Lung (1995) 173:209--221

Effects of Thromboxane A 2 Analogue on Vascular Resistance Distribution and Permeability in Isolated Blood-perfused Dog Lungs T. Shibamoto, H.-G. Wang, Y. Yamaguchi, T. Hayashi, Y. Saeki, S. Tanaka, and S. Koyama Department of Physiology, Division 2, Shinshu University School of Medicine, Matsumoto, Nagano 390, Japan

Abstract. This study was designed to determine the effects of thromboxane A2 (TxA2) on the distribution of vascular resistance, lung weight, and microvascular permeability in isolated dog lungs perfused at a constant pressure with autologous blood. The stable TxAz analogue (STA2; 30 I~g, n = 5) caused an increase in pulmonary capillary pressure (Pc) assessed as doubleocclusion pressure to 14.0 --- 0.4 mmHg from the baseline of 7.9 --- 0.3 mmHg with progressive lung weight gain. Pulmonary vascular resistance increased threefold exclusively due to pulmonary venoconstriction. Pulmonary venoconstriction was confirmed in lungs perfused in a reverse direction from the pulmonary vein to the artery (n = 5), as evidenced by marked precapillary vasoconstriction and a sustained lung weight loss. Furthermore, in lungs perfused at a constant blood flow (n = 5), STA2 also caused selective pulmonary venoconstriction. Vascular permeability measured by the capillary fdtration coefficient and the isogravimetric Pc at 30 and 60 min after STA2 infusion did not change significantly from baseline in any lungs studied. Moreover, elevation of Pc by raising the venous reservoir of the intact lobes (n = 5) to the same level as the STA2 lungs caused a greater or similar weight gain compared with the STA2 lungs. Thus, we conclude that TxAz constricts selectively the pulmonary vein resulting in an increase in Pc and lung weight gain without significant changes in vascular permeability in isolated bloodperfused dog lungs.

Offprint requests to: T. Shibamoto

210

T. Shibamotoet al.

Key words: Capillary filtration coefficient--Pulmonary edema--Pulmonary venoconstriction--Lung weight gain Isogravimetric capillary pressure. Introduction

Thromboxane A z (TxA2) has been implicated, at least in part, as a mediator of respiratory dysfunction in the adult respiratory distress syndrome [4] and in experimental animal models of pulmonary edema induced by endotoxin [5], microembolism [13], protamine [14], neutrophil activation [11], and limb ischemia and reperfusion [8]. The edematogenic effects of TxA z are thought to result from increased pulmonary capillary pressure (Pc) but not from increased vascular permeability [9, 25]. Using sheep lung lymph fistula models, Bowers and colleagues [3] reported that the stable T x A 2 analogue U46619 increases lung lymph flow with a decrease in the lung lymph to plasma protein ratio, a finding consistent with increased pulmonary microvascular pressure. However, there is disagreement concerning the effect of TxA 2 on pulmonary microvascular permeability [10, 24, 27]. Thromboxane synthetase inhibition prevents an increase in pulmonary vascular permeability in sheep after thrombin infusion [7] or after limb ischemia and reperfusion [8]. These findings suggest that T x A 2 contributes primarily to increased lung vascular permeability. Moreover, a stable TxA z analogue has been reported to increase pulmonary vascular permeability in isolated lamb lungs [26]. However, in rabbit lungs, the TxA 2 analogue does not increase the pulmonary capillary filtration coefficient [24] or transvascular albumin flux [10], which is consistent with the absence of increased vascular permeability. One possible explanation for this discrepancy may be ascribed to the species difference. We are not aware of any reports examining the effects of TxAz on the pulmonary vascular permeability of dog lungs despite the vast literature on pulmonary hemodynamics in this species. TxA2 has recently been shown to increase pulmonary microvascular pressure in isolated lamb lungs [26] and rabbit lungs [10, 24]. However, the effect of TxA2 on the longitudinal distribution of pulmonary vascular resistance is not consistent. Pulmonary venoconstriction was reported to comprise 89% of increased total pulmonary vascular resistance produced by stable T x A 2 in isolated lamb lungs perfused with blood-free buffer [26]. However, in isolated rabbit lungs [24] and isolated rat lungs [2], the magnitude of pulmonary arterial resistance was increased more than pulmonary venous resistance after infusion of the T x A 2 analogue U46619. However, these studies were conducted using isolated lungs peffused under constant flow. Different modes of perfusion could change the results of the pulmonary vasoconstrictor response to T x A 2. Furthermore, no studies were reported which adopted the retrograde perfusion technique to confirm the preferential pulmonary vasoconstrictive site for TxA2. Therefore, we examined the effect of 9,11-epithio-11,12-methano-TxAz (STAz), a stable TxA 2 analogue [12], on pulmonary microvascular permeability and changes in the distribution of pulmonary vascular resistance in isolated canine lungs perfused with autologous blood under either constant pressure or con-

