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Vol. 283, Issue 3, 1453-1459, 1997
Biochemical Pharmacology, University of Konstanz, Konstanz, Germany (S.U., R.L.F., H.-D.H., A.W.); Byk Gulden, Ltd., Konstanz, Germany (C.S.); and Medical Center of Pediatrics, University Hospital Marburg, Marburg, Germany (R.N.)
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Abstract |
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Abstract
Introduction
Methods
Results
Discussion
References
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Exposure of perfused rat lungs to lipopolysaccharides (LPS) causes induction of cyclooxygenase-2 followed by thromboxane (TX)-mediated bronchoconstriction (BC). Recently, phosphodiesterase (PDE) inhibitors have received much interest because they not only are bronchodilators but also can suppress release of proinflammatory mediators. In the present study, we investigated the effect of three different PDE inhibitors on TX release and BC in LPS-exposed perfused rat lungs. The PDE inhibitors used were motapizone (PDE III specific), rolipram (PDE IV specific), and zardaverine (mixed PDE III and IV specific). At 5 µM, a concentration at which all three compounds selectively block their respective PDE isoenzyme, rolipram (IC50 = 0.04 µM) and zardaverine (IC50 = 1.8 µM) largely attenuated the LPS-induced BC, whereas motapizone was almost ineffective (IC50 = 40 µM). In contrast to LPS, BC induced by the TX-mimetic U46619 was prevented with comparable strength by motapizone and rolipram. In LPS-treated lungs, the TX release was reduced to 50% of controls by rolipram and zardaverine but was unaltered in the presence of 5 µM motapizone. Increasing intracellular cAMP through perfusion of db-cAMP or forskolin (activates adenylate cyclase) also reduced TX release and BC. We conclude that PDE inhibitors act via elevation of intracellular cAMP. Although both PDE III and PDE IV inhibitors can relax airway smooth muscle, in the model of LPS-induced BC, PDE IV inhibitors are more effective because (in contrast to PDE III inhibitors) they also attenuate TX release.
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Introduction |
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Abstract
Introduction
Methods
Results
Discussion
References
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Exposure
of blood-free perfused rat lungs to LPS from Gram-negative bacteria
causes bronchoconstriction that is independent of blood-derived
leukocytes (Uhlig et al., 1995
). We have recently shown that
this effect of LPS depends on induction of the enzyme COX-2 and
subsequent formation of TX (Uhlig et al., 1996). Because Gram-negative septicemia is frequently associated with
bronchoconstriction (Pelosi et al., 1995; Wright et
al., 1994), perfusion of LPS in isolated rat lungs may serve to
study possible therapeutic interventions aimed at preventing
bronchoconstriction in Gram-negative sepsis. However, it is emphasized
that perfusion of rat lungs with LPS is only an incomplete model of the
acute respiratory distress syndrome or sepsis in general because it
lacks a number of important characteristics of this syndrome, such as
neutrophil infiltration, edema formation and pulmonary hypertension.
Still, its advantage is that a defined set of parameters allows an
assessment of a response under controlled conditions followed by an
interpretation of the results that is not confounded by interactions of
the lung with other organs such as the liver, central nervous system or blood.
PDE inhibitors have long been known for their potential to relax airway
smooth muscle (Rabe et al., 1995). In addition, they have
some anti-inflammatory properties, a finding that further stimulated
the interest in these compounds as drugs for the treatment of asthma
(Schudt et al., 1995) or for prevention of the
overactivation of the nonspecific immune system such as in septic shock
(Fischer et al., 1993) or acute respiratory distress
syndrome (Turner et al., 1993). At least seven major groups
of PDE enzymes are classified (Beavo and Reifsnyder, 1990). Of these,
PDE type III and type IV appear to have an important role in regulation
of the cellular inflammatory response. The present study investigates
the effect of selective or combined PDE III and PDE IV inhibition on
endotoxin-induced TX release and subsequent bronchoconstriction in
isolated perfused rat lungs.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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Materials. Female Wistar rats (220-250 g, Zentralinstitut, Hannover, Germany) were used as lung donors. Pentobarbital sodium (Nembutal) was purchased from the Wirtschaftsgenossenschaft Deutscher Tierärzte (Hannover, Germany). Bovine albumin (fraction V) from Serva (Heidelberg, Germany). db-cAMP and forskolin were from Sigma (Deisenhofen, Germany). Rolipram was a gift from Schering (Berlin, Germany), and zardaverine was from Byk Gulden (Konstanz, Germany). Motapizone was from Nattermann-Rhone Poulenc-Rorer (Cologne, Germany)
Isolated perfused rat lung preparation.
