What leads to different mediators of alkalosis-induced vasodilation in isolated and in situ pulmonary vessels?

John B. Gordon1, Michele A. VanderHeyden1, Ted R. Halla1, Edmundo P. Cortez1, Guillermo Hernandez1, Steven T. Haworth2, Christopher A. Dawson2, and Jane A. Madden3

Departments of 1 Pediatrics (Critical Care), 2 Physiology, and 3 Neurology, Medical College of Wisconsin and Zablocki Veterans Administration Medical Center, Milwaukee, Wisconsin 53226


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We previously found that nitric oxide synthase (NOS) inhibition fully blocked alkalosis-induced relaxation of piglet pulmonary artery and vein rings. In contrast, NOS inhibition alone had no effect on alkalosis-induced pulmonary vasodilation in isolated piglet lungs. This study sought to identify factors contributing to the discordance between isolated and in situ pulmonary vessels. The roles of pressor stimulus (hypoxia vs. the thromboxane mimetic U-46619), perfusate composition (blood vs. physiological salt solution), and flow were assessed. Effects of NOS inhibition on alkalosis-induced dilation were also directly compared in 150-350-µm-diameter cannulated arteries and 150-900-µm-diameter, angiographically visualized, in situ arteries. Finally, effects of NOS inhibition on alkalosis-induced vasodilation were measured in intact piglets. NOS inhibition with Nomega -nitro-L-arginine fully abolished alkalosis-induced vasodilation in all cannulated arteries but failed to alter alkalosis-induced vasodilation in intact lungs. The results indicate that investigation of other factors, such as perivascular tissue (e.g., adventitia and parenchyma) and remote signaling pathways, will need to be carried out to reconcile this discordance between isolated and in situ arteries.

newborn piglet; pressor stimulus; flow; perfusate; cannulated arteries; isolated lungs; angiography


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RELATIVE SIMPLICITY of isolated pulmonary vessel preparations compared with isolated lungs or intact animals has facilitated delineation of some aspects of pulmonary vasomotor control (10, 16, 18, 19, 21). However, determining which mechanism or mechanisms identified in the more reductionist preparations are relevant to the whole organ or organism can be difficult (5). Pulmonary vascular responses to several physiologically important stimuli, including flow (3, 33), pressure (24), and hypoxia (19, 27, 28, 36, 41, 42), appear to differ between isolated vessel and intact lung preparations. In addition, we found that the mediators of alkalosis-induced pulmonary vasodilation differ between isolated and in situ vessels. In preconstricted pulmonary artery (10) and vein (16) rings, alkalosis-induced relaxation could be accounted for completely by the nitric oxide-cGMP (NO-cGMP) pathway. In contrast, in hypoxic piglet lungs, nitric oxide synthase (NOS) inhibition alone had no effect on alkalosis-induced pulmonary vasodilation (38). Instead, combined inhibition of NOS, cyclooxygenase, and calcium-dependent potassium (KCa) channels was required to block vasodilation. These data suggest a need for an isolated vessel preparation that more closely mimics in situ vessels to better delineate mechanisms underlying pulmonary vasomotor responses that are relevant to the whole organ.

As a step toward developing such a preparation, we sought to identify factors contributing to the discordant responses of intact lungs and current isolated vessel preparations. We took advantage of the differing effects of NOS inhibition on alkalosis-induced vasodilation seen in our previous studies of isolated vascular rings (10, 16) and lungs (38) to test the hypothesis that differences in pressor stimulus (the thromboxane mimetic U-46619 in isolated vessels vs. hypoxia in lungs), perfusate composition [physiological salt solution (PSS) in isolated vessels vs. blood in lungs], or flow (absent in isolated vessels vs. present in lungs) lead to the discordance between preparations. Because tension measurements made in the relatively large (500-µm-diameter) vascular rings used in our previous studies (10, 16) may not predict the diameter changes of smaller resistance arteries, the effect of NOS inhibition on alkalosis-induced vasodilation was also measured in small (150- to 350-µm external diameter), cannulated pulmonary arteries in this study. In addition, since even these small cannulated arteries are not necessarily those responsible for resistance changes measured in the intact lung, we also measured the effects of NOS inhibition on alkalosis-induced vasodilation of in situ pulmonary arteries in the same diameter range with X-ray angiography. Finally, the effect of NOS inhibition on alkalosis-induced vasodilation in intact piglets was measured to ascertain that in vivo responses to alkalosis and Nomega -nitro-L-arginine (LNA) were similar to those described in isolated lungs (38).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The study was approved by the Institutional Animal Care and Use Committees of the Medical College of Wisconsin and the Zablocki Veterans Administration Medical Center in Milwaukee. A total of 46 piglets was used in the following protocols: intact animals (n = 4), isolated lungs vasoconstricted with U-46619 (n = 4), isolated lungs perfused with a blood-free solution (n = 8), isolated lungs studied at different flow rates (n = 8), isolated lungs studied with angiography to identify in situ artery responses (n = 28 arteries from 6 piglets), cannulated arteries pressurized with blood or blood-free solutions (n = 17 arteries from 12 piglets), and cannulated arteries perfused with PSS to study the effects of flow (n = 4 arteries from 4 piglets). All piglets were initially anesthetized with 40 mg/kg im ketamine, 1.5 mg/kg im acepromazine, and 25 mg/kg ip pentobarbital as previously described (10, 12, 38). Piglets used for isolated lung or vessel studies were then heparinized (5000 units iv) and exsanguinated through a carotid artery catheter. Piglets used for intact animal studies were given an additional dose of 5 mg/kg iv pentobarbital and then 2-4 mg/kg iv every hour for the duration of the study.

