Effects of a novel prostaglandin, in rabbit lung in situ
8-epi-PGF2,,
MUKUL BANERJEE, KYUNG HO KANG, JASON D. MORROW, L. JACKSON ROBERTS, AND JOHN H. NEWMAN Center for Lung Research and Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University School of Medicine, Nashville 37223; and Department of Physiology, Mehurry Medical College, Nashville, Tennessee37208 Banerjee, Mukul, Kyung Ho Kang, Jason D. Morrow, L. Jackson Roberts, and John H. Newman. Effects of a novel prostaglandin,&epi-PGF,,, in rabbit lung in situ. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H660-H663, 1992.-We determined the effects of Sepiprostaglandin (PG) FPcu, a noncyclooxygenase free radical-catalyzed product of arachidonic acid, on pulmonary vascular tone, its potency, and its mechanism of action. 8Epi-PGF2, (0.5-20 pg) was injected into the pulmonary artery (PA) catheter of 10 rabbits whoselungswere perfused in situ with Krebs-Henseleit buffer solution with 3% bovine serumalbumin. PA pressureincreasedfrom a baselineof 13.5k 0.6 to 25.6 t 2.0 cmH,O with 20 pugSepi-PGF,,. 8-EpiPGF2, causeda rapid rise in PA pressurefollowed by a gradual decline over 40-60 min to baselinelevels. Double vascular occlusion revealeda twofold increasein arterial resistanceat peak rise in PA pressure.The rise in PA pressurewith 20 pg 8-epiPGF2, was fivefold greater than with 20 pg of the cyclooxygenase-derivedprostaglandin PGF,,. The PA pressureresponse to 8-epi-PGF,, wasnot altered by either cyclooxygenaseblockadewith 150PM meclofenamateor a-receptor blockadewith 70 PM phentolamine,but was fully prevented by 40 PM SQ 29548, a thromboxane receptor antagonist.We concludethat in rabbits 8-epi-PGF,, is a potent vasoconstrictor of the pulmonary vasculature, which appearsto be due to the activation of SQ 29548responsivethromboxane receptors. pulmonary artery; endothelium-derivedrelaxing factor II-EPIPROSTAGLANDIN (PG) FPa is a recently discovered
prostaglandin-like compound produced by humans and rats in vivo (14). It is one of a group of unique PGF2, compounds created in biological fluids by nonenzymatic, free radical-catalyzed peroxidation of arachidonic acidcontaining lipids in plasma and cell membranes (13). These PGF2 compounds appear to be found in high concentrations during oxidative stress such as in diquat poisoning of rats (14). Little is known about the biological roles of these compounds except that 8-epi-PGF2, causes marked reduction of renal blood flow and glomerular filtration in euvolemic rats (14). Because the pulmonary circulation is sensitive to prostaglandins (3) and because oxidant stress is a common mechanism of lung injury, we hypothesized that 8-epi-PGF2, might be an effective modulator of pulmonary vascular tone. The purpose of this study, therefore, was to test this compound for its actions, its relative potency, and mechanisms of effect in isolated perfused rabbit lung.
665) with a tidal volume of 15 ml, breathing frequency of 40 min-l, and end-expiratory pressureof 3 cmHaO. Ventilatory gasconcentrations were 21% 02, 5% COz, and balanceNP. The chest was openedby midline incision, and the residualpulmonary blood wasslowly washedout with 500 ml Krebs-Henseleit buffer (KHB) solution at 37.5OC.Lungs were then perfusedat 100 ml/min with a Masterflex pump for the duration of the experiment. Catheters were securedinto the pulmonary artery and in the left ventricle and were connectedto pressuretransducersto continuously record the pulmonary arterial and outflow pressures.The perfusion reservoir was maintained at 3739°C and had a volume of 200 ml. The height of the reservoir wasadjustedto -4 cmH,O relative to the left atrium. Thus the lungswere perfusedunder a zone II condition. A ZO-min equilibration period was allowed to establish stable baselinepulmonary vascularpressuresbeforestarting experiments. Effects of 8-Epi-PGF,, on Pulmonary and Microvascular Pressure
Arterial
Pressure
8-Epi-PGF2, wasprepared for injection by adding 1 mg dry powder with 1 ml pure ethanol and then diluted with 19 ml saline. 8-Epi-PGF2, wasprovided by Upjohn. Progressivelyincreasingbolus (1 ml) doses(0.5, 1.0, 5.0, 10.0, and 20 pg) of 8-epi-PGF,, were injected into the afferent pulmonary artery (PA) catheter of 10rabbits. There wasa minimum interval of 10 min between dosesof prostaglandin or until the PA pressure returned to the baseline. To get an estimate of the microvascular pressureof the experimental rabbit, a doublevascular occlusiontechnique (8, 23) wasapplied at the end of exhalation by simultaneouslyoccluding the PA and left atria1 catheters. The vascular occlusion technique was applied in 10 rabbits first under baselineconditions and at the peak PA pressureresponseto 20 pg 8-epiPGF2,. Potency and Mechanism
of 8-Epi-PGF,,
Effects
