EDITORIAL FOCUS
Hypoxic constriction of porcine distal pulmonary arteries: endothelium and endothelin dependence

Q. Liu, J. S. K. Sham, L. A. Shimoda, and J. T. Sylvester

Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the role of endothelium in hypoxic pulmonary vasoconstriction (HPV), we measured vasomotor responses to hypoxia in isolated seventh-generation porcine pulmonary arteries < 300 µm in diameter with (E+) and without endothelium. In E+ pulmonary arteries, hypoxia decreased the vascular intraluminal diameter measured at a constant transmural pressure. These constrictions were complete in 30-40 min; maximum at PO2 of 2 mmHg; half-maximal at PO2 of 40 mmHg; blocked by exposure to Ca2+-free conditions, nifedipine, or ryanodine; and absent in E+ bronchial arteries of similar size. Hypoxic constrictions were unaltered by indomethacin, enhanced by indomethacin plus NG-nitro-L-arginine methyl ester, abolished by BQ-123 or endothelial denudation, and restored in endothelium-denuded pulmonary arteries pretreated with 10-10 M endothelin-1 (ET-1). Given previous demonstrations that hypoxia caused contractions in isolated pulmonary arterial myocytes and that ET-1 receptor antagonists inhibited HPV in intact animals, our results suggest that full in vivo expression of HPV requires basal release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.

vascular smooth muscle; internal diameter; calcium; acetylcholine; U-46619; potassium chloride


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE LAST DECADE, two fundamentally different hypotheses about the mechanisms of hypoxic pulmonary vasoconstriction (HPV) have emerged. According to one (73), hypoxia inhibits voltage-dependent potassium (KV) channels in plasma membranes of pulmonary arterial myocytes, leading to depolarization, Ca2+ influx through voltage-dependent Ca2+ channels, and contraction. According to the other (50), hypoxia acts on endothelium to stimulate production and release of the potent vasoconstrictor peptide endothelin (ET)-1, which mediates HPV by activating ETA receptors on smooth muscle. Both hypotheses have considerable experimental support. On one hand, hypoxia inhibited KV currents, increased membrane potential and intracellular Ca2+ concentration ([Ca2+]i), and caused contraction in isolated pulmonary arterial smooth muscle cells (7, 8, 15, 42, 47, 53, 54, 56, 70, 82). On the other, ET-1 receptor antagonists blocked HPV in intact animals (23, 24, 50, 71, 78).

The major site of HPV is thought to be small distal pulmonary arteries (1, 21, 59). In myocytes isolated from distal pulmonary arteries of the pig, we found that hypoxia inhibited KV channels, increased [Ca2+]i, and decreased cell length; however, these effects were quite small (58). If the cells were pretreated with a low concentration of ET-1 (10-10 M), which itself had no effect on cell length or [Ca2+]i, the magnitude of hypoxic contraction increased eightfold. Because ET-1 receptor antagonists inhibited HPV in intact animals (23, 24, 50, 71, 78), these results suggested that full expression of HPV in vivo might require release of ET-1 from the endothelium to facilitate mechanisms of hypoxic reactivity in pulmonary arterial smooth muscle.

The purpose of the present study was to test this possibility. First, we characterized the time course, PO2 dependence, and Ca2+ dependence of hypoxic constriction in small [intraluminal diameter (ID) < 300 µm] distal porcine pulmonary arteries and determined whether hypoxic vasoconstriction occurred in bronchial arteries of similar size. Next, we determined whether hypoxic constriction in pulmonary arteries was endothelium dependent and performed experiments to clarify the mechanism of this dependence.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of isolated arteries. Male pigs (20-25 kg) were anesthetized with ketamine (30 mg/kg im) followed by pentobarbital sodium (12.5 mg/kg iv). After the animal was exsanguinated from the femoral arteries, the lungs were rapidly removed and placed in ice-cold Krebs-Ringer bicarbonate (KRB) solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose. Under a dissecting microscope, 1-mm segments of pulmonary arteries (OD 100-150 µm) were isolated from seventh-generation branches in the left upper lobe. Bronchial arteries of similar size were isolated from the outer surface of segmental bronchi. The vessels were placed in a chamber filled with KRB solution, cannulated at one end with a glass micropipette, and secured with 12-0 nylon monofilament suture. KRB solution was infused slowly through the pipette until the artery was completely filled. In some pulmonary arteries, endothelial cells were disrupted by gently rubbing the intraluminal surface with a steel wire (diameter 70 µm). These vessels were then perfused with 2 ml of air bubbles followed by 2 ml of KRB solution (perfusion pressure < 5 mmHg). The other end of the vessel was cannulated with a second micropipette filled with KRB solution. Both cannulas were connected to a reservoir that was adjusted to set the transmural pressure (Ptm) to the desired value. Ptm was measured with a pressure transducer positioned at the level of the vessel lumen. The chamber was covered, and the vessels were superfused at 20 ml/min with recirculating KRB solution (total volume 50 ml) gassed with 16% O2-5% CO2-79% N2 and maintained at 37°C. The same gas mixture flowed over the surface of the superfusate in the covered chamber. To measure the vascular ID, the chamber was placed on the stage of an inverted microscope (Nikon TMS-F) connected to a video camera (Panasonic CCTV camera). The vascular image was projected onto a video monitor, and the ID was determined with a video dimension analyzer (Living Systems Instrumentation, Burlington, VT). ID and Ptm were recorded continuously (Gould, Cleveland, OH).

Experimental protocols. Initially, isolated arteries were allowed to equilibrate for 20 min at a Ptm of 10 mmHg. Ptm was then increased to 20 (pulmonary arteries) or 40 (bronchial arteries) mmHg and held constant. To test viability, the vessels were exposed first to KCl (60 mM) and then to the thromboxane A2 agonist U-46619 (10-8 M) followed by acetylcholine (ACh; 10-6 M). After the responses had stabilized (5-10 min), the agonists were washed out of the chamber by 15-20 min of nonrecirculating superfusion with fresh KRB solution. Vessels in which the ID decreased <30% after KCl and U-46619 were excluded. Endothelium-intact (E+) vessels were excluded if ACh reversed the U-46619 contraction by <50%. These criteria were not applied to E+ pulmonary arteries subjected to Ca2+-free conditions or nifedipine (see below). Endothelium-denuded (E-) vessels were excluded if ACh caused any vasodilation. In some preparations, viability tests were repeated at the end of the experiment.

