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1 Division of Pulmonary and Critical Care Medicine, The Asthma and Allergy Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21224; and 2 Department of Anesthesiology, St. Louis University, St. Louis, Missouri 63110
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We previously
found that injection of 15-µm microspheres into the bronchial artery
of sheep decreased bronchial artery resistance. This effect was
inhibited partially by indomethacin or 8-phenyltheophylline, suggesting
that microspheres caused release of a dilating prostaglandin and
adenosine. To identify the prostaglandin and confirm adenosine release,
we perfused the bronchial artery in anesthetized sheep. In 12 sheep,
bronchial artery blood samples were obtained before and after the
infusion of 1 × 106
microspheres or microsphere diluent into the bronchial artery. Microspheres, but not diluent, decreased bronchial artery resistance by
40% and increased bronchial artery plasma 6-ketoprostaglandin F1
(194.7 ± 45.0 to 496.5 ± 101.3 pg/ml), the stable metabolite of prostacyclin, and
prostaglandin (PG) F2 (28.1 ± 4.4 to 46.2 ± 9.7 pg/ml). There were no changes in
PGD2,
PGE2, thromboxane B2, adenosine, inosine, or
hypoxanthine. Pretreatment with dipyridamole, an adenosine uptake
inhibitor, did not affect bronchial artery nucleoside concentrations
(n = 7). Microsphere-induced
vasodilation was not enhanced by dipyridamole
(n = 9) and was not inhibited by
either the adenosine receptor antagonist xanthine amine congener (n = 4) or the nitric oxide (NO)
synthase inhibitor
NG-monomethyl-L-arginine
(n = 8). These results do not support
a role for either adenosine or NO and suggest that microspheres caused
bronchial artery vasodilation through release of prostacylin and an
unidentified vasodilator.
xanthine amine congener; NG-monomethyl-L-arginine; indomethacin; sheep
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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FOR MORE THAN 20 years, the intravascular injection of labeled microspheres has been used to measure regional blood flow in experimental animals (29). Carbonized microspheres labeled with radioactive elements, color, or fluorescence can be injected into the heart where they mix with blood, flow through conduit arteries, and lodge in capillaries in proportion to the flow received. The microspheres are quantified in the tissue by measurement of the tracer, and tissue flow is calculated by comparison with a reference flow (8). Alternatively, a known number of microspheres may be injected into a cannulated, perfused artery to study flow within a single organ.
This technique provided the ability to make serial measurements of regional blood flow in studies where other methods were either too invasive or anatomically impractical. For example, the use of microspheres has been considered the standard method of measuring bronchial artery blood flow (1-3, 10, 22) because of the relative inaccessibility of the bronchial circulation and the presence of multiple inflowing bronchial arteries (12). One of the major prerequisites of this technique is that microsphere injections do not significantly alter regional arterial resistance (8).
We recently tested the assumption that microspheres do not alter regional arterial tone by injecting 15-µm polystyrene microspheres into the bronchial artery of anesthetized sheep (27). We found that microspheres caused large, transient, dose-dependent decreases in bronchial artery resistance lasting up to 30 min in duration. This response could be elicited by injecting microspheres either into the cannulated pump-perfused bronchial artery or into the left heart of sheep with uncannulated bronchial arteries in which flow was measured by external flow probe (27). Pretreatment with 8-phenyltheophylline, an adenosine receptor antagonist, blocked 79% of the microsphere effect, suggesting that microspheres caused release of adenosine. Indomethacin pretreatment attenuated the vasodilating effect of microspheres by 37%, suggesting that microspheres also caused the production of a vasodilating product of cyclooxygenase metabolism (27). The purpose of the present study was to make direct measurements of adenine nucleosides and cyclooxygenase-derived prostanoids in bronchial artery blood after microsphere injection and to determine the role of nitric oxide (NO) in the microsphere-induced vasodilation.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Perfused Bronchial Artery Preparation
Anesthesia was induced in young sheep (20-30 kg) with intramuscular ketamine (30 mg/kg) and subsequently maintained by intravenous pentobarbital sodium (15 mg/kg loading dose and 20 mg · kg
1 · h1
infusion). Mechanical ventilation was begun via tracheostomy with
oxygen-supplemented, warmed, humidified room air at a tidal volume of
12 ml/kg, respiratory rate of 10-15
min1, and positive
end-expiratory pressure of 4 mmHg to maintain an arterial
PCO2 of ~40 mmHg and arterial
PO2 >80 mmHg. Femoral veins and
arteries were cannulated to place Swan-Ganz and systemic arterial
catheters and to supply arterial blood for bronchial artery perfusion.