To determine the relative potency of 8-epi-PGF,, and to examine the possiblemechanismsof its action on the pulmonary vasculature, we compared it with the effects of several vasoactive compoundsand hypoxia. The following vasoconstrictor agentswere studied. PGF,,. PGFzcy(20pg; CaymanChemical;20pg PGF2, in 1 ml perfusate), a cyclooxygenase-derivedvasoconstrictor prostaglandin, wasinjected into the PA catheter of 12 rabbits following the doseresponsewith 8-epi-PGF,,. Hypoxia. Before the injection of the second ZO-pgdose of 8-epi-PGF,,, eight rabbits wereexposedto hypoxia (0% 02, 5% C02, and 95% N2) three times each. Each hypoxic exposure MATERIALS AND METHODS lastedfor 5 min with an interval of 10 min betweeneachexpoAnimal Preparation sure. PO,, Pco~, and pH of the perfusate withdrawn from the We usedfemaleNew ZealandWhite rabbits weighing3-5 kg. left atria1catheter under control conditions before hypoxia were The rabbits were anesthetized by injecting into an ear vein 144 t 6 mmHg, 33 t 3 mmHg, and 7.43 + 0.03, respectively. During hypoxia, the perfusatePO, was36 t 5 mmHg, PCO~ was thiamylal sodium(30 mg/kg) and heparin sodium(1,000IU/kg). Through a tracheostomy and the insertion of a tracheal can- 32 + 1 mmHg, and pH was 7.43 t 0.01. To control for time and nula, animalswere ventilated with a Harvard respirator (model the-priming effect, we injected in another set of eight rabbits H660
0363-6135/92
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8-EPI-PGFz,,
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doses(5 and 20 pg) of 8-epi-PGF,,, as a priming stimulus. After an interval of 1 h, we injected again 20 pg 8-epi-PGF*,. KCL.Progressivelyincreasingdosesof KC1 (5, 10, 15, and 20 mM) wereaddedto the perfusatereservoir asthe last procedure in eachexperiment with 10 rabbits. If there wasno responseto a particular doseof KCl, we waited 10 min beforethe next dose. IO MIN Otherwise, as the PA pressurereached a peak at a particular dose of KCl, the next higher dose of KC1 was added to the Fig. 1. Photostatic enlargement of a pulmonary artery pressure tracing perfusate. taken after a bolus infusion of 20 N-g8-epi-PGF2,y into affluent cannula two
Effects of Diff erent Blocking Response to 6- Epi- PGF,,,
Agents
on PA Pressure
The following blocking agents were added in the perfusate reservoir in different experimentsusing separategroupsof rabbits. Meclofenamate. In four rabbits, 150 PM meclofenamate,a cyclooxygenaseinhibitor, wasaddedbefore repeating the doseresponseexperiment with five dosesof 8-epi-PGF,,. After the PA pressurereturned to the baselineafter the last dose(20 pg) of 8-epi-PGF,,,, 20 pugof PGF2,* were also injected to the PA catheter. Phentolamine. In four rabbits, 70 PM phentolamine,an cu-receptor blocker, was added before an injection of 20 pg 8-epiPG%* SQ 29548.In four rabbits, 10 and 20 pg of 8-epi-PGF*,, were injected into the PA catheter before and after adding in the perfusate 40 PM SQ 29548, a thromboxane receptor blocker, provided by Squibb. NW-nitro-L-arginine (L-IVIVA). In six rabbits, after two consecutivedosesof 20 pg 8-epi-PGF,,,, 20 PM L-NNA, a blocker of endothelial-derived relaxing factor (EDRF), was added to the perfusate. Three minutes later, another dose of 20 pg 8-epiPGFB,,was injected into the PA. [1-(5-isoquinolinesulfonyl)piperazine] (CI). In four rabbits, 20 pg 8-epi-PGF,,, were injected into the PA before and after adding in the perfusate0.2 mM CI, an inhibitor of protein kinase C (9, 21). Atria1 natriuretic factor (ANF). Two setsof experimentswere performed with four rabbits in each group. In one set, 40 rug rANF (synthetic rat ANF, 28 amino acids, Peninsula Laboratories) were injected into the PA catheter 2 min before the injection of 20 pug8-epi-PGF,,,. In the secondset, 40 pg rANF were added when the PA pressurereached its peak after the injection of 20 pg 8-epi-PGF,,. ANF is a potent short-lived pulmonary vasodilator used to determine reversibility of the pressorresponseto 8-epi-PGF,,,. Statistical
Analyses
Data are presentedas means t SE. The statistical significance of differences among the meanswas analyzed by Student’s t test and paired t test where applicable. The effect of meclofenamateon PA pressureresponseto 8-epi-PGF,,, was analyzed by a two-way analysis of variance (dose and drug group) and Duncan’smultiple range test. P < 0.05 wasconsidered significant. RESULTS
Effects of 8-Epi-PGF,,,
on PA Pressure
A typical pulmonary pressor response following the injection of 20 pg 8-epi-PGF,, is shown in Fig. 1. There was a rapid rise in PA pressure, peaking in 3 min, followed by a gradual decline to baseline levels over a period of 40-60 min. In response to increasing doses of 8-epi-PGF,, from 0.5 to 20 pg, the PA pressure rose in a dose-dependent manner from the baseline value of 13.5 t 0.6 to 25.6 t 2.0 cmH20 (Fig. 2).
of rabbit pulmonary artery perfused in situ.
