Wall stretch and thromboxane A2 activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation

Abstract

Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 μM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli.

Downregulation of Soluble Guanylate Cyclase and Protein Kinase G With Upregulated ROCK2 in the Pulmonary Artery Leads to Thromboxane A2 Sensitization in Monocrotaline-Induced Pulmonary Hypertensive Rats

“…The U46619-induced arterial contraction was significantly attenuated by Y-27632, an inhibitor of ROCK (Fu et al, 1998). In addition to contractile signaling, our recent study suggested that TP receptor-mediated stimulation of eNOS in the PA smooth muscle layer, counterbalances the potent vasoconstrictive effect of TXA 2 via the NO/cGMP-dependent signaling pathway (Kim et al, 2016).…”

Role of muscular eNOS in skeletal arteries: Endothelium-independent hypoxic vasoconstriction of the femoral artery is impaired in eNOS-deficient mice

“…However, in this study, we observed neither HVC nor L-NAME-induced contraction in mouse CAs. In our hands, functionally significant expression of eNOS in VSMCs seems to be restricted to specific types of vessels, such as skeletal and pulmonary arteries (24). Experimental damage (e.g., mechanical abrasion) of the endothelial layer evokes production of ROS, which convert myogenic NO into nonfunctional forms.…”

“…Because both contractile signals and eNOS activation require Ca 2ϩ /CaM signaling, eNOS in VSMCs may serve as an intrinsic counterbalance. We recently showed that mechanical stretching of the pulmonary arterial wall facilitates muscular eNOS activation, which would lower the resistance of pulmonary circulation induced by intrinsic contractile signals from thromboxane A 2 and mechanical stimuli (24). Thus the physiological role of eNOS in vascular smooth muscle is not limited to skeletal arteries.…”

Unconventional eNOS in pulmonary artery smooth muscles: why should it be there?

“…Since the large amount of pulmonary circulation is operating with distinctively low arterial pressure, the relatively prominent role of VSMC eNOS might be underlying the characteristic low resistance of PA resistance. The activation of intrinsic eNOS by the vasoactive agonists may counterbalance the excessive constriction of PA. Experimentally, the compromised contraction could be acutely revealed with the pharmacological inhibition of eNOS as shown in their studies [9,10]. Also, Kim et al demonstrated that the nNOS (NOS1)-and iNOS (NOS2)-specific inhibitors did not affect the contractile response to AngII and thromboxane A2, indicating the specific role of eNOS along with the expression patterns proven by immunohistochemistry [9].…”

“…The putative physiological role of VSMC eNOS in PA does not seem to be restricted to the modulation of AngII contraction. In their previous study, it was demonstrated that the combined stimulation of PA with increased wall tension (stretch) and thromboxane A2 may have also activated the VSMC eNOS [10].…”