Title page Carbon Monoxide-Mediated Activation of Large...
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Title page
Carbon Monoxide-Mediated Activation of Large-Conductance Calcium-Activated Potassium
Channels (BKCa) Contributes to Mesenteric Vasodilatation in Cirrhotic Rats.
Massimo Bolognesi, David Sacerdoti, Anna Piva, Marco Di Pascoli, Francesca Zampieri, Santina
Quarta, Roberto Motterlini, Paolo Angeli, Carlo Merkel, Angelo Gatta.
Department of Clinical and Experimental Medicine, University of Padova, Italy (M.B., D.S., A.P.,
M.D.P., F.Z., S.Q., P.A., C.M., A.G.), Department of Surgical Research, Vascular Biology Unit,
Northwick Park Institute for Medical Research, Harrow, Middlesex, UK (R.M.).
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Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.
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Running title page
Running title: Mesenteric Vasodilatation in Cirrhosis
Corresponding author: Massimo Bolognesi, MD, PhD, Clinica Medica 5, Dipartimento di Medicina
Clinica e Sperimentale, Policlinico Universitario, Via Giustiniani 2, 35128 Padova – Italy.
Telephone: +39 049 821 2300. Fax: +39 049 875 4179. e-mail: [email protected]
Number of text pages: 31
Number of tables: 0
Number of figures: 8
Number of references: 39
Number of words in the Abstract: 202
Number of word in the Introduction: 528
Number of words in the Discussion: 1463
List of nonstandard abbreviations: ACh, acetylcholine; ANOVA, analysis of variance; BKCa, large-
conductance calcium-activated K+ channels; CO, carbon monoxide; CORM, carbon monoxide
releasing molecule; COX, cyclooxygenase; CrMP, Chromium mesoporphyrin; EC50, molar
concentration of ACh causing 50% of the maximal vasorelaxant effect; EET, epoxyeicosatrienoic
acids; 20-HETE, 20-hydroxyeicosatetraenoic acid; EDHF; endothelial derived hyperpolarizing
factor; HO, heme oxygenase; IbTx, iberiotoxin; Indo, indomethacin; L-NAME, NG-nitro-L-
arginine-methyl-ester; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-
[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PE, phenylephrine; pEC50, log EC50; PSS, physiological
salt solution; Rmax, maximum relaxation of the artery; sGC, soluble guanylyl cyclase; SNP, sodium
nitroprusside.
Recommended section: Gastrointestinal, Hepatic, Pulmonary, and Renal
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ABSTRACT
Large-conductance calcium-activated potassium channels (BKCa) are important regulators of arterial
tone and represent a mediator of the endogenous vasodilator carbon monoxide (CO). As an
upregulation of heme oxygenase (HO)/CO system has been associated with mesenteric
vasodilatation of cirrhosis, we analyzed the interactions of BKCa and of HO/CO in the endothelium-
dependent dilatation of mesenteric arteries in ascitic cirrhotic rats. In pressurized mesenteric arteries
(diameter 170-350 µm) of ascitic cirrhotic rats, we evaluated: a) the effect of inhibition of BKCa,
HO and guanylyl-cyclase on dilatation induced by acetylcholine and by exogenous CO; b) HO-1
and BKCa subunits protein expression. Results: Inhibition of HO and of BKCa reduced acetylcholine-
induced vasodilatation more in cirrhotic than in control rats, while inhibition of guanylyl-cyclase
had similar effect in the two groups. CO was more effective in cirrhotic than in control rats, and the
effect was hindered by BKCa inhibition. The expression of HO-1 and of BKCa α−subunit were
higher in mesenteric arteries of cirrhotic rats compared to those of control animals, while the
expression of BKCa β1−subunit was lower. In conclusion, an overexpression of BKCa α−subunits,
possibly due to HO upregulation with increased CO production, participates in the endothelium-
dependent alterations and mesenteric arterial vasodilatation of ascitic cirrhotic rats.
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INTRODUCTION
Mesenteric arterial vasodilation is a key mechanism in the pathophysiology of the hyperdynamic
circulatory syndrome of cirrhosis. This syndrome is responsible for serious complications, such as
ascites, hepatorenal syndrome and gastrointestinal hemorrhage. The pathophysiological mechanism
that supports the vasodilation of mesenteric arteries in cirrhosis is a decrease in the response of the
arteries to vasoconstricting agents (Sieber et al., 1993), caused by an increase in vasodilating
substances of endothelial origin, such as nitric oxide (NO), prostacyclin and, as recently
demonstrated, carbon monoxide (CO) (Wiest and Groszmann, 1999; Fernandez et al., 2001;
Gonzales-Abraldes et al., 2002).
We have recently shown that an increased action of the heme oxygenase (HO)/CO system plays a
role in the hyporesponsiveness of small resistance mesenteric arteries to phenylephrine (PE) only in
the advanced stage of experimental cirrhosis (Bolognesi et al., 2005). Therefore, the increased
activity of the HO/CO system may participate in the evolution of cirrhosis from compensated to
decompensated.
HO is a microsomal enzyme with two main distinct isoforms: the inducible isoenzyme HO-1 and
the constitutive one HO-2 (Zhang et al., 2001; Johnson et al., 2003a). It is the rate-limiting enzyme
in the degradation of heme to biliverdin, CO and free iron (Motterlini et al., 1998). CO, generated
by HO in endothelial and smooth muscle layers of arterial vessels, modulates vascular tone by
inducing relaxation of vascular smooth muscle cells through stimulation on soluble guanylyl
cyclase (sGC) and opening of large-conductance calcium-activated potassium channels (BKCa)
(Zhang et al., 2001). It also inhibits the formation of 20-hydroxyeicosatetraenoic acid (20-HETE)
(Zhang et al., 2001; Kaide et al. 2004), a vasoconstrictor which inhibits potassium channels.
