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Am J Physiol Heart Circ Physiol 279: H2947-H2953, 2000;
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Vol. 279, Issue 6, H2947-H2953, December 2000

Hemoglobin and red blood cells alter the response of expired nitric oxide to mechanical forces

John T. Berg, Steven Deem, Mark E. Kerr, and Erik R. Swenson

Departments of Medicine and Anesthesiology, Veterans Affairs Puget Sound Health Care System and the University of Washington, Seattle, Washington 98108


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expired nitric oxide (NOe) varies with hemodynamic or ventilatory perturbations, possibly due to shear stress- or stretch-stimulated NO production. Since hemoglobin (Hb) binds NO, NOe changes may reflect changes in blood volume and flow. To determine the role of blood and mechanical forces, we measured NOe in anesthetized rabbits, as well as rabbit lungs perfused with buffer, red blood cells (RBCs) or Hb following changes in flow, venous pressure (Pv), and positive end-expiratory pressure (PEEP). In buffer-perfused lungs decreases in flow and Pv reduced NOe, but NOe rose when RBCs and Hb were present. These findings are consistent with changes in vascular NO production, whose detection is obscured in blood-perfused lungs by the more dominant effect of Hb NO scavenging. PEEP decreased NOe in all perfused lungs but increased NOe in live rabbits. The NOe fall with PEEP in isolated lungs is consistent with flow redistribution from alveolar septal capillaries to extra-alveolar vessels and decreased surface area or a direct, stretch-mediated depression of lung epithelial NO production. In live rabbits, increased NOe may reflect blood flow reduction and decreased Hb NO scavenging and/or autonomic responses that increase NO production. We conclude that blood and systemic responses render it difficult to use NOe changes as an accurate measure of lung tissue NO production.

shear stress; pulmonary circulation; positive end-expiratory pressure; pulmonary blood flow; pulmonary venous pressure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VASCULAR ENDOTHELIAL CELLS in culture release nitric oxide (NO) when mechanically deformed or stretched (8) or exposed to flow-induced shear stress (7). NO is present in the expired gas of humans and animals (11), and changes in its concentration have been used to gauge alterations in its production in the lung (2), although the source and contributions (vascular vs. alveolar/bronchial epithelial) to expired NO (NOe) are debated (10, 15). It has been shown that manipulations of hemodynamic parameters and positive end-expiratory pressure (PEEP) alter NOe in anesthetized rabbits and isolated lungs (5, 6, 13, 20). However, blood was present in those studies and, since hemoglobin (Hb) avidly binds NO (14, 21), observed NOe changes may arise from changes in blood volume and Hb-NO uptake. These factors, and systemic hemodynamic consequences of PEEP in the live animal could dominate and obscure any direct effect of mechanical forces on NO production by various lung cells.

This study was conducted to determine the changes in NOe in isolated buffer-perfused rabbit lungs in response to mechanical deformation and shear stress associated with alveolar pressure and vascular pressure-flow changes without the NO scavenging effect of Hb. NOe was measured in expired gas of buffer-perfused lungs following changes in perfusate flow, venous pressure (Pv), and end-expiratory pressure. Identical studies were conducted in lungs with red blood cells (RBC) or Hb in the perfusate. If responses in live animals and blood-perfused lungs (5, 6, 13, 20) result from changes in capillary Hb content, blood flow, or distribution, then we reasoned that blood-free perfusion would more accurately reflect the lungs' responses in vivo to mechanical forces. We show that mechanical forces do alter NO production as measured by NOe, but these changes cannot be accurately determined with blood perfusion in the isolated lung or in the live animal.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A total of 31 New Zealand White rabbits (Western Oregon Rabbitry, Philomath, OR), weighing 3.0 to 3.5 kg, were used in this study. The Animal Care Committee of the Seattle VA Medical Center approved all procedures.

