Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 88: 1520-1528, 2000;
8750-7587/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deem, S.
Right arrow Articles by Swenson, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deem, S.
Right arrow Articles by Swenson, E. R.
Vol. 88, Issue 5, 1520-1528, May 2000

Effects of the RBC membrane and increased perfusate viscosity on hypoxic pulmonary vasoconstriction

Steven Deem1, John T. Berg1, Mark E. Kerr1, and Erik R. Swenson1,2

1 Departments of Anesthesiology and Medicine, University of Washington, 98195; and 2 Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in part by scavenging of nitric oxide (NO) by Hb (Deem S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ, Am J Respir Crit Care Med 157: 1181-1186, 1998). We studied the contribution of the RBC compartmentalization of Hb to augmentation of HPV and scavenging of NO in isolated perfused rabbit lungs. Lungs were initially perfused with buffer; HPV was provoked by a 5-min challenge with hypoxic gas (inspired O2 fraction 0.05). Expired NO was measured continuously. Addition of free Hb to the perfusate (0.25 mg/ml) resulted in augmentation of HPV and a fall in expired NO that were similar in magnitude to those associated with a hematocrit of 30% (intracellular Hb of 100 mg/ml). Addition of dextran resulted in a blunting of HPV after free Hb but no change in expired NO. Blunting of HPV by dextran was not prevented by NO synthase inhibition with Nomega -nitro-L-arginine and/or cyclooxygenase inhibition. RBC ghosts had a mild inhibitory effect on HPV but caused a small reduction in expired NO. In conclusion, the RBC membrane provides a barrier to NO scavenging and augmentation of HPV by Hb. Increased perfusate viscosity inhibits HPV by an undetermined mechanism.

anemia; nitric oxide; hypoxia; red blood cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN A PREVIOUS INVESTIGATION, we found that red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in a concentration-dependent fashion (8). The mechanism for RBC-mediated augmentation of HPV appears to be related to the ability of Hb to scavenge nitric oxide (NO) from the pulmonary circulation. Expired NO concentration falls in a linear fashion as hematocrit (Hct) increases, and RBC dependence of HPV is eliminated when NO synthase (NOS) is inhibited by administration of the L-arginine analog Nomega -nitro-L-arginine (L-NA; Ref. 8).

Although it appears that binding of NO by Hb is involved in RBC augmentation of HPV, it is less obvious how or whether the intact RBC and the RBC membrane modulate these phenomena. Previous investigators have found that addition of small amounts of free Hb to blood (23) or blood-free (16, 34) perfusate in isolated rat lungs results in an increase in the pressor response to hypoxia. The goal of the current study was to further define the amount of free Hb necessary to augment HPV in buffer-perfused lungs challenged by moderate hypoxia and to document the effect of free Hb on expired NO. In addition, we wished to establish the role of the isolated RBC membrane in the form of RBC ghosts on HPV, and to determine whether increased perfusate viscosity as seen at higher Hcts has an inhibitory effect on HPV. Finally, we studied the effects of NOS and cyclooxygenase (COX) inhibition on HPV in the presence of increased perfusate viscosity to help define the mechanism by which increased viscosity inhibits HPV.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The protocol was approved by the Animal Care Committee of the Seattle Veterans Affairs Medical Center.

Experimental preparation. A total of 43 New Zealand White rabbits weighing 3-3.5 kg were anesthetized with ketamine (15 mg/kg) and xylazine (0.33 mg/kg). Mechanical ventilation was initiated after tracheostomy with room air using a ventilatory rate of ~30 breaths per minute and a tidal volume of 10 ml/kg (peak airway pressure of ~15 cmH2O). A carotid arterial catheter was placed, intravenous heparin (300 U/kg) was administered, and the rabbits were rapidly exsanguinated. After median sternotomy, the pulmonary artery and left atrium were cannulated in situ. The pulmonary circulation was then perfused by use of a closed circuit connected to a Masterflex pump (Cole-Palmer, Barrington, IL) with a total circuit volume of 250 ml at a constant flow of 150 ml/min (perfusate composition described below). The perfusate was warmed to 38°C with a countercurrent exchange heater. The ventilatory gas was switched to a mixture of 21% O2-5% CO2, with the balance made up by N2. The rabbit's chest was covered with plastic wrap to maintain humidity and surface temperature.

Pulmonary arterial pressure (Ppa) and left atrial pressure (Pla) were continuously measured via pressure tubing placed inside the perfusion cannulas and positioned at the inflow and outflow sites. Pla was maintained constant at 5 mmHg by adjusting the height of the venous reservoir. Fractions of inspired O2 (FIO2) and CO2 (FICO2) were measured near the tracheal tube. Mixed expired NO was measured in all experiments by use of a chemiluminescence detector (Sievers Instruments, Boulder, CO) by continuously sampling from a 50-ml reservoir placed in the expired gas line (31). The sample flow rate was 120 ml/min out of a fixed minute ventilation of 900 ml/min. Calibration was performed by using NO-free gas from the normoxic and hypoxic tanks and a certified tank containing NO at a concentration of 1.9 ppm (Air Liquide, Long Beach, CA) and sampled at 120 ml/min. Recalibration was performed at ~30-min intervals throughout the experiments. Pressure and NO data were continuously recorded with an analog-to-digital converter, data acquisition software (Strawberry Tree, Sunnyvale, CA), and a personal computer (Macintosh, Cupertino, CA).

