Published online before print December 12, 2005, doi: 10.1161/01.HYP.0000196276.29211.6f
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(Hypertension. 2006;47:19.)
© 2006 American Heart Association, Inc.
Editorial Commentaries |
NOS3 Regulation
Renal Tubular Epithelial Cells Are Not Simply Large Endothelial Cells
From the Vascular Biology Center (J.S.P.), Department of Pharmacology, Medical College of Georgia, Augusta, and the Department of Cellular & Integrative Physiology (P.K.C.), University of Nebraska College of Medicine, Omaha.
Correspondence to Jennifer S. Pollock, PhD, Professor, Vascular Biology Center, CB 3213, 1459 Laney-Walker Blvd, Medical College of Georgia, Augusta, GA 30912. E-mail jpollock{at}mcg.edu
Endothelial nitric oxide synthase (NOS3) was first purified and characterized in 1991 as the enzyme responsible for the production of the endothelium-dependent relaxation factor. NOS3 converts L-arginine and molecular oxygen to L-citrulline and NO. Over the last 14 years, regulation of NOS3 enzymatic activity has been shown to be mediated by transcription and translation, substrate and cofactor availability, subcellular localization, protein–protein interactions, and phosphorylation.
NOS3 is a homodimer with each monomer possessing a reductase domain (FMN, FAD, and NADPH binding region) and an oxygenase domain (arginine, heme, Fe, and tetrahydrobiopterin binding region) that are "linked" by the calmodulin-binding domain (CBD). NOS3 activity is Ca2+/calmodulin-dependent, and it has been proposed that calmodulin binds to the CBD and relieves the inhibition from the auto-inhibitory loop, thereby enabling the flow of electrons from the reductase domain to the oxygenase domain and the production of NO. "Uncoupled NOS" refers to the condition in which O2 is the final electron acceptor and thus NOS produces O2·–. NOS3 is also known to be myristoylated and palmitoylated at the amino-terminal region, which renders the enzyme membrane-bound and has been described to be active in the plasma membrane and golgi. However, there is evidence that under pathological conditions, NOS3 is upregulated and expressed in the cytosolic compartment.1 NOS3 is known to interact with numerous proteins that modulate enzymatic activity under normal and pathological conditions.
The overwhelming majority of studies regarding regulation, and especially phosphorylation, of NOS3 have been described in the vasculature and particularly in cultured aortic endothelial cells. Thus far, 5 serine/threonine phosphorylation sites and, most recently, a tyrosine phosphorylation site (Ser114, Thr495, Ser617, Ser633, Ser1177, and Tyr83) have been elucidated on NOS3 with varying effects on the status of activity (for review, see Reference 2). Phosphorylation of Ser1177 (the most studied site, located in the carboxy-terminal NADPH-binding region of the reductase domain) and Ser633 (located within the autoinhibitory loop) are critical in the positive regulation of NOS3 activity and for sustaining NO production after the transient rise in [Ca2+]i.3,4 Phosphorylation at Ser1177 and Ser633 has been shown to increase NO production at least 3-fold above basal levels. Thr495 is phosphorylated constitutively and functions as a negative regulatory site, such that phosphorylation is associated with low enzymatic activity. Phosphorylation at this site has been proposed to impede calmodulin binding because Thr495 is located in the CBD and substantially more calmodulin binds to NOS3 when Thr495 is dephosphorylated.