BKCa are by far the most abundant K+ channel expressed in vascular smooth muscle cells; their
importance as physiological regulators of blood flow has long been recognized (Clapp et al., 2003,
Kotlikoff and Hall, 2003). The opening of these channels, in response to localized intracellular
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Ca2+ transients (Ca2+ sparks) (Jaggar et al., 2000), leads to hyperpolarization of smooth muscle
cell and, thus, to relaxation. Their conductance is increased by membrane depolarization (Barriere
et al., 2001), NO (Barriere et al., 2001; Wu et al., 2002), CO (Wu et al., 2002, Jaggar et al., 2005)
and by other substances, such as epoxyeicosatrienoic acids (EET)(Archer et al., 2003).
In this study we tried to analyze if the alteration of HO/CO system detected in ascitic cirrhosis is
linked to alterations in the mechanisms transducing the CO signal in the smooth muscle cell.
Therefore, aim of the study was to analyze the role of BKCa, of sGC and of HO/CO system in the
response to Acetylcholine (ACh) of small resistance mesenteric arteries of rats with CCl4 induced
cirrhosis. In cirrhotic rats with ascites, we preliminarily analyzed the effect of Chromium
mesoporphyrin (CrMP), a non selective HO inhibitor, on the endothelium-dependent vasodilation
induced by ACh. Then, we examined the effect of inhibition of sGC and of BKCa, alone and
combined with CrMP, on ACh-induced vasodilation. Finally, the hemodynamic effect of a CO-
donor, alone and after the inhibition of HO, sGC and BKCa, and the expression of HO-1 and of the
two subunits of BKCa (α and β1) in small resistance mesenteric arteries of cirrhotic rats were
evaluated.
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MATERIALS AND METHODS
Animals.
The study was performed on 65 adult male Wistar rats (Charles River Laboratories, Calco, Italy);
body weight 200-225 g. The experiments were carried out in accordance with the legislation of
Italian authorities (D.L. 27/01/1992 no. 116), which complies with European Community guidelines
(CEE Directive 86/609) for the care and use of experimental animals. The experimental protocol
was approved by the Institutional Animal Care and Use Committee.
Cirrhosis was induced with the CCl4 inhalation method in 35 rats drinking phenobarbital (0.30 g/L
in the drinking water) as previously described (Bolognesi et al., 2005). Treatment was followed for
16 weeks, and animals were free of treatment for the last week before the experiment. Under
anesthesia with ketamine hydrochloride (100 mg/kg body wt intramuscularly), a midventral
laparotomy was performed and a section of small intestine was removed. The presence of ascites
was confirmed by visual examination at laparotomy. If the presence of ascites was not certain, the
rat was not considered ascitic. After laparotomy, cirrhotic rats were classified as cirrhotic with or
without ascites. Age-matched animals were used as controls.
Isolated Microvessel Preparation.
As the low splanchnic vascular resistance observed in portal hypertension depends mostly on
mesenteric resistance arteries, which are pre-capillary arteries with diameters narrower than 500 µm
(Mulvany and Aalkiaer, 1990), we studied the endothelial function, the response to CO and protein
expression in small mesenteric resistance arteries. The no-flow model was chosen to avoid
interference from shear stress.
The clamped section of the small intestine was placed in a chilled oxygenated modified Krebs
bicarbonate buffer (physiological salt solution: PSS) containing 118.5 mM NaCl, 4.7 mM KCl, 1.2
mM KH2PO4, 1.2 mM MgSO4, 2.8 mM CaCl2, 25 mM NaHCO3 and 11 mM dextrose.
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Third/fourth-order branches of the superior mesenteric artery (170-350 µm in diameter, 1-2 mm in
length), were isolated from surrounding perivascular tissue, removed from the mesenteric vascular
bed and mounted on glass micropipettes in a water-jacketed perfusion chamber (Living Systems
Instrumentation, Burlington, VT, USA) in warmed (37°C), oxygenated (95% O2 and 5% CO2) PSS.
The vessels were mounted on a proximal micropipette connected to a pressure servo controller.
Subsequently, the lumen of the vessel was flushed to remove residual blood and the end of the
vessel was mounted on a micropipette connected to a three-way stopcock. After the stopcock was
closed, the intraluminal pressure was allowed to increase slowly until it reached 80 mmHg. The
vessel was superfused with PSS (4 ml/min) at 37°C gassed with 95% O2 and 5% CO2 for a 45 min
period of equilibration (Bolognesi et al., 2005). Intraluminal pressure was maintained at 80 mmHg
throughout the experiment. After the equilibration period, the vessels were challenged with PE, an
alpha1-adrenoreceptor agonist (1 µM). An artery was considered unacceptable for experimentation
if it demonstrated leaks or failed to constrict by more than 20% to PE. The presence of a functional
endothelium was determined on the basis of a prompt relaxation to ACh (1 µM), in the vessel
precontracted with PE (1 µM). To remove the endothelium, 2 mL of air were flushed through the
lumen (Sun et al., 1994). In these arteries, the absence of a functional endothelium was confirmed
after preconstriction with PE by the absence of response to ACh with a normal response to sodium
nitroprusside (SNP), an endothelium-independent vasodilator. The effects of ACh and CO
administration were evaluated as variations in the internal diameter of the vessels preconstricted
with PE 10 µM; all responses were reported as percent inhibition of the contraction induced by PE.
Evaluation of the response to ACh of small mesenteric arteries preconstricted with
phenylephrine in CCl4 cirrhotic rats.
