Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors

Abstract

Kinins are vasoactive peptides that play important roles in cardiovascular homeostasis, pain and inflammation. After release from their precursor kininogens, kinins or their C-terminal des-Arg metabolites activate two distinct G protein-coupled receptors (GPCR), called B2 (B2R) or B1 (B1R). The B2R is expressed constitutively with a wide tissue distribution. In contrast, the B1R is not expressed under normal conditions but is upregulated by tissue insult or inflammatory mediators. The B2R is considered to mediate many of the acute effects of kinins while the B1R is more responsible for chronic responses in inflammation. Both receptors can couple to Galphai and Galphaq families of G proteins to release mediators such as nitric oxide (NO), arachidonic acid, prostaglandins, leukotrienes and endothelium-derived hyperpolarizing factor and can induce the release of other inflammatory agents. The focus of this review is on the different transduction events that take place upon B2R and B1R activation in human endothelial cells that leads to generation of NO via activation of different NOS isoforms. Importantly, B2R-mediated eNOS activation leads to a transient ( approximately 5min) output of NO in control endothelial cells whereas in cytokine-treated endothelial cells, B1R activation leads to very high and prolonged ( approximately 90min) NO production that is mediated by a novel signal transduction pathway leading to post-translational activation of iNOS.

Keywords: bradykinin; eNOS; endothelial cells; iNOS; kinin B1 receptor; kinin B2 receptor; nitric oxide; nitric oxide synthase.

Fig. 1. Schematic diagram showing the generation of kinin peptide agonists for the B2R and B1R and downstream signaling

Bradykinin and kallidin, generated by the action of plasma or tissue kallikrein on precursor high-molecular-weight (HMW) or low-molecular-weight (LMW) kininogen, are ligands of the B2R. They are converted to corresponding agonists of the B1R by removal of the C-terminal Arg by membrane-bound carboxypeptidase M (CPM), which interacts with the B1R, or soluble plasma carboxypeptidase N (CPN). The B2R is constitutively expressed whereas B1R expression is induced by injury or inflammatory conditions. Both the B2R and B1R can couple through either Gαq/11 or Gαi/o to release downstream mediators such as intracellular Ca²⁺, NO and arachidonic acid, which leads to generation of prostaglandins and other metabolites such as epoxyeicosatrienoic acids (which can act as endothelial derived hyperpolarizing factor). On endothelial cells, activation of B2Rs results in Gαq/11 and Ca²⁺–calmodulin–dependent activation of eNOS as well as Akt activation and phosphorylation of Ser¹¹⁷⁷ (as well as other sites not shown), dephosphorylation of Thr⁴⁹⁵ and generation of NO. However, in endothelial cells under inflammatory conditions, B1R stimulation results in much higher and prolonged NO production via Gα_i, Gβγ and Src-dependent activation of the ERK/MAP kinase pathway leading to activation of iNOS via phosphorylation at Ser⁷⁴⁵. See text for further details.

Fig. 2. Comparison of B2R and B1R - mediated NO generation in control and cytokine-treated HLMVECs

HLMVECs were pre-incubated in normal medium (A; Control) or with IL-1β (5 ng/ml) and IFN-γ (100 U/ml) for 16–24 h (B; Cytokine-treated). A. eNOS was activated with Ca²⁺ ionophore A23187 (10 μM), BK (100 nM) or 10 μM A23187 followed by 100 nM BK as indicated by the arrows. Measurements were made in the continuous presence of agonists. B. Cytokine-treated HLMVECs were treated with 100 nM des-Arg¹⁰-kallidin (DAKD) alone or after preincubation (20 min) with 1 μM B1R antagonist des-Arg¹⁰-Leu⁸-KD (DALKD) or iNOS inhibitor 1400W (4 μM). In both A & B, NO production was measured in real time with a porphyrinic electrode in the continuous presence of agonist. Data shown in B are from (Zhang et al., 2007).