Surgical preparation. Animals were anesthetized by ear-vein injection of ketamine (15 mg/kg) and xylazine (0.33 mg/kg). Following cannulation of the carotid artery and trachea, rabbits were ventilated (21% O2-5% CO2-balance N2) at a rate of 20 breaths/min and a tidal volume of 14 ml/kg and peak airway pressure (Paw) <20 cmH2O. Tidal volume and ventilatory rate were not changed during the experiment. Rabbits were then heparinized (1,000 U via ear vein) and exsanguinated immediately before medial sternotomy and in situ placement of pulmonary artery and left atrial cannulas. The pulmonary circulation was then perfused with Krebs-Henseleit 4% Hetastarch buffer (buffer; pH 7.4 at 38°C) through an open circuit connected to a Masterflex pump (Cole-Parmer, Barrington, IL) with a circuit volume of 270 ml and a constant flow rate of 120 ml/min. Pulmonary artery pressure (Ppa) and venous pressure (Pv) were continuously monitored via saline-filled polyethylene tubing (PE-90) positioned at the respective inflow or outflow sites of each cannula. Pv was maintained at 5 mmHg by the adjustment of the height of the venous reservoir. PEEP and Paw were measured at the juncture of the ventilator circuit and tracheal tube. Blood (perfusate) gases were also determined periodically, and sodium bicarbonate was added if needed to maintain pH within prescribed limits (7.40-7.45).

Measurement of NO. Mixed NOe was measured with a chemiluminescence detector (model 270B; Sievers Instruments, Boulder, CO) by continuous sampling from a 50-ml reservoir placed in the expired gas line (9, 19). The sample flow rate was 120 ml/min, with a fixed minute ventilation of 800 ml/min. Calibration was done using NO-free room air (<0.5 ppb) as a zero reference and a certified tank of NO (6.5 ppb; Air Liquide, Long Beach, CA) at a sampling rate of 120 ml/min as the second reference point. NOe measurements at baseline were averaged over 1 min just before a vascular or PEEP manipulation (each described below) was initiated. The resultant NOe changes following these challenges were averaged over the last minute of the 5-min challenge.

Preparation of RBCs and Hb. The RBCs were filtered through a high-efficiency leukocyte filter (Pall Biomedical, Fajardo, PR) to remove leukocytes and then added to the buffer-perfusion circuit (groups B and C). Bovine Hb (2.64 g; Sigma) was dissolved in 39 ml distilled water (DW), reduced with sodium dithionate (0.28 g in 1 ml DW) and dialyzed overnight against nitrogen and EDTA (60 mg/6 l DW) at room temperature. Hb concentration was then determined (Co-oximeter, model 682; Instrumentation Laboratory, Milan, Italy), and aliquots were stored with sodium dithionate (3 mg/4 ml Hb) at -10°C for up to 3 wk before use (group D).

Experimental protocol. Isolated lung preparations for all experimental protocols were prepared and perfused with buffer as described above. Following initiation of perfusion, baseline measurements were taken for Ppa, Pv, Paw, NOe, perfusate temperature, and blood gases (pH, PCO2, PO2, and HCO3-). A 20-min period was then allowed for stabilization. At this point, perfusates were adjusted to establish one of four different experimental groups: group A (n = 8) buffer perfused (no change in perfusate); group B (n = 5) 1% hematocrit (Hct) perfused (1% RBCs added to perfusate); group C (n = 5) 5% Hct perfused (5% RBCs added to perfusate); and group D (n = 5) Hb perfused (0.75 mg Hb/ml perfusate). A further 10-min period of stabilization followed the Hb or RBC addition. At this point blood gas and baseline measurements were taken in all groups. All experimental manipulations were therefore started at ~30 min following completion of surgical preparation.