Intact piglet protocol. In our previous studies, NOS inhibition alone blocked alkalosis-induced relaxation in isolated vessel rings (10, 16) but had no effect on alkalosis-induced vasodilation in whole lungs (38). This protocol was designed to test the hypothesis that responses of intact animals to NOS inhibition were similar to those of isolated lungs. After anesthesia, a tracheostomy tube was placed, and a carotid artery catheter and a flow-directed pulmonary artery catheter (model 132F5, Baxter Healthcare) were inserted. Piglets were ventilated (model 713, Harvard Apparatus) with a tidal volume of 20 ml/kg (peaked inspiratory pressure 14-18 cmH2O), rate of 20 breaths/min, and initial gas mixture of 28% O2 with 3-4% CO2, to achieve a PaO2approx 140 Torr, PaCO2approx 40 Torr, and pH approx 7.40 (termed normocapnic normoxia in this study). In this protocol and all isolated lung studies, the inhaled and/or exhaled gas mixtures were monitored (model CD3A CO2 monitor and S3A O2 monitor, AEI Technologies) and blood gases measured at each study condition (model 278 blood gas analyzer, Ciba-Corning). After 40-60 min, stable control normocapnic normoxic hemodynamics were measured as described below. The inspired O2 was then reduced to 12% achieving a PaO2approx 40 Torr without altering the PaCO2 and pH. After 15 min, stable normocapnic hypoxic hemodynamics were measured. The inspired CO2 was then reduced to 0%, resulting in a decrease in PaCO2 to ~22 Torr with a concomitant increase in pH to ~7.60. Neither tidal volume nor ventilator rate was altered. After 15 min, stable alkalotic hypoxic hemodynamics were measured. The gas mixture was then returned to the normocapnic normoxic mixture, and 30-100 mg/kg of LNA were infused intravenously over a 10- to 20-min period to block NOS activity. After a further 20-min stabilization period, the sequence of normocapnic normoxia, normocapnic hypoxia, and alkalotic hypoxia was repeated, and hemodynamic measurements were made. Responses to the endothelium-dependent vasodilators acetylcholine (ACh, 10-6 M) or bradykinin (BK 10-8 M) were measured before and after LNA to confirm NOS inhibition. After completion of each experiment, 100 mg/kg of pentobarbital were administered intravenously, and the piglets were euthanized with supersaturated KCl.

The hemodynamic data collected were mean pulmonary artery pressure (Ppa), mean systemic arterial pressure, central venous pressure, pulmonary capillary wedge pressure (PCWP), and cardiac output (CO, measured in duplicate or triplicate) as previously described (12). All pressures were measured in mmHg (Statham Gould model P23id), and CO was measured in milliliters per minute (model 9520, Edwards Laboratories). Cardiac index (CI) was calculated as mean CO/wt and used in subsequent calculations of pulmonary vascular resistance index (PVRI) = (Ppa - PCWP)/CI.

Isolated lung protocols. The isolated lung preparation has been described before (1, 27, 28, 38). Briefly, a tracheostomy tube was placed and the chest opened by a midline sternotomy. The ductus arteriosus was ligated, and the pulmonary artery and left atrium were cannulated. The lungs were then removed from the chest, and the cannulas were attached to a perfusion system. Perfusate exited the left atrium to a reservoir from where it was pumped (Masterflex model 7523-20) through a heat exchanger (Sci Med Pediatric, Sci Med Life Systems) and bubble trap and then into the pulmonary artery. Lungs were ventilated at a tidal volume of ~15 ml/kg (peak inspiratory pressure of 11-16 cmH2O) at a rate of 20 breaths/min. The gas mixtures were 21% O2 for normoxia and 6% O2 for hypoxia with 6-6.5% CO2 for normocapnia (pH ~7.40) or 3% CO2 for alkalosis (pH ~7.60).

Vasoconstriction with U-46619 in lungs. In our previous studies, NOS inhibition fully blocked alkalosis-induced relaxation of vessel rings that were preconstricted with the thromboxane mimetic U-46619 (10, 16) but had no effect on alkalosis-induced vasodilation in isolated lungs that were preconstricted with hypoxia (38). To test the hypothesis that differences in pressor stimulus contributed to this discordance, the effect of NOS inhibition on alkalosis-induced vasodilation was measured in lungs preconstricted with U-46619. Lungs were perfused with a 50:50 mixture of autologous blood and Ringer's lactate containing 3% dextran 70 (hematocrit ~15%). Perfusate flow was gradually increased to 100 ml · kg-1 · min-1 over the first 40 min, and when the Ppa had reached a steady value for >5 min, the stable normoxic normocapnic Ppa was measured. U-46619 was then infused at 0.01 µg · kg-1 · min-1 to induce vasoconstriction during normoxic normocapnia. After 15 min, the Ppa had reached a stable plateau, and we switched the gas mixture to normoxic hypocapnia while maintaining the U-46619 infusion. After 15 min of alkalosis, Ppa reached a steady nadir, and the stable alkalotic Ppa was measured. The U-46619 infusion was then stopped, and the lungs returned to normoxic normocapnia. LNA (10-3 M) was added to the reservoir to block NOS activity as previously described (11, 38), and after 15 min, the stable normoxic normocapnic Ppa was measured again. The U-46619 infusion was then resumed, and a stable increase in normoxic normocapnic Ppa was measured after 15 min. Finally, hypocapnia was resumed, and the alkalotic Ppa was measured during U-46619 infusion. As with the intact piglet protocol, responses to ACh were measured before and after adding LNA to confirm NOS inhibition in several experiments.

Blood-free perfusate in lungs. In our previous studies, NOS inhibition fully blocked alkalosis-induced relaxation of rings that were bathed in PSS (10, 16) but had no effect on alkalosis-induced vasodilation in lungs that were perfused with the autologous blood Ringer's lactate mixture (38). To test the hypothesis that differences in perfusate contributed to the discordant responses, this protocol measured the effect of NOS inhibition on alkalosis-induced vasodilation in lungs perfused blood-free perfusate. Lungs were prepared as described above but were perfused with a blood-free perfusate consisting of Ringer's lactate with 3% dextran 70. At the beginning of each experiment, flow was initiated and perfusate discarded as it exited the left atrium until it was clear and the hematocrit was <0.1%. Recirculation of the perfusate was then initiated and slowly increased to 100 ml · kg-1 · min-1 over 30-40 min. Lungs were divided into control (n = 4) and LNA (10-3 M, n = 4) groups. In both groups, stable normocapnic normoxic Ppa was measured after ~60 min, the lungs were made hypoxic, and the normocapnic hypoxic Ppa was measured after 15 min. Finally, the lungs were made alkalotic, and the alkalotic hypoxic Ppa was measured after 15 min. Responses to ACh were measured to confirm NOS inhibition in LNA-treated lungs.

