Erythrocytes Possess an Intrinsic Barrier to Nitric Oxide Consumption
2000; Elsevier BV; Volume: 275; Issue: 4 Linguagem: Inglês
10.1074/jbc.275.4.2342
ISSN1083-351X
AutoresMark W. Vaughn, Kuang‐Tse Huang, Lih Kuo, James C. Liao,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoIt has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K Hb∼107m−1 s−1) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500–1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant,K RBC, would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K RBC approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500–1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs. It has been reported that free hemoglobin (Hb) reacts with NO at an extremely high rate (K Hb∼107m−1 s−1) and that the red blood cell (RBC) membrane is highly permeable to NO. RBCs, however, react with NO 500–1000 times slower. This reduction of NO reaction rate by RBCs has been attributed to the extracellular diffusion limitation. To test whether additional limitations are also important, we designed a competition test, which allows the extracellular diffusion limitation to be distinguished from transmembrane or intracellular resistance. This test exploited the competition between free Hb and RBCs for NO generated in a homogenous phase by an NO donor. If the extracellular diffusion resistance is negligible, then the results would follow a kinetic model that assumes homogenous reaction without extracellular diffusion limitation. In this case, the measured effective reaction rate constant,K RBC, would remain invariant of the hematocrit, extracellular-free Hb concentration, and NO donor concentration. Results show that the K RBC approaches a constant only when the hematocrit is greater than 10%, suggesting that at higher hematocrit, the extracellular diffusion resistance is negligible. Under such a condition, the NO consumption by RBCs is still 500–1000 times slower than that by free Hb. This result suggests that intrinsic RBC factors, such as transmembrane diffusion limitation or intracellular mechanisms, exist to reduce the NO consumption by RBCs. hemoglobin red blood cell hematocrit Despite the well documented importance of nitric oxide, the transfer of NO from the producing cell to the target is poorly understood, because the free radical NO can be degraded in a variety of reactions. In particular, NO reacts with deoxy- and oxyhemoglobin (deoxyHb and oxyHb, respectively)1 at a very high rate (1.Cassoly R. Gibson Q.H. J. Mol. Biol. 1974; 91: 3301-3313Google Scholar, 2.Eich R.F. Li T. Lemon D.D. Doherty D.H. Curry S.R. Aitken J.F. Mathews A.J. Johnson K.A. Smith R.D. Phillips G.N.J. Olson J.S. Biochemistry. 1996; 35: 6976-6983Crossref PubMed Scopus (571) Google Scholar) to form nitrosyl Hb (HbNO) and met hemoglobin (metHb), respectively. If Hb in the RBC behaved like Hb in dilute solution, the half-life of NO in the blood (which contains about 12–15 mm heme) would be only about 1 μs. For such a rapid reaction, it would seem likely that a large portion of NO produced from the endothelium would be scavenged by the blood. Indeed, in vivo and in vitro evidence suggests that free Hb is an effective NO scavenger that can deplete NO. For example, infusion of free Hb solution into experimental animals or human subjects results in hypertension (3.Doherty D.H. Doyle M.P. Curry S.R. Vali R.J. Fattor T.J. Olson J.S. Lemon D.D. Nature Biotechnol. 1998; 16: 672-676Crossref PubMed Scopus (364) Google Scholar, 4.Pohl U. Lamontagne D. Basic Res. Cardiol. 1991; 86 (suppl.): 105Google Scholar), most likely due to the reaction of NO with oxyHb in the circulation (3.Doherty D.H. Doyle M.P. Curry S.R. Vali R.J. Fattor T.J. Olson J.S. Lemon D.D. Nature Biotechnol. 1998; 16: 672-676Crossref PubMed Scopus (364) Google Scholar). Modeling analyses (5.Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8137-8141Crossref PubMed Scopus (625) Google Scholar, 6.Lancaster Jr., J.R. Methods Enzymol. 1996; 268: 31-50Crossref PubMed Google Scholar, 7.Vaughn M.W. Kuo L. Liao J.C. Am. J. Physiol. 1998; 274: H1705-H1714Crossref PubMed Google Scholar) also showed that if endothelium-produced NO reacted as rapidly with blood as it does with free Hb, the NO concentrations in vascular smooth muscle would be too low to activate soluble guanylate cyclase, the primary target of NO. Mathematical modeling based on diffusion theory and in vitro measurements of kinetic constants (5.Lancaster Jr., J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8137-8141Crossref PubMed Scopus (625) Google Scholar, 6.Lancaster Jr., J.R. Methods Enzymol. 1996; 268: 31-50Crossref PubMed Google Scholar, 7.Vaughn M.W. Kuo L. Liao J.C. Am. J. Physiol. 