Vol. 280, Issue 2, 770-773, 1997
Effect of Lobaric Acid on Cysteinyl-Leukotriene Formation and
Contractile Activity of Guinea Pig Taenia Coli1
Stefan R.
Gissurarson,
Stefan B.
Sigurdsson,
Hildebert
Wagner and
Kristin
Ingolfsdottir
Department of Pharmacy, University of Iceland, Hagi/Hofsvallagata,
107 Reykjavik (S.R.G., K.I.),
Department of Physiology, University of
Iceland, Vatnsmyrarvegur 16, 101 Reykjavik, Iceland (S.B.S.) and
Institute of Pharmaceutical Biology, University of Munich, Karlstrasse
29, 80333 Munich, Germany (H.W.)
 |
Abstract |
Lobaric acid, a constituent of the lichen Stereocaulon
alpinum, was investigated for effects on the smooth muscle
taenia coli from guinea pigs. Inhibitory effects of lobaric acid on
spontaneous contractile activity and on contractile activity stimulated
by ionophore A23187 were studied. In addition, the activity of lobaric acid on ionophore-induced generation of cysteinyl-leukotrienes in
taenia coli was determined by enzyme immunoassay. Lobaric acid significantly reduced spontaneous contractile activity of the muscle
and inhibited contractions caused by ionophore A23187 with an effective
dose of 5.8 µM. Increased contractility caused by leukotriene
D4 was not influenced by lobaric acid. Lobaric acid inhibited the formation of cysteinyl-leukotrienes as determined by
enzyme immunoassay with an effective dose of 5.5 µM.
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Introduction |
Lobaric acid, a constituent of
the lichen Stereocaulon alpinum Laur., belongs to the class
of phenolic compounds known as depsidones (fig. 1). The
known anti-inflammatory activity exhibited by many phenolic compounds
prompted an investigation of possible inhibitory effects of lobaric
acid on arachidonic acid metabolism.
Leukotrienes are products of arachidonic acid metabolism through the
5-lipoxygenase pathway (Piper, 1984
; Samuelsson, 1983). This enzyme is
considered to be a promising therapeutic target because of its role in
a variety of conditions including asthma, psoriasis, rheumatoid
arthritis and ulcerative colitis (Davies et al., 1984; Higgs
and Moncada, 1985; Musser and Kreft, 1992). In an earlier investigation
lobaric acid, isolated from S. alpinum, showed inhibitory
effects on 5-lipoxygenase in an in vitro assay in which
porcine leukocytes were used as a source of the enzyme (Ingolfsdottir
et al., 1996). The inhibitory concentration of lobaric acid
in this assay was reported as 7.3 µM.
Since the discovery of leukotrienes, intensive research has been
undertaken to assess their importance as mediators of physiological and
pathological processes in various tissues and organs (Salmon and
Garland, 1991). Leukotriene B4 is one of the most potent
chemotactic agents known (Henderson, 1994). The cysteinyl-leukotrienes
(leukotrienes C4, D4 and E4) are
potent inflammatory agents and potent constrictors of smooth muscles in
man and many animals (Piper, 1984; Lewis et al., 1980). It
is known that leukotriene D4 interacts through specific
membrane receptors, and that many smooth muscles which are sensitive to
cysteinyl-leukotrienes have receptors to leukotriene D4
(Ford-Hutchinson et al., 1991). There is also some evidence for the existence of receptors for leukotriene C4 and
E4, but their action is often confused because of rapid
metabolism of leukotriene C4 
D4 E4 (Salmon and Garland, 1991; Lewis et al., 1980; Snyder and Krell, 1984).
Ionophore A23187 has been used in various tissue preparations to
examine nonimmunological release of leukotrienes and other mediators
(Stengel and Silbough, 1988). The ionophore induces leukotriene
generation through a rise in intracellular concentrations of calcium
ions (Wong et al., 1991). To examine the nature of the
in vitro inhibitory effect of lobaric acid on 5-lipoxygenase observed earlier, investigations were undertaken to study the effects
of lobaric acid on ionophore A23187 induced contractions in the smooth
muscle taenia coli from guinea pigs. This muscle shows stable
spontaneous contractile activity and is sensitive to leukotriene
D4 which causes marked increase in contractile activity.
EIA was used to determine the effects of lobaric acid on the release of
cysteinyl-leukotrienes from taenia coli.