To expose E+ pulmonary arteries to hypoxia, the O2 concentration of the gas mixture bubbling the reservoir and flowing over the superfusate surface in the vascular chamber was decreased from 16 to 7 (n = 7), 4 (n = 27), or 0% (n = 6) for 30 or 60 min. After hypoxic exposure, O2 concentration was returned to 16%. Arteries continuously exposed to 16% O2 served as time-matched controls (n = 36). Bronchial arteries were exposed to 4% (n = 9) or 16% (n = 5) O2 in a similar manner. Responses were quantified as changes in ID (Delta ID) from the values at the onset of exposure. To determine the degree of hypoxia at the outer surface of the vessels, we measured O2 tension with a microelectrode (Microelectrodes, Londonderry, NH) positioned at the level of the vessel in the chamber. In other experiments, we used a blood gas analyzer (Synthesis 15, Instrumentation Laboratory, Lexington, MA) to measure pH and PCO2 in superfusate samples obtained anaerobically from the chamber.

To evaluate the role of Ca2+ in HPV, we determined the effects of hypoxia (4% O2 for 30 min) in untreated E+ pulmonary arteries and E+ pulmonary arteries exposed to 1) Ca2+-free conditions (Ca2+-free KRB solution containing 10-3 M EGTA; n = 5), 2) the L-type Ca2+ channel antagonist nifedipine (10-6 M; n = 7), or 3) the sarcoplasmic reticulum (SR) Ca2+ channel agonist ryanodine (10-5 M; n = 5). Ca2+-free conditions and nifedipine were instituted before viability was tested to document effects on responses to KCl, U-46619, and ACh. Ryanodine was given after viability testing, and its effects were documented by a bolus injection of caffeine into the reservoir (final superfusate concentration 10 mM). The caffeine was then washed out by 15 min of nonrecirculating superfusion with KRB solution containing 10-5 M ryanodine.

To evaluate the role of the endothelium in HPV, we measured the responses to 4% O2 in 1) E- pulmonary arteries (n = 15); 2) E+ pulmonary arteries treated with the cyclooxygenase inhibitor indomethacin (Indo; 10-5 M; n = 5), indomethacin plus the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME; 3 × 10-5 M; n = 12), or the ETA receptor antagonist BQ-123 (3 × 10-6 M; n = 6); and 3) E- pulmonary arteries pretreated with a low superfusate concentration of ET-1 (10-10 M; n = 13). Responses were compared with those of time-matched control vessels not exposed to hypoxia but otherwise treated similarly (n = 17, 5, 6, 6, and 7, respectively). Exposure to Indo began with superfusion. L-NAME, BQ-123, and ET-1 were given 20-30 min before hypoxia.

Drugs. ACh, Indo, and L-NAME were purchased from Sigma (St. Louis, MO); BQ-123 and ET-1 were from American Peptide (Sunnyvale, CA); U-46619 was from Cayman Chemical (Ann Arbor, MI); and nifedipine, caffeine, and ryanodine were from Calbiochem (La Jolla, CA). Stock solutions of ACh, L-NAME, ET-1, and caffeine were prepared each day in deionized water and stored at 4°C until used. Indo was dissolved each day in an aqueous solution of NaHCO3 (2.35 × 10-2 M). Stock solutions of BQ-123 and nifedipine in ethanol and ryanodine in dimethyl sulfoxide were stored at -60°C and diluted with deionized water before use. Concentrations are expressed as final molar concentrations in the superfusate.

Data analysis. t-Tests and one-, two- or three-factor analysis of variance were used for statistical analysis of the data, taking repeated measures into account when appropriate. When analysis of variance yielded a significant F-ratio, the least significant difference was calculated to permit pairwise comparison among means or Dunnett's test was used to compare means to a control value. P values <=  0.05 were taken to indicate significance. Values are means ± SE. On each experimental day, two vessels from the same animal were subjected to different protocols; therefore, n is the number of both animals and vessels in each group.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Viability tests. Under normoxic conditions at the beginning of the experiment, the ID measured at a Ptm of 20 mmHg (ID20) was greater in E+ than in E- pulmonary arteries (Table 1). KCl and U-46619 caused vigorous constriction in both E+ and E- arteries. ACh reversed constrictions induced by U-46619 in E+ arteries but had no effect in E- arteries. Responses of E+ bronchial arteries to KCl, U-46619, and ACh were similar to those of E+ pulmonary arteries.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Responses of pulmonary and bronchial arteries with and without endothelium to KCl, U-46619, and ACh

Viability tests were repeated under normoxic conditions at the end of the experiment in 18 E+ pulmonary arteries exposed to 4% O2 for 30 min and 13 E+ pulmonary arteries exposed to 16% O2 throughout the experiment (Fig. 1). By the end of the experiment, vasoconstrictor responses to KCl decreased 74% in arteries exposed to hypoxia but only 26% in arteries not exposed to hypoxia. Vasoconstrictor responses to U-46619 did not change in either group. Vasodilator responses to ACh decreased 78% in arteries exposed to hypoxia but were unaltered in arteries not exposed to hypoxia. The effects of hypoxic exposure on vasoconstrictor responses to KCl and U-46619 in E- pulmonary arteries (data not shown) were similar to those in E+ arteries.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Vasomotor responses of isolated pulmonary arteries determined during normoxia (16% O2) at the beginning and end of the experiment. Control arteries were exposed only to 16% O2. Hypoxic arteries were exposed to 4% O2 for 30 min during the middle of the experiment and 16% O2 at all other times. Vasomotor responses are shown as the change in vascular intraluminal diameter (ID) at a transmural pressure of 20 mmHg (Delta ID20) induced by KCl (60 mM), U-46619 (10-8 M), or ACh (10-6 M). ACh was given during stable vasoconstriction induced by U-46619.

Responses of pulmonary and bronchial arteries to hypoxia. Decreasing the O2 concentration from 16 to 7, 4, or 0% for 30 min caused PO2 in the chamber superfusate to decrease rapidly from 114 ± 1 to 49 ± 1, 29 ± 1, and 2 ± 3 mmHg, respectively (Fig. 2A). On reoxygenation with 16% O2, PO2 increased rapidly, reaching 113 ± 2 mmHg at 60 min. When the O2 concentration was maintained at 16% throughout the experiment, PO2 averaged 116 ± 2 mmHg. PCO2 and pH were constant at 33 ± 0.2 mmHg and 7.40 ± 0.003, respectively.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   A: time course of superfusate PO2 in the vascular chamber and Delta ID20 from time 0 in pulmonary arteries exposed to 16, 7, 4, or 0% O2. LSD0.05, least significant difference between the mean values of Delta ID20 at the 0.05 level. B: relationship between mean superfusate PO2 and mean Delta ID20 measured at the end of exposure (30 min). Solid line, least squares fit of the Hill equation to the data. P50, PO2 at which Delta ID20 was half-maximal.

