Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways

Tadahiro Oonishi, Kanako Sakashita, and Nobuhiro Uyesaka

Nippon Telegraph and Telephone Corporation Medical Research Institute, Kanto Teishin Hospital, Tokyo 141; and Department of Physiology, Nippon Medical School, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To investigate the mechanism of the regulation of human red blood cell deformability, we examined the deformability under mechanical stress. Washed human red blood cells were rapidly injected through a fine needle, and their filterability was measured using a nickel mesh filter. The decrease in filterability showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at ~50 µM. The decreased filterability was accompanied by no change in cell morphology and cell volume, indicating that the decrease in filterability can be ascribed to alterations of the membrane properties. Ca2+ entry blockers (nifedipine and felodipine) inhibited the impairment of filterability under mechanical stress. Prostaglandins E1 and E2, epinephrine, and pentoxifylline, which are thought to modulate the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level of red blood cells, improved or worsened the impaired filterability according to their expected actions on the cAMP level of the cells. These results strongly suggest that the membrane properties regulating red blood cell deformability are affected by the signal transduction system, including Ca2+-dependent and cAMP-mediated signaling pathways.

calcium entry blockers, prostaglandins, epinephrine, pentoxifylline; adenosine 3',5'-cyclic monophosphate; calcium ion

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE DEFORMABILITY OF red blood cells plays a crucial role in the maintenance of normal blood flow in the microcirculation (2, 4, 7, 20). Aspects of red blood cell deformability have become increasingly important in various clinical situations (6, 10, 21, 25, 28, 32). However, the precise molecular mechanism of the regulation of red blood cell deformability, and, in particular, the mechanism responsible for changes in the membrane properties associated with the Ca2+-dependent signal transduction system in red blood cells (5, 9) remain to be solved. The membrane properties and thus the deformability of red blood cells are largely determined by a submembrane reticulum of proteins referred to as the membrane skeleton. The membrane skeleton of red blood cells has been characterized in greater detail than that of any other cells, and considerable information is available regarding the skeleton composition, ultrastructures, protein-to-protein interactions, membrane anchorage, and molecular defects (5, 20, 29). Nevertheless, such information has not been examined in relation to the overall physiological functions of red blood cells such as deformability, except for red blood cells from patients with hereditary hemolytic diseases (20). In the case of platelets, such information is closely related to the aggregability of platelets through the signal transduction system in the cell (3).

It is known that red blood cells show a decrease in deformability in response to a transient elevation of intracellular Ca2+ under mechanical stress (12, 28). To investigate the relationships between changes in red blood cell deformability, the Ca2+-dependent signaling pathways, and the corresponding adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signaling pathways in the cells, we have developed a new in vitro system that imposes mechanical stress on red blood cells to decrease their deformability. The whole cell deformability (filterability) was measured with a recently developed nickel mesh filtration method that has been found to be useful in investigating the red blood cell deformability, both physiologically and clinically (2, 10, 25, 32, 34). In the present study, using these methods, we examined the effects of extracellular Ca2+ and Ca2+ entry blockers on red blood cell deformability with the aim of determining the manner of transient Ca2+ entry under mechanical stress. Furthermore, to study the corresponding cAMP-mediated signaling pathways, we examined the effects of drugs such as catecholamines, prostaglandins, and xanthine derivatives on red blood cell deformability; these effects have long been a topic of some controversy among physiologists (9, 11, 14, 16, 31).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of cells. Venous blood from the anticubital veins of healthy young male volunteers was collected into disposable syringes with a 22-gauge needle, with a 1/10 volume of 3.3% trisodium citrate used as an anticoagulant. After centrifugation at 1,300 g for 10 min at 25°C, the plasma and buffy coat were removed and replaced with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid sodium salt (HEPES-Na)-buffered NaCl solution (HBS; 133.5 mM NaCl, 2 mM glucose, and 15 mM HEPES-Na buffer, 287 mosM/kgH2O, pH 7.40) supplemented with 1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid-4Na and 0.1% bovine serum albumin (BSA). After centrifugation at 800 g for 10 min at 25°C, the packed red blood cells were resuspended with HBS supplemented with 0.01% BSA (HBS-BSA) and subsequently washed two times in HBS-BSA by repeated resuspension and centrifugation at 600 and 500 g for 10 min at 25 °C, respectively. The red blood cell suspension was prepared with HBS-BSA, the cell density of which was adjusted to 8 × 109 cells/ml. The red blood cell suspension thus prepared contained leukocytes at <5 × 105 cells/ml. The sample was stored at 22°C in an isothermal water bath. All experiments were performed within 6 h of the collection of the blood.