Application of mechanical forces. In one sequence of manipulations the responses of NOe to changes in perfusate flow (Q) were determined. Baseline Q values were determined at Q = 120 ml/min, then Q was reduced to 30 ml/min for 5 min, and measurements were repeated. At the 5-min time point the perfusion pump was turned off for 5 min, and measurements were again taken (Q = 0). Flow was then restored to 120 ml/min for 10 min and values recorded. After 10-min stabilization, the pump was again turned off, but this time the pulmonary artery and venous perfusion lines were simultaneously clamped [double occlusion (DO)] so that microvascular transmural pressure remained unchanged. After 5 min, measurements were again taken (Q = 0, DO) and flow was returned to 120 ml/min.

A second set of mechanical manipulations involved subjecting the lung to changes in Pv while flow remained constant. The initial step in this sequence involved lowering the venous reservoir so that Pv = 0 mmHg. After 5 min, measurements were taken, then the reservoir was raised until Pv = 10 mmHg. Again, measurements were taken after 5 min and the reservoir returned to its initial position where Pv = 5 mmHg.

A final hemodynamic manipulation involved application of PEEP. After stabilization the lung was subjected to 10 cmH2O PEEP for 5 min, and NOe was measured. PEEP was then removed, and the lung was allowed to stabilize for 10 min before final measurements were taken.

In eight separate anesthetized rabbits, NOe was measured while the animal was alive before and after applying 10 cmH2O PEEP for 5 min. After these measurements, rabbits were heparinized and prepared for isolated lung perfusion as described above. Once the buffer-perfused lung was stable, NOe was measured with and without perfusate flow and with and without 10 cmH2O PEEP. Then RBCs were added to the perfusate to yield a Hct of 5%, and all four combinations of flow and no flow and PEEP and no PEEP were repeated.

Statistical applications. All within-group comparisons were done using a paired t-test. All intergroup comparisons were conducted using one-way ANOVA and (if significance achieved) the Student-Newman-Keuls Test (Primer Statistical Package, McGraw-Hill, Version 3.0). Statistical significance was accepted with P < 0.05. All values are expressed as means ± SE, with n = number of data points within a group.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of perfusate composition on baseline levels of NOe and Ppa. Addition of RBCs or Hb to the perfusate of isolated rabbit lungs caused concentrations of NO to decrease in the expired gas. Baseline levels (in parts per billion, ppb) of NOe for rabbits perfused with buffer were 64.7 ± 4.9 ppb (n = 8); for 1% RBC perfusate, 33.4 ± 5.9 ppb (n = 5); for 5% RBC, 19.2 ± 3.2 ppb (n = 5); and for rabbit lungs perfused with buffer containing 0.75 mg Hb/ml, 10.6 ± 0.9 ppb (n = 5). In two sets of 5% RBC-perfused lungs, flushing with buffer at the end of the experiment caused NOe to increase (from 17 to 31 ppb in one set, and from 9 to 32 ppb in the second set). All differences between groups (except for the comparisons between 5% RBCs vs. Hb or 1% RBCs) were significantly different (P < 0.05, ANOVA). Because of intergroup differences in baseline levels of NOe, we selected to analyze absolute changes in NOe following response to a given variable.

Increases were also observed in Ppa following addition of RBCs or Hb to the perfusate. Addition of 1% RBCs to the perfusate caused Ppa to increase by 0.4 ± 0.2 mmHg (P = 0.178, paired t-test; n = 5); 5% RBCs caused Ppa to increase by 1.2 ± 0.4 mmHg (P = 0.033; n = 5); and Hb addition produced a 2.8 ± 0.7 mmHg increase in Ppa (P = 0.019; n = 5).

Effects of flow changes and perfusate composition on NOe. When lungs were perfused with buffer, a decrease in flow reduced NOe (Fig. 1); with flow reduced to 30 ml/min, NOe decreased by 4.8 ± 1.1 ppb (P < 0.05), and when flow was stopped, NOe decreased by 12.3 ± 1.0 ppb (P < 0.05). In contrast, when RBCs or Hb were present in the perfusate, NOe increased when flow was reduced or stopped, but none of the respective values for the three groups (1% RBC, 5% RBC, or free Hb) differed significantly from each other (Fig. 1). NOe responses to reducing or stopping flow were reversed in all lungs when flow was resumed. Figure 2 gives a representative example of the changes with flow cessation and resumption in a lung perfused with buffer and 5% Hct. It shows the rapid speed at which the NOe is altered and that the value averaged in the last minute of the 5-min challenge represents a new steady state. In two sets of rabbit lungs, RBCs were flushed from the perfusate at the end of the experiment and the lung was subjected to flow reduction (120 to 0 ml/min). The NOe response in both sets of lungs was similar to that observed in other buffer-perfused lungs, and the directional change was opposite to that observed earlier in each rabbit when RBCs were present.


