Biphasic response of the ductus arteriosus to combined administration of indomethacin and L-NAME in fetal rats.

We studied the combined effects of indomethacin, a cyclooxygenase inhibitor, and N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on the patency of the ductus arteriosus (DA) in fetal rats. Pregnant rats were administered indomethacin (3 mg/kg) orally 3 h before cesarean section, and then L-NAME (5 and 50 mg/kg), D-NAME, an enantiomer of L-NAME (50 mg/kg), or methylene blue, a soluble guanylate cyclase inhibitor (100 mg/kg), was injected intraperitoneally at various times before the cesarean section. Using rapid freezing and shaving methods, the calibers of the DA were measured. Compared with the indomethacin alone group, L-NAME caused a rapid and marked increase in the DA caliber 0.5 h after injection in fetal rats in which the DA was constricted by treatment with indomethacin. Subsequently, the transient ductal dilatation was completely reversed by 3 h after the L-NAME injection. Methylene blue also caused a biphasic response, but D-NAME did not affect the DA caliber. We conclude that the inhibition of both cyclooxygenase (indomethacin) and NO synthase (L-NAME) or NO action (methylene blue) causes a biphasic effect on the DA caliber in fetal rats.

Effects of TAK-044, a nonselective endothelin receptor antagonist, on the spontaneous and indomethacin- or methylene blue-induced constriction of the ductus arteriosus in rats.

We studied the effects of TAK-044, a nonselective endothelin (ET) receptor antagonist, on the indomethacin- or methylene blue-induced constriction of the ductus arteriosus (DA) in rats and compared them with the effects on spontaneous DA constriction. Injection of TAK-044 into 21-day-old fetuses in utero was performed through the uterine wall of laparotomized mother rats under light ether anesthesia. The fetuses were autopsied 3 hr after treatment with TAK-044 (10 mg/kg) in utero and simultaneous administration to the laparotomized mother rats of indomethacin (3 mg/kg, p.o.) or methylene blue (100 mg/kg, i.p.). In the second experiment, pregnant rats were decapitated on day 21 of gestation to obtain newborn rats by cesarean delivery. Newborn rats which were given TAK-044 (2, 10 mg/kg) immediately after or 1 hr before cesarean delivery were autopsied at various times after birth. In both experiments, pups were rapidly frozen in an acetone-dry ice mixture at autopsy to evaluate the DA constriction by the whole-body freezing and shaving method. TAK-044 injection into the fetus 3 hr before autopsy completely inhibited the DA constriction induced by maternal treatment with indomethacin or methylene blue. TAK-044 caused dose-dependent inhibition of the spontaneous closure of the DA after birth. The inhibitory effect was more pronounced in pups which were given TAK-044 in utero 1 hr before birth; however, the inhibitory effect was incomplete in newborn pups. These results, together with the previous finding that BQ-123, an ETA-specific receptor antagonist, inhibits the ductal constriction induced by oxygen in vitro [Coceani et al., 1992], indicate that the ETA receptor plays a significant role in the indomethacin- or methylene blue-induced DA constriction as well as in the spontaneous DA constriction after birth, and also indicate that the inhibition of ETA receptor by TAK-044 was more easily achieved in fetuses than in neonates.

Quantitative alterations of hyaluronan and dermatan sulfate in the hairless mouse dorsal skin exposed to chronic UV irradiation.

The quantitative alterations of hyaluronan and dermatan sulfate in the upper dermis (fibrous tissue) and the lower dermis (adipose tissue) of the hairless mouse skin chronically exposed to the UV irradiation as solar-simulating irradiation (lambda(max) 352 nm, UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) were evaluated. Hyaluronan and dermatan sulfate contents in each part of dermis were determined as follows: skin sections on a glass slide prepared by histological technique were processed into the upper dermis and the lower dermis with a small surgical knife, and treated with chondroitinase ABC and ACII in the presence of bacterial collagenase. The resulting unsaturated disaccharides were determined by HPLC method. By applying this method to the UV-irradiated hairless mouse skin, it was found that the chronic UV irradiation increased dermatan sulfate in the upper dermis, whereas an increase of hyaluronan content was not statistically significant. In the lower dermis, on the contrary, both hyaluronan and dermatan sulfate contents remarkably increased as compared with the control mice. Furthermore, the histological study showed the accumulation of the collagen fibers in the lower dermis of the UV-irradiated hairless mouse skin following the disappearance of adipocytes. These findings indicate that the increases of glycosaminoglycan contents in the UV-irradiated skin are related to the accumulation of the extracellular matrix components in the lower dermis.

Quantification of hyaluronan and chondroitin/dermatan sulfates in the tissue sections on glass slides.

The method for the determination of hyaluronan and chondroitin/dermatan sulfates in the tissue sections on a glass slide, which were prepared by histological technique, was established by applying to porcine skin. The degradation of these glycosaminoglycans to the unsaturated disaccharides in porcine skin sections on a glass slide was achieved by chondroitinase ABC and ACII in the presence of highly purified bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. So far, the determination of the glycosaminoglycans in the tissues has taken up more than 5 days, whereas the determination of the glycosaminoglycans in the frozen sections by the present method was completed within a day. In addition, applications of the present method to the serial polyester wax sections processed with a small surgical knife made it possible to determine the glycosaminoglycans in a local part in the tissue section. The present method should open a way for the clinical analysis of glycosaminoglycans in the pathological tissue samples.

Role of the nitric oxide-cGMP system in the regulation of ductus arteriosus patency in fetal rats.

The purpose of this study was to examine the role of the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) system in the regulation of the ductus arteriosus (DA) patency in fetal rats. Pregnant rats were administered N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg, ip), an NO synthase (NOS) inhibitor; methylene blue (30, 50 and 100 mg/kg, ip), a soluble guanylate cyclase inhibitor; or indomethacin (3 mg/kg, po), a cyclooxygenase inhibitor, at various times before cesarean section. Dams were decapitated to obtain the fetuses by cesarean section, and fetuses were rapidly frozen in an acetone-dry ice mixture. Using rapid freezing and shaving methods, the calibers of the DA, pulmonary artery (PA) and descending aorta (Ao) were measured to evaluate the effects of treatment. L-NAME reduced the DA calibers to 86% of the initial values, but recovery to the control levels occurred 6 hr after the injection. Indomethacin decreased the DA calibers to 34% of the control values and sustained the DA constriction until 24 hr after the treatment. Methylene blue caused DA constriction to almost the same degree as indomethacin, but the levels normalized within 24 hr after the treatment. We conclude that L-NAME caused a slight constriction of the DA, whereas methylene blue and indomethacin caused marked constriction of the vessels, suggesting that the NO-cGMP system as well as prostaglandins contribute to the DA patency.