ROLE OF KUPFFER CELLS IN THE RELEASE OF NITRIC OXIDE AND CHANGE OF PORTAL PRESSURE AFTER ETHANOL PERFUSION IN THE RAT LIVER

Hiroshi Matsumoto*, Yoko Nishitani, Yasushi Minowa and Yuko Fukui

Department of Legal Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan

Received 17 May 1999; in revised form 27 July 1999; accepted 16 August 1999


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The objective of this study was to elucidate the role of Kupffer cells during the increase of portal vein pressure caused by ethanol. We measured nitric oxide (NO) in the perfused rat liver using a commercial NO meter. Ethanol perfusion increased NO release and portal vein pressure. Gadolinium chloride pretreatment reduced the increase in portal vein pressure during the early phase of ethanol perfusion, but did not affect the release of NO after ethanol infusion. These findings suggest that Kupffer cells play an important role in liver microcirculation during the early stage of ethanol intake, but that the mechanism may not be regulated by NO.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Nitric oxide (NO) is a highly reactive oxidant produced by liver parenchymal and non-parenchymal cells from L-arginine via the inducible form of nitric oxide synthase (iNOS) (Moncada and Higgs, 1993Go). In the liver, overproduction of NO has been suggested as an important factor in endotoxic shock as well as in other models of hepatic inflammation and injury (Knowles et al., 1990Go; Spitzer, 1994Go; Gross and Wolin, 1995Go; Nanji et al., 1995Go; Wang et al., 1995aGo). Also, the smooth muscle-relaxing properties of NO may affect sinusoidal haemodynamics. In rat hepatic lipocytes (Ito cells or fat-storing cells), NO is a potent modulator of lipocyte contractility and may regulate this function via autocrine (or intracrine) mechanisms (Rockey and Chung, 1995Go). Recently, hepatic vascular tone was suggested to be regulated by hepatic stellate cells (Bauer et al., 1994Go; Suematsu et al., 1995Go). However, whether NO regulates sinusoidal blood flow in normal liver via its effects on lipocytes or on smooth muscle cells (pre- or postsinusoidal) remains to be clarified.

On the other hand, it is known that ethanol perfusion causes an increase in portal vein pressure in rat livers (Oshita et al., 1993Go, 1994Go). These studies reported that portal vein pressure was decreased by ethanol perfusion with inhibitors of NOS, but increased by its perfusion with endothelin-1 (Oshita et al., 1993Go). When ethanol enters the liver, Kupffer cells produce NO and many kinds of cytokines, and stellate cells respond directly to the cytokines released from Kupffer cells and contract to regulate hepatic blood flow.

In the present study, we examined NO release due to ethanol in perfused rat livers and changes in portal vein pressure, to evaluate the role of Kupffer cells on these phenomena.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Male Wistar rats (Shimidzu Experimental Material Inc., Kyoto, Japan) weighing 200–250 g were used. Animals were starved for 12 h before the experiments. All chemicals were purchased from Nacalei Tesque Inc. (Kyoto, Japan).

Liver perfusion
The in situ liver perfusion technique previously described (Matsumoto et al., 1996Go) was used with some modification. The rat was anaesthetized with pentobarbital (50 mg/kg body weight), and the liver was perfused in situ via the portal vein, using a non-recirculating system. Briefly, the abdomen was opened, and the bile duct was cannulated with a polyethylene PE-10 tubing (i.d. 0.28 mm). The hepatic artery was ligated. A ligature was passed around the inferior vena cava (IVC) above the renal vein. The portal vein was then cannulated with a polyethylene PE-205 catheter. The liver was immediately perfused with Krebs–Henseleit bicarbonate solution (KHS) oxygenated with 95% O2–5% CO2 through silastic tubing (Hamilton et al., 1974Go) at 37°C. The KHS had the following composition (mM): 118 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3, and 11.0 glucose; pH 7.4. The IVC was cut below the ligature, thus allowing the perfusate to escape. Thereafter, the thorax was opened and the supradiaphragmatic part of IVC was cannulated using a PE-240 catheter, and a ligature around the infrarenal IVC was tied. The liver was then perfused at 36 ml/min through the portal vein, and the effluent escaped through the IVC cannula. The preparation was allowed to stabilize for 15–20 min before the experiments were performed.

Gadolinium chloride (GdCl3) pretreatment
Rats were anaesthetized with pentobarbital (50 mg/kg) and injected with 7.5 mg/kg body weight GdCl3 (75 mg dissolved in 10 ml of saline, pH 3) into a tail vein 24 h prior to the perfusion experiment. GdCl3 was previously shown to abolish Kupffer cell-mediated processes such as the intrahepatic phagocytosis of colloidal particles (Husztik et al., 1980Go).

Monitoring of portal vein pressure
The perfusion pressure was measured, continuously monitoring the height of perfusate in an open vertical capillary column (i.d. = 2 mm) attached to the perfusion system just proximal to the inflow cannula when the perfusate was infused (Oshita et al., 1993Go, 1994Go; Matsumoto et al., 1996Go).