All preparations were perfused with buffered Krebs-Henseleit solution (in mM: 11 D-glucose, 1.2 MgSO4, 1.2 KH2PO4, 4 KCl, 120 NaCl, 2.5 CaCl2, 25 NaHCO3; 4% hydroxyethylstarch) for 20-30 min to allow stabilization before experimental intervention. Normoxic values of Ppa, Pla, peak inspiratory pressure, and expired NO were recorded, and the preparations were then challenged with hypoxic gas (FIO2 0.05, FICO2 0.05, balance N2) for 5 min. Data were again recorded, and ventilation with normoxic gas was resumed. HPV was defined as the change in Ppa with hypoxia from the immediate normoxic baseline. After this initial hypoxic challenge, preparations were entered into one of five intervention groups: 1) RBCs (n = 5), 2) free Hb (n = 9), 3) free Hb plus dextran (n = 14), 4) NOS and COX inhibition plus dextran (n = 9), and 5) RBC ghosts plus free Hb (n = 7). Experiments in groups 1 and 2 were performed concurrently, with experiments randomized on a daily basis. Experiments in groups 3, 4, and 5 were performed after completion of the previous two groups. Perfusate pH, PO2, and PCO2 were measured periodically during the experimental protocols, and sodium bicarbonate was added as necessary to maintain perfusate pH at 7.40 ± 0.05.

Group 1: RBC perfusates. To provide RBCs for this experimental group, blood obtained during exsanguination and whole blood collected from donor rabbits were centrifuged at 3,000 g for 8 min. The plasma and buffy coat were discarded, and the RBCs were resuspended in saline and centrifuged again for 8 min. The supernatant was again discarded, and the wash and centrifugation were repeated. The RBCs were then filtered through a Pall high-efficiency leukocyte filter (Pall Biomedical, Fajardo, PR) to remove residual leukocytes.

After the initial hypoxic challenge during Krebs-Henseleit perfusion, packed RBCs (Hct ~80%) were added to the venous reservoir of the perfusion circuit in an amount sufficient to provide the desired Hct. Preparations were perfused sequentially with Hcts of 1%, 10%, and 30% and were challenged at each Hct with a 5-min period of hypoxia, as described above. A 10-min stabilization period with normoxic ventilation was allowed after each addition of RBCs. Hct was measured in all preparations by use of a microcapillary centrifuge (International Equipment, Needham Heights, MA).

Group 2: Free Hb. Powdered bovine Hb obtained from Sigma Chemical (St. Louis, MO) was dissolved in distilled water and dialyzed overnight. Sodium dithionate was added to the Hb solution, and it was bubbled with nitrogen during dialysis to minimize methemoglobin formation. The concentration and percentage of reduced and methemoglobin were measured spectrophotometrically as previously described (11, 22).

After the initial hypoxic challenge during perfusion with Krebs-Henseleit solution, the purified and reduced Hb product was added to the circulating perfusate by slow injection into the venous reservoir in an amount sufficient to produce a concentration of ~0.25 mg/ml (3.6 µM). A 10-min period of normoxic ventilation was allowed after addition of free Hb, and the lungs were then challenged with hypoxic gas as described above. After return to normoxia, additional free Hb was added to produce a 10-fold increase in Hb concentration (2.5 mg/ml); after another 10-min stabilization, the lungs were again subjected to a hypoxic challenge.

Group 3: Dextran plus free Hb. Free bovine Hb was prepared as described for group 2. After the initial hypoxic challenge during Krebs-Henseleit perfusion, free Hb was added to the perfusate, and a hypoxic challenge was performed as described for group 2. After return to normoxic ventilation, 3.9 g of dextran (Sigma Chemical; average molecular weight = 69,000) were added to the venous reservoir. This amount was sufficient to produce a perfusate viscosity similar to that seen in RBC perfusates at 10% Hct (Dex 10). After a 10-min stabilization period, a hypoxic challenge was performed as described under Experimental preparation. This sequence was repeated with addition of a second aliquot of dextran (5.6 g) to produce a perfusate viscosity similar to that seen in RBC perfusates at 30% Hct (Dex 30). In total, six experiments were conducted in this fashion. In three additional experiments, three sequential hypoxic challenges separated by 10-min intervals were performed after addition of free Hb to the perfusate to establish a stable response over time. After the third hypoxic challenge, 5.6 g dextran were added to the perfusate, and a final hypoxic challenge was performed. In a final five experiments, 3.9 g and 5.6 g dextran were serially added to the perfusate, in the absence of Hb, with each dose followed by hypoxic challenges. Hb (0.25 mg/ml) was added to the perfusate after the final dose of dextran in three experiments, and a final hypoxic challenge was performed.

The viscosity of the perfusates was measured by a rotational viscometer (Brookfield Engineering Laboratories, Stoughton, MA) at 38°C and a shear rate of 45 s-1. Viscosity measurements are reported in Table 1.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Perfusate viscosity

Group 4: NOS and COX inhibition. After the initial hypoxic challenge during buffer perfusion, L-NA was added to the perfusate to produce a concentration of 0.1 mM. We have previously documented the competitive nature of NOS inhibition with this agent at this concentration in our model (8). After 15 min of normoxic perfusion, a hypoxic challenge was performed as described under Experimental procedure. After return to normoxic ventilation, dextran (9.5 g) was added to the venous reservoir, and the hypoxic challenge was repeated. The preparation was again returned to normoxic ventilation, and 0.7 mM of the nonselective COX inhibitor indomethacin was added to the perfusate (n = 7). After 15 min of normoxic perfusion, a final hypoxic challenge was performed. In two experiments, 5 mg indomethacin were administered intravenously to the rabbit before exsanguination, and 0.7 mM indomethacin was added to the perfusate before perfusion. Hypoxic challenges during Krebs perfusion, after addition of L-NA and after addition of dextran to the perfusate, were then performed.