Red blood cell filterability. The filtration through a nickel mesh was carried out by the gravity-based, vertical tube method (2, 25). The nickel mesh (Dainippon Printing, Tokyo, Japan) is a porous thin metal film produced by means of a photofabrication technique. The average pore diameter of the nickel mesh used in this study was 4.4 µm. As described previously (2, 25), a height (pressure)-time curve was obtained during filtration by gravity using a pressure transducer (P100; Tsukasa Sokken, Tokyo, Japan). The tangent of the height-time curve, made by drawing points corresponding to different heights (pressures), gives the rate of the drop of the meniscus in the tube. By multiplying the cross-sectional area of the tube, the complete set of flow rates and corresponding pressures, i.e., the pressure-flow rate relationship, is given. We used a personal computer to collect the data automatically. The experiments were carried out at 36°C by circulating isothermal water through the jacket surrounding the vertical tube, the inner diameter of which was 4 mm. The filtration of the red blood cell suspension was started at a pressure of 15.0 cmH2O. The ratio (%) of the flow rate (ml/min) of the red blood cell suspension to that of the suspending medium (HBS-BSA) at 10 cmH2O was used as the index of red blood cell filterability. Before the filtration experiments, the suspending medium was filtered through a Millipore filter with a pore size of 0.1 µm to avoid the otherwise inevitable microdust contamination. Because the nickel mesh was subject to ultrasonic washing and was then reused at least 100 times, each experiment was done with the same nickel mesh.

Injection method for mechanical stress. Figure 1 illustrates the scheme of our prototype apparatus for imposing the mechanical stress on red blood cells. Five hundred microliters of the red blood cell suspension (cell density of 4 × 109 cells/ml) were packed into a disposable 1-ml tuberculin syringe (4 mm inner diameter; Terumo); air bubbles were carefully expelled. The cell suspension was then injected through a 25-gauge disposable needle (30 mm in length and 0.5 mm inner diameter; Terumo). The injection was performed by placing a weight on the upper end of the inner piston of the syringe, which was fixed vertically. The injected red blood cells were caught by a disposable 50-ml polypropylene centrifuge tube (Iwaki Glass, Funabashi, Japan). The distance from the tip of the needle to the bottom of the centrifuge tube was 15 mm.


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Fig. 1.   Schematic illustration of injection method for imposing mechanical stress on red blood cells.

An aliquot of the red blood cell suspension (8 × 109 cells/ml) was mixed with the same amount of HBS-BSA with an appropriate concentration of calcium chloride and then was incubated at 36°C for 5 min. Pretreatment with drugs was done by adding an appropriate concentration of the drug to the red blood cell suspension and by incubating the cell suspension at 36°C for 15 min before mixing with the Ca2+ solution. After undergoing the mechanical stress, the red blood cells were incubated at 36°C for 7 min and then diluted with HBS-BSA with an appropriate concentration of calcium chloride to 1 × 108 cells/ml; next, the cells were subjected to filterability measurement. The filtration was started at 10 min after the mechanical stress.