NO measurement
Nitric oxide was measured using a commercially available NO meter (Model NO-501 monitoring device, Intermedical Co. Inc., Nagoya, Japan). The principles behind these measurements and the design of the electrodes have been described previously (Malinski et al., 1993Go; Ichimori et al., 1994Go; Wang et al., 1995bGo). The model NO-501 NO monitoring device consisted of an ammeter with a built-in power supply and electrodes for the detection of NO. The electrodes were composed of a working electrode and a carbon fibre counter-electrode. To check the sensitivity and selectivity of the electrodes, they were calibrated before the experiments were begun. The electrodes were immersed in a small chamber supplied with 0.1 M phosphate-buffered saline (PBS, pH 7.4). A stock solution of 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP, Doujin Co. Inc., Kumamoto, Japan) was used as a stable standard solution (Tsukahara et al., 1993Go) to determine the sensitivity of the electrodes toward NO. It was prepared by dissolving 2.2 mg SNAP in 10 ml of PBS aerated with a 95% O2–5% CO2 gas mixture. The SNAP solution was stored in a cool, dark place and used within 2–5 h. For calibration, the 1 mM stock solution was added to PBS in the chamber to expose the NO electrode to a graded series of SNAP concentrations, from 1 x 10–3 to 1 x 10–6 M.

In vivo measurement of NO release from the in situ isolated perfused rat liver
NO release was continuously monitored with the working electrode placed in the central lobe of the liver. For readings in the liver, the working electrode was inserted with the catheter into the liver and the counter-electrode was fixed to touch the surface of the liver tissue. The two electrodes were positioned within 10 mm of each other to minimize the electrical noise, but care was taken to ensure that they did not touch. Once the basic current had become stable, the NO response current was continuously recorded.

Experimental design
The changes that occurred in the portal vein pressure and in NO production were monitored during 10 min of ethanol perfusion. For ethanol perfusion, its perfusate concentration was 50 or 100 mM. In GdCl3-pretreated rat liver, 50 mM ethanol was perfused and NO release and portal vein pressure were monitored.

Statistics
Values were expressed as means ± SD from four or five independent experiments. Two-way ANOVA and paired Student's t-test were used for statistical significance analysis; P < 0.05 was considered significant.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Calibration of electrodes using (SNAP)
The NO electrode required daily calibration with an NO standard prior to use, because the sensitivity of each electrode varied and was subjected to time-dependent decay. We chose the stable donor, SNAP, for this purpose. Figure 1Go shows a typical standard curve of a sensor. In this example, increasing the SNAP concentration from 1 x 10–5 and 1 x 10–4 to 1 x 10–3 M caused the currents to increase from 210 and 238 to 1030 pA. We obtained significant regression lines (Fig. 1Go). A level of 1 x 10–3 M SNAP corresponded to 1.3 µM NO (Ichimori et al., 1994Go). Therefore, a 1 pA response in this electrode corresponded to 1.5 nM of NO.



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Fig. 1. A typical calibration curve for the nitric oxide (NO)-sensitive electrode. SNAP is S-nitroso-N-acetyl-DL-penicillamine, a donor of NO. The regression line is: Current (pA) = 0.085 x SNAP (µM) + 16.8(r = 0.9965, P < 0.01).

 
Effects of ethanol perfusion on NO release and portal vein pressure
Portal pressure under control conditions without ethanol perfusion was 5.8 ± 0.2 cmH2O, which was similar to the perfused value (Oshita et al., 1993Go, 1994Go; Bauer et al., 1994Go). Figure 2Go shows typical tracings of portal vein pressure (A) and NO release (B), respectively after 50 mM ethanol perfusion. Portal pressure reached maximal levels after 2–4 min, followed by a gradual decrease over the period of ethanol perfusion. This finding corresponds with a previous report (Oshita et al., 1993Go). The changes in portal vein pressure, averaged over the 5-min period after initiation of ethanol infusion of 50 and 100 mM, were 1.86 ± 0.32 and 3.34 ± 0.40 cmH2O, respectively. NO release after ethanol infusion was raised after 2–3 min to reach a plateau (Fig. 2BGo). The changes in NO release after ethanol infusion of 50 and 100 mM were 26.8 ± 2.2 (n = 5, electrode A) and 51.5 (n = 2, electrode A) or 90.2 ± 7.9 pA (n = 3, electrode B), respectively.



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Fig. 2. Kinetics of portal vein pressure and nitric oxide (NO) release in the liver after ethanol perfusion (50 mM). (A) Comparison of portal vein pressure during ethanol perfusion between no-pretreatment (•) and GdCl3 pretreatment ({blacksquare}) in typical experiments. *Portal vein pressures at 3 s. (B) Recordings of NO production (in pA) with the monitor placed in the central lobe of the liver, before and during the 10 min ethanol perfusion (50 mM) in a typical experiment.

