Endothelial Nitric-oxide Synthase (Type III) Is Activated and Becomes Calcium Independent upon Phosphorylation by Cyclic Nucleotide-dependent Protein Kinases (original) (raw)
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Role of Nitric Oxide and Its Intracellular Signalling Pathways in the Control of Ca2 Homeostasis
Biochemical Pharmacology, 1998
Ca 2ϩ , a primary regulator of physiological functions in all cells, is involved in a variety of intracellular signalling pathways; control of Ca 2ϩ homeostasis is, therefore, a fundamental cell activity. To this end, cells have developed a variety of mechanisms to ensure the buffering of Ca 2ϩ , its influx and extrusion from the plasma membrane, and its release/accumulation within specific intracellular storage compartments. Over the last few years, evidence gathered from a number of cell systems has indicated that one of the key messengers governing the overall control of Ca 2ϩ homeostasis is nitric oxide (NO), which may be produced intracellularly or may originate from neighboring cells. The aim of the present commentary is to concentrate on the biochemical steps in Ca 2ϩ homeostasis that are controlled by NO and to describe what is known thus far concerning the molecular mechanisms of its action. Particular attention will be given to the effects of NO on: (i) inositol 1,4,5-trisphosphate and cyclic ADP ribose generation; (ii) Ca 2ϩ release from both inositol 1,4,5-trisphosphate-sensitive and ryanodine-sensitive Ca 2ϩ stores; and (iii) Ca 2ϩ influx via both store-and second messenger-operated Ca 2ϩ channels. The evidence discussed here documents the complexity of the interactions between the Ca 2ϩ and the NO signalling systems, which represent an extraordinary example of cross-talk operating at multiple sites and which are continuously active in the regulation of cytosolic Ca 2ϩ (and NO) levels. BIOCHEM PHARMACOL 55;6:713-718, 1998. † Abbreviations: NO, nitric oxide; [Ca 2ϩ ] i , intracellular Ca 2ϩ concentration; IP 3 and IP 3 R, inositol 1,4,5-trisphosphate and its receptor; RyR, ryanodine receptor; PLC, phospholipase C; G kinase, cGMP-dependent protein kinase I; cADP ribose, cyclic ADP ribose; SOCC and SMOC, store-operated and second messenger-operated Ca 2ϩ channels; and cGMP, cyclic GMP.
Journal of Biological Chemistry, 2001
Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2012
Background: Argininosuccinate synthase (AS) is critical for endothelial nitric oxide production, yet little is known about its regulation. Results: AS Ser-328 phosphorylation increased with calcium stimulation and decreased with PKC␣ interference. Conclusion: PKC␣ phosphorylates AS at Ser-328 under calcium-dependent stimulatory conditions to support nitric oxide production. Significance: Knowledge of how AS is regulated is essential in understanding nitric oxide homeostasis. Endothelial nitric-oxide synthase (eNOS) utilizes L-arginine as its principal substrate, converting it to L-citrulline and nitric oxide (NO). L-Citrulline is recycled to L-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase C␣ (PKC␣) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKC␣. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells. Nitric oxide (NO) production is strictly regulated in vascular endothelial cells. Impairment of this control is associated with risk factors that compromise endothelial function. Arginine
An Inducible Nitric-oxide Synthase (NOS)-associated Protein Inhibits NOS Dimerization and Activity
Journal of Biological Chemistry, 1999
A variety of transcriptional and post-transcriptional mechanisms regulate the expression of the inducible nitric-oxide synthase (iNOS, or NOS2). Although neurons and endothelial cells express proteins that interact with and inhibit neuronal NOS and endothelial NOS, macrophage proteins that inhibit NOS2 have not been identified. We show that murine macrophages express a 110-kDa protein that interacts with NOS2, which we call NOS-associated protein-110 kDa (NAP110). NAP110 directly interacts with the amino terminus of NOS2, and inhibits NOS catalytic activity by preventing formation of NOS2 homodimers. Expression of NAP110 may be a mechanism by which macrophages expressing NOS2 protect themselves from cytotoxic levels of nitric oxide.
Cellular signalling and regulation of nitric oxide synthesis
1996
Murine macrophages synthesise nitric oxide (NO) from L-arginine, and the reaction is catalysed by NO synthases (NOS). Macrophages stimulated by interferon-gamma (IFN-gamma) and low dose of lipopolysaccharide (LPS) or a high concentration of LPS alone can produce large amounts of NO. Using a murine macrophage cell line, J774, we have investigated the signalling mechanisms for NO production. Transient increases in cyclic adenosine monophosphate (cAMP) did not affect the production of NO. However, prolonged elevation of intracellular cAMP levels, using the prostaglandin E2 and phosphodiesterase inhibitors isobutylmethylxanthine or rolipram, was found to inhibit NO production. This inhibition did not occur at the level of gene transcription, but at the post transcriptional level. Interferon-gamma and LPS treatment resulted in protein kinase C (PKC) activation and translocation into the plasma membrane, which correlated with NO production and NOS activity. The activation and the transloc...