0 basal KCI
1 I 5
5 I 10
(mM)
or 8-epi-PGF2a
10 I 15
20 20
I
c19 mM
(pg)
Fig. 2. Progressive dose-response relationship of bolus infusions of 8-epi-PGFpfl or KC1 on pulmonary arterial pressure (P& in rabbit lung perfused in situ. * P < 0.05 between lungs pretreated with meclofenamate and control. + P < 0.05 from baseline.
Effects of 8-Epi-PGF,,
on Microvascular
Pressure
The results are shown in Table 1 and Fig. 3. PA pressure rose from 12.6 t 0.7 to 21.7 t 1.5 cmH20 with 20 pg 8-epi-PGF,,, and microvascular pressure (P,,) increased from 6.5 t 0.3 to 8.5 t 0.3 cmH20. The upstream pressure accounted for 78% of the total rise in PA pressure (Table 1). The pressure drops from PA to microvascular pressure (P PA - P,,) before and after injecting 20 pg 8-epi-PGF2, were 6.1 t 0.7 and 13.2 t 1.6 cmH20, respectively (P < 0.01). Venous resistance could not be calculated because the lungs were under zone II conditions (outflow pressure less than alveolar pressure). Effects of Vasoactive Compounds and Hypoxia on PA Pressure Response to 8- Epi- PGF,,
At 20 pg, the rise in PA pressure with 8-epi-PGF2, was sixfold greater than with 20 pg PGFzcy (Table 2). Meclofenamate appeared to partially reduce the response to 8-epi-PGF,, at the highest dose (Fig. 2). As shown in Fig. 2, the peak PA pressure response to 5,10,15, and 20 mM KC1 corresponded to the peak response to 1,5,10, and 20 pg 8-epi-PGF,,, respectively. Injection of 40 pg rANF before the injection of 20 pg 8-epi-PGFza caused ~50% Table 1. 8-Epi-PGF,, on the arterial side
acts primarily kmH20
P PA P
pri - p
9.1k1.2 2.0t0.2 7.1kl.l
Percent Change 72.6t10.2 31.3k5.0 117.4t15.5
Values ar:“means t SE; n = 10. PpA, pulmonary arterial pressure; P mv9 microvascular pressure.
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H662
8-EPI-PGFz,, 25 -
20 5 I” 5 a
15 -
2
10 -
ON
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c
Basal
Table 4. Effects of phentolamine, ANF, hypoxia,
n
8-epi-PGF2a
SQ 29548, and L-NNA on pulmonary arterial pressure response to 20 pg 8-epi-PGF,,,
*: PxO.05
from
basal
PPA,
cm&O
n Control *
ii AI 5-
OpPA
Pmv
PpA+mv
Fig. 3. Change in longitudinal vascular segment pressure to a bolus dose of 20 ,ug 8-epi-PGFz,, in 10 rabbit lungs perfused in situ, measured by double occlusion. P pA, pulmonary artery pressure; P,,, microvascular pressure; PI,;\ - P,,,,., difference. Most of the pressure change was in the upstream segment.
Table 2. Comparative
Phentolamine (70 PM) ANF (40 a) Hypoxia SQ 29548 (40 PM) L-NNA (20 ,uM) Values are means & SE; n, no. arginine. * P < 0.05.
4 14.8k4.5 4 11.9k4.5 8 1 l.Ot2.8 4 17.8t2.1 6 11.2k3.0 of experiments.
With
agent
17.3k4.2 6.5&2.2* 25.2t5.2* 0* 23.3t4.5* L-NNA,
NW-nitro-L-
both SQ 29548 and CI to the perfusate fully blocked the PA pressor response to 8-epi-PGF,,, (Table 4; CI data not shown). DISCUSSION
potency of 20 pg 8-epi-PGF,,, and PGF,,, with and without meclofenamate
8-Epi-PGF,,, causes a marked increase in PA pressure in rabbits whose lungs were perfused in situ with KHB solution. We limited the maximum dose to 20 pg because PGF260 cmH*O) in PA pressure. The addition of showed an increase in pulmonary vascular tone in both normoxic and hypoxic lungs following the administration of N-methyl-L-arginine, an EDRF blocker. In our experTable 3. ANF caused a more rapid decay of pulmonary iments, both hypoxia and L-NNA potentiated the PA arterial pressure following 20 pg 8-epi-PGFzcy pressure response to 8-epi-PGF,,. This suggests that these two pressors, hypoxia and 8-epi-PGF2,,, are moduANF Control lated by release of EDRF. 105~1.0 Basal Pi),z, cmH,O 10.9kl.O Meclofenamate partially altered the dose response of 33.7k4.4 25.0t2.9 Peak P1jll, cmHzO the PA pressure to only 20 pg 8-epi-PGF2,,, suggesting 1.8&0.1* 4.5k0.6 Time to half of peak, min that most of the effect of 8-epi-PGF,,, was not caused by Values are means t SE of 4 experiments. ANF, atria1 natriuretic the production of another intermediary arachidonate factor. * P < 0.05. Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 1, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
8EPI-PGFZty ON RABBIT
