@misc{oai:az.repo.nii.ac.jp:00003234,
 author = {富樫, 宏史 and Togashi, Hirofumi},
 month = {2013-02-19, 2014-08-18},
 note = {The ductus arteriosus (DA) bypasses most of the blood from the pulmonary circulation to the placenta, where oxygenation occurs. This vessel unlike the pulmonary artery (PA) and the aorta, which are both elastic in type, is equipped with the media abundant in smooth muscles as a type of muscular artery (Jones et al., 1969). After birth, with the beginning of respiration, the DA is rapidly closed and transformed into a fibrous cord. Since the survival of newborn placental mammals depends on the closure of the DA, the cessation of the umbilical circulation and the occlusion of the foramen ovale, the DA plays a significant role in the neonatal adaptation required immediately after birth.
 The patency of the DA is regulated by a balance of opposing actions of prostaglandin (PG) E_2 and oxygen (Clyman, 1987). Aspirin, indomethacin and other nonsteroidal anti-inflammatory drugs (NSAIDs) are potential inhibitors of cycloxygenase, a limiting enzyme of the biosynthesis of PGE_2.
NSAIDs are observed to constrict dose-dependently the fetal DA after administration to pregnant mothers and animals, and cause fetal pulmonary hypertension, persistent pulmonary hypertension and persistent fetal circulation in infants (Momma and Takao, 1987).
 Closure of the DA after birth occurs in two stages, i.e., initial closure and anatomical (permanent) closure. The initial closure is brought about by contraction of the smooth muscle cells (SMCs) in the wall of the DA (Clyman, 1987). The anatomical closure depends on a series of structural changes. Endothelial cells detach from the internal elastic lamina, and the subendothelial region begins to accumulate extracellular matrix materials and fluid. The internal elastic lamina becomes fragmented, allowing SMCs to migrate into the subendothelial region and obstructing the vessel lumen.
Eventually the DA degenerates into the fibrous cord referred to as the ligamenum arteriosus (Jarkovska et al., 1992). A DA not completely closed after birth is referred to as a patent ductus arteriosus (PDA). The treatment of PDA is important to the survival of newborns. PGE_1 and PGE_2 have been shown to induce a re-opening of the DA in humans and rabbits (Heymann and Rudolph,1977 ; Olley et al., 1976 , 1980 ; Momma et al., 1980). However, the dilating effect of PGs has not been examined in detail in rats, even though rats are often used in studies of reproductive biology.
 Since 1976, indomethacin has been used successfully to treat PDA (Heyman et al., 1976) based on its inhibitory effect on PGE_2 synthesis. Re-opening of the DA after indomethacin treatment, however, is quite common, although the reason is unclear (Clyman et al., 1985). In this connection, it is significant that premature infants who have been exposed in utero to indomethacin for threatened labor tend to show a higher incidence of PDA (Norton et al., 1993).
 Angiotensin-converting enzyme (ACE) inhibitors (ACEIs) belong to a relatively new class of antihypertensive drugs. They have been used in women of reproductive age, because compared with conventional antihypertensive agents, they have the advantage of leaving sympathetic functions intact and of enhancing the distribution of blood flow to the kidneys, heart and brain without altering cardial output (Johnston, 1984 ; Lund-Johansen, 1984). However, some studies have suggested that the continued use of ACEIs during pregnancy may not be safe to the fetus (Kreft-Jais et al., 1988 ; Pryde et al., 1993). It has been noted that the use of ACEIs during the second and third trimesters of pregnancy is associated with PDA (Kreft-Jais et al., 1988 ; Pryde et al., 1993).
 As mentioned above, the patency and the closure of the DA are not completely understood. The re-opening of the DA is of particular interest in part because it is thought to be related to the mechanism of PDA. In the present research, therefore, the re-opening of the DA and the effect of NSAIDs and ACEIs on the re-opening of the rat DA induced by PGE_2 were examined. The findings obtained may contribute to make clear the mechanism of the PDA and the relation of PDA and drugs.

 Summary of results
 1. The dilating effects of PGE_1 and PGE_2 on the once-constricted DA in newborn rats were investigated. Wistar rats were mated and the day on which sperm was found in a vaginal smear was designated as day 0 of gestation. Each newborn pup received subcutaneous injections of PG (4, 10μg/pup) at intervals after Cesarean delivery. The ratio of the inner diameter of the DA to that of the pulmonary artery (PA) was determined at intervals after the injection as an index of the dilating effect on the DA (Arishima et al., 1991 ; Takizawa et al., 1992).
 Both PGE_1 and PGE_2 dilated the DA for over 90 min. The maximal effect appeared 15 or 30 min after the injection. The dilating effect was significantly higher in the PGE_2 injected pups than in the PGE_1 injected pups. The dilating effect was significantly decreased in the 6-hr-old pups compared to the 3-hr-old pups. These results indicate that both PGE_1 and PGE_2 have a dilating effect on the once-constricted DA, that the effect decreases with time, and that the dilating activity of PGE_2 was stronger than that of PGE_1.

 2. It was also determined whether an NSAID administered prenatally to mother rats could potentiate a re-opening of the neonatal DA induced by PGE_2 after postnatal closure. A subcutaneous injection of PGE_2 (4 μg/pup) was given to newborn rats 3 hr after a Cesarean delivery from pregnant females which had orally received indomethacin (1, 3 mg/kg), aspirin (100, 300 mg/kg) or ibuprofen (10, 30 mg/kg) from day 18 to day 20 of gestation. The DA/PA ratio was determined at intervals after the injection.
 The indomethacin, aspirin and ibuprofen treatments each significantly increased the re-opening of the DA and prolonged the duration of re-opening induced by PGE_2. These results suggest that increased ductal responsiveness to PGE_2 in newborns rats seems to be a common response to maternal treatment with an NSAID such as indomethacin, aspirin and ibuprofen.

