Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells JANE A. MADDEN,
MAITHILI
SUBRAMANIAN
VADULA,
AND VISWANATH
P. KURUP
Research Service, Zablocki Veterans Affairs Medical Center, and the Departments of Neurology, Neurosurgery, Pediatrics, and Medicine, The Medical College of Wisconsin, Milwaukee, Wisconsin Madden, Viswanath
Jane A., Maithili Subramanian Vadula, and P. Kurup. Effects of hypoxia and other vasoactive
agentson pulmonary and cerebral artery smooth musclecells. 263 (Lung Cell. Mol. Physiol. 7): L384-L393, 1992.- Smooth musclecells (SMC) were isolated from cat cerebral arteries and three sizesof pulmonary artery (800-pm diameter) and used within 72-96 h. Changein cell length in responseto hypoxia and other vasoactive agentswasmeasuredin a specially constructedcell chamber on an inverted microscope.Pulmonary artery SMC responded to hypoxia differently accordingto artery size. SMC from 800-pm-diameterpulmonary arteries shortened0.81 -+0.44%. Cerebralartery SMC plated on a flexible polydimethyl siloxane membraneshowedlossof tension during exposureto hypoxia. The shortening of SMC from the 200- to 600-pm pulmonary arteries was accompaniedby myosin phosphorylation. SMC from >800-pm-diameterpulmonary arteries and cerebral arteries contained myosin that did not phosphorylate during hypoxia. The SMC from both artery types respondedto norepinephrine, serotonin, prostaglandin FZcX,and indomethacin and exhibited a-adrenergicreceptor population patterns similar to those of intact arteries. The pattern of hypoxic responsesexhibited by these nondedifferentiated pulmonary and cerebral artery SMC supports the idea that, at least in the cat, the hypoxic sensoris located within the SMC. vascular smooth musclecells; endothelium; adrenergic receptors; myosin phosphorylation
Am. J. Physiol.
DESPITE A CONSIDERABLE research effort to elucidate the mechanism underlying vascular hypoxic responses, neither the sensor nor the sequence of cellular events leading to relaxation or constriction has been identified. In the pulmonary circulation, reducing partial pressure of O2 (POT) results in constriction of arteries with diameters ~600 pm (21, 36). In the cerebral circulation, low PO, results in arterial dilation throughout the cerebrovascular tree (20). Whether the pulmonary and cerebral arteries sense hypoxia directly or whether they are reacting to mediators released by other vascular or nonvascular tissue in response to hypoxia is a matter of some controversy. It has been suggested that vascular muscle cells sense oxygen levels (22) and that oxygen directly affects vascular tone by changing ion conductances across the cell membrane (8, 37, 42). However, most commonly, hypoxic vasodilation and constriction are attributed to endothelium-derived relaxing and contracting factors and/or to arachidonic acid metabolites (4,5, 11, 15, 29). Some investigators have found that hypoxic vasoconstriction in isolated pulmonary arteries and lungs could be abolished either by inhibiting endothelial factors (32) or by removing endothelial ceils (33). However, others (l-3, 13, 43) have found contrary results. Similar dis-
53295
crepancies exist with regard to the role of the endothelium in cerebral vasodilation (24,28). The persistence of hypoxic dilation in indomethacin (Indo) -treated cerebral arteries suggests that prostacyclin produced by the endothelial cells is not solely responsible for the response (28a, 35, 42) Many studies have used isolated arteries to study hypoxic responses without the influence of extravascular tissue. Whereas much information has been gained from this experimental preparation, contributions from the vascular endothelium can still complicate interpretation of the results. Endothelial cells can be mechanically removed or chemically inactivated, but using these methods raises the possibility of smooth muscle cell (SMC) damage. The development of techniques to isolate and study SMC offers a useful alternative to endothelium removal (38, 40). The hypoxic response has not yet been extensively studied in isolated SMC, although Murray et al. (26, 27) have recently shown that serially cultured SMC from fetal calf main pulmonary artery could contract to hypoxia (26) and other vasoactive stimuli (27). However, serial culture of other SMC types has been shown to result in the loss of cellular morphology and characteristic responses (6,7,23). The hypoxic response of freshly isolated, nondedifferentiated SMC has not been studied. Therefore, this work was designed to determine whether nondedifferentiated SMC isolated from pulmonary and cerebral arteries of adult cats would respond to hypoxia and other vasoactive agents similarly to intact arteries. MATERIALS