Thromboxane in Isolated Dog Lungs

211

stant flow. In addition, we used reverse perfusion to clarify more accurately the pulmonary vascular response to the TxA2 analogue. Methods

Isolated Lung Preparation Twenty-five mongrel dogs (6-13 kg) were anesthetized with sodium pentobarbitai (25 mg/kg, i.v.), intubated, and ventilated mechanically with room air. The isolated lung preparation was described previously [17, 19, 20]. Ten minutes after heparin treatment (500 units/kg, i.v.), the dog was bled rapidly through the carotid artery catheter. After left thoracotomy, the left lower lobe was excised and weighed. Plastic cannulas were secured in the pulmonary artery and vein and the lobar bronchus. Thereafter, perfusion was begun within 10 min after excision of the lung. In the constant-pressure perfusion system, the cannulated lobe was suspended from an electric balance (LF-600, Murakami Koki, Japan) and perfused with 300 ml of shed blood that was pumped from the outflow (venous) reservoir through a heat exchanger (38 °C) into the inflow (arterial) reservoir. Airway pressure (Paw) was maintained at a constant level of 3 cm of H20. An overflow tube was connected to the two reservoirs to maintain a constant arterial perfusion pressure. The height of each reservoir could be adjusted independently to maintain arterial pressure (Pa) and venous pressure (Pv) at any steady level. The perfused blood was oxygenated in the outflow reservoir by continuous bubbling with 95% 02, 5% CO2. In the constant-flow perfusion system, the blood was pumped from a venous reservoir through a heat exchanger into the pulmonary artery. The remaining perfusion setup was the same as the constant-pressure perfusion system, as described above. Pulmonary Pa and Pv were measured using pressure transducers placed on the reference points at the level of the lung hilum. Blood flow (0) was measured with an electromagnetic flow meter (MFV 1200; Nihon Kohden, Tokyo, Japan), and the flow probe was positioned in the venous outflow line. Lung weight was monitored continuously and displayed on the physiograph. Pa and Pv were adjusted initially to a level within the normal perfusion range in zone 3 (Pa > Pv > Paw) to obtain an isogravimetric state (no weight gain or loss).

Measurements of Pulmonary Vascular Permeability Capillary Filtration Coefficient. The capillary filtration coefficient (Kf.c) was used as an index of microvascular permeability (6) and assessed by increasing Pa and Pv simultaneously by 6--8 mmHg from an isogravimetric state and observing the lung weight gain. The sudden increase in vascular pressure caused a rapid weight gain of the lobe because of an increase in the blood volume of the lung. This was followed by a gradual and prolonged weight gain that was attributed to transcapillary fluid filtration [6]. The weight gain rate (AWt/At) at each minute after the increase in pressure (t = 0) was plotted as a semilogarithmic function with time, and the slow phase of the weight transient was extrapolated to time zero. When the lung was not isogravimetric but was gaining weight (as described below), this extrapolated rate was subtracted by the weight gain rate of the last 1 man before Kf, e determination. The extrapolated rate of weight gain (AWt/At)t=o w a s then divided by the increase in Pc (APc). Capillary pressure was measured before and after the increase in vascular pressure using the double-vascular occlusion technique [23]. Kr,~ was normalized to the initial lung weight of 100 g to yield Kf,c in ml/min/cm H20/100 g, wet lung weight. (A Wt/At)t=O Kf,c

APc

(1)

Isogravimetric Capillary Pressure. The isogravimetric capillary pressure (Pc,i) is the Pc at which the lung neither gains nor loses weight. The Pc,i is determined by the following equation

