Role of PGI2 in the formation and maintenance of hyperdynamic circulatory state of portal hypertensive rats

Abstract

AIM: To investigate the role of prostacyclin (PGI₂) and nitric oxide (NO) in the development and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats.

METHODS: Ninety male Sprague-Dawley rats were divided into three groups: intrahepatic portal hypertension (IHPH) group by injection of CCl₄, prehepatic portal hypertension (PHPH) group by partial stenosis of the portal vein and sham-operation control (SO) group. One week after the models were made, animals in each group were subdivided into 4 groups: saline controlled group (n = 23), Nω-nitro-L-arginine (L-NNA)group (n = 21) group, indomethacin (INDO) group (n = 22) and high-dose heparin group (n = 24). The rats were administrated 1mL of saline, L-NNA (3.3 mg/kg·d) and INDO (5 mg/kg·d) respectively through gastric tubes for one week,then heparin (200 IU/Kg/min) was given to rats by intravenous injection for an hour. Splanchnic and systemic hemodynamics were measured using radioactive microsphere techniques. The serum nitrate/nitrite(NO₂^-/NO₃^-) levels as a marker of production of NO were assessed by a colorimetric method, and concentration of 6-keto-PGF1α, a stable hydrolytic product of PGI₂, was determined by radioimmunoassay.

RESULTS: The concentrations of plasma 6-keto-PGF1α (pg/mL) and serum NO₂^-/NO₃^- (μmol/L) in IHPH rats (1123.85±153.64, 73.34±4.31) and PHPH rats (891.88±83.11, 75.21±6.89) were significantly higher than those in SO rats(725.53±105.54, 58.79±8.47) (P<0.05). Compared with SO rats, total peripheral vascular resistance (TPR) and splanchnic vascular resistance (SVR) decreased but cardiac index (CI) and portal venous inflow (PVI) increased obviously in IHPH and PHPH rats (P<0.05). L-NNA and indomethacin could decrease the concentrations of plasma 6-keto-PGF1α and serum NO₂^-/NO₃^-in IHPH and PHPH rats (P<0.05) .Meanwhile, CI, FPP and PVI lowered but MAP,TPR and SVR increased(P<0.05). After deduction of the action of NO, there was no significant correlation between plasma PGI₂ level and hemodynamic parameters such as CI, TPR, PVI and SVR. However, after deduction of the action of PGI₂, NO still correlated highly with the hemodynamic parameters, indicating that there was a close correlation between NO and the hemodynamic parameters. After administration of high-dose heparin, plasma 6-keto-PGF₁α concentrations in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle (P<0.05). On the contrary, levels of serum NO₂^-/NO₃^- in IHPH, PHPH and SO rats were significantly lower than those in rats administrated Vehicle (P<0.05). Compared with those rats administrated vehicle, the hemodynamic parameters of portal hypertensive rats, such as CI and PVI, declined significantly after administration of high-dose heparin (P<0.05), while TPR and SVR increased significantly (P<0.05).

CONCLUSION: It is NO rather than PGI₂ that is a mediator in the formation and maintenance of hyperdynamic circulatory state of chronic portal hypertensive rats.

Key Words: Portal hypertension; Prostacyclin; Nitric oxide; Hyperdynamic circulatory

INTRODUCTION

It is well established that systemic and splanchnic hyperdynamic circulatory state plays an important role in maintaining and aggravating the high portal venous pressure. However, the underlying mechanisms have not been completely understood. Recent studies suggest that vasodilators such as nitric oxide (NO) and prostacyclin (PGI₂) contribute much to hyperdynamic circulation[1,2,].

NO may be a mediator in the pathogenesis of hyperdynamic circulatory state, but whether PGI₂ plays the same role is still controversial[3,4]. In order to elucidate the relative contribution of PGI₂, NO and the possible interaction between these two vasodilators in the development of hyperdynamic circulatory state of chronic portal hypertensive rats, we designed the experiment to detect the plasma PGI₂, NO level and hemodynamic effects of NO inhibitor (L-NNA ), COX2 inhibitor (indomethacin) and high-dose heparin on IHPH, PHPH and SO rats.

MATERIALS AND METHODS

Experimental model

Ninety adult male Sprague-Dawley rats(weighing 300±50 g) were used in all experiments. Animals were housed in an environmentally controlled vivarium with light control (12 h light-dark cycle) and allowed free access to standard pellet diet and water. Survival surgery and hemodynamic studies were performed in strict sterile conditions under ketamine hydrochloride anesthesia (100 mg/kg, im). The temperature of rats was maintained at 37±0.5 °C by a heating lamp and monitored by a rectal probe. Experimental animals were randomly divided into three groups: IHPH, PHPH and SO.