product. The lack of influence of phentolamine on the effect of 8-epi-PGF,, indicates that a-receptors were not involved in this vasoconstriction. However, the pressor effect of 8epi-PGF,, was fully prevented by SQ 29548, an endoperoxide receptor blocker. This strongly suggests that the mechanism of 8epi-PGFz,-induced pulmonary vasoconstriction is due to endoperoxide receptor activation. We have shown earlier in sheep that SQ 29548 antagonized the effect of PGDB and PGF2, on the pulmonary vasculature, with no effect on systemic vasculature (lo), suggesting that SQ 29548 may not be specific for thromboxane receptors. Evidence for thromboxane receptor-mediated contraction of guinea pig and human airways in vitro by PGD2, PGE2, and PGFzcy has been provided by Featherstone et al. (7), Coleman and coworkers (4, 5), and Armour et al. (2). Finally, at the cellular level, the contraction of the PA smooth muscle by 8-epi-PGFza may be due to the involvement of protein kinase C, since the use of CI, an inhibitor of protein kinase C, completely blocked the PA pressure response to 8-epi-PGF2,. Morimoto et al. (12) have recently provided evidence for the participation of both intracellular free calcium ions and protein kinase C in tonic vasoconstriction of rat thoracic aorta induced by PGF2,. Participation of protein kinase C in the tonic phase of smooth muscle contraction has been suggested by other investigators (15, 18, 19). Because CI is not a specific inhibitor of protein kinase C, we cannot assert that these data are sufficient to prove a role of protein kinase C in the observed response. In summary, 8-epi-PGFz,,, a noncyclooxygenase free radical-catalyzed product of arachidonic acid, is a powerful pulmonary vasoconstrictor in in situ perfused lungs of rabbits. The vasoconstrictor effect is potentiated by preexposure to hypoxia and by EDRF blockade, while the vasoconstriction is reversed by the use of atria1 natriuretic peptide. The mechanism of action of 8-epi-PGF2, on pulmonary vasculature appears to be due to the activation of SQ 29548-responsive receptors and the participation of protein kinase C. The role of this substance in normal physiology and in disease states remains to be determined. This work was supported by National Institutes of Health Grants K14-02138, HL-19153, GM-42056, and HL-02499. Address for reprint requests: M. Banerjee, Dept. of Physiology, Meharry Medical College, Nashville, TN 37208. Received 19 August 1991; accepted in final form 8 April 1992. HL-39952,
REFERENCES 1. Adnot, S., S. Eddahibi, and B. Raffestin. Loss of endotheliurn-dependent relaxant activity in the pulmonary circulation of chronically hypoxic rats: recovery on return to room-air or after treatment with L-arginine (Abstract). Am. Rev. Respir. Dis. 143: A182, 2.
1991.
Armour, C. L., P. R. A. Johnson, M. L. Alfredson, and J. L. Black. Characterization of contractile prostanoid receptors on human airway smooth muscle. Eur. J. Pharmacol. 165: 215-222, 1989.
3. Brigham, K. L. Mediators in the pulmonary circulation. In: The Pulmonary Circulation: Normal and Abnormal, edited by A. P. Fishman. Philadelphia, PA: Univ. of Pennsylvania Press, 1990, chapt. 8, p. 91-107. 4. Coleman, R. A., L. Feniuk, and I. Kennedy. A study of the prostanoid receptors mediating bronchoconstriction in the anaes-
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thetized guinea-pig and dog (Abstract). Proc. Br. Pharmacol. Sot., Oxford, UK, 1981, p. 913. 5. Coleman, R. A., and R. L. G. Sheldrick. Prostanoid-induced contraction of human bronchial smooth muscle is mediated by TP-receptors. Br. J. Pharmacol. 96: 688-692, 1989. G., T. Higenbottam, A. T. D. Xuan, S. Stewart, 6. Cremona, S. Large, F. Wells, and J. Wallwork. Basal release of endothelium-derived relaxing factors (EDRF) regulates the pulmonary vascular resistance (PVR) in isolated perfused porcine, sheep and human lungs (Abstract). Am. Rev. Respir. Dis. 143: A771, 1991. 7. Featherstone, R. L., C. Robinson, S. T. Holgate, and M. K. Church. Evidence for thromboxane receptor mediated contraction of guinea-pig and human airways in vitro by prostaglandin (PG) D2, 9 alpha, 11 beta-PGF2 and PGF-alpha. Life Sci. 46: 1301-1307,
1990.
Hakim, T. S., R. P. Michel, and H. K. Chang. Partitioning of pulmonary vascular resistance in dogs by arterial and venous occlusion. J. Appl. Physiol. 52: 710-715, 1982. 9. Harvath, L. Inhibition of human chemotaxis by the protein kinase inhibitor [ l-(5-isoquinolinesulfonyl)piperazine]. J. Immunol. 139: 3055-3061, 1987. 10. King, L. S., M. Fukushima, M. Banerjee, K. H. Kang, J. H. Newman, and I. Biaggioni. Pulmonary vascular effects of prostaglandin D2, but not its systemic vascular or airway effects, are mediated through thromboxane receptor activation. Circ. Res. 68: 8.
352-358,
1991.
