Mechanisms underlying beta2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells

Abstract

Endothelial beta(2)-adrenoceptor (beta(2)AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in beta(2)AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l-arginine uptake, NOS activity (from l-arginine to l-citrulline conversion), membrane potential (using [(3)H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with betaAR agonists or cyclic AMP-elevating agents. beta(2)AR stimulation increased l-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of l-arginine uptake by l-lysine inhibited NOS activity and, conversely, blockade of NOS using N(omega)-nitro-l-arginine methyl ester (l-NAME) inhibited l-arginine transport. beta(2)AR stimulation also caused a membrane hyperpolarization inhibitable by l-NAME, suggesting that the increase in l-arginine uptake occurred in response to NO-mediated hyperpolarization. beta(2)AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that beta(2)AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l-arginine uptake resulting from NO-mediated membrane hyperpolarization.

Figure 1. Effect of βAR stimulation or cAMP elevation on l-arginine uptake in HUVEC

A, effect of isoproterenol 1 μm, forskolin 1 μm or dibutyryl cAMP 1 mm on l-arginine uptake as compared with basal values (vehicle treatment), by HUVEC. *, **: P < 0.05 and < 0.01, respectively, versus basal. B, effect of isoproterenol on percentage increase in l-arginine uptake above basal, in HUVEC, in the presence of the selective β₁AR antagonist CGP 20712A 300 nm, the selective β₂AR antagonist ICI 118551 100 nm, or neither antagonist. ***P < 0.001 as compared with no antagonist. C, effect of isoproterenol 1 μm or forskolin 1 μm, as compared with basal values (vehicle treatment), on l-arginine uptake by HUVEC, in the absence (filled bars) or presence (open bars) of 0.2 mmN-ethylmaleimide. **, ***: P < 0.01 and < 0.001 as compared with absence of N-ethylmaleimide. Data are mean ±s.e.m. of 8–10 experiments in different cell cultures.

Figure 2. Interdependency of l-arginine uptake and NOS activity on βAR- and cAMP-mediated NO biosynthesis in HUVEC

A, effect of isoproterenol 1 μm or forskolin 1 μm, as compared with basal values (vehicle treatment), on NOS activity in HUVEC, in the absence (filled bars) or presence (open bars) of 0.1 mml-lysine. **P < 0.01 as compared with basal; ##, ###P < 0.01 and < 0.001 as compared with absence of l-lysine. B, effect of isoproterenol 1 μm or forskolin 1 μm, as compared with basal values (vehicle treatment), on cGMP accumulation in HUVEC, in the absence (filled bars) or presence (open bars) of 0.1 mml-lysine. ***P < 0.001 as compared with basal; #, ###: P < 0.05 and < 0.001 as compared with absence of l-lysine. C, effect of isoproterenol 1 μm or forskolin 1 μm, as compared with basal values (vehicle treatment), on NOS activity in HUVEC, in the absence (filled bars) or presence (open bars) of 0.1 mml-NAME. ***P < 0.001 as compared with basal; ###P < 0.001 as compared with absence of l-NAME. D, effect of isoproterenol 1 μm or forskolin 1 μm, as compared with basal values (vehicle treatment), on l-arginine uptake by HUVEC, in the absence (filled bars) or presence (open bars) of 0.1 mml-NAME. ***P < 0.001 as compared with basal; ##, ###: P < 0.01 and < 0.001 as compared with absence of l-NAME. Data are mean ±s.e.m. of 8–12 experiments in different cell cultures.

Figure 3. NO dependency of β₂AR-mediated membrane hyperpolarization in HUVEC

Effect of isoproterenol 1 μm on the time course of uptake of the membrane potential-sensitive probe [³H]TPP⁺ by HUVEC in the presence of the selective β₁AR antagonist CGP 20712A and the absence or presence of 0.1 mml-NAME. *, **: P < 0.05 and < 0.01 as compared with basal. Data are expressed as counts min⁻¹ (cpm) corrected for cellular protein, and are mean ±s.e.m. of eight experiments in different cell cultures.

Figure 4. Role of the PKA and PI3K/Akt pathways in β₂AR-mediated NOS activation in HUVEC

Percentage increase above basal (vehicle treatment) in NOS activity in HUVEC treated with albuterol 1 μm, in the absence or presence of: l-NAME 0.1 mm; the selective PKA inhibitor H-89 100 nm; the selective PI3K inhibitor wortmannin 500 nm; Akt inhibitor 10 μm; or the combination of H-89 100 nm and Akt inhibitor 10 μm. **, ***: P < 0.01 and < 0.01 as compared with no antagonist; ##P < 0.01 as compared with H-89 alone. Data are mean ±s.e.m. of six experiments in different cell cultures.

