Received for publication September 8, 2006.
Revised November 23, 2006.
Accepted for publication December 18, 2006.

Role of NF-B and protein kinase C signaling in the expression of the kinin B₁ receptor in human vascular smooth muscle cells

Abstract

Kinin B₁ receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of 3 previously proposed pathways (NF-B, protein kinase C, agonist autoregulation) that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real time RT-PCR with an optional actinomycin D treatment period and NF-B 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-B nuclear translocation and both mRNA concentration and stability (IL-1, 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 (siRNA) assays indicated that NF-B p65 is necessary for the effective expression of the cell surface B₁ receptor under basal or IL-1, FBS, or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1-, FBS-, or PMA-induced stabilization of B₁ receptor mRNA was inhibited by the addition of the protein kinase C inhibitor GF109203x, which also diminished the B_max under FBS or PMA treatment. Lys-des-Arg⁹-bradykinin had little effect on NF-B activation, the B_max, or receptor mRNA abundance or stability. Both NF-B and protein kinase C signaling are required for the effective expression of the kinin B₁ receptor in human umbilical artery smooth muscle cells.

Key words: Bradykinin, Interleukins, Protein Kinase C, Receptor synthesis/trafficking, NFkappaB

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