Altered endothelin receptor expression in prehepatic portal hypertension predisposes the liver to microcirculatory dysfunction in rats.

BACKGROUND/AIMS: Endothelin (ET) is one of the most active vascular regulators in the liver. It is unknown how partial portal vein ligation (PPVL) induced prehepatic portal hypertension influences the response of the liver to ET and its agonists. Therefore, this study was conducted to determine the expression of ET receptors and its functional significance after PPVL. METHODS: Competitive receptor binding study and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were performed using liver homogenates after 2 weeks of PPVL or sham operation in rats. Hepatic microcirculation was evaluated in vivo using intravital microscopy. RESULTS: Although there was no significant difference in dissociation constant (Kd) and total amount of receptors (Bmax) between sham and PPVL, the proportion of ET(B) receptor was significantly increased in PPVL. RT-PCR analysis confirmed the up-regulation of ET(B) receptors demonstrated by the competitive receptor binding assay. In the functional study, infusion of ET(B) agonist (IRL 1620) in a low dosage did not change the hepatic microcirculation in sham but strongly constricted the sinusoids leading to a reduction of sinusoidal perfusion in PPVL. CONCLUSIONS: These results suggest that prehepatic portal hypertension may predispose the hepatic microcirculation to dysregulation in stress conditions where ET is upregulated.

In vivo assessment of endothelin-induced heterogeneity of hepatic tissue perfusion.

Specific vasoactive substances such as endothelin (ET) have been proposed to induce heterogeneity of tissue perfusion and thus the oxygen delivery at the sinusoidal level in the liver, but a direct method for testing this hypothesis has not been available. Our objective was to develop a method to test the hypothesis that functional heterogeneity of blood flow can be induced at the sinusoidal level by mediators such as endothelin-1, which act at the sinusoidal level. We constructed oxygen-sensitive membranes using tris (1,10-phenanthroline) ruthenium (II) chloral hydrate, a dye whose fluorescence is quenched by oxygen incorporated into a silicon rubber membrane. The membrane (less than 40 microm thick) was formed on a glass coverslip that served as the viewing window of the system for in vivo fluorescence microscopy and allowed determination of the PO2 distribution in rat liver acini during intraportal infusion of ET or phenylephrine (PE) in vivo. Heterogeneity was quantified by comparing the coefficient of variation (CV) of the fluorescence intensity within the zone 1 before, during, and after drug infusion. PE and ET doses were matched to produce a similar increase in portal pressure. PE caused a gradient of PO2 across zones, but within zone 1 no significant increase in CV was observed. In contrast, ET produced a patchy pattern of both an increase and decrease in PO2 resulting in doubling (P < 0.01) in CV of fluorescence intensities within zone 1. These results indicate that PE, which acts at presinusoidal sites, results in a homogeneous decrease in tissue PO2 within a zone, while ET, which additionally acts at sinusoidal sites, induces significant microheterogeneity of tissue PO2. The oxygen-sensitive membrane provides a useful tool for oxygen mapping in vivo.

Hepatic neovascularization after partial portal vein ligation: novel mechanism of chronic regulation of blood flow.

The present study was undertaken to investigate hepatic microcirculatory response following partial portal vein ligation (PPVL) in rats. Portal pressure was markedly increased 2-6 wk after PPVL, but no significant reduction in sinusoidal perfusion and hepatocellular injury were detected. However, marked neovascularization was observed in PPVL rats using intravital microscopy and scanning electron microscopy (SEM). Extremely high red blood cell velocity (2,000-4,900 microm/s) was seen in these vessels. Injection of fluorescein sodium via the carotid artery revealed that the neovessels originated from the hepatic arterial vasculature. This was further confirmed by clamping the common hepatic artery and phenylephrine injection from the carotid artery. These vessels maintained sufficient flow after massive sinusoidal shutdown elicited by the portal infusion of endothelin receptor B agonist IRL-1620. SEM also showed extensive neovascularization at the hilum. Additionally, clamping the portal vein decreased sinusoidal perfusion only by 9.5% in PPVL, whereas a 71.2% decrease was observed in sham. These results strongly suggest that the liver maintains its microcirculatory flow by vascular remodeling from the hepatic arterial vasculature following PPVL.