212

T. Shibamoto et al. Pc,i = Pi + cr(arc - aft)

(2)

where axc and ~ri are plasma and interstitial protein oncotic pressure, respectively, Pi is interstitial pressure, and cr is the capillary osmotic reflection coefficient for proteins. Thus, a decrease in Pc,i would indicate a decrease in cr or an increase in Hi, indicating increased vascular permeability to proteins because Pi tends to increase with increased filtration. Therefore, Pc,i can be used as an index of microvascular permeability to proteins. The total vascular (Rt), arterial (Ra), and venous (Rv) resistances were calculated as follows. Rt = (Pa - Pv)/(~

(3)

Ra = (Pa - Pc)/0

(4)

Rv = (Pc - Pv)/Q

(5)

Experimental Protocol At approximately 20 min after the start of perfusion, an isogravimetric state was obtained at a Pa of 10--16 mmHg, Pv of 2.5-3.5 mmHg, and a blood flow above 0.7 i/rain/100 g. A stock solution of STA 2 was made by dissolving 1 mg of STA2 in 1 ml of 99.5% ethanol; it was stored at - 20 °C. The dose-response relationship between STA 2 and Rt was determined using the doses of 1, 3, 10, and 30 ~g of STAz diluted with 1 ml of physiologic saline in lung lobes (n = 5) perfused under constant pressure. Ten minutes was allowed between doses after each variable returned to the preinjection level. To evaluate the effects of STA2 on pulmonary vascular permeability, STA2 doses of 30 ~g were adopted. Kf.c and Pc,i were determined twice, and the mean values were used as the baseline values. Following the baseline measurement, all of the blood-perfused lobes were divided into the following five groups.

Control Group (n = 5). A 30-1~1bolus of 99.5% ethanol diluted with 1 ml of saline, the vehicle, was injected into the pulmonary artery under constant-pressure perfusion. STA2 Group (n = 5). A 30-1~g dose of STA 2 was injected as a bolus into the pulmonary artery of a lobe under constant-pressure perfusion. This group was examined following determination of the dose response of STA2 (1-10 I~g). STAz Group with Reverse Perfusion (n = 5). After establishment of constant-pressure perfusion, the inflow tube was connected to the pulmonary vein and the outflow tube to the pulmonary artery. Thus, the blood entered the lung lobe via the pulmonary vein and was withdrawn from the pulmonary artery. After stabilization, 30 v~g of STA 2 was injected into the pulmonary vein. STA2 Group under Constant-flow Perfusion (n = 5). The lung lobes were perfused under constant flow, 1.13 -+ 0.11 I/rain/100 g, and 30 I~g of STA2 was injected into the pulmonary artery. Kf, o was measured at 30 and 60 rain after the injection of STA2 or the vehicle in each group. Until 30 min after injection, the height of either reservoir was not changed to observe the response of the natural course. In some of the STA2-treated lungs, which were perfused at zone 2 (Pa > Paw > Pv) or Pv < 2.3 mmHg, due to a vasoconstriction-induced decrease in 0 , Kf.¢ at 30 min was measured at zone 3 following an increase in Pv by 3 mmHg for 3 min by raising the height of the outflow reservoir because Kf.¢ depends on vascular surface area [6, 19]. This last l-rain weight gain was subtracted from the extrapolated weight gain rate for calculation of Kf, c. Pc was determined at 2, 3, 6, 10, 20, 30, and 60 min after STA2 or the vehicle. At 60 min after the injection in the lobes that were gaining or losing weight, vascular pressures were adjusted to obtain an isogravimetric state and Pc,i. Pv Elevation Group under Constant-pressure Perfusion (n = 5). In this group, after an injection of the vehicle into the intact lobes, Pv was increased by raising only the venous (outflow)

Thromboxane in Isolated Dog Lungs

213

35"

o

30

¢-

25 =

E

20

~ E E

15

r,-

s

o

~k

10

0

I

a

I

I

1

3

10

30

S T A 2 d o s e (IJg)

Fig. 1. Peak change in Rt as a function of doses of STA 2 (n = 5). *P < 0.05 from baseline. Open and closed circles indicate the values before and after STA 2, respectively.

reservoir, keeping the height of the arterial (inflow) reservoir constant so that Pc might be increased continuously to the same levels as the STA 2 group for 30 rain. This weight gain was compared with that of the STA2 group to assess the permeability change of the STA2 group during the first 30 min.

Statistical Analysis All values were expressed as means -+ SE. Comparison among all groups and within each group for a given variable was performed by using analysis of variance followed by Duncan's multiple range test. Statistical significance was defined as P < 0.05.