1998; 274: H1705-H1714Crossref PubMed Google Scholar, 8.Vaughn M.W. Kuo L. Liao J.C. Am. J. Physiol. 1998; 274: H2163-H2176PubMed Google Scholar) have confirmed that Hb could effectively scavenge endothelial produced NO and mitigate its effect. Because 3–10 μm free Hb can abolish NO-mediated vasodilation in vitro (4.Pohl U. Lamontagne D. Basic Res. Cardiol. 1991; 86 (suppl.): 105Google Scholar, 9.Liao J.C. Hein T. Vaughn M.W. Huang K.-T. Kuo L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8757-8761Crossref PubMed Scopus (262) Google Scholar), it is unclear how NO can exercise its vasoregulatory function with 12–15 mm Hb concentration in the blood. This discrepancy is described as the “NO-Hb paradox,” and is one of the most important questions regarding the physiological and pathological functions of NO. NO produced from the endothelium must go through four steps to react with the RBC-enclosed Hb (Fig. 1): (a) diffusion through the RBC-free (or depleted) region, created by intravascular flow (10.Schmid-Schonbein H. Fisher T. Driessen G. Rieger H. Hwang N.H.C. Gross D.R. Patel D.J. Quantitative Cardiovascular Studies: Clinical Research Application of Engineering Principles. University Park Press, Baltimore1979: 353-417Google Scholar), to the bulk solution; (b) diffusion from the bulk solution to the RBC surface; (c) diffusion across RBC membrane; and (d) diffusion and reaction inside RBC cytosol. The first two are affected by extracellular factors, whereas the last two steps are affected by intracellular components intrinsic to RBC itself. In discussing the diffusion flow of molecules, it is customary to define the diffusional resistance as a measure of the diffusional barrier. Resistance is defined as driving force divided by flux; where, for diffusion, the driving force is the concentration difference, and the flux is the molar flow/unit area. Although this definition can be made precise, the term diffusional resistance is often most useful as a concept. The diffusional resistance, if significant, results in the formation of layer around the RBC, within which the NO concentration is much smaller (e.g. less than 10%) than the bulk of the solution (Fig. 1 b). This layer is termed the diffusion (or unstirred) layer, and the thickness of this layer is proportional to the diffusional resistance. In reality, the layer has no actual boundary, and the concentration profile is continuous. However, the use of the diffusion layer in discussion is conceptually convenient. It is now recognized that the NO consumption rate by RBCs is much slower than that expected based on the in vitro reaction rate of NO with free Hb (9.Liao J.C. Hein T. Vaughn M.W. Huang K.-T. Kuo L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8757-8761Crossref PubMed Scopus (262) Google Scholar, 11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). The in vivo quenching of NO by RBC is first reduced by the RBC-free zone (Fig. 1 a) created by the flow field (7.Vaughn M.W. Kuo L. Liao J.C. Am. J. Physiol. 1998; 274: H1705-H1714Crossref PubMed Google Scholar, 9.Liao J.C. Hein T. Vaughn M.W. Huang K.-T. Kuo L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8757-8761Crossref PubMed Scopus (262) Google Scholar, 12.Butler A.R. Magson I.L. Wright P.G. Biochim. Biophys. Acta. 1998; 1425: 168-176Crossref PubMed Scopus (178) Google Scholar). However, even without the RBC-free zone, the NO consumption by RBC is still about 500–1000 times slower than the NO reaction rate with free Hb (9.Liao J.C. Hein T. Vaughn M.W. Huang K.-T. Kuo L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8757-8761Crossref PubMed Scopus (262) Google Scholar, 11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). This low reaction rate has been attributed to diffusion resistance from the bulk solution to the surface of RBC (11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). It has been suggested that the diffusional resistance is mainly associated with a diffusion or “unstirred” layer around each RBC (Fig. 1 b). Unfortunately, experimental difficulties have prevented distinguishing the contribution of the extracellular diffusion resistance(Fig. 1 b) from transmembrane (Fig. 1 c) or intracellular (Fig. 1 d) resistance. For in vitro experiments measuring the uptake of NO (11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 13.Carlsen E. Comroe Jr., J.H. J. Gen. Physiol. 1958; 42: 83-107Crossref PubMed Scopus (120) Google Scholar) or oxygen (14.Coin J.T. Olson J.S. J. Biol. Chem. 1979; 254: 1178-1190Abstract Full Text PDF PubMed Google Scholar, 15.Merchuk J.C. Tzur Z. Lightfoot E.N. Chem. Eng. Sci. 1983; 38: 1315-1321Crossref Scopus (12) Google Scholar, 16.Hartridge H. Roughton F.J.W. J. Physiol. (Lond.). 