Group 5: RBC ghosts. RBC ghosts were prepared as originally described by Marchesi and Palade (21) and as modified by Baldwin and Wilson (1). Briefly, rabbit blood was washed and filtered as described for group 1 and then subjected to hypotonic hemolysis and repeated high-speed centrifugation to remove free Hb. This procedure resulted in the production of RBC membrane, which retained some structural integrity when examined under interference-contrast microscopy (Fig. 1). The final ghost concentrate was assayed spectrophotometrically at three wavelengths to verify the absence of Hb (sensitivity of assay <0.1 mg/ml) (10). The volume of packed RBCs at the beginning of ghost synthesis was similar to that required to produce a Hct of 30% when added to the perfusion circuit (~70 ml), although the final volume of ghost concentrate varied between 15 and 20 ml.


View larger version (130K):
[in this window]
[in a new window]
  		Fig. 1.   Red blood cell (RBC) ghosts as viewed by interference-contrast microscopy at ×630.

After the initial hypoxic challenge during Krebs-Henseleit perfusion, in five experiments the prepared RBC ghosts were slowly added to the venous reservoir of the perfusion circuit. After a 10-min stabilization period, a hypoxic challenge was performed as described above. After return to normoxic ventilation, 3.6 µM Hb was added to the venous reservoir, and the sequence was repeated. In two experiments, the sequence was reversed, with addition of free Hb followed by RBC ghosts.

Statistical analysis. Statistical analysis was performed with the StatView software package (Abacus Concepts, Berkeley, CA). Data are presented as means ± SE. Within-group comparisons were performed by using a paired Student's t-test, with P values adjusted for multiple comparisons by using the Bonferroni correction. P < 0.05 was accepted as statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General. Baseline (normoxic) Ppa before hypoxic challenges and expired NO in major subgroups of experiments are reported in Table 2. HPV was weak during Krebs perfusion in all groups (Figs. 2-4, 7, and 8).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Normoxic pulmonary arterial pressure and expired nitric oxide in major experimental sets

Group 1. Addition of RBCs to the perfusate resulted in an increase in normoxic Ppa that approached statistical significance at 30% Hct (Table 2; P = 0.07, Krebs vs. 30% Hct). Expired NO fell at each level of Hct and was significantly lower than during Krebs perfusion at Hct 30% (Table 2; P < 0.05). Likewise, the addition of RBCs resulted in an increase in HPV at each level of Hct, although the difference from HPV with Krebs was statistically significant only at Hct 30% (P < 0.05; Fig. 2).


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 2.   Hypoxic pulmonary vasoconstriction (HPV; change in pulmonary artery pressure from immediately preceding normoxic baseline pressure) in preparations to which RBCs were added to perfusate; n = 5. Values (%) represent approximate hematocrit (Hct) of perfusate. * P < 0.05 vs. perfusion with Krebs-Henseleit buffer (Krebs).

Group 2. Addition of 3.6 µM Hb to the perfusate resulted in a nonsignificant increase in normoxic Ppa (Table 2), a marked fall in expired NO (Table 2), and dramatic augmentation of HPV (Fig. 3). Addition of 10-fold more free Hb to the perfusate resulted in an increase in normoxic Ppa, a further fall in expired NO, and further augmentation of HPV; however, all but three of the preparations developed intractable pulmonary hypertension and/or pulmonary edema during this intervention and did not complete the hypoxic challenge. No statistical analysis was performed for this intervention because of the large dropout.


View larger version (9K):
[in this window]
[in a new window]
  		Fig. 3.   HPV in preparations to which free Hb was added to perfusate; n = 9. Hb was added to achieve a concentration of 0.25 mg/ml (3.6 µM). * P < 0.05 vs. perfusion with Krebs-Henseleit buffer.

Group 3. Addition of 3.6 µM Hb to the perfusate resulted in a nonsignificant increase in normoxic Ppa and a fall in expired NO (Table 2) and again resulted in augmentation of HPV (P < 0.05; Fig. 4). Addition of dextran, 3.9 (Dex 10) and 5.6 g (Dex 30), to the perfusate after free Hb resulted in increases in normoxic Ppa (P < 0.05 for Dex 10 and Dex 30 vs. Krebs) and no change in expired NO (Table 2). However, addition of dextran resulted in a diminution of HPV (P < 0.05 for Hb vs. Dex 30; Fig. 4). In contrast, three successive hypoxic challenges after addition of a single dose of free Hb to the perfusate resulted in augmentation of the pressor response (Fig. 5; P < 0.05, challenge 1 vs. challenge 2). Addition of dextran after three successive hypoxic challenges after a single dose of free Hb in two preparations resulted in a 50% reduction in the pressor response to hypoxia.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4.   HPV in preparations to which free Hb (3.6 µM) and then dextran were added to perfusate; n = 6. Dextran was added in an amount sufficient to increase perfusate viscosity to that approximating perfusates of Hct 10% (Dex 10) and Hct 30% (Dex 30). * P < 0.05 vs. perfusion with Krebs-Henseleit buffer; dagger  P < 0.05 vs. perfusion with buffer containing free Hb.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5.   HPV during repeated hypoxic challenges (1, 2, and 3) after addition of a single dose of free Hb (3.6 µM) to perfusate; n = 3. * P < 0.05 vs. challenge 1.