Results

Effects of STA 2 on Pulmonary Hemodynamics and Lung Weight Figure 1 shows the summary data of changes in Rt in each dose. STA 2 produced a dose-dependent increase in Rt. Rt showed a threefold increase approximately 6 min after 30 txg of STA2. Figure 2 shows a representative example of the response to 30 ~g of STA 2 of the lobe perfused under constant pressure. An injection of STA2 caused marked pulmonary vasoconstriction as reflected by decreases in 0 and Pv and an increase in Pa. At the same time the lung weight decreased transiently followed by a gradual weight gain. A double-occlusion maneuver, performed 2 min after STA2, revealed a higher Pc than the corresponding baseline value, suggesting that predominant pulmonary venoconstriction occurred. Figure 3 shows an example of a recording of a reverse-perfused lobe that was injected with 30 ~g of STA2. Marked pulmonary vasoconstriction was also observed as evidenced by decreases in t) and Pa, the outflow pressure, and an increase in Pv, the inflow pressure, a finding similar to the STA2-treated lobe perfused in a normal direction. In contrast, lung weight decreased progressively after STA 2 and was sustained below the preinjection level. Pc was decreased below the

214

T. Shibamoto et al.

STA2

Pulmonary Venous Pressure (mmHg) Pulmonary Arterial Pressure (mmHg)

20 10

__;~

'i

!i

L_-i

0

~

20 10 - - - ~

/ .....

i

,,

V - ' - " V ~ %

v-'-

0

Pulmonary 600 I ~ _ ~ _ Blood 400 Flow 200 (ml/min) 0 Weight (g)

3 rain

f

~

C

10

7 -t

~

i

Fig. 2. Representativerecording of a response of lung weight, Pa, Pv, and pulmonary blood flow to an injection of 30 ~g of STA2in the constant-pressure perfusion group. baseline level after STA2, suggesting that increased upstream vascular resistance, that is, pulmonary venoconstriction, occurred. This STA2-induced selective pulmonary venoconstriction was also observed in a lobe perfused under constant flow, as shown in Figure 4, where the pressure gradient between Pc and Pv was increased dramatically after 30 I~g of STA2, whereas the gradient between Pa and Pc was not changed. This figure shows a slight dip in lung weight immediately after injection followed by a marked weight gain. Pa was increased markedly without changes in Pv or 0 because of constant-flow perfusion. The injection of 30 ~g of STA 2 into the lobes under constant-pressure perfusion caused pulmonary vasoconstriction that lasted 60 min so that 0 decreased from 1.34 ± 0.16 to 0.56 ± 0.081/min/100 g at 6 min and then returned to 1.17 ± 0.181/min/100 g at 60 min after injection. Pa was increased, transiently and slightly, from 12.1 ± 0.3 mmHg to a peak level of 15.8 ± 0.5 mmHg at 6 min followed by a return to the preinjection level, 12.7 +- 0.6 mmHg at 60 min. Pv was decreased from 3.3 ± 0.3 to 1.6 ± 0.3 mmHg at 6 min and returned to 2.4 --- 0.3 mmHg at 30 min and 3.2 ± 0.3 mmHg at 60 min after STA2 injection. In the reverse STA2 group, similar pulmonary vasoconstriction was observed, although the changes in Pa and Pv were opposite. On the other hand, in the constant-flow group, 30 Ixg of STA2 caused a marked increase in Pa with a peak value of 25.2 --- 2.4 mmHg from the baseline values of 12.6 - 0.42 mmHg. Pv and 0 were not changed significantly from the baseline values of 3.2 --- 0.2 mmHg and 1.13 ± 0.11 l/min/100 g, respectively, throughout the experimental period.

Thromboxane in Isolated Dog Lungs

215

STA2 Pulmonary Venous Pressure (mmHg)

Pulmonary Arterial Pressure (mmHg)

3 rain 20 10 0

~

-

"-LT-~--

20 10 0

Pulmonary 600[ ~ Blood 400 Flow (ml/min)

200 0

Lung

Weight (g)

10

Fig. 3. Representative recording of a response of lung weight, Pa, Pv, and pulmonary blood flow to an injection of 30 i~g of STA2 in the reverse-perfusion group.