1927; 62: 232-242Crossref Scopus (36) Google Scholar) by dilute erythrocyte suspensions, it has been impossible to separate the effect of the extracellular from transmembrane or intracellular diffusion resistances. Therefore, it has been reasonable to make the simplest assumption and assign all differences between RBCs and the equivalent solution of free Hb to extracellular diffusional resistance (11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 15.Merchuk J.C. Tzur Z. Lightfoot E.N. Chem. Eng. Sci. 1983; 38: 1315-1321Crossref Scopus (12) Google Scholar). In the dilute RBC suspensions that are accessible experimentally, the extracellular diffusional barrier around the cell is likely to be quite significant and perhaps dominant whether or not additional cellular factors are involved. Nonetheless, the RBC may possess a specific intrinsic (transmembrane or intracellular) barrier that limits the NO consumption rate. To probe the existence of such mechanisms, the external diffusion resistance must be distinguished from any intrinsic barrier that reduces the NO consumption rate. To accomplish this, we designed an experiment that exploited the competition for NO between RBC and extracellular Hb. This design allowed us to use high concentrations of RBC and, most importantly, avoid much of the extracellular diffusional resistance and concentration limitations of traditional kinetic measurements. In studies of oxygen uptake, about half of the resistance is associated with oxygen transfer from the bulk solution to the surface of RBC, and the other half is attributed to the diffusion resistance from oxygenated hemoglobin within the RBC (17.Vandegriff K.D. Olson J.S. Biophys. J. 1984; 45: 825-835Abstract Full Text PDF PubMed Scopus (32) Google Scholar). With our technique, the extracellular diffusional resistance can be eliminated, because NO is generated uniformly throughout the continuous phase and a high hematocrit can be used to reduce the distance that NO needs to travel. In addition, only a small quantity of NO is used, and thus the intracellular diffusional resistance through previously reacted Hb is minimized. The NO consumption in the extracellular phase is monitored directly by measuring the NO/Hb reaction product; NO oxidizes oxyHb to metHb. The uptake rate of NO by RBCs is calculated by the use of a kinetic equation derived for this experimental condition from the extracellular metHb and oxyHb concentrations with or without RBCs present. The NO donor, spermine NONOate (N-[4[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3-propanediamine) was purchased from Alexis Corp. (San Diego, CA). Some tests used SIN-1 (3-morpholinosydnonimine HCl) purchased from Alexis Corp. or DPTA NONOate (3-3′-(hydroxynitrosohydrazono) bis 1-propanamine) purchased from Cayman Chemical (Ann Arbor, MI). Dulbecco's phosphate-buffered saline, Sephadex G-25 (Amersham Pharmacia Biotech), DEAE-Sephadex A-50 (Amersham Pharmacia Biotech), Drabkin's solution, and Tris base were purchased from Sigma. The mixed bed ion exchange resin AG 501-X8 was purchased from Bio-Rad. Bovine blood was collected in heparinized (5 IU/ml) tubes. The plasma and the buffy coat were removed following centrifugation at 800 × g for 20 min. The cells were resuspended and immediately washed four times in Dulbecco's phosphate-buffered saline (0.122 molar NaCl, 0.030 molar KH2PO4 + Na2HPO4, 2 mg/ml glucose, pH 7.4, 290 mosmol/kg). After each wash, the cells were centrifuged at 800 × g for 10 min to separate the RBCs from the supernatant. The erythrocytes were purified by filtration through a mixture of microcrystalline cellulose and α cellulose (18.Beutler M.D. Red Cell Metabolism: A Manual of Biochemical Methods. Grune & Stratton, Orlando, FL1984Google Scholar). OxyHb solution was prepared from bovine RBCs using the modification of Riggs' procedure (19.Riggs A. Methods Enzymol. 1981; 76: 5-29Crossref PubMed Scopus (244) Google Scholar). Purified bovine RBCs were centrifuged (800 × g, 10 min), and the supernatant discarded. The cells were lysed by diluting with 2 volumes of ice-cold deionized water, freezing in liquid nitrogen, and then thawing at room temperature. Cell debris was removed by centrifugation at 15,000 × g for 30 min in a refrigerated centrifuge. Salts were removed by passing through a bed of AG 501-X8 resin and then eluting at 4 °C through a column of Sephadex G-25 that had been pre-equilibrated with 20 mmTris acetate + 0.5 mm EDTA, pH 8.3. The Hb was stored on ice and used within three days. Spermine NONOate was prepared as a stock solution of 1–2 mm. Approximately 0.4 mg of spermine NONOate was measured into a 1.5-ml micro-centrifuge tube. The donor was diluted in 1 ml of ice-cold, isotonic