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Methods |
Materials.
Lobaric acid was isolated from the lichen
S. alpinum Laur. and purified by use of medium pressure
liquid chromatography as described earlier (Ingolfsdottir et
al., 1996). Purity of lobaric acid was confirmed by infrared, mass
spectral, 1H and 13C NMR spectroscopic
analysis. Arachidonic acid, ionophore A23187, indomethacin and
leukotriene D4 were obtained from Sigma Chemical Company
(St. Louis, MO) and dissolved in DMSO (Merck Art 2931) for initial
stock solutions followed by appropriate dilutions in Krebs solution.
Stock solutions of lobaric acid in DMSO were prepared at a
concentration of 1 × 10
2 M.
A cysteinyl-leukotriene EIA kit was obtained from Cayman Chemicals (Ann
Arbor, MI). Ultrapure water was prepared by passing deionized and
distilled water through an activated carbon filter (Carbon-Cap 75, Whatman, Clifton, NJ). All buffers and reagents for EIA were
reconstituted in ultrapure water. The EIA plates were read at 405 nm
(Thermo max, supported by Softmax version 2.02; Molecular Devices,
Menlo Park, CA).
All other solvents and materials were obtained from Merck, Darmstadt,
Germany.
Muscle preparation.
Eighteen female guinea pigs (515-876 g)
obtained from Keldur (Institute for Experimental Pathology, University
of Iceland) were anesthetized in CO2 and sacrificed by
stunning followed by exsanguination. The abdomen were immediately
opened and the smooth muscle taenia coli removed, transferred to Krebs
solution at room temperature, pH 7.4, and rinsed gently.
The composition of the Krebs' solution was: 112.60 mM NaCl; 5.91 mM
KCl; 24.90 mM NaHCO3; 1.19 mM MgCl; 1.18 mM
NaH2PO4; 2.00 mM CaCl2; 11.50 mM
glucose; pH 7.4.
The taenia coli muscle was cut in approximately 1.5- to 2-cm strips and
prepared by tying together the two ends with silk suture to make a
ring. The ring was transferred to a small organ bath (4 ml) with
Krebs' solution aerated with 95% O2 and 5%
CO2. The muscle was held vertical in the organ bath by
connection to a glass oxygen tube and the other end connected with silk
suture to a force transducer (Grass, Grass Instrument Co., Quincy, MA), which allowed monitoring of changes in contractile activity of the
muscle. The isometric force was quantitated by integrating the active
tension curve with an electronic integrator device. The heat in the
organ bath was kept constant at 37°C by placing the organ bath in a
thermostated water bath and by having continuous flow of
O2/CO2 bubbling through the Krebs' solution.
During the equilibration period (60-90 min) the Krebs' buffer was
changed every 15 min. After the equilibration period the muscles were
incubated for 15 min with either: a) Krebs' solution containing DMSO
(3 µl/ml) as control solution; b) Krebs' solution containing
arachidonic acid (10 µM); c) Krebs' solution containing DMSO (3 µl/ml), arachidonic acid (10 µM) and indomethacin (3 µM); d)
Krebs' solution containing DMSO (3 µl/ml), arachidonic acid (10 µM), indomethacin (3 µM) and lobaric acid (2.5, 5, 10, 20 and 30 µM). The incubation time of 15 min was sufficient to obtain maximal
effects of the test compounds. At the end of the incubation, the
muscles were challenged with 1 µM ionophore A23187, which is the
concentration needed for release of leukotrienes (Wong et
al., 1991; Hoult et al., 1994).
The tissues were removed from the bath and weighed after 30 min (wet
weight). The whole physiological solution was then transferred from the
organ bath to polypropylene test tubes and immediately placed in a
freezer until EIA determination of the cysteinyl-leukotrienes.
Recording of contractions.
The spontaneous contractile
activity of taenia coli was monitored for 10 min before treatment with
the test compounds (a-d, under "Muscle Preparation") and in the
recovering period after treatment with the test compounds. Changes in
contractile activity were also recorded after treatment with ionophore
A23187, in the absence or presence of the test compounds (a-d, under
"Muscle Preparation"). In addition contractile activity was
monitored for 10 min before and after treatment of the muscle
preparation with leukotriene D4 (1 nm), with and without 20 µM lobaric acid.