Although the injection device has a disadvantage in that there is no completely satisfactory way of characterizing the shear stress, some estimate of the level of the shear stress can be given by using the following analysis. The flow rate (Q) in the needle is calculated as Q = pi VR2, where V is the falling velocity of the weight and R (0.2 cm) is the radius of the syringe; this requires a pressure (P) in the syringe by Poiseuille's law, P = 8Leta Q/pi r4 = 8Leta VR2/r4, where L (3 cm) is the length of the needle, eta  (0.008 dyn/cm2) is the fluid viscosity at 30°C, and r (0.025 cm) is the radius of the needle. As the red blood cell suspension at 36°C was placed in the syringe at room temperature (25°C), it is assumed that the cells were mechanically stressed at ~30°C. The resultant upward force (F) on the syringe plunger is calculated as F = pi PR2 = 8pi Leta R4/Mr4 where M is mass. The net downward force on the weight is Mg - F, where M is the mass placed on the syringe and g (980 cm/s2) is the acceleration due to gravity; therefore, dV/dt = g - KV, where K = 8pi Leta R4/Mr4. The resultant velocity is V = (g/K)[1 - exp(-Kt)] (where t is time), and the distance fallen (d) is d = (g/K){t - [1 - exp(-Kt)]/K}. The mean velocity in the needle is VR2/r2, and the wall shear stress in the needle is tau  = Pr/2L = 4eta VR2/r3 by Stokes relation. For the case in which M = 1,000 g, K = 2.47 s-1, and the plunger reaches the bottom of the syringe (d = 4 cm) at about t = 0.094 s (94 ms) when the velocity is 80 cm/s and the wall shear stress is ~6,600 dyn/cm2. In this case, the mean velocity in the needle reaches ~5,100 cm/s, and the transit time is then ~0.6 ms. Thus the cells are exposed to relatively high stress for a very short time.

Other laboratory methods. An aliquot of the red blood cell suspension was fixed for microscopy with an isotonic 1% glutaraldehyde solution containing 50 mM phosphate buffer (pH 7.4) and 24.5 mM NaCl. Specimens were observed in a differential interference contrast microscope (TMD, Nikon, Tokyo) at ×400 magnification. Cell counting was performed with a hemocytometer (Coulter Counter model DN; Coulter Electronics, Luton, Beds., UK). The hematocrit (Hct) value was determined by centrifuging red blood cell suspensions in the capillary tubes at 14,400 g for 5 min on a Hct centrifuge (model MC-201; Hitachi Koki, Hitachi, Japan). The leukocyte count of the red blood cell suspensions and mean corpuscular volume (MCV) of the red blood cells were measured by an automated hematology analyzer (model NE-800; TOA Medical Electronics, Kobe, Japan). The osmolality of the suspending medium was measured with a freezing-point depression-type osmometer (DigiMatic Osmometer model 3D II; Advanced Instruments, Needham Heights, MA).

Materials. Felodipine [(±)ethyl methyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate] and pentoxifylline were provided by Hoechst (Tokyo, Japan). BSA (essentially fatty acid free) and prostaglandin (PG) E2 methyl ester (solution in methyl acetate) were purchased from Sigma (St. Louis, MO). Nifedipine was purchased from RBI (Natick, MA). PGE1 and epinephrine were obtained as the commercial preparations Prostandin (Ono Yakuhin Kogyo, Osaka, Japan) and Bosmin (Daiichi, Seiyaku, Tokyo), respectively. Other chemicals used were of reagent grade.

Statistical analysis. The data are expressed as means ± SD. Comparisons between the filterability of red blood cell suspensions treated with and without drugs were made by paired t-test. P values <0.01 were considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Decrease in filterability of mechanically stressed red blood cells and its dependency on extracellular Ca2+. The filterability of the mechanically stressed red blood cells decreased depending on the weight used for injecting red blood cells from the syringe in the presence of extracellular Ca2+; an increase in weight resulted in a decrease in filterability, which was apparently saturated when a weight of >1,000 g was used (Fig. 2). Because the motion in the case of the weight of >1,000 g may be almost as fast as a free fall and because increasing weight may not change the expected level of the shear stress much, this apparent saturation may be attributable to a property of the test system, not the red blood cells. For the subsequent experiments, a weight of 1,000 g was used. The filterability of the mechanically stressed red blood cells decreased depending on the concentration of Ca2+ added to the suspending medium. The decrease in filterability showed a V-shaped dependence curve against the extracellular Ca2+ concentration; the maximum decrease was observed at ~50 µM (Fig. 3). At extracellular Ca2+ concentrations ranging from 0 to 1 mM, no change in cell morphology was observed. The Hct values and the cell numbers before and after the mechanical stress were the same. The cell volume of the mechanically impaired red blood cells as evaluated by their Hct and MCV values did not change until 60 min after the mechanical stress was imposed. Without mechanical stress, no decrease in filterability was induced by the extracellular Ca2+. As we reported previously (2, 25), the filtration experiments were not influenced by contaminated leukocytes.


