low-dose phosphodiesterase inhibition improves responsiveness to inhaled nitric oxide in isolated...

Journal of Surgical Research 138, 224–230 (2007)

Low-Dose Phosphodiesterase Inhibition Improves Responsiveness toInhaled Nitric Oxide in Isolated Lungs From Endotoxemic Rats1

Alexandra Klein, M.D.,*,2 Ulrich Zils, M.D.,† Christian Bopp, M.D.,* André Gries, M.D.,*Eike Martin, M.D., FANZCA,* and René Gust, M.D.*

*Department of Anaesthesiology, University of Heidelberg, Heidelberg, Germany; †Department of Neurosurgery, Campus VirchowHospital, Charité University of Berlin, Berlin, Germany

Submitted for publication June 7, 2005

doi:10.1016/j.jss.2005.12.010

Background. Inhalation of nitric oxide (NO) and in-hibition of phosphodiesterase type 5 (PDE5) selec-tively dilate the pulmonary circulation in patientswith acute lung injury (ALI) associated with pulmo-nary hypertension. PDE5 inhibitors administered atdoses that decrease pulmonary artery pressures havebeen shown to worsen arterial oxygenation. We inves-tigated the efficacy of doses of PDE5 inhibitors that donot reduce pulmonary artery pressure alone (sub-threshold doses) to improve the response to inhaledNO in an animal model of ALI.

Materials and methods. Adult Sprague-Dawley ratswere pre-treated with 0.5 mg/kg Escherichia coli0111:B4 endotoxin and 16 to 18 h later, their lungswere isolated perfused and ventilated. The thrombox-ane mimetic U46619 was used to induce pulmonaryhypertension. After the determination of subthresholddoses of two different PDE5 inhibitors, either 50 �gzaprinast or 10 ng sildenafil was added to the perfus-ate and the decrease of pulmonary artery pressuremeasured in the presence and absence of inhaled NO.

Results. In the presence of 4 or 10 ppm NO, zaprinast(�1.6 � 0.4 and �2.9 � 0.6 mmHg, respectively) andsildenafil (�1.9 � 0.4 and �2.4 � 0.3 mmHg, respec-tively) improved responsiveness to inhaled NO com-pared to lungs from rats treated with LPS only (0.7 �0.1 and �1.0 � 0.1 mmHg, respectively; P < 0.05). Nei-ther zaprinast nor sildenafil prolonged the pulmonaryvasodilatory response to inhaled NO.

Conclusions. Subthreshold doses of PDE5 inhibitorsimproved responsiveness to inhaled NO. Combining

1 Alexandra Klein, Ulrich Zils, and Christian Bopp contributedequally to this work.

2 To whom correspondence and reprint requests should be ad-dressed at Department of Anaesthesiology, University of Heidelberg,Im Neuenheimer Feld 110, 69120 Heidelberg, Germany. E-mail:
[email protected].
2240022-4804/07 $32.00© 2007 Elsevier Inc. All rights reserved.

inhaled NO with subthreshold doses of PDE5 inhibi-tors may offer a therapeutic strategy with minimalside-effects in ALI associated with pulmonaryhypertension. © 2007 Elsevier Inc. All rights reserved.

Key Words: nitric oxide; pulmonary hypertension;sildenafil; zaprinast; acute lung injury; endotoxin;isolated-perfused lung.

INTRODUCTION

Pulmonary hypertension is an unresolved clinicalchallenge in a variety of congenital and acquiredlung diseases. Inhalation of vasodilators, in particu-lar prostacyclin and nitric oxide (NO) selectively di-late the pulmonary circulation in patients with hy-poxic respiratory failure associated with pulmonaryhypertension [1–3]. In pulmonary vascular cells, NOstimulates soluble guanylate cyclase (sGC), an en-zyme that promotes the conversion of GTP toguanosine 3=,5=-cyclic monophosphate (cGMP) [4].Activation of cGMP-dependent protein kinases in-duces smooth muscle relaxation. A family of enzymescalled phosphodiesterases (PDE) inactivates cGMPby converting it to GMP [5, 6]. The cGMP-bindingphosphodiesterase type 5 (PDE5) is known to bespecific for cGMP and is selectively inhibited by za-prinast and sildenafil [6, 7].