AND METHODS
Adult mongrel cats (2.5-4.0 kg) of either sex were anesthetized with ketamine (10 mg/kg) and pentobarbital sodium (30 mg/kg) and decapitated. The brain and lungs were removed. The middle cerebral arteries and severalof their sidebranches were dissectedfrom the brain. Becausewe (21) and others (36) have shown that in adult cat pulmonary arteries the hypoxic responseis dependent on size, pulmonary arteries of various diameters (~200, 200-600, and 800 pm) were dissectedfrom portions of both lung lobes. Cell Isolation
Procedure
Immediately after dissectionthe arteries were placed in cold Puck’s saline solution containing penicillin-streptomycin (pH 7.4, 4°C). The arteries were then transferred to 5 ml of an enzyme mixture (describedin Enzyme solution) and incubated for 90 min at 37°C to isolate SMC. Enzyme digestion was stoppedby adding an equalvolume of medium199 (M-199) plus 10%fetal bovine serum.The solution wasfiltered through three staccups in series (38) to remove connective tissue and cell debris. The supernatant containing a mixture of SMC, endothelial cells,and fibroblasts was centrifuged at 360g for 6 min. The pellet was suspendedin 1 ml of M-199 (GIBCO, Grand
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Island, NY) plus 10% fetal bovine serum (Hyclone). The total viable cell population was determined with the use of a hemacytometer and the trypan blue exclusion test (39). If the total viable cell population was 5 x lo5 cells/ml or greater, the solution was diluted, and the cell concentration was adjusted to I x 10” cells/ml. Approximately 0.1 ml of the cell mixture was plated on sterilized glass cover slips previously coated with 0.1 mg/ml poly+lysine (Sigma, St. Louis, MI). A culture of 3T3 cells (fibroblast cell line, American Type Culture Collection) was also plated on cover slips and treated in the same manner as the other cells. The cover slips were then put in culture dishes containing 2.5 ml of M-199 plus 10% fetal bovine serum and placed in a water-jacketed incubator at 37°C for 24 h to allow the cells to attach to the cover slips. After the 24-h incubation, the total SMC per cover slip was estimated based on cover slip surface area and the total viable cell population plated. The SMC were distinguished from fibroblasts by their spindle-shape morphology and distinct plasma membrane. To further ensure that the cells were SMC, two randomly chosen cover slips from early and late platings, along with a control cover slip of the 3T3 fibroblast cells for comparison, were subjected to immunofluorescent staining. This was done as follows: the cover slips were rinsed in physiological saline solution (PSS) and then stained with smooth musclespecific mouse anti-myosin antibody (1:200 dilution, Sigma) for 1 h. The cover slips were washed three times with PSS for a total of 15 min, and fluorescein isothiocyanate (FITC)-labeled anti-rabbit immunoglobulin G (IgG, 1:80 dilution, Sigma) was added for 1.5-2 h at 37°C. Cover slips were rinsed three times in PSS with a final wash in deionized water. The cover slips were mounted with Permount and observed at x40 or Xl00 magnification on an inverted microscope (Nikon diaphot) equipped with fluorescence optics. Background staining as seen in the 3T3 cells was used to differentiate SMC from fibroblasts. Contraction
Studies
The system used for studying cell contraction has been described previously in detail (43). Briefly, a specially constructed double-walled chamber is placed on the stage of an inverted microscope. The inside of the chamber is fitted with a 60 x 15-mm disposable culture dish. A glass cover slip containing a 24- to 48-h primary culture of SMC is placed inside the dish. The lid of the petri dish covering the cells has slits on its side for insertion of two pieces of polyethylene tubing (PE-190), which serve as ports for gas and PSS. PSS flows via gravity from a reservoir into the culture dish at a rate of 0.5 ml/min. A recirculating pump returns the PSS to the reservoir at a rate such that the dish always has 9 ml of fluid in it. Both the reservoir and the chamber are held at 37°C by a constant-temperature circulating water bath, and both are aerated with calibrated mixtures of O2 and CO, delivered from a weather balloon. Precalculated amounts of drugs