Abstract
Kinin B1 receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-κB (NF-κB), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-κB immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-κB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1β, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B1 receptor agonist [3H]Lys-des-Arg9-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-κB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1β, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1β-, FBS-, or PMA-induced stabilization of B1 receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-κB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-κB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.
Footnotes
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This work was supported by Canadian Institutes of Health Research operating grant MOP-14077 (to F.M. and A.A.) and a Canada Graduate Scholarships Doctoral Award (to G.M.).
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M.E.M. and M.-T.B. contributed equally to this work.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.030684.
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ABBREVIATIONS: BK, bradykinin; siRNA, small interfering RNA; IL, interleukin; NF-κB, nuclear factor-κB; HUA, human umbilical artery; SMC, smooth muscle cell; FBS, fetal bovine serum; PCR, polymerase chain reaction; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DMSO, dimethyl sulfoxide; GF109203x, 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; CHX, cycloheximide.
- Received September 7, 2006.
- Accepted December 18, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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