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Vol. 284, Issue 3, 1156-1164, March 1998
Department of Medicine (H.S., X.-p.G, I.R.), University of Illinois at Chicago and West Side Department of Veterans Affairs Medical Center, Chicago, Illinois, and Cardiovascular Research Institute and Department of Medicine (G.H.C.), University of California, San Francisco, California
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study investigated whether short-term exposure to Escherichia coli lipopolysaccharide (LPS) elicits vasomotor dysfunction in skeletal muscle in vivo and, if so, whether perivascular mast cell proteases partly modulate this response. With intravital microscopy, we found that suffusion of E. coli LPS on the in situ hamster spinotrapezius muscle for 60 min elicits immediate vasoconstriction followed by vasodilation. Vasoconstriction is abrogated by SK&F 108566, a selective, nonpeptide angiotensin II (AT II) subtype 1 receptor antagonist, chymostatin and soybean trypsin inhibitor. These compounds also attenuate E. coli LPS-induced vasodilation. By contrast, superoxide dismutase, catalase and indomethacin attenuate only E. coli LPS-induced vasodilation. Endothelin receptor antagonists, lisinopril, leupeptin, Bestatin and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid are ineffective. Histochemical analysis of the spinotrapezius muscle reveals abundant perivascular mast cells with chymostatin-inhibitable chymase-like activity. Pretreatment of hamsters with compound 48/80 for 4 days curtails E. coli LPS-induced vasoconstriction and converts vasodilation to vasoconstriction. On balance, these data indicate that E. coli LPS stimulates perivascular mast cells in the in situ hamster spinotrapezius muscle to release an AT II-producing chymase-like protease(s). AT II thus produced elicits local vasoconstriction and elaborates reactive oxygen species which, in turn, generate vasodilator prostaglandins.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Despite
recent advances in medical care, Gram-negative bacterial sepsis
syndrome remains a major cause of morbidity and mortality among
hospitalized patients (Centers for Disease Control and Prevention, 1990
; Natanson, 1994). A characteristic feature of this syndrome is
vasomotor dysfunction consisting of profound peripheral vasodilation, refractory hypotension and end-organ failure that evolves several hours
after exposure to the offending pathogen(s) (Hess et al., 1981; Natanson, 1994; Wurster et al., 1994). The emergence
of this triad is considered an ominous prognostic sign associated with
high mortality rate (Natanson, 1994). However, the mechanisms underlying the evolution of vasomotor dysfunction in Gram-negative sepsis syndrome are uncertain.
Current concepts suggest that LPS, a macromolecular glycolipid
component of Gram-negative bacterial walls, plays an important role in
the genesis of vasomotor dysfunction in sepsis syndrome (Natanson,
1994; Neviere et al., 1996; Shenep and Morgan, 1984; Shenep
et al., 1988). To this end, skeletal muscle contains a large
proportion of resistance arterioles in the peripheral circulation and
contributes appreciably to regulation of peripheral vascular resistance
under pathophysiological conditions, such as sepsis syndrome (Baker
et al., 1992; Cryer et al., 1987; Neviere
et al., 1996; Roswell, 1986).
It is well established that resistance arterioles in skeletal muscle
and other organs are surrounded by mast cells that release potent
phlogistic proteases, including chymase and tryptase, upon stimulation
(Gao et al., 1993; Huntley et al., 1985; Li
et al., 1993; Raud, 1989; Rubinstein et al.,
1990; Shepherd and Duling, 1996; Urbaschek and Urbaschek, 1979).
However, the role these proteases play in the pathophysiology of
vasomotor dysfunction observed in sepsis syndrome is uncertain
(Svensjö et al., 1990; Urbaschek and Urbaschek, 1979).
Hence, the purpose of this study was to begin addressing this issue by
determining whether short-term exposure to Escherichia coli
LPS elicits vasomotor dysfunction in skeletal muscle in vivo
and, if so, whether perivascular mast cell proteases partly modulate
this response.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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General Methods
Preparation of animals. Adult male golden Syrian hamsters (n = 80) weighing 137 ± 4 g were anesthetized with pentobarbital sodium (6 mg/100 g b.wt. i.p.). A tracheostomy was performed to facilitate spontaneous breathing. A femoral vein was cannulated to inject supplemental anesthesia during the experiment (2-4 mg/100 g b.wt./h). A femoral artery was cannulated to monitor systemic arterial pressure and heart rate. Both did not change significantly during the course of the experiments. Body temperature was monitored during the experiments and maintained constant (37-38°C) using a feedback controller and heating pad.