Inhibition of phosphodiesterase type 5 (PDE5) is anew approach to reduce pulmonary artery pressurethereby improving arterial oxygenation in patientswith respiratory failure associated with severe pulmo-nary hypertension [8–11]. Recent data, however, sug-gests that PDE5 inhibition at doses which affectpulmonary artery pressure and/or vascular resistancemay worsen arterial oxygenation by increasing in-
trapulmonary shunt in infants with congenital heart

225KLEIN ET AL.: PDE5 INHIBITION IMPROVES NO-RESPONSIVENESS IN LUNG INJURY

diseases or animal models of acute lung injury (ALI)[11–14]. In contrast, inhaled NO up to 20 ppm de-creased or did not alter shunt. Combination of PDE5inhibitors with NO inhalation resulted in reduction ofpulmonary artery pressure but worsened arterial oxy-genation [12, 13]. Because worsening of arterial oxy-genation after PDE5 inhibition is dependent on theability of sildenafil or zaprinast to reduce pulmonaryartery pressure, we used subthreshold doses of both toevaluate whether these doses would be able to improvethe response to inhaled NO. We report here, that sub-threshold doses of zaprinast and sildenafil improve the inresponse to inhaled NO in isolated, perfused and venti-lated lungs obtained from LPS-treated rats. Further-more, we evaluated the prolonged effects of zaprinast andsildenafil after inhalation of NO.

MATERIALS AND METHODS

The Committee for Research Animal Studies of the University ofHeidelberg approved these investigations.

Isolated, Perfused Rat Lungs

Lungs obtained from rats were isolated, perfused, and ventilatedas described previously [15]. Briefly, adult Sprague-Dawley rats(Charles River Laboratories, Sulzfeld, Germany), weighing 400 to450 g were killed by intraperitoneal injection of sodium pentothal(100 mg/kg body wt.). After a midline thoracotomy, the pulmonaryartery and left atrium were cannulated. Lungs were perfused withHank’s balanced salt solution containing 5% dextran, 5% bovineserum, using a roller pump (Abimed, Langenfeld, Germany) at apulsatile flow of 0.03 mL/g body wt/min in a recirculating system at37°C as well as 30 �M indomethacin. The perfusate contained indo-methacin and N-methyl-L-arginine-esther (L-NAME) to avoid syn-thesis of vasoactive mediators during lung perfusion and was con-tinuously aerated with CO2 in the presence of NaHCO3 to adjust andmaintain pH, PCO2 and PO2 between 7.35 to 7.40, 35 to 40 mmHg,and 150 to 250 mmHg, respectively.

Pulmonary artery pressure (PAP) and left atrial pressure weremeasured via small catheters (PE-50 tubing) placed within the twolumen of inflow and outflow perfusion catheters. Left atrial pressurewas set at 2 cm/H2O. The sensitivity of the PAP measurement in ourpreparation was 0.01 mmHg. Because in isolated, perfused, and ven-tilated lungs response to vasodilators can only be observed after induc-tion pulmonary vasoconstriction, we used the thromboxane analogueU46619 to increase pulmonary artery pressure by 6 to 8 mmHg.

Dose-Response Curves of Zaprinast and Sildenafil

To determine subthreshold doses of zaprinast and sildenafil, de-fined as the dose at which no pulmonary vasodilation occurs withinthe first 3 min, 27 rats were injected intraperitoneally with 0.5 mg/kgEscherichia coli 0111:B4 LPS (Difco Laboratories, Detroit, MI)(groups 1-5, Table 1). Sixteen to 18 h later, lungs were isolated,perfused and ventilated, and the thromboxane mimetic U46619 wasused to increase the PAP by 6 to 8 mmHg. The infusion rate wasadjusted to the minimum dose required to maintain an elevatedPAP. In 20 rats randomly assigned into four groups (1 to 4), twodifferent doses of either zaprinast (100 �g; n � 4 or 50 �g; n � 8) orsildenafil (25 ng; n � 3 or 10 ng; n � 5) were added to the perfusateand the change of pulmonary artery pressure was measured 3, 6, 9,and 11 min later. In seven additional rats (group 7), PAP wasmeasured up to 11 min to demonstrate that pulmonary hypertension
remains stable over that observation time.
Pulmonary Vasoreactivity to Inhaled NO in the Absence andPresence of PDE5 Inhibitor

Nitric oxide gas was obtained from AGA (Bottrop, Germany) as amixture of 800 ppm NO in pure N2. Variable concentrations of NOwere mixed with air just before entering the ventilator. NO levelswere measured continuously during administration by chemilumi-nescence analysis (CLD 55AM; Eco Physics, Dürnten, Switzerland).