NaCl solution containing 0.001 N NaOH. The concentration was verified by diluting the concentrate in 0.01 N NaOH and measuring the absorbance at 250 nm (extinction coefficient 7500 cm−1m−1). The spermine NONOate solution was prepared fresh daily and kept in the dark and on ice until use. Some additional tests used DPTA NONOate and 3-morpholinosydnonimine HCl prepared in the same manner. Each test consisted of four solutions, run simultaneously: oxyHb in buffer, oxyHb with NO donor in buffer, free oxyHb in a suspension of RBCs, and free oxyHb in a suspension of RBCs with NO donor. A solution of oxyHb was prepared by adding oxyHb concentrate to Dulbecco's phosphate-buffered saline to produce the desired Hb concentration (normally 10 μm). This solution was used without further dilution for the buffer samples. RBC suspensions were produced by centrifuging purified RBCs (800 × g, 10 min), removing the supernatant, and diluting the packed cells with the oxyHb solution to produce the desired hematocrit (normally 15%). The samples were equilibrated (1 h) at 25 °C, and then 15 ml of each solution were loaded into a 20-ml syringe (Becton-Dickenson, Inc.). For the samples containing NO donor, spermine NONOate was added immediately before loading into the syringe. The NO donor concentration was computed based on the total volume of solution. Immediately after the solutions were added to the syringes, the initial 1-ml sample was taken, and the syringes were placed on a rocking mixer (Clay Adams, Fisher Scientific) to keep the cells uniformly dispersed. The initial sample was centrifuged (20 s at 10,000 ×g) to separate the RBCs, and the supernatant was assayed for oxyHb and metHb using a Beckman DU 640 spectrophotometer. Subsequent 1-ml samples taken at time intervals were assayed in the same manner. The concentrations of metHb and oxyHb were determined spectrophotometrically by fitting a set of “basis spectra” to the measured spectra by means of linear regression. All samples and basis spectra were scanned from 380 to 700 nm using a Beckman DU 640 spectrophotometer. To obtain the pure components for the basis spectra, six samples of oxyHb were obtained as above using freshly drawn bovine blood. Residual metHb was removed by loading the Hb onto a DEAE-Sephadex A-50 column (2.5 by 50 cm), maintained at 4 °C and previously equilibrated with 50 mm Tris acetate, pH 7.6/0.5 mm EDTA (19.Riggs A. Methods Enzymol. 1981; 76: 5-29Crossref PubMed Scopus (244) Google Scholar). Hb was eluted with this buffer, and the concentration of the eluent was determined using Drabkin's solution to convert the Hb to cyano-metHb (19.Riggs A. Methods Enzymol. 1981; 76: 5-29Crossref PubMed Scopus (244) Google Scholar). Each sample was serially diluted to 10, 5, 2.5, and 1.25 μm and scanned, and the digitized spectra were stored on computer. The averaged spectrum was obtained by use of linear regression. These same oxyHb samples were converted to metHb (without change in concentration) by adding a slight excess of NO gas to each sample, and the average spectrum was obtained in the same way. The solutions that do not contain NO donor ensured that the Hb autooxidation and RBC lysis were negligible. The ratio of the reaction rates between the NO-Hb and the NO-RBC reactions is determined by comparing the NO production rate with the metHb production rate in the oxyHb plus RBC suspension with NO donor. The rate of NO production can be determined from the control experiment, free oxyHb plus NO donor in buffer. Because only free oxyHb is present (in excess) in this control experiment, the total amount of metHb produced is the total amount of NO generated. Because the NO-oxyHb reaction rate is known, the ratio can be used to compute the NO-RBC reaction rate. For each solution, the oxyHb consumed and the metHb produced in the extra erythrocyte space are measured at various time points. The NO-RBC reaction rate constant is determined from kinetic equations describing NO uptake by oxyHb and RBCs. These equations assume that the extracellular diffusional resistance is negligible, and thus the NO concentration in the solution is homogeneous. Therefore, deviations from the model may suggest that the NO concentration is nonhomogeneous, which in turn indicates the significance of extracellular diffusional resistance. By using the pseudo-steady-state approximation, d[NO]/dt ≈ 0, solution of these equations can he simplified to the following equation, ([metHb] c−(1−Hct)[metHb] ex)/[oxyHb] RBC=(KRBC/KHb)ln ([totalHb] ex/[oxyHb] ex)Equation 1 in which square brackets denote concentration. Here Hct is hematocrit, [metHb]c is the metHb concentration in the cell-free control, [metHb]ex is the metHb concentration in