Moderate exercise promotes human RBC-NOS activity, NO production and deformability through Akt kinase pathway
Frank Suhr, Julian Brenig, Rebecca Müller, Hilke Behrens, Wilhelm Bloch, Marijke Grau, Frank Suhr, Julian Brenig, Rebecca Müller, Hilke Behrens, Wilhelm Bloch, Marijke Grau
Abstract
Background: Nitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs.
Methods/findings: Fifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer(1177) was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr(495) were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability.
Conclusion/significance: This human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
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References
- Gladwin MT, Crawford JH, Patel RP (2004) The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation. Free Radic Biol Med 36: 707–717.
- Malan D, Ji GJ, Schmidt A, Addicks K, Hescheler J, et al. (2004) Nitric oxide, a key signaling molecule in the murine early embryonic heart. FASEB J 18: 1108–1110.
- Kroncke KD, Fehsel K, Kolb-Bachofen V (1997) Nitric oxide: cytotoxicity versus cytoprotection–how, why, when, and where? Nitric Oxide 1: 107–120.
- Cho HJ, Xie QW, Calaycay J, Mumford RA, Swiderek KM, et al. (1992) Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med 176: 599–604.
- Ghafourifar P, Schenk U, Klein SD, Richter C (1999) Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation. J Biol Chem 274: 31185–31188.
- Giulivi C, Poderoso JJ, Boveris A (1998) Production of nitric oxide by mitochondria. J Biol Chem 273: 11038–11043.
- Jubelin BC, Gierman JL (1996) Erythrocytes may synthesize their own nitric oxide. Am J Hypertens 9: 1214–1219.
- Kleinbongard P, Schulz R, Rassaf T, Lauer T, Dejam A, et al. (2006) Red blood cells express a functional endothelial nitric oxide synthase. Blood 107: 2943–2951.
- Mihov D, Vogel J, Gassmann M, Bogdanova A (2009) Erythropoietin activates nitric oxide synthase in murine erythrocytes. Am J Physiol Cell Physiol 297: C378–C388.
- Ulker P, Yaras N, Yalcin O, Celik-Ozenci C, Johnson PC, et al. (2011) Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells. Nitric Oxide 24: 184–191.
- Green DJ, Maiorana A, O’Driscoll G, Taylor R (2004) Effect of exercise training on endothelium-derived nitric oxide function in humans. J Physiol 561: 1–25.
- Yalcin O, Bor-Kucukatay M, Senturk UK, Baskurt OK (2000) Effects of swimming exercise on red blood cell rheology in trained and untrained rats. J Appl Physiol 88: 2074–2080.
- Ozuyaman B, Grau M, Kelm M, Merx MW, Kleinbongard P (2008) RBC NOS: regulatory mechanisms and therapeutic aspects. Trends Mol Med 14: 314–322.
- Bor-Kucukatay M, Wenby RB, Meiselman HJ, Baskurt OK (2003) Effects of nitric oxide on red blood cell deformability. Am J Physiol Heart Circ Physiol 284: H1577–H1584.
- Suhr F, Porten S, Hertrich T, Brixius K, Schmidt A, et al. (2009) Intensive exercise induces changes of endothelial nitric oxide synthase pattern in human erythrocytes. Nitric Oxide 20: 95–103.
- Senturk UK, Gunduz F, Kuru O, Aktekin MR, Kipmen D, et al. (2001) Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats. J Appl Physiol 91: 1999–2004.
- Szygula Z (1990) Erythrocytic system under the influence of physical exercise and training. Sports Med 10: 181–197.
- Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, et al. (1999) The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 9: 845–848.
- Ulker P, Sati L, Celik-Ozenci C, Meiselman HJ, Baskurt OK (2009) Mechanical stimulation of nitric oxide synthesizing mechanisms in erythrocytes. Biorheology 46: 121–132.
- Ludolph B, Bloch W, Kelm M, Schulz R, Kleinbongard P (2007) Short-term effect of the HMG-CoA reductase inhibitor rosuvastatin on erythrocyte nitric oxide synthase activity. Vasc Health Risk Manag 3: 1069–1073.
- Kabanova S, Kleinbongard P, Volkmer J, Andree B, Kelm M, et al. (2009) Gene expression analysis of human red blood cells. Int J Med Sci 6: 156–159.
- Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, et al. (1998) Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 70: 2446–2453.
- Itoh Y, Ma FH, Hoshi H, Oka M, Noda K, et al. (2000) Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal Biochem 287: 203–209.
- Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, et al. (1998) Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett 427: 263–266.
- Espey MG, Miranda KM, Thomas DD, Wink DA (2001) Distinction between nitrosating mechanisms within human cells and aqueous solution. J Biol Chem 276: 30085–30091.