IHPH was induced in 22 rats by injection of CCl₄ according to a previously reported method[5]. Briefly, the rats were injected 15 times intramuscularly with (0.3 mL/100 g, first time injection of 0.5 mL/100 g) 60% CCl₄ in mineral oil, once every four days, and were given 10% alcohol instead of water. PHPH was induced in 20 rats by partial portal vein ligation according to a previously reported method[6]. In brief, the portal vein was isolated and a calibrated stenosis was performed with a single 3-0 silk ligature around a 20-gauge blunt-tipped needle. The needle was then removed, and the portal vein was allowed to reexpand. The viscera were placed back into the abdomen, and the incision was closed in two layers with suture. Antibiotic ointment was applied to the surgical wound. SO was maded in 24 rats.

Experimental scheme

One week after the models were made, animals in each group were subdivided into 4 groups: saline controlled group(n = 23), Nω-nitro-L-arginine (L-NNA) group (n = 21), indomethacin(INDO) group (n = 22) and high-dose heparin group (n = 24). Rats were administrated 1mL of saline, L-NNA (dissolved in 1 mL saline at the dose of 3.3 mg/kg·d) and INDO (dissolved in 1 mL saline at the dose of 5 mg/kg·d) respectively through gastric tubes for one week, then 1mL heparin solution at the dose of 200 IU/Kg/min and 1mL saline were given to high-dose heparin group rats and saline controlled rats respectively by intravenous injection for an hour.

Hemodynamic study

Splanchnic and systemic hemodynamic parameters including mean arterial blood pressure (MAP), free portal pressure (FPP), cardiac index (CI), portal venous inflow (PVI), total peripheral vascular resistance (TPR) and splanchnic vascular resistance (SVR) were measured using radioactive microsphere techniques[7].

Assay of 6-keto-PGF1α and NO₂^-/NO₃^-

The concentration of 6-keto-PGF_1α in the plasma, a stable metabolite of PGI₂, was measured by radioimmunoassay as described previously, and NO₂^-/NO₃^- were determined in serum by a colorimetric method according to a previous method[8].

Statistical analysis

All data were expressed as mean±SD. Statistical analysis was performed using SPSS10.0 package of statistical programs. Comparisons of means in three models and subgroups were performed by one-way ANOVA. Pearson’s correlation analysis was performed for selected variables. P<0.05 was taken as statistically significant.

RESULTS

Effects of L-NNA and INDO on plasma PGI₂ and NO concentrations

The concentrations of plasma 6-keto-PGF_1α (pg/mL) and NO₂^-/NO₃^- in both IHPH and PHPH rats were significantly higher than those in SO rats (P<0.05). Compared with controlled group, both L-NNA and indomethacin reduced the plasma 6-keto-PGF_1α (pg/mL) and NO₂^-/NO₃^- concentrations (Table 1) (P<0.05).

Table 1 Effects of L-NNA and INDO on plasma PGI₂ and NO concentrations (mean±SD).

Group	Vehicle (n = 23)	L-NNA (n = 21)	Indo (n = 22)	Heparin (n = 24)
6-keto-PGF1α (pg/mL)
SO	725.53±105.54	748.48±67.68	336.91±37.05c	3965.96±976.82c
IHPH	1123.85±153.64a	494.74±145.98ac	342.86±104.79ac	2930.61±1400.38c
PHPH	891.88±83.11a	386.54±98.44ac	266.94±57.63c	2766.47±506.95c
NO2-/NO3- (μmol/L)
SO	58.79±8.47	21.31±1.76c	55.72±5.33	45.28±4.398c
IHPH	73.34±4.31a	40.17±10.32ac	46.42±7.43ac	54.02±11.89ac
PHPH	75.21±6.89a	30.45±3.28ac	54.74±4.39c	62.06±3.56ac

^aP<0.05 vs SO;

^cP<0.05 vs control.

Effects of L-NNA and INDO on systemic and splanchnic hemodynamics

In basal state, MAP and TPR were significantly decreased but CI was increased in IHPH and PHPH rats when compared with SO rats (P<0.05). Compared with controlled group, the CI decreased while the MAP and TPR in IHPH and PHPH rats elevated after L-NNA or INDO was given (Table 2) (P<0.05). INDO had no impacts on the CI and TPR in SO rats.

Table 2 Effects of L-NNA and INDO on systemic hemodynamics (mean±SD).