Figure 5 shows the changes in Pc and weight in all five groups. In both of the STA2 groups perfused under constant pressure and constant flow, Pc increased significantly. However, as expected, the increase in Pc of the constantflow group was significantly greater than that of the constant pressure with the peak value of 21.9 --- 3.2 mmHg and 14.0 --- 0.4 mmHg, respectively, 6 min after 30 ~g of STA2. Then, Pc decreased gradually but still remained elevated at 30 min after STA2. The weight in these two groups was increased in response to STA2. The lung weight gain at the early stage was significantly greater in the constant-flow group than in the constant-pressure group. However, at 20 rain there was no difference between these two group. In contrast, Pc in the reverse group decreased significantly to 5.7 - 0.7 mmHg from the baseline of 8.4 - 0.6 mmHg. The weight in this group showed a rapid and sustained decrease after STA2 injection. We compared the lung weight change between the STA2 group and the Pv elevation group. In the Pv elevation group Pc was increased to the same level as that in the STAz group under constant pressure perfusion to address the permeability change of the latter group. At 6 min after the injection of the vehicle, Pv was elevated to 11.7 --- 0.3 mmHg with a resultant increase in Pc to 14.2 --- 0.5 mmHg, equivalent to the corresponding Pc of the STA 2 group, 14.0 -+ 0.4 mmHg. The weight gain in the Pv elevation group was greater than that in the STA z constant-pressure group within 10 min after STA z, although Pc, one of the determinants of transvascular fluid filtration [21], was increased similarly. However, this weight gain in the Pv elevation group was similar to

216

T. Shibamoto et al. STA2

Pulmonary Venous Pressure

(mmHg)

3 min

t

40 30 20 10 0 40

Pulmonary Arterial Pressure

(mmHg)

30 20 10

400E ~ 0

Pulmonary Blood

200

Weight

10

Flow (ml/min)

"

(g)

~

I

Fig. 4. Representative recording of a response of each variable to an injection of 30 wg of STA2 in the constant-flow perfusion group.

that in the S T A 2 constant-flow group. There were no significant changes in lung weight gain among these three groups at 20 rain and thereafter. These findings suggest that the increased extravascular fluid filtration caused by an increase in Pc in the STA 2 lungs is comparable to that in the intact lungs of the Pv elevation group at the early stage of STA2 administration. Figure 6 shows the summary data of Ra and Rv in lobes treated with 30 p~g of STA 2 or the vehicle. Rv increased approximately sixfold in all three STAEtreated groups. However, there were no significant changes in Ra from the baseline in any groups studied. The selective venous constriction in response to STA2 was clearly demonstrated regardless of the direction of perfusion or mode of perfusion.

Effect o f STA 2 on Pulmonary Microvascular Permeability Figure 7 shows the results of Kf, c and Pc,i. There were no significant differences in the baseline Kf,c or Pc,i values among any groups studied. A 30-~g STA2 did not significantly change the Kf,~ measured at 30 or 60 min after injection in any group. There were no significant differences in Pc,i between the baseline values and those at 60 min after STA2 or the vehicle in any group, although the Pc,i at 60 min in the STA 2 constant-pressure group tended to be greater than its baseline value and the corresponding values in the other two groups. In the control group there was no significant change in Kf,~ or Pc,i.

Thromboxane in Isolated Dog Lungs

217

26

22 18

-r

E

g

14-

0 a.

10" 6" 2

5

-5

1'0

1'5

2'0

2'5

3()

Time (mln)

+ 0 ¢ m =,. 0

30 20 10

Fig. 5. Time course of Pc and lung weight change in the control (open circles, n = 5), 30 Ixg of STA 2 constant-pressure perfusion (closed circles, n = 5), 30 ~g of STA 2 reverse perfusion (closed triangles, n = 5), 30 p~g of STA2 constant-flow perfusion (closed squares, n = 5), and Pv elevation (open triangles, n = 5) groups. *P < 0.05 from baseline.

0 -10" -20"

3 J

-30" -40

0

-5

5

10

15

20

25

30

Time (mln)

30

r-I Control v * * J_

-,°

l,l, l+J...

-+

+ ,oi+,+.+ ' ++ + 0

++ +

• 0

[ ] STA2 reverse Ill STA2 constant Q

T II *.

0 3 T l260(m el n)

60

0

3

6

20

Time (mln)

60

Fig. 6. Time course of Ra and Rv in the control (n -- 5), 30 p~gof STA 2 constant-pressure peffusion (n = 5), 30 ~g of STA 2 reverse-perfusion (n = 5), and 30 ~g of STA2 constant-flow perfusion (n = 5) groups. *P < 0.05 from baseline.