are delivered to the cells via a syringe in a side arm port on the tubing connecting the PSS reservoir to the chamber. A reticle in the eyepiece of the microscope is used to measure changes in SMC length prior to and after agonist stimulation. Contractile responses to hypoxia and other agonists were measured. Various antagonists were also used to determine the presence of functional receptors on the SMC. The effects of all agents on SMC length were measured and recorded every 30 s for at least 10 min. Each dish used in a contraction study contained many SMC. Within the microscope’s visual field we could normally see five or six SMC. These cells were measured throughout the study. Although SMC outside the visual field were not measured, they were visually examined before and after stimulation with hy-
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L385
poxia or other agents to determine if any of them had changed in length. In this way we could ascertain whether the rest of the SMC in the dish were exhibiting responses similar to those seen in the measured cells. Silastic
Membrane
Studies
The cells were plated on a cover slip coated with a flexible membrane that wrinkled due to tension generated by the cells attached to it. The membrane was made by plating polydimethy1 siloxane (PDS; 60,000 centistokes, Dow Corning) onto clean cover slips to form an - IOO-pm-thick layer on the surface (10). The cover slip was then turned and held coated side down over the flame of a Bunsen burner for 2 s. This timing is critical to form a smooth surface. The flaming also sterilizes the surface of the membrane. A drop of media containing 1 x 10” cells was then placed on these cover slips. The cells were allowed to attach for -10 min, and then 5 ml of media were added to the dish to completely cover the membrane. The cells were placed in the incubator for 96 h. This incubation was longer than for the cells grown on the polylysine-coated cover slips because the SMC grew more slowly on the PDS membranes. Polyacrylamide
Gel Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the presence and molecular weight of myosin in the SMC. The method followed was modified from Weber et al. (41) and used a gradient gel as described previously (19). The cells were grown to confluency (- 72 h), scraped off the cover slips, and placed into a centrifuge tube containing a cell disruption solution (described below) for 1 h. The cells were homogenized in a polytron homogenizer at 10,000 rpm for 15 min. The supernatant was saved, and the pellet was washed twice for 30 min at 4°C in disruption solution minus Triton X-100. The supernatant was saved from each wash. All supernatants, washes, and the pellets were stored at -70°C before analysis. Because feline SMC myosin is not commercially available, bovine myosin was used as a standard for comparison. Myosin
Light
Chain Phosphorylation
To determine if myosin light chain phosphorylation occurred in conjunction with responses to hypoxia, particularly in the cells from the 200- to 600-pm-diameter pulmonary arteries, we used the O’Farrell method (30) as modified by Kurup et al. (19). The procedures used were enzyme-linked immunoabsorbent assay (ELISA) and two-dimensional gel electrophoresis. Monolayers of SMC from the 200- to 600~pm-diameter pulmonary arteries were exposed to hypoxia for 10 min. Monolayers not exposed to hypoxia served as controls. SMC from >800pm-diameter pulmonary arteries and cerebral arteries were treated in a similar fashion. The cells were scraped off the cover slips, and samples were prepared as described above for the PAGE. The first dimension of the two-dimensional gel electrophoresis enables the determination of the isoelectric focusing points (PI) of the myosin protein and the phosphorylated myosin. Isoelectric focusing in the first dimension was carried out in gels containing ampholytes in the range of pH 5.0-9.5. This was followed by electrophoresis in SDS-PAGE in the second dimension. The pH range was chosen based on Murray et al. (27), who found that the PI of bovine myosin was -5.5. The presence of myosin and its percent concentration in the SMC as determined by the ELISA method were compared with a bovine myosin standard containing both the light and heavy chains. The ELISA was carried out according to the method described previously (17). Briefly, this consisted of adding 100 ~1 of the cell extract to each well of a microtiter plate and then