High and low flow in lungs. In our previous studies, lungs were perfused at a flow rate of 100 ml · kg body wt-1 · min-1 (38). In contrast, vessel rings were bathed in PSS and exposed to no flow. To test the hypothesis that differences in flow may alter the contribution of NO to the response to alkalosis, this protocol measured the effects of alkalosis in control (n = 5) and LNA (10-3 M, n = 3) lungs at high and low flow rates. After 40 min of perfusion at 150 ml · kg-1 · min-1, the gas mixture was switched to normocapnic hypoxia. The flow was set at either 150 ml · kg-1 · min-1 or 50 ml · kg-1 · min-1 while Ppa was maintained similar at either flow rate by raising or lowering the reservoir, thus achieving a higher or lower left atrial pressure (Pla). The effects of alkalosis were then measured by reducing the CO2 to 3%. Lungs were then returned to normocapnic hypoxia, and the other flow rate was initiated. The normocapnic and alkalotic Ppa during hypoxia were then measured again. Because flow rate and Pla differed in this protocol, data are expressed as PVRI, which was calculated as (Ppa - Pla)/flow rate indexed to body weight.

Angiography of in situ arteries in isolated lungs. We previously measured the effects of alkalosis with and without LNA on resistance changes in whole lungs (38) and on tension changes in single, 500-µm-diameter vascular rings (10, 16). To provide a more direct comparison between isolated and in situ arteries, X-ray angiography was used to determine whether NOS inhibition alone blocks alkalosis-induced vasodilation of in situ 150-900-µm diameter arteries in the intact lung. Lungs from six piglets were isolated as described above and perfused with autologous blood with Ringer's lactate and 3% detran 70 mixture. In four of the lungs (2 control, 2 LNA), only the left lobe was perfused (50 ml · kg-1 · min-1), whereas in the other two (2 LNA), the whole lung was perfused at 100 ml · kg-1 · min-1. After perfusion was initiated, lungs were divided into control (n = 2 lungs, 10 arteries) and LNA (10-3 M, n = 4 lungs, 18 arteries). After 40-60 min of ventilation with normocapnic normoxia, Ppa reached a steady pressure, and the stable Ppa was measured. Angiographic images were collected as previously described (1). Briefly, the ventilator was stopped in end expiration, and a bolus of 2-5 ml of nonionic, radiopaque contrast medium, 61% iopamidol (Isovue-300; Bracco Diagnostics, Princeton, NJ), was introduced into the pulmonary artery without altering flow. Digital images (8 bit, 512 × 512) were captured at 30 frames/s with a Silicon Mounted Digital 1M-15 CCD camera and Imaging Technologies IM-PCI frame grabber. U-46619 was then infused at 0.003-0.005 µg · kg-1 · min-1 in control lungs and 0.0015-0.003 µg · kg-1 · min-1 in LNA lungs to cause a small increase in perfusion pressure (2-4 mmHg) and a decrease in artery diameters. After 10 min, a stable Ppa plateau occurred, and angiographic images were again collected. Alkalosis was then induced with 3% CO2, and after 10-15 min, a stable nadir in Ppa occurred, and another bolus was administered. Finally, lungs were returned to normocapnia, and after a stable increase in Ppa was seen, a final bolus was administered. The change in Ppa in response to ACh was then measured to confirm NOS inhibition in the LNA-treated lungs.

The angiographic data were analyzed offline. For each lung, four or five vessels were identified, depending on the number in the X-ray field, and their diameters were measured under the four study conditions using methods previously described (1, 4). Mean responses of all control and LNA arteries under each condition were calculated. In addition, mean responses of LNA arteries <350- or >350-µm-diameter were calculated to determine whether there was any effect of vessel size on the response to LNA in situ.

Isolated vessel protocols. After administering heparin and exsanguinating the piglets, we opened the chests and removed the lungs (10, 16). The lobar pulmonary arteries were identified and traced until small, 150-350-µm external diameter branches (4th-5th generation) were seen. These were carefully dissected, and 2-cm-long segments were attached at each end to glass cannulas. The side branches were then ligated, and the intravascular pressure was raised to 10 mmHg by raising the inflow reservoir as previously described (24). Arteries were initially pressurized and bathed in a PSS solution. The bath PSS was initially bubbled with a 6% CO2 and 21% O2 solution, and vessel diameter was measured with a color video camera (Panasonic Digital 5000) mounted on a stereo microscope (Olympus SZ-STB1) that projected the artery on a video monitor (Sony PVM-1390). The external diameter was measured using a video scaler (FORA-IV 550). Increases or decreases in diameter reflect vasodilation or vasoconstriction, respectively. In all studies, vascular smooth muscle function was first established by documenting vasoconstriction to three challenges with 40 mM KCl, and endothelial function was then documented by demonstrating relaxation to 10-7 M ACh. Vessels failing to demonstrate both vascular smooth muscle and endothelial integrity were excluded from further study.

Cannulated arteries pressurized with PSS or blood. In our previous study, NOS inhibition blocked alkalosis-induced relaxation of 500-µm-diameter artery and vein rings bathed in PSS (10, 16) but not in whole lungs perfused with blood (38). This protocol tests the hypotheses that the size of the vessels, the eccentric tension applied by wires stretching rings, and/or the use of PSS instead of blood may have contributed to the discordant effects of NOS inhibition by measuring the effects of NOS inhibition in small, cannulated, 150-350-µm external diameter arteries pressurized with PSS or blood.

