Vascular Smooth Muscle Thromboxane A2 Receptors Mediate Arachidonic Acid-Induced Sudden Death in Rabbits
We recently identified a subgroup of rabbits (called nonresponders) that were deficient in vascular thromboxane A2 receptors. Thromboxane A2-mediated platelet aggregation was not different between responders and nonresponders. In the present study, we utilized these nonresponders as a model to study the relative contribution of the platelet and vascular thromboxane A2 receptors to the observed hemodynamic responses associated with arachidonic acid-induced sudden death. Mean arterial pressure was slightly but not significantly lower in the nonresponders compared with the responders. However, nonresponders were protected from arachidonic acid-induced sudden death. While 100% of the responders died at the 2.0 mg dose of arachidonic acid, only 27% of nonresponders died at this same dose. Administration of the thromboxane A2 mimetic U46619 (5 μg/kg IV) decreased blood pressure by 41±6 mm Hg in responders but had no effect in the nonresponders. The affinity and density of thromboxane A2 receptors in cultured aortic vascular smooth muscle cells obtained from both responders and nonresponders were assessed using radioligand binding. The Kd values were not different (4.4±1.0 versus 2.4±0.6 nmol/L, responder versus nonresponder). However, there was a significant decrease in the density of receptors from vascular smooth muscle cells of nonresponders (Bmax=397±59 versus 157±59 fmol/106 cells, responder versus nonresponder, P<.01). U46619 produced a concentration-dependent increase in [3H]-thymidine incorporation into responder vascular smooth muscle cells but had no effect in the nonresponder cells. Using an anti-thromboxane A2 receptor antibody, we compared the amount of receptor expressed in aortic tissue obtained from responders and nonresponders. Consistent with the results observed with [3H]-thymidine uptake and radioligand binding assays, the expression of thromboxane A2 receptor protein was decreased in nonresponder compared with responder vascular tissue. Platelet thromboxane A2 receptor expression was not different. These studies demonstrate that the vascular smooth muscle cells of nonresponder rabbits are deficient in the thromboxane A2 receptor. Furthermore, the reduction in arachidonic acid-induced sudden death in nonresponders indicates that the vascular smooth muscle thromboxane A2 receptor mediates this effect.
- thromboxane A2
- vascular smooth muscle cells
- arachidonic acid
- mean arterial pressure
- radioligand binding
- HR = heart rate
- I-BOP = [1S-(1α,2β(5Z),3α(1E,3R*),4α)]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1]heptan-2-y1]-5-heptenoic acid
- MAP = mean arterial pressure
- PAGE = polyacrylamide gel electrophoresis
- PG = prostaglandin
- PRP = platelet-rich plasma
- TTBS = Tris-buffered saline (20 mmol/L TRIZMA hydrochloride; 500 mmol/L NaCl, pH 7.5) with Tween-20
- TX = thromboxane
An increased synthesis of thromboxane (TX) A2 is associated with a number of cardiovascular diseases including pulmonary hypertension, unstable angina and myocardial ischemia.1 TXA2 and its immediate precursor, PG H2 (PGH2), induce platelet aggregation and vascular smooth muscle vasoconstriction.2 TXA2 is also mitogenic for vascular smooth muscle.3–5⇓⇓ TXA2 and PGH2 share a common membrane-bound receptor, and these receptors are found in a variety of tissues, including platelets and vascular smooth muscle cells.6 Using methods of equilibrium ligand binding, platelets and blood vessels reversibly bind TX agonists and antagonists and have saturable binding sites that are specific for TX agonists and antagonists.7–13⇓⇓⇓⇓⇓⇓ The receptor from the human platelet has been purified, and the receptors from the placenta and megakaryocyte have been cloned.14,15⇓ A splice variant of this placental receptor has been described in endothelial cells.16 The vascular receptor has not been purified or cloned to date.
