A novel pathway for receptor-mediated post-translational activation of inducible nitric oxide synthase

Abstract

Inducible nitric oxide synthase (iNOS) is a major source of nitric oxide during inflammation whose activity is thought to be controlled primarily at the expression level. The B1 kinin receptor (B1R) post-translationally activates iNOS beyond its basal activity via extracellular signal regulated kinase (ERK)-mediated phosphorylation of Ser(745) . Here we identified the signalling pathway causing iNOS activation in cytokine-treated endothelial cells or HEK293 cells transfected with iNOS and B1R. To allow kinetic measurements of nitric oxide release, we used a sensitive porphyrinic microsensor (response time = 10 msec.; 1 nM detection limit). B1Rs signalled through Gαi coupling as ERK and iNOS activation were inhibited by pertussis toxin. Furthermore, transfection of constitutively active mutant Gαi Q204L but not Gαq Q209L resulted in high basal iNOS-derived nitric oxide. G-βγ subunits were also necessary as transfection with the β-adrenergic receptor kinase C-terminus inhibited the response. B1R-dependent iNOS activation was also inhibited by Src family kinase inhibitor PP2 and trans-fection with dominant negative Src. Other ERK-MAP kinase members were involved as the response was inhibited by dominant negative H-Ras, Raf kinase inhibitor, ERK activation inhibitor and MEK inhibitor PD98059. In contrast, PI3 kinase inhibitor LY94002, calcium chelator 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), protein kinase C inhibitor calphostin C and protein kinase C activator PMA had no effect. Angiotensin converting enzyme inhibitor enalaprilat also directly activated B1Rs to generate high output nitric oxide via the same pathway. These studies reveal a new mechanism for generating receptor-regulated high output nitric oxide in inflamed endothelium that may play an important role in the development of vascular inflammation.

Fig 1

B1R agonist stimulates iNOS-dependent high output nitric oxide in cytokine-treated HLMVEC. (A) Control HLMVEC were pre-incubated for 2 hrs in L-Arg-free medium (top) or maintained in medium containing 1 mM Arg (bottom) and then 1 mM Arg (top) or 100 nM DAKD (bottom) was added and real-time nitric oxide was measured with a porphyrinic microsensor for 80 min. (B) HLMVEC were treated with cytokines for 16 hrs to induce iNOS and B1R expression. Cells were either pre-incubated for 2 hrs in L-Arg-free medium (dashed lines) or maintained in medium containing 1 mM Arg (solid lines). Where indicated, cells were also pre-incubated for 20 min. with either B1R antagonist DALKD (1 μM) or iNOS inhibitor 1400 W (4 μM). To stimulate basal or B1R-dependent nitric oxide production, 1 mM Arg or 100 nM DAKD was added and real-time nitric oxide was measured with a porphyrinic microsensor for 80 min. (C) HLMVEC were pretreated with cytokines and pre-incubated in L-Arg-free medium for 2 hrs. Some cells were pre-treated for 20 min. with 25 μM BAPTA-AM, 1 μM DALKD or 4 μM 1400 W min. as indicated. 1 mM L-Arg was added first to activate basal iNOS-mediated nitric oxide, followed 5 min. later by 100 nM DAKD to stimulate B1R-dependent activation of iNOS (except tracing labelled ‘L-Arg alone’). (D) Immunoblotting of iNOS in lysates of control or cytokine-treated HLMVEC. Data are representative of three experiments.

Fig 2

eNOS expression and Ca²⁺-dependent activation are not affected by cytokine treatment. (A) Control or cytokine-treated HLMVEC were lysed and eNOS expression was assessed by Western analysis using eNOS-specific monoclonal antibody. Results are representative of two experiments done in duplicate. (B) Ca²⁺-dependent eNOS activity was stimulated in control or cytokine-treated HLMVEC by adding 10 μM calcium ionophore A23187 at time 0 and nitric oxide generation was measured with a por-phyrinic electrode. Tracings shown are representative of 4 experiments. (C) Quantitation of maximal nitric oxide concentration achieved in experiments described in B. Results shown are mean ± S.E.M for n= 4.

Fig 3

Basal and B1R-activated nitric oxide release in cytokine treated HLMVEC or in HEK cells or HLMVEC transfected with B1R and iNOS. (A) HLMVEC were pre-treated with cytokines to induce iNOS and B1R expression or electroporated with iNOS and B1R cDNAs as described in ‘Materials and methods’. Basal Arg-dependent nitric oxide generation was measured by incubating cells for 2 hrs in L-Arg-free media and then adding 1 mM L-Arg to initiate the response. To measure B1R-dependent iNOS activation and nitric oxide production, cells were maintained in L-Arg-containing media and B1R agonist (100 nM DAKD) was added to stimulate the response. In both cases, nitric oxide production was measured continuously with a porphyrinic electrode, and the nitric oxide concentration achieved at 20 min. used to quantitate the results. Results shown are mean values ± S.E.M for n= 3. *P < 0.05 as compared with control; #P < 0.05 as compared with cytokine-treated cells. (B) HEK293 cells were transfected with B1R or iNOS alone or with iNOS + B1R cDNAs. Basal Arg-dependent or B1R-activated nitric oxide production was measured as above. Results shown are mean values ± S.E.M for n= 3. *P < 0.05 as compared with control.

Fig 4

B1R-mediated iNOS activation is PTX sensitive. HLMVEC were pre-treated with cytokines and HEK293 cells were transfected with iNOS and B1R as described in ‘Materials and methods’. Cells were pre-treated with PTX (1.5 μg/ml) for 3 hrs. Basal Arg-mediated (A) or B1R-activated (B) iNOS-dependent nitric oxide production was measured as described (see Fig. 3A legend and ‘Materials and methods’). Data shown are mean values ± S.E.M for n= 3. *P < 0.05 compared with control.