The rat lungs were
prepared and perfused essentially as described recently (Uhlig and
Wollin, 1994). Briefly, lungs were perfused at constant hydrostatic
pressure (12 cm H2O) through the pulmonary artery, which resulted in a flow rate of 
20 ml/min. As a perfusion medium, we used Krebs-Henseleit buffer (37°C), which contained 2%
albumin, 1% glucose and 3% HEPES. The total amount of recirculating buffer was 100 ml. The lungs were suspended by the trachea and were
ventilated by negative pressure ventilation with 80 breaths/min and a
tidal volume between 1.6 and 2 ml. Every 5 min, a hyperinflation (
16
cm H2O) was performed. Artificial thorax chamber
pressure was measured with a differential pressure transducer, and air flow velocity was measured with a pneumotachograph tube connected to a
differential pressure transducer. The lungs respired humidified air.
The perfusate flow and arterial and venous pressures were continuously
monitored. The pH of the perfusate before entering the lung was kept at
7.35 by automatic bubbling of the buffer with CO2
as soon as the pH exceeded this value. All data were transmitted to a
computer (Compaq Deskpro 286) via an A/D converter (Metrabyte 16) or an RS232 serial interface and analyzed by
self-written software (programming language ASYST 3.1). Simultaneously,
chamber pressure, tidal volume (by electronic integration), perfusate flow rate, pulmonary artery pressure and pulmonary vein pressure were
recorded on a Graphtec Linearrecorder WR 3310 (Tokyo, Japan).
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Measurement of TX.
Samples taken from the perfusate were
stored at 20°C. TXA2 was assessed as the
stable byproduct TXB2 via enzyme
immunoassay (Cayman, Ann Arbor, MI). The cross-reactivity of the
detecting antibody was TXB2, 100%; 2,3-dinor
TXB2, 8.2%; and prostaglandins (e.g.,
PGD2, PGE2,
6-keto-PGF1
), <0.5%.
RT-PCR analysis.
RNA was isolated from lung tissue by using
Chaosolv solution (Biotecx Laboratories, Houston, TX; supplier's
manual) and analyzed as described previously (Uhlig et al.,
1996). Then, 4 µg of RNA were used for target-specific RT with
Superscript RT and specific primers (GC content of 50-60%). The
primers used have been described previously (Uhlig et al.,
1996). The reactions were cycled 32 times (30 sec at 94°C, 30 sec at
56°C and 30 sec at 72°C after a 5 min denaturing step at 95°C).
Products were analyzed by 2% agarose gel electrophoresis and ethidium
bromide staining. Without specific primer or with the PCR reaction
lacking the template, no amplification products were found. Samples
were assayed in various dilutions to ensure proportionality in the
yield of PCR products. The identity of the fragments was evaluated by
their molecular mass and restriction enzyme analysis.
PDE inhibition.
PDE activity was determined with some
modifications (Bauer and Schwabe, 1980) as described by Thompson and
Appleman (1979). PDE I [Ca2+ (1 mM)/calmodulin
(100 nM) dependent] from bovine brain was kindly provided by Dr.
Gietzen (Ulm, Germany). PDE II [cGMP stimulated (5 µM)] was
purified from rat heart (Schudt et al., 1991b). PDE III
(cGMP inhibited) and PDE V (cGMP specific) were assayed in homogenates
of human platelets as described by Schudt et al. (1991a). PDE IV (cAMP specific) was tested in the cytosol of human
polymorphonuclear as described by Schudt et al. (1991a).
IC50 values were calculated from
concentration-inhibition curves by nonlinear regression analysis using
GraphPAD software (GraphPAD, Sorrento Valley, CA).
Experimental design. db-cAMP, rolipram and motapizone were dissolved in 0.9% NaCl. Forskolin was dissolved in warmed cremophor EL (Sandoz AG, Basel, Switzerland) and further diluted in PBS; LPS was dissolved in PBS/0.005% hydroxylamine and subsequently sonified for 1 min; and zardaverine was dissolved in 1 M NaOH at 70°C and further diluted in PBS. U46619 was prepared as 1 mM stock solution in ethanol. None of the solvents alone had any effect on the LPS-induced bronchoconstriction.
To obtain a stable base line, all lungs were perfused for 40 min before a bolus of 5 mg of LPS was injected into the pulmonary artery. The following values (n = 82, mean ± S.D.) were obtained after 40 min of perfusion and ventilation: tidal volume, 1.87 ± 0.21 ml; pulmonary resistance, 0.25 ± 0.03; and pulmonary compliance, 0.38 ± 0.15. All PDE inhibitors were added 10 min before the administration of LPS. Within these 10 min, none of them induced any alterations in lung function. After administration of LPS, the lungs were perfused and ventilated for an additional 110 min. To mimic the time course of the LPS-induced bronchoconstriction, the TX agonist U46619 was added as a bolus 70 min after the beginning of the experiment; also, in these experiments, PDE inhibitors were added 40 min before the administration of U46619.Statistics.