- Hardeman MR, Dobbe JG, Ince C (2001) The Laser-assisted Optical Rotational Cell Analyzer (LORCA) as red blood cell aggregometer. Clin Hemorheol Microcirc 25: 1–11.
- Baskurt OK, Boynard M, Cokelet GC, Connes P, Cooke BM, et al. (2009) New guidelines for hemorheological laboratory techniques. Clin Hemorheol Microcirc 42: 75–97.
- Lincoln TM (1989) Cyclic GMP and mechanisms of vasodilation. Pharmacol Ther 41: 479–502.
- Hofmann F, Ammendola A, Schlossmann J (2000) Rising behind NO: cGMP-dependent protein kinases. J Cell Sci 113 (Pt 10): 1671–1676.
- Petrov V, Amery A, Lijnen P (1994) Role of cyclic GMP in atrial-natriuretic-peptide stimulation of erythrocyte Na+/H+ exchange. Eur J Biochem 221: 195–199.
- Petrov V, Lijnen P (1996) Regulation of human erythrocyte Na+/H+ exchange by soluble and particulate guanylate cyclase. Am J Physiol 271: C1556–C1564.
- Bateman RM, Sharpe MD, Ellis CG (2003) Bench-to-bedside review: microvascular dysfunction in sepsis–hemodynamics, oxygen transport, and nitric oxide. Crit Care 7: 359–373.
- Pries AR, Secomb TW (2003) Rheology of the microcirculation. Clin Hemorheol Microcirc 29: 143–148.
- Forstermann U, Munzel T (2006) Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113: 1708–1714.
- Forstermann U, Nakane M, Tracey WR, Pollock JS (1993) Isoforms of nitric oxide synthase: functions in the cardiovascular system. Eur Heart J 14 Suppl I10–15.
- Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R (2001) Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88: E68–E75.
- Fleming I, Busse R (2003) Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 284: R1–12.
- Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, et al. (1999) Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601–605.
- Zhang QJ, McMillin SL, Tanner JM, Palionyte M, Abel ED, et al. (2009) Endothelial nitric oxide synthase phosphorylation in treadmill-running mice: role of vascular signalling kinases. J Physiol 587: 3911–3920.
- Chmiel B, Cierpka L (2003) Organ preservation solutions impair deformability of erythrocytes in vitro. Transplant Proc 35: 2163–2164.
- de Oliveira S, Silva-Herdade AS, Saldanha C (2008) Modulation of erythrocyte deformability by PKC activity. Clin Hemorheol Microcirc 39: 363–373.
- Huang Q, Yuan Y (1997) Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability. Am J Physiol 273: H2442–H2451.
- Sprague RS, Ellsworth ML, Stephenson AH, Lonigro AJ (1996) ATP: the red blood cell link to NO and local control of the pulmonary circulation. Am J Physiol 271: H2717–H2722.
- Busse R, Ogilvie A, Pohl U (1988) Vasomotor activity of diadenosine triphosphate and diadenosine tetraphosphate in isolated arteries. Am J Physiol 254: H828–H832.
- Bogle RG, Coade SB, Moncada S, Pearson JD, Mann GE (1991) Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells. Biochem Biophys Res Commun 180: 926–932.
- Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376.
- Wan J, Ristenpart WD, Stone HA (2008) Dynamics of shear-induced ATP release from red blood cells. Proc Natl Acad Sci U S A 105: 16432–16437.
- Sprague RS, Ellsworth ML, Stephenson AH, Kleinhenz ME, Lonigro AJ (1998) Deformation-induced ATP release from red blood cells requires CFTR activity. Am J Physiol 275: H1726–H1732.
- Sprague RS, Stephenson AH, Ellsworth ML, Keller C, Lonigro AJ (2001) Impaired release of ATP from red blood cells of humans with primary pulmonary hypertension. Exp Biol Med (Maywood ) 226: 434–439.
- Sprague RS, Stephenson AH, Bowles EA, Stumpf MS, Lonigro AJ (2006) Reduced expression of G(i) in erythrocytes of humans with type 2 diabetes is associated with impairment of both cAMP generation and ATP release. Diabetes 55: 3588–3593.
- Kaneko FT, Arroliga AC, Dweik RA, Comhair SA, Laskowski D, et al. (1998) Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension. Am J Respir Crit Care Med 158: 917–923.
- Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, et al. (2002) Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 106: 987–992.
- Robertson BE, Schubert R, Hescheler J, Nelson MT (1993) cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am J Physiol 265: C299–C303.
- Armstead WM (1996) Role of ATP-sensitive K+ channels in cGMP-mediated pial artery vasodilation. Am J Physiol 270: H423–H426.
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