Responses to increasing doses of ACh (10-9 to 10-4 M) were determined in arteries superfused with
PSS containing vehicles for the inhibitors tested. Inhibitors were added to freshly prepared PSS, and
20 to 30-min drug-tissue contact time was allowed before retesting the response to ACh in the same
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vessel. ACh was added to the bath (extraluminal application), and cumulative dose-response curves
were generated, with 2 to 3 min intervals between doses. After each dose-response test, the tissues
were washed with fresh PSS for at least 20 min. Vascular diameters were measured 1 to 3 min after
the addition of ACh with the use of a video system consisting of a microscope with a CCD
television camera (Eclipse TS100-F, Nikon, Tokyo, Japan), a television monitor (Ultrak Inc.,
Lewisville, Tx, USA) and a video measuring system (Systems Instrumentation, Burlington, VT,
USA). In control rats and in ascitic cirrhotic rats, concentration-response curves to ACh were
evaluated:
a) before and after 20 min superfusion with the HO inhibitor CrMP (15 µM).
b) before and after 20 min superfusion with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-
a]quinoxalin-1-one (ODQ) (10 µM) in arteries treated with indomethacin (Indo) (2.8 µM) and NG-
nitro-L-arginine-methyl-ester (L-NAME) (1 mM); and then after a further 20 min of CrMP (15 µM)
plus ODQ (10 µM) superfusion in arteries already evaluated after ODQ superfusion alone.
c) before and after 20 min superfusion with the BKCa inhibitor iberiotoxin (IbTx) (25 nM) in
arteries treated with Indo (2.8 µM), L-NAME (1 mM) and ODQ (10 µM); and then after a further
20 min of CrMP (15 µM) plus IbTx (25 nM) superfusion in arteries already evaluated after IbTx
superfusion alone.
Concentration-response curves to ACh were also evaluated in small mesenteric arteries of control
rats before and after 20 min superfusion with three different amount of the BKCa inhibitor IbTx: 25
nM, 50 nM, 100 nM.
Only one experiment was performed in each artery.
Evaluation of the response to a CO donor.
Small mesenteric arteries of control rats and of rats with cirrhosis and ascites were prepared and
mounted on glass micropipettes in a water-jacketed perfusion chamber as already described. After
the equilibration period and after verifying their viability, the arteries were preconstricted with the
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addition of PE to the superfusion PSS.
A CO donor (200 µM) (CORM-3 - CO releasing molecule (Clark et al., 2003; Foresti et al., 2004)
was then added to the superfusion PSS for 15 minutes and the change in lumen diameter was
recorded. Chemical composition of CORM-3 is: Tricarbonylchloro(glycinato)ruthenium(II)
([Ru(CO)3Cl(glycinato)]). The concentration of CORM-3 was chosen after testing the
hemodynamic effect of four different CORM-3 concentrations (25 µM, 50 µM, 100 µM and 200
µM) (Foresti et al., 2004) in a preliminary experiment on small mesenteric arteries of control rats.
Only with the higher dose (200 µM) we obtained a clear and measurable effect. Therefore, 200 µM
was the minimum efficacious dose in our experimental system. In this preliminary experiment, we
also verified that the hemodynamic effect of CORM-3 on small mesenteric arteries is slow, as it
could only be detected a few (3 to 4) minutes after administration, and could be fully evaluated only
after an exposure of 10-15 minutes. For this reason, to evaluate CORM-3 effect in our experimental
set, the changes in lumen diameter were recorded for 15 min, during a continuous exposure to
CORM-3. Taking all this into account, we decided to test a single dose of CORM-3, using the
minimum dose that was efficacious in our control animals: 200 µM.
The effect of CORM-3 was also verified: a) in arteries superfused with CrMP (15 µM) for 30
minutes; b) in arteries superfused with CrMP (15 µM) plus ODQ (10 µM) for 30 minutes; c) in
arteries superfused with CrMP (15 µM) plus IbTx (25 nM) for 30 minutes. In a second series of
experiments, the effect of CORM-3 was verified in arteries preconstricted with PE after removal of
the endothelium. In these arteries, after verifying CO effect, CORM-3 was removed by the
superfusing PSS and, when a stable baseline was obtained, the NO donor SNP (100 nM to produce
a detectible dilation) was added. When a stable baseline was observed, CORM-3 was added again
to the superfusing PSS, and the dilating effect was measured again.
Chemicals
CrMP was obtained from Porphyrin Products (Logan, UT, USA). CORM-3 was synthesized by
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Brian E. Mann (Clark et al., 2003) and stock solutions were prepared using deionized water. All
other chemicals were obtained from Sigma Chemical (Saint Louis, MO, USA). PE and L-NAME
were dissolved in deionized water and diluted with PSS. CrMP was dissolved in a solution of 50
mM NaCO3.
Western blot analysis of HO-1 and of subunits α and β1 of BKCa protein expression in
mesenteric arteries of CCl4 cirrhotic rats.
Standard techniques were used to evaluate protein expression. After removal of veins and adipose
tissue, small mesenteric arteries (30-40 arteries with diameter <500 µm) were collected from each
rat, snap frozen in liquid N2 and stored at -80°C until analyzed. The vessels were homogenized in
urea lysis buffer. Protein extracts were assayed for protein content using the BCA protein assay kit
(Pierce, Rockford, IL, USA). Sodium dodecyl sulphate - polyacrilammide gel elettrophoresis (SDS-
PAGE) and immunoblotting were performed on 50 µg of total protein extracts. HO-1 protein
expression was detected using a monoclonal murine antibody against HO-1 (StressGen
Biotechnologies Corp., Victoria, BC, Canada). Only HO-1, and not HO-2, was tested in the present
study, because we had already demonstrated, in similar experimental condition, that among the two
main HO isoforms (HO-1 and HO-2), only HO-1 is overexpressed in small mesenteric arteries of
CCl4-induced cirrhotic rats (Bolognesi et al., 2005).