As previously observed (38), ID20 in normoxic E+ pulmonary arteries decreased gradually due to development of intrinsic tone [change in ID20 (Delta ID20) = -13 ± 7 µm at 30 min; Fig. 2A]. Compared with these vessels, arteries exposed to 4 and 0% O2 developed vigorous constriction (Delta ID20 at 30 min = -57 ± 10 and -62 ± 8 µm, respectively; P < 0.0001), whereas arteries exposed to 7% O2 did not (Delta ID20 at 30 min = -23 ± 10 µm; P = 0.999). The relationship between mean Delta ID20 (expressed as a fraction of maximum Delta ID20) and mean PO2 at 30 min appeared to be sigmoid (Fig. 2B). Using least squares iteration to fit these data to the Hill equation (r2 = 0.999996), we estimated that hypoxic vasoconstriction was half-maximal at a PO2 of 40 mmHg. After 30 min of reoxygenation (Fig. 2A), Delta ID20 in arteries exposed to 7 and 4% O2 (-23 ± 10 and -23 ± 10 µm, respectively) returned to the value observed in arteries exposed to 16% O2 throughout the experiment (-25 ± 10 µm); however, arteries exposed to 0% O2 continued to constrict (Delta ID20 = -78 ± 17 µm after 30 min of reoxygenation).

Although 4% O2 elicited vigorous reversible vasoconstriction in pulmonary arteries (P < 0.0001), it had no effect on vasomotor tone in bronchial arteries (P = 0.95; Fig. 3).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Vasomotor tone in pulmonary (PA) and bronchial (BA) arteries during exposure to 4 and 16% O2. Values are expressed as a percentage of ID20 at time 0 (control).

Ca2+ dependence of HPV. Vasoconstrictor responses to KCl were virtually abolished in pulmonary arteries subjected to Ca2+-free conditions or nifedipine (Delta ID20 = -9 ± 6 and -7 ± 2% of control ID20, respectively, compared with -69 ± 2% in untreated arteries; P < 0.0001; Table 1). Vasoconstrictor responses to U-46619 were also reduced but less severely (Delta ID20 = -26 ± 5 and -18 ± 4% of control ID20, respectively, compared with -46 ± 2% in untreated arteries; P < 0.0001; Table 1). Vasodilator responses to ACh were not altered (Delta ID20 = 91 ± 14 and 74 ± 16% of U-46619-induced constrictions, respectively, compared with 112 ± 5% in untreated arteries; P > 0.05; Table 1). Caffeine did not alter the vasomotor tone in arteries treated with ryanodine (Delta ID20 = 11 ± 7%; P = 0.13).

As shown in Fig. 4, exposure to Ca2+-free conditions, nifedipine, or ryanodine eliminated the vasoconstrictor responses to 4% O2 (Delta ID20 at 30 min = 9 ± 4, -6 ± 4 and -2 ± 12 µm, respectively, compared with -57 ± 10 µm in untreated arteries; P < 0.0001). After 30 min of reoxygenation (16% O2), the vasomotor tone in arteries treated with nifedipine or ryanodine (Delta ID20 = -4 ± 5 and -27 ± 27 µm, respectively) was not different from that in untreated arteries (Delta ID20 = -23 ± 18 µm) but was decreased in arteries exposed to Ca2+-free conditions (Delta ID20 = 26 ± 11 µm). Baseline ID20 measured before hypoxic exposure in E+ arteries subjected to Ca2+-free conditions (223 ± 9 µm) or ryanodine (196 ± 17 µm) did not differ from the baseline ID20 in untreated E+ arteries (193 ± 7 µm); however, baseline ID20 in nifedipine-treated arteries was increased (252 ± 21 µm; P < 0.05).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 4.   Vasomotor responses to hypoxia (4% O2) in pulmonary arteries exposed to Ca2+-free conditions (Ca2+-free Krebs-Ringer bicarbonate solution containing 10-3 M EGTA), nifedipine (10-6 M), or ryanodine (10-5 M) and in untreated (control) pulmonary arteries.

Endothelium and endothelin dependence of HPV. As shown in Fig. 5, ID20 in E+ pulmonary arteries exposed to 4% O2 for 60 min decreased relative to ID20 in normoxic E+ arteries (Delta ID20 at 60 min = -71 ± 14 and -25 ± 10 µm, respectively; P < 0.0001). In E- pulmonary arteries, this hypoxic vasoconstriction was abolished (Delta ID20 at 60 min = -4 ± 10 and -6 ± 14 µm in normoxic and hypoxic E- arteries, respectively; P = 0.999). Compared with untreated E+ arteries, ID20 measured before hypoxic exposure in untreated E- arteries was decreased (162 ± 9 µm; P < 0.01).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   Vasomotor tone in pulmonary arteries with (E+) and without (E-) endothelium during 60-min exposures to 4 and 16% O2.

To determine which endothelium-derived factors were responsible for the endothelium dependence of HPV, we exposed E+ pulmonary arteries to 4% O2 for 30 min after treatment with Indo, Indo plus L-NAME, or BQ-123 (Fig. 6). Compared with untreated arteries (Delta ID20 at 30 min = -44 ± 8 µm), hypoxic vasoconstriction was unaltered in arteries treated with Indo (Delta ID20 at 30 min = -40 ± 10 µm; P = 0.9), enhanced in arteries treated with Indo plus L-NAME (Delta ID20 at 30 min = -124 ± 6 µm; P < 0.0001), and abolished in arteries treated with BQ-123 (Delta ID20 at 30 min = -3 ± 4 µm; P = 0.0006). Baseline ID20 measured before hypoxic exposure averaged 198 ± 20, 168 ± 5, and 196 ± 23 µm in E+ arteries treated with Indo, Indo plus L-NAME, or BQ-123, respectively. These values did not differ from baseline ID20 in untreated E+ arteries (193 ± 7 µm).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of indomethacin (Indo; 10-5 M), Indo plus NG-nitro-l-arginine methyl ester (L-NAME; 3 × 10-5 M), and BQ-123 (3 × 10-6 M) on vasomotor tone in E+ pulmonary arteries during exposure to 4 and 16% O2. Data from untreated (control) arteries are shown for comparison.