We recently reported that a subgroup of rabbits was deficient in vascular TXA2 receptors.17 Pulmonary arteries obtained from these rabbits, termed nonresponders, failed to contract to TXA2 agonists. However, the vascular responses to other vasoconstrictor agents such as norepinephrine, KCl, and endothelin were not altered in the nonresponder rabbits. When we prepared membranes from vessels of responder and nonresponder rabbits, there was a significant decrease in TXA2 receptor number in the nonresponders. These findings were not restricted to the pulmonary vasculature as the decrease in TXA2 receptors and lack of vasoactive effect of the TXA2 mimetics were also observed in the aorta, supporting the results of Keith and Salama.18 In contrast to the absence of vasoconstrictor effects of TXA2 agonists in vascular tissue from nonresponder rabbits compared with responder rabbits, we also reported that the platelet aggregatory response to TXA2 agonists was similar between the two groups.17 Platelet receptor number was not different in responders and nonresponders. Thus, the nonresponder rabbits were deficient in vascular but not platelet TXA2 receptors. The rank-order potency of activity and binding of TX agonists differs between platelets and blood vessels, suggesting that the platelet and vascular TXA2 receptors are not the same.11,12⇓ A difference in the potency of TX antagonists was also reported between these tissues. In contrast, other studies provided pharmacological evidence that the platelet and vascular receptors are similar.9,19,20⇓⇓
One limitation to studying the role of TXA2 in cardiovascular disease has been the inability to differentiate the contributions of the platelet and the vascular smooth muscle to the observed hemodynamic responses. For example, Lefer and coworkers21,22⇓ showed that arachidonic acid (2.0 mg/kg IV) caused death within 5 minutes in rabbits. This arachidonic acid-induced sudden death has been observed in a number of other species.23,24⇓ Pretreatment with an inhibitor of TXA2 synthase or with a TXA2 receptor antagonist protects the animals from arachidonic acid-induced sudden death,22 suggesting that the sudden death is due to an action of TXA2 at the platelet to cause intravascular platelet aggregation, at the vascular smooth muscle to cause vasoconstriction, or at the bronchioles to cause bronchoconstriction. Since there is no evidence that rabbit airway smooth muscle cells have TXA2 receptors,25 arachidonic acid-induced sudden death in rabbits is mediated through either the platelet or vascular smooth muscle receptor. Because the TXA2 antagonists are not selective and cannot distinguish between vascular and platelet receptors,11,12⇓ the nonresponder rabbits provide an effective model to examine the role of the vascular versus the platelet receptor. We hypothesized that because the nonresponder rabbits lacked vascular but not platelet TXA2 receptors, responses to arachidonic acid would be reduced in the intact animal. Specific experiments were designed to first characterize the vascular smooth muscle cell TXA2 receptor in responder and nonresponder rabbits and to then examine the contribution of this vascular receptor to arachidonic acid-induced sudden death in responder and nonresponder rabbits.
Arachidonic acid (sodium salt) was from Nu-Chek Prep, Inc. U46619, I-BOP, and TXB2 were from Cayman Chemical Co. [3H]-TXB2 was from Amersham. [3H]-Thymidine was from DuPont NEN. SQ 29548 was provided by S.J. Lucania of Bristol-Meyers-Squibb (Princeton, NJ). All cell culture reagents were purchased from GIBCO. Flasks used in cell culture were from Corning. All other chemicals were of reagent grade.
Two- to three-month-old male New Zealand White rabbits (New Franken Rabbitry, New Franken, Wis) were anesthetized with sodium pentobarbital (120 mg/kg IV), and the aorta was removed and placed immediately in Krebs' bicarbonate buffer of the following composition (mmol/L): NaCl 118, KCl 4, CaCl2 3.2, NaHCO3 24, KH2PO4 1.4, MgSO4 1.2, glucose 11, pH 7.4. The aorta was cleaned of adherent fat and connective tissue using care not to disturb the endothelial layer. Rings of aorta (3 to 4 mm) were obtained and suspended in 15 mL organ baths containing Krebs’ bicarbonate buffer, which was warmed to 37°C and continuously aerated with a 95% O2/5% CO2 mixture. Isometric tension was measured with force-displacement transducers and recorded with a Grass model 7D polygraph. Resting tension was adjusted to 2 g and the vessels were allowed to equilibrate for 1 hour. The KCl concentration of the baths was increased to 40 mmol/L until stable, reproducible contractions were produced. After the vessels reached peak contraction to 40 mmol/L KCl, tissue baths were rinsed and the vessels allowed to return to resting tension. Responses to the TXA2 mimetic U46619 (10−7 mol/L) were obtained. Results were expressed as the percent contraction of the maximal response to 40 mmol/L of KCl. Animals in which the aortic rings contracted to KCl but not to U46619 were called nonresponders.17
Vascular smooth muscle cells were isolated and cultured from responder and nonresponder rabbit aortas using previously described methods.26 After collection of endothelial cells,26 strips of denuded vessels were placed into gelatin-coated flasks with M199 medium containing 10% fetal calf serum with l-glutamine (1%) and antibiotics (1% antibiotic/antimycotic solution; 10 000 U penicillin, 10 mg streptomycin, and 25 ng amphotericin B [Sigma Chemical Co]). Smooth muscle cells migrated to the flasks within 3 to 5 days. Once growth was established on the coated flasks, then the vessels were removed and the cells resuspended in M199 medium containing 20% fetal calf serum. Purity of smooth muscle cells was confirmed by positive staining for smooth muscle cell α-actin. For radioligand binding and [3H]-thymidine uptake assays, cells in second or third passages were used.