Values in the figures are given as mean ± S.E.M. For analysis of the pulmonary resistance data, the percentage
data in the figures were transformed by the arcsin transformation (Zar,
1984) and multiple comparisons performed by the Tukey-Kramer Test (SAS software, release 6.11, SAS Institute, Cary, NC). The data for TX
release were analyzed by repeated-measures analysis with orthogonal polynomials (SAS) and Dunnett's test against the LPS-induced TX release.
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Results |
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Abstract
Introduction
Methods
Results
Discussion
References
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The selectivity of the three inhibitors used in the present study on different PDE isoenzymes was quantitatively compared on the basis of their IC50 values (table 1). These data confirmed that motapizone is predominantly PDE III specific and rolipram is PDE IV specific and that zardaverine acts on PDE III as well as PDE IV.
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Perfusion of rat lungs with 50 µg/ml LPS increased pulmonary
resistance (fig. 1 and see fig. 5) as
described recently (Uhlig et al., 1995, 1996). Pretreatment
of lungs with zardaverine (fig. 1A), motapizone (fig. 1B) and rolipram
(fig. 1C) dose-dependently prevented the LPS-induced increase in
pulmonary resistance. The IC50 values were 1.8 µM for zardaverine, 40 µM for motapizone and 0.04 µM for
rolipram.
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Because it is known that the LPS-induced bronchoconstriction in this
model is mediated by TX (Uhlig et al., 1996), we
investigated whether the PDE inhibitors acted by interfering with the
release of this mediator. With respect to rolipram and zardaverine, we studied only concentrations that provided maximum protection against the LPS-induced bronchoconstriction. Both 5 µM rolipram and 50 µM
zardaverine markedly reduced but did not completely prevent the
LPS-induced TX release (fig. 2).
Motapizone had no effect on TX release at 5 µM but attenuated it at
25 µM and completely prevented it at 250 µM
(IC50 = 20 µM, fig.
3). By quantifying the area under the
curve, we found that TX release was attenuated by 50% in the presence
of 5 µM rolipram compared with control lungs perfused with LPS alone.
The corresponding data were attenuation by 76% of LPS-induced TX
release for 50 µM zardaverine, 3% for 5 µM motapizone, 76% for 25 µM motapizone and 93% for 250 µM motapizone. To further explore
the mechanism(s) by which PDE inhibitors reduce the TX release in
LPS-treated rat lungs, we checked whether they affected the expression
of the message for COX-2, the enzyme responsible for TX formation in
this model (Uhlig et al., 1996). Figure
4 shows that neither rolipram (5 µM)
nor motapizone (250 µM) decreased the induction of COX-2 by LPS. In
contrast, both rolipram and motapizone appeared to enhance COX-2 mRNA.
We also examined whether rolipram or motapizone affected the activity
of TX synthase or COX; however, neither rolipram nor motapizone had an
effect on these enzyme activities (data not shown).
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We further pursued the obvious hypothesis that the reduction in LPS-induced TX release and bronchoconstriction in the presence of PDE inhibitors is related to an increase in cAMP. We investigated the effects of db-cAMP and the adenylate cyclase activator forskolin. Both db-cAMP and forskolin prevented a major part of the increased airway resistance in lungs exposed to LPS (fig. 5). The LPS-induced TX release was reduced by 74% in the presence of db-cAMP and by 67% in the presence of forskolin (fig. 6).
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Finally, we examined whether inhibition of PDE as such prevents
bronchoconstriction induced by the spasmogen TX using the stable
agonist U46619. To create a time course comparable to the experiments
shown above in which bronchoconstriction started after 30 min after
injection of LPS, 5 µM rolipram or 5 µM motapizone was present in
the perfusate 40 min before the addition of 5 nmol of the TX receptor
agonist U46619. At these concentrations, both PDE inhibitors attenuated
but did not fully prevent the U46619-induced bronchoconstriction (fig.
7).
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Discussion |
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Abstract
Introduction
Methods
Results
Discussion
References
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Perfusion of LPS in rat lungs causes constriction of terminal
bronchioles (Uhlig et al., 1995) that is mediated by
COX-2-dependent formation of TX (Uhlig et al., 1996). In the
present study, we demonstrated that PDE inhibitors can interfere with
this LPS-induced bronchoconstriction via two separate
mechanisms: blockade of TX release and direct bronchodilation. Because
cAMP-elevating compounds acted in a similar fashion as the PDE
inhibitors, elevation of cytosolic cAMP probably represents their
common mechanism of action.