For electrical field stimulation taenia coli was isolated as described
above and cut in approximately 1- to 1.5-cm strips. Each muscle strip
was mounted in an organ bath for measurement of isometric contractions
and allowed to equilibrate for at least 60 min. Electrical field
stimulation by use of pairs of platinum electrodes was performed in the
presence of: a) lobaric acid (2.5, 5, 10 and 20 µM), b) vehicle
(DMSO, 1 µl/ml), c) pure Krebs' solution (control). Electrical field
stimulation was constant for all measurements (40 V, 100 ms).
Preparation for EIA determination.
The physiological
solutions which had been taken from the organ bath and frozen were
thawed at room temperature and acidified to pH 4 with HCl. They were
subsequently passed through C18 cartridges (Spe-edTM, 500 mg/6 ml) which had been prewashed with 5 ml
ethanol and activated with 5 ml ultrapure water. Each cartridge was
then washed with 5 ml ultrapure water followed by 5 ml hexane.
Cysteinyl-leukotrienes were eluted with 5 ml ethanol/water (90:10). The
eluted samples were evaporated to dryness under a stream of dry
nitrogen, reconstituted in 1 ml EIA buffer for EIA analysis. The
cysteinyl-leukotriene concentration was quantitated by EIA (Pradelles
et al., 1985) and calculated as picograms of
cysteinyl-leukotrienes per milligram of tissue.
Statistical analysis.
Data and charts are expressed as the
mean ± S.E. or ± S.E.M. ED50 was determined by
linear regression. Statistical difference was evaluated by the
Student's t test. P values of less than 0.05 were
considered to represent significant differences.
| |
Results |
Spontaneous and A23187-induced contractions.
To stimulate
generation of lipoxygenase products in taenia coli the muscle was
challenged with ionophore A23187 in the presence of indomethacin and
arachidonic acid. The ionophore forms a stable complex with
Ca++ and carries the ion across cell membranes and leads to
a rise in intracellular concentrations of Ca++ (Kaufmann
et al., 1980). The rise in calcium concentration activates 5-lipoxygenase and is followed by increased generation of
5-lipoxygenase metabolites including cysteinyl-leukotrienes (Wong
et al., 1991).
As previously described (Wong et al., 1991), ionophore
A23187 causes generation of eicosanoids via both
cyclooxygenase and 5-lipoxygenase pathways. The cyclooxygenase
inhibitor indomethacin was therefore used to suppress cyclooxygenase
activity in taenia coli (Mion et al., 1994). Arachidonic
acid was added to prevent depletion.
Lobaric acid depressed the taenia coli muscle preparation in two ways.
First, when lobaric acid was added to the organ bath it reduced the
spontaneous contractile activity in a dose-dependent way (fig.
2). The amount of DMSO used did not affect spontaneous contractile activity. Second, lobaric acid inhibited the contractile response caused by ionophore A23187, also in a dose-dependent way (fig.
3). When the muscle was challenged with ionophore in the
presence of indomethacin the contractile activity of the muscle increased 4-fold (fig. 3) compared with a control preparation in which
the muscle was treated solely with indomethacin and vehicle (DMSO/Krebs' solution). Arachidonic acid, when added alone to the
muscle preparation at a concentration of 10 µM, caused an increase in
base-line tension of 13% ± 7% (S.E., n = 4) and an increase in contractile activity from the control set as 100% to 124% ± 11% (SE, n = 4). These effects disappeared in the
presence of indomethacin.
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Fig. 2.
Effect of lobaric acid (± S.E.M.) on spontaneous
contractile activity of taenia coli. Contractile activity before
administration of the test compound is set as 100% (control).
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Fig. 3.
Effect of lobaric acid on contractile response (± S.E.M.) of taenia coli induced by ionophore A23187 (1 µM).
Spontaneous contractile activity prior to administration of ionophore
A23187 is set as 100% (control). n = 6 (n* = 3).
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The concentration of lobaric acid that reduces the effect of ionophore
A23187 by 50% is 5.8 µM (ED50).
Leukotriene D4-induced contraction.
Leukotriene D4 at a concentration of 1 nM increased
contractile activity of the muscle approximately 4-fold. Lobaric acid (20 µM) did not significantly prevent increase in contractile activity caused by leukotriene D4 (fig. 4).
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Fig. 4.