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  		Fig. 1.   Effect of reducing flow rate on expired NO (NOe). Flow was reduced from 120 to 30 ml/min (left) or from 120 to 0 ml/min (right). Values are means ± SE (n = 5-8 animals per group). Hct, hematocrit. *P < 0.05, change from baseline value. aP < 0.05 compared with blood- or hemoglobin (Hb)-perfused lungs.



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  		Fig. 2.   A representative example of the opposite directional changes in NOe with flow cessation and resumption (turning pump off and on) in a lung perfused with buffer and a 5% Hct; ppb, parts per billion.

In addition to stopping flow by turning off the pump (data presented above), flow was also stopped by DO (Fig. 3). In buffer-perfused lungs, stopping flow by DO resulted in a significant fall in NOe that was similar to that seen when flow was stopped by turning off the pump (Fig. 3). In comparison, in RBC- or Hb-perfused lungs DO resulted in a smaller increase in NOe than did turning off the pump (Fig. 3). In all cases, the effect of flow interruption on NOe, whether by turning off the pump or by DO, was rapid, and a plateau in NOe was evident by the fourth minute of flow changes.


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  		Fig. 3.   Effect of flow cessation on NOe. Flow was stopped by either turning off the perfusion pump (pump off) or by simultaneous clamping of arterial and venous cannula (double occlusion). Values are means ± SE (n = 5-8 animals per group). *P < 0.05 change from baseline value. aP < 0.05 compared with blood- or Hb-perfused lungs.

Effects of Pv and perfusate composition on vascular hemodynamics and NOe. Increased Pv raised Ppa. For example, increasing Pv by 5 mmHg in buffer-perfused rabbit lungs (from 5 to 10 mmHg) caused Ppa to increase by 2.3 mmHg (from 10.5 ± 1.0 to 12.8 ± 0.6 mmHg, n = 8). The pressure increase of only 2.7 mmHg (5-2.3 mmHg) rather than 5 mmHg suggests an increase in microcirculatory blood volume as a consequence of increased Pv. With decreasing Pv (from 5 to 0 mmHg), the opposite occurred: Ppa decreased from 10.5 ± 1.0 to 9.6 ± 1.2 mmHg (n = 8).

Figure 4 shows that Pv changes caused opposite NOe responses in buffer- and RBC- or Hb-perfused lungs. Reducing Pv from 5 to 0 mmHg resulted in a small fall in NOe in buffer-perfused lungs and a small increase in RBC- or Hb-perfused lungs. In contrast, increasing Pv from 0 to 10 mmHg resulted in an increase in NOe in buffer-perfused lungs and decreases in RBC- or Hb-perfused lungs. The latter changes were all significantly different from baseline (P < 0.05). In all cases, the effect of Pv changes on NOe was rapid, and a plateau was evident by the fourth minute.


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  		Fig. 4.   Effect of changing venous pressure (Pv) on NOe. Pv was reduced from 5 mmHg to 0 (left) or was increased from 0 to 10 mmHg (right). Values are means ± SE (n = 5-8 animals per group). *P < 0.05 change from baseline value. aP < 0.05 compared with blood- or Hb-perfused lungs.

Effects of PEEP on Ppa and NOe. Application of PEEP produced an elevation in Ppa. In buffer-perfused lungs 10 cmH2O PEEP increased Ppa by 5.2 ± 1.0 mmHg (n = 6). NOe decreased following application of PEEP, and these changes were significant (P < 0.05) in all groups (Fig. 5). In all cases, the effect of PEEP on NOe was rapid, and a plateau in NOe was evident by the fourth minute.