Six groups of rats treated with LPS were studied (groups 6-11,Table 1). In isolated-perfused lungs, U46619 was administered toincrease PAP by 6 to 8 mmHg. After a stably elevated PAP wasachieved, lungs were ventilated with either 4 (n � 8) or 10 (n � 6)ppm NO alone for 3 min (groups 6 and 7, respectively). In groups8 and 9, lungs were ventilated with 4 ppm NO and either 50 �gzaprinast (n � 8) or 10 ng sildenafil (n � 6) was added to theperfusate. In groups 9 and 10, lungs were ventilated with 10 ppm NOand either 50 �g zaprinast (n � 4) or 10 ng sildenafil (n � 5) was addedto the perfusate. PAP was measured after 3 min and after 11 min.

Prolonged Effects of Zaprinast and Sildenafil onPulmonary Vasoreactivity

To evaluate whether zaprinast or sildenafil prolong the vasodila-tory effect of inhaled NO once it is turned off the change of PAP ingroups 2 and 4 in response to 50 �g zaprinast or 25 ng sildenafil inthe absence of inhaled NO was measured at 11 min. In all groupsventilated with NO (groups 6-11, Table 1), change of PAP was mon-itored up to 11 min.

Statistical Analysis

All data are expressed as mean � standard deviation. The mea-surements of the pulmonary response to zaprinast and sildenafil arethe absolute changes in PAP caused by increasing doses of PDE5inhibitors. Multivariate analysis of variance followed by a pairedt-test with Bonferroni correction was used to examine dose-responsecurves to zaprinast and sildenafil. The measurements of the pulmo-

TABLE 1

Overview of the Study Groups

Groups

1 Zaprinast 100 �g N � 42 Sildenafil 25 ng N � 33 Zaprinast 50 �g N � 84 Sildenafil 10 ng N � 55 U46619 N � 76 NO 4 ppm N � 87 NO 10 ppm N � 68 NO 4 ppm � zaprinast 50 �g N � 89 NO 4 ppm � sildenafil 10 ng N � 6

10 NO 10 ppm � zaprinast 50 �g N � 411 NO 10 ppm � sildenafil 10 ng N � 5

Rats were treated with 0.5 mg/kg LPS and lungs isolated, perfusedand ventilated 16 to 18 h later. Thromboxane analogue U46619 wasused to elevate pulmonary artery pressure by 6 to 8 mmHg. Ingroups 1 to 4, dose-response curves in response to two different dosesof either zaprinast or sildenafil were performed. Then, responsive-ness to 4 or 10 ppm inhaled NO in the absence (groups 6 and 7) andpresence of zaprinast (groups 8 and 10) or sildenafil (groups 9 and 11)were studied. End of observation time was either after 3 min or 11 min.One group (group 5) served as untreated controls to demonstratethat elevated pulmonary artery pressure remained stable over theobservation time of 11 min.

nary response to thromboxane mimetic U46619, inhaled NO, and

226 JOURNAL OF SURGICAL RESEARCH: VOL. 138, NO. 2, APRIL 2007

inhaled NO in combination with zaprinast or sildenafil are expressedas the absolute changes in PAP. Simple t-test was used to examinethe effects of inhaled NO versus treatment at 3 min. The stability oflung preparation was evaluated by using the t-test to examine theeffects of U46619 alone versus U46619 in combination with inhaledNO or inhaled NO plus treatment at 11 min. Statistical significancewas reached when P � 0.05.

RESULTS

Dose Response Curves of Zaprinast and Sildenafil

To determine subthreshold doses of PDE5 inhibitorstwo different doses of either zaprinast or sildenafilwere added to the perfusate of isolated lungs obtainedfrom LPS-pretreated rats. Subthreshold doses weredefined as the dose of a given PDE5 inhibitor that didnot cause vasodilation over a period of 3 min. Inisolated-perfused lungs from LPS-treated rats, 100 �gzaprinast decreased PAP by �0.3 � 0.1 mmHg after 3min compared to baseline values (P � 0.05 versus base-line; Fig. 1). Addition of 50 �g zaprinast to the perfus-

