(Hypertension. 1998;31:206.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Methacholine-Induced Contraction of Rabbit Pulmonary Artery: Role of Platelet-Endothelial Transcellular Thromboxane Synthesis
From the Departments of Pharmacology and Toxicology and Internal Medicine, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.
Correspondence to Sandra L. Pfister, PhD, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail spfister{at}post.its.mcw.edu
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Abstract |
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Arachidonic acid- and methacholine-induced contractions of rabbit pulmonary arteries are mediated by thromboxane (TX) A2. Although removal of the endothelium abolishes the contractions, endothelial cells isolated from pulmonary arteries do not synthesize TXA2. Further studies described here showed that the expression of TX synthase was evident in platelets and intact pulmonary artery but not in endothelial cells. These studies examined the role of platelet TXA2 production in the vasoconstrictor response to methacholine. Endothelial cells were incubated with platelets in the presence or absence of methacholine. Methacholine caused an increase in TXB2 production. Pretreatment of endothelial cells with aspirin (100 µmol/L) before the addition of platelets did not impair the ability of methacholine to increase TXB2 synthesis. Conversely, if platelets were pretreated with aspirin, methacholine failed to stimulate TXB2. Using endothelial cells with their cellular lipids labeled with [3H]arachidonic acid, methacholine did not stimulate the production of [3H]TXB2. When the endothelial cells were incubated with methacholine and control platelets, [3H]TXB2 was detected. If aspirin-treated platelets were incubated with endothelial cells, methacholine did not increase the production of [3H]TXB2. However, pretreatment of the endothelial cells with aspirin did not affect the ability of methacholine to induce [3H]TXB2 release. This suggests that methacholine stimulated the endothelial cell to release arachidonic acid, which was transferred to the platelets and metabolized to TXA2. To test whether this cell-cell interaction is necessary for methacholine-induced contractions, rabbits were administered aspirin (20 mg/kg) for 2 days. On day 4, methacholine-induced contractions of pulmonary arteries were depressed in aspirin-treated compared with control subjects. Control arteries synthesized 6-keto-prostaglandin F1
and TXB2. Aspirin treatment inhibited both pulmonary artery and platelet TXB2 production but had no effect on vessel 6-keto-prostaglandin F1. These studies implicate platelets as a vascular source of TXA2 and indicate that both endothelial cells and platelets may be required for methacholine-induced TXA2 synthesis and vasoconstriction.
Key Words: thromboxane A2 • cyclooxygenase • platelets • arachidonic acid • endothelial cells • endothelium-derived contracting factor
Abbreviations: PG = prostaglandin • PMN = polymorphonuclear leukocyte • RIA = radioimmunoassay • RP-HPLC = reverse-phase high-pressure liquid chromatography • TX = thromboxane
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Introduction |
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In recent years, the importance of various factors synthesized and released from the blood vessel endothelium that contribute to the regulation of vascular tone has become apparent. In pulmonary vessels, we have identified an endothelium-derived contracting factor as thromboxane (TX) A2.1 The control of pulmonary vascular resistance involves the interaction of various vasoconstrictors, such as TXA2, and vasodilators, such as prostacyclin. Because both prostacyclin and TXA2 are cyclooxygenase metabolites of arachidonic acid, it is postulated that a balance in these two compounds contributes to the regulation of vascular tone. Abnormalities in the balance of these factors may then have a role in certain pathological states. For example, an increased synthesis of TXA2 is associated with pulmonary disease,2–8 and TXA2 has been shown to be involved in pulmonary vasoconstriction observed in a number of animal models of pulmonary hypertension.5–8
The major source of TXA2 is the platelet, polymorphonuclear leukocyte (PMN), and monocyte.9–11 Although there are numerous reports of TXA2 production by blood vessels,10,12,13 there is still controversy as to whether endothelial cells synthesize TXA2. Several researchers have reported that cultured endothelial cells produce not only prostacyclin but also TXA2.14–16 In contrast, we and others have failed to detect TXB2 synthesis by endothelial cells.1,17–21 Cultured rabbit pulmonary arterial endothelial cells synthesized 6-keto-prostaglandin (PG) F1, the stable metabolite of prostacycline, but not TXB2.1 Immunohistochemical studies indicated the presence of cyclooxygenase, but not TX synthase, in pulmonary artery endothelial cells. Campbell and coworkers16 reported that primary cultures of umbilical endothelial cells produced TXB2. However, when the cells were passaged, TXB2 production was lost. The synthesis of TXB2 in the primary cultures was associated with adherent platelets. With passage, adherent platelets were lost, as was TXB2 synthesis. Because it is important to identify the cellular source of TXA2 in pulmonary vessels to assess its role in pulmonary function, the present study was designed to test the hypothesis that TXA2 synthesis by intact pulmonary arteries requires an interaction between the endothelial cells and adherent platelets. Additional experiments are described that use aspirin-treated rabbits to study the role of the platelet in the vasoconstrictor response to arachidonic acid and methacholine in the intact pulmonary artery.