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  		Fig. 5.   Effect of positive end-expiratory pressure (PEEP) on NOe in isolated lungs. Values are means ± SE (n = 5-8 animals per group). *P < 0.05 change from baseline value.

Effects of PEEP and flow changes on NOe in lungs studied in vivo and ex vivo. Identical to the response of NOe to PEEP reported above (Fig. 5), application of 10 cmH2O PEEP in this series of lungs also reduced NOe in buffer- and 5% RBC-perfused lungs (Fig. 6). Interestingly, before the lungs in these rabbits were isolated, PEEP in vivo caused NOe to increase (Fig. 6), but in contrast to the time-course response in the isolated perfused lung there was no evident plateau in the rising NOe with PEEP at the fifth minute. Also, similar to the results above (Fig. 1), flow cessation caused NOe to decrease in buffer-perfused lungs and increase in 5% RBC-perfused lungs. In buffer-perfused lungs the NOe response to combined PEEP and flow cessation was greater than the response to either perturbation alone. However, in 5% RBC-perfused lungs the response to PEEP and flow cessation was similar to the effect of PEEP alone.


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  		Fig. 6.   Effect of PEEP on NOe in the lungs of live rabbits, whose lungs were then studied in isolation for their responses to PEEP and flow interruption, with and without red blood cells in the perfusate. Values are means ± SE (n = 5-8 animals per group). *P < 0.05 change from baseline value. aP < 0.05 compared with buffer-perfused lungs receiving 10 cmH2O PEEP or flow cessation + 10 cmH2O PEEP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The most significant findings in the present study are that mechanical forces, in the form of vascular flow and pressure changes, and alveolar pressure changes alter NOe in blood-free (buffer-perfused) rabbit lungs. Reducing flow or Pv in buffer-perfused lungs decreased NOe, and increasing Pv increased NOe (Figs. 1 and 4). Interestingly and importantly, addition of RBCs or Hb reversed the polarity of NOe responses to changes in vascular pressure and flow observed in the buffer-perfused lungs. Finally, increased lung volume and higher mean alveolar pressure caused by PEEP reduced NOe in both buffer- and Hb/RBC-perfused lungs but had the opposite effect in live anesthetized animals. These findings will be discussed in light of changes in perfused vascular volume, vascular shear stress and alveolar stretch, Hb-NO affinity, and nonpulmonary systemic factors in the live animal.

Effects of experimental maneuvers on the lung vasculature and parenchyma. The effects of the experimental hemodynamic maneuvers on the pulmonary microcirculation can be summarized as follows. 1) A reduction in perfusate flow causes derecruitment and/or underfilling of pulmonary capillaries, a decrease in blood volume in close contact with alveolar air, and a decrease in shear stress in the pulmonary vascular endothelium. 2) Turning off the perfusion pump causes capillaries to empty, whereas DO (simultaneous arterial and venous clamps) allows them to remain filled. Both maneuvers stop flow and eliminate shear stress, but the DO maintains more stretch. 3) Increasing and decreasing Pv causes capillary blood volume to increase and decrease, respectively. 4) PEEP compresses alveolar septal capillaries and diverts blood to nonseptal corner vessels while also increasing lung volume, alveolar surface area, and mean alveolar pressure. 5) PEEP in the live animal reduces cardiac output and may cause arterial hypotension with strong autonomic nervous system responses.

Interpretation of observations. The observation that addition of RBCs or Hb to the perfusate of isolated perfused lungs decreases NOe and increases Ppa confirms previous reports by our laboratory (9). The explanation is that Hb-binding of NO reduces the amount available for pulmonary vascular relaxation.