Constitutively active protein kinase C (PKC) has been shown to phosphorylate Thr495. Specifically, activation of PKC 
has recently been shown to activate NOS3 and increase arterial blood flow in vivo,5 whereas inhibition of the activation of PKC results in dephosphorylation of Thr495 and NOS3-derived O2·–.6 The serine/threonine phosphatases PP1 and PP2B (calcineurin) have been implicated in activation of NOS3 activity via effects on the Thr495 site.4 Lin et al7 recently proposed that the phosphorylation of Thr495 may function as a "switch" to regulate the production of O2·– and/or NO. These investigators found that a mutant NOS3 construct (T495A) that is unable to be phosphorylated at the Thr495 site exhibited high enzymatic activity; however, cellular production of NO was very low and large amounts of O2·– were generated. Thus, on dephosphorylation of Thr495, NOS3 may generate both O2·– and NO. Thus, high enzymatic activity may translate into high O2·– and low NO production or high NO and low O2·– production. It has been proposed that phosphorylation of Ser1177 and Ser633 and dephosphorylation of the Thr495 sites on NOS3 are most likely not regulated independently but may be coordinated. We would extend this to include that the coordination of the phosphorylation and dephosphorylation events may depend on the redox status of the cellular and/or subcellular environment. Further research is needed to explore these hypotheses.
Ser114 and Ser617 are the least studied sites on NOS3. Ser114 is located within the oxygenase domain, near the tetrahydrobiopterin (H4B) binding site, and is thought to be a negative regulatory site similar to the Thr495 site. Ser617 is located near the autoinhibitory loop, and the function of this site remains controversial. Phosphorylation at Tyr83 was recently reported to activate NOS3 via Src kinase activation by hydrogen peroxide in cultured endothelial cells.8
Phosphoregulation of NOS3 in renal tubular epithelial cells has only recently begun to be elucidated, and information is available only for specific segments of the nephron. Garvin and associates9 have previously demonstrated the importance of NOS3-mediated NO production in inhibiting sodium reabsorption in the medullary thick ascending limb (mTAL). In this issue of Hypertension, Herrera et al10 extend their observations to demonstrate the temporal changes in phosphorylation of NOS3 in the mTAL from salt-loaded rats in conjunction with the quantification of NOS3-derived NO and O2·– production. These authors found that NOS3 expression and NO production are dissociated in the mTAL under salt loading and that the changes in NO production were mainly attributed to the phosphorylation status of NOS3 at the Thr495 site. However, the dephosphorylation at Thr495 did not result in NOS uncoupling, despite increased O2·– production within the mTAL of the salt-loaded rats. This study is highlighted in this commentary to underscore the contrasting phosphoregulation of NOS3 from vascular aortic endothelial and renal epithelial cells (Figure). Specifically, changes in NO production during high salt in the mTAL are attributable mainly to changes in NOS3 activity due to phosphorylation at Thr495, with no evidence for NOS3-derived O2·– production. Thus, although uncoupled NOS3 in the vascular endothelium appears to be linked to the phosphorylation status of Thr495, this phenomenon is not apparent in the mTAL. These differences may be related to the cellular and subcellular environment and/or the protein-protein interactions within the various cell types. The functional consequences of the products of NOS3 activity in the nephron may elicit opposite effects. NO inhibiting sodium reabsorption9 and O2·– stimulating sodium reabsorption in the mTAL,11 although no evidence has been provided thus far that NOS3 mediates O2·– production in the mTAL. Further research is clearly indicated to discern the molecular mechanisms of epithelial NOS3-mediated NO and O2·– production. We could envision that these mechanisms would provide novel targets for the regulation of sodium and water homeostasis along the nephron.
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Garvin’s laboratory previously reported that introducing flow through isolated mTAL activates NOS3 via PI3 kinase-dependent phosphorylation at Ser1177.12 In addition, this group has proposed that a portion of the NOS3 pool acutely translocates to the apical membrane on introducing flow from a static level in the mTAL. However, it is unknown whether the translocation involves a phosphorylation/dephosphorylation of NOS3 or whether the effects of salt loading on the mTAL are also associated with a translocation of NOS3.