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Animals
Two-month-old male New Zealand White rabbits were obtained from New Franken Rabbitry (New Franken, WI). The animals were housed in the Medical College of Wisconsin Animal Care Facilities and maintained on a standard rabbit chow diet and given tap water ad libitum. Rabbits were anesthetized with intravenous sodium pentobarbital (120 mg/kg), and the heart and lungs were removed as a unit and placed immediately in Krebs-bicarbonate buffer of the following composition (mmol/L): NaCl 118, KCl 4, CaCl2 3.3, NaHCO3 24, KH2PO4 1.4, MgSO4 1.2, glucose 11, pH 7.4. The main pulmonary artery was identified at its origin from the right ventricle, and both left and right pulmonary arteries were dissected to their most distal end. The pulmonary artery distal to the first branching of the left or right pulmonary artery was used, and this is referred to as the intrapulmonary artery.
Platelet-Endothelial Cell Experiments
Blood was collected from New Zealand White rabbits in 3.2% citrate and centrifuged at 150g for 10 minutes.10,22 The supernatant contains platelet-rich plasma. Platelets were sedimented by centrifugation of platelet-rich plasma at 2000g for 10 minutes, washed, and resuspended in buffer of the following composition (mmol/L): HEPES 10, NaCl 150, KCl 5, CaCl2 2, MgCl2 1, and glucose 11, pH 7.4. Endothelial cells were isolated and cultured from the rabbit pulmonary arteries by a modification of methods described previously.1 Endothelial cells were grown in 24-well plates. Washed platelets (6x108 platelets/well) or HEPES buffer were layered over the endothelial cells or added to empty wells. Cells were immediately treated with buffer or methacholine (10-4 mol/L) and incubated for 30 minutes at 37°C. The buffer was removed, and TXB2 and 6-keto-PGF1 production was measured by specific radioimmunoassays (RIA) using the method of Campbell and Ojeda.1,23 In an additional experiment, platelet-rich plasma was treated with 100 µmol/L aspirin for 30 minutes at 37°C. The platelets were washed twice to remove the unreacted aspirin, and the above experimental protocol was repeated. Finally, endothelial cells were treated with 100 µmol/L aspirin for 30 minutes at 37°C. The cells were repeatedly washed to remove the unreacted aspirin, and the above experimental protocol was repeated.
These studies were confirmed with [3H]arachidonic acid. Endothelial cells were incubated overnight with 0.5 µCi [3H]arachidonic acid to label their phospholipids. Under these labeling conditions, only 10% of the added radioactivity remains in the labeling buffer and the remainder is incorporated into endothelial cell lipids.24 After the prelabeling period, cells were washed four times with HEPES buffer containing 1% fatty acid-free BSA. The washed cells were incubated in fresh protein-free HEPES buffer in the presence or absence of platelets (1.2x109 platelets/ incubation) and treated with methacholine (10-4 mol/L) or its vehicle for 15 minutes at 37°C. Radioactivity released into the incubation media was extracted and analyzed by reverse-phase high-pressure liquid chromatography (RP-HPLC) as described previously.1 Column eluate was collected in 0.5 mL fractions, and radioactivity was determined by liquid scintillation spectrometry or the radioactivity in the eluate was detected with a Ramona-D radioactivity detector (Raytest, Pittsburgh, PA). Elution times of radioactive peaks were compared with retention times of known PG standards. These experiments were repeated with aspirin-treated platelets and aspirin-treated endothelial cells as described above.