To further test the role of ET-1 in hypoxic pulmonary vasoconstriction, we "primed" E- pulmonary arteries by exposing them to a low concentration of ET-1 (10-10 M) for 20-30 min before hypoxia (4% O2 for 30 min; n = 13) or continued normoxia (16% O2; n = 7). Responses in these vessels were compared with those of untreated normoxic (n = 17) and hypoxic (n = 15) E- arteries (Fig. 7). During normoxia, ET-1 priming decreased ID20 18 ± 4% compared with a 4 ± 2% increase over a similar time period in unprimed E- arteries (P < 0.0001). Hypoxia did not alter the vasomotor tone in unprimed E- arteries (Delta ID20 at 30 min = -1 ± 5 and -9 ± 5 µm in normoxic and hypoxic arteries, respectively; P = 0.14); however, hypoxic vasoconstriction was restored in E- arteries primed with ET-1 (Delta ID20 at 30 min = -2 ± 9 and -39 ± 8 µm in normoxic and hypoxic arteries, respectively; P < 0.0001).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of endothelin (ET)-1 priming on vasomotor tone in E- pulmonary arteries during exposure to 4 and 16% O2. Primed arteries were pretreated with ET-1 (10-10 M) for 20-30 min before exposure.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HPV has been thoroughly studied in intact animals and isolated lungs. These preparations provide consistent and physiologically relevant responses but impose significant limitations on mechanistic investigation. More reduced preparations permit more precise intervention but may yield inconsistent responses of uncertain relevance (63). In the present study, we used small distal porcine pulmonary arteries to determine whether HPV was endothelium dependent. We chose the pig as our experimental animal because HPV is vigorous in this species (9, 62) and porcine lungs are large enough to permit dissection of small distal pulmonary arteries. We studied seventh-generation pulmonary arteries < 300 µm in diameter because small distal pulmonary arteries are thought to be the major site of HPV (1, 21, 59). We measured changes in vasomotor tone as changes in ID at a constant Ptm because HPV normally occurs locally and does not alter pulmonary arterial pressure and because this measurement may more accurately reflect properties of in vivo arteries in which contraction is neither isometric nor isotonic (17).

As shown in Fig. 2, physiological decreases in PO2 caused vigorous sustained vasoconstriction in our preparation. Relative to the gradual vasoconstriction caused during normoxia by intrinsic tone (38, 40), hypoxic vasoconstriction began 5-10 min after the onset of exposure and appeared to be complete within 30-40 min (Figs. 2A and 5). Maximum HPV decreased ID20 62 µm or ~40% of the constrictor response to 60 mM KCl (Fig. 2A, Table 1). HPV was half-maximal at a PO2 of 40 mmHg (Fig. 2B). In isolated and intact lungs, HPV occurred somewhat more rapidly, requiring 10-15 min to achieve initial plateau responses, which were half-maximal at O2 tensions of 30-60 mmHg (3, 9, 61, 62). In isolated ferret lungs, maximum HPV was 38% of the maximum vasoconstrictor response to KCl (16). Thus, in terms of magnitude, PO2 dependence, and temporal characteristics, hypoxic vasoconstriction in our isolated arteries was similar to that in intact and isolated lungs.

Although severe hypoxia caused sustained vasoconstriction in arteries (Fig. 2, 0% O2), it caused vasoconstriction followed by vasodilation in isolated lungs (62). Vasodilation to severe hypoxia was probably caused by deterioration of the vascular smooth muscle energy state, activation of ATP-dependent K+ (KATP) channels, hyperpolarization, decreased Ca2+ influx through voltage-dependent Ca2+ channels, and relaxation (33, 76, 77). This sequence may have not have occurred in our arteries because unlike in isolated lungs perfused at a constant flow, intraluminal pressures (and, therefore, Ptm) did not increase during HPV, resulting in a lower workload on pulmonary arterial smooth muscle and better maintenance of smooth muscle energy state and tone during severe hypoxia.

Vasoconstriction elicited by severe hypoxia (0% O2) was not reversible in isolated arteries (Fig. 2). Indeed, reoxygenation after 0% O2 caused further constriction. Even in arteries exposed to 4% O2, where HPV was reversible, transient constriction was observed on reoxygenation. The mechanism of this constriction remains to be determined; however, because it occurred with reoxygenation, it may have been mediated by reactive oxygen species (27, 79). Injury due to reactive oxygen species may explain depression of the vasoconstrictor responses to KCl and the vasodilator responses to ACh seen after reoxygenation (Fig. 1). Because vasoconstrictor responses to U-46619 (and, therefore, contractile ability) were unchanged, these results suggest that voltage-dependent Ca2+ channels and NO responsiveness in smooth muscle and/or ACh-induced NO production in endothelium were dysfunctional after reoxygenation. Further studies are needed to confirm these results and determine the underlying mechanisms. Because of the possibility of reoxygenation injury, we utilized single exposures to 4% O2 to elicit HPV in subsequent experiments.

In more proximal pulmonary arteries, hypoxic responses were typically triphasic (early transient contraction followed by relaxation and late sustained contraction) and did not occur unless the arteries were subjected to 0% O2 after some special treatment such as precontraction with vasoactive agonists or prolonged exposure to moderate hypoxia (22, 30, 32, 63). In small (OD < 300 µm) distal pulmonary arteries of the cat, increases in isometric tension caused by decreases in PO2 from >400 to 250, 150, 100, 50, or 20 mmHg were transient, and the peaks of these contractions were half-maximal at a PO2 of 100 mmHg (41). These differences may be attributable to species, arterial size or locus, or method of measurement. Because increases in vasomotor tone were measured as increases in isometric tension, deterioration of energy state may have contributed to hypoxic relaxation in proximal pulmonary arteries (33) and the transience of hypoxic constriction in small distal pulmonary arteries (41). We are unaware of previous attempts to assess hypoxic responses in pulmonary arteries from measurements of ID at a constant Ptm.

HPV in intact animals and isolated lungs and hypoxia-induced increases in [Ca2+]i in pulmonary arterial myocytes were inhibited by L-type Ca2+ channel antagonists (2, 8, 28, 45, 67) and potentiated by agonists of these channels (8, 44, 66), indicating that HPV required influx of extracellular Ca2+ through sarcolemmal L-type Ca2+ channels. Our findings that Ca2+-free conditions or nifedipine prevented HPV in isolated pulmonary arteries (Fig. 4) support this view and demonstrate further similarity between the hypoxic responses of our preparation and those of intact and isolated lungs. It has been suggested that inhibition of HPV by Ca2+ channel antagonists could be due to nonspecific decreases in baseline tone (55). Because baseline ID20 in nifedipine-treated E+ arteries was higher than in untreated E+ arteries, this possibility cannot be ruled out.

Some investigators reported that depletion of Ca2+ stores in the SR with ryanodine and caffeine, which activate SR ryanodine receptors, or thapsigargin and cyclopiazonic acid, which inhibit SR Ca2+ uptake, inhibited hypoxia-induced increases in myocyte [Ca2+]i (15, 56, 70). Our observations that ryanodine blocked HPV in isolated pulmonary arteries are consistent with these findings and support the proposal that HPV is triggered by release of SR Ca2+, which then acts on sarcolemmal ion channels to cause depolarization and Ca2+ influx through voltage-dependent Ca2+ channels (15, 52). Alternatively, HPV could be mediated by an initial influx of Ca2+ through voltage-dependent Ca2+ channels, leading to Ca2+-induced Ca2+ release from the SR and sustained elevation of [Ca2+]i (8). Further work is required to evaluate these possibilities.