Effect of lobaric acid (20 µM) on contractile
response (± S.E.M.) of taenia coli induced by 1 nM leukotriene
D4 (LTD4). Spontaneous contractile activity
before administration of test compounds is set as 100% (control).
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Electrical field stimulation.
Lobaric acid (2.5, 5, 10 and 20 µM) did not significantly depress the response of taenia coli to
electrical field stimulation (fig. 5). DMSO (1 µl/ml)
did not influence contractile activity induced by electrical field
stimulation.
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Fig. 5.
Effect of lobaric acid on contractile response (± S.E.) of taenia coli to electrical field stimulation (40 V, 100 ms),
n = 4.
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Quantification of cysteinyl-leukotrienes.
To establish the
extent of cysteinyl-leukotriene generation in taenia coli induced by
ionophore A23187, in the presence and absence of lobaric acid, the
physiological solutions were taken from the organ baths and the
concentration of cysteinyl-leukotrienes was determined by using EIA.
The amount of cysteinyl-leukotrienes that was generated was compared
with control samples taken from muscles that were neither treated with
lobaric acid nor with ionophore A23187. Results showed that test
concentrations of lobaric acid ranging from 0 to 20 µM gradually
inhibited the generation of cysteinyl-leukotrienes caused by ionophore
in the muscle (table 1). Test concentrations of lobaric
acid greater than 20 µM totally inhibited generation of
cysteinyl-leukotrienes in the muscle compared with control samples. The
ED50 value of lobaric acid, i.e., the concentration at which generation of cysteinyl-leukotrienes was inhibited by 50% was 5.5 µM (table 1).
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TABLE 1
Effect of lobaric acid on ionophore A23187-induced
cysteinyl-leukotriene generation in taenia coli as determined by EIA
Controls represent muscle preparations neither treated with ionophore A
23187 nor lobaric acid.
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Discussion |
The aim of this study was to determine the effect of a natural
product, lobaric acid from the lichen S. alpinum, on
leukotriene-related mechanisms in taenia coli from guinea pigs.
A method was developed whereby effects of lobaric acid on
ionophore-induced contractions were investigated and whereby effects of
lobaric acid on the generation of cysteinyl-leukotrienes were determined.
Taenia coli was chosen for the muscle preparation because it is
sensitive to cysteinyl-leukotrienes, shows spontaneous contractility and the quantification of muscular activity is relatively easy to
perform.
Results, which show that lobaric acid inhibits ionophore
A23187-induced contractile activity and generation of
cysteinyl-leukotrienes in taenia coli, might correlate with the
in vitro 5-lipoxygenase blocking activity of lobaric acid
reported previously (Ingolfsdottir et al., 1996). However,
it must be borne in mind that other mechanisms of action are possible,
e.g., inhibition of phospholipase A2 and leukotriene C4 synthase. Further studies are needed to
substantiate the mode by which lobaric acid exerts activity in this
model.
It can be assumed, nevertheless, that the mode of action of lobaric
acid is not attributable to effects on leukotriene receptors (at least
leukotriene D4 receptors), because the compound did not
influence increased contractility caused by leukotriene D4. Furthermore, it can be presumed that lobaric acid does not affect voltage-sensitive Ca++ channels. First, it has been shown
that blocking of Ca++ channels inhibits increased
Ca++ uptake and therefore activity caused by
cysteinyl-leukotrienes (Oliva et al., 1994). Second, taenia
coli treated with lobaric acid in our study did not show decreased
response to electrical field stimulation (Zschaufer et al.,
1988).
In addition to the effects of lobaric acid discussed above, it was
found that the compound depresses spontaneous activity of taenia coli.
The reason for this effect is not known, but lipophilic properties of
the molecule are likely to be an important factor. Further studies are
needed for clarification.
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Footnotes |
Accepted for publication October 1, 1996.
Received for publication May 22, 1996.
1
Supported by grants from the Icelandic Council of
Science, University of Iceland Research Fund and NM Pharma Research
Fund.
Send reprint requests to: Kristin Ingolfsdottir, University
of Iceland, Department of Pharmacy, Hagi/Hofsvallagata, 107 Reykjavik,
Iceland.
| |
Abbreviations |
EIA, enzyme immunoassay;
ED50, effective dose;
DMSO, dimethyl sulfoxide;
S.E., standard error;
S.E.M., standard error of the mean.
| |
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Abstract
Introduction