Vascular Reactivity
Two-month-old New Zealand White rabbits were treated with aspirin (5, 10, or 20 mg/kg PO) or its vehicle on days 1 and 2. On day 4, the pulmonary vessels and platelets were isolated as described above. Strips of artery (30 mg, wet weight) or isolated platelets (500x106/ mL) obtained from the control and aspirin-treated rabbits were placed in HEPES buffer and incubated at 37°C for 15 minutes with [14C]arachidonic acid (0.05 µCi, 10-7 mol/L) and the calcium ionophore A23187 (20 µmol/L). The incubation buffer was extracted and analyzed using RP-HPLC.1 To quantitate 6-keto-PGF1 and TXB2 production, vessels (3 mg, wet weight) from control and aspirin-treated rabbits were incubated at 37°C in HEPES buffer containing vehicle or arachidonic acid (10-5 mol/L) for 15 minutes. The synthesis of 6-keto-PGF1 and TXB2 was measured by RIA.23 Additionally, rings (2 to 3 mm) of pulmonary arteries were obtained and suspended in 15 mL organ baths containing Krebs-bicarbonate buffer at 37°C and continuously aerated with 95% O2/5% CO2.1 Isometric tension was measured with Grass force-displacement transducers and recorded with a Grass polygraph (model 7D). Resting tension was adjusted to its length tension maximum of 1 g. The vessels were allowed to equilibrate for 1 hour. Contractions were produced by increasing the KCl concentration of the bath to 40 mmol/L. KCl-induced contractions were repeated until maximal reproducible responses were obtained. Concentration-response curves were obtained by the cumulative addition of either arachidonic acid (10-8 to 10-5 mol/L), methacholine (10-8 to 10-3 mol/L), or U46619 (10-12 to 10-7 mol/L). Because KCl contractile responses remain stable throughout the experiment, results were expressed as a percentage of the KCl contraction. Methacholine was dissolved in distilled water, and a volume of 0.05 mL was added to the tissue baths. Arachidonic acid and U46619 were dissolved in ethanol and given in a volume that yielded a final ethanol concentration of the bath of <0.07%.
Polyacrylamide Gel Electrophoresis and Western Blotting
Rabbit pulmonary arteries, rabbit platelets, and rabbit pulmonary artery endothelial cells were obtained as described above. Platelets, endothelial cells, and pulmonary artery lysates were prepared by homogenizing samples in a buffer containing 20 mmol/L HEPES, 255 mmol/L sucrose, 1 mmol/L EDTA, and 100 µmol/L phenylmethylsulfonylfluoride, pH 7.4. The protein lysates were analyzed by SDS-PAGE by the method of Laemmli25,26 using a 4% acrylamide stacking gel and a 10% acrylamide resolving gel.27 The protein concentration of the pulmonary artery lysates, pulmonary endothelial cell lysates, and platelet lysates was 10 µg. Platelet lysates were also analyzed at a protein concentration of 25 µg. Human platelet microsomes (Biomol Research Laboratories, Inc., Plymouth Meeting, PA) enriched in TX synthase (4 µg) were included as a positive control. The purity of the human microsomal preparation is approximately 47%. The proteins were electrophoretically transferred to nitrocellulose, and the nitrocellulose membrane was blocked for 4 hours at 4°C with 2% nonfat dry milk in Tris-buffered saline (20 mmol/L TRIZMA hydrochloride; 500 mmol/L NaCl, pH 7.5) with Tween-20 (TTBS) before incubation with a polyclonal TX synthase antibody. This polyclonal antibody was made in our laboratory against a unique peptide sequence of TX synthase (NH2-Cys-Ser-Lys-Ser-Ala-Leu-Gly-Pro-Lys-Asn-Gly-Val-COOH). The peptide sequence was synthesized by the Protein and Nucleic Acid Analytical Facility located at the Medical College of Wisconsin. The peptide was conjugated covalently to keyhole limpet hemocyanin and injected with adjuvant into rabbits.28 Sera from the rabbits was screened for antibody production using an enzyme-linked immunoassay.29 Preliminary results indicated that rabbits produced an antibody that selectively recognized rabbit platelet TX synthase. The primary antibody was used at a dilution of 1:1000 for 1 hour at 4°C. After washing, the blot was incubated for 30 minutes with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG antibody) at a dilution of 1:3000. After again washing with TTBS, the blot was incubated for 1 minute with DuPont Renaissance Chemiluminescen: reagents. The membrane was subsequently exposed to Kodak Biomax MR imaging film and developed. Prestained protein markers were used for molecular mass determination.
Statistics
Data are expressed as the mean±SEM. Statistical analysis of the data was performed with an analysis of variance to determine differences within the groups followed by Dunnet’s modification of the t test to determine differences between groups. A value of P<.05 was considered statistically significant.
