Endojen ve Eksojen Adenozin Tri-Fosfat?ın Eritrosit Deformabilitesine Etkisi

Öz

Amaç: Eritrosit deformabilitesi eritrositlerin dolaşım sisteminde karşılaştıkları kuvvetlerin etkisi altında şekil değiştirebilme yeteneğidir. Bu parametre deneysel ve klinik hemoreoloji çalışmalarında sıklıkla kullanılarak eritrositlerin mekanik özellikleri değerlendirilmektedir. Eritrositler plazma içinde endojen ve eksojen adenozin tri-fosfat ATP ʼye sürekli maruz kalmaktadır. Eksojen ATPʼnin eritrositlerde hücre içi iyon dengesinde ve hücre hacminde değişmeye neden olduğu daha önce gösterilmiştir. Oysa hücre geometrisinden büyük oranda etkilenen eritrosit deformabilitesinin ATPʼden etkilenip etkilenmediği bilinmemektedir. Çalışmanın amacı eritrosit deformabilitesinin endojen ve eksojen ATPʼye yanıtlarının incelenmesidir. Gereç ve Yöntemler: Çalışmada gönüllü erkeklerden alınan venöz kan örnekleri kullanılmıştır. Kan örneklerinden eritrosit izolasyonu yapıldıktan sonra otolog plazma içinde hematokrit %40 olarak ayarlanmıştır. Eksojen ATP çalışmalarında purinerjik reseptör inhibitörü varlığında ve yokluğunda eritrosit süspansiyonlarına 100, 300 ve 500 µM ATP eklenmiş ve yarım saat inkübasyonun ardından deformabilite ölçümleri yapılmıştır. Endojen ATP çalışmalarında ATP kanal inhibitörü varlığında ve yokluğunda eritrositlere mekanik stres uygulanmış ve mekanik stresin hemen ardından eritrosit deformabilitesi ölçülmüştür.Bulgular: 300 ve 500 µM ATP eritrosit deformabilitesinde düşmeye neden olurken bu düşüş purinerjik reseptör inhibitörü varlığında ortadan kalkmıştır. Öte yandan mekanik stres eritrositlerden ATP salınımını uyarmış ve bu salınım ATP kanal inhibitörü ile ortadan kalkmıştır. Bu koşullar altında endojen ATP eritrosit deformabilitesinde bir değişikliğe neden olmamıştır.Sonuç: Çalışmanın sonuçları eksojen ATP'nin hücre membranında bulunan purinerjik reseptörler aracılığı ile eritrosit deformabilitesinde azalmaya neden olduğunu ilk defa göstermiştir. Öte yandan dolaşım sisteminde gözlenen mekanik stres düzeylerinde eritrositlerden salınan endojen ATPʼnin eritrosit deformabilitesini etkilemediği gösterilmiştir

Anahtar Kelimeler

• Eritrosit, Deformabilite, ATP, Mekanik stres

Effect of Endogenous and Exogenous Adenosine-Three Phosphate on Erythrocyte Deformability

Öz

Objective: Erythrocytes respond to forces by extensive changes in their shape, with the degree of deformation under a given force known as erythrocyte deformability. Erythrocytes are consistently exposed to endogenous and exogenous ATP in the plasma. Studies have shown that exogenous adenosine-three phosphate ATP causes the intracellular ionic balance to change and reduces the cell volume. Although it is well known that cell volume is one of the regulators of erythrocyte deformability, there is no study on the effect of ATP on erythrocyte deformability. The aim of this study was to investigate the effects of endogenous and exogenous ATP on erythrocyte deformability. Material and Methods: Erythrocytes isolated from volunteers were used in this study. After erythrocyte isolation, the hematocrit of erythrocyte suspensions was set to 40%. The effect of 100, 300 and 500 µM ATP on erythrocyte deformability was evaluated in the presence or absence of purinergic receptor antagonist. The effect of endogenous ATP on erythrocyte deformability was evaluated after mechanical stress application in the presence or absence of ATP channel inhibitor.Results: 300 and 500 µM ATP caused a decrease in erythrocyte deformability while purinergic receptor antagonist ameliorated this effect. Moreover, mechanical stress application resulted in increased ATP release from erythrocytes. This increase was shown to be abolished in the presence of an ATP channel blocker. Endogenous ATP exerted no effect on erythrocyte deformability. Conclusion:The results of this study demonstrated that exogenous ATP decreases erythrocyte deformability through purinergic receptors. Moreover, endogenous ATP released under mechanical stress conditions had no effect on red blood cell deformability.