Elevation of cAMP reduces LPS-induced TX-release.
In our
studies, we focused on TX as the principal mediator responsible for the
LPS-induced bronchoconstriction (Uhlig et al., 1996).
However, other COX metabolites, such as PGD2 and
PGF2, could also be formed and act on TP
receptors in LPS-treated lungs. Our data clearly show that increasing
intracellular cAMP by various means reduced the LPS-induced release of
TX. This finding is in line with the concept that an increase in
intracellular cAMP reduces the release of various other proinflammatory
mediators, such as TNF or eicosanoids (Rola-Pleszczynski and Stankova,
1992; Schudt et al., 1995). It was shown that db-cAMP can
reduce the release of TX from endotoxin-treated (Koyama et
al., 1992) or air-infused (Kobayashi et al., 1987)
sheep in vivo and from murine hepatocytes (Mandl et
al., 1988) in vitro. Similar effects are obtained by treatment with PDE inhibitors. For instance, PDE inhibitors prevented the LPS-induced formation of TNF in vivo (Fischer et
al., 1993; Turner et al., 1993; Kips et al.,
1993), in perfused mouse liver (Leist et al., 1996) and in
isolated peritoneal macrophages (Fischer et al., 1993).
Interestingly, human monocytes contain almost only PDE IV (Schudt
et al., 1995), and PDE III inhibitors did not suppress LPS-induced TNF-production from these cells (Seldon et al.,
1995). However, it appears that further differentiation of monocytes induces other PDE isoenzymes, among them PDE III (Gantner et
al., 1997; Schudt et al., 1995).
, table
1). At this concentration, both PDE
inhibitors attenuated U46619- and endothelin-1-induced (Held et
al., 1997) bronchoconstriction, suggesting that this concentration
was also effective under our conditions. However, at this concentration
only rolipram, not motapizone, reduced the LPS-induced TX release.
These findings suggest that the TX-producing lung cells, which are
unknown in this model (Uhlig et al., 1996; Uhlig and Wendel,
1995), contain predominantly PDE IV. In agreement with these
considerations, we observed that the mixed PDE III/IV inhibitor
zardaverine was no more effective than rolipram alone. In addition, we
recently found that only rolipram, not motapizone, suppressed the
endothelin-1-induced TX release (Held et al., 1997). There
are only a few reports in the literature on the effects of PDE
inhibitors on TX synthesis. In piglets in vivo, amrinone
(PDE III specific) had no effect on TX and TNF release in
Streptococcus-induced pulmonary hypertension (Berger
et al., 1993). Conflicting in vivo results were
reported with two nonspecific PDE inhibitors: pentoxifylline and the
closely related compound HWA 138. Although pentoxifylline was found to reduce LPS-induced TX formation in piglets (Li et al.,
1995), HWA 138 failed to do so in sheep (Masouye et al.,
1992). Studies in perfused rat lungs demonstrated that milrinone (PDE
III specific) attenuated TX release and bronchoconstriction elicited by
antigen challenge (Post et al., 1989). However, the
concentration of milrinone required, such as 100 µM, was high. On a
cellular level, it was shown that milrinone diminished TX release from
stimulated human platelets, although the efficacy of milrinone was
stimulus dependent (Barradas et al., 1993; Jeremy et
al., 1993). And, finally, rolipram and ibudilast (a nonselective
PDE inhibitor) prevented the leukotriene B4-induced release of TX from guinea pig
eosinophils (Souness et al., 1994).
Mechanism of action.
As far as smooth muscle relaxation is
concerned, the relaxant properties of cAMP-elevation are well known and
understood (Rabe et al., 1995; Souness and Giembycz, 1994).