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[14C]Arachidonic acid was obtained from DuPont NEN (Boston, MA); [3H]6-keto-PGF1
and [3H]TXB2 were from Amersham (Arlington Heights, IL); arachidonic acid was from Nu-Check Prep, Inc. (Elysian, MN); ß-methacholine, A23187, and aspirin were from Sigma; U46619, 6-keto-PGF1 and TXB2 were from Cayman Chemical Company (Ann Arbor, MI). Thromboxane synthase was from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). All cell culture reagents were purchased from GIBCO (Grand Island, NY). Flasks used in cell culture were from Corning (Corning, NY) All other chemicals were of reagent grade. |
Results |
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Using a specific polyclonal anti-TX synthase antibody, Western blot analysis showed the presence of immunoreactive bands in lysates prepared from rabbit platelets and rabbit pulmonary arteries (Fig 1). These bands corresponded to the 60-kDa TX synthase protein.30–32 Human platelet microsomes enriched in TX synthase were included as a positive control. In pulmonary endothelial cells, the presence of TX synthase was not detected. This experiment was repeated 3 times, and similar results were observed.
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We determined whether platelets synthesized TXB2 in response to methacholine. When platelets were incubated with varying concentrations of methacholine, we failed to detect any stimulation of TXB2 synthesis by RIA (data not shown). In contrast, arachidonic acid caused a concentration-related increase in TXB2 synthesis by platelets (data not shown). To determine whether the production of TXB2 by intact pulmonary artery required an interaction between endothelial cells and adherent platelets, endothelial cells and platelets were incubated as described under Methods. As shown previously, endothelial cells alone did not produce TXB2 under either basal (Table 1) or methacholine-stimulated conditions.1 In the presence of endothelial cells and platelets, TXB2 was enhanced approximately 2-fold by the addition of methacholine (Table 1). Because aspirin irreversibly inactivates cyclooxygenase, it was used as a tool to investigate the interaction between these cells. Results showed that when endothelial cells were coincubated with aspirin-treated platelets, methacholine failed to induce TXB2 synthesis (Table 1). In contrast, when aspirintreated endothelial cells were coincubated with platelets, methacholine-induced TXB2 production was not impaired (Table 1). The production of 6-keto-PGF1 was measured in the coincubation studies (Table 1). The addition of platelets to endothelial cells did not alter the production of 6-keto-PGF1. If the endothelial cells were pretreated with aspirin, 6-keto-PGF1 production decreased (data not shown). In the presence of normal platelets, aspirin-treated endothelial cell production of 6-keto-PGF1 was still attenuated (Table 1).
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These studies were confirmed with [3H]arachidonic acid and the results shown in Fig 2. In radiolabeled endothelial cells incubated with methacholine (10-4 mol/L), there was no production of [3H]TXB2 (Fig 2A). When the endothelial cells were incubated with methacholine in the presence of control platelets, [3H]TXB2 was a major synthetic product (Fig 2B). If aspirin-treated platelets were added to normal endothelial cells, methacholine failed to stimulate the production of [3H]TXB2 (Fig 2C). In contrast, aspirin pretreatment of endothelial cells blocked the production of the cyclooxygenase products 6-keto-PGF1 and -PGF2; however, pretreatment of the endothelial cells with aspirin did not affect the ability of methacholine to induce TXB2 release in the presence of normal platelets (Fig 2D). The production of [3H]6-keto-PGF1 by endothelial cells was not affected by the addition of platelets (Fig 2B). When the endothelial cells were pretreated with aspirin and coincubated with normal platelets, no production of [3H]6-keto-PGF1 was observed (Fig 2D).
