Title page Carbon Monoxide-Mediated Activation of Large …jpet.aspetjournals.org/content/jpet/early/2007/01/17/... · 2007. 1. 17. · JPET #116665 page 2 Running title page Running

JPET #116665 page 1

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.).

JPET Fast Forward. Published on January 17, 2007 as DOI:10.1124/jpet.106.116665

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 17, 2007 as DOI: 10.1124/jpet.106.116665

at ASPE

T Journals on A

pril 26, 2021jpet.aspetjournals.org

Dow

nloaded from

http://jpet.aspetjournals.org/

JPET #116665 page 2

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


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 3

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.


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 4

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


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 5

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.


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 6

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.


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 7

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


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 8

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


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 9

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


at ASPE

T Journals on A


Dow

nloaded from


JPET #116665 page 10

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,


at ASPE

T Journals on A


Dow

nloaded from



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.


at ASPE

T Journals on A


Dow

nloaded from



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


at ASPE

T Journals on A


Dow

nloaded from



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.


at ASPE

T Journals on A


Dow

nloaded from



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).


at ASPE

T Journals on A


Dow

nloaded from



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


at ASPE

T Journals on A


Dow

nloaded from



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


at ASPE

T Journals on A


Dow

nloaded from



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


at ASPE

T Journals on A


Dow

nloaded from



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


at ASPE

T Journals on A


Dow

nloaded from



arterial vasodilatation of ascitic cirrhotic rats.


at ASPE

T Journals on A


Dow

nloaded from



Acknowledgments

We thank Doctor Brian E. Mann for the synthesis of CORM-3 and Antonietta Sticca for her expert

technical assistance.


at ASPE

T Journals on A


Dow

nloaded from



REFERENCES

Amberg GC and Santana LF (2003a) Downregulation of the BK channel β1 subunit in genetic

hypertension. Circ Res 93:965-971.

Amberg GC, Bonev AD, Rossow CF, Nelson MT and Santana LF (2003b) Modulation of the

molecular composition of large conductance, Ca2+ activated K+ channels in vascular smooth muscle

during hypertension. J Clin Invest 112:717-724.

Archer SL, Gragasin FS, Wu X, Wang S, McMurtry S, Kim DH, Platonov M, Koshal A, Hashimoto

K, Campbell WB, Falk JR and Michelakis ED (2003) Endothelium-derived hyperpolarizing factor

in human internal mammary artery is 11,12-epoxyeicosatrienoic acid and causes relaxation by

activating smooth muscle BKCa channels. Circulation 107:769-776.

Barkoudah E, Jaggar JH and Leffler CW (2004) The permissive role of endothelial NO in CO-

induced cerebrovascular dilation. Am J Physiol Heart Circ Physiol 287:H1459-H1465.

Barriere E, Tazi KA, Pessione F, Heller J, Poirel O, Lebrec D and Moreau R (2001) Role of small-

conductance Ca2+-dependent K+ channels in in vitro nitric oxide-mediated aortic hyporeactivity to

alpha-adrenergic vasoconstriction in rats with cirrhosis. J Hepatol 35:350-357.

Bolognesi M, Sacerdoti D, Di Pascoli M, Angeli P, Quarta S, Sticca A, Pontisso P, Merkel C and

Gatta A (2005) Haeme oxygenase mediates hyporeactivity to phenylephrine in the mesenteric

vessels of cirrhotic rats with ascites. Gut 54:1630-1636.

Bratz IN, Dick GM, Partridge LD and Kanagy NL (2005) Reduced molecular expression of K+


at ASPE

T Journals on A


Dow

nloaded from



channel proteins in vascular smooth muscle from rats made hypertensive with N-omega-nitro-L-

arginine. Am J Physiol Heart Circ Physiol 289:H1284-H1290.

Clapp LH and Jabr RI (2003) The BK Channel. Protective or detrimental in genetic hypertension?

Circ Res 93:893-895.

Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, Foresti R and Motterlini R

(2003) Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res

93:e2-e8.

Dubuis E, Gautier M, Melin A, Rebocho M, Girardin C, Bonnet P and Vandier C (2002) Chronic

carbon monoxide enhanced IbTx-sensitive currents in rat resistance pulmonary artery smooth

muscle cells. Am J Physiol Lung Cell Mol Physiol 283:L120–L129.

Dubuis E, Potier M, Wang R and Vandier C (2005) Continuous inhalation of carbon monoxide

attenuates hypoxic pulmonary hypertension development presumably through activation of BKCa

channels. Cardiovasc Res 65:751–761.