Addition of dextran to the perfusate had no effect on expired NO whether added after (Table 2) or before (Fig. 6) addition of free Hb to the perfusate. Addition of dextran, 3.9 and 5.6 g, to the perfusate before Hb increased normoxic Ppa from 10.4 ± 0.5 to 11.0 ± 0.5 and 12.0 ± 0.7 Torr, respectively (P < 0.05 for both concentrations of dextran vs. Krebs) but had no effect on HPV, and addition of free Hb after dextran resulted in only a small increase in HPV (2.3 ± 0.6 Torr).


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6.   Effects of increased perfusate viscosity on expired nitric oxide (NO). Dextran was added in Dex 10 and Dex 30 amounts; n = 5.

Group 4. Addition of L-NA to the perfusate resulted in a fall in expired NO and augmentation of HPV (P < 0.05 vs. HPV with Krebs perfusion; Table 2, Fig. 7). An increase in perfusate viscosity by addition of dextran after L-NA resulted in an increase in normoxic Ppa and a reduction in HPV (P < 0.05 vs. HPV after L-NA alone; Table 1, Fig. 7). Indomethacin added either after (Fig. 7) or before (data not shown) L-NA and dextran did not prevent blunting of HPV by dextran.


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7.   Effects of NO synthase and cyclooxygenase inhibition on attenuation of HPV by increased perfusate viscosity; n = 7. L-NA, Nomega -nitro-L-arginine (0.1 mM) added to perfusate; Dex, dextran added to increase viscosity of perfusate to that approximating Hct 30%; Indo, 0.7 mM indomethacin added to perfusate. * P < 0.05 vs. response during Krebs-Henseleit perfusion; dagger  P < 0.05 vs. response after L-NA.

Group 5. Addition of RBC ghosts to the perfusate had no effect on normoxic Ppa but resulted in a small but statistically significant fall in expired NO (Table 2). RBC ghosts resulted in a significant reduction in the already weak pressor response to hypoxia (P < 0.05 vs. HPV with Krebs perfusion). Addition of 3.6 µM Hb after ghosts resulted in augmentation of HPV (P < 0.05 vs. HPV with ghosts alone), although HPV in the presence of ghosts plus Hb was not significantly different from that during Krebs-Henseleit perfusion (Fig. 8). In the two experiments in which Hb was added to the perfusate before ghosts, addition of ghosts resulted in a reduction in HPV from 3.5 ± 0.5 to 0.5 ± 0.5 Torr.


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 8.   Effects of RBC ghosts on HPV; n = 5. Approximately 15 ml of ghosts synthesized from packed RBCs were added to the perfusate, followed by free Hb (3.6 µM). * P < 0.05 vs. perfusion with Krebs-Henseleit buffer.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The current study builds on our previous work documenting the Hct dependence of HPV and expired NO in an isolated lung model (8). Our data also confirm that addition of free Hb to an isolated, perfused system results in dramatic augmentation of HPV and a reduction of expired NO. The Hb concentration that resulted in augmentation of HPV (3.6 µM or 0.25 mg/ml) is more than 100-fold less than that contained within an RBC perfusate at Hct 30%, although the pressor response to hypoxia was similar in these two groups (Figs. 2 and 3). This study also provides evidence that increasing perfusate viscosity is in fact inhibitory of HPV and is not the mechanism by which increasing Hct augments HPV. Finally, these data suggest that the RBC membrane has a mild inhibitory effect on HPV by an as-yet-undefined mechanism.

Limitations of the model. The isolated, perfused lung model is well established for investigation of HPV and provides the opportunity to study and control multiple variables over a relatively short period of time. Conditions in this model will admittedly not entirely reflect conditions in the intact animal.

In our previous work (8), we showed that HPV at different levels of Hct was relatively stable with repeated hypoxic challenges over time. HPV is a variable biological response, however, both between and within species. Although it has not been reported in the literature, we have observed variability in HPV in isolated rabbit lungs depending on the season of the year. This finding may explain some of the observed variability in HPV between groups. In particular, the HPV response in group 2 is much stronger than in groups 3 and 5 after addition of free Hb to the perfusate. These experiments were subjected to identical baseline conditions, with the only difference being that the experiments were conducted consecutively over the course of a year. It is because of this variability in response that we have emphasized within-group analysis, with the exception of groups 1 and 2, which were performed concurrently and in random order. We also attempted to control for varying HPV response by performing subgroup experiments, with reversal of the order of interventions (groups 3-5).

In the current study, Hct was varied in individual experiments rather than maintained at a constant level over the course of each experiment as in our previous work (8). The observation that HPV is augmented by increasing Hct in a single preparation supplements the previous data by eliminating the possibility of unmeasured between-group differences.

Hb and HPV. Previous studies that have shown a salutary effect of free Hb on HPV in isolated lung models differ from ours in several ways (16, 23, 35). In the study by Mazmanian et al. (23), isolated rat lungs were perfused with blood before addition of free Hb at a concentration twice that of ours (0.5 mg/ml). This resulted in an approximate doubling of the pressor response to FIO2 0.02. Studies by Hasunuma et al. (16) and Voelkel et al. (35), in buffer-perfused rat lungs, showed that addition of 20 µM free Hb resulted in a two- to threefold increase in the pressor response to ventilation at FIO2 0. Thus our investigation complements previous work in that we used a more moderate hypoxic challenge (FIO2 0.05) in buffer-perfused lungs and used a much smaller concentration of Hb to augment HPV.