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The next series of experiments was designed to use aspirin-treated rabbits to further study the role of platelets in the vasoconstrictor response to arachidonic acid and methacholine in the intact pulmonary artery. Arachidonic acid and methacholine produced concentration-related contractions in the control rabbits (Fig 3). However, in the rabbits treated with the highest dose of aspirin (20 mg/kg), there was a significant attenuation of both arachidonic acid-(maximal contraction 66.1±5.4% versus 15.2±3.8%, control versus aspirin-treated; P<.05) and methacholine-(maximal contraction 55.4±5.8% versus 16.3±4.0%, control versus aspirin-treated; P<.05) induced contractions (Fig 3). The vasoconstrictor response to the TX-mimetic U46619 was the same in the control and high-dose aspirin-treated animals (data not shown). To assess cyclooxygenase inhibition, pulmonary arteries and platelets from the control and aspirin-treated rabbits were incubated with [14C]arachidonic acid and extracted metabolites analyzed by RP-HPLC. Both control (Fig 4A) and high-dose aspirin-treated (Fig 4B) pulmonary arteries synthesized [14C]6-keto-PGF1; however, its synthesis was less in the vessels from the aspirin-treated rabbits. When measured by RIA, it also seemed that 6-keto-PGF1 production was reduced in the aspirin-treated rabbit pulmonary arteries (Table 2). High-dose aspirin treatment inhibited the production of [14C]TXB2 (Fig 4B). By HPLC analysis, TXB2 production was 45.3±18 cps/mg for the control pulmonary arteries and 23.0±8 cps/mg tissue for the aspirin-treated pulmonary arteries. When measured by RIA, there was an approximate 60% reduction in TXB2 production in the pulmonary arteries obtained from aspirin-treated rabbits compared with the control rabbits (Table 2). Likewise, in the platelets from the high-dose aspirin-treated rabbits, [14C]TXB2 production was depressed compared with control rabbits (Fig 4C and 4D).
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Because 6-keto-PGF1 production was lower in the high-dose aspirin-treated rabbits compared with control rabbits, additional experiments were performed in which rabbits were treated with lower doses of aspirin. The vascular reactivity responses to methacholine in rabbits administered the low-dose regimen (5 mg/kg, PO) or the medium-dose regimen (10 mg/kg, PO) is shown in Fig 3 (bottom). The lower dose of aspirin did not reduce the contractile response to methacholine compared with the control rabbits, whereas the medium dose produced an approximate 50% reduction in methacholine-induced contractions. The low-dose aspirin treatment did not alter either the pulmonary artery or platelet production of [14C]TXB2 compared with the control rabbits (data not shown). In pulmonary arteries obtained from rabbits treated with the 10 mg/kg dose of aspirin, [14C]6-keto-PGF1 production was similar when compared with the control rabbits (Fig 5); however, the platelet [14C]TXB2 production was decreased in the treated rabbits compared with the control rabbits.
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Discussion |
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Our original study reported that endothelium-dependent contractions are produced by arachidonic acid and methacholine in rabbit intrapulmonary artery. Using a variety of techniques, including vascular reactivity studies with inhibitors, bioassay, HPLC, RIA, and gas chromatography/mass spectrometry analysis, our previous work conclusively showed that TXA2 is the mediator of these contractions. However, the question remained as to which cell type(s) in the intact pulmonary artery produce TXA2. Although there are numerous reports of TXA2 production by blood vessels,10,12,13 controversy exists as to whether endothelial cells synthesize TXA2. Some researchers have reported that cultured endothelial cells produce not only prostacyclin but also TXA2.14,15 In contrast, other studies did not detect TXA2 synthesis by endothelial cells.17–21 In our previous study,1 immunofluorescence of rabbit pulmonary endothelial cells failed to detect the presence of TX synthase, whereas the presence of cyclooxygenase was clearly shown. The present study further supported the absence of TX synthase in cultured rabbit pulmonary artery endothelial cells. Using a specific polyclonal anti-TX synthase antibody, Western blot analysis showed the presence of immunoreactive bands in lysates prepared from rabbit platelets and intact rabbit pulmonary artery. These bands corresponded to the 60-kD TX synthase protein purified from human platelets30–32 and comigrated with a human platelet microsome enriched in TX synthase. However, in lysates prepared from the rabbit pulmonary artery endothelial cells, no immunoreactive band was observed. Our previous experiments also showed that endothelial cells incubated with [14C]arachidonic acid did not produce [14C]TXB2. When a more sensitive RIA method was used to quantitate TXB2 production under both basal and stimulated conditions, no detectable TXB2 was measured. Taken collectively, these studies would be consistent with some cell type other than endothelial cells releasing TXA2. Because TXA2 is the major arachidonic acid metabolite in platelets, we tested the hypothesis that TXA2 synthesis by intact pulmonary arteries requires an interaction between endothelial cells and platelets.