Observations related to flow changes. NOe is flow dependent in buffer-perfused lungs as demonstrated by a progressive but reversible reduction in NOe when flow is reduced or stopped (Figs. 1-3). The results presented in Fig. 3 suggest that flow-mediated shear stress is a more important determinant than vessel stretch in the NOe response, because the addition of maintained vessel stretch with the DO technique of flow interruption was equal to the effect of turning off the pump alone, in which vessel stretch diminishes as the blood vessels empty.

Our results, however, differ from Carlin et al. (5), who subjected buffer-perfused rabbit lungs to experimental flow interruption and observed no change in NOe with cessation of flow. A possible explanation is that those lungs were ventilated with 35% oxygen (rather than the 21% O2 used in the present study), and baseline values for NOe were considerably higher. Accordingly, the higher levels of NOe may have obscured changes that were apparent at lower concentrations of NOe. Another possibility is that these investigators pretreated the lungs with indomethacin, which, like other prostaglandin synthase inhibitors and products, are known to alter NOe and in vivo NO metabolism (23).

The fact that NOe decreases in buffer-perfused lungs when flow is reduced may be explained by two potential mechanisms. The first possibility relates to stimulation of NO production by pulmonary vascular endothelial cells in response to physiological shear stress. Increases have been observed in cultured endothelial cells (7) and inferred in blood- or buffer-perfused rat lungs following exposure to increased shear stress (4, 25). Nevertheless, other data suggest that vascular stress is not an important determinant of NO production during buffer perfusion. In isolated, perfused rat (22, 24) and rabbit (18) lungs, inhibition of NO synthase by L-arginine analogs had no statistically significant effect on pulmonary vascular resistance or pressure-flow relationships unless there was a perfusate of high viscosity or RBCs were present. However, it is possible that the small changes in NOe with changes in flow we observed are of insufficient magnitude to alter vascular resistance when tone is low, as during buffer perfusion.

A second possible mechanism to explain the observed flow-associated changes in NOe in buffer-perfused lungs (Fig. 1) relates to the effect of flow on blood volume. Pulmonary blood volume varies directly with changes in flow when Pv is held constant (16). In our model, a reduction in perfusate flow therefore might result in vascular derecruitment and a decrease in perfused surface area, across which vascularly produced NO can diffuse into alveolar gas. If lung vascular endothelial cell NO production is flow dependent, then a reduction in total perfused vascular surface area could cause a reduction in NOe, as we observed (Fig. 1). Even though the lung was placed in a zone 3 condition, which likely ensured full capillary recruitment, a reduction in capillary distension (i.e., volume) with reduction in flow would reduce surface area.

The observed opposite increases in NOe with flow reduction in RBC- and Hb-perfused lungs (Fig. 1) is best explained by the decreased amount of Hb scavenging of NO from alveolar gas subsequent to vascular underfilling and derecruitment. This observation may have physiological significance. It is known that addition of NO to inspired gas decreases pulmonary vascular resistance and presumably increases capillary blood volume. The observed increase in NO excretion in blood-perfused lungs during flow reduction may therefore reflect physiological events that occur within areas of the lung that become subjected to reduced blood flow. Increased alveolar and small airway NO concentration in these areas (resulting from a decreased amount of Hb to bind NO) would vasodilate adjacent vessels and increase blood flow to that area. If true, the observed association between flow and NOe suggests that Hb-binding of NO may participate in regulation of regional blood flow distribution.

Observations related to Pv. An increase in Pv at constant flow in buffer-perfused rabbit lungs caused NOe to increase, and a decrease in Pv caused NOe to decrease (Fig. 4). This observation is consistent with Pv-related changes in vascular volume and associated changes in alveolar capillary area, presumably by small vessel and capillary distension. Similar to the responses with flow changes (Fig. 1), NOe responses to changes in Pv were reversed by addition of RBCs or Hb to the perfusate. This observation can be explained by the dominant effect of Hb binding NO in the face of increased vascular volume.