Arteries were divided into two groups: those pressurized with PSS (n = 7 arteries from 5 piglets) and those pressurized with blood (n = 10 arteries from 7 piglets). In all arteries, baseline normocapnic, normoxic diameters were measured, U-46619 was added to the bath to achieve a concentration of 10-8-5 × 10-8 M, and diameters were measured again after a stable constriction had occurred. The gas mixture was then switched from 6% to 2% CO2 with 21% O2, and the vessel diameters were measured during alkalosis. After demonstrating intact alkalosis-induced vasodilation in all arteries, the gas mixture was returned to normocapnic normoxia, and the U-46619 was washed out by replacing the bath solution three times. The stable baseline diameters were again measured, and the arteries were divided into control and LNA (10-4 M) groups. Normocapnic normoxic, normocapnic U-46619, and alkalotic U-46619 diameters were again measured. In addition, responses to 10-7 M ACh were measured at the end of each experiment in the control and LNA arteries.

Arteries with flow. The final protocol further tests the hypothesis that flow contributes to the discordant responses of arteries and lungs seen in our previous studies. The responses to alkalosis were compared with and without 10-4 M LNA in cannulated arteries perfused at a constant flow rate with PSS as previously described by Madden and Christman (23). Arteries (n = 4 from 4 piglets) were cannulated as described above. After verifying smooth muscle and endothelial function with KCl and ACh, flow was initiated at 0.2-0.5 ml/min using a microprocessor-controlled roller pump (Masterflex model 7524-10) while maintaining an intraluminal pressure of 10 mmHg. After measuring the baseline normoxic, normocapnic diameter, we added U-46619 to the bath, and diameter was again measured. The gas mixture was then made alkalotic, and the diameter was measured again. The vessels were then washed, and 10-4 M LNA was added to the bath. The normocapnic, normoxic baseline diameter, U-46619 diameter, and alkalotic diameter were then measured again. NOS inhibition was documented by demonstrating blockade of ACh-induced dilation after the addition of LNA.

Chemicals and solutions. Lungs were perfused with autologous blood mixed with 3% dextran 70 (Sigma) in Ringer's lactate or with the 3% dextran 70 and Ringer's lactate alone. The angiographic studies were performed with 61% iopamidol (Isovue-300). Arteries were bathed in PSS composed of (in mM) 120 NaCl, 4.7 KCl, 1.7 NaH2PO4, 0.72 MgSO4 7 H2O, 2.5 CaCl2 2 H2O, 20 NaHCO3, and 10 glucose. KCl challenges were achieved by modifying the NaCl and KCl in the PSS (80 mM NaCl and 40 mM KCl), thus maintaining isoosmolar conditions. Arteries were pressurized with either PSS or with autologous blood. ACh (Sigma Chemical) was prepared in normal saline, and LNA (Sigma Chemical) was dissolved in normal saline, to which a few drops of 12 N HCl were added, and then the pH was titrated back to ~7.4 with 1 N NaHCO3. U-46619 (Biomol Research Laboratories) was initially prepared in ethanol and then diluted further with normal saline. All drug concentrations are expressed as final molar concentrations in the vessel chamber or perfusion system.

Statistical analysis. All data are expressed as means ± SE. Comparisons within single groups were made by repeated measures ANOVA, and when P < 0.05, the least significant difference (LSD) test was used for post hoc identification of differences between specific conditions. For comparisons among groups, either simple ANOVA or a two-way repeated measures ANOVA was used as appropriate. Again, the LSD was used for post hoc testing when the ANOVA was significant. In addition to raw data comparisons, the percent alkalosis-induced decrease in the pressor response was calculated for the isolated arteries as (diameter during normocapnic U-46619 - diameter during alkalosis U-46619) divide  (diameter during normocapnic U-46619 - diameter during normocapnic baseline conditions) × 100%. All data analyses were performed using SigmaStat statistical software version 2.03 (SPSS, Chicago, IL).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In all protocols, blood gases measured during normocapnia, hypocapnia, normoxia, or hypoxia were similar during their respective control and LNA-treated conditions (Table 1). The concentrations of LNA used have previously been shown to block NOS activity (10, 38), but to demonstrate NOS inhibition in this study, responses to ACh or BK were measured in the 4 intact piglets, in 16 of the isolated lung experiments, and in 17 of the cannulated artery studies. LNA significantly reduced the endothelium-dependent responses in both the intact lungs and isolated vessels (Table 2).

                              
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Table 1.   Blood gases during the different protocols


                              
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Table 2.   Change in Ppa, PVRI, or diameter in response to ACh or BK

Intact piglets. Under control conditions (i.e., before LNA infusion), hypoxia increased Ppa and PVRI, and alkalosis attenuated the hypoxic response (Fig. 1). Infusion of LNA caused a decrease in CI (Fig. 1) and an increase in Ppa and PVRI compared with control conditions (Fig. 1). However, the responses to hypoxia and alkalosis were similar to those occurring under control conditions (Fig. 1). Thus as in hypoxic isolated lungs (38), NOS inhibition alone failed to block alkalosis-induced vasodilation in hypoxic intact piglets.


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Fig. 1.   Effects of hypoxia and alkalosis (ALK) before and after nitric oxide synthase (NOS) inhibition in intact piglets. * Significant increase in pulmonary artery pressure (Ppa) and pulmonary vascular resistance index (PVRI) during normocapnic hypoxia; ** significant decrease in Ppa during ALK; dagger  significant increase in Ppa and PVRI during Nomega -nitro-L-arginine (LNA); dagger dagger significant decrease in cardiac index (CI) during LNA. PCWP, pulmonary capillary wedge pressure.

Effects of pressor stimulus in lungs. Because flow was normalized to body weight and maintained constant throughout the experiments, changes in Ppa reflect changes in PVRI. Under control conditions, U-46619 increased normocapnic Ppa and alkalosis attenuated the pressor response (Fig. 2). The results were essentially the same after adding LNA, although all Ppa was higher than its respective control values (Fig. 2). Thus as in the hypoxic intact piglets (Fig. 1) and our previous study of hypoxic piglet lungs (38), NOS inhibition alone failed to block alkalosis-induced vasodilation in U-46619-treated lungs.


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Fig. 2.   Effects of U-46619 and ALK before and after NOS inhibition in isolated lungs. * Significant increase in Ppa during normocapnic hypoxia; ** significant decrease in Ppa during ALK; dagger  significant increase in Ppa during LNA compared with control conditions.