We showed that methacholine stimulated TXB2 synthesis in intact pulmonary artery.1 However, incubation of platelets with methacholine does not stimulate TXB2 production, and cholinergic receptors have not been reported to exist on platelets. Therefore, a question remains as to how methacholine enhances the release of TXA2 from the platelet in the intact pulmonary artery. PGH2 from stimulated platelets may be taken up by endothelial cells and used to produce PGI2; however, this transfer of PGH2 does not operate in the reverse direction, ie, platelets do not take up PGH2 from endothelial cells for TXA2 synthesis.17,19 It is possible that endothelial cells will, however, transfer arachidonic acid to platelets and promote TXB2 synthesis. We investigated this interaction in cultured endothelial cells and platelets during methacholine stimulation. Methacholine stimulated TXB2 synthesis in platelets and endothelial cells but not in platelets alone or in endothelial cells alone, supporting our hypothesis that an interaction between these two cell types is required for methacholine-induced TXA2 synthesis. Additional experiments used aspirin as a tool to confirm that arachidonic acid and not the cyclooxygenase metabolite PGH2 was transferred to the platelet and further metabolized to TXA2. Aspirin irreversibly inactivates cyclooxygenase by acetylating the enzyme.33 Pretreatment of platelets with aspirin blocks methacholine-induced TXB2 synthesis when platelets and endothelial cells are coincubated, indicating that platelet cyclooxygenase is required. These data suggest that endothelial cells release arachidonic acid on cholinergic stimulation and that the platelets convert this arachidonic acid to TXB2. To further support this conclusion, aspirin-treated endothelial cells and platelets produced TXB2 in response to methacholine, indicating that endothelial cell cyclooxygenase is not required and suggesting that endothelial cell-derived arachidonic acid is the source of TXB2. This conclusion is represented schematically in Fig 6.
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The last series of experiments used aspirin-treated rabbits to study the role of the platelet in the vasoconstrictor response to arachidonic acid and methacholine in the intact pulmonary artery. It has been documented that aspirin can selectively inhibit platelet cyclooxygenase because of the inability of platelets to regenerate their cyclooxygenase. Thus, aspirin has a longer duration of action in platelets than in endothelial cells.34 This treatment regimen was designed to selectively inhibit platelet, but not endothelial, cyclooxygenase. In rabbits treated with high and medium doses of aspirin, platelet and pulmonary artery [14C]TXB2 synthesis was inhibited. Pulmonary artery production of [14C]6-keto-PGF1 was reduced in the high-dose aspirin-treated rabbits compared with the control rabbits. However, at the lower doses of aspirin, pulmonary artery 6-keto-PGF1 production was similar between the treated and control rabbits. The vasoconstrictor responses to methacholine and arachidonic acid were reduced in vessels from the medium- and high-dose aspirin-treated rabbits but not in the vessels from control rabbits or in rabbits treated with the low-dose aspirin regimen. The response to U46619 was the same in the vessels from control and aspirin-treated animals. Because platelet TXA2 production is decreased in the aspirin-treated rabbits that exhibited decreased contractile response to methacholine, these results would implicate platelets as the cellular source of TXA2 production. It is important to note that a recent report by Barry et al35 has shown that microparticles released from platelets also play a role in the transcellular metabolism of arachidonic acid. Specifically, platelet microparticles were shown to transfer arachidonic acid to the platelets to be used for TXA2 synthesis. Although this mechanism was not explored in the present study, it gives further support for how platelets can influence vascular function.
Although the major source of TXA2 is the platelet, PMN and monocyte also produce TXA2.9–11 Therefore, PMNs or monocytes may also be possible sources of TX synthase in the rabbit pulmonary artery. Because this potential mechanism was not examined in the present study, the role of PMNs and monocytes cannot be eliminated.
Therefore, we propose that methacholine-induced contractions of rabbit pulmonary artery are mediated by the release of arachidonic acid from endothelial cells and its transfer to adherent platelets that subsequently synthesize the contracting factor TXA2. TXA2 is released by the platelet and has a direct effect on vascular smooth muscle TXA2 receptors. These studies support the concept of transcellular metabolism of arachidonic acid in the pulmonary vasculature. Because TXA2 is an important mediator of pulmonary hypertension caused by a variety of pathophysiological conditions,2–8 the identity of the platelet, and not the endothelium, as a cellular source of TXA2 may provide further insight into the role of platelets in the regulation of pulmonary vascular tone.
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Acknowledgments |
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We thank Joseph James and Donna Kotulock for technical assistance and Gretchen Barg for secretarial assistance. These studies were supported by grants from the National Heart, Lung, Blood Institute (HL-37981 and HL-57895).
Received September 17, 1997; first decision October 7, 1997; accepted October 30, 1997.
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