FIG. 1. Determination of subthreshold doses of zaprinast andsildenafil in lungs obtained from LPS-treated rats. Rats were treatedwith 0.5 mg/kg LPS and lungs isolated, perfused, and ventilated 16to 18 h later. The PAP was increased by 6 to 8 mmHg using U46619,and change of PAP in response to zaprinast or sildenafil, respec-tively, was measured. In LPS-treated rats perfused with U46619(n � 7) alone, elevated PAP was stable over a period of 11 min (p �NS.). High doses of zaprinast (n � 4) or sildenafil (n � 3) caused adecrease of PAP after 3 min (#P � 0.05 versus baseline). Zaprinastcaused a continuous decline of PAP over the observation time of 11min (*P � 0.05 versus baseline). Administration of 25 ng sildenafil,however, decreased PAP after 3 min without further change over theobservation time (p � NS for 6, 9, and 11 min, respectively versus3 min). Because 3 min after administration of 50 �g zaprinast (n � 8)or 10 ng sildenafil (n � 5) did not change PAP, these doses were
considered as subthreshold doses (p � NS).
ate of LPS-pretreated rats caused no change of PAPafter 3 min compared with baseline values (�0.1 � 0.1mmHg versus �0.1 � 0.1; NS). Up to 11 min, however,both doses of zaprinast caused a continuing decrease ofPAP by �1.0 � 0.2 mmHg and �1.2 � 0.4 mmHg,respectively (P � 0.05 versus baseline values).

In contrast to zaprinast, addition of 25 ng sildenafilto the perfusate of isolated-perfused lungs from LPS-treated rats caused a sudden decrease of PAP, whichdid not further change over the observation time of11 min (�0.6 � 0.3 mmHg versus baseline values;P � 0.05). There was 10 ng sildenafil added to theperfusate that caused no pulmonary vasodilation at 3and 11 min (�0.1 � 0.1 mmHg and �0.1 � 0.1 mmHg,respectively versus baseline values; NS).

Pulmonary Vasoreactivity to Inhaled NO in the Presence ofSubthreshold Doses of Zaprinast or Sildenafil

To investigate whether subthreshold doses of PDE5inhibitor augment the pulmonary vasodilatory responseto inhaled NO in isolated-perfused lungs of LPS-treatedrats, decrease of PAP in the absence and presence ofeither 50 �g zaprinast or 10 ng sildenafil was measured.In lungs from LPS-treated rats, 4 ppm NO decreasedPAP by 0.7 � 0.1 mmHg (Fig. 2). Zaprinast and sildenafiladded to the perfusate of isolated lungs from LPS pre-treated rats and ventilated with 4 ppm NO caused afurther decrease of PAP (�1.6 � 0.4 mmHg and �1.9 �

FIG. 2. Zaprinast or sildenafil improve the response to 4 ppm NOin lungs from LPS-treated rats. Rats were treated with 0.5 mg/kgLPS and lungs were isolated-perfused 16 to 18 h later. The PAP wasincreased by 6 to 8 mmHg using U46619 and the response to 4 ppmNO in the absence and presence of 50 �g zaprinast (n � 8 and 8,respectively) or 10 ng sildenafil (n � 5 and 6, respectively) wasmeasured. The decrease of PAP in response to 4 ppm NO (n � 8) wasaugmented by administration of zaprinast or sildenafil into the per-fusate (*P � 0.05 versus NO alone).

0.4 mmHg, respectively; P � 0.05; Fig. 2). In lungs from


LPS pre-treated rats, 10 ppm NO caused a decrease ofPAP by �1.0 � 0.1 mmHg (Fig. 3). This decrease wasaugmented by addition of zaprinast or sildenafil to theperfusate of isolated lungs (�2.9 � 0.6 mmHg and �2.4� 0.3 mmHg, respectively; P � 0.05).

Prolonged Effects of Zaprinast and Sildenafil on PulmonaryVasoreactivity in Isolated Perfused Lungs

To investigate whether the augmentation of pulmo-nary vasodilatory response to inhaled NO in the pres-ence of PDE5 inhibitor persists after NO inhalationhas been turned off after a period of 3 min, PAP wasfurther measured for up to 11 min.

In lungs obtained from LPS-treated rats perfusedwith 50 �g zaprinast alone, PAP was decreased com-pared with lungs that had been ventilated with 4 or 10ppm NO alone (�0.9 � 0.2 mmHg versus �0.1 � 0.1and 0.2 � 0.2 mmHg, respectively; P � 0.05; Fig. 4). Inlungs perfused with 50 �g zaprinast combined with theventilation of 4 or 10 ppm NO, pulmonary vasodilatoryresponse was increased compared with lungs venti-lated with 4 or 10 ppm NO alone (�0.9 � 0.2 mmHgand �1.0 � 0.2 mmHg, respectively, versus �0.1 � 0.1and 0.2 � 0.2 mmHg, respectively; P � 0.05). Improve-ment of vasodilatory response to inhaled NO after ad-ministration of zaprinast in isolated-perfused lungswas not different to isolated lungs perfused with zap-rinast alone.