Discussion The present study demonstrated that STA 2 causes an increase in pulmonary microvascular pressure which in part accounts for the STA2-induced lung

218

T. Shibamoto et al. 0.25

E E X

E

£

[ ] Baseline [ ] 30 min ~1 60 min

0.20" 0.15"

O.lO-

E 0.05 --o= 0,00

Control

STA2

12-

~

8

~

6

0 a.

4"

STA2 STA2 reverse constant Q

[ ] Baseline [ ] 60 min

~

-,-

20

Control

STA2

STA2 STA2 reverse constant Q

Fig. 7. Kf, c and Pc,i in the control (n = 5), 30 I~gof STA2constant-pressure perfusion (n = 5), 30 I~g of STA2 reverse-perfusion (n = 5), and 30 p~gof STA2 constant-flow (Q) perfusion (n = 5) groups. weight gain. This finding was subsequently reinforced and confirmed by the results of the reverse-perfusion group. In this group a decrease in Pc was accompanied by concomitant sustained lung weight reduction. The second finding of the present study is that STA 2 constricts selectively the pulmonary vein but not artery regardless of the direction or modes of perfusion. These findings are consistent with the previous observation that an infusion of arachidonate, the precursor of TxA2, increases Rt and Pc by increasing Rv but not Ra in blood-perfused canine lungs [22]. Indeed, pulmonary vasoconstriction induced by arachidonate is ascribed primarily to the cyclooxygenase products, particularly TxA2 [10, 16, 18]. Moreover, Barman and colleagues [1] have suggested recently that a ~table TxA2 analogue, U46619, constricts all pulmonary segments with greater constriction of the small vein in isolated dog lungs. Although a different TxA 2 analogue, STA2, was used in the present study [12], we confirmed the previous suggestion of U46619-induced preferential venoconstriction in dog lungs [1] and reinforced it by the reverse-perfusion procedure. However, the predominance of pulmonary venoconstriction over arterial constriction in response to TxA 2 is not consistent among animal species. Arterial vasoconstriction rather than venoconstriction is a predominant response

Thromboxanein IsolatedDog Lungs

219

of the isolated rabbit lungs to U46619; the increase in Ra was greater than that in Rv (Ra, 3.8-fold vs Rv, 2.6-fold) [24]. In rat lungs, U46619 increased Rt by increasing only small artery resistance [2]. Furthermore, using STAz, as used in the current study, Yoshimura and co-workers [27] reported that this analogue increased pulmonary arterial resistance but not venous resistance in the blood-perfused lamb lungs. Surprisingly, in the isolated lamb lungs, when the buffered saline was used as perfusate instead of blood, this TxA2 analogue induced primarily pulmonary venoconstriction [26]. The reasons for the different results of these studies compared with the present studies are not known. However, we have shown that a change in modes of perfusion, that is, constant flow or constant pressure, does not account for this difference. It may be ascribed to species difference and the different perfusate. The mechanism whereby STA2 constricts only the pulmonary vein in dog lungs is not known. One possibility may be related to TxAz-induced platelet aggregation because TxA2 stimulates platelet activation and is an important, albeit not exclusive, contributor to intravascular aggregation of platelets [15]. However, this assumption seems unlikely because the platelet aggregate should have obliterated the inflow vessel, the pulmonary artery, but not the outflow one, the pulmonary vein. In addition, the reverse-perfusion study also demonstrated that pulmonary vasoconstriction is not dependent on the direction of perfusion but rather the vascular site, the pulmonary vein. A more plausible explanation may be that functional TxA2 receptors distribute abundantly in the dog pulmonary vein compared with the arterial side. However, there is no direct evidence to support the presence of nonhomogeneous distribution of TxA2 receptors in canine pulmonary vessels. In the present study, lung weight decreased initially after STA2 injection in the constant-pressure or constant-flow perfusion groups. This weight loss might be caused by a rapid reduction of intravascular volume, which might be due to pulmonary arterial constriction, an inflow block. This possible weak arterial constriction could not be detected by a double-occlusion maneuver performed at 2 min and might have been overwhelmed by the subsequent stronger venoconstriction. This initial weight loss was followed by a progressive weight gain that might be caused by increased Pc, which could increase fluid filtration and extravascular lung water. In contrast, in the reverseperfusion group, weight continued to decrease after STAz. This weight loss was caused by the upstream constriction, which could reduce intravascular volume, and the decrease in Pc, which could reduce transcapillary fluid filtration. The other main finding in the present study is the Kf,c and Pc,i did not change significantly at 30 or 60 min, respectively, after STA2. These findings suggest that TxA2 does not increase pulmonary microvascular permeability in dog lungs 30 min after injection. The absence of increased pulmonary vascular permeability was also demonstrated in isolated rabbit lungs that were infused with U46619 [10, 24]. However, a stable TxA2 analogue produced an increase in Ke, c and a decrease in the osmotic reflection coefficient in the isolated newborn lamb lungs [27]. This discrepancy may be attributed to an age difference rather than species difference because adult sheep lung vasculature integrity was demonstrated to be resistant to TxAz [3].