Group	Vehicle (n = 23)	L-NNA (n = 21)	Indo (n = 22)	Heparin (n = 24)
MAP (mmHg)
SO	141.86±3.02	159.29±1.98c	145.86±3.72	157.33±3.77c
IHPH	134.00±1.83a	161.50±6.14c	161.14±4.45ac	137.88±2.17a
PHPH	130.29±1.89a	142.50±4.37ac	147.71±2.75c	138.29±4.39ac
CI (mL/min)
SO	28.46±0.58	25.34±2.22c	27.20±0.61	23.52±0.72c
IHPH	32.03±1.34a	25.03±1.76c	28.66±1.63c	28.39±2.25a
PHPH	33.19±0.66a	28.13±0.51ac	29.62±1.16ac	26.67±0.66ac
TPR (mmHg/mL·min)
SO	4.99±0.13	6.33±0.60c	5.36±0.13	6.70±0.33c
IHPH	4.19±0.18a	6.48±0.53c	5.64±0.42c	4.88±0.36a
PHPH	3.93±0.06a	5.07±0.19ac	5.00±0.33ac	5.18±0.21ac

^aP<0.05 vs SO;

^cP<0.05 vs control.

In basal state, FPP and PVI were significantly increased but SVR was lowered in IHPH and PHPH rats when compared with those in SO rats. Both L-NNA and INDO reduced the PVI but enhanced the SVR in IHPH, PHPH and SO rats (Table 3) (P<0.05).

Table 3 Effects of L-NNA and INDO on systemic and splanchnic hemodynamics (mean±SD).

Group	Vehicle (n = 23)	L-NNA (n = 21)	Indo (n = 22)	Heparin (n = 24)
FPP (mmHg)
SO	6.93±0.35	7.29±0.39	7.07±0.35	7.06±0.39
IHPH	10.29±0.39a	8.75±0.46ac	9.28±0.57ac	9.25±0.65ac
PHPH	13.71±0.49a	10.05±0.45ac	11.57±0.53ac	11.07±0.79a
PVI (mL/min)
SO	2.35±0.27	1.06±0.20c	1.63±0.17c	1.57±0.31c
IHPH	3.83±0.64a	2.34±0.50ac	2.16±0.59c	2.05±0.62ac
PHPH	7.37±1.56a	3.71±0.53ac	3.77±0.81ac	2.35±0.25ac
SVR (mmHg/mL·min)
SO	58.06±6.95	96.18±13.71c	85.56±8.13c	99.36±20.26c
IHPH	32.96±5.09a	67.83±14.33ac	74.75±19.34c	68.51±22.26ac
PHPH	16.39±3.23a	36.21±5.21ac	37.52±8.10ac	54.85±6.71ac

^aP<0.05 vs SO;

^cP<0.05 vs control.

Effects of high-dose heparin on plasma PGI₂ and NO concentrations and systemic and splanchnic hemodynamics

After administration of high-dose heparin, plasma 6-keto-PGF₁α concentrations (pg/mL) in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle. On the contrary, serum NO₂^-/NO₃^- (μmol/L) concentrations in IHPH, PHPH and SO rats were significantly lower than those in rats administrated vehicle (Table 1) (P<0.05). Compared with the rats administrated vehicle, the hemodynamic parameters of portal hypertensive rats such as CI and PVI were declined significantly after the administration of high-dose heparin, while TPR and SVR were increased significantly (Tables 2 and 3) (P<0.05).

Correlation analysis

There was a significant positive correlation between plasma 6-keto-PGF₁α and NO₂^-/NO₃^- (r = 0.3939, P<0.01). There were no significant correlations between plasma PGI₂ level and hemodynamics parameters such as CI, TPR, PVI and SVR after deduction of the action of NO, but after deduction of the action of PGI₂, NO still correlated highly with those hemodynamic parameters (Table 4).

Table 4 Pearson partial correlations between PGI₂, NO levels and hemodynamic parameters.

Hemodynamic parameters	PGI2(deduction of the action of NO)		NO (deduction of the action of PGI2)
	R	P	R	P
CI	-0.0259	0.8218	0.552	0.0001
MAP	-0.1335	0.2436	-0.4572	0.0001
TPR	0.0122	0.9154	-0.6053	0.0001
FPP	0.2598	0.0216	0.4659	0.0001
PVI	0.1536	0.1794	0.3579	0.0013
PVR	-0.0523	0.6493	-0.228	0.0446
SVR	-0.1216	0.2889	-0.4325	0.0001

DISCUSSION

Whether PGI₂ plays a role in formation and development of hyperdynamic circulatory state in portal hypertensive rats has not been specifically verified. Hamilton et al[9] found that the plasma concentration of 6-keto-PGF1α, a stable hydrolytic product of PGI₂, was markedly elevated in PHPH rats by partial stenosis of the portal vein and was positively correlated to portal venous pressure (PVP). Sitzmann et al[10] found that the concentration of PGI₂ in systemic artery circulation had a close relationship to the enhanced PVP, the increased mesenteric artery flow (MAF) and the decreased resistance of mesenteric artery in portal hypertensive rats. The hyperdynamic circulatory state in portal hypertensive rats was significantly alleviated after administration of COX inhibitor (indomethacin), which could be reversed after infusion of extrinsic PGI₂, thus postulating that PGI₂ contributes to the formation of hyperdynamics as a systemic mediator via escaping the hepatic hydrolysis through portal systemic shunt. The following findings that PGI₂ is positively related to PVP in portal hypertension and Budd-chiari syndrome patients and COX-1 mRNA transcription is elevated in superior mesenteric artery and thoracic aortic artery in PHPH rats also support the above hypothesis[11].