The right spinotrapezius muscle was prepared for intravital microvascular observation as described previously (Gray, 1973; Lash and
Bohlen, 1987). A median skin incision was made along the spine, and the
loose connective tissue beneath the skin was cut away to expose the
muscle surface. The animal was placed on its left side and the lateral
side of the right spinotrapezius muscle was carefully pulled out with
blunt dissection. The muscle was spread, ventral surface up, over a
plastic baseplate and its edge fixed in the horizontal position using a
silk thread. Care was taken to maintain a physiological length of the
muscle during the procedure. An upper plastic chamber was placed above
the muscle and contained the suffusate. The chamber was connected
via a three-way valve to a reservoir that allowed continuous
suffusion of the muscle with warm (37-38°C) bicarbonate buffer
(composition, in mM: NaCl, 131.9; KCl, 2.95;
CaCl2, 1.48; MgCl2, 0.76;
NaHCO3,11.87) bubbled continuously with 95%
N2-5% CO2 (pH 7.4). The
chamber was also connected via a three-way valve to an
infusion pump that allowed controlled administration of E. coli LPS and drugs into the suffusate.
Determination of arteriolar diameter.
The spinotrapezius
muscle microcirculation was transilluminated with a fiberoptic light
source (Nikon) and viewed through a Nikon microscope with a water
immersion lens. The image was projected through the microscope and into
a closed-circuit television system consisting of camera (Panasonic
WV-1500), monitor (Panasonic TR-124 MA) and videotape recorder
(Panasonic AG-1230). The luminal diameter of second-order arterioles in
the spinotrapezius muscle (base-line diameter, 51 ± 2 µm)
(Fronek and Zwiefach, 1975) was measured from the video display of the
microscope image by a videomicrometer (VIA 100; Boeckeler Instruments,
Tucson, AZ) as described previously in our laboratory (Gao et
al., 1994, 1995; Mayhan and Rubinstein, 1995; Rubinstein et
al., 1991). This system was calibrated against a precision
line-width standard. In each animal, the same arteriolar segment was
used to measure changes in diameter during the experiment.
Experimental Protocols
Effects of E. coli LPS on arteriolar diameter.
These studies determined the effects of short-term suffusion of
E. coli LPS on the spinotrapezius muscle on arteriolar
diameter. After suffusing the bicarbonate buffer for 45 min
(equilibration period), increasing concentrations of E. coli
LPS (0.3, 3.0 and 30.0 µg/ml) were suffused in random order. Each
concentration was suffused for 60 min. Arteriolar diameter was measured
before, every minute during the first 15 min of suffusion and every 5 min for the next 90 min. At least 45 min elapsed between subsequent suffusions of E. coli LPS. In preliminary studies, we
determined that repeated suffusions of E. coli LPS were
associated with reproducible results. The concentrations of E. coli LPS used in these studies are similar to those used
previously in the in situ hamster cheek pouch (Gao et
al., 1995; Svensjö et al., 1990).
Mechanisms of E. coli LPS-induced changes in
arteriolar diameter.
Role of angiotensin II. These
studies determined whether local production of AT II partly mediates
E. coli LPS-induced vasoconstriction in the spinotrapezius
muscle (Baker et al., 1992; Dunn and Horton, 1993). After
suffusing the bicarbonate buffer for 45 min, SK&F 108566 (0.1 µM), a
selective, nonpeptide AT1 RA (Edwards et
al., 1991), was suffused on the spinotrapezius muscle 30 min
before and during suffusion of E. coli LPS (3.0 µg/ml) for
60 min. In another series of experiments, SOD (60 U/ml) and SK&F 108566 (0.1 µM) were suffused together 30 min before and during suffusion of
E. coli LPS (3.0 µg/ml) for 60 min. Last, AT II (0.05 µM) was suffused for 10 min before and after suffusing SK&F 108566 (0.1 µM) for 30 min. Arteriolar diameter was determined during each intervention.
).
The concentrations of SOD and AT II used in these studies previously
were used in the in situ hamster cheek pouch (Cornish
et al., 1979; Del Maestro et al., 1981; Edwards
et al., 1991; Erlansson et al., 1990).