Effects of blood-free perfusate in lungs. In this protocol, isolated lungs were studied under either control or LNA conditions. Again, flow was constant, so changes in Ppa reflect changes in PVRI. Normoxic Ppa was similar in both groups (Fig. 3). Hypoxia increased Ppa more in the LNA compared with control lungs, but alkalosis attenuated the hypoxic responses similarly in both groups (Fig. 3). Thus NOS inhibition alone did not block alkalosis-induced vasodilation in lungs perfused with a blood-free perfusate.


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Fig. 3.   Effects of hypoxia and ALK in blood-free lungs studied under control or NOS-inhibited conditions. * Significant increase in Ppa during normocapnic hypoxia; ** significant decrease in Ppa during ALK; dagger  significantly higher hypoxic Ppa in LNA compared with control lungs.

Responses of in situ arteries. In this protocol, both Ppa and in situ artery diameters were measured during constant flow in control and LNA-treated lungs. All Ppa were significantly higher in LNA than in control lungs (Fig. 4). In both groups, U-46619 caused a small, but significant, increase in Ppa that was abolished during alkalosis and restored when the lungs were returned to normocapnia (Fig. 4). Baseline normocapnic artery diameters were greater in control than LNA arteries, indicating relative vasoconstriction of the LNA arteries (Fig. 4). In both groups, U-46619 significantly decreased artery diameters, indicating vasoconstriction. This was attenuated by alkalosis and restored when the lungs were returned to normocapnia (Fig. 4). To assess whether the effects of LNA on alkalosis-induced vasodilation differed between larger and smaller arteries, they were divided into >350- or <350-µm-diameter groups. Alkalosis attenuated the vasoconstrictor response to U-46619 in both size ranges of LNA-treated arteries (Fig. 4). Thus NOS inhibition alone did not block the decrease in resistance of whole lungs or the increase in diameter of large and small in situ arteries during alkalosis.


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Fig. 4.   Effects of U-46619 and ALK on Ppa and in situ arterial diameter in isolated lungs. * Significant increase in Ppa or decrease in diameter during normocapnic U-46619 infusion compared with baseline normocapnic conditions.

Responses of cannulated arteries pressurized with PSS or blood. Responses to U-46619 and alkalosis were initially measured under control conditions in all arteries to confirm alkalosis-induced vasodilation (data not shown). They were then randomly assigned to control or LNA groups, and responses to U-46619 and alkalosis were again measured. The fact that baseline diameter was greater in PSS LNA arteries than PSS control arteries (Fig. 5A), but baseline diameter was greater in blood control than blood LNA arteries (Fig. 5C), was a matter of chance since there was either a decrease or no change in diameter in all arteries treated with LNA. In PSS-pressurized (Fig. 5A) and blood-pressurized (Fig. 5C) control arteries, U-46619 decreased vessel diameter, indicating vasoconstriction. This response was attenuated by alkalosis. In contrast, PSS- and blood-pressurized arteries treated with LNA constricted to U-46619 but did not dilate during alkalosis. The % decrease in the U-46619 response in cannulated arteries was significantly reduced by LNA, regardless of whether they were pressurized with PSS or blood (Fig. 5, B and D). Thus in contrast to the in situ arteries (Fig. 4), LNA completely blocked the vasodilator response to alkalosis in the cannulated arteries.


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Fig. 5.   Effects of U-46619 and ALK on cannulated, PSS- (A and B) or blood- (C and D) pressurized artery diameter. * Significant decrease in diameter in response to U-46619; ** significant increase in diameter during ALK. dagger  ALK caused a significantly greater decrease in U-46619-induced constriction in control compared with LNA arteries.

Effects of flow in isolated lungs. Alkalosis attenuated the normocapnic, hypoxic PVRI measured at flow rates of either 50 or 150 ml · kg-1 · min-1 to a similar extent in both the control and LNA lungs (Fig. 6A). Thus NOS inhibition alone did not block alkalosis-induced attenuation of hypoxic PVRI at high or low flow rates in whole lungs.


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Fig. 6.   A: effects of flow on responses to hypoxia and ALK in lungs. Hypoxic PVRI was greater at flow = 50 than 150 ml · kg-1 · min-1 in control lungs (*). ALK reduced hypoxic Ppa at both flow rates in control and LNA lungs (**). B and C: dagger  ALK caused a significantly greater decrease in U-46619-induced constriction in control compared with LNA arteries.

Effects of flow in cannulated arteries. In cannulated, perfused arteries (Fig. 6B), neither the pressor responses to U-46619 nor the dilator responses to alkalosis were as great as in pressurized arteries (Fig. 5, A and C). However, as in the PSS-pressurized arteries, alkalosis decreased the U-46619 response under control conditions, but not after adding LNA (Fig. 6C). Thus as in the arteries studied without flow (Fig. 5), NOS inhibition appeared to block alkalosis-induced vasodilation of cannulated arteries studied with flow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that alkalosis-induced relaxation in piglet pulmonary artery and vein rings was mediated solely by the NO-cGMP pathway (10, 16). Neither cyclooxygenase products nor K+ channel opening contributed to the response (10, 16). These studies and others showing that alkalosis 1) enhanced Ca2+ entry into cultured endothelial cells (39), 2) increased in vitro NOS activity at a pH of 7.50 (8), and 3) enhanced NO synthesis in cultured human pulmonary artery endothelial cells (26) are consistent with the hypothesis that alkalosis-induced vasodilation is mediated by NO. However, in a subsequent study of isolated newborn piglet lungs, we found that NOS inhibition alone failed to alter alkalosis-induced vasodilation (38). Instead, combined inhibition of NOS, cyclooxygenase, and KCa channels was required to block the response in intact lungs (38). The primary goal of the current study was to identify factor(s) contributing to the apparent discordance in mediators of alkalosis-induced vasodilation between isolated and in situ pulmonary vessels.

