The Effect And Mechanism Of Endoplasmic Reticulum Ca²⁺ Release And Endothelial Nitric Oxide Synthase In The Regulation Of Endothelial Function

Nitric oxide (NO) is a chief signaling molecule in cardiovascular regulation. In addition to relaxing vascular tone, NO regulates cardiac contractility, platelet aggregation, angiogenesis, and vascular smooth muscle proliferation. Thus, physiological NO formation is essential for cardiovascular homeostasis. Abnormalities of NO production, on the other hand, are found in almost all cardiovascular diseases. In endothelial cells, NO is primarily produced by endothelial NO synthase (eNOS). Since NO is a diffusible gas and cannot be stored in an intracellular compartment, the onset of NO signaling is triggered by eNOS activation. The intensity and time-span of NO signaling are largely dictated by the functional status of eNOS. Thus, understanding the mechanisms of eNOS activation and regulation has been the focus of cardiovascular NO research.The catalytic activity of eNOS is triggered by the binding of calmodulin (CaM). In a classic view, the binding between eNOS and CaM is initiated by the increased intracellular Ca2+ concentrations. A canonical eNOS activation process begins with the binding between agonists and their respective receptors, most of which are coupled with G-protein. Activation of these receptors results in the increases of intracellular free Ca2+. The increases of Ca2+ concentrations by some agonists, such as acetylcholine and bradykinine, are dependent on influx of extracellular Ca2+. In other cases, such as the activation of purine receptor (P2Y) receptors by ATP, Ca2+ are primarily released from intracellular Ca2+ repertoire-endoplasmic reticulum (ER), though the refill of ER Ca2+ relies on extracellular Ca2+ supply. Increased cytosolic Ca2+ bind with CaM forming a Ca2+/CaM complex, the latter subsequently binds with eNOS. Binding with CaM results in a conformational change of eNOS. This enables the electron transfer from eNOS reductase domain to the oxygenase domain, where L-arginine, oxygen, and NADPH are converted to NO and L-citrulline.Besides Ca+, eNOS is also regulated by protein phosphorylation. eNOS is known being phosphorylated at several of its serine and threonine residues. The first site where phosphorylation was found to critically influence eNOS function is the serine 1179/1177 (bovine/human). eNOS Ser1179 was initially reported to be phosphorylated by Akt. Subsequent studies showed that other kinases, such as AMPK, PKG, CaMKII, also phosphorylate eNOS Ser1179. Ser1179 phosphorylation enhances eNOS function. A variety of extracellular stimuli are now known to influence eNOS activity by modulating its Ser1179 phosphorylation. eNOS is also phosphorylated at its Ser635 residues. Ser635 phosphorylation augments eNOS activity and this has been shown to be an important mechanism underlying the response of endothelial cells to share stress. So far, studies show that protein kinase A (PKA) phosphorylates eNOS Ser635. In contrast to the positive effect of Ser1179 and Ser635 phosphorylation, phosphorylation of Thr497 attenuates eNOS activity. PKC has been shown to be responsible for eNOS Thr497 phosphorylation in endothelial cells. Beyond Serll79/Ser635/Thr497, eNOS has been reported to be phosphorylated at its Ser116 and Ser617 residues. Compared to the extensively studied roles of eNOS Ser1179/Ser635/Thr497 phosphorylation in endothelial regulation and diseases, the in vivo significance of eNOS Serl 16 and Ser617 phosphorylation remains to be established.Although both Ca2+ and protein phosphorylation play crucial roles in eNOS regulation, the interrelationship between Ca2+ mobilization and eNOS phosphorylation is understood in much less detail. Nevertheless, recent studies show that while CaM binding remains essential for eNOS activation, whether or not the binding between CaM and eNOS requires the increases of cytosolic Ca2+ is heavily influenced by the phosphorylation status of eNOS. For example, phosphorylation of Ser1179 or Ser635 was reported to render eNOS activation in resting intracellular Ca2+ concentrations. This is apparently because phosphorylation of the two sites enhances the binding affinity of eNOS with CaM. On the other hand, how intracellular Ca2+ mobilization influences eNOS phosphorylation is largely unclear.Our studies suggested that with bovine endothelial cells, discharging ER Ca2+ by thapsigargin induced a dose-dependent increase of eNOS Ser635 phosphorylation leading to elevated NO production. This effect was independent of extracellular Ca2+, and selective to Ser635 because the phosphorylation status of eNOS Ser1179 or Thr497 was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2+ release-induced eNOS Ser635 phosphorylation; whereas inhibiting protein kinase A or Ca2+/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK directly phosphorylated eNOS Ser635 residues in vitro. Further studies demonstrated that ER Ca2+ release-induced ERK1/2 activation mediated the enhancing action of purine receptor stimulation on eNOS Ser635 phosphorylation in endothelial cells. Mutating the Ser635 to non-phosphorylatable alanine prevented ATP from activating eNOS in cells.In conclusion, In the present study we report that discharging Ca2+ from ER results in a selective upregulation of eNOS phosphorylation on its Ser635 residue. Our studies further reveal that the action of ER Ca2+ release on eNOS Ser635 phosphorylation is mediated by ERK1/2. Moreover, we demonstrate ER Ca2+ release-elicited ERK1/2 activation and subsequent Ser635 phosphorylation account for the regulation of ATP on eNOS in endothelial cells.