Role of reactive oxygen species. These studies determined
whether superoxide and hydrogen peroxide, which are elaborated by AT II
(McKechnie et al., 1986), partly mediate E. coli
LPS-induced responses. After the equilibration period, SOD (60 U/ml) or
catalase (60 U/ml) was suffused on the spinotrapezius muscle for 30 min before and during suffusion of E. coli LPS (3.0 µg/ml) for
60 min. Arteriolar diameter was determined during each intervention. In
preliminary studies, we determined that suffusion of SOD and catalase
alone for 90 min has no significant effects on arteriolar diameter. The
concentrations of SOD and catalase used in these studies are similar to
those previously used in the in situ hamster cheek pouch
(Del Maestro et al., 1981; Edwards et al., 1991;
Erlansson et al., 1990).
Role of endothelin. These studies determined whether ET,
which activates the vascular renin-angiotensin system (Rakugi et al., 1990), mediates E. coli LPS-induced responses.
After the equilibration period, PD 142893 (1.0 µM), an
ETA receptor antagonist (Mayhan and Rubinstein,
1995), or BQ-485 (1.0 µM), an ETAB receptor antagonist (Itoh et al., 1993), was suffused for 30 min
before and during suffusion of E. coli LPS (3.0 µg/ml) for
60 min. Arteriolar diameter was determined during each intervention. In
preliminary studies, we determined that suffusion of PD 142893, BQ-485
and dimethyl sulfoxide (1.0 µM), the vehicle of PD 142983 and BQ-485, alone for 90 min had no significant effects on arteriolar diameter. The
concentrations of PD 142983 and BQ-485 used in these studies previously
abrogated ET-induced vasoconstriction in hamsters and dogs,
respectively (Itoh et al., 1993; Mayhan and Rubinstein, 1995).
Role of prostaglandins. These studies determined whether
prostaglandins, which are thought to play a role in the pathophysiology of vasomotor dysfunction in sepsis syndrome (Fujimura and Ebihara, 1990; Natanson, 1994; Warren et al., 1991), mediate E. coli LPS-induced responses. After the equilibration period,
indomethacin (10 mg/kg) was administered i.v. for 30 min by an infusion
pump followed by suffusion of E. coli LPS (3.0 µg/ml) on
the spinotrapezius muscle for 60 min. Arteriolar diameter was
determined during each intervention. In preliminary studies, we
determined that indomethacin (10 mg/kg i.v.) alone had no significant
effects on arteriolar diameter. The concentration of indomethacin used
in these studies previously inhibited cyclooxygenase in the hamster
cheek pouch (Gao et al., 1993, 1995; Raud, 1989; Rubinstein
et al., 1991).
Role of mast cells and chymase-like proteases. These studies
determined whether perivascular mast cells and chymase-like proteases, which convert angiotensin I to AT II in the peripheral circulation (Cornish et al., 1979; Okamuara et al., 1990;
Reilly et al., 1982; Wintroub et al., 1984),
modulate E. coli LPS-induced immediate biphasic vasomotor
dysfunction in the in situ spinotrapezius muscle. To
accomplish this goal, we adopted four strategies. First, we determined
the presence of perivascular mast cells and chymase-like activity in
the spinotrapezius muscle and cheek pouch. The latter tissue was used
as a positive control (Pearce et al., 1985; Raud, 1989;
Shepherd and Duling, 1996; Takai et al., 1996). Hamsters were killed with an overdose of pentobarbital (50 mg/100 g b.wt. i.p.)
and the spinotrapezius muscle and cheek pouch were carefully excised
and placed in Mota's lead acetate solution (1% lead acetate, 50%
ethanol and 0.5% acetic acid) for 24 h (Martin et al.,
1992). Tissues were then dehydrated in graded solutions of ethanol,
embedded in paraffin, cut into 5-µm sections and placed onto glass
slides. Tissue sections were deparaffinized in xylene and rehydrated in graded ethanol solutions. To identify mast cells, tissue sections were
immersed in toluidine blue solution (0.5% in 0.5 N HCl) for 5 min,
rinsed in water, air-dried and coverslipped.
To identify cells containing chymase-like activity, tissue sections
adjacent to or near those stained with toluidine blue were subjected to
enzyme histochemical staining with NASDCA with minor modifications as
described previously (Martin et al., 1992). In this staining
reaction, active chymase-like enzyme within mast cells precipitates the
azo dye Fast Garnet GBC at the site of production. In toluidine
blue-stained sections, extravascular cells containing metachromatic
(purple) cytoplasm or granules were considered to be mast cells. In
sections stained with NASDCA-Fast Garnet GBC, extravascular cells
containing reddish-brown cytoplasm or granules were considered to
contain chymase-like activity. To compare the total number of mast
cells (represented by metachromatic cells) with the number of cells
exhibiting chymase-like (NASDCA-positive) activity, the number of cells
in each category were counted and compared in identical regions of
adjacent tissue sections. All mast cells in each section were counted
using objectives of 10× to 40×.