However, Blanchart et al[4] did not find the above-mentioned effects of INDO on hyperdynamics of portal hypertensive rats, considering that the formation of collateral circulation in PHPH rats was a result of vascular dilation adapted to the high PVP. Our previous studies have shown that the magnitude of systemic and splanchnic hyperdynamics as well as portal systemic shunt was in the order of PCS>PHPH>IHPH, whereas the concentration of 6-keto-PGF₁α is in the order of PHPH>IHPH>PCS. Moreover, the concentration of 6-keto-PGF₁α was higher in PCS rats than in SO rats, but was lower than in PHPH and IHPH rats, namely the dynamics in PCS rats increased most but PGI₂ elevated least. After administration of NOS inhibitors, hyperdynamic state in PCS, PHPH and IHPH rats was reversed to the basic state of SO rats while PGI₂ level was ascended, especially in PCS and SO rats (there was no statistic difference when compared with PHPH and IHPH rats).

In our previous experiments, we also found that 3 and 7 d after orthotopic liver transplantation in IHPH rats, plasma PGI₂ concentration was obviously lower, but was still higher than in normal controlled rats 3 d after operation and so did the PVP. Seven days after operation, there was no difference in PGI₂ concentration between PHPH and normal controlled rats. Nevertheless, 3 and 7 d after orthotopic liver transplantation in IHPH rats, hyperdynamics still existed, verifying that it is the enhanced PVP that causes the increase of PGI₂, and PGI₂ does not play a role in hyperdynamic circulatory state. Recent data also show that PGI₂ could not modulate the vascular tension to the normal level in eNOS deleted mice[12].

The findings of this study demonstrate that there are some hyperdynamic characteristics in IHPH and PHPH rats such as the increase of cardiac output, PVI, and the decrease of SVR and MAP. L-NNA and INDO could lower the CI and PVI, but elevate the MAP and TPR in both IHPH and PHPH rats, thereby improving the hyperdynamic circulatory state, which seems that both NO and PGI₂ are mediators in the pathogenesis of hyperdynamics. However, Hardy et al[13] found that while INDO inhibited the synthesis of PGI₂ , it could simultaneously inhibit the release of NO, which is consistent with our results in this study (Table 1). In another word, when INDO reduces the plasma PGI₂ level in IHPH and PHPH rats, it decreases the serum NO level as well. Pearson partial correlation analysis between PGI₂, NO levels and hemodynamic parameters manifests that after deduction of the action of NO, there is no significant correlation between plasma PGI₂ level and hemodynamic parameters such as CI, TPR, PVI and SVR. However, after deduction of the action of PGI₂, NO still correlates highly with those hemodynamic parameters. Therefore, it is NO rather than PGI₂ that is a mediator in the formation and development of hyperdynamic circulatory state in chronic portal hypertensive rats.

It was reported that high-dose heparin could make physical changes of endothelial cell membranes to enhance intracellular PLA₂ activity, thus increasing the production of PGI₂[14,15]. Meanwhile, high-dose heparin could decrease the production of NO in endothelial cells by decreasing expression of eNOS or NOS activity as an effect on cell signaling pathways[16,17]. These results are consistent with our results in this study that after administration of high-dose heparin, plasma 6-keto-PGF1α concentrations (pg/mL) in IHPH, PHPH and SO rats were significantly higher than those in rats administrated vehicle while serum NO₂^-/NO₃^- (μmol/L) concentrations in IHPH, PHPH and SO rats were significantly lower than those in rats administrated vehicle (Table 1). However, the most important finding after application of high-dose heparin is that heparin extenuates the hyperdynamic circulatory state of portal hypertensive rats while facilitate the production of PGI₂. After administration of high-dose heparin, the CI and PVI in IHPH, PHPH and SO rats attenuated significantly while TPR and SVR significantly increased (Tables 2, 3). These results indicate that high-dose heparin can extenuate the hyperdynamic circulation in portal hypertensive rats by decreasing the production of NO which further surports that PGI₂ is not a mediator of hyperdynamic circulation in portal hypertensive rats.