Our laboratories13 have shown that NOS3 phosphorylation at Thr495 appears prominent by immunohistochemistry in the apical aspect of the mTAL in normal rat kidney, but is markedly reduced in kidneys from rats with streptozotocin-induced type 1 diabetes. Interestingly, pThr495 immunostaining is also evident in the medullary collecting duct; however, it is distributed throughout the cells and appears to be unchanged in diabetes. It is unclear whether this difference between the mTAL and collecting duct reflects differential activity of PKC or the phosphatases, PP1 and PP2B. Thus, the changes in renal medullary NOS3 phosphorylation at Thr495 that occur during type 1 diabetes vary between nephron segments. The myocardium also expresses NOS3, and recently Brixius et al14 demonstrated differential mechanisms of phosphoregulation of agonist-mediated activation of NOS3 between ventricular and atrial myocardium. This further indicates the differential molecular mechanisms of NOS3 phosphoregulation within various cell types.
These studies indicate that the regulation of NOS3 activity within vascular endothelial cells, myocardium, renal mTAL, and renal medullary collecting duct will most likely have contrasting outcomes under various physiological and pathophysiological conditions. Many unanswered questions remain about the regulation of NOS3 in renal epithelial cells and possible dysfunction under pathophysiological conditions.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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 Top References |
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1. Sullivan JS, Pollock DM, Pollock JS. Altered nitric oxide synthase 3 distribution in mesenteric arteries of hypertensive rats. Hypertension. 2002; 39: 597–602.
2. Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1–R12.
3. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papaetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999; 339: 597–601.
4. Michell BJ, Chen Z, Tiganis T, Stapleton D, Katsis F, Power DA, Sim AT, Kemp BE. Coordinated control of endothelial nitric oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem. 2001; 276: 17625–17628.
5. Partovian C, Zhuang Z, Moodie K, Lin M, Ouchi N, Sessa WC, Walsh K, Simons M. PKC alpha activates eNOS and increases arterial blood flow in vivo. Circ Res. 2005; 97: 482–487.
6. Fleming I, Mohamed A, Galle J, Turchanowa L, Brandes RP, Fisslthaler B, Busse R. Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKC alpha. Cardiovasc Res. 2005; 65: 897–906.
7. Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA, Sessa WC. Phosphorylation of threonine 497 in endothelial nitric oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem. 2003; 278: 44719–44726.
8. Fulton D, Church JE, Ruan L, Li C, Sood SG, Kemp BE, Jennings IG, Venema RC. SRC kinase activates endothelial nitric oxide synthase by phosphorylating TYR-83. J Biol Chem. 2005; 280: 35943–35952.
9. Ortiz PA, Hong NJ, Garvin JL. NO decreases thick ascending limb chloride absorption by reducing Na(+)-K(+)-2Cl(–) cotransporter activity. Am J Physiol Renal Physiol. 2001; 281: F819–F825.
10. Herrera M, Silva GB, Garvin JL. A high salt diet dissociates NO synthase-3 expression and NO production by the THAL. Hypertension. 2006; 47: 95–101.
11. Juncos R, Garvin JL. Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb. Am J Physiol Renal Physiol. 2005; 288: F982–F987.
12. Ortiz PA, Hong NJ, Garvin JL. Luminal flow induces eNOS activation and translocation in the rat thick ascending limb. Role of PI3-kinase and Hsp90. Am J Physiol Renal Physiol. 2004; 287: F281–F288.
13. Lee DL, Sasser JM, Hobbs JL, Boriskie A, Pollock DM, Carmines PK, Pollock JS. Posttranslational regulation of NO synthase activity in the renal medulla of diabetic rats. Am J Physiol Renal Physiol. 2005; 288: F82–F90.
14. Brixius K, Bloch W, Pott C, Napp A, Krahwinkel A, Ziskoven C, Koriller M, Mehlhorn U, Hescheler J, Fleischmann B, Schwinger RH. Mechanisms of beta 3-adrenoceptor-induced eNOS activation in right atrial and left ventricular human myocardium. Br J Pharmacol. 2004; 143: 1014–1022.[CrossRef][Medline] [Order article via Infotrieve]
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