Liu, S., D. E. Crawley, P. J. Barnes, and T. W. Evans. Endothelium-derived relaxing factor inhibits hypoxic pulmonary vasoconstriction in rats. Am. Rev. Respir. Dis. 143: 32-37, 1991. 12. Morimoto, S., S. Kim, K. Fukuo, E. Koh, R. Morita, S. Kitano, Y. Miyashita, S. Imanaka, and T. Ogihara. Participation of both intracellular free Ca2+ and protein kinase C in tonic vasoconstriction induced by prostaglandin F2a. Eur. J. Pharmacol. 188: 369-378, 1990. 13. Morrow, J. D., T. M. Harris, and L. J. Roberts. Noncyclooxygenase oxidative formation of a series of novel prostaglandins: analytical ramifications for measurement of eicosanoids. Anal. Biochem. 184: l-10, 1990. 14. Morrow, J. D., K. E. Hill, R. F. Burk, T. M. Nammour, K. F. Badr, and L. J. Roberts. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical catalyzed mechanism. Proc. Natl. Acad. Sci. USA 11.
87: 9383-9387,
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Nakaki, T., B. L. Roth, D. Chuang, and E. Costa. Phasic and tonic components in 5-HT2 receptor-mediated rat aorta contraction: participation of Ca++ channels and phospholipase Cl. J. Pharmacol. Exp. Ther. 234: 442-446, 1985. 16. Peach, M. J., R. A. Johns, and C. E. Rose, Jr. The potential role of interactions between endothelium and smooth muscle in pulmonary vascular physiology and pathophysiology. In: Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves. New York: Dekker, 1989, p. 643-697. 17. Pison, U., F. Lopez, C. F. Heidelmeyer, M. K. Falke, and W. M. Zapol. Inhaled nitric oxide selectively reverses hypoxic pulmonary vasoconstriction in a mechanically ventilated sheep model (Abstract). Am. Rev. Respir. Dis. 143: A772, 1991. 18. Rasmussen, H. The calcium messenger system. N. Engl. J. Med. 15.
314: 1094-1101, 19.
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H. The calcium messenger system. N. Engl. J. Med. 1986.
Rodman, D. M., T. Yamaguchi, K. Hasunuma, R. F. O’Brien, and I. F. McMurtry. Effects of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L207-L214, 1990. 21. Salzer, W., C. Gerard, and C. McCall. Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation. Biochem. Biophys. Res. Commun. 148: 747-754, 1987. 22. Thomas, H. M., R. Carson, G. Gupta, E. D. Fried, and R. S. Novitch. The effect of N-methyl arginine (NMA) on the pulmonary circulation in the presence of regional hypoxia (Abstract). Am. Rev. Respir. Dis. 143: A775, 1991. 23. Townsley, M. I., R. J. Korthuis, B. Rippe, J. C. Parker, and A. E. Taylor. Validation of double vascular occlusion method for Pc,i in lung and skeletal muscle. J. Appl. Physiol. 61: 20.
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8-epi-PGF2,,
MUKUL BANERJEE, KYUNG HO KANG, JASON D. MORROW, L. JACKSON ROBERTS, AND JOHN H. NEWMAN Center for Lung Research and Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University School of Medicine, Nashville 37223; and Department of Physiology, Mehurry Medical College, Nashville, Tennessee37208 Banerjee, Mukul, Kyung Ho Kang, Jason D. Morrow, L. Jackson Roberts, and John H. Newman. Effects of a novel prostaglandin,&epi-PGF,,, in rabbit lung in situ. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H660-H663, 1992.-We determined the effects of Sepiprostaglandin (PG) FPcu, a noncyclooxygenase free radical-catalyzed product of arachidonic acid, on pulmonary vascular tone, its potency, and its mechanism of action. 8Epi-PGF2, (0.5-20 pg) was injected into the pulmonary artery (PA) catheter of 10 rabbits whoselungswere perfused in situ with Krebs-Henseleit buffer solution with 3% bovine serumalbumin. PA pressureincreasedfrom a baselineof 13.5k 0.6 to 25.6 t 2.0 cmH,O with 20 pugSepi-PGF,,. 8-EpiPGF2, causeda rapid rise in PA pressurefollowed by a gradual decline over 40-60 min to baselinelevels. Double vascular occlusion revealeda twofold increasein arterial resistanceat peak rise in PA pressure.The rise in PA pressurewith 20 pg 8-epiPGF2, was fivefold greater than with 20 pg of the cyclooxygenase-derivedprostaglandin PGF,,. The PA pressureresponse to 8-epi-PGF,, wasnot altered by either cyclooxygenaseblockadewith 150PM meclofenamateor a-receptor blockadewith 70 PM phentolamine,but was fully prevented by 40 PM SQ 29548, a thromboxane receptor antagonist.We concludethat in rabbits 8-epi-PGF,, is a potent vasoconstrictor of the pulmonary vasculature, which appearsto be due to the activation of SQ 29548responsivethromboxane receptors. pulmonary artery; endothelium-derivedrelaxing factor II-EPIPROSTAGLANDIN (PG) FPa is a recently discovered
prostaglandin-like compound produced by humans and rats in vivo (14). It is one of a group of unique PGF2, compounds created in biological fluids by nonenzymatic, free radical-catalyzed peroxidation of arachidonic acidcontaining lipids in plasma and cell membranes (13). These PGF2 compounds appear to be found in high concentrations during oxidative stress such as in diquat poisoning of rats (14). Little is known about the biological roles of these compounds except that 8-epi-PGF2, causes marked reduction of renal blood flow and glomerular filtration in euvolemic rats (14). Because the pulmonary circulation is sensitive to prostaglandins (3) and because oxidant stress is a common mechanism of lung injury, we hypothesized that 8-epi-PGF2, might be an effective modulator of pulmonary vascular tone. The purpose of this study, therefore, was to test this compound for its actions, its relative potency, and mechanisms of effect in isolated perfused rabbit lung.
