cells.
We investigated the effect of the glucocorticoid methylprednisolone on the modulation of the expression of the bradykinin B2 receptors in cultured, guinea-pig, tracheal, smooth muscle cells. These receptors are implicated in the pathogenesis of human asthma. Untreated cells expressed a single population of binding sites for [3H]bradykinin with a dissociation constant, Kd, of 87.7+/-12.0 pM and a maximum binding site density, Bmax, of 245.4+/-71 fmol/mg protein. Treatment of the cultured guinea-pig tracheal smooth muscle cells with methylprednisolone 10(-5) M for 6 h increased the number of bradykinin receptors; this response reached a maximum of 78% and returned to the basal value after 12 h. Bradykinin (10(-12) M) elicited a six-fold higher calcium level in treated cells than in control cells. To investigate bradykinin B2 receptor mRNA expression in guinea-pig cells, we used the reverse transcription polymerase chain reaction (RT-PCR) technique to synthesize a specific bradykinin B2 cDNA probe of 296 bp corresponding to nucleotides 456-751 of the human sequence. This guinea-pig cDNA had 88%, 86% and 83% homology with the corresponding human, mouse and rat sequences, respectively, but no homology with any other known sequences. Following methylprednisolone treatment, Northern blot hybridization indicated that mRNA increased fourfold after 3 h compared with control cells, and returned to basal level within 7 h. The rate of gene transcription, assessed by nuclear run-on assays, increased fourfold after 3 h treatment with 10(-5) M methylprednisolone. These results indicate that glucocorticoids induce early up-regulation of bradykinin B2 receptors in cultured guinea-pig tracheal smooth muscle cells by increasing the rate of transcription of the bradykinin B2 receptor gene.
Scherrer
D
D
Laboratoire de Neuroimmunopharmacologie, INSERM U 425, Université Louis Pasteur Strasbourg I, Faculté de Pharmacie, Illkirch, France.
Schmidlin
F
F
Haddad
E B
EB
Kassel
O
O
Landry
Y
Y
Gies
J P
JP
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000494:pharmacology*; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D008775:Methylprednisolone / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018002:Receptors, Bradykinin / Q000187:drug effects* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014132:Trachea / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
D005938:Glucocorticoids; D012333:RNA, Messenger; D018002:Receptors, Bradykinin; D002118:Calcium; D008775:Methylprednisolone