After the animals were paralyzed with pancuronium bromide (2 mg iv), a
left lateral thoracotomy was performed and heparin sodium (20,000 USP
units iv) was administered. A balloon-tipped catheter was placed in the
left atrium, the tracheal and esophageal branches of the
bronchoesophageal artery were ligated, and the bronchial branch was
cannulated with an 18-gauge angiocatheter as previously described (28).
The bronchial artery was perfused with a constant flow of blood
withdrawn from a femoral artery catheter by a calibrated,
variable-speed pump (Gilson Minipuls 2). Bronchial artery pressure was
measured from a fluid-filled side port just proximal to the 18-gauge
angiocatheter. Bronchial artery resistance was defined as bronchial
artery inflow pressure divided by bronchial artery flow. We previously
showed that microsphere injections did not alter the back pressure to
flow of the bronchial circulation, and bronchial artery pressure flow
relationships in this preparation were linear over a wide range of
pressures and flows (27).
Tracheal, left atrial, pulmonary artery, bronchial artery, and aortic pressures were measured with Statham P50 transducers referenced to the level of the left atrium and recorded on a Grass model 7D polygraph. Arterial O2 and CO2 tensions and pH were measured at regular intervals using standard electrode techniques.
Microsphere Injections
Colored 15-µm microspheres (Dye-Trak, Triton Technology) were suspended in 0.01% Tween in normal saline (diluent) and vortexed before injection. The absence of microsphere aggregation was confirmed microscopically before injection. For injections into the bronchial artery, all doses of microspheres were suspended in 0.5 ml of diluent before infusion at 1 ml/min through a side port just proximal to the 18-gauge catheter. All injections were followed by a 1-ml flush of normal saline at the same infusion rate.Bronchial Arterial Blood Collection
Blood samples were obtained from the bronchial artery microcirculation by simultaneously interrupting forward flow, opening a side port of the bronchial artery cannula, and increasing left atrial pressure to 15 mmHg by inflating the balloon-tipped catheter in the left atrium. Under these conditions, retrograde blood flow occurred from the pulmonary to the bronchial circulation through bronchopulmonary anastomoses, the main drainage pathway of the bronchial circulation (12). Bronchial artery blood (4 ml) was collected in a chilled test tube containing 0.4 ml of "stopping solution" consisting of 4 mM 8-azaguanine to inhibit adenosine deaminase, 100 µM dipyridamole to block adenosine uptake, 4 mM EDTA to inactivate adenosinetriphosphatase and 5'-nucleotidase, and 5 µg/ml indomethacin to inhibit cyclooxygenase (6). The blood samples were immediately centrifuged and the plasma frozen at
70°C until later analysis.
Adenine Nucleoside, Prostaglandin, and Lactate Measurements
Plasma concentrations of adenosine, inosine, and hypoxanthine were measured by high-pressure liquid chromatography (HPLC) with a System Gold HPLC (model 338, Beckman, Fullerton, CA) as previously described (32). Concentrations of adenine nucleosides were determined by peak integration and comparison with chromatograph peak areas from known standard concentrations and corrected for recovery that was measured by addition of known concentrations of 2-chloroadenosine to each sample before sample processing. Percent recovery ranged from 50 to 100%.Plasma concentrations of thromboxane (Tx)
B2, the stable metabolite of
TxA2, 6-ketoprostaglandin
F1, the stable metabolite of
prostacyclin, prostaglandin (PG)
F2,
PGD2, and
PGE2 were measured by combined
capillary gas chromatography-mass spectroscopy by a modification of the
method described by Liu et al. (24).