In lungs from LPS-pretreated rats ventilated with 4

FIG. 3. Zaprinast or sildenafil improve the response to 10 ppmNO in lungs from LPS-treated rats. Rats were treated with 0.5 mg/kgLPS and lungs were isolated-perfused 16 to 18 h later. The PAP wasincreased by 6 to 8 mmHg using U46619 and the response to 10 ppmNO in the absence and presence of 50 �g zaprinast (n � 8 and 8,respectively) or 10 ng sildenafil (n � 5 and 5, respectively) wasmeasured (Table 1). The decrease of PAP in response to 10 ppm NO(n � 6) was augmented by administration of zaprinast or sildenafilinto the perfusate (*P � 0.05 versus NO alone).

or 10 ppm NO for 3 min, 10 ng sildenafil caused no

vasodilatory response at the end of observation time of11 min compared with NO ventilation alone (0.1 � 0.1mmHg and �0.3 � 0.2 mmHg, respectively versus �0.1� 0.1 and 0.2 � 0.2 mmHg, respectively; Fig. 5).U46619-induced pulmonary hypertension was stableduring the observation time of 11 min (Figs. 4 and 5).

DISCUSSION

Inhalation of NO selectively dilates the pulmonarycirculation in patients with ALI [16, 17]. In 60% ofseptic patients with ALI, however, inhaled NO failed toreduce pulmonary hypertension and/or to improve ar-terial oxygenation [18, 19]. The precise mechanisms forNO hyporesponsiveness to inhaled NO are unknown butevidence suggest that endotoxemia, the most commoncause of Gram-negative bacterial sepsis associated withALI, induces dysfunction of the pulmonary NO/cGMPsignal transduction pathway [15, 20]. Inhibition ofPDE5 is a new therapeutical approach to reduce pri-

FIG. 4. Zaprinast does not prolong the vasodilatory effect of 4 ppmNO in lungs from LPS-treated rats. Lungs obtained from rats pre-treated with 0.5 mg/kg LPS 16 to 18 h earlier were isolated, perfused,and ventilated. Then, PAP was increased by 6 to 8 mmHg usingU46619 and the change of PAP in the presence and absence of 50 �gzaprinast after ventilation with and without 4 or 10 ppm NO for 3min was measured at the end of observation time of 11 min. In lungsfrom LPS-treated rats, addition of 50 �g zaprinast (n � 8) to theperfusate decreased PAP compared with lungs that had been venti-lated with 4 (n � 14) or 10 (n � 4) ppm NO alone (�P � 0.05). In lungsthat had been ventilated with 4 or 10 ppm NO for 3 min and perfusedwith zaprinast, vasodilatory response was increased when comparedwith lungs ventilated with NO alone (n � 8 and n � 6, respectively;�P � 0.05). The decrease of PAP in lungs ventilated with NO in thepresence of zaprinast was the same than that observed in lungs
treated with zaprinast alone (p � NS).


mary or secondary pulmonary hypertension [9, 11, 14].Growing evidence, however, suggest that doses ofPDE5 inhibitors that reduce PAP in patients with pul-monary hypertension may worsen arterial oxygenation[11, 14]. The fact that PDE5 inhibitors at doses thatdecrease PAP may worsen gas exchange has also beendemonstrated in animal models of ALI [12, 13]. Incontrast, inhaled NO at doses up to 20 ppm decreasedPAP without affecting arterial oxygenation in the samestudies. We therefore investigated whether the combi-nation of subthreshold PDE5 inhibition with low-doseinhaled NO augments its response in a rat model ofALI. To examine the effects of PDE5 inhibition in theabsence and presence of inhaled NO in ALI, we studiedpulmonary vasodilatory response patterns of zaprinastand sildenafil and their ability to improve responsive-ness to inhaled NO isolated-perfused lungs from ratspre-treated with endotoxin. Prior studies have demon-strated that although rats are relatively resistant toendotoxin, administration of LPS produces interstitialand alveolar edema, thickening of lung microvascularwalls, and leukocyte adherence and infiltration [21,22]. Although zaprinast and sildenafil showed differentpatterns of pulmonary vasodilation both augmented theresponse to inhaled NO. In contrast to sildenafil, zapri-nast caused a continuous decline of PAP. Different