To this end, we first sought to exclude any effect of pressor stimulus on the discordance. Alkalosis-induced pulmonary vasodilation can be appreciated only when vessels have tone. Therefore, responses have generally been measured after inducing acute vasoconstriction (9, 12, 27, 31, 40). In our previous studies, hypoxia was used to induce acute vasoconstriction in lungs (38), but the thromboxane mimetic U-46619 was used to preconstrict arterial and venous rings (10, 16). Because acute hypoxia has been shown to reduce NOS activity (20) and a decrease in basal NOS synthesis might minimize the effects of NOS activation in response to alkalosis, we hypothesized that the differences in pressor stimuli might contribute to the discordance between preparations. However, in the current study, NOS inhibition failed to inhibit alkalosis-induced vasodilation in lungs preconstricted with either U-46619 (Figs. 2 and 4) or hypoxia (Figs. 1, 3, and 6A). Thus it does not appear that differences in pressor stimulus contributed to the discordance in mediators of alkalosis-induced vasodilation between lungs and vessels.

A second candidate factor that we investigated was the type of perfusate. In our previous study of isolated lungs (38) as well as intact piglets (Fig. 1) and several isolated lung protocols in the current study (Figs. 3, 4, and 6A), lungs were perfused with blood. In contrast, in most isolated vessel studies, including our own, vessels are bathed in PSS (10, 16, 19, 24, 33, 36, 41, 42). Because NO is rapidly inactivated in the presence of red blood cells, we hypothesized that the effects of increased NO synthesis might be more pronounced in preparations bathed or perfused with PSS, thus accounting for the discordance between vessels and lungs. However, despite reducing the hematocrit <1%, NOS inhibition alone failed to block alkalosis-induced vasodilation in blood-free isolated lungs (Fig. 3). Moreover, LNA inhibited alkalosis-induced vasodilation to a similar degree in cannulated arteries pressurized with PSS (Fig. 5, A and B) or blood (Fig. 5, C and D). Thus the presence or absence of blood did not appear to contribute to the discordance in mediators of alkalosis-induced vasodilation between preparations. This observation is consistent with studies of hypoxic pulmonary vasoconstriction in isolated piglet lungs showing that the response was unaffected by hematocrit (15). However, it should be noted that the influence of blood on pulmonary vascular responses may vary with species, because the same investigators found that the vigor of hypoxic pulmonary vasoconstriction did vary with hematocrit in cats and rats (15). Moreover, red blood cells seemed necessary for NO-mediated responses in isolated rabbit lungs (35).

Another candidate factor considered in this study was vessel size and type of isolated vessel preparation. In our previous studies, we examined the mediators of alkalosis-induced relaxation in 500-µm-diameter arterial and venous rings (10, 16). However, isolated vessel ring preparations have been criticized on the basis of: 1) their relatively large size compared with small resistance arteries in the lung, 2) the eccentric resting tension applied to the arteries by wires compared with the circumferential tension applied by intravascular pressure in vivo, and 3) the fact that the PSS bathing the luminal and abluminal sides of rings are the same. In an effort to address some of these criticisms, we measured the effects of NOS inhibition on alkalosis-induced dilation in 150- to 350-µm external diameter, cannulated arteries pressurized with PSS or blood. However, as in the isolated vascular rings (10, 16), LNA completely blocked responses to alkalosis in all of the cannulated artery preparations (Figs. 5, A-D, and 6B). Thus the discordance between isolated vessels and intact lungs does not appear to be related to the type of isolated vessel preparation.

One problem with comparing responses of isolated vessels and whole lungs is that changes in diameter or tension in individual vessels of a particular size range may not reflect the composite effects responsible for resistance changes across the whole lung. Therefore, in this study we measured the effects of alkalosis with and without NOS inhibition on in situ arteries within the intact lung. A lower U-46619 infusion rate was used in this experiment because the venous constriction that occurs with higher doses causes upstream distension of arteries, which complicates interpretation of vessel diameter changes. In contrast to the cannulated arteries (Figs. 5, A-D, and 6B), NOS inhibition had no effect on the vasodilatory effects of alkalosis on either 150-350 or 350- to 900-µm-diameter in situ arteries (Fig. 4). Thus direct comparison of similarly sized isolated and in situ arteries continued to demonstrate markedly different mediators of alkalosis-induced vasodilation.

A final candidate factor considered in this study was the influence of flow on the mediators of alkalosis-induced vasodilation. Because flow increases NO synthesis, we hypothesized that NOS activation associated with alkalosis might not be apparent under conditions of flow. To test this hypothesis, we measured the effects of NOS inhibition on alkalosis-induced vasodilation at high and low flow in isolated lungs (Fig. 6A). As in previous studies by others (14), PVRI was higher at low flow, and NOS inhibition accentuated hypoxic pulmonary vasoconstriction more at high than low flow, indicating that NO synthesis was greater at high flow (Fig. 6A). However, LNA did not attenuate alkalosis-induced vasodilation at either flow rate. To further evaluate the effects of flow, we also measured responses to alkalosis in a perfused cannulated arteries. With or without flow, LNA blocked the response to alkalosis in the cannulated arteries (Fig. 6B). Thus flow does not appear to be a significant contributor to the discordance in mediators of alkalosis-induced vasodilation between isolated and in situ vessels.

In summary, LNA (30-100 mg/kg in intact piglets or 10-3 M in isolated lungs) failed to attenuate the alkalosis-induced decrease in PVRI in intact piglets or isolated lungs studied under a variety of conditions (Figs. 1-4, and 6A). Nor did it alter alkalosis-induced vasodilation of in situ arteries (Fig. 4). In contrast, 10-4 M LNA completely blocked alkalosis-induced vasodilation in all cannulated artery protocols (Figs. 5, A-D, and 6B). The mediators of endothelium-dependent vasodilation also appeared to differ between isolated and in situ vessels in this study. In arteries, endothelium-dependent vasodilation was completely abolished by LNA (Table 2). In contrast, although vasodilation was significantly reduced by LNA in the isolated lungs and intact piglets, ACh and BK still elicited an ~50% decrease in Ppa or PVRI (Table 2). This residual vasodilator response could indicate that LNA failed to fully block NOS activity in the lungs. However, this seems unlikely since LNA 1) significantly increased normoxic PVRI or Ppa (Figs. 1-4 and 6A), 2) significantly increased responses to hypoxia and U-46619 (Figs. 1-3 and 6A), and 3) abolished the flow-dependent decrease in hypoxic PVRI (Fig. 6A). Moreover, the concentration of LNA used in the current study has been shown to block NOS activity (7, 25, 29) without completely eliminating endothelium-dependent vasodilation, which, at least in some cases, is apparently mediated by prostacyclin or hyperpolarization (6, 17). Other physiologically important stimuli also result in discordant responses between in situ and isolated vessels. For example, increased flow typically causes acute vasodilation in intact lungs due to shear-induced increases in NO and PG synthesis (2, 3, 37), but it causes constriction of cannulated pulmonary vessels (33). Similarly, increased pressure caused dilation of cat lungs but constriction of cannulated arteries (24). Finally, hypoxia (30-50 Torr O2) alone typically causes a brisk and sustained increase in PVRI in the whole lung (27, 28) but requires preconstriction with another agonist and is often biphasic or unsustained in isolated vessels (19, 36, 41, 42).