665) with a tidal volume of 15 ml, breathing frequency of 40 min-l, and end-expiratory pressureof 3 cmHaO. Ventilatory gasconcentrations were 21% 02, 5% COz, and balanceNP. The chest was openedby midline incision, and the residualpulmonary blood wasslowly washedout with 500 ml Krebs-Henseleit buffer (KHB) solution at 37.5OC.Lungs were then perfusedat 100 ml/min with a Masterflex pump for the duration of the experiment. Catheters were securedinto the pulmonary artery and in the left ventricle and were connectedto pressuretransducersto continuously record the pulmonary arterial and outflow pressures.The perfusion reservoir was maintained at 3739°C and had a volume of 200 ml. The height of the reservoir wasadjustedto -4 cmH,O relative to the left atrium. Thus the lungswere perfusedunder a zone II condition. A ZO-min equilibration period was allowed to establish stable baselinepulmonary vascularpressuresbeforestarting experiments. Effects of 8-Epi-PGF,, on Pulmonary and Microvascular Pressure
Arterial
Pressure
8-Epi-PGF2, wasprepared for injection by adding 1 mg dry powder with 1 ml pure ethanol and then diluted with 19 ml saline. 8-Epi-PGF2, wasprovided by Upjohn. Progressivelyincreasingbolus (1 ml) doses(0.5, 1.0, 5.0, 10.0, and 20 pg) of 8-epi-PGF,, were injected into the afferent pulmonary artery (PA) catheter of 10rabbits. There wasa minimum interval of 10 min between dosesof prostaglandin or until the PA pressure returned to the baseline. To get an estimate of the microvascular pressureof the experimental rabbit, a doublevascular occlusiontechnique (8, 23) wasapplied at the end of exhalation by simultaneouslyoccluding the PA and left atria1 catheters. The vascular occlusion technique was applied in 10 rabbits first under baselineconditions and at the peak PA pressureresponseto 20 pg 8-epiPGF2,. Potency and Mechanism
of 8-Epi-PGF,,
Effects
To determine the relative potency of 8-epi-PGF,, and to examine the possiblemechanismsof its action on the pulmonary vasculature, we compared it with the effects of several vasoactive compoundsand hypoxia. The following vasoconstrictor agentswere studied. PGF,,. PGFzcy(20pg; CaymanChemical;20pg PGF2, in 1 ml perfusate), a cyclooxygenase-derivedvasoconstrictor prostaglandin, wasinjected into the PA catheter of 12 rabbits following the doseresponsewith 8-epi-PGF,,. Hypoxia. Before the injection of the second ZO-pgdose of 8-epi-PGF,,, eight rabbits wereexposedto hypoxia (0% 02, 5% C02, and 95% N2) three times each. Each hypoxic exposure MATERIALS AND METHODS lastedfor 5 min with an interval of 10 min betweeneachexpoAnimal Preparation sure. PO,, Pco~, and pH of the perfusate withdrawn from the We usedfemaleNew ZealandWhite rabbits weighing3-5 kg. left atria1catheter under control conditions before hypoxia were The rabbits were anesthetized by injecting into an ear vein 144 t 6 mmHg, 33 t 3 mmHg, and 7.43 + 0.03, respectively. During hypoxia, the perfusatePO, was36 t 5 mmHg, PCO~ was thiamylal sodium(30 mg/kg) and heparin sodium(1,000IU/kg). Through a tracheostomy and the insertion of a tracheal can- 32 + 1 mmHg, and pH was 7.43 t 0.01. To control for time and nula, animalswere ventilated with a Harvard respirator (model the-priming effect, we injected in another set of eight rabbits H660
0363-6135/92
$2.00 Copyright 0 1992 the American Physiological
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Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 1, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
8-EPI-PGFz,,
ON RABBIT
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doses(5 and 20 pg) of 8-epi-PGF,,, as a priming stimulus. After an interval of 1 h, we injected again 20 pg 8-epi-PGF*,. KCL.Progressivelyincreasingdosesof KC1 (5, 10, 15, and 20 mM) wereaddedto the perfusatereservoir asthe last procedure in eachexperiment with 10 rabbits. If there wasno responseto a particular doseof KCl, we waited 10 min beforethe next dose. IO MIN Otherwise, as the PA pressurereached a peak at a particular dose of KCl, the next higher dose of KC1 was added to the Fig. 1. Photostatic enlargement of a pulmonary artery pressure tracing perfusate. taken after a bolus infusion of 20 N-g8-epi-PGF2,y into affluent cannula two
Effects of Diff erent Blocking Response to 6- Epi- PGF,,,
Agents
on PA Pressure
