Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells

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ÜLKER P., YARAŞ N., Yalcin O., Celik-Ozenci C., Johnson P. C., Meiselman H. J., ...Daha Fazla

NITRIC OXIDE-BIOLOGY AND CHEMISTRY, cilt.24, sa.4, ss.184-191, 2011 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 24 Sayı: 4
  • Basım Tarihi: 2011
  • Doi Numarası: 10.1016/j.niox.2011.03.003
  • Dergi Adı: NITRIC OXIDE-BIOLOGY AND CHEMISTRY
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.184-191
  • Anahtar Kelimeler: Nitric oxide synthase, Erythrocyte, Shear stress, Activation, Intracellular calcium, FLUORESCENT INDICATORS, S-NITROSOHEMOGLOBIN, HUMAN-ERYTHROCYTES, ENDOTHELIAL-CELLS, MECHANISMS, FLOW, DICHLOROFLUORESCIN, STIMULATION, OXYGENATION, RELEASE
  • Akdeniz Üniversitesi Adresli: Evet

Özet

Red blood cells (RBC) play an important role in the balance between generation and scavenging of nitric oxide (NO) and hence its local bioavailability and influence on vasomotor control. Previous studies have reported increased NO levels in RBC suspensions subsequent to exposure to shear forces; the present study was designed to further investigate changes in intracellular NO concentration and possible mechanisms involved for RBC exposed to well-controlled shear forces. Attached human RBC were subjected to shear stresses up to 0.1 Pa in a parallel-plate flow channel; fluorescent methods were used to monitor changes in intracellular NO and calcium concentrations. Intracellular NO concentration, estimated by the fluorescence level of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (OAF-FM), increased sharply within 30 s following the application of shear stress between 0.013 and 0.1 Pa. This increase was only partially prevented by the absence of L-arginine and by the presence of L-N-acetyl-methyl-arginine (L-NAME), strongly suggesting that this response was in part related to the activation of NO-synthase (NOS) enzyme. The increase in intracellular NO concentration under shear stress was also inhibited by calcium chelation in the suspending medium, indicating the role of calcium entry for NOS activation. Increases of intracellular calcium concentrations under the same shearing conditions were demonstrated by monitoring Fluo-3/AM fluorescence in RBC exposed to shear stress. Serine 1177 phosphorylated NOS protein, the activated form of the enzyme determined by immunohistochemistry, was found to be significantly increased following the exposure of RBC to 0.1 Pa shear stress for 1 min. These data confirm that RBC possess a NOS enzyme that is actively synthesizing NO and activated by effective shear forces. The data also suggest that there may be additional (e.g., non-enzymatic) NO generating mechanisms in RBC that are also enhanced under shear stress. (C) 2011 Elsevier Inc. All rights reserved.

Shear stress activation of nitric oxide synthase and increased nitric oxide levels in human red blood cells

By:Ulker, P (Ulker, Pinar)[ 2 ] ; Yaras, N (Yaras, Nazmi)[ 3 ] ; Yalcin, O (Yalcin, Ozlem)[ 4 ] ; Celik-Ozenci, C (Celik-Ozenci, Ciler)[ 5 ] ; Johnson, PC (Johnson, Paul C.)[ 4 ] ; Meiselman, HJ (Meiselman, Herbert J.)[ 6 ] ; Baskurt, OK (Baskurt, Oguz K.)[ 1 ]

 

NITRIC OXIDE-BIOLOGY AND CHEMISTRY

 

Volume: 24

Issue: 4

Pages: 184-191

DOI: 10.1016/j.niox.2011.03.003

Published: MAY 31 2011

View Journal Information

NITRIC OXIDE-BIOLOGY AND CHEMISTRY

JCR® Category Quartile in Category
BIOCHEMISTRY & MOLECULAR BIOLOGY Q2
CELL BIOLOGY Q2

Data from the 2012 edition of Journal Citation Reports®

Publisher ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA

ISSN: 1089-8603

Research Domain Biochemistry & Molecular Biology
Cell Biology

Abstract

Red blood cells (RBC) play an important role in the balance between generation and scavenging of nitric oxide (NO) and hence its local bioavailability and influence on vasomotor control. Previous studies have reported increased NO levels in RBC suspensions subsequent to exposure to shear forces; the present study was designed to further investigate changes in intracellular NO concentration and possible mechanisms involved for RBC exposed to well-controlled shear forces. Attached human RBC were subjected to shear stresses up to 0.1 Pa in a parallel-plate flow channel; fluorescent methods were used to monitor changes in intracellular NO and calcium concentrations. Intracellular NO concentration, estimated by the fluorescence level of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (OAF-FM), increased sharply within 30 s following the application of shear stress between 0.013 and 0.1 Pa. This increase was only partially prevented by the absence of L-arginine and by the presence of L-N-acetyl-methyl-arginine (L-NAME), strongly suggesting that this response was in part related to the activation of NO-synthase (NOS) enzyme. The increase in intracellular NO concentration under shear stress was also inhibited by calcium chelation in the suspending medium, indicating the role of calcium entry for NOS activation. Increases of intracellular calcium concentrations under the same shearing conditions were demonstrated by monitoring Fluo-3/AM fluorescence in RBC exposed to shear stress. Serine 1177 phosphorylated NOS protein, the activated form of the enzyme determined by immunohistochemistry, was found to be significantly increased following the exposure of RBC to 0.1 Pa shear stress for 1 min. These data confirm that RBC possess a NOS enzyme that is actively synthesizing NO and activated by effective shear forces. The data also suggest that there may be additional (e.g., non-enzymatic) NO generating mechanisms in RBC that are also enhanced under shear stress. (C) 2011 Elsevier Inc. All rights reserved.