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incubating the plate for 3 h at 37°C. After overnight storage at 4°C the wells were washed three times with a phosphate-buffered saline (PBS)-Tween buffer. The wells were then filled with 1% bovine serum albumin containing PBS-Tween buffer at room temperature for 1 h. The extracts were then treated for 1 h with 100 ~1 of mouse anti-myosin monoclonal antibody (1:200 dilution) at 37°C and then washed three times with PBSTween. Subsequently, the cell extracts were challenged with goat anti-mouse immunoglobulin M (IgM, l:l,OOO dilution) at 37°C for 1 h and again washed three times with the PBS-Tween buffer. They were then challenged with biotinylated rabbit antigoat IgG antibodies (l:l,OOO dilution) for I h at 37°C. After washing the wells as before, streptavidin peroxidase (l:lO,OOO dilution/well) was added to each well, left for 30 min, and then washed as above. This was followed by orthophenyldiamine for color development. After lo-15 min the reaction was stopped with 2 N H2S04, and the optical density (OD) was read at 490 nm using an automatic ELISA reader (Dynatech Lab). From the OD values of the known myosin, a standard curve was plotted and the OD values of the samples were converted to absolute values. Two-Dimensional Gel Electrophoresis A homogenized cell extract prepared as above for PAGE and ELISA was also used in two-dimensional gels to determine whether phosphorylated myosin was present in SMC exposed to hypoxia. The two-dimensional PAGE consists of isoelectric focusing in one dimension and a molecular sieving using SDSPAGE in the second dimension. The first-dimension gel was cast in glass capillary tubes with the use of a Bio-Rad system according to the manufacturer’s directions. Tube gels were prefocused for 15 min each at 200, 300, and 400 V, respectively, before the samples were added. Samples and pH standards (BioRad) were loaded in separate gels with the use of an ultrafine pipette tip and run at 750 V for 3.5 h. The gels were stained with Coomassie brilliant blue 250 (Bio-Rad), and the proteins were compared with the pH standards for their isoelectric points. The first-dimension gels were removed and bridged onto the second dimension with an agarose bridge (19). The second dimension, consisting of 820% polyacrylamide gel (Bio-Rad) containing SDS, was prepared using a gradient gel casting system (Bio-Rad). The electrophoresis was carried out at 7.5 mA/ gel in a minigel chamber (Bio-Rad). After the second-dimension electrophoresis, the gels were blotted onto polyvinyldifluoride membranes (PVDF) for 8-12 min according to the manufacturer’s semi-dry electroblot protocol. The membranes were stained with colloidal gold for protein and immunostained for specific reactivity anti-myosin sera (18, 25). Solutions Puck’s saline solution. Puck’s saline solution was composed of the following (in mM): 0.1 CaC1,.2H20, 4.7 KCl, 1.18 KH2PO*, 7 MgS04, 1.19 HZO, 120.0 NaCl, 0.116 Na,HPO,*7H,O, 5.5 D-glucose, and 0.013 phenol red (pH 7.34). Penicillin-streptomycin solution. The penicillin-streptomycin solution contained 10,000 U/ml sodium penicillin and IO mg/ml streptomycin sulfate mixture (GIBCO 600-5140). Enzyme solution. The enzyme solution contained the following ingredients added to 10 ml PSS: 157 U/ml collagenase (type II), 50 U/ml elastase, 5 U/ml deoxyribonuclease, 1.5% wt/vol bovine serum albumin, 4 mM ATP, 0.1% wt/vol soybean trypsin inhibitor, and 1 PM isoproterenol. The solution was maintained at pH 7.4 by gassing with 95% 02-5%C02. All enzymes were obtained from Worthington Biomedical, Newark, NJ. All other chemicals were obtained from Sigma. PSS. The PSS was composed of (in mM) 141 Na+, 4.7 K+,
AND
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2.5 Ca”+, 0.72 Mg 2+ , 124 Cl- 17 H2P04 , 22.5 HCO,, and 11 glucose. Cell disruption solution. The disruption solution consisted of PSS with the detergents Triton X-100 (2% vol/vol), ethylene glycol-bis(a-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA, 2 mM), and dithiothreitol (DTT, 0.2 mM; all from Sigma). Polyacrylamide gels. First-dimension gels consisted of 31.8% acrylamide, 5.7% bisacrylamide, ampholytes in the pH range of 2-10, Nonidet phosphate-40 (NP-40), and 2.2% urea added to the gel mixture. Second-dimension gels used were SDS-polyacrylamide consisting of 30% acrylamide, 2.5% bisacrylamide, and 20% SDS with pH 8.8 tris(hydroxymethyl)aminomethane hydrochloride for the running and pH 6.8 for the stacking. The gel mixture was poured into the plates as an 8-20% gradient. ?