The protein expression of the two subunits of BKCa, β1- and α-subunits, were detected using
polyclonal rabbit anti-slo β1 (KCNMB1) and anti-KCa1.1 (alpha subunit 1) (KCNMA1) antibody
(Alomone Labs Ltd., Jerusalem, Israel). The secondary antibodies, anti-rabbit conjugated to
horseradish peroxidase, were diluted 1:1,000 in Phosphate-buffered saline containing 2% non-fat
dry milk. Antigenic detection was visualized by standard ECL-enhanced chemiluminescence
(Amersham, Arlington Heights, IL, USA) with exposure to X-ray film. Control antigens (Alomone
Labs Ltd., Jerusalem, Israel) were used as positive controls. Protein expression was determined by
densitometric analysis using the VersaDoc Imaging System (Bio-Rad Laboratories, Hercules, CA,
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USA). After stripping, the blots were assayed for β-actin content as standardization of sample
loading. The quantitative densitometric values of each protein of interest were normalized to β-actin
and displayed in histograms.
Protein expression of HO-1 was evaluated in 4 control rats, in 6 cirrhotic rats with ascites and also
in 4 cirrhotic rats without ascites. Protein expression of BKCa subunits was evaluated in 5 control
rats, in 4 cirrhotic rats with ascites and also in 4 cirrhotic rats without ascites.
Data Analysis
Data were expressed as mean±SE. Vasorelaxant responses were expressed as percent inhibition of
the contraction induced by PE. Concentration-response data derived from each vessel were fitted
separately to a logistic function by nonlinear regression and EC50 (molar concentration of ACh
causing 50% of the maximal vasorelaxant effect) was calculated and expressed as log [M] (pEC50).
From the same regression the maximum relaxation (Rmax) was also calculated. Two-way ANOVA
was used to compare dose-response curves from controls and treated groups. Other data were
analyzed by one-way ANOVA or Student’s t test for paired or unpaired observations when
appropriate. The n values quoted indicate the number of experiments and animals used. The null
hypothesis was rejected at p<0.05.
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RESULTS
All rats treated with CCl4 included in the study had macronodular or micronodular cirrhosis.
In 27 out of 35 cirrhotic rats the presence of ascites was confirmed by visual examination at
laparotomy. The presence of ascites was dubious in two cirrhotic rats. These “intermediate” rats
were classified as non ascitic, but they were not used for any experiment. Control rats had no
appreciable alteration in liver appearance. At the time of the study no difference in body weight
between cirrhotic (nonascitic rats: 570±24 g, ascitic rats: 548±11 g) and control rats (575±20 g) was
observed.
Evaluation of the response to ACh.
Comparing dose-response curves to ACh in baseline condition, ascitic cirrhotic rats (n.6) showed a
higher sensitivity to ACh in respect of control rats (p=0.036) (Fig. 1).
Effect of HO inhibition:
Inhibition of HO with CrMP caused a slight shift of the concentration-response curve to ACh in
control rats (F=8.61, p<0.001, two-way ANOVA), EC50 (Fig. 1). On the contrary, a marked
rightward shift of the concentration-response curve to ACh was detected after CrMP in cirrhotic rats
with ascites (F=4.38, p=0.0027, two-way ANOVA), with a significant increase in EC50 (Fig. 1), The
increase in pEC50 after CrMP was significantly higher in cirrhotic rats with ascites (p=0.03) (Fig.
1). Following CrMP, sensitivities of control and cirrhotic rats with ascites to ACh were the same
(Fig. 1).
Effect of sGC inhibition and of HO inhibition:
After treatment with indo and L-NAME, mesenteric arteries of cirrhotic rats with ascites maintained
a higher sensitivity to ACh in respect of control rats (p=0.05) (Fig. 2). In arteries treated with Indo
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and L-NAME, the inhibition of sGC with ODQ provoked a shift of the dose-response curve to ACh,
both in control rats (F=3.58, p=0.01, two way ANOVA) and in cirrhotic rats with ascites (F=8.09,
p=0.0005, two-way ANOVA) (Fig. 2). The addition of CrMP to ODQ provoked a further decrease
in the response to ACh, both in control rats (F=9.77, p<0.001, two way ANOVA) and in cirrhotic
rats with ascites (F=15.64, p<0.001, two-way ANOVA) (Fig. 2). The effect of ODQ and of
CrMP+ODQ on Rmax was slightly but not significantly greater in control rats than in cirrhotic rats
with ascites (Fig. 2).
Effect of BKCa inhibition:
Even after treatment with indo, L-NAME and ODQ, mesenteric arteries of cirrhotic rats with ascites
maintained a higher sensitivity to ACh in respect of control rats (p=0.024) (Fig. 3). The addition of
IbTx provoked a decrease in the response to ACh both in control rats (F=7.74, p=0.007, two-way
ANOVA) and in cirrhotic rats with ascites (F=11.59, p=0.001, two way ANOVA) (Fig. 3). The
effect of IbTx was more evident in ascitic cirrhotic rats than in control rats (the increase in pEC50
after IbTx was significantly higher in cirrhotic rats with ascites in respect of control rats: p=0.010,
while the decrease in maximum relaxation was not different between the two groups: p: NS). The
addition of CrMP to IbTx did not provoke a further decrease in the response to ACh both in control
rats and in cirrhotic rats with ascites (p:NS, two-way ANOVA) (Fig. 3). Final sensitivity and
maximum relaxation to ACh were similar in controls and in ascitic cirrhotic rats (Fig. 3).
In control rats, 25 nM of IbTx did not change the response of mesenteric arteries to ACh, while a
significant rightward shift of the concentration-response curve to ACh was evident after incubating
the arteries with the higher concentrations of IbTx (50 nM and 100 nM) (F=6.77, p<0.001, two way
ANOVA) (Fig. 4).
Evaluation of the response to CORM-3 of small mesenteric arteries preconstricted with
phenylephrine in CCl4 cirrhotic rats.