The current study excluded several factors hypothesized to contribute to the discordant responses of isolated vessels and intact lungs. Neither pressor stimulus, type of perfusate, size, or vessel (in a range of 150- to 900-µm diameter) type of isolated vessel preparation (ring vs. cannulated) nor flow contributed to the differing effects of NOS inhibition on alkalosis-induced vasodilation in vessels and whole lungs. Nor could the anesthetics we used be implicated in the discordance because the same agents were used in all preparations. Several other factors may play a role. For example, perivascular tissue may be disrupted in isolated vessels, and the intact adventitia (13) and/or pulmonary parenchyma (22) may contribute to responses of in situ arteries. Distant signaling along the vascular tree may also contribute to the responses of in situ vessels (30, 32, 34). In addition, rhythmic distension of lungs could result in different mediators than occur in vessels simply bathed in PSS. Further investigation of these and other factors potentially contributing to the discordant responses of current isolated vessel and intact lung preparations is needed to develop an isolated vessel preparation with which to identify physiologically relevant mechanisms underlying pulmonary vasomotor responses.


    ACKNOWLEDGEMENTS

This work was supported by an American Heart Association (Northland Affiliate) Grant-in-Aid awarded to J. B. Gordon and by the Department of Pediatrics of the Medical College of Wisconsin. M. A. VanderHeyden and E. P. Cortez were supported by the Elaine Kohler Fund and the Steigleder Endowment Fund. C. A. Dawson was supported by National Heart, Lung, and Blood Institute Grant HL-19298, the W. M. Keck Foundation, and the Department of Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: J. B. Gordon, Children's Hospital of Wisconsin, Critical Care Section, MS 681, 9000 W. Wisconsin Ave., Milwaukee, WI 53226 (E-mail: jgordon{at}mcw.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.

First published January 24, 2003;10.1152/ajplung.00402.2002

Received 25 November 2002; accepted in final form 15 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bentley, J, Rickaby D, Haworth S, Hanger C, and Dawson C. Pulmonary arterial dilation by inhaled NO: arterial diameter, no concentration relationship. J Appl Physiol 91: 1948-1954, 2001[Abstract/Free Full Text].

2.   Buga, GM, Gold ME, Fukuto JM, and Ignarro LJ. Shear stress-induced release of nitric oxide from endothelial cells grown on beads. Hypertension 17: 187-193, 1991[Abstract].

3.   Chammas, J, Rickaby D, and Guarin M. Flow-induced vasodilation in ferret lung. J Appl Physiol 83: 495-502, 1997[Abstract/Free Full Text].

4.   Clough, A, Krenz G, Owens M, Al-Tinawa A, Dawson C, and Linehan J. An algorithm for angiographic estimation of blood vessel diameter. J Appl Physiol 71: 2050-2058, 1991[Abstract/Free Full Text].

5.   Dumont, JE, Dremier S, Pirson I, and Maenhaut C. Cross signaling, cell specificity, and physiology. Am J Physiol Cell Physiol 283: C2-C28, 2001[Abstract/Free Full Text].

6.   Feddersen, CO, Mathias MM, McMurtry IF, and Voelkel NF. Acetylcholine induces vasodilation and prostacyclin synthesis in rat lungs. Prostaglandins 31: 973-987, 1986[Medline].

7.   Fineman, JR, Heymann MA, and Soifer SJ. Nomega -nitro-L-arginine attenuates endothelium-dependent pulmonary vasodilation in lambs. Am J Physiol Heart Circ Physiol 260: H1299-H1306, 1991[Abstract/Free Full Text].

8.   Fleming, I, Hecker M, and Busse R. Intracellular alkalinization induced by bradykinin sustains activation of the constitutive nitric oxide synthase in endothelial cells. Circ Res 74: 1220-1226, 1994[Abstract].

9.   Gao, Y, Tassiopoulos A, McGraw D, Hauser M, Camporesi E, and Hakim T. Segmental pulmonary vascular responses to changes in pH in rat lungs: role of nitric oxide. Acta Anaesthesiol Scand 43: 64-70, 1999[ISI][Medline].

10.   Gordon, JB, Halla TR, Fike CD, and Madden JA. Mediators of alkalosis-induced relaxation in pulmonary arteries from normoxic and chronically hypoxic piglets. Am J Physiol Lung Cell Mol Physiol 276: L155-L163, 1999[Abstract/Free Full Text].

11.   Gordon, JB, Moreira GA, O'Donnell DC, Aldinger AM, and Tod ML. Relative effects of cyclooxygenase and nitric oxide synthase inhibition on vascular resistances in neonatal lamb lungs. Pediatr Res 42: 738-743, 1997[Abstract].

12.   Gordon, JB, Rehorst-Paea LA, Hoffman GM, and Nelin LD. Pulmonary vascular responses during acute and sustained respiratory alkalosis or acidosis in intact newborn piglets. Pediatr Res 46: 735-741, 1999[Abstract].

13.   Gutterman, D. Adventitia-dependent influences on vascular function. Am J Physiol Heart Circ Physiol 277: H1265-H1272, 1999[Free Full Text].

14.   Hakim, TS. Flow-induced release of EDRF in the pulmonary vasculature: site of release and action. Am J Physiol Heart Circ Physiol 267: H363-H369, 1994[Abstract/Free Full Text].