The mechanism by which an increase in intracellular cAMP can affect the
production of TX is far less clear. In the case of LPS-stimulated
monocytes/macrophages, the cAMP-mediated suppression of TNF release has
been ascribed to reduced transcription of the TNF gene (Giroir and
Beutler, 1992; Scales et al., 1989; Spriggs et
al., 1992). However, the LPS-induced induction/stabilization of
COX-2 mRNA, which is mandatory for formation of TX in our model (Uhlig
et al., 1996), was not diminished but rather increased by
the PDE inhibitors. This finding is in agreement with the presence of a
cAMP-responsive element in the COX-2 gene (Appleby et al.,
1994) and with cellular studies on the effect of cAMP on COX-2 mRNA
levels (Nüsing et al., 1996). We also excluded a
direct effect of the PDE inhibitors on the activity of COX or TX
synthase. Moreover, neither of these two enzymes appears to be
regulated by PKA. Therefore, the target for cAMP, or rather PKA,
probably is upstream of COX. In line with this, the liberation of
arachidonic acid by PLA2 is suppressed in the
presence of PDE IV inhibitors in neutrophils (Hichami et al., 1995) and monocytes (Nakashimura et al., 1995), an
effect that appears to be mediated by PKA (Nakashimura et
al., 1995). The substrate for PKA, however, is not known. The
85-kDa cytosolic PLA2 appears to be the enzyme
responsible for agonist-induced arachidonic acid release (Mukherjee
et al., 1994), although the involvement of other
PLA2 isoenzymes cannot be excluded. Although phosphorylation of cPLA2 by mitogen-activated
protein kinase, protein kinase C or G proteins appears to be a control
mechanism for this enzyme (Mukherjee et al., 1994), we found
no evidence in the literature that PKA may phosphorylate
PLA2. Because PKA appears to not directly affect
PLA2, alternative explanations must be
considered: PKA could be regulating one of the kinases that
phosphorylate PLA2, or, alternatively, PKA may
control the increase in intracellular calcium that is essential for
activation of cPLA2. Of relevance to the current
discussion may be the recent findings that PDE inhibitors reduced the
secondary influx of extracellular calcium in human neutrophils (Schudt
et al., 1991a) and that in smooth muscle cells, calcium
influx through L-type calcium channels was inhibited by cAMP-mediated
PKA activation (Orlov et al., 1996).
Effect of PDE inhibitors on airway smooth muscle.
Ample
evidence exists that cAMP PDE inhibitors relax airway smooth muscle
(Rabe et al., 1995; Souness and Giembycz, 1994). In general,
it is thought that airway smooth muscle contains both PDE III and PDE
IV (de Boer et al., 1992; Rabe et al., 1993). PDE
inhibitors appear to be particularly effective in relaxing smaller
airways (Souness and Giembycz, 1994). This is of interest to the
present study because we have previously shown that the bronchoconstriction in response to LPS and TX occurs predominantly at
smaller airways (Martin et al., 1996; Uhlig et
al., 1995).
).
Thus, our data demonstrate that in the present model, inhibition of PDE
IV is more effective than inhibition of PDE III. Interestingly, rolipram was also more effective than a PDE III inhibitor in preventing antigen-induced bronchoconstriction in sensitized guinea pigs (Underwood et al., 1994). In summary, PDE IV inhibitors
possess both antiinflammatory and spasmolytic properties that encourage further studies in the treatment of bronchoconstriction in inflamed lung tissue.
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Footnotes |
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Accepted for publication August 18, 1997.
Received for publication December 30, 1996.
1 This work was supported by the Deutsche Forschungsgemeinschaft Grant We 686/15-1 within the Sonderforschungsbereich 156.
Send reprint requests to: Dr. Stefan Uhlig, Biochemical Pharmacology, University of Konstanz, Fach M668, D-78457 Konstanz, Germany. E-mail: SUhlig{at}fz-borstel.de
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Abbreviations |
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COX, cyclooxygenase; IC50, median inhibitory concentration; LPS, lipopolysaccharide; PDE, phosphodiesterase; PKA, protein kinase A; PLA2, phospholipase A2; db-cAMP, di-butyryl; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; RT, reverse transcription (transcriptase); PCR, polymerase chain reaction; PBS, phosphate-buffered saline; TNF, tumor necrosis factor; TX, thromboxane.
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References |
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Abstract
Introduction
Methods
Results
Discussion
References
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,5-nucleotide phosphodiesterase with QAE Sephadex A25.
Naunyn-Schmiedeberg's Arch. Pharmacol.
311: 193-198, 1980[Medline].
release by PDE inhibitors.
Br. J. Pharmacol.
121: 221-231, 1997[Medline]. or 
-mRNA and cell-associated bioactivity.
J. Leukocyte Biol.
45: 416-421, 1989[Abstract]. generation from peripheral blood monocytes by inhibitors of phosphodiesterase 4: Interaction with stimulants of adenylyl cyclase.
J. Pharmacol. Exp. Ther.
48: 747-757, 1995.This article has been cited by other articles:
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, n = 12) and its attenuation by pretreatment
with PDE inhibitors 10 min before injection of LPS (50 µg/ml). A,
Zardaverine: 0.5 µM (
, n = 3), 5 µM (
,
n = 3) or 50 µM (
, n = 6).
B, Motapizone: 5 µM (
, P < .05 vs. control (
, n = 6). *,
P < .05 vs. LPS.