Plasma lactate concentrations were measured by standard spectrophotometric assay (Sigma Chemical, St. Louis, MO).
Experimental Protocols
Mediator measurements. In 12 sheep, 1 × 106 microspheres and an equal volume of microsphere diluent alone were injected into the bronchial artery in random order after setting baseline bronchial artery pressure to 140 mmHg. Bronchial blood flow was kept constant after each injection so that changes in bronchial artery pressure reflected changes in bronchial artery resistance. All bronchial pump settings were noted on each record and converted to values of flow based on a four-point pump calibration curve that was generated before each experiment. A retrograde sample of bronchial artery blood was obtained just before and ~6 min after each injection of microspheres or diluent because this time corresponded to the peak microsphere-induced vasodilation. In two sheep, multiple microsphere and diluent injections were performed. The data from these injections were averaged to provide a single set of microsphere and diluent data for each sheep.
In seven of these sheep, blood samples were obtained before and after an additional injection of 106 microspheres, which was performed during an intrabronchial artery infusion of dipyridamole (104 M at 1 ml/min) to
block the uptake of adenosine into erythrocytes and endothelium (6).
Because 1 × 106 microspheres
may cause near-maximal bronchial artery vasodilation (27), it was
possible that dipyridamole could increase measurable levels of
adenosine or its metabolites without causing further enhancement of
vasodilation. Therefore, in two other sheep, 1 × 105 microspheres were injected
before and during the dipyridamole infusion to better assess the
ability of dipyridamole to enhance the microsphere-induced
vasodilation. In five of the nine sheep treated with dipyridamole, the
efficacy of the dipyridamole infusion was tested by determining the
effect of dipyridamole on the bronchial artery vasodilation from an
infusion of adenosine. Adenosine was infused into the bronchial artery
(105 M at 0.5 ml/min for 3 min) before and 10 min after initiation of dipyridamole. The adenosine
(Sigma Chemical) was dissolved in normal saline. The dipyridamole
(102 M solution; the
generous gift of Du Pont Pharma, North Billerica, MA) was diluted in
normal saline to the desired concentration.
Effect of xanthine amine congener.
To examine further the role of adenosine, 5 × 105 microspheres were injected in
four sheep during an infusion of xanthine amine congener (XAC), a
selective adenosine receptor antagonist (38). Changes in bronchial
artery pressure at constant bronchial artery flow were measured under
the following conditions in each sheep: 1)
105 M adenosine infused (1 ml/min) into the bronchial artery,
2) XAC vehicle infused (1 ml/min)
into the bronchial artery, 3)
105 M adenosine and XAC
vehicle, 4)
105 M adenosine and XAC
(105 M at 1 ml/min),
5) 5 × 105 microspheres and XAC vehicle,
and 6) 5 × 105 microspheres and XAC. XAC or
XAC vehicle was always infused for 10 min before administration of
either adenosine or microspheres. The XAC (Research Biochemical,
Natick, MA) solution was prepared by diluting a stock solution
containing 103 M XAC in 0.1 M NaOH with normal saline.
Effect of
NG-monomethyl-L-arginine.
To determine the role of NO, microspheres (5 × 105 to 1.1 × 106) were injected in eight
sheep before and after an infusion of NG-monomethyl-L-arginine
(L-NMMA,
103 M at 1 ml/min), a NO
synthase inhibitor. Because
L-NMMA increased baseline
bronchial artery resistance, sodium nitroprusside (SNP), a NO donor,
was infused (104 M at ~1
ml/min) with L-NMMA to reverse
the L-NMMA-induced increase in
resistance before the second microsphere dose was infused. Because NO
is capable of inhibiting (5) or stimulating (11) prostacyclin
production, it was possible that either NO synthase inhibition or SNP
could indirectly alter the microsphere-induced vasodilation through
changes in prostacyclin concentration. Therefore, four of these sheep
were pretreated with indomethacin (5 mg/kg iv over 30 min followed by 3 mg · kg1 · h1)
before the microsphere injections. The indomethacin-treated sheep were
injected with larger microsphere doses to produce a comparable level of
microsphere-induced vasodilation to that seen in the
nonindomethacin-treated animals. We previously showed in this
preparation that this dose of indomethacin completely inhibited the
transient pulmonary hypertension caused by intrapulmonary artery
injection of arachidonic acid (27).