Evaluating contraceptive choice through the method-mix approach. An Indian Council of Medical Research (ICMR) task force study.

The method-mix approach was used to evaluate informed contraceptive choices in the present study. A total of 8,077 potential clients were given a balanced presentation of all available contraceptive methods in the national program, ie, the CuT 200 intrauterine device (IUD), low-dose combined oral pills (OC), condom, and sterilization (female/male) along with a new method, Norplant(R).(1) The majority of women opted for spacing methods; among them, the IUD was preferred by about 60% of clients, followed by condoms (9%), OC (6%), and Norplant (5%). Sterilization, mainly female, was accepted by about 17% of the women making an informed choice. The economic status of couples did not influence the contraceptive choices, as all the methods were offered free of cost in the present study, which is the current practice in the national program. Illiterate women more often accepted sterilization (about 25%) than did literate women (15%). This is because illiterate women had more children; about 30% of illiterate women had three or more children, as opposed to 16.2% of literate women. However, literacy status did not influence the choice of any specific spacing method. The study also revealed that, by encouraging potential clients to make an informed choice, they could override the provider's bias while accepting a particular type of spacing method. This is evident from the observation that Norplant was the first choice of the provider for 35% of the women, whereas only 5% of women preferred and accepted Norplant. The present study stresses an urgent need to promote the practice of informed choices in the national program with a variety of contraceptive options-especially, spacing methods for improving contraceptive prevalence and reproductive health in the country.

Functional significance of endothelin B receptors in mediating sinusoidal and extrasinusoidal effects of endothelins in the intact rat liver.

Endothelins (ET) are important regulators of the hepatic microcirculation that act through different receptor subtypes. We investigated functional significance of ET(B) receptors in mediating microhemodynamic effects of ETs in normal and endotoxin (lipopolysaccharide [LPS])-primed rat liver. LPS priming (Escherichia coli O26:B6; 1 mg. kg(-1)) selectively increased ET(B) mRNA and led to a shift in available receptors to the ET(B) subtype. IRL 1620 (an ET(B) agonist) increased portal pressure in a dose-dependent manner, and the increase in ET(B) expression was associated with prolonged portal pressor response in isolated livers. However, lactate dehydrogenase (LDH) release was attenuated and sinusoidal blood flow was better maintained upon ET(B) stimulation in vivo. In isolated livers, portal constriction as well as release of LDH, were substantially increased in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS). In vivo microscopic assessment of sinusoidal perfusion during ET(B) stimulation revealed a disruption of the flow pattern including frequent reversal of the flow direction without significant sinusoid constriction. Sinusoidal flow decreased even further after discontinuation of IRL 1620. Both effects were mediated at extrasinusoidal sites that probably included postsinusoidal sites. However, after pretreatment with L-NAME, IRL 1620 evoked a significant sinusoidal constriction that colocalized with the body of the stellate cell. We propose that ET(B1)-induced NOS activity attenuates ET(B2) (and presumably ET(A))-mediated portal pressor response and stellate cell constriction. Transcriptional activation of the ET(B) gene may have a permissive effect on liver blood flow and protect against hepatocellular damage under pathophysiological conditions associated with endotoxemia.

Altered endothelin receptor subtype expression in hepatic injury after ischemia/reperfusion.