l
Drugs Norepinephrine (NE), serotonin (5-HT), prostaglandin F,, (PGF2cY), and Indo were prepared in PSS, adjusted to a pH of 7.4, and used at 1 x 10Y4 M. The adrenergic blockers, prazosin and yohimbine, were also prepared in PSS and used at 1 x 10e4 M. The blockers were added to the culture dish at least 20 min before adding NE. Gases Weather balloons filled with calibrated gas mixtures were pumped from the balloon through dispersion stones into the PSS reservoir and the contraction chamber. The gas mixtures were control or normoxia (Po2, 140 Torr; Pco,, 37 Torr) and hypoxia (Po2, 50 Torr; Pco~, 37 Torr). The PSS from the reservoir and the chamber were sampled at regular intervals, and the PO,, Pco2 and pH were determined with a Corning model 278 blood gas analyzer. Statistical Analyses Cell lengths are expressed as means t SE. Paired and unpaired Student’s t test was used for statistical analysis. A value of P < 0.05 was considered statistically significant. RESULTS
Morphological Studies
Approximately 85% of the ceils isolated from the pulmonary and cerebral arteries were viable as determined by the trypan blue exclusion test. These cells were a mixture of SMC, fibroblasts, and endothelial cells. The endothelial cells died almost immediately after plating and did not pose a contamination problem. The SMC could be distinguished from fibroblasts by their spindle shape and by their ability to fluoresce to FITC-labeled anti-myosin antibody. With the use of the 3T3 fibroblast cells as controls, it was determined that the SMC comprised at least 55% of a given cell mixture. Responses to Hypoxia by Pulmonary and Cerebral SMC
Figure 1 shows the percent change in length during hypoxia by pulmonary and cerebral artery SMC. These data were obtained from at least 15 different cats and 56 separate cultures. Within 1 min after exposure to hypoxia the SMC from the 200- to 600-pm-diameter pulmonary arteries began to decrease in length. After 5 min they had shortened 24.2 t 2.7% from their initial length. This contraction was similar to that seen in cells from the
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CONTRACTILE
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OF PULMONARY
AND CEREBRAL
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that was present in the normoxic ing exposure to hypoxia. SIZE OF PULMONARY ARTERY SUPPLYING SMC
q q
~200 pm 200-600 pm >800 pm
w
CEREBRAL
m
L387
SMC
state disappeared
dur-
Responses of Pulmonary and Cerebral SMC to NE, 5-HT, PGF,,, and Indo
Because the SMC from cerebral and >800-pm-diameter pulmonary arteries did not contract to hypoxia, we wished to determine if these SMC were capable of responding to other vasoactive agents. The pulmonary artery SMC that did contract to hypoxia were also examined. The SMC from all sizes of pulmonary arteries contracted significantly to NE, PGF2,, and Indo (P < 0.05, Fig. 4). Contractions to 5-HT were statistically significant from zero in SMC from 200- to 600- and >800pm-diameter pulmonary arteries but not in cells from the 800-pm-diameter pulmonary arteries and cerebral arteriea did not show any phosphorylated myosin (Fig. 6). Thus, even though these cells had myosin available, the myosin was not phosphorylated during exposure to hypoxia.