The observation that a small amount of free Hb added to buffer perfusate results in a dramatic fall in expired NO is consistent with the presumed mechanism by which Hb augments HPV. NO binds avidly to oxyhemoglobin, with the resulting reaction products of methemoglobin and nitrate (3). The removal of NO from the circulation will result in conditions favorable for vasoconstriction to various stimuli. This hypothesis is supported by the observation by Voelkel et al. (35) that addition of free Hb to buffer perfusate results in a fall in perfusate cGMP, the second messenger by which NO exerts its vasodilatory effect.

In isolated, buffer-perfused lungs, expired NO declines over time, probably because of substrate limitation, as illustrated by data collected but not reported in a previous study from our laboratory (Fig. 9; Ref. 8). The expired NO immediately preceding four consecutive hypoxic challenges falls only ~10% (total time of ~45 min), however. Although this change is statistically significant (P < 0.01), the decrement is much smaller than the immediate decreases in expired NO seen with addition of RBCs, free Hb, or L-NA to the perfusate in the current study (Table 2), which was conducted over a similar time period.


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 9.   Decline in baseline (normoxic) NO before 4 successive hypoxic challenges in buffer-perfused rabbit lungs; n = 8. * P < 0.01 compared with value at time 0 (unpublished data from Ref. 8).

We have assumed that the mechanism by which RBCs augment HPV is through removal of NO from the circulation and away from vascular smooth muscle (VSM). Indeed, the inverse relationship between expired NO and Hct and the strength of HPV is unlikely to be coincidental. Liu et al. (19) have shown that the half-life of perfusate NO is inversely proportional to the concentration of RBCs, and multiple studies have documented the importance of NO in regulation of HPV (5, 13, 18, 23, 27, 30). Previously, we documented that NOS inhibition resulted in a dramatic increase in HPV in buffer-perfused lungs but had little effect on the strength of HPV in lungs perfused with RBCs at 30% Hct (8).

A reasonable explanation for the greater effect of free Hb than intact RBCs on expired NO and HPV is that the RBC membrane provides a diffusion-limited barrier to binding of NO by Hb. Although Hakim et al. (15) found that "packaging" of Hb in RBCs did not affect the in vitro half-life of NO in aqueous solution, Liu et al. (19) found that NO disappearance from an in vitro system was ~650 times faster in the presence of free oxyhemoglobin compared with a comparable concentration of intra-RBC Hb. These authors used a NO-sensitive electrode that contacts directly with the NO-containing solution, giving it a much faster response time than that of Hakim et al. (15). The relative diffusion barrier provided by the RBC membrane may be magnified in vivo, where a layer of cell-free plasma exists next to the endothelium during blood perfusion (6, 34). Work by Liao et al. (17) supports this hypothesis; they have shown that free Hb (10 µM) abolishes NO-mediated vasodilation in the presence or absence of flow in isolated microvessels. RBCs at 50% Hct reduced NO-mediated vasodilation only when flow was absent, however. Other work suggests that modulation of NO scavenging by Hb may even be peculiar to the type of RBC. Calatayud et al. (7) have found that fetal RBCs have increased capacity to scavenge NO in comparison to adult RBCs, an effect that was not explained by differences in the type of contained Hb (HbF vs. A) (7).

Free Hb has additional characteristics that may affect its potential for scavenging of NO and augmentation of HPV. Unmodified bovine Hb exhibits a high degree of endothelial permeability (26). The movement of free Hb into close approximation with VSM may increase the potential for abluminal scavenging of NO that might otherwise be destined for VSM sites (25, 26). Free Hb has cytotoxic effects on endothelial cells that may also result in a reduction of NO production (12, 24). The propensity of free Hb to produce lung injury was overtly manifest in our preparations when we added larger amounts (500 mg) of Hb to the perfusate. The majority of preparations developed intractable pulmonary hypertension and edema after this intervention. It is possible that more subtle endothelial injury occurred after administration of the smaller dose of Hb (50 mg) and was responsible, in part, for the observed enhanced pressor response to hypoxia.

Perfusate viscosity and HPV. We studied changes in perfusate viscosity in an attempt to explain the potent effect of acellular Hb in enhancing HPV and in reducing expired NO in comparison to intact RBCs. The viscosity of blood or perfusates containing high concentrations of RBCs is much higher than that of an acellular perfusate (Table 1). Increased perfusate viscosity and flow, as well as reduced vessel diameter, result in increased vascular shear stress, which may in turn result in a reduction in pulmonary vascular resistance mediated by endothelial production of NO (2, 36, 37) or possibly prostacyclin (2, 33). It is therefore conceivable that perfusion with intact RBCs may result in viscosity-mediated blunting of HPV relative to the potential NO-scavenging capability of the contained Hb. Indeed, increasing perfusate viscosity by additing dextran resulted in blunting of the HPV response to free Hb (Fig. 3). However, the blunting of HPV by increased perfusate viscosity was not mirrored by an increase in expired NO (Table 2, Fig. 6), and inhibition of NO and prostacyclin synthesis did not eliminate the inhibitory effect of increased viscosity on HPV (Fig. 7).