The following blocking agents were added in the perfusate reservoir in different experimentsusing separategroupsof rabbits. Meclofenamate. In four rabbits, 150 PM meclofenamate,a cyclooxygenaseinhibitor, wasaddedbefore repeating the doseresponseexperiment with five dosesof 8-epi-PGF,,. After the PA pressurereturned to the baselineafter the last dose(20 pg) of 8-epi-PGF,,,, 20 pugof PGF2,* were also injected to the PA catheter. Phentolamine. In four rabbits, 70 PM phentolamine,an cu-receptor blocker, was added before an injection of 20 pg 8-epiPG%* SQ 29548.In four rabbits, 10 and 20 pg of 8-epi-PGF*,, were injected into the PA catheter before and after adding in the perfusate 40 PM SQ 29548, a thromboxane receptor blocker, provided by Squibb. NW-nitro-L-arginine (L-IVIVA). In six rabbits, after two consecutivedosesof 20 pg 8-epi-PGF,,,, 20 PM L-NNA, a blocker of endothelial-derived relaxing factor (EDRF), was added to the perfusate. Three minutes later, another dose of 20 pg 8-epiPGFB,,was injected into the PA. [1-(5-isoquinolinesulfonyl)piperazine] (CI). In four rabbits, 20 pg 8-epi-PGF,,, were injected into the PA before and after adding in the perfusate0.2 mM CI, an inhibitor of protein kinase C (9, 21). Atria1 natriuretic factor (ANF). Two setsof experimentswere performed with four rabbits in each group. In one set, 40 rug rANF (synthetic rat ANF, 28 amino acids, Peninsula Laboratories) were injected into the PA catheter 2 min before the injection of 20 pug8-epi-PGF,,,. In the secondset, 40 pg rANF were added when the PA pressurereached its peak after the injection of 20 pg 8-epi-PGF,,. ANF is a potent short-lived pulmonary vasodilator used to determine reversibility of the pressorresponseto 8-epi-PGF,,,. Statistical
Analyses
Data are presentedas means t SE. The statistical significance of differences among the meanswas analyzed by Student’s t test and paired t test where applicable. The effect of meclofenamateon PA pressureresponseto 8-epi-PGF,,, was analyzed by a two-way analysis of variance (dose and drug group) and Duncan’smultiple range test. P < 0.05 wasconsidered significant. RESULTS
Effects of 8-Epi-PGF,,,
on PA Pressure
A typical pulmonary pressor response following the injection of 20 pg 8-epi-PGF,, is shown in Fig. 1. There was a rapid rise in PA pressure, peaking in 3 min, followed by a gradual decline to baseline levels over a period of 40-60 min. In response to increasing doses of 8-epi-PGF,, from 0.5 to 20 pg, the PA pressure rose in a dose-dependent manner from the baseline value of 13.5 t 0.6 to 25.6 t 2.0 cmH20 (Fig. 2).
of rabbit pulmonary artery perfused in situ.
0 basal KCI
1 I 5
5 I 10
(mM)
or 8-epi-PGF2a
10 I 15
20 20
I
c19 mM
(pg)
Fig. 2. Progressive dose-response relationship of bolus infusions of 8-epi-PGFpfl or KC1 on pulmonary arterial pressure (P& in rabbit lung perfused in situ. * P < 0.05 between lungs pretreated with meclofenamate and control. + P < 0.05 from baseline.
Effects of 8-Epi-PGF,,
on Microvascular
Pressure
The results are shown in Table 1 and Fig. 3. PA pressure rose from 12.6 t 0.7 to 21.7 t 1.5 cmH20 with 20 pg 8-epi-PGF,,, and microvascular pressure (P,,) increased from 6.5 t 0.3 to 8.5 t 0.3 cmH20. The upstream pressure accounted for 78% of the total rise in PA pressure (Table 1). The pressure drops from PA to microvascular pressure (P PA - P,,) before and after injecting 20 pg 8-epi-PGF2, were 6.1 t 0.7 and 13.2 t 1.6 cmH20, respectively (P < 0.01). Venous resistance could not be calculated because the lungs were under zone II conditions (outflow pressure less than alveolar pressure). Effects of Vasoactive Compounds and Hypoxia on PA Pressure Response to 8- Epi- PGF,,
At 20 pg, the rise in PA pressure with 8-epi-PGF2, was sixfold greater than with 20 pg PGFzcy (Table 2). Meclofenamate appeared to partially reduce the response to 8-epi-PGF,, at the highest dose (Fig. 2). As shown in Fig. 2, the peak PA pressure response to 5,10,15, and 20 mM KC1 corresponded to the peak response to 1,5,10, and 20 pg 8-epi-PGF,,, respectively. Injection of 40 pg rANF before the injection of 20 pg 8-epi-PGFza caused ~50% Table 1. 8-Epi-PGF,, on the arterial side
acts primarily kmH20
P PA P
pri - p
9.1k1.2 2.0t0.2 7.1kl.l
Percent Change 72.6t10.2 31.3k5.0 117.4t15.5
Values ar:“means t SE; n = 10. PpA, pulmonary arterial pressure; P mv9 microvascular pressure.