15.   Hakim, TS, and Malik AB. Hypoxic vasoconstriction in blood and plasma perfused lungs. Respir Physiol 72: 109-122, 1988[ISI][Medline].

16.   Halla, TR, Madden JA, and Gordon JB. Mediators of alkalosis-induced relaxation of piglet pulmonary veins. Am J Physiol Lung Cell Mol Physiol 278: L968-L973, 2000[Abstract/Free Full Text].

17.   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].

18.   Hwa, JJ, Ghibaudi L, Williams P, and Chatterjee M. Comparison of acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed. Am J Physiol Heart Circ Physiol 266: H952-H958, 1994[Abstract/Free Full Text].

19.   Leach, RM, Robertson TP, Twort CHC, and Ward JPT Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries. Am J Physiol Lung Cell Mol Physiol 266: L223-L231, 1994[Abstract/Free Full Text].

20.   LeCras, T, and McMurtry I. Nitric oxide production in the hypoxic lung. Am J Physiol Lung Cell Mol Physiol 280: L575-L582, 2001[Abstract/Free Full Text].

21.   Levy, M, Tulloh RMR, Komai H, Stuart-Smith K, and Haworth SG. Maturation of the contractile response and its endothelial modulation in newborn porcine intrapulmonary arteries. Pediatr Res 38: 25-29, 1995[Abstract].

22.   Lloyd, TC, Jr. Hypoxic pulmonary vasoconstriction: role of perivascular tissue. J Appl Physiol 25: 560-565, 1968[Free Full Text].

23.   Madden, J, and Christman N. Integrin signaling, free radicals, and tyrosine kinase mediate flow constriction in isolated cerebral arteries. Am J Physiol Heart Circ Physiol 277: H2264-H2271, 1999[Abstract/Free Full Text].

24.   Madden, JA, Al-Tinawi A, Birks E, Keller PA, and Dawson CA. Intrinsic tone and distensibility of in vitro and in situ cat pulmonary arteries. Lung 174: 291-301, 1996[ISI][Medline].

25.   McMahon, TJ, Hood JS, Bellan JA, and Kadowitz PJ. Nomega -nitro-L-arginine methyl ester selectively inhibits pulmonary vasodilator responses to acetylcholine and bradykinin. J Appl Physiol 71: 2026-2031, 1991[Abstract/Free Full Text].

26.   Mizuno, S, Demura Y, Ameshima S, Okamura S, Miyamori I, and Ishizaki T. Alkalosis stimulates endothelial nitric oxide synthase in cultured human pulmonary endothelial cells. Am J Physiol Lung Cell Mol Physiol 283: L113-L119, 2002[Abstract/Free Full Text].

27.   Moreira, GA, O'Donnell DC, Tod ML, Madden JA, and Gordon JB. Discordant effects of alkalosis on elevated pulmonary vascular resistance and vascular reactivity in lamb lungs. Crit Care Med 27: 1838-1842, 1999[ISI][Medline].

28.   Peake, MD, Harabin AL, Brennan AJ, and Sylvester JT. Steady-state vascular responses to graded hypoxia in isolated lungs of five species. J Appl Physiol 51: 1214-1219, 1981[Abstract/Free Full Text].

29.   Perreault, T, and De Marte J. Maturational changes in endothelium-derived relaxations in newborn piglet pulmonary circulation. Am J Physiol Heart Circ Physiol 264: H302-H309, 1993[Abstract/Free Full Text].

30.   Rivers, R.J. Remote effects of pressure changes in arterioles. Am J Physiol Heart Circ Physiol 268: H1379-H1382, 1995[Abstract/Free Full Text].

31.   Schreiber, MD, Heymann MA, and Soifer SJ. Increased arterial pH, not decreased PaCO2, attenuates hypoxia-induced pulmonary vasoconstriction in newborn lambs. Pediatr Res 20: 113-117, 1986[Abstract].

32.   Segal, S, and Duling B. Propagation of vasodilation in resistance vessels of the hamster: development and review of a working hypothesis. Circ Res 61: II20-II25, 1987[Medline].

33.   Shimoda, L, Norins N, and Madden J. Flow-induced responses in cat isolated pulmonary arteries. J Appl Physiol 83: 1617-1622, 1997[Abstract/Free Full Text].

34.   Shirai, M, Ninomiya I, and Sada K. Constrictor responses of small pulmonary arteries to acute pulmonary hypertension during left atrial elevation. Jpn J Physiol 41: 129-142, 1991[ISI][Medline].

35.   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].

36.   Teng, GQ, and Barer GR. In vitro responses of lung arteries to acute hypoxia after NO synthase blockade or chronic hypoxia. J Appl Physiol 79: 763-770, 1995[Abstract/Free Full Text].

37.   Van Grondelle, A, Worthen GS, Ellis D, Mathias MM, Murphy RC, 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].

38.   VanderHeyden, M, Halla T, Madden J, and Gordon J. Multiple calcium-dependent modulators mediate alkalosis-induced vasodilation in newborn piglet lungs. Am J Physiol Lung Cell Mol Physiol 280: L519-L526, 2001[Abstract/Free Full Text].

39.   Wakabayashi, I, and Groschner K. Divergent effects of extracellular and intracellular alkalosis on Ca2+ entry pathways in vascular endothelial cells. Biochem J 323: 567-573, 1997[ISI][Medline].

40.   Yamaguchi, T, O'Brien RF, Hanson WL, Wagner WW, Jr, and McMurtry IF. Prostacyclin contributes to inhibition of hypoxic pulmonary vasoconstriction by alkalosis. Prostaglandins 38: 53-63, 1989[Medline].

41.   Yuan, XJ, Tod ML, Rubin LJ, and Blaustein MP. Contrasting effects of hypoxia on tension in rat pulmonary and mesenteric arteries. Am J Physiol Heart Circ Physiol 259: H281-H289, 1990[Abstract/Free Full Text].

42.   Zhao, Y, Packer CS, and Rhoades RA. Pulmonary vein contracts in response to hypoxia. Am J Physiol Lung Cell Mol Physiol 265: L87-L92, 1993[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 284(5):L799-L807