Modulation of density-fractionated RBC deformability by ... - IOS Press

Clinical Hemorheology and Microcirculation 33 (2005) 363–367 IOS Press

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Modulation of density-fractionated RBC deformability by nitric oxide Melek Bor-Kucukatay a , Herbert J. Meiselman b and Oguz K. Ba¸skurt c,∗ a

Department of Physiology, Pamukkale University Faculty of Medicine, Denizli, Turkey Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, CA, USA c Department of Physiology, Akdeniz University Faculty of Medicine, Antalya, Turkey b

Abstract. The role of nitric oxide (NO) in maintaining normal mechanical behavior of red blood cell (RBC) has been previously demonstrated. The effects of NO donor and NOS inhibitor on the mechanical properties of density fractionated RBC were tested in this study. A non-specific NOS inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) at a concentration of 10−3 M and sodium nitroprusside (SNP), a nitric oxide donor at a concentration of 10−6 M was added to blood samples with hematocrit adjusted to 0.4 l/l and RBC deformability was measured by an ektacytometer in the density fractionated RBC after one hour incubation at 37◦ C. There was no significant effect of the NO donor SNP on cellular deformability in the older (denser) RBC fraction in contrast with the younger (least dense) fraction. Alternatively, the sensitivity of cellular deformability to competitive NOS inhibition by L-NAME was greater in the older fraction. These findings suggest that older RBC are characterized by diminished internal NO synthesis and are also less sensitive to external NO indicating that the target mechanisms for NO may also be deteriorated.

1. Introduction In addition to its major role in cardiovascular regulation via modifying vascular smooth muscle tone, nitric oxide (NO) has also been demonstrated to affect the cellular elements of blood including red blood cells (RBC) [8]. There is now experimental evidence indicating that NO modulates RBC mechanical properties and that this effect is, in part, mediated by particulate guanylate cyclase [9]. It has also been shown that NO may interfere with potassium flux through RBC membrane [2,9,10], and mimics the effect of potassium channel blockers in its effect on RBC mechanical properties. Both external and internally-synthesized NO modulates RBC mechanical properties, and blockage of internal NO synthesis by both constitutional and inducible nitric oxide synthase (NOS) results in deterioration of RBC deformability [9]. RBC do not have protein synthesis mechanisms, and therefore the activity of most enzymes diminishes throughout the life-span of these cells [12]. Both NO synthesizing enzymatic mechanisms and target mechanisms of NO (e.g., guanylate cyclase and related molecular mechanisms) are thus expected to be affected by cellular aging. Therefore, it might be expected that the role of NO in modulating RBC mechanical properties differs between RBC of different in vivo ages [1]. The goal of the present *

Corresponding author: Oguz K. Baskurt, Department of Physiology, Akdeniz University Faculty of Medicine, Kampus, Antalya 07070, Turkey. Tel.: +90 242 310 1560; Fax: +90 242 310 1561; E-mail: [email protected]. 1386-0291/05/$17.00  2005 – IOS Press and the authors. All rights reserved

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M. Bor-Kucukatay et al. / Modulation of density-fractionated RBC deformability by nitric oxide

study was to test this hypothesis via evaluating the effects of a NO-donor (sodium nitroprussid) and a non-specific NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME) on the deformability of density-fractionated RBC.

2. Materials and methods 2.1. Blood samples and experimental protocol Venous blood samples were obtained from healthy, adult male volunteers and anticoagulated with sodium heparin (15 IU/ml). The hematocrit of the samples was adjusted to 0.4 l/l by removing or adding autologous plasma, following which the samples were divided into three aliquots: A non-specific NOS inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) at a concentration of 10−3 M was added to one aliquot, 10−6 M sodium nitroprusside (SNP), a nitric oxide donor, was added to the second one, and the third without any addition served as the control. The three aliquots were then incubated at room temperature for one hour. Following the one-hour incubation period, RBC were separated according to their density using disR continuous Iodixanol (Optiprep ) density gradients [17]. Five Iodixanol layers (2 ml each) with densities of 1.075, 1.085, 1.095, 1.105 and 1.115 g/ml were carefully layered on top of each other in a test tube with the densest layer at the bottom. One ml of an aliquot described above was gently layered on the least dense layer and the tube was centrifuged at 2500g for 25 minutes at 22◦ C. RBC were then harvested from specific density fractions: least dense cells were those that accumulated in 1.095 g/ml layer and most dense cells were those that accumulated in the 1.105 g/ml layer. RBC deformability was assessed for cells in these fractions as well as non-fractionated RBC. 2.2. Assessment of RBC deformability RBC deformability at 37◦ C was determined at various fluid shear stresses by laser diffraction analysis using an ektacytometer (LORCA, RR Mechatronics, Hoorn, The Netherlands). The system has been described elsewhere in detail [13]. Briefly, a low hematocrit suspension of RBC in an isotonic viscous medium (4% polyvinylpyrrolidone 360 solution, MW = 360 kDa) is sheared in a Couette system composed of a glass cup and a precisely fitting bob, with a gap of 0.36 mm between the cylinders. A laser beam is directed through the sheared sample and the diffraction pattern produced by the deformed cells is analyzed by a computer, which also controls the stepping motor that generates the pre-determined shear stresses. Based upon the geometry of the elliptical diffraction pattern, an elongation index (EI) is calculated as: EI = (L − W )/(L + W ), where L and W are the length and width of the diffraction pattern. An increased EI at a given shear stress indicates greater cell deformation and hence greater RBC deformability. EI values were determined for nine shear stresses between 0.5–50 Pa and the shear stress required for half-maximal deformation (SS1/2 ) was calculated from the data set for each measurement by using a Lineweaver–Burk analysis procedure [3]. Briefly, the shear stress–EI curve was linearized by plotting the reciprocal of EI versus the reciprocal of shear stress, with the x-intercept of this line corresponding to the negative reciprocal value of shear stress causing half-maximal deformation (SS1/2 ). Obviously, impaired RBC deformability leads to increased SS1/2 values; SS1/2 values are used herein since presentation and comparison of data via this approach are more convenient than via shear stress–EI curves.