Anahtar Kelimeler

• Erythrocyte, Deformability, ATP, Mechanical stress

Kaynakça

1. 1. Chien S. Red cell deformability and its relevance to blood flow. Annu Rev Physiol 1987; 49: 177-92.
2. 2. Shiga T. Maeda N, Kon K. Erythrocyte rheology. Crit Rev Oncol Hematol 1990; 10(1): 9-48.
3. 3. Gordon RJ, Ravin MB. Rheology and anesthesiology. Anesth Analg 1978; 57(2): 252-61.
4. 4. Mohandas N, Chasis JA, Shohet SB. The influence of membrane skeleton on red cell deformability, membrane material properties, and shape. Semin Hematol 1983; 20(3): 225-42.
5. 5. Heath BP, Mohandas N, Wyatt JL, Shohet SB. Deformability of isolated red blood cell membranes. Biochim Biophys Acta 1982; 691(2): 211-9.
6. 6. Mohandas N, Shohet SB. The role of membraneassociated enzymes in regulation of erythrocyte shape and deformability. Clin Haematol 1981; 10(1): 223-37.
7. 7. Mohandas N. Chasis JA. Red blood cell deformability, membrane material properties and shape: Regulation by transmembrane, skeletal and cytosolic proteins and lipids. Semin Hematol 1993; 30(3): 171-92.
8. 8. Evans EA, La Celle PL. Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation. Blood 1975; 45(1): 29-43.