In a second group of animals, the right spinotrapezius muscle was
prepared for intravital microscopy experiments as outlined above. After
the equilibration period, chymostatin (10 µg/ml) or soybean trypsin
inhibitor (100 µg/ml), two relatively selective and potent inhibitors
of mast cell chymase (Martin et al., 1992; Reilly et
al., 1982), was suffused for 30 min before and during suffusion of
E. coli LPS (3.0 µg/ml) for 60 min. Arteriolar diameter was determined during each intervention. In preliminary studies, we
determined that suffusion of chymostatin (10 µg/ml) and soybean trypsin inhibitor (100 µg/ml) alone for 90 min had no significant effects on arteriolar diameter. The concentrations of chymostatin and
soybean trypsin inhibitor used in these studies previously inhibited
mast cell chymase (Martin et al., 1992; Okamura et
al., 1990; Reilly et al., 1982; Rubinstein et
al., 1990).
A third group of animals was treated with compound 48/80 (1.5 mg/kg
diluted in 0.2 ml saline, once daily i.p.) to deplete mast cells from
preformed mediators (Gao et al., 1993; Raud, 1989; Urbaschek
and Urbaschek, 1979) or saline (0.2 ml) for 4 days. Thereafter, the
right spinotrapezius muscle was prepared for intravital microscopy
experiments. After the equilibration period, E. coli LPS
(3.0 µg/ml) was suffused for 60 min. Arteriolar diameter was determined during each intervention. The concentration of compound 48/80 used in these studies previously depleted mast cells from preformed mediators in the hamster cheek pouch (Gao et al.,
1993; Raud, 1989).
In a fourth group of animals, we determined whether exogenous AT II
elicits vasoconstriction by releasing chymase-like protease(s) in the
spinotrapezius muscle. After the equilibration period, AT II (0.05 µM) was suffused on the spinotrapezius muscle for 10 min. Once
suffusion of AT II was stopped and arteriolar diameter returned to
baseline, a mixture of chymostatin (10 µg/ml) and soybean trypsin
inhibitor (100 µg/ml) was suffused 30 min before and during suffusion
of AT II (0.05 µM) for 10 min. In another group of animals, AT II
(0.05 µM) was suffused for 10 min before and after suffusing SK&F
108566 (0.1 µM) for 30 min. Arteriolar diameter was determined during
each intervention. The concentration of AT II used in these studies
previously elicited potent vasoconstriction in the in situ
hamster cheek pouch (Cornish et al., 1979).
Role of other proteases. These studies determined whether
proteases other than chymase modulate E. coli LPS-induced
responses (Gao et al., 1994; Okamura et al.,
1990). In one group of animals, after the equilibration period,
lisinopril (10 µM), an ACE inhibitor, was suffused for 30 min before
and during suffusion of E. coli LPS (3.0 µg/ml) for 60 min. In a second series of experiments, a mixture of protease
inhibitors consisting of leupeptin, Bestatin and
DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid
(each, 10 µM), to inhibit aminopeptidases, thiol proteinases and
carboxypeptidases N, respectively, was suffused for 30 min before and
during suffusion of E. coli LPS (3.0 µg/ml) for 60 min. In
preliminary studies, we determined that suffusion of lisinopril and the
mixture of proteinase inhibitors alone for 90 min had no significant
effects on arteriolar diameter. The concentration of lisinopril used in these studies previously inhibited ACE in the hamster cheek pouch (Rubinstein et al., 1995). The mixture of proteinase
inhibitors used in these studies was used previously in the in
situ hamster cheek pouch (Gao et al., 1994).
Data and Statistical Analyses
When an agent was suffused on the spinotrapezius muscle, we determined the maximal steady-state change in arteriolar diameter and used this as the response to that agent. Arteriolar diameter was expressed as a percentage of the diameter during the control period. Data were expressed as mean ± S.E.M. except for body weight and arteriolar diameter, which were expressed as mean ± S.D. because they characterize the entire sample group and are not compared with another group. Differences between variables were assessed by two-way analysis of variance and the Newman-Keuls multiple range test. A P value of < .05 was considered statistically significant.