For measurement of TXA2 receptors, confluent aortic vascular smooth muscle cells obtained from responder and nonresponder rabbits were detached with 0.04% trypsin in PBS. The equilibrium binding studies were performed by incubating aliquots of cells (2.5×105) for 30 minutes at 37°C with 2×104 cpm of the TXA2 agonist 125I-BOP (2000 Ci/mmol)13 and varying concentrations of 127I-BOP in a final volume of 0.2 mL. The reaction was terminated by the addition of 4 mL of ice-cold 50 mmol/L Tris HCl (pH 7.4) followed by the filtration of the sample through Whatman GF/C glass fiber filters. The filters were then washed with three additional 4-mL volumes of buffer, and bound radioactivity was counted in a gamma counter (Packard Instrument Co). Nonspecific binding was determined in the presence of the TXA2 receptor antagonist SQ 29548 (10−5 mol/L).
Measurement of [3H]-Thymidine Incorporation
To measure [3H]-thymidine incorporation,3 vascular smooth muscle cells from responder and nonresponder rabbit aortas were grown to 60% confluence in 24-well plates. The cells were exposed to DMEM containing antibiotics (1% antibiotic/antimycotic solution) and 0.5% fetal bovine serum to arrest cell growth. After 48 hours, new DMEM was added containing 0.5% fetal bovine serum, and the cells were stimulated with increasing concentrations of U46619 for 24 hours. In some experiments, the TXA2 receptor antagonist SQ 29548 (10−5 mol/L) was included in the incubation. [3H]-Thymidine (1 μCi/well) was added during the final 4 hours. The medium was removed, and cells were washed with ice-cold PBS, followed by 0.3 mL of ice-cold perchloric acid (0.3 mol/L) and PBS. In order to solubilize the cells, 0.5 mL of 0.1% SDS/0.1N NaOH was added to each well. Radioactivity in the solubilized monolayer was determined by liquid scintillation spectrometry.
PAGE and Western Blotting
Rabbit aorta was obtained from responder and nonresponder rabbits as described above. Whole blood from responder and nonresponder rabbits was collected in 3.2% sodium citrate (pH 7.4, 9:1). PRP was prepared by centrifuging the blood at 150g for 10 minutes. The PRP was centrifuged at 1000g for 20 minutes to obtain the platelets. Platelet and aortic 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 (10 μg/lane) were resolved by SDS-PAGE by the method of Laemmli27 and Coligan et al28 using a 4% acrylamide stacking gel and 10% acrylamide resolving gel.29 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 receptor antibody (kindly provided by Dr Guy Le Breton, University of Illinois, Chicago). Incubation with the primary antibody was at a dilution of 1:1000 for 1 hour at 4°C. Following 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 chemiluminescent reagents. The membrane was subsequently exposed to Kodak Biomax MR scientific imaging film to visualize protein separation. Prestained protein markers (Sigma) were used for molecular mass determination.
The central ear artery from rabbits was cannulated with an Exel SafeLet catheter (24 gauge by 3/4″) under local anesthesia using lidocaine/prilocaine cream (Astra Pharmaceutical Products, Inc). MAP was measured in conscious rabbits using a Narco pressure transducer and a Grass model 7D polygraph. HR was obtained from the arterial blood pressure tracing. Rabbits were monitored in a quiet room for ≈30 minutes before drug administration. Following the measurement of MAP and HR in the conscious rabbits, the animals were anesthetized with sodium pentobarbital (30 mg/kg IV). Sodium arachidonic acid was diluted in 1 mmol/L Na2CO3 just prior to use and injected into the ear vein at varying concentrations (vehicle, 0.5, 1.0, and 2.0 mg/kg) over a 15- to 20-second period. MAP was continually monitored. The vehicle had no effect on MAP or HR in either group of rabbits. In a separate experiment, animals were administered U46619 (5 μg/kg IV; in saline) and hemodynamic responses monitored. Following both the arachidonic acid and U46619 experiments, aortas were removed and suspended in tissue baths, and contractile responses to U46619 were determined as described above.