Phosphorylation and subcellular translocation of endothelial nitric oxide synthase
Proceedings of the National Academy of Sciences, 1993
In the vascular endothelium, diverse cell surface receptors are coupled to the Ca2+/calmodulin-dependent activation of nitric oxide (NO) synthase. We now report that, in intact cultured endothelial cells, several drugs and agonists are associated with increased serine phosphorylation of the endothelial NO synthase. We biosynthetically labeled bovine aortic endothelial cells with [32P]orthophosphoric acid, exposed the cells to various drugs and hormones, and then immunoprecipitated the enzyme from cell extracts using a highly specific anti-peptide antibody. The marked endothelial NO synthase phosphorylation induced by bradykinin is maximal only after 5 min of agonist exposure and is stable for at least 20 min. Basal and agonist-induced phosphorylation of the NO synthase in endothelial cells is completely inhibited by the calmodulin antagonist compound W-7. We prepared subcellular fractions of endothelial cells that had been biosynthetically labeled with [35S]methionine or [32P]orthop...
Nitric oxide synthases: regulation and function
2012
Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize L-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH 4). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin-and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins.
Free Radical Biology and Medicine, 2003
Shear stress stimulates NO production involving the Ca 2ϩ-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A-(PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca 2ϩ ] i conditions. More interestingly, S635D cells produced NO even when [Ca 2ϩ ] i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca 2ϩ ] i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca 2ϩ-independent manner may play an important role in regulating vascular biology and pathophysiology.
Nitric oxide signalling in cardiovascular health and disease
Following the discovery of nitric oxide (NO) as one of the endothelium-derived relaxing factors (for which the Nobel Prize in Physiology or Medicine was awarded in 1998), a substantial body of evidence has confirmed the involvement of NO endogenously prod uced by the nitric oxide synthases (NOSs) in both the protection from and initiation of human vascular and cardiac disease. This apparent paradox stems from the more recently appreciated complexity of the distinctive regulation of NOS and NO bio availability. In particular, transcriptional and post-translational regulation of the NOSs and redox modulation of NOS and NO bio-activity, as well as of downstream elements of NO signalling (such as soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (also known as protein kinase G; PKG)), dictate the final effect on target organs. The actions of NO are not limited to the control of cardiac or vascular contractility, but have been extended to the regulation of cardio vascular tissue remodelling (for example, fibrosis) and metabolism (such as in brown and perivascular fat). Intriguingly, NO can also be gener ated from nitrite and dietary nitrate through nitrate reductase expressed in com-mensal bacteria, further linking the microbiota to cardiovascular homeostasis. To untangle this complex regulation, we first review the distribution and molecular regulation of NOS isoforms in cardiovascular tissues as well as important elements of downstream NO signalling. We next summarize the latest experimental paradigms of NOS-mediated regulation of cardiac and vascular physiology. The following sections cover the mechanisms of dys-function of NOS signalling and the consequences of this dysfunction on cardiovascular pathophysiology. Finally, we focus on emerging paradigms in human diseases and review the latest developments in therapeutic strategies to modulate NO signalling. NOS isoforms in cardiovascular tissues NO is synthesized by three isoforms of NOS encoded by distinct genes: brain or neuronal NOS (nNOS; encoded by NOS1), inducible NOS (iNOS; encoded by NOS2), and endothelial NOS (eNOS; encoded by NOS3). The constitutively expressed nNOS and eNOS are highly regu lated by transcriptional, post-transcriptional (including microRNA), and post-translational mechanisms , including phosphorylation, acetyl ation, protein-protein interaction, S-nitrosylation, and S-gluta thio-nyl ation. Conversely, the inducible isoform, iNOS, is mainly regulated through gene transcription under pro-inflammatory and oxidative stress conditions. NOSs are oxidoreductase homodimer enzymes, composed of an amino-terminal oxygenase domain that contains binding sites for the substrate l-arginine, the cofactor tetrahydrobiopterin (BH 4), and a ferric haem cluster, as well as a reductase domain with binding sites for the electron donors nicotinamide adenine dinucleo-tide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN). Both domains are connected by a sequence that binds calcium (Ca 2+)-complexed calmodulin. Upon NOS activation, the flavins in the reductase domain transfer NADPH-derived electrons to the haem in the oxygenase domain of the other mono mer, allowing oxygen (O 2) binding on the reduced haem iron (Fe 2+) and the conversion of l-arginine to HO-l-arginine, then NO and l-citrulline 1. Abstract | Nitric oxide (NO) signalling has pleiotropic roles in biology and a crucial function in cardiovascular homeostasis. Tremendous knowledge has been accumulated on the mechanisms of the nitric oxide synthase (NOS)-NO pathway, but how this highly reactive, free radical gas signals to specific targets for precise regulation of cardiovascular function remains the focus of much intense research. In this Review, we summarize the updated paradigms on NOS regulation, NO interaction with reactive oxidant species in specific subcellular compartments, and downstream effects of NO in target cardiovascular tissues, while emphasizing the latest developments of molecular tools and biomarkers to modulate and monitor NO production and bioavailability. NATURE REVIEWS | CARDIOLOGY ADVANCE ONLINE PUBLICATION | 1 REVIEWS © 2 0 1 8 M a c m i l l a n P u b l i s h e r s L i m i t e d , p a r t o f S p r i n g e r N a t u r e. A l l r i g h t s r e s e r v e d .