Fernandez M, Lambrecht RW and Bonkovsky HL (2001) Increased heme oxygenase activity in

splanchnic organs from portal hypertensive rats: role in modulating mesenteric vascular reactivity. J

Hepatol 34:812-817.

Foresti R, Hammad J, Clark JE, Johnson TR, Mann BE, Friebe A, Green CJ and Motterlini R

(2004) Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing

molecule. Br J Pharmacol 142:453-460.


at ASPE

T Journals on A


Dow

nloaded from



Gonzales-Abraldes J, Garcia-Pagan JC and Bosch J (2002) Nitric oxide and portal hypertension.

Metab Brain Dis 17:311-324.

Jaggar JH, Leffler CW, Cheranov SY, Tcheranova D, E S and Cheng X (2002) Carbon monoxide

dilates cerebral arterioles by enhancing the coupling of Ca2+ sparks to Ca2+-activated K+ channels.

Circ Res 91:610-617.

Jaggar JH, Li A, Parfenova H, Liu J, Umstot ES, Dopico AM and Leffler CW (2005) Heme is a

carbon monoxide receptor for large-conductance Ca2+-activated k+ channels. Circ Res 97:805-812.

Jaggar JH, Porter VA, Lederer WJ and Nelson MT (2000) Calcium sparks in smooth muscle. Am J

Physiol Cell Phsyiol 278:C235-C256.

Johnson FK, Durante W, Peyton KJ and Johnson RA (2003a) Heme oxygenase inhibitor restores

arteriolar nitric oxide function in Dahl rats. Hypertension 41:149-155.

Johnson FK and Johnson RA (2003b) Carbon monoxide promotes endothelium-dependent

constriction of isolated gracilis muscle arterioles. Am J Physiol Regul Integr Comp Physiol

285:R536-R541.

Kaide JI, Zhang F, Wei Y, Wang W, Gopal VR, Falck JR, Laniado-Schwartzman M and Nasjletti A

(2004) Vascular CO counterbalances the sensitizing influence of 20-HETE on agonist-induced

vasoconstriction. Hypertension 44:1-7.

Kotlikoff M and Hall I (2003) Hypertension: β testing. J Clin Invest 112:654-656.


at ASPE

T Journals on A


Dow

nloaded from



Kozma F, Johnson RA, Zhang F, Yu C, Tong X and Nasjletti A (1999) Contribution of endogenous

carbon monoxide to regularion of diameter in resistance vessels. Am J Physiol Regul Integr Comp

Physiol 276:R1087-R1094.

Liu Y, Hudetz AG, Knaus HG and Rusch NJ (1998) Increased expression of Ca2+-sensitive K+

channels in the cerebral microcirculation of genetically hypertensive rats: evidence for their

protection against cerebral vasospasm. Circ Res 82:729-737.

Liu Y, Pleyte K, Knaus HG and Rusch NJ (1997) Increased expression of Ca2+-sensitive K+

channels in aorta of hypertensive rats. Hypertension 30:1403-1409.

Motterlini R, Gonzales A, Foresti R, Clark JE, Green CJ and Winslow RM (1998) Heme

oxygenase-1-derived carbon monoxide contributes to the suppression of acute hypertensive

responses in vivo. Circ Res 83: 568-577.

Mulvany MJ and Aalkiaer C (1990) Structure and function of small arteries. Physiol Rev 70:921-

961.

Naik JS, O'Donaughy TL and Walker BR (2003a) Endogenous carbon monoxide is an endothelial-

derived vasodilator factor in the mesenteric circulation. Am J Physiol Heart Circ Physiol

284:H838-H845.

Naik JS and Walker BR (2003b) Heme oxygenase-mediated vasodilation involves vascular smooth

muscle cell hyperpolarization. Am J Physiol Heart Circ Physiol 285:H220-H228.

Sacerdoti D, Bolognesi M, Di Pascoli M, Gatta A, McGiff JC, Laniado Schwartzman M and


at ASPE

T Journals on A


Dow

nloaded from



Abraham NG (2006) The rat mesenteric arterial dilator response to 11,12-epoxyeicosatrienoic acid

(EET) is mediated by activating heme oxygenase (HO). Am J Physiol Heart Circul Physiol

291:1999-2002.

Schrier RW, Arroyo V, Bernardi M, Epstein M, Henriksen JH and Rodes J (1988) Peripheral

arterial vasodilation hypothesis: a proposal fort he initiation of renal sodium and water retention in

cirrhosis. Hepatology 8:1151-1157.

Sieber C, Lopez-Talavera JC and Groszmann RJ (1993) Role of nitric oxide in the in vitro

splanchnic hyporeactivity in ascitic cirrhotic rats. Gastroenterology 104:1750-1754.