FIG. 5. Sildenafil does not prolong the vasodilatory effect of 10ppm NO in lungs from LPS-treated rats. Lungs obtained from ratspre-treated with 0.5 mg/kg LPS 16 to 18 h earlier were isolated,perfused, and ventilated. Then, PAP was increased by 6 to 8 mmHgusing U46619 and the change of PAP in the presence and absence of10 ng sildenafil after ventilation with and without 4 or 10 ppm NOfor 3 min was measured at the end of observation time of 11 min. Inlungs that had been ventilated with 4 (n � 6) or 10 (n � 5) ppm NO,sildenafil showed no increased vasodilatory response when comparedwith lungs ventilated with NO alone (n � 8 and n � 6, respectively)or perfused with sildenafil alone (n � 5).

inhibitors of PDE5 have been demonstrated to be ef-

fective to improve arterial oxygenation and to decreasepulmonary artery pressure in pulmonary hypertension[23–25]. In awake lambs with U46619-induced pulmo-nary hypertension, intravenous infusion of zaprinastor sildenafil administered by nasogastric tube, havebeen shown to selectively dilate the pulmonary circu-lation [23, 26]. Pulmonary vasodilation to PDE5 inhibi-tion has also been demonstrated in isolated-perfusedlungs from mice and rats [27, 28]. It is possible thatinhibition of other PDE isoforms or yet not identifiedcGMP-metabolizing PDE is responsible for the differentpatterns of pulmonary vasodilation in response to zapri-nast and sildenafil. For example, human PDE1 isoformsHcam1 and Hcam3A, both present in the lung are in-hibited by zaprinast but only very high concentrationsof sildenafil inhibit PDE1 [29, 30]. Moreover, zapri-nast, but not sildenafil, inhibits PDE9 that is ex-pressed in lung and does not show high homology withPDE5 [31, 32].

However, study design and route of administrationmay be important whether sildenafil augments theresponse to inhaled NO or not, because sildenafil failedto augment the response to inhaled NO when given inincreasing doses during NO ventilation [27]. Further-more, administration of zaprinast intravenously or sil-denafil by nasogastric tube failed to augment the re-sponse to inhaled NO [23, 26]. In contrast, in awakelambs with U46619-induced pulmonary hypertension,nebulized sildenafil, or zaprinast augmented the re-sponse to inhaled NO [33, 34]. In humans with pulmo-nary arterial hypertension, oral administration of 75 mgsildenafil in combination with 80 ppm NO further de-creased PAP and pulmonary vascular resistance indexwhen compared with NO inhalation alone [10].

To examine whether inhibition of PDE5 prolongs theduration of action of inhaled NO, PAP was measuredup to 11 min after 4 and 10 ppm NO have been turnedoff. In awake lambs with U46619-induced pulmonaryhypertension, it has been demonstrated that zaprinastbut not sildenafil caused prolonged vasodilatory effectin response to 2.5 to 40 ppm NO [23, 26]. In our study,zaprinast deceased PAP in lungs from LPS-treatedrats up to 11 min after an initial 3-min-period of NOventilation. However, this decrease was similar to thatobserved when zaprinast was added to the perfusatewithout NO inhalation. Therefore prolonged decreaseof PAP by zaprinast combined with inhalation of NOwas rather caused by the zaprinast-induced pulmonaryvasodilation. Similar effects of zaprinast on pulmonaryhemodynamics were also demonstrated by Adrie et al.who investigated the single and combined effects ofintravenous infusion of zaprinast and inhaled NO inlambs with lung injury induced by saline lavage [12]. Itappears that this observation is dependent on lunginjury because in contrast to saline-induced lung injury
in lambs and LPS-induced lung injury in rats, in lambs


with U46619-induced pulmonary hypertension zapri-nast prolonged the vasodilatory effect of inhaled NO[12, 23, 26].

Sildenafil in combination with inhaled NO for 3 mindid not cause any change in PAP after NO has beenturned off. This observation was also demonstrated inawake lambs with U46619-induced pulmonary hyper-tension in which sildenafil did not cause prolongedpulmonary vasodilation after discontinuation of 2, 5,10, and 40 ppm NO [23].

In summary, the inhibition of pulmonary PDE5 bytwo different inhibitors at subthreshold doses aug-mented the response to low doses of inhaled NO inisolated-perfused lungs obtained from LPS-pretreatedrats. Because subthreshold doses of sildenafil areable to augment the response to inhaled NO in thismodel of ALI, these data offer a new approach toadminister sildenafil to patients with ALI-inducedpulmonary hypertension without potential side ef-fects.

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