Drugs and Reagents
E. coli LPS (serotype 0111:B4), SOD, catalase, indomethacin, chymostatin, soybean trypsin inhibitor, AT II, naphthol AS-D chloroacetate and Fast Garnet GBC were obtained from Sigma Chemical Co. (St. Louis, MO). Leupeptin and Bestatin were obtained from Peninsula Laboratories (Belmont, CA). DL-2-Mercaptomethyl-3-guanidinoethylthiopropanoic acid was obtained from Calbiochem (San Diego, CA). Lisinopril was a gift from Merck & Co. Research Laboratories (Rahway, NJ). SK&F 108566 was a gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). BQ-485 was a gift from Banyu Pharmaceutical Co. (Tsukuba, Japan). PD 142893 was a gift from Parke-Davis Pharmaceutical Research (Ann Arbor, MI). All other chemicals were of the highest analytical grade available. SK&F 108566 and indomethacin were dissolved in Na2CO3 and diluted in saline to the desired concentrations on the day of the experiment. BQ-485 and PD 142893 were dissolved in dimethyl sulfoxide and diluted in saline to the desired concentrations on the day of the experiment. All other drugs were dissolved in saline on the day of the experiment.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effects of E. coli LPS on Arteriolar Diameter
Suffusion of E. coli LPS onto the spinotrapezius muscle for 60 min elicited a significant, concentration-dependent, immediate biphasic vasomotor response consisting of vasoconstriction followed by vasodilation (figs. 1 and 2; P < .05). Maximal vasoconstriction was observed within 4 min of the start of suffusion and maximal vasodilation within 45 min (fig. 1). Arteriolar diameter returned to baseline within 20 min after suffusion of E. coli LPS was stopped. Suffusion of 3.0 µg/ml E. coli LPS elicited a 10.7 ± 0.4% decrease in arteriolar diameter from baseline at 4 min and a 15.8 ± 1.1% increase in arteriolar diameter from baseline at 45 min (fig. 1; n = 37; P < .05 in comparison with baseline). A similar immediate biphasic vasomotor response was observed in larger (A1) and smaller (A3) arterioles of the spinotrapezius muscle (data not shown). Based on these data, we used arteriolar diameter at 4 and 45 min after the start of suffusion of E. coli LPS (3.0 µg/ml) in all subsequent data analysis. Suffusion of saline (vehicle) for the entire duration of the experiment was not associated with significant changes in arteriolar diameter from base line (fig. 1; n = 4; P > .5).
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Mechanisms of E. coli LPS-Induced Changes in Arteriolar Diameter
Role of angiotensin II. SK&F 108566 (0.1 µM) abrogated E. coli LPS (3.0 µg/ml)-induced immediate biphasic vasomotor response (fig. 3A; each group, n = 7; P < .05). Arteriolar diameter increased by 4.8 ± 0.8% from baseline at 4 min and by 2.9 ± 1.8% from baseline at 45 min during suffusion of SK&F 108566 (0.1 µM) and E. coli LPS (3.0 µg/ml). Suffusion of Sk&F108566 (0.1 µM) together with SOD (60 U/ml) had similar effects on E. coli LPS (3 µg/ml)-induced responses (fig. 3B; each group, n = 5; P < .05). SK&F 108566 (0.1 µM) also abrogated AT II (0.05 µM)-induced vasoconstriction (n = 4; P < .05). Arteriolar diameter decreased by 22.7 ± 1.9% from base line during suffusion of AT II (0.05 µM) alone and by 1.0 ± 1.0% from baseline during suffusion of SK&F 108566 (0.1 µM) and AT II (0.05 µM).
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Role of superoxide and hydrogen peroxide. SOD (60 U/ml) had no significant effects on E. coli LPS (3.0 µg/ml)-induced vasoconstriction (fig. 4A; P > .5). However, it reverted E. coli LPS-induced vasodilation to significant vasoconstriction (fig. 4A; n = 7; P < .05). Arteriolar diameter decreased by 12.3 ± 2.6% from baseline at 45 min during suffusion of SOD (60 U/ml) and E. coli LPS (3.0 µg/ml). Catalase (60 U/ml) had no significant effects on E. coli LPS (3.0 µg/ml)-induced vasoconstriction (fig. 3B; P > .5). However, it significantly attenuated E. coli LPS (3.0 µg/ml)-induced vasodilation (fig. 4B; n = 4; P < .05). Arteriolar diameter increased only by 5.8 ± 2.0% from baseline during suffusion of catalase (60 U/ml) and E. coli LPS (3.0 µg/ml).