L-NMMA and SNP (Sigma Chemical)
were dissolved in normal saline. The indomethacin (Sigma Chemical)
solution was prepared by dissolving 100 mg in 50 ml normal saline and
10 ml of 1 M NaHCO3.
Statistics
The effect of microspheres on bronchial artery resistance and the effects of dipyridamole and L-NMMA on microsphere-induced vasodilation were analyzed by paired t-test. The baseline pressures and flows, the XAC data, and the effect of L-NMMA and SNP on baseline bronchial artery tone were analyzed by one-factor analysis of variance with one repeated measure. The measurements of adenine nucleoside metabolites and prostaglandins were analyzed by a two-factor (microsphere, microsphere diluent) analysis of variance with two repeated measures (34). When significant variance ratios (P
0.05) were obtained,
least significant differences were calculated to allow comparison of
individual means. Values presented in the text are means ± SE.
Differences were considered significant when P 0.05.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A total of 22 sheep (27.6 ± 1.0 kg body wt) was studied. Initial
bronchial artery blood flow averaged for all animals was 0.98 ± 0.05 ml · min1 · kg1
and resulted in a bronchial artery pressure of 140.2 ± 0.8 mmHg and
a bronchial artery resistance of 151.8 ± 11 mmHg · ml1 · min · kg.
Baseline aortic, pulmonary artery, left atrial, and tracheal pressures
averaged 107 ± 3.4, 13.5 ± 1.0, 5.9 ± 0.5, and 11.6 ± 0.5 mmHg, respectively.
Effect of Microspheres on Bronchial Artery Adenine Nucleosides and Lactate
Figure 1 is a record from a representative experiment showing the effect of microspheres and the retrograde bronchial artery blood collection technique on vascular pressures. Injection of 1 × 106 microspheres caused a rapid 40% decrease in bronchial artery pressure (140 to 84 mmHg) within the first 6 min of injection without affecting left atrial, systemic artery, or pulmonary artery pressures. Bronchial artery flow was kept constant so that changes in bronchial artery pressure directly reflected changes in resistance. Bronchial artery pressure spontaneously returned to baseline 35 min after injection. To collect bronchial artery blood, the bronchial artery pump was stopped, a side port from the bronchial artery cannula was opened (indicated by bronchial artery pressure of 0 mmHg), and left atrial pressure was increased to 15 mmHg by inflating the left atrial balloon catheter. As shown in Fig. 1, this maneuver had little effect on systemic artery pressure but increased pulmonary artery pressure to 20 mmHg and pulmonary capillary pressure to a value between 15 and 20 mmHg, thereby increasing the gradient for retrograde bronchial blood flow. Interestingly, interruption of antegrade bronchial artery flow after microsphere injection had no apparent effect on the subsequent course of the microsphere-induced vasodilation.
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Figure 2 shows the mean decrease in
bronchial artery resistance after the injection of either 1 × 106 microspheres or the same
volume of diluent into the bronchial artery as well as the level of
baseline bronchial artery resistance measured just before injection.
Microspheres caused a 36.9 ± 2.6% decrease in bronchial artery
resistance, whereas resistance did not change after diluent injection.
Baseline values of bronchial artery pressure, flow, and resistance
measured just before injection of either microspheres or diluent did
not differ, averaging 141.5 ± 1.2 mmHg, 1.01 ± 0.6 ml · min1 · kg1,
and 153.4 ± 11 mmHg · ml1 · min · kg,
respectively.
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As shown in Fig. 3, neither microspheres nor microsphere diluent caused detectable changes in bronchial artery concentrations of adenosine or its metabolites in the presence or absence of dipyridamole. Microspheres also had no effect on bronchial artery lactate concentration, which averaged 1.2 ± 0.1 mM (data not shown).