 
Effects of GdCl3 pretreatment on NO release and portal vein pressure in ethanol-perfused rat liver
Figure 2AGo (closed squares) shows a typical tracing of the change in portal vein pressure after 50 mM ethanol infusion in GdCl3-pretreated rat liver. Portal pressure changed only slightly after 5–6 min and increased to the same level as in the no-pretreatment group (closed circles) 8–10 min after the initiation of ethanol perfusion. No differences between rats with and without GdCl3 pretreatment were observed. Portal pressure profiles by pretreatment of GdCl3 (10 mg/kg) were very similar to the above tracing (data not shown). This finding suggests there was no effect of pretreatment with GdCl3. The value of NO release in GdCl3-pretreated rat liver was 25.6 ± 3.3 pA.


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
In the present study, intrahepatic NO production was quantitatively analysed by amperometric analysis using a commercial NO meter, and portal vein pressure was simultaneously monitored. Ethanol stimulates vascular endothelium in animals to release NO (Greenberg et al., 1995Go). Our findings suggest that ethanol stimulates hepatocytes and sinusoidal cells other than Kupffer cells to release NO. Shah et al. (1997) reported that sinusoidal endothelial cells from normal rat liver express the endothelial form of NO synthase (ecNOS), which has been recognized to play an important role in vascular regulation in most tissues. Rockey and Cheng (1998) confirmed this finding and identified altered regulation of ecNOS in one animal model of cirrhosis. Accordingly, the sinusoidal endothelial cells may release NO after ethanol infusion.

The role of NO in the regulation of basal vascular resistance in the hepatic portal circulation has been discussed, although findings have been equivocal; some studies show clear evidence of a role of NO (Mittal et al., 1994Go), whereas others have failed to show any response (Suematsu et al., 1995Go), or a response only after upregulation of iNOS by endothelin (Pannen et al., 1998Go). We examined the role of Kupffer cells in hepatic haemodynamics using GdCl3, which is well known to inactivate Kupffer cells (Adachi et al., 1994Go; Puschel et al., 1996Go). Our findings showed that the depletion of Kupffer cells by GdCl3 pretreatment caused no change in NO release after ethanol infusion, although a clear change in portal vein pressure during the early phase of ethanol infusion was observed. Therefore, Kupffer cells play an important role in regulation of portal vein pressure.

Shah et al. (1997) also demonstrated that sinusoidal cells in vitro and in vivo express ecNOS and produce NO basally, and increase their production in response to flow. An increase in portal vein pressure concomitant with the blockage of NO release demonstrates that endogenous endothelium-derived NO modulates portal pressure (Shah et al., 1997Go). Their observations of the perfused rat liver were under normal perfusion. Our findings, that no change of portal pressure and NO release were observed after 10 min of ethanol intake in the GdCl3 pretreatment group, are in agreement with these reports. However, Kupffer cells play an important role in sinusoidal haemodynamics, at least during the early stage after ethanol intake. Oshita et al. (1993) reported that an NO inhibitor did not affect the increase in portal vein pressure after ethanol perfusion and that endothelin-1 markedly increased portal vein pressure. Therefore, the contraction during the initial phase of ethanol perfusion may be regulated by molecules other than NO, e.g. endothelin.

Nitric oxide has been reported to be synthesized by hepatocytes (Curran et al., 1989Go). The precise mechanism by which ethanol causes NO release and the sites where NO acts in the presence of ethanol are unknown. Roland et al. (1996) reported that administration in vivo of GdCl3 to rats decreases the release of NO by isolated rat Kupffer cells in response to lipopolysaccharide (LPS). They also showed that the LPS-stimulated Kupffer cells from GdCl3-treated rats demonstrated an 86% decrease in iNOS synthesis (Roland et al., 1996Go). Shiratori et al. (1998) reported that NO production by hepato-cytes and iNOS mRNA levels were markedly enhanced by the LPS-activated Kupffer-cell-conditioned medium. However, we observed no change in NO release after Kupffer cell depletion. Therefore, the NO release could have been produced mainly by hepatocytes and sinusoidal cells other than Kupffer cells under ethanol perfusion. Vos et al. (1999) reported that, 6 h after endotoxin administration, iNOS expression is maximally induced in hepatocytes, Kupffer cells and inflammatory cells, notably neutrophils. Double staining revealed that most neutrophils (90%) were also iNOS-positive. In GSH-depleted endotoxaemic rats, iNOS protein and iNOS mRNA contents in liver tissue were strikingly diminished, as were plasma NOx levels compared with endotoxaemic rats not subjected to GSH depletion. This was because of a decreased expression of iNOS in hepatocytes.

In conclusion, NO release and portal vein pressure were raised simultaneously after ethanol perfusion of rat liver. In GdCl3-pretreated rats, the same phenomenon of NO was observed, but a different change in portal vein pressure occurred during the early stage of ethanol perfusion. These findings suggest that Kupffer cells play an important role in sinusoidal haemodynamics during the initial stage of ethanol intake.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This work was supported in part by Grants-in-Aid for Scientific Research of The Ministry of Education, Science, Sports and Culture of Japan. We thank Mr Tsukamoto and Mr Sudo, Intermedical Co. Inc., Nagoya, Japan for their technical advice.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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