Observations related to PEEP. PEEP affects both lung volume and flow in the pulmonary microvasculature so that interpretation of NOe responses is more difficult. In the current study, 10 cmH2O PEEP in isolated lungs decreased NOe independent of perfusate composition (Fig. 5). In contrast, Carlin et al. (6) observed small increases (~4 ppb) in NOe after PEEP was raised to 5 or 10 cmH2O in blood-perfused (Hct = 18%) isolated rabbit lungs. However, similar to their other study (5), these lungs were ventilated with 35% O2 and pretreated with indomethacin, and thus results may have differed because of these protocol differences.

Persson et al. (13) and Bannenberg and Gustafsson (3) applied graded PEEP to anesthetized live rabbits and guinea pigs ventilated with 21% oxygen. They found dose-dependent increases in NOe and interpreted their data to support the concept that airway and alveolar stretch with PEEP increases NO production, analogous to the stretch-mediated NO release of vascular endothelial cells (8). In the subset of rabbits we studied, in which the NOe responses to PEEP were measured first in vivo and then in the isolated perfused lung (Fig. 6), we confirm the PEEP-associated increase in NOe in the live animal. Yet in these same lungs studied within 15 min ex vivo, the same PEEP application reduced NOe. Our data thus do not support a stretch-mediated increased NO production by PEEP, since we failed to observe it in the isolated lung. Interestingly, Persson et al. (13) found that vagotomy and lung denervation reduced the PEEP-induced increases in NOe in live rabbits by 50%, and in a more recent study, the same group demonstrated in live rabbits and the lungs in isolation that epinephrine infusion rapidly increases NOe (2). Since PEEP of 7-10 cmH2O reduces cardiac output and causes marked hypotension in live rabbits (13), an increase in NOe in the live animal is better explained by a combination of 1) reduced lung blood flow and less alveolar NO uptake by Hb, 2) increased autonomic traffic via lung vagal efferent nerves, and 3) increased circulating epinephrine with hypotension.

In the isolated lung devoid of innervation and systemic autonomic neurohumoral influences, PEEP reduces NOe possibly by compressing alveolar septal capillaries and diverting flow to extra-alveolar vessels and reducing effective surface area for NO diffusion into alveolar gas. Another explanation may be that rates of NO synthesis may be less in extra-alveolar vessels, similar to the marked heterogeneity of vascular responses (including those involving NO) in vessels in different regions of the lung (12) and along the longitudinal length of lung blood vessels (17). Last, it is possible that alveolar and airway epithelia behave differently in response to stretch with PEEP with regard to NO production than vascular cells. To date, the only evidence for stretch-mediated NO release comes from vascular studies; such studies in airway or alveolar cells need to be performed.

Summary. We examined effects of changes in perfusate blood composition and flow, Pv, and PEEP on NOe levels in isolated rabbit lungs and of PEEP in the live rabbit. We found significant and reproducible changes in NOe in buffer-perfused lungs following manipulation of hemodynamic parameters (flow or Pv) that are most consistent with shear-stress stimulation of vascular NO production and/or changes in perfused capillary surface area. Both the magnitude and direction of these changes are altered by RBCs or Hb in the perfusate. In isolated lungs PEEP reduces NOe either by suppression of NO production or changes in effective surface area available for NO diffusion out of the vasculature. However, in live animals the effects of PEEP on systemic hemodynamics and lung blood flow increase NO production independent of mechanical stresses. Therefore, we conclude, in blood-perfused lungs or live animals, NOe cannot be taken as an accurate reflection of lung NO production, and changes in NOe must be interpreted cautiously in the context of blood or Hb-containing lung perfusates.


    ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health Grant HL-45571.


    FOOTNOTES

Address for reprint requests and other correspondence: J. T. Berg, Pulmonary Research Laboratories, 151-L, VA Medical Center, 1660 S. Columbian Way, Seattle, WA 98108 (E-mail: jtberg{at}u.washington.edu).

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. Section 1734 solely to indicate this fact.

Received 24 February 2000; accepted in final form 27 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(6):H2947-H2953


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