We used dextran to increase perfusate viscosity to isolate the pure mechanical effect of viscosity on the vasculature from other potential metabolic effects of RBCs. Unfortunately, increases in perfusate viscosity by dextran do not entirely reproduce the non-Newtonian viscosity effects of cellular (RBC-containing) perfusates. In addition, some investigators have found that shear stress-mediated changes in pulmonary vascular tone require the presence of RBCs (29, 32), perhaps through specific endothelial-RBC membrane interactions or effects of RBCs on metabolite flux. The latter studies differed from ours, however, in that they were performed under normoxic conditions. Hypoxia results in a fall in expired NO in isolated rabbit lungs perfused with buffer or RBCs, and NOS inhibition had no effect on pulmonary artery pressure during hypoxia in rabbit lungs perfused at 30% Hct (8). These data suggest that RBC-mediated shear stress production of NO is relatively unimportant in modulation of HPV in the rabbit.

In addition to possible differences in shear stress effects during normoxia vs. hypoxia, species differences also exist that may confound extrapolation of our findings. As noted above, Sprague et al. (29) found that increases in viscosity by RBCs but not dextran resulted in NO-mediated reduction in pulmonary vascular resistance in isolated rabbit lungs. In the rat, however, increased NO production induced by increased perfusate viscosity or by pharmacological vasoconstriction does not appear to require the presence of RBCs (36, 37). However, other investigators have found that RBCs are necessary for modulation of vascular tone in rat lungs (32). Finally, Barnard et al. (2) have shown that, although NO appears to be the primary modulator of vascular tone in rat lungs, in the dog lung cyclooxygenase products appear to be more important. Further investigation will be necessary to completely elucidate the effects of species on modulation of HPV and basal pulmonary artery tone by RBCs and shear stress.

When added in concentrations sufficient to increase perfusate viscosity to that of RBC perfusate at 10% Hct, dextran reduced HPV by ~50% and reduced HPV even further when added at a higher concentration (Fig. 4). It is difficult to predict whether this magnitude of reduction would occur if the baseline HPV response was higher, as in group 2. Further work will be required to define this issue.

The observed reduction of HPV by increased perfusate viscosity confirms earlier work by Benumof and Wahrenbrock (4), who found that HPV was blunted when vascular pressures were increased by infusion of dextran in the intact dog lung. The mechanism by which increased perfusate viscosity inhibits HPV is unclear.

RBC membrane and HPV. In addition to modulation of NO binding to Hb by the RBC membrane and/or cytoplasm, these cellular components may have effects on HPV and NO independent of Hb. To test this possibility, we perfused rabbit lungs with RBC ghosts devoid of Hb. HPV during perfusion with RBC ghosts was reduced in comparison to that seen with buffer perfusion, although in both instances the response was weak and of questionable physiological significance (Fig. 8). The combination of RBC ghosts and free Hb produced a hypoxic pressor response that was weaker than that seen with free Hb alone (Fig. 8). Addition of RBC ghosts to the perfusate also resulted in a small fall in expired NO. This is potentially explained by the capacity of biological membrane to accelerate the reaction of NO with O2 to form NO2- and hence speed the disappearance of NO from solution (20). It is also possible that there was a small amount of residual free Hb in the ghost preparation that was below the sensitivity of our assay. Whether the observed fall in expired NO in the current study is physiologically significant is uncertain, and it does not explain the blunting of HPV in the presence of RBC ghosts. On the other hand, the capacity of membranes to facilitate formation of reactive oxygen species from NO, or alternatively to provide antioxidant activity, may play a role in modulation of HPV.

Preparation of RBC ghosts in the described fashion will exclude analysis of some properties of the RBC that may act to modulate HPV. The absence of RBC cytoplasm results in a loss of enzymes and metabolites that may influence HPV in a variety of ways. Intact RBCs have the capacity to scavenge free radicals (38) and release ATP in response to hypoxia and deformation (9, 28). The lack of intact cytoskeleton in RBC ghosts also makes it unlikely that they will undergo changes in deformability in response to hypoxia or other stimuli in the same fashion as intact cells; a loss of RBC deformability in response to hypoxia may be an important component of the pulmonary hypoxic pressor response in some species (14). However, these issues are beyond the scope of this investigation, which was designed to isolate two components of the RBC, specifically Hb and the RBC membrane.

In conclusion, we have found that a subphysiological amount of free Hb dramatically augments HPV in buffer-perfused rabbit lungs, in association with a reduction in expired NO. These effects occur at a free Hb concentration that is ~400 times less than that found in RBCs at a Hct of ~30%, with comparable increases in HPV and reductions in expired NO. The observation that free Hb is a much more potent stimulator of HPV and scavenger of NO may be explained by the relative diffusion barrier to NO provided by the RBC membrane, a weak inhibitory effect on HPV by the RBC membrane, and a strong inhibitory effect on HPV of increased perfusate viscosity as seen during perfusion at increasing Hct. The mechanisms by which the RBC membrane and increased perfusate viscosity inhibit HPV are, at this point, undefined, although our data suggest that increased production of NO in the case of the RBC membrane and increased production of either NO or prostacyclin in the case of increased viscosity are not important factors in the rabbit.


    ACKNOWLEDGEMENTS

The authors thank Sunday Stray for assistance with viscosity measurements.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-03796 (S. Deem) and HL-45571 (E. R. Swenson).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Deem, Dept. of Anesthesiology, Box 359724, Harborview Medical Ctr., 325 Ninth Ave., Seattle, WA 98104-2499 (E-mail: sdeem{at}u.washington.edu).