Although hypoxia caused vigorous vasoconstriction in pulmonary arteries, it did not alter the ID in bronchial arteries (Fig. 3). These results are consistent with previous studies (6, 48) of systemic arteries in which hypoxia had no effect or caused vasodilation. We used bronchial arteries because these vessels could be isolated from the same organ and studied in the same manner as pulmonary arteries, thereby minimizing the influence of methodological factors on our results. The only methodological difference was Ptm, which was higher in bronchial arteries to reflect in vivo conditions; however, it is unlikely that the higher Ptm prevented hypoxic constriction in bronchial arteries because these vessels constricted vigorously to KCl and U-46619 (Table 1). It is more likely that the hypoxic responses of pulmonary and bronchial arteries were different due to intrinsic differences in reactivity.

The similarities in temporal characteristics, PO2 dependence, and Ca2+ dependence of hypoxic vasoconstriction between small porcine pulmonary arteries and isolated and intact lungs and the absence of this response in bronchial arteries suggested that studies in small porcine pulmonary arteries could help to clarify the mechanisms of in vivo HPV; therefore, we used this preparation to determine if HPV was endothelium dependent.

As shown in Fig. 5, endothelial denudation abolished HPV in these vessels. This effect was not due to denudation-induced injury to vascular smooth muscle because E- arteries constricted vigorously to KCl and U-46619 (Table 1). Neither was it due to decreased baseline vasomotor tone (55) because ID20 before hypoxic exposure was smaller than in E+ arteries, suggesting that baseline tone had increased. Rather, it must have been due to elimination of some endothelial influence on vascular smooth muscle. Endothelium can release relaxing factors, such as NO and prostacyclin, and contracting factors, such as endothelin and thromboxane; therefore, HPV could be due to a hypoxia-induced decrease in relaxing factor activity or a hypoxia-induced increase in contracting factor activity. Alternatively, basal release of endothelial factors might facilitate the mechanisms of HPV intrinsic to vascular smooth muscle. To evaluate these possibilities, we determined how Indo, L-NAME, and BQ-123 altered HPV in E+ arteries and whether ET-1 restored HPV in E- arteries.

Liu and Sylvester (38) have shown that L-NAME (3 × 10-5 M) blocked the vasodilator responses to ACh in this preparation, indicating inhibition of NO synthase, and that BQ-123 (3 × 10-6 M) blocked the vasoconstrictor responses to ET-1, indicating inhibition of ETA receptors. Measurement of prostaglandins D2, E2, and F2alpha , 6-keto prostaglandin F1alpha , and thromboxane B2 production by gas chromatography/mass spectrometry indicated that Indo (10-6 M) blocked cyclooxygenase in isolated proximal porcine pulmonary arterial rings (Shimoda T, Sylvester J, Undem B, and Hubbard W, unpublished data). Indo (10-5 M), L-NAME (3 × 10-5 M), or BQ-123 (3 × 10-6 M) did not alter the vasoconstrictor responses to KCl or U-46619 in small porcine pulmonary arteries (38). Finally, ID20 measured before hypoxic exposure was not different among untreated E+ arteries and E+ arteries treated with Indo, Indo plus L-NAME, or BQ-123, suggesting similar baseline vasomotor tone. These results indicate that our pharmacological interventions had the intended effects on the endothelium and did not have unintended nonspecific effects on vascular smooth muscle.

As shown in Fig. 6, HPV was unaltered by Indo, enhanced by Indo plus L-NAME, and abolished by BQ-123. Many studies in isolated and intact lungs (16, 19, 34, 39, 68, 72, 74) have demonstrated enhancement of HPV by inhibitors of cyclooxygenase and NO synthase alone or in combination. Like our results, these data indicate that cyclooxygenase products and NO modulated but did not mediate HPV. In contrast, antagonists of ETA receptors blocked HPV in intact and isolated lungs (11, 14, 23, 24, 50, 57, 71, 78) and acute hypoxia increased ET-1 production in intact and isolated lungs and cultured endothelium (20, 25, 29, 60, 64). These data are consistent with the effects of BQ-123 shown in Fig. 6 and suggest that ET-1 mediated HPV.

Other observations question this possibility. ET-induced pulmonary vasoconstriction, unlike HPV, reversed very slowly (4, 43), perhaps due to prolonged receptor binding (75). ET-1 caused transient vasodilation during HPV, perhaps by activating KATP channels in vascular smooth muscle or ETB receptors and NO production in endothelium (10, 18, 20, 35-37, 80). Some investigators (11, 46, 51) were unable to demonstrate that hypoxia increased pulmonary ET-1 production. Others (20, 64) found that hypoxia-induced increases in ET-1 concentration occurred much later than or did not correlate with HPV. Thus the role of ET-1 in HPV may be more subtle than direct concentration-dependent activation of smooth muscle contraction.

To reconcile these discrepancies as well as the observations that isolated pulmonary arterial myocytes contract to hypoxia (42, 47, 58), we hypothesized that the basal production of ET-1 by the endothelium allowed full expression of HPV in vivo by facilitating mechanisms of hypoxic reactivity in vascular smooth muscle. To test this hypothesis, we determined whether exposure to a low concentration of ET-1 (10-10 M) would restore HPV in E- pulmonary arteries. We used 10-10 M because Liu and Sylvester (38) previously found that this concentration was at the threshold for contraction in these vessels. As shown in Fig. 7, ET-1 priming almost completely restored HPV in E- arteries (Delta ID20 at 30 min = -39 ± 8 µm or ~70% of the response to 4% O2 in untreated E+ arteries). These results are supportive of our hypothesis and consistent with previous observations by Sham et al. (58) in freshly isolated smooth muscle cells from these vessels in which ET-1 priming potentiated an otherwise small but significant hypoxic contraction nearly eightfold.

ET-1 could facilitate HPV in several ways. In pulmonary arterial myocytes, ET-1 priming potentiated hypoxic contraction but did not alter hypoxia-induced increases in [Ca2+]i, suggesting an increase in myofilament Ca2+ sensitivity (58). ET-1 is known to have this effect in both systemic and pulmonary arterial smooth muscle (13, 49), and increased Ca2+ sensitivity has been proposed to contribute to hypoxic responses in precontracted rat pulmonary arteries (54, 72). Other possibilities include an increased open probability of Ca2+ channels (26) and a depolarized resting membrane potential (69). The latter could make sarcolemmal KV channels more susceptible to hypoxic inactivation, thought by some investigators to be an important early step in HPV (73). It was recently suggested that ET-1 might facilitate HPV by preventing hypoxic activation of KATP channels in pulmonary vascular smooth muscle (57); however, glibenclamide, an KATP antagonist, did not alter HPV in isolated ferret lungs exposed to moderate hypoxia (76). Further studies are needed to evaluate these possibilities.