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The addition of CORM-3 to the superfusion caused a slight but not significant dilatation (5±3%) in
mesenteric arteries of control rats (p: NS), while it caused a significant vasorelaxation (25±6%) in
cirrhotic rats with ascites (p=0.05) (Fig. 5). Vasorelaxation was more evident in ascitic cirrhotic rats
than in controls (p=0.012) (Fig. 5). In arteries treated with CrMP and preconstricted with PE,
CORM-3 provoked a significantly greater vasorelaxation both in controls and in ascitic cirrhosis
(p=0.016 and p=0.022, respectively), but in the latter group the vasorelaxation was more evident
(p=0.036) (Fig. 5). In arteries pretreated with CrMP and ODQ and preconstricted with PE, CORM-
3 did not provoke any significant dilatation, both in control and in cirrhotic rats with ascites (Fig.
5). In arteries pretreated with CrMP and IbTx and preconstricted with PE, CORM-3 caused a
significant dilatation in controls rats (14±3%, p=0.004), which was slightly but not significantly
lower than the dilatation obtained in the arteries pretreated only with CrMP (14±3% vs. 20±3%,
respectively, p: NS) (Fig. 5); on the contrary, in cirrhotic rats with ascites, pretreatment with CrMP
and IbTx markedly reduced the dilator effect of CORM-3 in respect of the effect obtained after
pretreatment with only CrMP (4±1% vs. 46±11, respectively, p=0.003) (Fig. 5).
After endothelium removal, CORM-3 provoked a significant but slight dilatation both in controls
(p=0.002) and in cirrhotic rats (p=0.056), but more evident in controls (p=0.015) (Fig. 6). After the
addition of a low concentration of SNP, which caused per se a modest dilation (25%±8 vs 25%±5,
p: NS, in controls and cirrhotic rats, respectively), the vasodilating effect of CORM-3 was more
evident, both in controls (p=0.0003) and in cirrhotic rats (p=0.037), but remained more evident in
control rats (p=0.0008) (Fig. 6).
Western Blot analysis.
In small mesenteric resistance arteries, HO-1 and BKCa α-subunit protein expression was increased
in rats with cirrhosis, particularly in those with ascites (Fig. 7, 8). On the contrary, BKCa β1-subunit
protein expression was significantly decreased both in cirrhotic rats with and without ascites (Fig.
8).
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DISCUSSION
This study demonstrates that in small mesenteric arteries of rats with CCl4 induced cirrhosis, the
response to ACh is increased and it is normalized by the inhibition of HO and BKCa. CO was more
effective in cirrhotic than in control rats, and the effect was hindered by BKCa inhibition. In
mesenteric arteries of cirrhotic rats there is an overexpression of the α-subunit of BKCa, which,
together with the increased expression of HO-1, and an increased production of CO, may cause the
increased response to ACh.
BKCa channels are composed of the pore-forming α-subunit and of the auxiliary regulatory β-
subunits that modulate channel gating (Tanaka et al., 2004). A change in the expression of BKCa
subunits has been reported in experimental arterial hypertension. Indeed, a decrease in β1-subunit
expression has been reported in genetic (Amberg and Santana, 2003a) and in acquired Angiotensin
II-induced hypertension (Amberg et al., 2003b), suggesting that it may contribute to the
development of hypertension. Bratz et al. (2005) reported a decrease in the expression of BKCa α-
subunit in superior mesenteric arteries from rats made hypertensive with N(omega)-nitro-L-
arginine. Liu et al. (1997; 1998) reported an increase in the expression of the pore-forming α-
subunit in aortas (Liu et al., 1997) and in cerebral arteries (Liu et al., 1998) of spontaneously
hypertensive rats, and they suggested that such an increase may be a compensatory vasodilatory
reaction in systemic hypertension (Liu et al., 1998).
Our data demonstrate that an altered expression of BKCa may participate in the exaggerated
mesenteric vasodilatation of cirrhosis. BKCa are stimulated by the CO locally produced by
endothelial HO (Naik et al., 2003a), which is also overexpressed in experimental cirrhosis
(Bolognesi et al., 2005).
We showed that inhibition of HO was more effective on the endothelium-dependent vasodilatation
in cirrhotic rats with ascites in respect of controls rats (Fig. 1). To analyze the mechanisms that
could link HO to the ACh-induced mesenteric vasorelaxation, we studied the relationship between
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HO and the effectors of CO on smooth muscle cells, i.e., sCG and BKCa (Zhang et al., 2001; Jaggar
et al., 2002; Wu et al., 2002).
In arteries treated with Indo and L-NAME, to inhibit any possible interfering action from
cyclooxygenase (COX) and nitric oxide synthase (NOS) respectively, both the inhibition of sGC
and, afterwards, of HO had a similar effect on ACh-induced vasorelaxation in control and in ascitic
cirrhotic rats. Hence, the action of HO on smooth muscle cells is not exclusively due to the
activation of sGC, and moreover, the action on sGC is not what differentiates the HO action in
ascitic cirrhotic rats.
We evaluated the effect of inhibition of BKCa in mesenteric arteries pretreated with the inhibitors of
COX, NOS and sGC. Sensitivity to ACh was increased in ascitic cirrhotic rats also after the
inhibition of these three systems. The inhibition of BKCa caused a decrease in the maximum
relaxation to ACh, particularly in control rats. But it provoked a marked decrease in the sensitivity
to ACh only in cirrhotic rats with ascites, suggesting a role of BKCa in the altered vasoactive
response that occurs in this pathological condition. Further inhibition of HO did not lead to any
modification in the response to ACh, both in controls and in cirrhotic rats with ascites, excluding
other mediators of HO.