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Effect of Dipyridamole
Dipyridamole pretreatment did not augment the microsphere-induced vasodilation, whereas the decreased bronchial artery resistance caused by exogenous adenosine was greatly enhanced in the presence of dipyridamole from20.9 ± 3.0 to 40 ± 4.1% of
baseline resistance (Fig. 4). Two of these
sheep were injected with a lower dose of microspheres (1 × 105) to more closely approximate
the vasodilation from 105 M
adenosine. As with the larger microsphere dose, dipyridamole failed to
enhance the microsphere-induced vasodilation from 1 × 105 microspheres (microspheres
alone, 23.2%; microspheres with dipyridamole, 24.4%).
Dipyridamole decreased baseline bronchial artery resistance, but the
decrease observed before the adenosine injection was not different from
that seen before the microsphere injection (Fig. 4).
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Effect of XAC
As shown in Fig. 5, XAC had no effect on the microsphere-induced decrease in bronchial artery resistance after 5 × 105 microspheres (microspheres plus XAC diluent,29.3 ± 2.7%; microspheres plus XAC, 28.8 ± 6.2%). As a positive control, the same
dose of XAC, but not its vehicle, blocked 81% of the bronchial artery vasodilation caused by infusion of
105 M adenosine into the
bronchial artery. XAC increased baseline bronchial artery resistance,
but the increase observed before the adenosine injection was not
different from the increase seen before the microsphere injection.
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Effect of Microspheres on Bronchial Artery Prostaglandins
Figure 6 shows the effect of 1 × 106 microspheres or microsphere diluent on bronchial artery plasma concentrations of 6-keto-PGF1, TxB2,
PGF2, and
PGE2
(PGD2 was not detected).
Microspheres, but not microsphere diluent, caused a significant,
2.5-fold increase in the concentration of
6-keto-PGF1 (194.7 ± 45.0 to 496.5 ± 101.3 pg/ml) and a nearly 2-fold increase in the level
of PGF2 (28.1 ± 4.4 to 46.2 ± 9.7 pg/ml). There were no changes in either TxB2 or
PGE2 concentrations.
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Figure 7 shows the average bronchial artery
6-keto-PGF1 concentrations and
the change in bronchial artery resistance associated with three serial
injections of 1 × 106
microspheres in the two sheep that received multiple injections. Each
injection of microspheres, but not diluent, caused a large decrease in
bronchial artery resistance associated with an increase in bronchial
artery plasma 6-keto-PGF1
concentration.
Effect of L-NMMA
In the absence of indomethacin, infusion of 103 M
L-NMMA at 1 ml/min into the
bronchial artery caused a 38% increase in bronchial artery resistance
(Fig. 8). The addition of SNP
(104 M at 0.1 ± 0.5 ml/min) reversed this increase in resistance, allowing the second
microsphere injection to occur at a comparable level of bronchial
artery resistance. As shown on Fig. 8,
left, L-NMMA did not inhibit
microsphere-induced vasodilation; in fact, the combination of
L-NMMA and SNP significantly
augmented the decrease in resistance by 50% (32.1 ± 4 to
47.3 ± 4.3%). In the presence of indomethacin,
L-NMMA still failed to block the vasodilation from microspheres, but the enhancing effect of
L-NMMA plus SNP was lost (Fig.