the extracellular space of the RBC suspension, [oxyHb]ex and [totalHb]ex are the oxyHb and initial total Hb concentrations in the extracellular space of the RBC suspension, respectively. K RBC andK Hb are the rate constants (m−1s−1) of the NO-RBC and NO-free Hb reactions, respectively. K RBC is defined based on the Hb concentration in the solution as if all the RBCs are lysed. The Hb concentration (mm, heme) in the solution can be calculated as 19.1 × Hct. By plotting the experimental data according to the above equation, we obtained a straight line of slop eK RBC/K Hb and an intercept of zero. This plot (termed the Kplot) allowed the verification of the experiment and the determination of the rate constant. During the experiment, if RBC lysis, determined by an increase in total Hb in extra-erythrocytespace, contributed more than 6% of the extraerythrocyte Hb concentration, then the data were discarded. If the extracellular diffusional resistance is negligible (which is the model assumption), Equation 1 will fully describe the experimental data, and the measured K RBC will be independent of hematocrit, extracellular Hb, and NO donor concentration. Otherwise, the measured K RBC will decrease as hematocrit decreases, as extracellular Hb increases, and as NO donor concentration decreases. As stated above, previous experimental techniques for measuring K RBC were limited to very dilute RBC suspension and could not distinguish external diffusion limitation from intrinsic barrier in the RBC. To avoid these limitations, we designed the following competition approach. In this set of experiments, known concentrations of RBC-contained oxyHb and free oxyHb competed for a limiting amount of NO generated by NO donor in solution. If oxyHb in the RBC reacted with NO as fast as free oxyHb in the solution, then NO would be consumed by the RBC and free oxyHb at the same specific rate. On the other hand, if free oxyHb consumed NO faster than the RBC-enclosed oxyHb, then NO would be consumed by the two species in a ratio determined by their reaction rates. This ratio was determined by measuring the metHb and oxyHb in the extra erythrocyte space. The metHb in the RBC was continuously regenerated by metHb reductase systems and could not be used to indicate NO consumption by RBC. However, the NO consumed by the RBCs could be calculated from mass balance using the total amount of NO generated and the amount consumed by the free Hb in the solution. The rate of NO generated was determined from a control experiment, where free oxyHb was present (without RBC) in excess to the NO donor. Kinetic equations were formulated to describe the experimental system, and the solution is shown in Equation 1. The results from a typical Hb-RBC competition experiment are shown in Fig. 2. In this experiment, the sample contained 10 μm spermine NONOate as the NO donor, 7.5 μm extracellular oxyHb (heme concentration), and 7.8% hematocrit (1.5 mm RBC heme). Fig. 2 a depicts the time course of metHb production in the extra erythrocyte space. Fig. 2 b shows the K plot of this and three additional experiments using the same sample of blood. The formation of the straight line in this plot indicates that the result was consistent with the kinetic model. This straight line also indicates that the reaction rate of the RBCs was constant throughout the test, suggesting that no internal diffusion layer formed during the test to slow NO uptake. The ratioK RBC/K Hb = 0.00089 ± 0.00005 (mean ± S.E., n = 4) is determined from slope of this line. The result indicates that RBC reaction with NO is about three orders of magnitude lower than free Hb, consistent with other evidence (9.Liao J.C. Hein T. Vaughn M.W. Huang K.-T. Kuo L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8757-8761Crossref PubMed Scopus (262) Google Scholar, 11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). The generation of NO from a homogeneous NO source, soluble NO donor, reduces the barrier of external diffusion. However, it cannot eliminate the barrier of external diffusion, particularly at low concentrations of RBC suspension. Because RBCs are in the particulate form, they compete less favorably with the free Hb, which is in the homogeneous phase. This disadvantage is amplified at low hematocrit, where the external diffusion barrier is larger. Therefore, we predicted that at low hematocrit the K RBC measured is not an intrinsic quantity of the NO-RBC reaction rate. Rather, it is reduced by external diffusion. Hence, the apparent K RBCmeasured will decrease as the hematocrit decreases. Conversely, the apparent K RBC will increase as the external diffusion limitation is reduced by increasing hematocrit, until it is eliminated. In