Received 10 June 1999; accepted in final form 7 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baldwin, AL, and Wilson LM. Stationary red blood cells induce a negative charge on mucosal capillary endothelium. Am J Physiol Gastrointest Liver Physiol 266: G685-G694, 1994[Abstract/Free Full Text].

2.   Barnard, JW, Wilson PS, Moore TM, Thompson WJ, and Taylor AE. Effect of nitric oxide and cyclooxygenase products on vascular resistance in dog and rat lungs. J Appl Physiol 74: 2940-2948, 1993[Abstract/Free Full Text].

3.   Beckman, JS. Biochemistry of nitric oxide and peroxynitrite. In: Nitric Oxide: A Modulator of Cell-Cell Interactions in the Microcirculation, edited by Kubes P.. Austin, TX: Landes, 1995, p. 1-17.

4.   Benumof, JL, and Wahrenbrock EA. Blunted hypoxic pulmonary vasoconstriction by increased lung vascular pressures. J Appl Physiol 38: 846-850, 1975[Abstract/Free Full Text].

5.   Brashers, VL, Peach MJ, and Rose CE, Jr. Augmentation of hypoxic pulmonary vasoconstriction in the isolated perfused rat lung by in vitro antagonists of endothelium-dependent relaxation. J Clin Invest 82: 1495-1502, 1998.

6.   Butler, AR, Megson IL, and Wright PG. Diffusion of nitric oxide and scavenging by blood in the vasculature. Biochim Biophys Acta 1425: 168-176, 1998[Medline].

7.   Calatayud, S, Beltran B, Brines J, Moncada S, and Esplugues JV. Foetal erythrocytes exhibit an increased ability to scavenge for nitric oxide. Eur J Pharmacol 347: 363-366, 1998[ISI][Medline].

8.   Deem, S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ. Red-blood-cell augmentation of hypoxic pulmonary vasoconstriction: hematocrit dependence and the importance of nitric oxide. Am J Respir Crit Care Med 157: 1181-1186, 1998[Abstract/Free Full Text].

9.   Ellsworth, ML, Forrester T, Ellis CG, and Dietrich HH. The erythrocyte as a regulator of vascular tone. Am J Physiol Heart Circ Physiol 269: H2155-H2161, 1995[Abstract/Free Full Text].

10.   Fairbanks, VF, and Klee GC. Biochemical aspects of hematology. In: Fundamentals of Clinical Chemistry, edited by Tietz NW.. Philadelphia, PA: Saunders, 1987, p. 789-812.

11.   Feelisch, M, Kubitzek D, and Werringloer J. The oxyhemoglobin assay. In: Methods in Nitric Oxide Research, edited by Stamler JS, and Feelisch M.. West Sussex, UK: Wiley, 1996, p. 455-478.

12.   Goldman, DW, Breyer RJ, III, Yeh D, Brockner-Ryan BA, and Alayash AI. Acellular hemoglobin-mediated oxidative stress toward endothelium: a role for ferryl iron. Am J Physiol Heart Circ Physiol 275: H1046-H1053, 1998[Abstract/Free Full Text].

13.   Grimminger, FR, Spriestersbach R, Weissman N, Walmrath D, and Seeger W. Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. J Appl Physiol 78: 1509-1515, 1995[Abstract/Free Full Text].

14.   Hakim, TS, and Macek AS. Role of erythrocyte deformability in the acute hypoxic pressor response in the pulmonary vasculature. Respir Physiol 72: 95-108, 1988[ISI][Medline].

15.   Hakim, TS, Sugimori K, Camporesi EM, and Anderson G. Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiol Meas 17: 267-277, 1996[ISI][Medline].

16.   Hasunuma, K, Yamaguchi T, Rodman DM, O'Brien RF, and McMurtry IF. Effects of inhibitors of EDRF and EDHF on vasoreactivity of perfused rat lungs. Am J Physiol Lung Cell Mol Physiol 260: L97-L104, 1991[Abstract/Free Full Text].

17.   Liao, JC, Hein T, Vaughn MW, Huang KT, and Kuo L. Intravascular flow decreases erythrocyte consumption of nitric oxide. Proc Natl Acad Sci USA 96: 8757-8761, 1999[Abstract/Free Full Text].

18.   Liu, SF, Crawley DE, Barnes PJ, and Evans TW. Endothelium-derived relaxing factor inhibits hypoxic pulmonary vasoconstriction in rats. Am Rev Respir Dis 143: 32-37, 1991[ISI][Medline].

19.   Liu, X, Miller MJS, Joshi MS, Sadowska-Krowicka H, Clark DA, and Lancaster JR, Jr. Diffusion-limited reaction of free nitric oxide with erythrocytes. J Biol Chem 272: 18709-18713, 1998[Abstract/Free Full Text].

20.   Liu, X, Miller MJS, Joshi MS, Thomas DD, and Lancaster JR, Jr. Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes. Proc Natl Acad Sci USA 95: 2175-2179, 1998[Abstract/Free Full Text].

21.   Marchesi, VT, and Palade GE. The localization of Mg-Na-K-activated adenosine triphosphatase on red cell ghost membranes. J Cell Biol 35: 385-404, 1967[Abstract/Free Full Text].

22.   Martin, W, Villani GM, Jothianandan D, and Furchgott RF. Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J Pharmacol Exp Ther 232: 708-716, 1985[Abstract/Free Full Text].

23.   Mazmanian, G, Baudet B, Brink C, Cerrina J, Kirkiacharian S, and Weiss M. Methylene blue potentiates vascular reactivity in isolated rat lungs. J Appl Physiol 66: 1040-1045, 1989[Abstract/Free Full Text].