As CO is the vasoactive molecule produced by HO (Zhang et al., 2001), we investigated the
response to exogenous CO in small mesenteric arteries of cirrhotic rats with ascites. We used
CORM-3, a CO-releasing water-soluble molecule (Clark et al., 2003; Foresti et al., 2004). In
mesenteric arteries of control rats the vasodilating response to CORM-3 was insignificant, while it
was evident after elimination of endogenous CO by pre-treatment with the HO inhibitor (Sacerdoti
et al., 2006) (Fig. 5). A similar trivial effect of exogenous CO in baseline conditions has been
reported by Kozma et al. (1999), who reported that in first-order gracilis muscle arterioles, CO does
not produce arteriolar dilatation unless the preparation is exposed previously to CrMP. One possible
explanation for the ineffectiveness of exogenous CO as a vasodilator in preparations not exposed to
an inhibitor to HO is that, in such a setting, the vasodilatory mechanism mediated by endogenous
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CO is maximally active (Kozma et al., 1999; Zhang et al. 2001). Another explanation may be that
inhibition of endogenous CO results in increased NOS activity (Johnson and Johnson, 2003b),
which, in turn, permits the exogenous CO to cause dilatation (Barkoudah et al., 2004). At any rate,
in control rats the vasodilatory effect of exogenous CO after HO inhibition was completely
abolished after inhibition of sGC, but only partially and not significantly reduced after inhibition of
BKCa (Fig. 4). These result seem in agreement with Naik and Walker (2003b), who have shown that
the effect of exogenous CO is sensitive to ODQ and IbTx, while the effect of endogenous CO is
cGMP-independent, via activation of BKCa. On the other hand, the lack of CO effect after sGC
inhibition, reported also by other authors (Villamor et al. 2000; Naik and Walker, 2003b), could be
explained by the finding of Barkoudah et al. (2004), who hypothesized a permissive role of cGMP
for effect of CO on BKCa. This interpretation is confirmed by the scarce effect of CO detected in
arteries after removal of the endothelium (Barkoudah et al., 2004; Foresti et al., 2004), an effect that
was restored after the administration of NO, which probably produces the minimum background
level of cGMP necessary for CO to cause vasodilatation by activating BKCa channels (Barkoudah et
al., 2004).
The administration of CORM-3 to mesenteric arteries of cirrhotic rats with ascites caused a
vasodilation evident even in baseline condition, which was amplified by pretreatment with CrMP.
The dilator effect of CO was abolished not only after sCG inhibition, but also after the inhibition of
BKCa (the final response was significantly lower in respect of control rats, p=0.015) underlining the
pivotal role of these channels for the action of CO in this condition. The increased vasodilating
effect of CO in ascitic cirrhotic rats was reversed in mesenteric arteries without endothelium, a
condition in which CO effect was lower in respect of control rats, even after SNP pretreatment.
These results, taken together, not only support the hypothesis that the HO/CO system plays a role in
the vasodilatation of mesenteric arteries in ascitic cirrhosis, but also suggest that in ascitic cirrhotic
rats the effect of CO is mainly through BKCa. They also underline that in cirrhotic rats there are
other endothelial factors, apart from NO, permitting the vasodilating effect of CO on mesenteric
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arteries. It is not clear what these factors are. It is possible that a role is played by the endothelial
derived hyperpolarizing factor (EDHF) and/or by the myoendothelial gap junctions. Further studies
are needed to clarify this issue.
Western blot data confirmed and supported the results of the hemodynamic experiments. There was
an increase in protein expression of HO-1 and of BKCa α-subunit in mesenteric arteries of cirrhotic
rats, particularly in those with ascites, associated to a decrease in the expression of β1-subunit. This
finding supports the hypothesis that CO could play a key-role in the regulation of mesenteric
vasorelaxation in cirrhosis, as the α-subunit is the target of CO on BKCa. CO action on BKCa has
been identified in the capacity of enhancing the coupling of Ca2+ sparks to BKCa (Jaggar et al.,
2002). More recently, Jaggar et al. (2005) have demonstrated that CO activates BKCa by binding to
heme and modifying its interaction with an α-subunit heme-binding domain. The effect of CO on
BKCa is independent from the regulatory β1-subunit (Wu et al., 2002; Jaggar et al. 2005). Indeed,
the presence of BKCa β-subunit is not necessary for the effect of CO, while, on the contrary, it is
essential for the stimulating effect of NO (Wu et al., 2002). The decreased expression of β1-subunit
may be due to activation of the Renin-Angiotensin-Aldosterone system present in ascitic cirrhosis
(Wilkinson and Williams, 1980; Schrier et al., 1988), as β1-subunit synthesis is inhibited by high
levels of Angiotensin II (Amberg et al., 2003b). However, the decreased presence of β1-subunit
seems not sufficient to inhibit the action of BKCa, because of the increased expression of the α-
subunits and of the increased expression of HO, which, through CO, stimulates BKCa independently
from β1-subunits. Why the expression of α-subunit is increased in cirrhotic rats remains to be
clarified. A few authors (Dubuis et al., 2002; Dubuis et al., 2005; Wu and Wang, 2005) have
hypothesized that the expression of BKCa channels may be induced by high CO levels, a condition
that might be present in the mesenteric circulation of cirrhotic rats.
In conclusion, an overexpression of BKCa α−subunits, together with HO-1 upregulation with
increased CO production, participates in the endothelium-dependent alterations and mesenteric
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arterial vasodilatation of ascitic cirrhotic rats.
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Acknowledgments
We thank Doctor Brian E. Mann for the synthesis of CORM-3 and Antonietta Sticca for her expert
technical assistance.
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Footnotes
Source of financial support: this work was supported by grants from the University of Padova, from
the Italian Ministry of Education and from the Center for the Study of the Liver Disease of the
Veneto Region.
Person to receive reprint request: Massimo Bolognesi, MD, PhD, Clinica Medica 5, Dipartimento di
Medicina Clinica e Sperimentale, Policlinico Universitario, Via Giustiniani 2, 35128 Padova – Italy.