Synthesis of TXA2
Blood was collected from responder and nonresponder rabbits in tubes containing heparin (1000 U/mL) and indomethacin (10 μmol/L) at 4°C. The plasma was obtained following centrifugation at 1500g for 10 minutes at 4°C. Plasma samples were extracted as previously described.30 Briefly, 1000 cpm of 3H-TXB2 was added to each plasma sample to be used for calculation of extraction recoveries. The sample was acidified to pH <2.0 and extracted over octadecylsilyl columns. The column was washed sequentially with 15% ethanol, water, and petroleum ether. The TXB2 was eluted with ethyl acetate, evaporated to dryness under a stream of nitrogen, and reconstituted in toluene/ethyl acetate/ methanol/glacial acetic acid (60:40:5:0.01). The sample was then purified over a silicic acid extraction column. The column was rinsed with toluene/ethyl acetate/methanol (60:40:3). TXB2 was eluted with toluene/ethyl acetate/methanol/water (60:40:4.5:0.1), evaporated under a stream of nitrogen, and dissolved in 0.1 mol/L sodium phosphate buffer containing 0.1% polyvinylpyrrolidone. TXB2 was measured in the extracted samples by specific radioimmunoassay.30 The antibodies for TXB2 were produced in rabbits in our laboratory. The sensitivity of the assay is less than 1 pg and the cross-reactivity of the TXB2 antisera with known arachidonic acid metabolites is less than 0.1%.
Data are expressed as the mean±SEM for observations in vessels obtained from different rabbits. When multiple comparisons were made, statistical evaluation was performed using a one-way ANOVA followed by the Sidak multiple-comparison test when significant differences were present. A Student’s t test was used to analyze the radioligand binding, radioimmunoassay, and hemodynamic data. A value of P<.05 was considered statistically significant. [125I]BOP saturation data and Scatchard plots were determined using a computer-assisted (PRISM) nonlinear regression analysis.
Vascular Reactivity Responses in Responder and Nonresponder Rabbits
Isolated aortic rings were obtained from rabbits and tested for U46619-induced contractions. In one group of rabbits, U46619 produced a concentration-dependent contraction (Fig 1). This group was referred to as responders. Aortic rings from a second group of rabbits exhibited no contractile activity to U46619 and were called nonresponders. A total of 121 rabbits were used for the vascular reactivity, hemodynamic, radioligand binding, and cell culture studies. Of that number, 35 (28.9%) were identified as nonresponders. The maximal contraction to KCl (40 mmol/L) was also compared in aortic rings from responder and nonresponder rabbits. No significant differences in the ability of vessels from responders and nonresponders to contract to KCI was observed (2.3±0.2 versus 2.5±0.2 g; responder versus nonresponder, NS).
Radioligand Binding in Cultured Vascular Smooth Muscle Cells Isolated From Responder and Nonresponder Aortas
The affinity and density of TXA2 receptors in cultured aortic vascular smooth muscle cells obtained from both responder and nonresponder rabbits were assessed using 125I-BOP. Vascular smooth muscle cells from responder rabbits exhibited specific and saturable binding. In those nonresponder smooth muscle cell preparations where there was measurable binding, it was saturable. A representative analysis of the equilibrium binding studies including saturation curves (top) and the Scatchard analysis (bottom) is shown in Fig 2. Table 1 shows the averaged results for responder and nonresponder vascular smooth muscle cells. The Kd values were not different. However, there was a significant decrease in the density of receptors from vascular smooth cells of nonresponder rabbits (Bmax=397±59 versus 157±59 fmol/106 cells, responder versus nonresponder, P<.01).
Effect of TXA2 Receptor Stimulation on [3H]-Thymidine Incorporation in Cultured Responder and Nonresponder Vascular Smooth Muscle
The ability of U46619 to stimulate the incorporation of [3H]-thymidine into responder and nonresponder vascular smooth muscle cells was examined as an index of receptor activation and vascular smooth muscle cell growth. In responder cells, U46619 produced a concentration-dependent increase in [3H]-thymidine incorporation (Fig 3). In contrast, in the vascular smooth muscle cells obtained from nonresponder rabbits, U46619 had no stimulatory effect.
Pretreatment with the TXA2 receptor antagonist SQ 29548 blocked [3H]-thymidine incorporation (data not shown) in the responder cells. The results shown in Fig 3 are representative of one experiment. The averaged results for four independent experiments were (in cpm/well) 2600±517, 6827±1686, and 9728±1914 versus 2699± 693, 5611±1601, and 4465±943 for basal, U46619 (10−7 mol/L), and U46619 (10−6 mol/L), respectively, responders versus nonresponders.
