Increased hepatic platelet activating factor (PAF) and PAF receptors in carbon tetrachloride induced liver cirrhosis

Abstract

Background and aims: The liver is a major site for the synthesis and actions of platelet activating factor (PAF), a potent hepatic vasoconstrictor and systemic vasodilator. As PAF is implicated in portal hypertension and hyperdynamic circulation associated with liver cirrhosis, we characterised changes in the hepatic PAF system in experimental cirrhosis.

Methods: In rats made cirrhotic by carbon tetrachloride (CCl(4)) administration for eight weeks, we determined hepatic levels of PAF and its cognate receptor, and the effects of PAF and PAF antagonist (BN52021) on portal and arterial pressure.

Results: Compared with control rats, cirrhotic rats had higher hepatic PAF levels, higher apparent hepatic efflux of PAF, and higher PAF levels in arterial blood (p<0.01, p<0.01, p<0.05, respectively). Relative to controls, cirrhotic livers had elevated hepatic PAF receptors (by mRNA and protein levels and [(3)H]PAF binding), higher (p<0.01) baseline hepatic portal pressure, and an augmented (p = 0.03) portal pressure response to PAF infusion (1 microg/kg). Portal infusion of BN52021 (5 mg/kg) showed that elevated endogenous PAF was responsible for 23% of the cirrhotic portal pressure increase but made no contribution to systemic hypotension. Finally, increased PAF receptor density was observed in the contractile perisinusoidal stellate cells isolated from cirrhotic livers relative to those from control livers.

Conclusions: In cirrhosis, increased hepatic release of PAF elevates systemic PAF; in combination with upregulated hepatic PAF receptors in stellate cells, this contributes to portal hypertension.

Figure 1

Morphometric analysis of the cirrhotic liver. After eight weeks of carbon tetrachloride or vehicle treatment, liver tissue was fixed and stained with haematoxylin-eosin. (A) Complete liver cirrhosis with intensive bridging fibrous septum formation is visible. Fibrous septa uniformly affected hepatic tissue, dividing liver parenchyma into small pseudonodules. (B) Liver tissue displayed necroinflammatory damage consisting of inflammatory cell infiltration, both acidophilic necrosis (green arrows) and apoptosis (black arrows), as well as hepatocyte hydropy (*) and fatty degeneration (blue arrows).

Figure 2

Effect of cirrhosis on circulating platelet activating factor (PAF) levels. Concentrations of PAF in blood drawn from the femoral artery, portal vein, and hepatic vein (suprahepatic vena cava) were determined, as described in the methods section. Values are means (SD) of 6–7 samples each. Differences among groups were significant at p<0.05 except for control portal vein versus control superior vena cava. *p<0.05 versus control; **p<0.01 versus control and p<0.05 versus portal vein.

Figure 3

Effect of cirrhosis on hepatic platelet activating factor (PAF) binding and PAF receptor mRNA and protein. (A) Results of the binding assay to control and cirrhotic membranes. Specific binding was calculated as (total binding−non-specific binding). Values are means (SD) of three separate experiments: differences from control were statistically significant (*p<0.05 or **p<0.01). (B) Reverse transcriptase-polymerase chain reaction (RT-PCR) for PAF receptor (PAF_R) mRNA was performed with cDNA prepared from RNA samples of control and cirrhotic rat livers. Expression of β-actin mRNA was assessed using the same amount of cDNA. PCR products of PAF and β-actin from control (CT) and cirrhotic (CR) rat livers are shown. (C) Protein expression of PAF receptor in control (CT) and cirrhotic (CR) liver. Expression of Erk 2 is shown to demonstrate equal loading.

Figure 4

Effect of cirrhosis on platelet activating factor (PAF) induced changes in arterial and portal pressure. Arterial and portal venous pressure, before (Basal) and after portal administration of PAF (PAF), were measured as described in the methods section. Values are means (SD) of four control determinations and six cirrhotic determinations. **p<0.01, ***p<0.001 versus basal value.

Figure 5

Effect of BN52021 on femoral arterial and portal venous pressure of cirrhotic rats. After eight weeks of vehicle or carbon tetrachloride (CCl₄) treatment, rats were anaesthetised and their femoral arterial and portal venous pressures determined. Following stabilisation (five minutes), BN52021 (5 mg/kg) was infused via the portal vein for one minute using a 23 gauge needle with the aid of a 975 Harvard compact infusion pump. Values shown are from six rats (mean (SD)) 5–6 minutes after introduction of BN52021 when the pressure stabilised to a constant value. *p<0.05 versus basal value.

Figure 6

Effect of cirrhosis on platelet activating factor (PAF) binding and PAF receptor mRNA in stellate cells. (A) Results of the binding assay in control and cirrhotic stellate cells. Specific binding was calculated as (total binding−non-specific binding). Values are means (SD) of three separate experiments: differences from control were statistically significant (p<0.05 or p<0.01). (B) Reverse transcriptase-polymerase chain reaction (RT-PCR) for PAF receptor (PAF_R) mRNA was performed with cDNA prepared from RNA samples of control and cirrhotic stellate cells. Expression of β-actin mRNA was assessed using the same amount of cDNA. PCR products of PAF and β-actin from control (CT) and cirrhotic (CR) rat livers are shown.