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Role of endothelin. PD 142893 (1 µM) and BQ-485 (1 µM) had no significant effects on E. coli LPS (3.0 µg/ml)-induced responses (data not shown; each group, n = 4; P > .5).
Role of prostaglandins. Indomethacin (10 mg/kg) had no significant effects on E. coli LPS (3.0 µg/ml)-induced vasoconstriction (fig. 5; P > .5). However, it curtailed E. coli LPS (3.0 µg/ml)-induced vasodilation (fig. 5; each group, n = 4; P < .05). Arteriolar diameter decreased by 9.1 ± 1.2% from baseline at 4 min and by 0.3 ± 2.5% from baseline at 45 min during suffusion of E. coli LPS (3.0 µg/ml) in the presence of indomethacin (10 mg/kg).
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Role of mast cells and chymase-like proteases. Sections of unexposed hamster spinotrapezius muscle and cheek pouch contain numerous perivascular mononuclear metachromatic cells with typical morphological appearance of mast cells (fig. 6, D and E, respectively). The distribution of cells with chymase-like activity (NASDCA-hydrolyzing activity) was similar to that of metachromatic cells (fig. 6, A and C). Detailed comparison of adjacent sections stained with NASDCA and toluidine blue, respectively, indicated that almost all cells containing chymase-like activity also stained metachromatically with toluidine blue (fig. 6, C and D). In nearby sections, 926 NASDCA-positive cells were enumerated in tissues containing 1705 metachromatic cells. The identity of NASDCA-cleaving activity as chymase was supported by virtual abolition of enzyme activity in tissue sections pre- and coincubated with chymostatin (fig. 6B). The proportion of mast cells exhibiting NASDCA-hydrolyzing activity was higher in the spinotrapezius muscle than in the cheek pouch. Specifically, 2928 NASDCA-positive cells were counted compared with 3218 metachromatic cells in identical regions of tissue sections. Thus, assuming that all NASDCA-positive cells are metachromatic, the percentage of mast cells that manifest chymase-like activity in the spinotrapezius muscle and cheek pouch is 91% and 54%, respectively. The number of NASDCA-positive cells was significantly lower in the spinotrapezius muscle of E. coli LPS-exposed (fig. 6F) than unexposed (fig. 6E) hamsters (data not shown).
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Role of other proteases. Lisinopril (10 µM) and a mixture of leupeptin, Bestatin and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (each, 10 µM) had no significant effects on E. coli LPS (3.0 µg/ml)-induced changes in arteriolar diameter (fig. 9, A and B, respectively; each group, n = 4; P > .5).
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study presents two new findings. First, suffusion of E. coli LPS on the in situ hamster spinotrapezius muscle,
at concentrations similar to circulating levels in sepsis syndrome
(Shenep and Morgan, 1984; Shenep et al., 1988), for 60 min
elicits an immediate biphasic vasomotor response, vasoconstriction
followed by vasodilation. This response is not related to nonspecific
damage to microvascular endothelium because arteriolar diameter returns
to baseline once suffusion of E. coli LPS is stopped.
Second, E. coli LPS-induced vasoconstriction is abrogated by
SK&F 108566, a selective, nonpeptide AT1 RA,
chymostatin and soybean trypsin inhibitor. These compounds also
attenuate E. coli LPS-induced vasodilation. By contrast, SOD, catalase and indomethacin attenuate E. coli LPS-induced
vasodilation and have no significant effects on E. coli
LPS-induced vasoconstriction. Endothelin receptor antagonists and the
protease inhibitors lisinopril, leupeptin, Bestatin and
DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid are
ineffective.
Histochemical analysis of hamster spinotrapezius muscle and cheek pouch
reveals abundant perivascular mast cells with chymostatin-inhibitable chymase-like activity. Pretreatment of hamsters with compound 48/80 for
4 days to deplete mast cell of preformed mediators, including
chymase-like protease(s) (Gao et al., 1993; Huntley et
al., 1985; Li et al., 1993; Raud, 1989; Rubinstein
et al., 1990; Shepherd and Duling, 1996; Urbaschek and
Urbaschek, 1979), curtails E. coli LPS-induced
vasoconstriction and converts vasodilation to vasoconstriction in the
spinotrapezius muscle. On balance, these data indicate that E. coli LPS stimulates perivascular mast cells in the hamster
spinotrapezius muscle to release an AT II-producing chymase-like
protease(s). Angiotensin II thus produced elicits local
vasoconstriction and elaborates reactive oxygen species which, in turn,
generate vasodilator prostaglandins. A proposed mechanism by which
E. coli LPS modulates immediate biphasic vasomotor dysfunction in the in situ hamster spinotrapezius muscle is
depicted schematically in figure 10.