Plasma TXA2 Concentrations
The plasma concentrations of TXB2 were measured in samples obtained from responder and nonresponder rabbits. TXB2 production was not different between the two groups of rabbits (183±37 versus 240±76 pg/mL, responder versus nonresponder, respectively; n=11, NS).
Expression of TXA2 Receptor in Tissue From Responder and Nonresponder Rabbits
Utilizing a specific polyclonal anti-TXA2 receptor antibody, Western blot analysis showed the presence of immunoreactive bands in lysates prepared from responder and nonresponder aortas and platelets (Fig 4). These bands corresponded to the 55 kD receptor protein previously described by Borg and coworkers.31 In platelets, there was no difference in the 55 kD protein expression between the responder and nonresponder rabbits. However, the vascular expression of the receptor was greatly reduced in nonresponders compared with responders. This experiment was repeated three times and similar results were observed.
Hemodynamic Responses in Responder and Nonresponder Rabbits
MAP and HR measurements obtained from both conscious and anesthetized responder and nonresponder rabbits are summarized in Table 2. Although MAP was slightly lower in the nonresponder rabbits than in the responder rabbits, the differences were not significant. When the 0.5 mg/kg dose of arachidonic acid was administered to responder rabbits, blood pressure decreased and 28% of the rabbits died (Fig 5). In contrast, this same dose of arachidonic acid had no effect on the survival of the nonresponder rabbits. The 1.0 mg/kg dose of arachidonic acid elicited sudden death in 64% of the responder rabbits but only in 13% of the nonresponder rabbits. All responder rabbits (100%) administered the 2.0 mg/kg dose of arachidonic acid died whereas only 27% of the nonresponder rabbits died at this dose.
Because studies showed that the systemic administration of the TXA2 mimetic U46619 induced a fall in blood pressure and cardiac output in rabbits,32,33⇓ we examined the effect of U46619 in responder and nonresponder rabbits. Twelve rabbits received U46619 (5 μg/kg IV). Blood pressure decreased in seven of the animals (decrease in MAP, 41±6 mm Hg), and in three of the seven rabbits, pressure did not recover, and the animals died (Fig 6, A and B). HR was not altered by U46619 in the surviving rabbits (245±6 versus 243±7.2 bpm; pre-U46619 versus post-U46619; n=4; data not shown). Aortic rings obtained from these seven rabbits contracted to U46619 (maximal contraction, 128.9±7.1%, Fig 6C), indicating that these rabbits were responders. In the additional five rabbits receiving U46619, there was no change in blood pressure (increase in MAP, 4.5±3.9 mm Hg) and none of these rabbits died (Fig 6A and 6B). Again, HR was unaltered (257±12 versus 250±15 bpm, pre-U46619 versus postU46619; n=5; data not shown). The contractile response to U46619 in isolated aortic rings obtained from these five rabbits was absent, indicating that these rabbits were nonresponders (Fig 6C).
TXA2 is the predominant metabolite of arachidonic acid in platelets and is a potent inducer of platelet aggregation and constriction of vascular smooth muscle.2 Alterations in synthesis or activity of TXA2 have been implicated as an important pathophysiological mediator of a number of cardiovascular diseases.1 Although the specific receptor for TXA2 has been characterized both pharmacologically and molecularly, less is known about the physiological regulation of the receptor. We have recently found that a subgroup of New Zealand White rabbits did not contract to TXA2 agonists.17 This subgroup of rabbits were referred to as nonresponder rabbits. Using a combination of physiological and pharmacological studies along with ligand binding, these animals were found to be deficient in the vascular but not the platelet TXA2 receptor.17 In the present study, we have utilized these nonresponder rabbits as a model to assess the relative contribution of the platelet and vascular TXA2 receptors to the observed hemodynamic responses associated with arachidonic acid-induced sudden death.