8, right).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Potential Effects of Microspheres on the Circulation
Theoretically, the injection of microspheres into a vascular bed results in two populations of vessels: occluded and unoccluded. The occlusion of vessels could lead to decreased vascular resistance if significant tissue ischemia causes release of diffusible vasodilator substances. For example, Hori et al. (18) showed that injection of 15-µm microspheres into the coronary circulation of dogs caused sustained coronary vasodilation that was inhibitable by theophylline and associated with increased coronary sinus adenosine and lactate concentrations, suggesting that injection of microspheres caused adenosine-induced vasodilation from ischemic heart injury (18). Although not considered by Hori et al. (18), events taking place in nonoccluded vessels could also be a source of vasodilation after microsphere injection. When a microsphere occludes a small arteriole, adjacent nonoccluded vessels are subjected to increased flow and, therefore, increased shear stress. This would occur either under conditions of constant arterial inflow (as in the current experiments) or constant arterial pressure; the former is because of flow diversion, the latter because of an increased pressure drop across the occluded region secondary to a decrease in downstream pressure (35). As discussed below, increased shear stress can cause release of vasodilating substances from the vessel wall (33).In addition to the indirect effects of increased shear stress in the nonoccluded vessels, video microscopy studies of in vivo microsphere behavior showed that a direct interaction between microspheres and vascular endothelium may persist in nonoccluded vessels (16, 17). Microspheres moved slowly through the microcirculation frequently lodging at branch points or on the walls of larger arterioles allowing erythrocytes to pass by (16, 17). These aberrant microspheres protruded into the vessel lumen causing long microsphere chains to form with only partial flow obstruction (17). Thus many microspheres appear to have prolonged direct contact with endothelial cells in vessels with continued blood flow.
On the basis of these considerations, there are two possible mechanisms to explain how microspheres caused vasodilation in the bronchial vascular bed. First, microspheres may have caused ischemia resulting in the release of adenosine and, presumably, lactate. Second, microspheres may have caused release of vasodilating mediators either from the indirect effects of increased shear stress in the nonoccluded vessels or the direct contact of microspheres with the endothelial surface of partially occluded vessels.
Methodological Considerations
To identify directly the presence of vasodilating substances in the bronchial circulation, we collected bronchial artery blood by transiently allowing retrograde flow from the pulmonary circulation to the bronchoesophageal artery. As shown in Fig. 1, we simultaneously increased left atrial and, therefore, pulmonary capillary pressure to increase the gradient for retrograde bronchial blood flow. We collected 4 ml of retrograde bronchial artery blood because we found that a 3-ml injection of contrast into the unperfused sheep bronchial artery caused enhancement of small airways by high-resolution computed tomography with little or no signal observed in the pulmonary circulation (unpublished data). We obtained a bronchial artery blood sample before each microsphere or diluent injection to provide baseline mediator concentrations and control for the potential effects of the transient increase in left atrial pressure on vasoactive mediators (36).Role of Adenosine
Based on our previous results with 8-phenyltheophylline (27), we were surprised to find no change in adenosine concentration after injection of microspheres (Fig. 3) despite a similar vasodilatory response (Fig. 2). There are two possible explanations for these contradictory data. First, adenosine could have mediated microsphere-induced vasodilation, but its rapid removal by cellular uptake or metabolism prevented us from detecting increased plasma levels. Adenosine is removed from the circulation by an active nucleoside transporter system present in erythrocytes and endothelial cells (4) and by metabolism via adenosine deaminase to inosine and hypoxanthine (6). This explanation seemed unlikely in our preparation, however, because we did not detect changes in inosine or hypoxanthine after microspheres, and infusion of dipyridamole, an adenosine uptake inhibitor, into the bronchial artery did not alter the concentrations of adenosine or its metabolites after microsphere injection (Fig. 3). Interestingly, Grantham et al. (15) found negligible adenosine uptake and metabolism in the isolated buffer-perfused bronchial circulation of sheep, suggesting that bronchial artery endothelium may lack the nucleoside transporter system.Second, it was possible that the previously demonstrated attenuation of the microsphere-induced vasodilation by 8-phenyltheophylline represented a nonspecific effect of the drug. To address this possibility, we examined the effects of dipyridamole, an adenosine uptake inhibitor, and the adenosine receptor antagonist XAC (38) on the vasodilation from microspheres and exogenous adenosine. XAC was selected because it has 100-fold greater affinity for adenosine receptors and is 2,500-fold more soluble than 8-phenyltheophylline (38). Bruns and Fergus (7) showed that adenosine receptor antagonists with a solubility to receptor affinity ratio of <100 had poor in vivo activity. The ratios for 8-phenyltheophylline and XAC are 6.3 and 3,300, respectively, for A2 receptor antagonism, suggesting that the in vivo effectiveness of 8-phenyltheophylline was inferior (7).