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Fig. 2.   Decrease in filterability under mechanical stress. Filterability (%) of mechanically stressed red blood cells in 50 µM extracellular Ca2+ concentration was plotted against the weight (g) used for pressing down the inner piston of the red blood cell-packed syringe. After mechanical stress, red blood cell suspensions at 40% hematocrit were incubated for 10 min at 36°C, diluted to 1% hematocrit, and then filtered through a nickel mesh, as described in METHODS. Filterability (%) was determined as the ratio of the flow rate (ml/min) of the red blood cell suspension to that of the suspending medium (HEPES-Na-bufered NaCl-bovine serum albumin) at 10 cmH2O. Data are means ± SD from 3 experiments.


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Fig. 3.   Dependency of the filterability of red blood cells under mechanical stress on the extracellular Ca2+ concentration. Red blood cell filterability with (open circle ) and without (bullet ) mechanical stress is shown, the latter of which was 95.5 ± 2.5%; filterability did not change when the extracellular Ca2+ concentration was increased to 1.5 mM. Data are means ± SD from 3 experiments.

Effects of Ca2+ entry blockers on the mechanically impaired filterability. Pretreatment with a Ca2+ entry blocker (nifedipine or felodipine) inhibited the impairment in filterability caused by 50 µM Ca2+ under mechanical stress in a dose-dependent manner (Figs. 4 and 5). Because the action of Ca2+ entry blockers was reported to be modified by solvents (22, 30), the red blood cells treated with the same concentration of ethanol (0.02% vol/vol) were used as a reference. The maximum inhibitory effect was observed at 0.5 µM and higher for nifedipine (from 24.3 ± 10.3 to 42.2 ± 15.6%, P < 0.01) and at 0.1 µM and higher for felodipine (from 23.3 ± 6.0 to 47.8 ± 14.8%, P < 0.01). The improvement of filterability by the Ca2+ entry blockers was not accompanied by any change in cell volume. Without mechanical stress, neither nifedipine nor felodipine changed the red blood cell filterability when the red blood cells were incubated at 36°C for 60 min at 50 µM or 1 mM of the extracellular Ca2+ concentration.


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Fig. 4.   Effects of nifedipine on mechanically impaired filterability. After a 0.02% volume of the indicated concentrations of nifedipine dissolved in ethanol was added to the red blood cell suspension and incubated at 36°C for 15 min, the cell suspension was mixed with the same amount of 100 µM Ca2+ solution and further incubated for 5 min at 36°C. After undergoing mechanical stress, the red blood cell suspension at 40% hematocrit was incubated for 10 min at 36°C, diluted to 1% hematocrit, and then filtered through the nickel mesh. Mechanically impaired filterability with 50 µM extracellular Ca2+ concentration without nifedipine (a), with 0.02% ethanol (b), and with 0.5 µM nifedipine in 0.02% ethanol (c). Inset: representative plot of filterability according to the effects of different concentrations of nifedipine on mechanically impaired filterability of red blood cells in 50 µM extracellular Ca2+ concentration. Data are means ± SD from 6 sets of experiments. * P < 0.01 vs. a and b. Comparison of data between 2 groups was made by paired t-test.


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Fig. 5.   Effects of felodipine on mechanically impaired filterability. Experimental conditions are as described in Fig. 4. *P < 0.01 vs. a and b.

Effects of PGE1, PGE2, epinephrine, and pentoxifylline. The effects of several drugs were examined on the mechanically impaired filterability at the 50 µM extracellular Ca2+ concentration. PGE1 improved the impaired filterability; the maximum improvement (from 21.2 ± 4.5 to 63.7 ± 11.9%, P < 0.01) was observed at the concentration of ~1 × 10-10 M, showing a bell-shaped improvement with concentrations ranging from 10-12 to 10-8 M (Fig. 6). Conversely, PGE2 worsened the impaired filterability; the maximum deterioration (from 26.3 ± 5.2 to 5.3 ± 1.3%, P < 0.01) was observed at the concentration of ~1 × 10-10 M, showing a V-shaped decrease with the concentrations ranging from 10-12 to 10-8 M (Fig. 7).