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8-EPI-PGFz,, 25 -
20 5 I” 5 a
15 -
2
10 -
ON
RABBIT
LUNG
c
Basal
Table 4. Effects of phentolamine, ANF, hypoxia,
n
8-epi-PGF2a
SQ 29548, and L-NNA on pulmonary arterial pressure response to 20 pg 8-epi-PGF,,,
*: PxO.05
from
basal
PPA,
cm&O
n Control *
ii AI 5-
OpPA
Pmv
PpA+mv
Fig. 3. Change in longitudinal vascular segment pressure to a bolus dose of 20 ,ug 8-epi-PGFz,, in 10 rabbit lungs perfused in situ, measured by double occlusion. P pA, pulmonary artery pressure; P,,, microvascular pressure; PI,;\ - P,,,,., difference. Most of the pressure change was in the upstream segment.
Table 2. Comparative
Phentolamine (70 PM) ANF (40 a) Hypoxia SQ 29548 (40 PM) L-NNA (20 ,uM) Values are means & SE; n, no. arginine. * P < 0.05.
4 14.8k4.5 4 11.9k4.5 8 1 l.Ot2.8 4 17.8t2.1 6 11.2k3.0 of experiments.
With
agent
17.3k4.2 6.5&2.2* 25.2t5.2* 0* 23.3t4.5* L-NNA,
NW-nitro-L-
both SQ 29548 and CI to the perfusate fully blocked the PA pressor response to 8-epi-PGF,,, (Table 4; CI data not shown). DISCUSSION
potency of 20 pg 8-epi-PGF,,, and PGF,,, with and without meclofenamate
8-Epi-PGF,,, causes a marked increase in PA pressure in rabbits whose lungs were perfused in situ with KHB solution. We limited the maximum dose to 20 pg because PGF260 cmH*O) in PA pressure. The addition of showed an increase in pulmonary vascular tone in both normoxic and hypoxic lungs following the administration of N-methyl-L-arginine, an EDRF blocker. In our experTable 3. ANF caused a more rapid decay of pulmonary iments, both hypoxia and L-NNA potentiated the PA arterial pressure following 20 pg 8-epi-PGFzcy pressure response to 8-epi-PGF,,. This suggests that these two pressors, hypoxia and 8-epi-PGF2,,, are moduANF Control lated by release of EDRF. 105~1.0 Basal Pi),z, cmH,O 10.9kl.O Meclofenamate partially altered the dose response of 33.7k4.4 25.0t2.9 Peak P1jll, cmHzO the PA pressure to only 20 pg 8-epi-PGF2,,, suggesting 1.8&0.1* 4.5k0.6 Time to half of peak, min that most of the effect of 8-epi-PGF,,, was not caused by Values are means t SE of 4 experiments. ANF, atria1 natriuretic the production of another intermediary arachidonate factor. * P < 0.05. Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 1, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
8EPI-PGFZty ON RABBIT
product. The lack of influence of phentolamine on the effect of 8-epi-PGF,, indicates that a-receptors were not involved in this vasoconstriction. However, the pressor effect of 8epi-PGF,, was fully prevented by SQ 29548, an endoperoxide receptor blocker. This strongly suggests that the mechanism of 8epi-PGFz,-induced pulmonary vasoconstriction is due to endoperoxide receptor activation. We have shown earlier in sheep that SQ 29548 antagonized the effect of PGDB and PGF2, on the pulmonary vasculature, with no effect on systemic vasculature (lo), suggesting that SQ 29548 may not be specific for thromboxane receptors. Evidence for thromboxane receptor-mediated contraction of guinea pig and human airways in vitro by PGD2, PGE2, and PGFzcy has been provided by Featherstone et al. (7), Coleman and coworkers (4, 5), and Armour et al. (2). Finally, at the cellular level, the contraction of the PA smooth muscle by 8-epi-PGFza may be due to the involvement of protein kinase C, since the use of CI, an inhibitor of protein kinase C, completely blocked the PA pressure response to 8-epi-PGF2,. Morimoto et al. (12) have recently provided evidence for the participation of both intracellular free calcium ions and protein kinase C in tonic vasoconstriction of rat thoracic aorta induced by PGF2,. Participation of protein kinase C in the tonic phase of smooth muscle contraction has been suggested by other investigators (15, 18, 19). Because CI is not a specific inhibitor of protein kinase C, we cannot assert that these data are sufficient to prove a role of protein kinase C in the observed response. In summary, 8-epi-PGFz,,, a noncyclooxygenase free radical-catalyzed product of arachidonic acid, is a powerful pulmonary vasoconstrictor in in situ perfused lungs of rabbits. The vasoconstrictor effect is potentiated by preexposure to hypoxia and by EDRF blockade, while the vasoconstriction is reversed by the use of atria1 natriuretic peptide. The mechanism of action of 8-epi-PGF2, on pulmonary vasculature appears to be due to the activation of SQ 29548-responsive receptors and the participation of protein kinase C. The role of this substance in normal physiology and in disease states remains to be determined. This work was supported by National Institutes of Health Grants K14-02138, HL-19153, GM-42056, and HL-02499. Address for reprint requests: M. Banerjee, Dept. of Physiology, Meharry Medical College, Nashville, TN 37208. Received 19 August 1991; accepted in final form 8 April 1992. HL-39952,
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