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The hamster is an established model to elucidate mechanisms underlying
the deleterious effects of E. coli LPS and preformed mast
cell mediators in the in situ peripheral microcirculation (Li et al., 1993; Shepherd and Duling, 1996; Svensjö
et al., 1990; Urbaschek and Urbaschek, 1979). Urbaschek and
Urbaschek (1979) showed that suffusion of E. coli LPS on the
in situ cheek pouch, at concentrations similar to those used
in this study, elicits transient vasoconstriction and mast cell
degranulation. However, the mechanisms underlying E. coli
LPS-induced vasoconstriction were not elucidated. It is well
established that chymase, a preformed mast cell serine protease, is
released upon mast cell degranulation and elaborates AT II in the
tissue (Huntley et al., 1985; Husain, 1993; Martin et
al., 1992; Okamura et al., 1990; Pearce et
al., 1985; Reilly et al., 1982; Takai et
al., 1996; Wintroub et al., 1984). To this end, Cornish
et al. (1979) identified an ACE-independent metabolic
pathway(s) in the in situ cheek pouch that produces AT II.
An AT II-generating chymase-like protease was detected recently, and
purified from, cheek pouch homogenates, although its cellular origin(s)
was not determined (Takai et al., 1996). This protease was
inhibited by chymostatin and soybean trypsin inhibitor and not by ACE
inhibitors (Takai et al., 1996).
The results of this study support and extend these observations by
showing that relatively large numbers of perivascular mast cells
containing AT II-producing chymase-like protease(s) are present in the
hamster spinotrapezius muscle. This protease(s) is likely to play a
role in modulating E. coli LPS-induced immediate biphasic
vasomotor dysfunction in the muscle microcirculation because
chymostatin and soybean trypsin inhibitor, two relatively selective and
potent chymase inhibitors (Martin et al., 1992; Reilly
et al., 1982) and SK&F 108566, a selective nonpeptide
AT1 RA, abrogate E. coli LPS-induced
responses. These effects are specific because inhibitors of other AT
II-forming proteases, including ACE, have no significant effects on
E. coli LPS-induced responses, and because chymostatin and
soybean trypsin inhibitor have no significant effects on
vasoconstriction elicited by exogenous AT II in the in situ
spinotrapezius muscle. Depletion of mast cells from preformed mediators
with compound 48/80 curtails E. coli LPS-induced
vasoconstriction and converts vasodilation to vasoconstriction.
Pretreatment of hamsters with compound 48/80 may also release histamine
and tryptase which are packed together with chymase in mast cell
granules (Martin et al., 1992; Raud, 1989; Rubinstein et al., 1990; Shepherd and Duling, 1996). Conceivably, both
phlogistic mediators could modulate E. coli LPS-induced
responses in the in situ hamster spinotrapezius muscle.
However, this possibility seems unlikely because tryptase, unlike
chymase, is not inactivated by soybean trypsin inhibitor (Martin
et al., 1992; Rubinstein et al., 1990; Takai
et al., 1996), which abrogates E. coli
LPS-induced responses in the spinotrapezius muscle, and because
histamine elicits immediate vasodilation in the in situ
peripheral microcirculation of hamsters (Raud, 1989). Taken together,
these data suggest that E. coli LPS stimulates perivascular
mast cells in the in situ hamster spinotrapezius muscle to
release an AT II-producing chymase-like protease(s). However, the
cellular origin(s) of AT II produced in the muscle was not elucidated.
Additional studies are warranted to address this issue.