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Fig. 6.   Effects of prostaglandin E1. Red blood cells were pretreated with the indicated concentrations of prostaglandin E1 at 36°C for 15 min. Incubation with Ca2+ solution and filtration of red blood cell suspensions were done in the same way as that employed in the experiments with nifedipine (Fig. 4). A: representative plot of filterability according to the effects of different concentrations of prostaglandin E2 on mechanically impaired filterability of red blood cells in 50 µM extracellular Ca2+ concentration. B: mechanically impaired filterability in 50 µM extracellular Ca2+ concentration without prostaglandin E1 (a) and with 10-10 M prostaglandin E1 (b). Data are means ± SD from 6 sets of experiments. * P < 0.01 vs. reference. Comparison of data between two groups was made by paired t-test.


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Fig. 7.   Effects of prostaglandin E2. Pretreatment with prostaglandin E2, incubation with Ca2+ solution, and filtration of red blood cell suspensions were done in the same way as that employed in the experiments with nifedipine (Fig. 4). A: representative plot of filterability according to effects of different concentrations of prostaglandin E2 on mechanically impaired filterability of red blood cells in 50 µM extracellular Ca2+ concentration. B: mechanically impaired filterability in 50 µM extracellular Ca2+ concentration without prostaglandin E2 (a), with 0.02% ethanol (b), and with 10-10 M prostaglandin E2 in 0.02% ethanol (c). Data are means ± SD from 4 sets of experiments. * P < 0.01 vs. a and b. Comparison of data between 2 groups was made by paired t-test.

Epinephrine showed a biphasic effect on the mechanically impaired filterability: an improvement at 10-10 M (from 22.7 ± 4.5 to 48.8 ± 15.5%, P < 0.01) and a deterioration at 10-8 M (from 22.7 ± 4.5 to 12.7 ± 7.8%, P < 0.01). Red blood cells from some individuals showed a marked improvement at 10-10 M and a small deterioration at 10-8 M, whereas those from other individuals showed a small improvement and a marked deterioration at the same concentrations (Fig. 8).


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Fig. 8.   Effects of epinephrine. Pretreatment with epinephrine, incubation with Ca2+ solution, and filtration of red blood cell suspensions were done in the same way as that employed in experiments with prostaglandin E1 (Fig. 6). A: representative plots of filterability according to effects of different concentrations of epinephrine on mechanically impaired filterability of red blood cells in 50 µM extracellular Ca2+ concentration. open circle , Typical improvement in impaired red cell filterability at 10-10 M epinephrine; Delta , typical deterioration at 10-8 M epinephrine. B: mechanically impaired filterability in 50 µM extracellular Ca2+ concentration without epinephrine (a) and with 10-10 M (b) and 10-8 M (c) epinephrine. Data are means ± SD from 6 sets of experiments. * P < 0.01 vs. a. Comparison of data between 2 groups was made by paired t-test.

Pentoxifylline, a potent inhibitor of phosphodiesterase, markedly improved the mechanically impaired filterability; the maximum improvement (from 29.2 ± 5.9 to 73.3 ± 9.3%, P < 0.01) was at the concentration of ~1 × 10-9 M, showing a bell-shaped improvement with the concentrations ranging from 10-12 to 10-7 M (Fig. 9).


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Fig. 9.   Effects of pentoxifylline. Pretreatment with pentoxifylline, incubation with Ca2+ solution, and filtration were done in the same way as that employed in the experiments with prostaglandin E1 (Fig. 6). A: representative plot of filterability according to the effects of different concentrations of pentoxifylline on mechanically impaired filterability of red blood cells in 50 µM extracellular Ca2+ concentration. B: mechanically impaired filterability in 50 µM extracellular Ca2+ concentration without (a) and with (b) 10-9 M pentoxifylline. Data are means ± SD from 6 sets of experiments. * P < 0.01 vs. a. Comparison of data between 2 groups was made by paired t-test.