It is possible that agonists other than ET-1 could facilitate HPV. This might explain why ET-1 receptor antagonists sometimes did not inhibit HPV in intact animals (81), isolated lungs (65), or pulmonary arteries (12, 31). In support of this possibility, Sato et al. (57) found that ETA receptor antagonists blocked HPV in intact and isolated rat lungs but not in isolated lungs costimulated with angiotensin II. Such data emphasize that our results should not be extended to other preparations, conditions, or species without experimental confirmation of applicability. Nevertheless, it is interesting to note that the inhibitory effect of ETA receptor antagonists on HPV in vivo has been reasonably consistent across species (5, 14, 23, 24, 50, 57, 71, 78), suggesting that ET-1 may play a pivotal physiological role.


    FOOTNOTES

Address for reprint requests and other correspondence: J. T. Sylvester, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.

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 20 September 2000; accepted in final form 15 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al-Tinawi, A, Krenz GS, Rickaby DA, Linehan JH, and Dawson CA. Influence of hypoxia and serotonin on small pulmonary vessels. J Appl Physiol 76: 56-64, 1994[Abstract/Free Full Text].

2.   Archer, SL, Yankovich RD, Chesler E, and Weir EK. Comparative effects of nisoldipine, nifedipine and bepridil on experimental pulmonary hypertension. J Pharmacol Exp Ther 233: 12-17, 1985[Abstract].

3.   Barer, GR, Howard P, and Shaw JW. Stimulus-response curves for the pulmonary vascular bed to hypoxia and hypercapnia. J Physiol (Lond) 211: 139-155, 1970[ISI][Medline].

4.   Chatfield, BA, McMurtry IF, Hall SL, and Abman SH. Hemodynamic effects of endothelin-1 on ovine fetal pulmonary circulation. Am J Physiol Regulatory Integrative Comp Physiol 261: R182-R187, 1991[Abstract/Free Full Text].

5.   Chen, SJ, Chen YF, Meng QC, Durand J, Dicarlo VS, and Oparil S. Endothelin-receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in rats. J Appl Physiol 79: 2122-2131, 1995[Abstract/Free Full Text].

6.   Coburn, RF, Grubb B, and Aronson RD. Effect of cyanide on oxygen tension-dependent mechanical tension in rabbit aorta. Circ Res 44: 368-378, 1979[ISI][Medline].

7.   Cornfield, DN, Stevens T, McMurtry IF, Abman SH, and Rodman DM. Acute hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 265: L53-L56, 1993[Abstract/Free Full Text].

8.   Cornfield, DN, Stevens T, McMurtry IF, Abman SH, and Rodman DM. Acute hypoxia causes membrane depolarization and calcium influx in fetal pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 266: L469-L475, 1994[Abstract/Free Full Text].

9.   De Canniere, D, Stefanidis C, Hallemans R, Delcroix M, Brimioulle S, and Naeije R. Stimulus-response curves for hypoxic pulmonary vasoconstriction in piglets. Cardiovasc Res 26: 944-949, 1992[ISI][Medline].

10.   Deleuze, PH, Adnot S, Shiiya N, Roudot Thoraval F, Eddahibi S, Braquet P, Chabrier PE, and Loisance DY. Endothelin dilates bovine pulmonary circulation and reverses hypoxic pulmonary vasoconstriction. J Cardiovasc Pharmacol 19: 354-360, 1992[ISI][Medline].

11.   Doi, S, Smedira N, and Murray PA. Pulmonary vasoregulation by endothelin in conscious dogs after left lung transplantation. J Appl Physiol 88: 210-218, 2000[Abstract/Free Full Text].

12.   Douglas, SA, Vickery-Clark LM, and Ohlstein EH. Endothelin-1 does not mediate hypoxic vasoconstriction in canine isolated blood vessels: effect of BQ-123. Br J Pharmacol 108: 418-421, 1993[ISI][Medline].

13.   Evans, AM, Cobban HJ, and Nixon GF. ETA receptors are the primary mediators of myofilament calcium sensitization induced by ET-1 in rat pulmonary artery smooth muscle: a tyrosine kinase independent pathway. Br J Pharmacol 127: 153-160, 1999[Abstract/Free Full Text].

14.   Franco-Cereceda, A, and Holm P. Selective or nonselective endothelin antagonists in porcine hypoxic pulmonary hypertension? J Cardiovasc Pharmacol 31, Suppl1: S447-S452, 1998[ISI][Medline].

15.   Gelband, CH, and Gelband H. Ca2+ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels. Circulation 96: 3647-3654, 1997[Abstract/Free Full Text].

16.   Gottlieb, JE, McGeady M, Adkinson NF, Jr, and Sylvester JT. Effects of cyclo- and lipoxygenase inhibitors on hypoxic vasoconstriction in isolated ferret lungs. J Appl Physiol 64: 936-943, 1988[Abstract/Free Full Text].

17.   Halpern, W, and Kelley M. In vitro methodology for resistance arteries. Blood Vessels 28: 245-251, 1991[ISI][Medline].

18.   Hasunuma, K, Rodman DM, O'Brien RF, and McMurtry IF. Endothelin 1 causes pulmonary vasodilation in rats. Am J Physiol Heart Circ Physiol 259: H48-H54, 1990[Abstract/Free Full Text].

19.   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].

20.   Helset, E, Kjaeve J, Bjertnaes L, and Lundberg JM. Acute alveolar hypoxia increases endothelin-1 release but decreases release of calcitonin gene-related peptide in isolated perfused rat lungs. Scand J Clin Lab Invest 55: 369-376, 1995[ISI][Medline].

21.   Herold, CJ, Wetzel RC, Robotham JL, Herold SM, and Zerhouni EA. Acute effects of increased intravascular volume and hypoxia on the pulmonary circulation: assessment with high-resolution CT. Radiology 183: 655-662, 1992[Abstract].

22.   Holden, WE, and McCall E. Hypoxia-induced contractions of porcine pulmonary artery strips depend on intact endothelium. Exp Lung Res 7: 101-112, 1984[ISI][Medline].

23.   Holm, P, Liska J, Clozel M, and Franco-Cereceda A. The endothelin antagonist bosentan: hemodynamic effects during normoxia and hypoxic pulmonary hypertension in pigs. J Thorac Cardiovasc Surg 112: 890-897, 1996[Abstract/Free Full Text].

24.   Holm, P, Liska J, and Franco-Cereceda A. The ETA receptor antagonist, BMS-182874, reduces acute hypoxic pulmonary hypertension in pigs in vivo. Cardiovasc Res 37: 765-771, 1998[ISI][Medline].