As shown in Fig. 4, dipyridamole had no effect on the magnitude of the vasodilation to microspheres, whereas it significantly potentiated the effect of exogenous adenosine. The lack of potentiation of the microsphere effect was not because of an inability of further bronchial artery dilation inasmuch as the vasodilation to a smaller dose of microspheres in two of these sheep was also not enhanced by dipyridamole. Dipyridamole alone caused a significant decrease in bronchial artery resistance (Fig. 4), presumably from a combination of adenosine uptake blockade and cGMP phosphodiesterase inhibition (39). This effect was small, however, and did not prevent the potentiation of the vasodilation from exogenous adenosine. It was therefore not an explanation for the inability of dipyridamole to enhance the microsphere effect.
XAC markedly inhibited the vasodilation from exogenous adenosine but had no effect on the decrease in bronchial artery resistance after microsphere injection (Fig. 5). XAC, but not its vehicle, increased baseline bronchial artery resistance, suggesting that endogenous adenosine was a determinate of baseline bronchial artery tone. The small increase in baseline resistance was not an explanation for the inability of XAC to block microsphere-induced vasodilation, however, because the same increase in resistance occurred before XAC successfully blocked the vasodilation from exogenous adenosine (Fig. 5). The dipyridamole, XAC, and lactate data support the negative results shown in Fig. 3 and suggest that ischemia-induced adenosine release does not mediate microsphere-induced vasodilation in the bronchial circulation.
Role of Prostaglandins and NO
It is well established that increased shear force from increased blood velocity causes vasodilation (33). Although some studies suggest that this mechanical stimulus may be capable of directly dilating vascular smooth muscle, the preponderance of evidence indicates that vasodilation occurs in this setting from the release of endothelium-derived vasodilators (33). Depending on the vascular bed and species, vasodilating prostaglandins (21), NO (23), or the combination of prostaglandins and NO (20) has been shown to be responsible for this phenomenon. Our previous results suggested that a dilator prostaglandin was partially responsible for the effect of microspheres on bronchial artery tone, but the identity of the prostanoid was not determined and the role of NO was not examined (27).As shown in Fig. 6, microspheres, but not microsphere diluent, caused a
large increase in the bronchial artery concentration of
6-keto-PGF1, the stable
metabolite of prostacyclin. Prostacyclin is a potent vasodilator that
is produced primarily by endothelial cells (25), suggesting that the
interaction of microspheres with the bronchial artery endothelium
resulted in the production and release of prostacyclin. These data
explain the previously observed inhibitory effect of indomethacin on
microsphere-induced vasodilation (27).
There was also a significant increase in
PGF2, a smooth muscle
constrictor, but the levels of this prostanoid were an order of
magnitude lower than the
6-keto-PGF1 concentrations. Although the cellular source of the
PGF2 increase is less clear,
the ratio of 6-keto-PGF1 to
PGF2 observed in this study was
similar to the ratios measured from isolated porcine coronary, carotid,
and pulmonary arteries (19), suggesting that the increased
6-keto-PGF1 and
PGF2 were both generated by the
bronchial artery wall rather than inflammatory cells.
As shown in Fig. 7, repetitive injections of microspheres produced
superimposable decreases in bronchial artery resistance accompanied by
reproducible increases in the concentration of bronchial artery
6-keto-PGF1. Each microsphere
injection was also associated with increases in
PGF2
(PGF2 = 25, 52, and 32 pg/ml,
respectively; data not shown). Although these data are derived from
only two sheep, they suggest that the mechanism causing activation of
cyclooxygenase was not subject to tachyphylaxis from either feedback
inhibition of prostacyclin synthase (25) or endothelial injury.
To examine the role of NO, we performed microsphere injections before and during an infusion of L-NMMA into the bronchial artery. As shown in Fig. 8, L-NMMA alone caused a 38% increase in bronchial artery resistance, suggesting a significant role for constitutive NO in the maintenance of normal bronchial artery tone. This result is similar to that of Sasaki et al. (31) who showed that NO synthase inhibition decreased baseline bronchial artery blood flow by ~60% in anesthetized sheep. This increased bronchial artery resistance could be reversed by L- but not D-arginine, supporting the specificity of the NO synthase inhibitor (31).