Sun D, Kaley G and Koller A (1994) Characteristics and origin of myogenic response in isolated

gracilis muscle arterioles. Am J Physiol 266:H1177-H1183.

Tanaka Y, Koike K, Alioua A, Stefani E and Toro L (2004) β1-subunit of maxiK channel in smooth

muscle: a key molecule which tunes muscle mechanical activity. J Pharmacol Sci 94:339-347.

Villamor E, Perez-Vizcaino F, Cogolludo AL, Conde-Oviedo J, Zaragoza-Arnaez F, Lopez-Lopez

JG and Tamargo J (2000) Relaxant effects of carbon monoxide compared with nitric oxide in

pulmonary and systemic vessels of newborn piglets. Pediatr Res 48:546-553.

Wiest R and Groszmann RJ (1999) Nitric oxide and portal hypertension: its role in the regulation of

intrahepatic and splanchnic vascular resistance. Semin Liver Dis 19:411-426.

Wilkinson SP and Williams R (1980) Renin-angiotensin-aldosterone system in cirrhosis. Gut

21:545-554.


at ASPE

T Journals on A


Dow

nloaded from



Wu L, Cao K, Lu Y and Wang R (2002) Different mechanisms underlying the stimulation of KCa

channels by nitric oxide and carbon monoxide. J Clin Invest 110:691-700.

Wu L and Wang R (2005) Carbon monoxide: endogenous production, physiological functions, and

pharmacological applications. Pharmacol Rev 57:585-630.

Zhang F, Kaide JI, Rodriguez-Mulero F, Abraham NG and Nasjletti A (2001) Vasoregulatory

function of heme-heme oxygenase-carbon monoxide system. Am J Hypertension 14:62S-67S.


at ASPE

T Journals on A


Dow

nloaded from



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]


at ASPE

T Journals on A


Dow

nloaded from



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.


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.


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


at ASPE

T Journals on A


Dow

nloaded from



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.


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)


at ASPE

T Journals on A


Dow

nloaded from



(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


at ASPE

T Journals on A


Dow

nloaded from



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.


at ASPE

T Journals on A


Dow

nloaded from


0

20

40

60

80

100

120

-9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120

-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])


at ASPE

T Journals on A


Dow

nloaded from


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

20

40

60

80

100

120

-9 -8 -7 -6 -5 -4

§

§

§

§

Indo+L-NAMEIndo+L-NAME+ODQIndo+L-NAME+ODQ+CrMP




Rel

axat

ion

(%)

ACh (log[M])R

elax

atio

n(%

)

ACh (log[M])

Control


Figure 2


at ASPE

T Journals on A


Dow

nloaded from


0

20

40

60

80

100

120

-9 -8 -7 -6 -5 -4

0

20

40

60

80

100

120

-9 -8 -7 -6 -5 -4

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


Rel

axat

ion

(%)

ACh (log[M])



Rel

axat

ion

(%)

ACh (log[M])

Control


Figure 3


at ASPE

T Journals on A


Dow

nloaded from


0

20

40

60

80

100

120

-9 -8 -7 -6 -5 -4

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)

§


at ASPE

T Journals on A


Dow

nloaded from


0

10

20

30

40

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


at ASPE

T Journals on A


Dow

nloaded from


0

4

8

12

16

20

CORM

CORM in arteriespretreated with

SNP

Rel

axat

ion

(%

)

Controls cirrhotic rats with ascites

#

#

*

*

Figure 6


at ASPE

T Journals on A


Dow

nloaded from


0

10

20

30

40

**

β-actin

ΗΟ−1~ 30

kDa

~ 42

Cirrhoticrats withascites

Cirrhoticrats

withoutascites

Control rats


Cirrhoticrats

withoutascites

Control rats

Figure 7

% H

O-1

/act

in


at ASPE

T Journals on A


Dow

nloaded from


0

5

10

15

20

25

30

35

40

45

0

10

20

30

40

50

60

70

80

A B

*

*

*

*

632

β-actin

BKCa

α subunit

β1 subunit~ 65

~ 125

kDa 541

~ 42


Cirrhoticrats

withoutascites

Control rats


Cirrhoticrats

withoutascites

Control rats

Figure 8%

BK

Ca

Alp

ha/

acti

n

% B

KC

aB

eta1

/act

in


Cirrhoticrats

withoutascites

Control rats


at ASPE

T Journals on A


Dow

nloaded from


Documents

Title page Carbon Monoxide-Mediated Activation of Large …jpet.aspetjournals.org/content/jpet/early/2007/01/17/... · 2007. 1. 17. · JPET #116665 page 2 Running title page Running