A number of in vitro experiments were performed to confirm that the vascular smooth muscle cell is the source of the TXA2 receptor in the responder rabbits. Dorn9 had previously characterized the TXA2 receptor in cultured aortic vascular smooth muscle cells obtained from rabbits. However, we examined [125I]-BOP binding in vascular smooth muscle cells cultured from responder and nonresponder rabbit aortas that were first identified by the ability of aortic segments to contract to the TXA2 agonist U46619. In this way, we could compare the responses in responder and nonresponder vascular smooth muscle cells. While our results indicated that the affinity of I-BOP for the receptor was not different between responder and nonresponders, there was a significant decrease in receptor number in the nonresponder vascular smooth muscle cells. Since the decrease in receptor number occurred in both acute vascular preparations17 and in cultured cells, these data would also suggest that the deficiency in receptors is related to a defect in the cell and is not related to a factor occurring in the intact animal. No differences in circulating plasma TXA2 concentration were measured between responder and nonresponders.
Because a deficiency in receptor density occurred between cells from responder and nonresponder animals, it was important to determine if a functional difference existed between the cells. Several recent studies have reported the ability of TXA2 mimetics to stimulate vascular smooth muscle cell growth via a TXA2 receptor-mediated process.3–5,34⇓⇓⇓ Cultured vascular smooth muscle cells obtained from responder and nonresponder aortas were incubated with varying concentrations of U46619 followed by a 4-hour labeling with [3H]-thymidine. This procedure showed that U46619 stimulated incorporation of [3H]-thymidine in a concentration-dependent manner only in the responder vascular smooth muscle cells. The effect observed in the responder cells was inhibited by pretreatment with SQ 29548. These findings indicate that the TXA2 receptors in responder vascular smooth muscle cells are functional and coupled to cell growth. No stimulation was observed in cells obtained from the nonresponders, indicating that a deficiency in receptors results in a similar defect in a functional response.
The next series of experiments was performed to determine if the receptor protein was present in the vascular tissue of nonresponder rabbits. Using a polyclonal antibody raised against native TXA2 receptor protein,31 we compared the expression of the vascular receptor in aortic tissue obtained from responder and nonresponder rabbits. Previous studies showed that the antibody recognized the vascular receptor in rabbits.31 Consistent with the results observed with [3H]-thymidine uptake and radioligand binding assays, the expression of TXA2 receptor protein was decreased in nonresponder compared with responder vascular tissue. There was no change in this receptor in platelets from responder and nonresponders.
Finally, an important observation from this study was the protection of nonresponder rabbits from arachidonic acid-induced sudden death. Previous studies indicated that TXA2 mediates arachidonic acid-induced sudden death. Sudden death could be blocked by TXA2 antagonists.21,22⇓ The antagonists are not selective and cannot distinguish between vascular and platelet receptors.11,12⇓ These rabbits provide an effective model to examine the role of the vascular versus the platelet receptor. While 100% of the responder rabbits died at the 2.0 mg dose of arachidonic acid, only 27% of nonresponder rabbits died at this same dose. The survival rate for nonresponder rabbits was 73%. The mechanism for the protection in the nonresponder rabbits must be related to the decrease in vascular TXA2 receptors since our previous study showed that the platelet receptor was unaltered in these rabbits.17 In addition, others reported that rabbit airway smooth muscle does not contract to TXA2 agonists,25 eliminating bronchoconstriction as a mechanism for arachidonic acid-induced sudden death in rabbits.
While the administration of U46619 to isolated vessels elicits vasoconstriction, the in vivo intravenous administration of U46619 to rabbits produces a decrease in MAP32,33⇓ and a fall in cardiac output.32 Because circulating platelet number also decreased in these rabbits,32 the authors hypothesized that U46619 stimulated platelet aggregation, and the consequent platelet entrapment in the pulmonary vasculature contributed to the reduction in cardiac output and fall in MAP. Alternatively, U46619 could directly stimulate the vascular TXA2 receptors to cause intense pulmonary or coronary vasoconstriction with an ensuing reduction in cardiac output. Our data obtained from the intravenous administration of U46619 to responder and nonresponder rabbits would support the second mechanism. Only the responder rabbits demonstrated a fall in MAP in response to U46619. In nonresponder rabbits that lack vascular TXA2 receptors but not platelet TXA2 receptors, U46619 did not lower MAP or affect survival of the animal.
In summary, the present study identifies the presence of functional TXA2 receptors on vascular smooth muscle cells of responder rabbits, whereas a deficiency in receptor protein, number, and function occurs in nonresponder rabbit cells. The additional finding that nonresponder rabbits do not die when administered arachidonic acid supports our original hypothesis that the vascular smooth muscle cell TXA2 receptor mediates arachidonic acid-induced sudden death.
These studies were supported by National Heart, Lung, and Blood Institute grant HL-37981. We thank Joseph James for technical assistance and Gretchen Barg for secretarial assistance.
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