Telephone: +39 049 821 2300. Fax: +39 049 875 4179. e-mail: [email protected]
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LEGENDS FOR FIGURES
Figure 1. Concentration-response curves to Acetylcholine (ACh) obtained in small resistance
mesenteric arteries, before (open circle) and after (closed square) the inhibition of heme oxygenase
with CrMP (15 µM). In baseline condition, the endothelium-dependent vasodilatation induced by
ACh was increased in cirrhotic rats with ascites in respect of control rats (difference in pEC50:
p=0.036). Inhibition of heme oxygenase decreased the effect of ACh more in cirrhotic rats with
ascites in respect of controls rats (difference in the change of pEC50: p=0.03). After CrMP the
response to ACh was similar in the two groups. §= significantly different (p<0.01) from baseline
(two-way ANOVA). #=p=0.036 in respect of control rats; *= p=0.05 from baseline; **= p<0.05
from baseline.
Figure 2. Concentration-response curves to Acetylcholine (ACh) obtained in small resistance
mesenteric arteries incubated with indomethacin (indo) (2.8 µM) and L-NAME (1 mM), to inhibit
COX and NOS, respectively. In the presence of vehicle (open circle) the endothelium-dependent
vasodilatation induced by ACh was increased in cirrhotic rats with ascites in respect of control rats
(difference in pEC50: p=0.05). In respect of vehicle (open circle), incubation with the sCG inhibitor
ODQ (10 µM) (closed square) decreased the effect of ACh both in control rats and in rats with
cirrhosis and ascites. The addition of CrMP (15 µM) to ODQ (10 µM) (closed triangle) further
reduced the effect of ACh in both groups. §= significantly different (p≤0.01) from the other two
curves (two-way ANOVA). #=p=0.05 in respect of control rats; *= significantly different (p<0.05)
from Indo+L-NAME; **=significantly different (p<0.05) from Indo+L-NAME+ODQ.
Figure 3. Concentration-response curves to Acetylcholine (ACh) obtained in small resistance
mesenteric arteries incubated with indomethacin (indo) (2.8 µM), L-NAME (1 mM) and ODQ (10
µM), to inhibit COX, NOS and sGC, respectively. In the presence of vehicle (open circle) the effect
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of ACh was increased in cirrhotic rats with ascites in respect of control rats (difference in pEC50:
p=0.024). In respect of vehicle (open circle), incubation with the BKCa inhibitor IbTx (25 nM)
(closed square) decreased the effect of ACh both in control rats and in rats with cirrhosis and
ascites. The inhibition of BKCa was more effective on the endothelium-dependent vasodilatation
induced by ACh in cirrhotic rats with ascites in respect of controls rats (difference in the change of
pEC50: p=0.010). The addition of CrMP (15 µM) to IbTx (25 nM) (closed triangle) did not further
modify the effect of ACh in both groups. §= significantly different (p<0.01) from indo+L-
NAME+ODQ (two-way ANOVA); #=p=0.024 in respect of control rats. *=significantly different
(p<0.05) from Indo+L-NAME+ODQ.
Figure 4. Concentration-response curves to Acetylcholine (ACh) obtained in small resistance
mesenteric arteries of control rats in the presence of vehicle (open circle) and after incubation with
three different amount of the BKCa inhibitor iberiotoxin (IbTx): 25 nM (closed square), 50 nM
(closed triangle), 100 nM (closed circle). §= significantly different (p<0.05) from baseline and from
IbTx 25 nM. *= significantly different (p<0.05) from baseline; **=significantly different (p<0.05)
from IbTx 25nM. The inhibitory effect of high concentrations of IbTx on ACh induced
vasodilatation underlines the physiological role of BKCa in the regulation of endothelium-dependent
vasodilatation of mesenteric arteries.
Figure 5. Effect of a CO releasing molecule (CORM-3, 200 µM) on the internal diameter of small
mesenteric arteries. The effect of CORM-3 was evaluated in the presence of vehicle (baseline, white
bars), and after elimination of endogenous CO by pre-treatment with the heme oxygenase inhibitor
CrMP (15 µM) (black bars). In arteries pre-treated with CrMP (15µM), the effect of CORM-3 was
also evaluated after incubation with the sGC inhibitor ODQ (10 µM) (gray bars) or with the BKCa
inhibitor IbTx (25 nM) (striped bars). In the presence of vehicle (white bars), the addition of
CORM-3 caused a vasorelaxation more evident in ascitic cirrhotic rats (n.6) than in controls (n.8)
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(p=0.012). In arteries incubated with CrMP (black bars), CORM-3 provoked a significantly greater
vasorelaxation both in controls (n.5) and in ascitic cirrhosis (n.4) (p=0.016 and p=0.022,
respectively), but in the latter group the vasorelaxation was more evident (p=0.036). In arteries
incubated with CrMP and ODQ (gray bars), CORM-3 did not provoke any significant dilatation,
both in control (n.4) and in cirrhotic rats with ascites (n.5). In arteries pretreated with CrMP and
IbTx (striped bars), CORM-3 caused a significant dilatation in controls rats (n.6) (p=0.004), which
was slightly but not significantly lower than the dilatation obtained in the arteries pretreated only
with CrMP; on the contrary, in cirrhotic rats with ascites (n.5), pretreatment with CrMP and IbTx
markedly reduced the dilator effect of CORM-3 (p=0.003) in respect of the effect obtained after
incubation with only CrMP. #= p<0.05 in respect of control rats; *= p<0.05 in respect arteries
pretreated with CrMP.
Figure 6. Effect of a CO releasing molecule (CORM-3, 200 µM) on the internal diameter of small
mesenteric arteries after endothelium removal. The effect was evaluated before and after the
administration of the NO donor SNP (100 nM). After endothelium removal, CORM-3 provoked a
significant but slight dilatation both in controls (n.4) (p=0.002) (closed diamond) and in cirrhotic
rats with ascites (n.5) (p=0.056) (open triangle); the dilatation was more evident in controls
(p=0.015). After the addition of a low concentration of SNP, which caused per se a modest dilation,
the vasodilating effect of CORM-3 was more evident, both in controls (p=0.0003) and in cirrhotic
rats (p=0.037), but remained more evident in control rats (p=0.0008). The effect of CORM after
SNP was measured when a new stable baseline was obtained after the administration of SNP. #=
p<0.05 in respect of control rats; *= p<0.05 in respect of values obtained before SNP
administration.