Current concepts suggest that generation of reactive oxygen species is
amplified in sepsis syndrome and contributes to vasomotor dysfunction,
partly by eliciting potent vasodilation in the peripheral circulation
(McKenchnie et al., 1986; Natanson, 1994). The results of
this study support this notion. We found that AT II produced in the
in situ hamster spinotrapezius muscle during suffusion of
E. coli LPS elaborates superoxide and hydrogen peroxide.
These mediators, in turn, activate cyclooxygenase to generate
vasodilator prostaglandins because indomethacin, at a concentration
known to inhibit cyclooxygenase in hamsters (Gao et al.,
1993, 1995; Raud, 1989; Rubinstein et al., 1991), abrogates
E. coli LPS-induced vasodilation without affecting the
initial vasoconstriction (Feng et al., 1995; Gao et
al., 1995; Warren et al., 1991). The magnitude of
vasodilation elicited by prostaglandins in the in situ
hamster spinotrapezius muscle during suffusion of E. coli
LPS corresponds to ~50% reduction in peripheral vascular resistance.
This figure is consistent with that observed in patients with sepsis
syndrome (Hess et al., 1981; Natanson, 1994). Overall, these
data suggest that E. coli LPS-induced release of
chymase-like protease(s) from perivascular mast cells in the in
situ skeletal muscle activates a local cascade of biologic
responses leading to AT II-dependent production of reactive oxygen
species which, in turn, elaborate vasodilator prostaglandins.
SK&F 108566, a selective, nonpeptide AT1 RA
(Edwards et al., 1991), abrogates E. coli
LPS-induced vasodilation in the in situ hamster
spinotrapezius muscle. Because this compound also inhibits the initial
E. coli LPS-induced vasoconstriction, the subsequent blockade of vasodilation could reflect the lack of stretch-induced, nitric oxide-mediated vasodilation (Natanson, 1994; Warren et al., 1991; Wurster et al., 1994). This possibility
seems unlikely, however, because Gao et al. (1995) showed
that NG-L-nitro arginine, a
nitric oxide synthase inhibitor, has no significant effects on E. coli LPS-induced immediate biphasic vasomotor dysfunction in the
in situ hamster cheek pouch.
Resident and migrant cells and phlogistic mediators other than
perivascular mast cells and AT II-producing chymase-like protease(s) could play a role in modulating vasomotor dysfunction in skeletal muscle microcirculation in sepsis syndrome (Natanson, 1994; Neviere et al., 1996; Shepherd and Duling, 1996; Svensjö
et al., 1990; Warren et al., 1991). The results
of this study support this contention partly by showing that suffusion
of E. coli LPS on the in situ spinotrapezius
muscle of hamsters depleted of mast cell chymase-like pro tease(s) by
compound 48/80 still elicits vasoconstriction. However, this response
is slower to evolve than that observed during suffusion of E. coli LPS in saline-treated hamsters. The putative role of other
cells and phlogistic mediators in modulating E. coli
LPS-induced immediate vasomotor dysfunction in in situ hamster spinotrapezius muscle should be further investigated.
In summary, we found that suffusion of E. coli LPS on the in situ hamster spinotrapezius muscle for 60 min elicits an immediate, reversible biphasic vasomotor response, vasoconstriction followed by vasodilation. This response is modulated by E. coli LPS stimulation of perivascular mast cells to release an AT II-producing chymase-like protease(s). The angiotensin II thus produced elicits local vasoconstriction and elaborates reactive oxygen species which, in turn, generate vasodilator prostaglandins. We suggest that inhibitors of mast cell chymase-like protease(s) could be beneficial in the treatment of early-phase E. coli sepsis syndrome.
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Acknowledgments |
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The expert technical assistance of Karen Koerber is gratefully acknowledged.
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Footnotes |
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Accepted for publication November 12, 1997.
Received for publication May 22, 1997.
1 This study was supported, in part, by grants from the National Institutes of Health (DE10347 and HL24136), American Heart Association of Metropolitan Chicago and Laerdal Foundation for Acute Medicine.
2 Recipient of a Career Investigator Award from the American Lung Association.
3 Recipient of a Research Career Development Award from the National Institutes of Health (DE00386) and a University of Illinois Scholar Award.
Send reprint requests to: Dr. Israel Rubinstein, Department of Medicine (M/C 787), University of Illinois at Chicago, 840 S. Wood Street, Chicago, IL 60612-7323.
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Abbreviations |
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LPS, lipopolysaccharide; AT II, angiotensin II; AT1 RA, angiotensin II subtype 1 receptor antagonist; SOD, superoxide dismutase; ACE, angiotensin I-converting enzyme; ET, endothelin; NASDCA, naphthol AS-D chloroacetate.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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0022-3565/98/2843-1156$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics
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P < .05 in comparison with E.
coli LPS (0.3 µg/ml); ¶P < .05 in comparison with
E. coli LPS (3.0 µg/ml).