9. 9. Burnstock G. Purinergic cotransmission. Brain Res Bull 1999; 50(5-6): 355-7.
10. 10. Burnstock G. Do some nerve cells release more than one transmitter? Neuroscience 1976; 1(4): 239-48.
11. 11. Burnstock G. Historical review: ATP as a neurotransmitter. Trends Pharmacol Sci 2006; 27(3): 166-76.
12. 12. Fitz JG. Regulation of cellular ATP release. Trans Am Clin Climatol Assoc 2007; 118: 199-208.
13. 13. Burnstock G. Knight GE. Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol 2004; 240: 31-304.
14. 14. McMillan MR, Burnstock G, Haworth SG. Vasodilatation of intrapulmonary arteries to P2-receptor nucleotides in normal and pulmonary hypertensive newborn piglets. Br J Pharmacol 1999; 128(3): 543-8.
15. 15. Burnstock G. Vessel tone and remodeling. Nat Med 2006; 12(1): 16-7.
16. 16. Yamamoto K, Sokabe T, Matsumoto T, Yoshimura K, Shibata M, Ohura N, Fukuda T, Sato T, Sekine K, Kato S, Isshiki M, Fujita T, Kobayashi M, Kawamura K, Masuda H, Kamiya A, Ando J. Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice. Nat Med 2006; 12(1): 133-7.
17. 17. Ohtani M, Ohura K. Oka T. Involvement of P2X receptors in the regulation of insulin secretion, proliferation and survival in mouse pancreatic beta-cells. Cell Physiol Biochem 2011; 28(2): 355-66.
18. 18. Kurashima Y, Amiya T, Nochi T, Fujisawa K, Haraguchi T, Iba H, Tsutsui H, Sato S, Nakajima S, Iijima H, Kubo M, Kunisawa J, Kiyono H. Extracellular ATP mediates mast cell-dependent intestinal inflammation through P2X7 purinoceptors. Nat Commun 2012; 3: 1034.
19. 19. Piccini A, Carta S, Tassi S, Lasiglié D, Fossati G, Rubartelli A. ATP is released by monocytes stimulated with pathogen-sensing receptor ligands and induces IL1beta and IL-18 secretion in an autocrine way. Proc Natl Acad Sci U S A 2008; 105(23): 8067-72.
20. 20. Okada SF, Ribeiro CM, Sesma JI, Seminario-Vidal L, Abdullah LH, van Heusden C, Lazarowski ER, Boucher RC. Inflammation promotes airway epithelial ATP release via calcium-dependent vesicular pathways. Am J Respir Cell Mol Biol 2013; 49(5): 814-20.
21. 21. Gourine AV, Dale N, Llaudet E, Poputnikov DM, Spyer KM, Gourine VN. Release of ATP in the central nervous system during systemic inflammation: Real-time measurement in the hypothalamus of conscious rabbits. J Physiol 2007; 585(Pt 1): 305-16.
22. 22. Bodin P, Burnstock G. Increased release of ATP from endothelial cells during acute inflammation. Inflamm Res 1998; 47(8): 351-4.
23. 23. Dale N, Frenguelli BG. Release of adenosine and ATP during ischemia and epilepsy. Curr Neuropharmacol 2009; 7(3): 160-79.
24. 24. Burnstock G, Williams M. P2 purinergic receptors: Modulation of cell function and therapeutic potential. J Pharmacol Exp Ther 2000; 295(3): 862-9.
25. 25. Hoffman JF, Alicia D, Amittha W, Sulayman D. Tetrodotoxin-sensitive Na+ channels and muscarinic and purinergic receptors identified in human erythroid progenitor cells and red blood cell ghosts. Proc Natl Acad Sci USA 2004; 101(33): 12370-4.
26. 26. Sprague RS, Ellsworth ML, Stephenson AH, Kleinhenz ME, Lonigro AJ. Deformation-induced ATP release from red blood cells requires CFTR activity. Am J Physiol 1998; 275(5 Pt 2): H1726-32.
27. 27. Wan J, Ristenpart WD, Stone HA. Dynamics of shearinduced ATP release from red blood cells. Proc Natl Acad Sci U S A 2008; 105(43): 16432-7.
28. 28. Forsyth AM, Wan J, Owrutsky PD, Abkarian M, Stone HA. Multiscale approach to link red blood cell dynamics, shear viscosity, and ATP release. Proc Natl Acad Sci U S A 2011; 108(27): 10986-91.
29. 29. Wan J, Forsyth AM, Stone HA. Red blood cell dynamics: From cell deformation to ATP release. Integr Biol (Camb) 2011; 3(10): 972-81.
30. 30. Sprague RS, Bowles EA, Achilleus D, Ellsworth ML. Erythrocytes as controllers of perfusion distribution in the microvasculature of skeletal muscle. Acta Physiol (Oxf) 2011; 202(3): 285-92.
31. 31. Sluyter R, Shemon AN, Barden JA, Wiley JS. Extracellular ATP increases cation fluxes in human erythrocytes by activation of the P2X7 receptor. J Biol Chem 2004; 279(43): 44749-55.
32. 32. Sluyter R, Dalitz JG, Wiley JS. P2X7 receptor polymorphism impairs extracellular adenosine 5'-triphosphate-induced interleukin-18 release from human monocytes. Genes Immun 2004; 5(7): 588-91.
33. 33. Parker JC, Snow RL. Influence of external ATP on permeability and metabolism of dog red blood cells. Am J Physiol 1972; 223(4): 888-93.
34. 34. Parker JC, Castranova V, Goldfinger JM. Dog red blood cells: Na and K diffusion potentials with extracellular ATP. J Gen Physiol 1977; 69(4): 417-30.
35. 35. Light DB, Capes TL, Gronau RT, Adler MR. Extracellular ATP stimulates volume decrease in Necturus red blood cells. Am J Physiol 1999; 277(3 Pt 1): C480-91.
36. 36. Rodriguez-Garcia R, López-Montero I, Mell M, Egea G, Gov NS, Monroy F. Direct cytoskeleton forces cause membrane softening in red blood cells. Biophys J 2015; 108(12): 2794-806.