Figure 7. Western blot analysis of heme oxygenase-1 (HO-1) in the small mesenteric arteries of
controls rats and of cirrhotic rats, with and without ascites. The reported blots are representative of
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4 to 6 experiments. Lower panel: densitometric analysis of HO-1. *= p<0.05 in respect of control
rats. In cirrhotic rats the expression of HO-1 was increased.
Figure 8. Western blot analysis of the α- and of β1-subunits of BKCa in the small resistance
mesenteric vessels of controls rats and of cirrhotic rats, with and without ascites. The reported blots
are representative of 3 to 5 experiments; lane 1-2: cirrhotic rats with ascites; lane 3-4: cirrhotic rats
without ascites; lane 5-6: control rats. Lower panel: densitometric analysis of α-subunit (graph A)
and of β1-subunit (graph B). *= p<0.05 in respect of control rats. In cirrhotic rats with and without
ascites, the expression of BKCa α-subunit was increased, while the expression of BKCa β1-subunit
was decreased.
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-9 -8 -7 -6 -5 -4
0
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-9 -8 -7 -6 -5 -4
Control
Cirrhosis with ascites
pEC50 Rmax
(log[M]) (%)
-6.76±0.21 88±7-6.36±0.31 70±8**
n= 9
pEC50 Rmax
(log[M]) (%)
-7.47±0.19# 104±2-6.19±0.43** 78±12*
n= 6§
§
Figure 1
BaselineCrMPBaselineCrMP
BaselineCrMPBaselineCrMP
Rel
axat
ion
(%)
ACh (log[M])
Rel
axat
ion
(%)
ACh (log[M])
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pEC50 Rmax
(log[M]) (%)
-6.09±0.26 81±8-5.90±0.42 53±12-5.55±0.33 20±13* **
n= 6
pEC50 Rmax
(log[M]) (%)
-7.02±0.31# 93±6-6.05±0.47* 82±12-5.58±0.27* 56±22
n= 4
0
20
40
60
80
100
120
-9 -8 -7 -6 -5 -4
0
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120
-9 -8 -7 -6 -5 -4
§
§
§
§
Indo+L-NAMEIndo+L-NAME+ODQIndo+L-NAME+ODQ+CrMP
Indo+L-NAMEIndo+L-NAME+ODQIndo+L-NAME+ODQ+CrMP
Indo+L-NAMEIndo+L-NAME+ODQIndo+L-NAME+ODQ+CrMP
Indo+L-NAMEIndo+L-NAME+ODQIndo+L-NAME+ODQ+CrMP
Rel
axat
ion
(%)
ACh (log[M])R
elax
atio
n(%
)
ACh (log[M])
Control
Cirrhosis with ascites
Figure 2
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pEC50 Rmax
(log[M]) (%)
-5.88±0.15 68±9-6.08±0.24 30±13*-5.67±0.10 29±12*
n= 6
§§
pEC50 Rmax
(log[M]) (%)
-6.52±0.19# 71±12-5.24±0.28* 43±14-5.46±0.24* 23±8*
n= 5
§
§
Indo+L-NAME+ODQIndo+L-NAME+ODQ+IbTxIndo+L-NAME+ODQ+IbTx+CrMP
Indo+L-NAME+ODQIndo+L-NAME+ODQ+IbTxIndo+L-NAME+ODQ+IbTx+CrMP
Rel
axat
ion
(%)
ACh (log[M])
Indo+L-NAME+ODQIndo+L-NAME+ODQ+IbTxIndo+L-NAME+ODQ+IbTx+CrMP
Indo+L-NAME+ODQIndo+L-NAME+ODQ+IbTxIndo+L-NAME+ODQ+IbTx+CrMP
Rel
axat
ion
(%)
ACh (log[M])
Control
Cirrhosis with ascites
Figure 3
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pEC50 Rmax
(log[M]) (%)
-6.87±0.28 94±2-6.60±0.16 88±5-5.99±0.23 * ** 58±10* **-5.69±0.23 * ** 46±13* **
n= 5
§
Rel
axat
ion
(%)
ACh (log[M])
Control
Figure 4
BaselineIbTx (25 nM)IbTx (50 nM)IbTx (100 nM)
BaselineIbTx (25 nM)IbTx (50 nM)IbTx (100 nM)
§
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50
60
control ratscirrhotic ratswith ascites
Rel
axat
ion
(%
)
baselinearteries pretreated with CrMParteries pretreated with CrMP+ODQarteries pretreated with CrMP+IbTx
*
#
#*
** *#
Figure 5
Effect of CORM administration
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8
12
16
20
CORM
CORM in arteriespretreated with
SNP
Rel
axat
ion
(%
)
Controls cirrhotic rats with ascites
#
#
*
*
Figure 6
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**
β-actin
ΗΟ−1~ 30
kDa
~ 42
Cirrhoticrats withascites
Cirrhoticrats
withoutascites
Control rats
Cirrhoticrats withascites
Cirrhoticrats
withoutascites
Control rats
Figure 7
% H
O-1
/act
in
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5
10
15
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25
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35
40
45
0
10
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40
50
60
70
80
A B
*
*
*
*
632
β-actin
BKCa
α subunit
β1 subunit~ 65
~ 125
kDa 541
~ 42
Cirrhoticrats withascites
Cirrhoticrats
withoutascites
Control rats
Cirrhoticrats withascites
Cirrhoticrats
withoutascites
Control rats
Figure 8%
BK
Ca
Alp
ha/
acti
n
% B
KC
aB
eta1
/act
in
Cirrhoticrats withascites
Cirrhoticrats
withoutascites
Control rats
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