25.   Horio, T, Kohno M, Yokokawa K, Murakawa K, Yasunari K, Fujiwara H, Kurihara N, and Takeda T. Effect of hypoxia on plasma immunoreactive endothelin-1 concentration in anesthetized rats. Metabolism 40: 999-1001, 1991[ISI][Medline].

26.   Inoue, Y, Oike M, Nakao K, Kitamura K, and Kuriyama H. Endothelin augments unitary calcium channel currents on the smooth muscle cell membrane of guinea-pig portal vein. J Physiol (Lond) 423: 171-191, 1990[Abstract].

27.   Katusic, ZS. Superoxide anion and endothelial regulation of arterial tone. Free Radic Biol Med 20: 443-448, 1996[ISI][Medline].

28.   Kennedy, T, and Summer W. Inhibition of hypoxic pulmonary vasoconstriction by nifedipine. Am J Cardiol 50: 864-868, 1982[ISI][Medline].

29.   Kourembanas, S, Marsden PA, McQuillan LP, and Faller DV. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 88: 1054-1057, 1991[ISI][Medline].

30.   Kovitz, KL, Aleskowitch TD, Sylvester JT, and Flavahan NA. Endothelium-derived contracting and relaxing factors contribute to hypoxic responses of pulmonary arteries. Am J Physiol Heart Circ Physiol 265: H1139-H1148, 1993[Abstract/Free Full Text].

31.   Lazor, R, Feihl F, Waeber B, Kucera P, and Perret C. Endothelin-1 does not mediate the endothelium-dependent hypoxic contractions of small pulmonary arteries in rats. Chest 110: 189-197, 1996[Abstract/Free Full Text].

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

33.   Leach, RM, Sheehan DW, Chacko VP, and Sylvester JT. Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries. Am J Physiol Lung Cell Mol Physiol 278: L294-L304, 2000[Abstract/Free Full Text].

34.   Leeman, M, de Beyl VZ, Biarent D, Maggiorini M, Melot C, and Naeije R. Inhibition of cyclooxygenase and nitric oxide synthase in hypoxic vasoconstriction and oleic acid-induced lung injury. Am J Respir Crit Care Med 159: 1383-1390, 1999[Abstract/Free Full Text].

35.   Lippton, HL, Cohen GA, Knight M, McMurtry IF, Gillot D, Arena F, Summer W, and Hyman AL. Evidence for distinct endothelin receptors in the pulmonary vascular bed in vivo. J Cardiovasc Pharmacol 17: S370-S373, 1991[ISI][Medline].

36.   Lippton, HL, Cohen GA, McMurtry IF, and Hyman AL. Pulmonary vasodilation to endothelin isopeptides in vivo is mediated by potassium channel activation. J Appl Physiol 70: 947-952, 1991[Abstract/Free Full Text].

37.   Liska, J, Holm P, Owall A, and Franco-Cereceda A. Endothelin infusion reduces hypoxic pulmonary hypertension in pigs in vivo. Acta Physiol Scand 154: 489-498, 1995[ISI][Medline].

38.   Liu, Q, and Sylvester JT. Development of intrinsic tone in isolated pulmonary arterioles. Am J Physiol Lung Cell Mol Physiol 276: L805-L813, 1999[Abstract/Free Full Text].

39.   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].

40.   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].

41.   Madden, JA, Dawson CA, and Harder DR. Hypoxia-induced activation in small isolated pulmonary arteries from the cat. J Appl Physiol 59: 113-118, 1985[Abstract/Free Full Text].

42.   Madden, JA, Vadula MS, and Kurup VP. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 263: L384-L393, 1992[Abstract/Free Full Text].

43.   Mann, J, Farrukh IS, and Michael JR. Mechanisms by which endothelin 1 induces pulmonary vasoconstriction in the rabbit. J Appl Physiol 71: 410-416, 1991[Abstract/Free Full Text].

44.   McMurtry, IF. BAY K 8644 potentiates and A23187 inhibits hypoxic vasoconstriction in rat lungs. Am J Physiol Heart Circ Physiol 249: H741-H746, 1985[Abstract/Free Full Text].

45.   McMurtry, IF, Davidson AB, Reeves JT, and Grover RF. Inhibition of hypoxic pulmonary vasoconstriction by calcium antagonists in isolated rat lungs. Circ Res 38: 99-104, 1976[Abstract].

46.   Medbo, S, Yu XQ, Asberg A, and Saugstad OD. Pulmonary hemodynamics and plasma endothelin-1 during hypoxemia and reoxygenation with room air or 100% oxygen in a piglet model. Pediatr Res 44: 843-849, 1998[Abstract].

47.   Murray, TR, Chen L, Marshall BE, and Macarak EJ. Hypoxic contraction of cultured pulmonary vascular smooth muscle cells. Am J Respir Cell Mol Biol 3: 457-465, 1990[ISI][Medline].

48.   Namm, DH, and Zucker JL. Biochemical alterations caused by hypoxia in the isolated rabbit aorta. Correlation with changes in arterial contractility. Circ Res 32: 464-470, 1973[Medline].

49.   Nishimura, J, Moreland S, Ahn HY, Kawase T, Moreland RS, and van Breemen C. Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery. Circ Res 71: 951-959, 1992[Abstract].

50.   Oparil, S, Chen SJ, Meng QC, Elton TS, Yano M, and Chen YF. Endothelin-A receptor antagonist prevents acute hypoxia-induced pulmonary hypertension in the rat. Am J Physiol Lung Cell Mol Physiol 268: L95-L100, 1995[Abstract/Free Full Text].

51.   Perreault, T, Stewart DJ, Cernacek P, Wu X, Ni F, De Marte J, and Giaid A. Newborn piglet lungs release endothelin-1: effect of alpha-thrombin and hypoxia. Can J Physiol Pharmacol 71: 227-233, 1993[ISI][Medline].

52.   Post, JM, Gelband CH, and Hume JR. [Ca2+]i inhibition of K+ channels in canine pulmonary artery. Novel mechanism for hypoxia-induced membrane depolarization. Circ Res 77: 131-139, 1995[Abstract/Free Full Text].

53.   Post, JM, Hume JR, Archer SL, and Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882-C890, 1992[Abstract/Free Full Text].

54.   Robertson, TP, Aaronson PI, and Ward JP. Hypoxic vasoconstriction and intracellular Ca2+ in pulmonary arteries: evidence for PKC-independent Ca2+ sensitization. Am J Physiol Heart Circ Physiol 268: H301-H307, 1995[Abstract/Free Full Text].

55.   Robertson, TP, Hague D, Aaronson PI, and Ward JP. Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat. J Physiol (Lond) 525: 669-680, 2000[Abstract/Free Full Text].