To allow the two microsphere injections to be made from the same level of baseline tone, we reversed the increased bronchial artery resistance caused by L-NMMA with SNP. We did not use an arterial constrictor to match the increased tone caused by L-NMMA because of the potential differences in the loci of vasoconstriction under the two conditions. We reasoned that vasodilating the constricted vascular bed with SNP would more accurately mimic the vascular hemodynamics that were present before administration of either drug. Moreover, NO is known to cause feedback inhibition of NO synthase (9), thus providing additional NO synthase blockade.
We were surprised to find that the combination of L-NMMA and SNP enhanced the vasodilation from microspheres (Fig. 8). Given the complex relationship between NO and cyclooxygenase (5, 11, 13, 30), we hypothesized that either NO synthase inhibition or SNP resulted in an upregulation of microsphere-induced prostacyclin generation. For example, endogenous NO inhibited prostacyclin synthesis in rat diaphragmatic arterioles (5), and exogenous NO caused a dose-dependent inhibition of bradykinin-induced generation of prostacyclin from endothelial cells (13). Alternatively, both endogenous (11) and exogenous NO (11, 30) have been shown to stimulate in vivo (30) and in vitro (11, 30) prostacyclin production, possibly through activation of prostaglandin H synthase (11). We therefore repeated the protocol in four additional sheep that were pretreated with indomethacin to block prostacyclin production. As shown in Fig. 8, indomethacin pretreatment prevented the enhanced vasodilation associated with L-NMMA and SNP, suggesting that additional stimulation of prostacyclin production was probably responsible. More importantly, microsphere-induced vasodilation was not inhibited by NO synthase blockade, suggesting that NO was not involved.
Although our results clearly indicate that prostacyclin plays a role in microsphere-induced vasodilation in the bronchial circulation, indomethacin blocked less than half of the response (27); thus the major source of the vasodilation remains unknown. A growing amount of literature has shown the existence of endothelium-derived vasodilating substances other than NO and prostacyclin in several vascular systems including sheep bronchial artery (31), rat basilar artery (14), and rat cremaster muscle artery (37). One possible candidate vasodilator that may explain these data is endothelium-derived hyperpolarizing factor (EDHF), an as yet uncharacterized substance (26) that is released from endothelium by muscarinic agonists, increased intracellular calcium concentration, and, possibly, increased shear stress (26). Interestingly, EDHF may be more important than NO in the vasomotor regulation of small peripheral arterioles (26), a finding which could explain why NO did not appear to contribute to microsphere-induced vasodilation in the bronchial circulation. Alternatively, much of the vasodilation from microspheres could result from a mediator-independent process through direct cell-to-cell communication between the endothelium and smooth muscle (33). Further studies to elucidate the effect of microspheres on bronchial artery tone may provide insight into mechanisms of microcirculatory control. On a more practical level, microsphere-induced vasodilation has the potential to cause an overestimation of true blood flow if large frequent injections of microspheres are utilized to estimate regional perfusion (27).
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ACKNOWLEDGEMENTS |
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We thank Teresa Privett and Won Lee for expert technical assistance and Wanda Moran for excellent secretarial support.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-50504 (to D. B. Pearse) and HL-10342 (to E. M. Wagner) and by a grant-in-aid (to D. B. Pearse) and an established investigator award (to E. M. Wagner) from the American Heart Association, with funds contributed in part by the American Heart Association, Maryland Affiliate.
Address for reprint requests: D. B. Pearse, Div. of Pulmonary and Critical Care Medicine, The Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.
Received 9 September 1997; accepted in final form 13 November 1997.
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Abstract
Introduction
Methods
Results
Discussion
References
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) and
after (
) 3 serial injections of 1 × 106 microspheres into bronchial
artery of anesthetized sheep. Microsphere injections were separated by
injections of microsphere diluent. Open bars represent maximal change
in bronchial artery resistance associated with each injection plotted
as a percentage of baseline resistance
(n = 2).