T
5-HT
NE
INDO
PGF2a
SMC
aration contained -9 pg/ml. A comparable number of cells from the 200-600 pm pulmonary arteries contained 27 pg/ml, and the cerebral arteries contained 0.35 kg/
**+I*
800-pmdiameter pulmonary arteries, there was no myosin phosphorylation in cerebral artery SMC exposed to hypoxia. The present study did not examine whether the differential responses to hypoxia exhibited by the various sizes of cat pulmonary artery SMC would persist throughout many cell passages. SMC isolated from fetal calf main pulmonary artery retained the ability to contract to hypoxia (26) and other agonists (27) for at least 9 wk in culture. Because other studies have found that SMC maintained in culture may exhibit phenotypic changes (6) or uncharacteristic responses to agonists (7, 23) responses in these serially cultured cells should be evaluated carefully. That the nondedifferentiated pulmonary and cerebral artery SMC used in the present study were viable and retained many of their in situ characteristics was further demonstrated by their responses to other vasoactive agonists. All SMC from the different sizes of pulmonary
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artery and from cerebral artery contracted in response to NE, 5-HT, PGF2 , and Indo. These contractile responses indicated that the cells retained functional receptors for these agents. Identification of the myosin phosphorylation characteristics in response to these vasoactive agents deserves study. The cerebral artery SMC contraction to Indo correlates with our finding of contraction in whole, isolated cerebral arteries exposed to Indo (28a). These data indicate that Indo may possess some nonspecific contractile effects in addition to inhibiting the synthesis of arachidonic acid metabolites. The use of the cyl- and a,-adrenergic receptor blockers, prazosin and yohimbine, revealed that the SMC possessed adrenergic receptor populations similar to those found by others in cat pulmonary and cerebral circulations (9, 14, 16). Although our studies are not conclusive regarding the types and distributions of adrenergic or other receptors, they further indicate that the isolated nondedifferentiated pulmonary and cerebral artery SMC retain many of their in situ properties and underscore their potential usefulness in investigating SMC phenomena. Although we found that cat pulmonary and cerebral artery SMC can respond to hypoxia in the absence of the endothelium, it is possible that this is a species-specific phenomenon. Dog, pig, and human pulmonary arteries appear to require an intact endothelium for a hypoxic response (11, 12, 31, 34). The response in rat pulmonary arteries has been shown to require (32) or be independent of (1, 43) the endothelium. Isolated bovine pulmonary arteries and their SMC respond in the absence of endothelium (2, 3, 26). The hypoxic vasodilation by cerebral arteries has been attributed to endothelium-produced prostacyclin (24), although this is not a universal finding (35, 42). In the present study cerebral artery SMC showed a decrease in tone under hypoxic conditions. In other studies we have found that cat isolated cerebral arteries retained their ability to dilate to hypoxia after indomethacin treatment (28a) and endothelium removal (28). Thus in this species the endothelium does not appear to be required. In conclusion, the differential pattern of hypoxic responses exhibited by nondedifferentiated SMC isolated from cat pulmonary and cerebral arteries supports the idea that, at least in the cat, the hypoxic sensor is located within the SMC. The hypoxic contraction of the SMC from the 200- to 600-pm-diameter pulmonary arteries appears to be mediated by myosin light chain phosphorylation. Additionally, the pulmonary and cerebral artery SMC responded to other vasoactive agonists and appeared to possess an adrenergic receptor population resembling that of the intact cat pulmonary and cerebral circulations. These similarities suggest that pulmonary and cerebral artery SMC are potentially useful experimental models for studying cellular physiology and pharmacology. The authors gratefully acknowledge the helpful advice of Dr. Thomas R. Murray regarding the polvdimethvl siloxane membranes.
AND CEREBRAL
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This study was supported by Veterans Affairs Medical Research funds. Address for reprint requests: J. A. Madden, Neurology Research, VAMC/151, Milwaukee, WI 53295. Received 20 December 1991; accepted in final form 28 April 1992. REFERENCES 1. Bennie, B. E., C. S. Packer, D. R. Powell, N. Jin, and R. A. Rhoades. Biphasic contractile response of pulmonary artery to hypoxia. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L156L163, 1991. 2. Burke-Wolin, T. M., and M. S. Wolin. Hz02 and cGMP may function as an 0, sensor in the pulmonary artery. J. Appl. Physiol. 66: 167-170, 1989. 3. Burke-Wolin, T. M., and M. S. Wolin. Inhibition of cGMPassociated pulmonary arterial relaxation to H202 and O2 by ethanol. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): H1267-H1273, 1990. 4. Busse R., and E. Bassenge. Endothelium and hypoxic responses. Bibl. Cardiol. 38: 21-34, 1984. 5. Busse, R., U. Pohl, C. Kellner, and U. Klemm. Endothelial cells are involved in the vasodilatory response to hypoxia. Pfluegers Arch. 397: 78-80, 1983. 6. Charnley, J. H., Groschel-Stewart.
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