This study was performed to determine whether ischemia/reperfusion (I/R) injury in rat liver results in alterations in endothelin receptor expression. Hepatic ischemia was produced in rats for 60 min followed by 6 or 24 h reperfusion. Portal inflow pressure was increased (7.38+/-0.60 mmHg) at 24 hours after reperfusion. Serum ALT increased significantly at both 6 and 24 h (6 h; 258.3+/-74.3, 24 h; 243.1+/-74.8 IU/L). Portal vascular response to an endothelin-B receptor agonist (IRL 1620) was significantly increased in the I/R livers compared to control and this was potentiated by L-NAME. IRL 1620 also caused LDH release from I/R livers but not controls. LDH release after IRL 1620 in I/R livers correlated with increased portal pressure response. To determine whether the altered response might be the result of altered endothelin receptor expression, livers were harvested after reperfusion and total endothelin binding sites were determined by competitive binding with ET-1. Proportion of endothelin receptor subtypes (ET(A)/ET(B)) was determined using the ET(A) antagonist BQ-610 (1 microM) and ET(B) agonist IRL-1620 (100 nM). There were no significant changes in Kd but Bmax for endothelin-1 was decreased in I/R group especially non-ischemic lobe at 24 h. ET(A) receptors were significantly decreased whereas ET(B) receptors were increased. These changes were more pronounced at 24 h after reperfusion than at 6 h. Interestingly, the changes in ET receptors was observed identically both in ischemic and non-ischemic lobes (ischemic lobe ET(A) 41.9%, ET(B) 51%; non-ischemic lobe ET(A) 38.8%, ET(B) 49.5%). These results indicate that the major functional endothelin receptor subtype upregulated in I/R is the ET(B) receptor and that this upregulation may contribute to microvascular dysregulation and hepatic injury.

A simple regression model for estimating gestational age in later pregnancy by ultrasonography.

Three hundred and six antenatal women with confirmed LMP underwent a single real time ultrasonographic examination between 32-40 weeks of gestation. Four parameters viz. BPD, HL, FL and AP having a positive correlation with gestational age were studied. A multiple regression model for predicting gestational age by a single ultrasonographic examination was developed by forward selection method after eliminating multicollinearity; as a high degree of intercorrelation was found between FL and HL, HL was eliminated from the data. The BPD does not have a significant role in predicting gestational age in later pregnancy. The proposed regression equation with FL and AP predicts gestational age 75% of time within +/- 10 days of 95% confidence limit.

Randomized clinical trial with intrauterine devices (levonorgestrel intrauterine device (LNG), CuT 380Ag, CuT 220C and CuT 200B). A 36-month study. Indian Council of Medical Research Task Force on IUD.

A total of 1905 subjects were randomly allocated to four types of intrauterine devices (IUDs) and were observed for 45,683 woman-months of use. While no method failure was observed with levonorgestrel (LNG) IUD, 11 women became pregnant with other devices; 4 with Copper T 380Ag, 1 with Copper T 220C, and 6 while using Copper T 200B, indicating method failure rates of 1.0, 0.3 and 1.6, respectively, at 36 months of use. These rates were within acceptable range. Continuation rates were significantly lower with LNG IUD (74.5, 58.7, 38.8 at 1 year, 2 years and 3 years, respectively) as compared to other copper devices, which ranged between 82.4 to 84.4 at 1 year, 66.6 to 69.9 at 2 years and 45.4 to 50.4 at 3 years. The difference in continuation rates was mainly due to menstrual disturbances (e.g. amenorrhoea, irregular bleeding) which were higher with LNG IUD (27.9 per 100 users) as compared to the copper devices (13.4-15.4 per 100 users) at 36 months of use. The risk of expulsion ranged between 8.3 to 10.6 per 100 users and was comparable for all the devices. The observations from the present study based on 36 months of experience with different intrauterine devices do not indicate the need to replace CuT 200, the device currently in use in the National Programme.

Congenital malformations in the newborn--a prospective study.

In a prospective study of 4098 births the incidence of congenital malformations was 1.464 per 100 births. Major malformations were seen in 1.1% births and minor malformations in 0.4% births. Patterns of congenital anomalies included multiple anomalies (37.68%), CNS malformations (13.33%), alimentary tract anomalies (6.66%), cardiovascular malformations (8.99%), genitourinary malformation (6.66%), limb anomalies (13.33%), and anomalies of skin and appendages (13.33%). Factors like maternal age, hormone testing and drug ingestion during pregnancy, radiation exposure and maternal infections were identified as possible risk factors for congenital malformations in the newborn.