Fig 5

Effect of transfection with constitutively active Gαi, ct-βARK or dominant-negative Src on iNOS-dependent nitric oxide production. (A) Representative tracings of real time nitric oxide measurement. HLMVEC were trans-fected with Gαi Q204L (top) or Gαq Q209L (bottom) and treated with cytokines. Basal Arg-mediated or B1R-activated iNOS-dependent nitric oxide production was measured as above (see Fig. 3A legend and ‘Materials and methods’). (B) HLMVEC or HEK293 cells were transfected with B1R, iNOS, and Gαi Q204L or Gαq Q209L and basal Arg-mediated or B1R-activated iNOS-dependent nitric oxide production was measured as above. Data shown are mean values ± S.E.M for n= 3; *P < 0.05 as compared with L-Arg dependent nitric oxide output. (C) HLMVEC or HEK293 cells were infected with adenovirus containing ct-βARK (ADV- ct-βARK) or empty vector (ADV-EV). HLMVEC were then treated with cytokines as above and HEK293 cells were transfected with iNOS and B1R. Basal Arg-mediated or B1R-activated iNOS-dependent nitric oxide production was measured. Data shown are mean values ± S.E.M for n= 3; *P < 0.05 compared with nitric oxide output without ct-βARK trans-fection. (D) HEK293 cells were transfected with iNOS and B1R, or iNOS, B1R and dominant negative (DN)-Src. Basal Arg-mediated or B1R-activated iNOS-dependent nitric oxide production was measured as above. For ‘m-SIRK’, cells were maintained in Arg-containing medium and 1 μM m-SIRK was pre-incubated for 15 min. Data shown are mean values ± S.E.M for n= 3; *P < 0.05 nitric oxide output without m-SIRK or DN-Src.

Fig 6

B1R agonist-induced iNOS activation is dependent on Ras, Raf and Erk. (A) HLMVEC were cytokine treated as before and HEK293 cells were trans-fected with iNOS + B1R. Some cells were transfected with DN-H-Ras or pretreated with Raf kinase inhibitor (100 nM, 20 min.) or Src family kinase inhibitor PP2 (15 μM, 30 min.). In all samples, B1R-dependent nitric oxide release was measured in response to 100 nM DAKD as described (see Fig. 3A legend and ‘Materials and methods’). (B) HLMVEC were cytokine treated and HEK293 cells were transfected with iNOS + B1R. Cells were pre-treated with ERK activation inhibitor (10 μM, 20 min.) or MEK inhibitor PD98059 (50 μM, 30 min.). Basal Arg-dependent or B1R-activated nitric oxide release were measured as above (see Fig. 3A legend and ‘Materials and methods’). (C) Cytokine-treated HLMVEC or HEK293 cells transfected with iNOS + B1R were pre-treated with PI3K inhibitor LY94002 (10 μM, 30 min.), PKC activator PMA (1 μM, 30 min.), PKC inhibitor calphostin C (1 μM, 30 min.) or Akt inhibitor SH6 (25 μM, 30 min.) and B1R-dependent nitric oxide release in response to 100 nM DAKD was measured as above. (D) HEK293 cells were transfected with iNOS, B1R, and constitutively active mutants Gαi Q204L or Gαq Q209L. Some cells were pre-treated with MEK inhibitor PD98059 (50 μM, 30 min.) and Arg-dependent nitric oxide release was measured as above. Data shown are mean values ± S.E.M for n= 3. *P < 0.05 as compared with control.

Fig 7

B1R-mediated ERK activation depends on Gαi, Src and MEK. (A) Representative Western blots of phospho- ERK (p-ERK) in cytokine-treated HLMVEC which were pre-incubated without or with PP2 (15 μM, 30 min.), PTX (1.5 μg/ml, 3 hrs), m-SIRK (1 μM, 30 min.), or PD98059 (50 μM, 30 min.) and stimulated with 100 nM DAKD for the indicated time. Cells were lysed and analyzed by Western blotting with polyclonal antibody to phospho- ERK. Blots were stripped and re-probed with monoclonal antibody to β-actin or total ERK as protein loading controls. (B) Graph shows quantitation of Western blots by densitometry, normalized for protein loading (β-actin or total ERK staining). Bars indicate mean ± S.E.M. (n= 3 each). *P < 0.05 versus DAKD treatment alone (20 min.).

Fig 8

ACE inhibitor generates iNOS-dependent high output nitric oxide through the B1R-Gαi-MAPK pathway. HLMVEC were pre-treated with cytokines and pre-incubated with or without B1R antagonist DALKD (1 μM, 30 min.), B2R antagonist HOE140 (1 μM, 30 min.), PTX (1.75 μg/ml, 3 hrs), PD98059 (50 μM, 30 min.) or 1400 W (4 μM, 30 min.). B1R peptide agonist DAKD (100 nM) or ACE inhibitor enalaprilat (100 nM) were added and nitric oxide production was recorded continuously for 20 min. Data show mean values ± S.E.M for n= 3. *P < 0.05 as compared with enalaprilat alone.

Fig 9

Schematic diagram of the signalling pathway activated by B1R agonists leading to iNOS-derived high output nitric oxide. Expression of iNOS and the B1R are induced under inflammatory conditions. iNOS has constitutive activity, which can be further enhanced by B1R agonists DABK, DAKD or ACE inhibitors via B1R-dependent activation of the ERK/MAP kinase pathway as shown. This results in phosphorylation of iNOS at Ser⁷⁴⁵, leading to generation of high output nitric oxide in endothelial cells.