24.   Motterlini, R, Foresti R, Vandegriff K, Intaglietta M, and Winslow RM. Oxidative-stress response in vascular endothelial cells exposed to acellular hemoglobin solutions. Am J Physiol Heart Circ Physiol 269: H648-H655, 1995[Abstract/Free Full Text].

25.   Motterlini, R, Vandegriff KD, and Winslow RM. Hemoglobin-nitric oxide interaction and its implications. Transfus Med Rev 10: 77-84, 1996[ISI][Medline].

26.   Nakai, K, Ichiro S, Ohta T, Ando J, Kitabatake A, Nakazato Y, and Takahashi TA. Permeability characteristics of hemoglobin derivatives across cultured endothelial cell monolayers. J Lab Clin Med 132: 313-319, 1998[ISI][Medline].

27.   Persson, MG, Gustafsson LE, Wiklund NP, Moncada S, and Hedqvist P. Endogenous nitric oxide as a probable modulator of pulmonary circulation and hypoxic pressor response in vivo. Acta Physiol Scand 140: 449-457, 1990[ISI][Medline].

28.   Sprague, RS, Ellsworth ML, Stephenson AH, and Lonigro AJ. ATP: the red blood cell link to NO and local control of the pulmonary circulation. Am J Physiol Heart Circ Physiol 271: H2717-H2722, 1996[Abstract/Free Full Text].

29.   Sprague, RS, Stephenson AH, Dimmitt RA, Weintraub NA, Branch CA, McMurdo L, and Lonigro AJ. Effect of L-NAME on pressure-flow relationships in isolated rabbit lungs: role of red blood cells. Am J Physiol Heart Circ Physiol 269: H1941-H1948, 1995[Abstract/Free Full Text].

30.   Sprague, RS, Thiemermann C, and Vane JR. Endogenous endothelium-derived relaxing factor opposes hypoxic pulmonary vasoconstriction and supports blood flow to hypoxic alveoli in anesthetized rabbits. Proc Natl Acad Sci USA 89: 8711-8715, 1992[Abstract/Free Full Text].

31.   Spriesterbach, R, Grimminger F, Weissmann N, Walmrath D, and Seeger W. On-line measurement of nitric oxide generation in buffer-perfused rabbit lungs. J Appl Physiol 78: 1502-1508, 1995[Abstract/Free Full Text].

32.   Uncles, DR, Daugherty MO, Frank DU, Roos CM, and Rich GF. Nitric oxide modulation of pulmonary vascular resistance is red blood cell dependent in isolated rat lungs. Anesth Analg 83: 1212-1217, 1996[Abstract].

33.   Van Grondelle, A, Worthen GS, Ellis D, Murphy MM, Strife RJ, Reeves JT, and Voelkel NF. Altering hydrodynamic variables influences PGI2 production by isolated lungs and endothelial cells. J Appl Physiol 57: 388-395, 1984[Abstract/Free Full Text].

34.   Vaughn, MW, Kuo L, and Liao JC. Effective diffusion distance of nitric oxide in the microcirculation. Am J Physiol Heart Circ Physiol 274: H1705-H1714, 1998[Abstract/Free Full Text].

35.   Voelkel, NF, Lobel K, Westcott JY, and Burke TJ. Nitric oxide-related vasoconstriction in lungs perfused with red cell lysate. FASEB J 9: 379-386, 1995[Abstract/Free Full Text].

36.   Wilson, PS, Khimendo P, Moore TM, and Taylor AE. Perfusate viscosity and hematocrit determine pulmonary vascular responsiveness to NO synthase inhibitors. Am J Physiol Heart Circ Physiol 270: H1757-H1765, 1996[Abstract/Free Full Text].

37.   Wilson, PS, Thompson WJ, Moore TM, Khimenko PL, and Taylor AE. Vasoconstriction increases pulmonary nitric oxide synthesis and circulating cyclic GMP. J Surg Res 70: 75-83, 1997[ISI][Medline].

38.   Winterbourn, CC, and Stern A. Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical. J Clin Invest 80: 1486-1491, 1987.

J APPL PHYSIOL 88(5):1520-1528
8570-7587/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
A. Godecke
On the impact of NO-globin interactions in the cardiovascular system
Cardiovasc Res, February 1, 2006; 69(2): 309 - 317.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Deem, S. S. Kim, J.-H. Min, R. Eveland, J. Moulding, S. Martyr, X. Wang, E. R. Swenson, and M. T. Gladwin
Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin
Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2561 - H2568.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. Deem, J.-U. Kim, B. N. Manjula, A. S. Acharya, M. E. Kerr, R. P. Patel, M. T. Gladwin, and E. R. Swenson
Effects of S-Nitrosation and Cross-Linking of Hemoglobin on Hypoxic Pulmonary Vasoconstriction in Isolated Rat Lungs
Circ. Res., October 4, 2002; 91(7): 626 - 632.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. DEEM, M. T. GLADWIN, J. T. BERG, M. E. KERR, and E. R. SWENSON
Effects of S-Nitrosation of Hemoglobin on Hypoxic Pulmonary Vasoconstriction and Nitric Oxide Flux
Am. J. Respir. Crit. Care Med., April 1, 2001; 163(5): 1164 - 1170.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deem, S.
Right arrow Articles by Swenson, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deem, S.
Right arrow Articles by Swenson, E. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online