Figure 5. Role of the PKA and PI3K/Akt pathways in β₂AR-mediated NOS-3 serine phosphorylation in HUVEC

A, Western blot depicting the presence of a 135 kDa band (the known molecular mass of NOS-3) in NOS-3 immunoprecipitates prepared from HUVEC lysates, probed with anti-NOS-3 antibody. B, Western blot depicting the presence of a 135 kDa band (the known molecular mass of NOS-3) in NOS-3 immunoprecipitates prepared from HUVEC lysates, probed with anti-phosphoserine IgG. Lanes: 1 = basal (vehicle treatment); 2 = albuterol 1 μm; 3 = albuterol +l-NAME 0.1 mm; 4 = albuterol + H-89 100 nm; 5 = albuterol + wortmannin 500 nm; 6 = albuterol + Akt inhibitor 10 μm; 7 = albuterol + H-89 + Akt inhibitor. C, densitometric ratio of 135 kDa phosphoserine/NOS-3 bands, in HUVEC treated as shown, expressed as mean ±s.e.m. of six experiments. *, **: P < 0.05 and < 0.01 as compared with basal; #, ##: P < 0.05 and < 0.01 as compared with albuterol alone.

Figure 6. Effect of β₂AR stimulation, and of concomitant inhibition of the PKA and PI3K/Akt pathways, on NOS-3 expression in HUVEC

Density of 135 kDa band detected by Western blotting for NOS-3 in HUVEC lysates, expressed as a percentage of basal (vehicle-treated HUVEC). Basal density is set at 100%, in order to allow interblot normalization of densities. Results are shown for HUVEC following treatment with albuterol 1 μm, in the absence or presence of: l-NAME 0.1 mm; H-89 100 nm; wortmannin 500 nm; Akt inhibitor 10 μm; and the combination of H-89 with Akt inhibitor. Data are mean ±s.e.m. of six experiments.

Figure 7. Role of the PKA and PI3K/Akt pathways, and of G_i protein, in β₂AR-mediated phosphorylation of NOS-3 specifically on serine-1177 in HUVEC

A, Western blot depicting the presence of a 135 kDa band (the known molecular mass of NOS-3) in NOS-3 immunoprecipitates prepared from HUVEC lysates, probed with anti-NOS-3 antibody (upper blot) and anti-phospho-NOS-3 (serine-1177-specific) antibody (lower blot). Lanes: 1 = basal (vehicle treatment); 2 = albuterol 1 μm; 3 = albuterol + H-89 100 nm; 4 = albuterol + Akt inhibitor 10 μm; 5 = albuterol + H-89 + Akt inhibitor. B, densitometric ratio of phosphoserine-1177-NOS-3/total NOS-3 bands, in HUVEC treated as shown, expressed as mean ±s.e.m. of six experiments. C, Western blot depicting the presence of a 135 kDa band in HUVEC lysates, probed with anti-phospho-NOS-3 (serine-1177-specific) antibody (upper blot) and anti-α-tubulin antibody (lower blot). Lanes: 1 = basal (vehicle treatment); 2 = albuterol 1 μm; 3 = albuterol + LY 294002 10 μm; 4 = albuterol + Rp-8-Br-MB-cAMPS 30 μm; 5 = albuterol + SH5 1 μm; 6 = albuterol + SH5 + Rp-8-Br-8-MB-cAMPS; 7 = albuterol + pertussis toxin 100 ng ml⁻¹D, densitometric ratio of phosphoserine-1177-NOS-3/tubulin bands, in HUVEC treated as shown, expressed as mean ±s.e.m. of three experiments. *, **, ***: P < 0.05, < 0.01 and < 0.001 as compared with basal; #, ##, ##: P < 0.05, < 0.01 and < 0.001 as compared with albuterol alone.

Figure 8. Phosphorylation sites of NOS-3 in HUVEC

LC-MS/MS result for trypsin digest of 135 kDa band seen on Coomassie Blue staining of gel following SDS-PAGE of NOS-3 immunoprecipitate from HUVEC. Phosphorylation sites are shown underscored.