Both the impairment and the deterioration in the impaired filterability produced by the drugs described above were accompanied by no changes in cell volume or morphology. Without mechanical stress, the drugs used did not change the red blood cell filterability when the red blood cells were incubated at 36°C for 60 min at 50 µM or 1 mM of the extracellular Ca2+ concentration.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

It is known that red blood cells have the capacity to change their deformability in response to various kinds of mechanical stress (12, 27, 28). Usually, the exposure of red blood cells to mechanical stress in vitro has been achieved by generating shear forces in a viscometer (12, 17, 27). O'Rear et al. (27) reported that red blood cells after exposure to subhemolytic shear stress (<1,500 dyn/cm2 for 2 min) in a concentric cylinder viscometer showed little change in cell morphology. Mechanical hemolysis occurred at >3,000 dyn/cm2 for 2 min exerted by a closed concentric cylinder viscometer (26). In contrast, the shear stress generated by the injection method is relatively high stress (~6,600 dyn/cm2) for a very short time (~0.6 ms) as described in METHODS. The actual shear stress experienced by the cells in the needle varies from zero on the center line of the flow up to the estimated level for cells near the wall of the needle and increases with time as the syringe is emptied. Therefore, the maximum shear stress generated by this method is much higher than that found in normal physiological conditions. However, it is noteworthy that the overall impact of the stress on the red blood cells may be attributable to the time of exposure to the shear stress, as well as the stress level. Leverett et al. (18) showed the threshold for extensive shear stress damage by dividing the shear stress-time domain into two distinct regimes, using the data from several different methods. Their shear stress-time diagram indicates that the shear stress of 6,600 dyn/cm2 for 0.6 ms may be a stress below the threshold for extensive cell damage. In fact, the decrease in red blood cell filterability caused by the injection method was accompanied by no morphological change or hemolysis. Thus a quantitative decrease in the filterability was obtained by this simple and reproducible method.

The role of intracellular Ca2+ in regulating the physiological function of red blood cells has been studied widely, and an elevation of intracellular Ca2+ has been shown to exert profound effects on the membrane properties that regulate red blood cell deformability (29). The mechanical stress given by shear stress of a physiological magnitude was shown to induce transient elevations of intracellular Ca2+ (17) and a decrease in deformability (12, 28). These findings were confirmed in the present study. Red blood cell deformability has been ascribed to three physical properties: the surface area-to-volume ratio, the internal viscosity or mean corpuscular hemoglobin concentration, and membrane viscoelastic properties (4, 7, 20, 34). The decrease in filterability without changes in cell volume and morphology obtained in the present study therefore indicates that the decreased filterability produced by our method is the result of neither a change in the surface area-to-volume ratio nor an increased intracellular viscosity from dehydration but rather of an alteration of the membrane properties.

Under the mechanical stress, the red blood cell filterability markedly decreased depending on the extracellular Ca2+ concentration, indicating that mechanical stress induces an influx of Ca2+, which causes a decrease in filterability (Fig. 3). Considering that the Ca2+ entry blockers inhibited the decrease in filterability under the mechanical stress (Figs. 4 and 5) and that, without mechanical stress, no decrease in filterability was induced by the extracellular Ca2+, it is possible that transient elevations of intracellular Ca2+ through a mechanically activated Ca2+ channel in the red blood cell membrane play a central role in decreasing the red blood cell filterability under mechanical stress. Nifedipine and felodipine did not return the decreased filterability to the level characteristic of cells mechanically stressed in the absence of extracellular Ca2+, suggesting that a dihydropyridine-insensitive channel may also facilitate the Ca2+ influx. To our knowledge, this inhibitory effect of Ca2+ entry blockers is the first documentation of the changes induced by mechanically generated intracellular Ca2+ elevations in red blood cells. The physiological meaning of the present observation that red blood cell filterability under mechanical stress decreased maximally at ~50 µM of the extracellular Ca2+, which is considerably lower than the Ca2+ concentration in normal plasma, is under further investigation. However, the V-shaped dependency of the impairment on extracellular Ca2+ suggests that there are threshold and delayed times of response to the intracellular Ca2+ elevations and that there are some physiological mechanisms that exert compensatory effects on the impairment of the filterability. We therefore investigated the role of cAMP-mediated signaling pathways in the impaired filterability.

Previous studies of the effect of PGE1 on red blood cell filterability have yielded somewhat contradictory results (9, 11, 14, 16, 31). In the present study, a marked improvement (from 21.2 ± 4.5 to 63.7 ± 11.9%) in filterability by PGE1 was demonstrated at very low concentrations (1 × 10-10 M; Fig. 6). It is noteworthy that this increase in filterability was strikingly large compared with increases in previous reports (14, 16, 31). The previous studies were performed on the "resting" red blood cells that are not exposed to mechanical stress. Because the filterability of "normal" red blood cells is essentially unimpaired in the resting state, it could be quite difficult to increase their filterability. In some reports (16, 31), a small increase in the filterability of resting red blood cells was obtained. It is plausible that those cells were unintentionally exposed to mechanical stress during experimental procedures such as washing, which is an integral process for measuring the red blood cell filterability.