220

T. Shibamoto et al.

STA2 might have transiently increased pulmonary vascular permeability at the early stage in the present study, and this permeability change might have been recovered at 30 min when normal Kf, c was observed. However, this possibility seems unlikely. The weight gain of the Pv elevation group was greater than that observed in the STA 2 constant-pressure group and was comparable to the STA2 constant-flow group within 10 min. The greater weight gain of the Pv elevation group than that of the STA 2 constant-pressure group may be attributed in part to the difference in intravascular blood volume between the two groups because the STA2 lungs were perfused in zone 2 (Pa > Paw > Pv), whereas the Pv elevation lungs were perfused in zone 3 (Pa > Pv > Paw). The Pc of the constant-flow STA 2 group was greater than the Pv elevation group within 10 rain after injection. From this finding, it could be expected that extravascular fluid filtration was more pronounced in the STA2 constant-flow group than the Pv elevation group, resulting in greater weight gain in the former group than the latter group. However, there were no differences in weight change between these two group. This unexpected finding may be explained as follows. The increase in intravascular blood volume distal to the pulmonary capillary in the Pv elevation group, as suggested by elevated Pv, might have counteracted the weight gain due to the enhanced fluid filtration caused by a marked increase in Pc in the constant-flow STA2 group. Indeed, Pv in the Pv elevation group at 6 min, 11.7 --- 0.3 mmHg, was much greater than the corresponding values in the constant-flow group, 3.1 +-- 0.2 mmHg. These findings suggest that the vascular permeability integrity of the STA2-treated canine lungs was not impaired at the initial period as well as at 30 and 60 min after the STA 2 injection. In conclusion, a stable TxA2 analogue produces increases in pulmonary microvascular pressure and pulmonary venous vascular resistance but not arterial vascular resistance in isolated blood-perfused dog lungs. No change in pulmonary microvascular permeability occurred at 30 or 60 min after the TxA 2 analogue infusion. Acknowledgments. This study was supported in part by Grant-in-aid for Scientific Research 05454420 from the Ministry of Education, Science, and Culture of Japan. The authors thank ONO Pharmaceutical Company (Osaka, Japan) for the gift of STA2.

References 1. Barman SA, Senteno E, Smith S, Taylor AE (1989) Acetylcholine's effect on vascular resistance and compliance in the pulmonary circulation. J Appl Physiol 67:1495-1503 2. Barnard JW, Ward RA, Adkins WK, Taylor AE (1992) Characterization of thromboxane and prostacyclin effects on pulmonary vascular resistance. J Appl Physiol 72:1845-1853 3. Bowers RE, Ellis EF, Brigham KL, Oates JA (1979) Effects of prostaglandin cyclic endoperoxides on the lung circulation of unanesthetized sheep. J Clin Invest 63:131-137 4. Brigham KL, Duke S (1985) Prostaglandins and lung disease: adult respiratory distress syndrome. Semin Respir Med 7:11-16 5. Brigham KL, Meyrick B (1986) State of the art: endotoxin and lung injury. Am Rev Respir Dis 133:913-927