56.   Salvaterra, CG, and Goldman WF. Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L323-L328, 1993[Abstract/Free Full Text].

57.   Sato, K, Morio Y, Morris KG, Rodman DM, and McMurtry IF. Mechanism of hypoxic pulmonary vasoconstriction involves ETA receptor-mediated inhibition of KATP channel. Am J Physiol Lung Cell Mol Physiol 278: L434-L442, 2000[Abstract/Free Full Text].

58.   Sham, JSK, Crenshaw BR, Shimoda LA, and Sylvester JT. Effects of hypoxia in porcine pulmonary arterial myocytes: roles of KV channel and endothelin-1. Am J Physiol Lung Cell Mol Physiol 279: L262-L272, 2000[Abstract/Free Full Text].

59.   Shirai, M, Sada K, and Ninomiya I. Effects of regional alveolar hypoxia and hypercapnia on small pulmonary vessels in cats. J Appl Physiol 61: 440-448, 1986[Abstract/Free Full Text].

60.   Shirakami, G, Nakao K, Saito Y, Magaribuchi T, Jougasaki M, Mukoyama M, Arai H, Hosoda K, Suga S, Ogawa Y, Yamada T, Mori K, and Imura H. Acute pulmonary alveolar hypoxia increases lung and plasma endothelin-1 levels in conscious rats. Life Sci 48: 969-976, 1991[ISI][Medline].

61.   Suggett, AJ, Mohammed FH, Barer GR, Twelves C, and Bee D. Quantitative significance of hypoxic vasoconstriction in the ferret lung. Respir Physiol 46: 89-104, 1981[ISI][Medline].

62.   Sylvester, JT, Harabin AL, Peake MD, and Frank RS. Vasodilator and constrictor responses to hypoxia in isolated pig lungs. J Appl Physiol 49: 820-825, 1980[Abstract/Free Full Text].

63.   Sylvester, JT, Sham JSK, Shimoda LA, and Liu Q. Cellular mechanisms of acute hypoxic pulmonary vasoconstriction. In: Respiratory-Circulatory Interactions in Health and Disease, edited by Scharf SM, Pinsky MR, and Magder S.. New York: Dekker, 2001, vol. 157, p. 315-359. (Lung Biol Health Dis Ser)

64.   Takeda, S, Nakanishi K, Inoue T, and Ogawa R. Delayed elevation of plasma endothelin-1 during unilateral alveolar hypoxia without systemic hypoxemia in humans. Acta Anaesthesiol Scand 41: 274-280, 1997[ISI][Medline].

65.   Takeoka, M, Ishizaki T, Sakai A, Chang SW, Shigemori K, Higashi T, and Ueda G. Effect of BQ123 on vasoconstriction as a result of either hypoxia or endothelin-1 in perfused rat lungs. Acta Physiol Scand 155: 53-60, 1995[ISI][Medline].

66.   Tolins, M, Weir EK, Chesler E, Nelson DP, and From AH. Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator. J Appl Physiol 60: 942-948, 1986[Abstract/Free Full Text].

67.   Tucker, A, McMurtry IF, Grover RF, and Reeves JT. Attenuation of hypoxic pulmonary vasoconstriction by verapamil in intact dogs. Proc Soc Exp Biol Med 151: 611-614, 1976[Abstract].

68.   Tucker, A, and Reeves JT. Nonsustained pulmonary vasoconstriction during acute hypoxia in anesthetized dogs. Am J Physiol 228: 756-761, 1975[ISI][Medline].

69.   Turner, JL, and Kozlowski RZ. Relationship between membrane potential, delayed rectifier K+ currents and hypoxia in rat pulmonary arterial myocytes. Exp Physiol 82: 629-645, 1997[Abstract].

70.   Vadula, MS, Kleinman JG, and Madden JA. Effect of hypoxia and norepinephrine on cytoplasmic free Ca2+ in pulmonary and cerebral arterial myocytes. Am J Physiol Lung Cell Mol Physiol 265: L591-L597, 1993[Abstract/Free Full Text].

71.   Wang, Y, Coe Y, Toyoda O, and Coceani F. Involvement of endothelin-1 in hypoxic pulmonary vasoconstriction in the lamb. J Physiol (Lond) 482: 421-434, 1995[Abstract].

72.   Ward, JP, and Robertson TP. The role of the endothelium in hypoxic pulmonary vasoconstriction. Exp Physiol 80: 793-801, 1995[Abstract].

73.   Weir, EK, and Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J 9: 183-189, 1995[Abstract/Free Full Text].

74.   Weir, EK, McMurtry IF, Tucker A, Reeves JT, and Grover RF. Prostaglandin synthetase inhibitors do not decrease hypoxic pulmonary vasoconstriction. J Appl Physiol 41: 714-718, 1976[Abstract/Free Full Text].

75.   Westcott, JY, Henson J, McMurtry IF, and O'Brien RF. Uptake and metabolism of endothelin in the isolated perfused rat lung. Exp Lung Res 16: 521-532, 1990[ISI][Medline].

76.   Wiener, CM, Dunn A, and Sylvester JT. ATP-dependent K+ channels modulate vasoconstrictor responses to severe hypoxia in isolated ferret lungs. J Clin Invest 88: 500-504, 1991[ISI][Medline].

77.   Wiener, CM, and Sylvester JT. Effects of glucose on hypoxic vasoconstriction in isolated ferret lungs. J Appl Physiol 70: 439-446, 1991[Abstract/Free Full Text].

78.   Willette, RN, Ohlstein EH, Mitchell MP, Sauermelch CF, Beck GR, Luttmann MA, and Hay DW. Nonpeptide endothelin receptor antagonists. VIII: attenuation of acute hypoxia-induced pulmonary hypertension in the dog. J Pharmacol Exp Ther 280: 695-701, 1997[Abstract/Free Full Text].

79.   Wolin, MS. Reactive oxygen species and vascular signal transduction mechanisms. Microcirculation 3: 1-17, 1996[Medline].

80.   Wong, J, Vanderford PA, Fineman JR, Chang R, and Soifer SJ. Endothelin-1 produces pulmonary vasodilation in the intact newborn lamb. Am J Physiol Heart Circ Physiol 265: H1318-H1325, 1993[Abstract/Free Full Text].

81.   Wong, J, Vanderford PA, Winters JW, Chang R, Soifer SJ, and Fineman JR. Endothelin-1 does not mediate acute hypoxic pulmonary vasoconstriction in the intact newborn lamb. J Cardiovasc Pharmacol 22, Suppl8: S262-S266, 1993[ISI][Medline].

82.   Yuan, XJ, Goldman WF, Tod ML, Rubin LJ, and Blaustein MP. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L116-L123, 1993[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 280(5):L856-L865
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society