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2.3. Statistics Data are presented as mean ± standard error. SS1/2 values of SNP or L-NAME treated RBC were compared with control RBC (fractionated or non-fractionated) using “paired t test” with Bonferroni correction for multiple comparisons. 3. Results RBC deformability for cells in the least dense fraction was significantly greater when compared to both non-fractionated cells and most dense fractions as indicated by lower SS1/2 values (Fig. 1). Figure 1 also demonstrates that RBC deformability was significantly improved (i.e., decreased SS1/2 values) after incubation with SNP at 10−6 M concentration. This effect of SNP effect was more pronounced for least dense RBC fraction, whereas no effect was observed for the densest RBC. In contrast with the SNP effect, incubation with L-NAME resulted in a deterioration of RBC deformability indicated by increased SS1/2 values (Fig. 2). Furthermore, the effect of L-NAME was more pronounced in the dense fraction of RBC whereas no effect was detected in the least dense fraction.

Fig. 1. Effect of 10−3 M sodium nitroprusside (SNP) on cellular deformability expressed as “shear stress at half-maximal deformation” (SS1/2 ) of density fractionated and non-fractionated RBC. (Data are mean ± standard error; ∗ : difference from the control of the same fraction, p < 0.05; ∗∗ : difference from “non-fractionated control”, p < 0.01; ∗∗∗ : difference from “least dense control”, p < 0.01.)

Fig. 2. Effect of 10−3 M N-omega-nitro-L-arginine methyl ester (L-NAME) on cellular deformability expressed as “shear stress at half-maximal deformation” (SS1/2 ) of density fractionated and non-fractionated RBC. (Data presented as mean ± standard error; ∗ : difference from the control of the same fraction, p < 0.05.)

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4. Discussion The major findings of this study include: (1) impaired deformability for the dense (i.e., older) RBC fraction when compared to either the least dense fraction or to unfractionated cells; (2) no significant effect of the NO donor SNP on cellular deformability for the older RBC fraction; (3) greater sensitivity of cellular deformability to competitive NOS inhibition by L-NAME in the older fraction. The role of NO in maintaining normal mechanical behavior of RBC has been previously demonstrated by various studies [4,6,9,14–16]. These studies suggested that NO modulates RBC deformability partly through a guanylate cyclase dependent mechanism [9]. NO may also influence potassium leakage and the associated mechanical deterioration [2,4,9,10]. Obviously, these modulator effects require intact pathways in order to be able to influence their target (e.g., RBC deformability). Therefore, alterations in these modulator effects with the in vivo age of RBC indicate that these intermediary pathways might be affected by the aging process of RBC. In fact, it is well documented that the function of many enzymes and other biological mediators deteriorate throughout the RBC life span [11,12]: this deterioration is related to the RBC being incapable of protein synthesis since it lacks the genetic code and the necessary molecular machinery for this function. Additionally, RBC are among the cells that are most prone to oxidative damage [5], with oxidatively damaged proteins and other bio-molecules first becoming non-functional, then destroyed by proteolytic enzymes [7]. The in vivo age dependent effect of NO donor and NOS-inhibitor should be the direct result of such alterations. The diminished effect of the NO donor SNP on older, most dense RBC deformability (Fig. 1) should reflect deteriorated target mechanism(s), including the intermediary pathways. The final target of NO in modulating RBC deformability has not yet been clearly demonstrated, but there is evidence indicating that potassium channels may play a role [9]. Note that the lack of effect of L-NAME on deformability should indicate the significantly higher NOS activity in the younger RBC, which could not be totally inhibited by the same concentration of L-NAME that was effective on the older RBC. In overview, this preliminary study suggests that older (i.e., denser) RBC are characterized by diminished internal NO synthesis and are also less sensitive to external NO. These properties most likely play a role in the impaired deformability characterizing the in vivo aging of RBC. However, further studies are required to specify the target and intermediary mechanisms that are down-regulated by the aging process and thus the exact biochemical pathways that mediate age-associated deterioration of deformability. Acknowledgements This study was supported by NIH Research Grants HL 15722, HL 70595 and FIRCA IR03 TW01295 and Akdeniz University Research Projects Unit. References [1] C. Acquaye and R.M. Johnson, Modified celluloses for erythrocyte deformability fractionation, Exp. Hematol. 21 (1993), 1358–1360. [2] N.C. Adragna and P.K. Lauf, Role of nitrite, a nitric oxide derivative, in K–Cl cotransport activation of low-potassium sheep red blood cells, Memb. Biol. 166 (1998), 157–167. [3] O.K. Baskurt and H.J. Meiselman, Analyzing shear stress-elongation index curves: comparison of two approaches to simplify data presentation, Clin. Hemorheol. Microcirc. 31 (2004), 23–30. [4] O.K. Baskurt, M. Uyuklu and H.J. Meiselman, Protection of erythrocytes from sub-hemolytic mechanical damage by nitric oxide mediated inhibition of potassium leakage, Biorheology 41 (2004), 79–89.


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