The increase in filterability produced by PGE1 and the decrease produced by PGE2 in this study (Figs. 6 and 7) are consistent with the findings of previous reports (14, 31). Mechanisms of the changes in filterability produced by these drugs have been proposed (9). Allen (1) proposed that PGE2 increases the cell volume by increasing the water content, resulting in the deterioration of filterability, and that PGE1 has an effect directly opposite to that of PGE2. A recent study by Li et al. (19) suggested that PGE2 impairs the filterability by decreasing the cell volume through a stimulation of a Ca2+-dependent K+ channel. However, the present results show that PGE1 and PGE2 are able to alter the filterability without a significant change in cell volume, indicating that the effects of these drugs on the filterability under mechanical stress are exerted by an alteration of the membrane properties.

Adenylate cyclase is reported to be present in human red blood cells (13, 24), the activity of which is thought to be affected by PGE1 and PGE2, as it is in platelets (3, 8, 23). Therefore, it is quite possible that the intracellular cAMP level is involved in the regulation of red blood cell filterability by changing the membrane properties through an activation of cAMP-dependent kinases (5). We tested this hypothesis by observing the effects of pentoxifylline, which is a potent inhibitor of phosphodiesterase and hence is expected to increase the intracellular cAMP level in human red blood cells. We found that pentoxifylline markedly improved the mechanically impaired filterability with an incubation at a very low concentration for only 15 min at 36°C (Fig. 9).

The effects of epinephrine on the impaired filterability (Fig. 8) are consistent with the presence of adrenergic receptors (33) and adenylate cyclase in human red blood cells (13, 24), also supporting the possibility that cAMP-dependent kinases affect the membrane properties for regulating red blood cell filterability. Furthermore, the biphasic effect of epinephrine on the impaired filterability is interesting as to the pathophysiology of the microcirculation (Fig. 8A). Epinephrine worsened the impaired filterability at the concentration of 1 × 10-8 M, as in the study by Rasmussen et al. (31). In the present study, at 1 × 10-10 M, which is within the normal concentration range in plasma (>2.7 × 10-10 M; see Ref. 15), epinephrine improved the filterability (Fig. 8B), suggesting that epinephrine plays a beneficial role in maintaining normal microcirculation in vivo by improving red blood cell filterability. Another important observation is that the bi-phasic effect of epinephrine differed somewhat from person to person (Fig. 8A), indicating a difference in the responsiveness to epinephrine among individuals, which may be related to predisposing conditions to certain clinical situations such as hypertension.

It has long been postulated that red blood cell deformability is regulated by the two main pathways of the intracellular signal transduction system (5, 9), as in many other cell types (3, 23); these Ca2+-dependent and cAMP-dependent pathways may evoke various kinds of interacting kinases and phosphatases, which lead to the phosphorylation and dephosphorylation of key proteins in the membrane cytoskeleton and regulate red blood cell filterability. The present results showed that posttranslational or regulatory modifications of the membrane properties of red blood cells can be reflected in their filterability, as in platelet aggregability. In conclusion, the present study clearly demonstrated the existence of several signal transduction pathways in human red blood cells.

    ACKNOWLEDGEMENTS

We express appreciation to Dr. H. Shio (Shiga Medical Center for Adult Diseases) and Dr. E. A. O'Rear (University of Oklahoma) for critical comments and helpful discussion and to K. Kanezaki (Kanto Teishin Hospital) for excellent technical assistance. We thank the journal reviewers especially for helpful suggestions concerning the manuscript.

    FOOTNOTES

This work was supported in part by grant 05305006 from the Ministry of Education, Science, and Culture of Japan to N. Uyesaka.

Address for reprint requests: T. Oonishi, NTT Medical Research Institute, Kanto Teishin Hospital, 5-9-22, Higashi-Gotanda, Shinagawa-ku, Tokyo 141, Japan.

Received 9 April 1997; accepted in final form 1 August 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C1828-C1834
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