that region, the K RBC measured is independent of hematocrit, and it represents the intrinsic rate constant of the NO-RBC reaction. In this high hematocrit region, the NO-RBC reaction is limited by intrinsic factors such as RBC membrane permeability or intra-erythrocytic conditions. This reasoning is supported by experimental results. In Fig.3, we varied the hematocrit in the competition experiments and showed the effect of external diffusion limitation exactly as predicted. When the hematocrit was greater than 10%, K RBC calculated from the kinetic model (Equation 1) is constant, suggesting that the RBC consumption rate is fully described by the kinetic model that does not include any extracellular diffusion resistance. On the other hand, when hematocrit was smaller than 5%, K RBC calculated from the kinetic model decreased as hematocrit decreased. This decrease in the rate constant suggests that extracellular diffusional resistance, which is not considered in the model, becomes significant. These experiments concluded that when the hematocrit is higher than about 10%, the K RBC measured is essentially free of external diffusion limitation. Under this condition, the thickness of the diffusion layer (Fig. 1 b) surrounding the RBC is very small, much smaller than the intercellular distance. Therefore, external diffusion is no longer the limiting process. When the hematocrit is lower than 10%, the diffusion layer thickens, and thus external diffusion (Fig. 1 b) is more important compared with other processes (Fig. 1, c and d). Note that this conclusion does not necessarily imply that external diffusion is unimportant under physiological conditions. The competition experiment uses a homogeneous NO generating system (soluble NO donor), where external diffusion can be eliminated at high hematocrit. However, in blood vessels, NO is generated from endothelium, which has a very different mass transfer barrier. It is interesting to note that under high hematocrit (without external diffusion limitation), theK RBC is still about 800 times lower thanK Hb, indicating that the NO-RBC reaction rate is intrinsically lower than the NO-free Hb reaction rate. Therefore, the limitation is attributed to intrinsic factors in RBCs, such as transmembrane or intracellular limitations. Varying the hematocrit provides a sensitive probe of the near RBC space. In a very dilute RBC suspension, much of the extracellular Hb and NO production is far from the cell. As the hematocrit increases, the average spacing between cells decreases. If a diffusion layer exists, the proportion of extracellular volume associated with the diffusion layer increases as the hematocrit increases. The average NO concentration in the diffusion layer is low so less metHb will be produced. Thus, the apparent rate would depend on hematocrit. On the other hand, if NO uptake is controlled by factors intrinsic to the RBC, then the NO concentration in the extracellular space will be nearly uniform. Then there would be little change in apparent rate as hematocrit increases. This behavior is reflected in Fig. 3,a and b, where, as the hematocrit increases beyond 7.5%, there is little change in the apparent reaction rate coefficient. If the apparent RBC reaction rate is independent of RBC concentration, as it would be if the resistance of the diffusion layer were negligible, then the plot ofK RBC/K Hb × Hctversus Hct will be a straight line with slopeK RBC/K Hb. This is indeed the behavior seen in Fig. 3 c. Here the presence of a non-zero intercept indicates that the apparent reaction rate depends on Hct in dilute suspensions, where the diffusion layer becomes important (11.Liu X. Miller M.J.S. Joshi M.S. Sadowska-Krowicka H. Clark D.A. Lancaster Jr., D.A. J. Biol. Chem. 1998; 273: 18709-18713Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). It is expected that increasing extracellular Hb or NO donor concentrations will increase the metHb formation in the extracellular space. However, the extracellular NO consumption will follow the kinetic model (Equation 1) only if the system is homogeneous, namely, no extracellular diffusion resistance, as the kinetic model assumes. In this case,K RBC derived from the kinetic equation will be independent of extracellular Hb or NO donor concentration, as the model states. On the other hand, if the diffusion layer outside of RBC becomes significant, the system is nonhomogeneous, which means that the NO concentration in the layer is much smaller than the bulk solution, and the homogeneous kinetic model fails. In this case, theK RBC derived from Equation 1 will decrease when the extracellular Hb increases or NO donor decreas
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