Thromboxane in Isolated Dog Lungs

221

6. Drake R, Gaar KA, Taylor AE (1978) Estimation of the fdtration coefficient of pulmonary exchange vessels. Am J Physiol 234:H266--H274 7. Garcia-Szabo RR, Peterson MB, Watkins WD, Bizios R, Kong DL, Malik AB (1983) Thromboxane generation after thrombin: protective effect of thromboxane synthetase inhibition on lung fluid balance. Circ Res 53:214-222 8. Klausner JM, Paterson IS, Goldman G, Kobzik L, Valeri CR, Shepro D, Hechtman HB (1989) Thromboxane A2 mediates increased pulmonary microvascular permeability following limb ischemia. Circ Res 64:1178-1189 9. Kubo K, Kobayashi T (1985) Effects of OKY-046, a selective thromboxane synthetase inhibitor, on endotoxin-induced lung injury in unanesthetized sheep. Am Rev Respir Dis 132:494499 I0. Littner MR, Lott FD (1989) Edema from cyclooxygenase products of endogenous arachidonic acid in isolated lung. J Appl Physiol 67:846--855 11. Loyd JE, Newman JH, English DE, Ogletree ML, Meyrick BD, Brigham KL (1983) Lung vascular effects of phorbol myristate acetate in awake sheep. J Appl Physiol 54:267-276 12. Mais DE, Saussy DL Jr, Chaikhouni A, Kochel PJ, Knapp KR, Hamanaka N, Halushka PV (1985) Pharmacologic characterization of human and canine thromboxane A2/prostaglandin H2 receptors in platelets and blood vessels: evidence for different receptors. J Pharmacol Exp Ther 233:418--424 13. Malik AB (1983) Pulmonary microembolism. Physiol Rev 63:1114-1207 14. Morel DR, Zapol WM, Thomas SJ, Kitain EM, Robinson DR, Moss J, Chenoweth DE, Lowenstein E (1987) C5a and thromboxane generation associated with pulmonary vaso- and bronchoconstriction during protamine reversal of heparin. Anesthesiology 65:597-604 15. Oates JA, FitzGerald GA, Branch RA, Jackson EK, Knapp HR, Roberts LJ (1988) Clinical implication of prostaglandin and thromboxane A2 formation. N Engl J Med 319:689-698 16. Ogletree ML, Brigham KL (1980) Arachidonate raises vascular resistance but not permeability in lungs of awake sheep. J Appl Physiol 48:581-586 17. Parker JC, Townsley MI, Rippe B, Taylor AE, Thigpen J (1984) Increased microvaseular permeability in dog lungs, due to high peak airway pressures. J Appl Physiol 57:1809-1816 18. Seeger W, Ernst CH, Walmrath D, Neuhof H, Roka L (1985) Influence of the thromboxane antagonist BM 13.177 on the arachidonic acid-induced increase in pulmonary vascular resistance and permeability in rabbit lungs. Thrombosis Res 40:793-805 19. Shibamoto T, Parker JC, Taylor AE, Townsley MI (1990) Derecruitment of f'dtration surface area in paraquat-injured isolated dog lungs. J Appl Physiol 68:1581-1589 20. Shibamoto T, Hayashi T Jr, Sawano F, Saeki Y, Matsuda Y, Kawamoto M, Koyama S (1992) Pulmonary vascular response to anaphylaxis in isolated canine lungs. Am J Physio1263:RI024R1029 21. Taylor AE, Parker JC (1985) Pulmonary interstitial spaces and lymphatics. In: Handbook of Physiology. The Respiratory System. Circulation and Nonrespiratory Functions. American Physiological Society, Bethesda, MD, pp 162-230 22. Townsley MI, Korthuis RJ, Taylor AE (1985) Effects of arachidonate on permeability and resistance in canine lungs. J Appl Physiol 58:206--210 23. Townsley MI, Korthuis RJ, Rippe B, Parker JC, Taylor AE (1986) Validation of double vascular occlusion method for Pc,i in the lung and skeletal muscle. J Appl Physiol 61:127-132 24. Wakerlin GE, Benson GV, Pearl RG (1991) A thromboxane analog increases pulmonary capillary pressure but not permeability in the perfused rabbit lung. Anesthesiology 75:475-480 25. Win R, Nadir HB, Harker L, Hilderbrandt J (1983) Thromboxane A2 mediates lung vasoconstriction but not permeability after endotoxin. J Clin Invest 72:911-918 26. Yoshimura K, Todd ML, Pier KG, Rubin LJ (1989) Role of venoconstrictionin thromboxaneinduced pulmonary hypertension and edema in lambs. J Appl Physiol 66:929--935 27. Yoshimura K, Todd ML, Pier KG, Rubin LJ (1989) Effects of a thromboxane A: analogue and prostacyclin on lung fluid balance in newborn lambs. Circ Res 65:1409-1416 Accepted for publication: 12 July 1994