 3. Whether an ACEIs administered prenatally to mother rats could potentiate a re-opening of the neonatal DA induced by PGE_2 after postnatal closure was investigated. A subcutaneous injection of PGE_2 (4 μg/pup) was given to newborn rats 3 hr after a Cesarean delivery from pregnant females which had orally received enalapril (0.1, 1 or 10 mg/kg) from day 14 to day 20 of gestation. The DA/PA ratio was determined at intervals after the injection.
 The enalapril treatment significantly increased the re-opening of the DA, except in the low dose (0.1 mg/kg) group, but the duration of re-opening was not prolonged.

 4. Early studies of prostaglandin metabolism have revealed that the lung is a major site of prostaglandin inactivation. Piper et al. (1970) showed that the bioactivity of PGEs and PGF_2α almost completely disappeared after a single transit through the pulmonary circulation in guinea-pigs. It has been established that 15-hydroxy prostaglandin dehydrogenase (15-PGDH) is a key enzyme catalyzing the initial reactions in converting the biologically active PGE_2 to its inactive metabolite 15-keto-PGE_2 (Änggård et al., 1964, 1966).
 In the present research, the hypothesis that maternal treatment with indomethacin or enalapril would inhibit the catabolism of PGE_2 and that PDA would be partly due to this inhibition was tested. Also, the perinatal changes of lung 15-PGDH activity and the effects of prenatal indomethacin or enalapril treatment on lung 15-PGDH activity were investigated.
 The 15-PGDH activity in the lungs of non-treated rats was increased from day 20 of gestation to 3 hrs after birth; however, the activity was transiently decreased in 6 hr-old pups, and at 12 hrs had recovered to the level of 3 hr-old pups. Indomethacin treatment (3 mg/kg) from day 18 to day 20 of gestation significantly decreased the activity of 15-PGDH in neonatal lungs, whereas enalapril treatment did not affect the 15-PGDH activity. These results suggest that the decrease in the catabolism of PGE2_ in the lungs is partly responsible for the failure of indomethacin therapy for PDA, and that the catabolism does not contribute to the PDA after enalapril treatment.

 5. SMC proliferation and apoptosis in the media of the DA and the effect of enalapril on this cell proliferation and apoptosis were studied. Proliferating cell nuclear antigen (PCNA)-positive cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells (Gavriel et al., 1992) were observed as indices of cells proliferation and apoptosis, respectively.
 The PCNA indices of the DA and aorta were both approximately 75% in 19-day-old fetuses and significantly declined to about 20% in 20-day-old fetuses. In 3-day-old newborn pups, the PCNA index in the DA further declined from that of 20-day-old fetuses, whereas the PCNA index in the aorta was significantly increased compared to that in the DA.
 The effect of enalapril on the PCNA indices of the DA and aorta was examined in perinatal rats. In 19-day-old control fetuses, the PCNA indices of the DA and aorta were about 75%, whereas the indices of these vessels were significantly decreased when enalapril (10 mg/kg for 7 days) was transplacentally administered. The decline of the PCNA index induced by enalapril was more prominent in the DA compared to the aorta. The PCNA indices of these vessels in 20-day-old fetuses were not altered by the maternal treatment with enalapril. These results indicate that enalapril inhibits the proliferative activity of the fetal vessels, and that this effect was age-dependent and more prominent in the DA compared to the aorta.
 TUNEL-positive cells were not observed in the DA of fetuses. In 1-day-old pups, in contrast, TUNEL-positive cells in the DA were regularly observed, as clusters in the center of the DA and as circular arrangements in the outer layer of the media. In 3- and 5-day-old pups, the regular localization of TUNEL-positive cells observed in 1-day-old pups had been lost ; the TUNEL-positive cells in the DA were now irregularly scattered in the vessel's wall. In the enalapril-treated group of 3-day-old pups, remarkable TUNEL-positive cells were seen in the center of the DA, and the positive cells were significantly increased in number. In 5-day-old pups, the number and the pattern of localization of the TUNEL-positive cells were the same as in the control group.

 Thus, based on the foregoing observations, PGE_1 and PGE_2 have a dilating effect on the once-constricted DA. NSAID or enalapril treatment increased the ductal responsiveness to PGE_2 in newborns rats. Since the neonatal lung 15-PGDH activity was significantly decreased after maternal indomethacin treatment, the decrease in the catabolism of PGE_2 in the lung is partly responsible for the failure of indomethaicn therapy for PDA. However, 15-PGDH activity was not affected after maternal enalapril treatment. The SMC proliferation in the prenatal DA was decreased after maternal enalapril treatment, and the onset of apoptosis in the neonatal DA was delayed, suggesting that the maturation of the SMC in the DA was suppressed after maternal enalapril treatment and that the DA then had an immature response to PGE_2., 動脈管は胎生期に特有の血管であり、肺動脈と大動脈を結び肺への血液流入の大部分を大動脈へと迂回させている。動脈管の収縮閉鎖は卵円孔の閉鎖、臍循環の停止と並んで胎生期循環から成体循環への移行に重要な役割を果たしており、動脈管の収縮が生後速やかに非可逆的に起ることは新生児の生存にとって極めて重要である。
　出生後、動脈管収縮が順調に進行せず、開存したままとなると動脈管開存症と呼ばれる。また、胎生末期に動脈管が収縮すると胎児肺高血圧が生じ、出生後、新生児肺高血圧持続症あるいは胎児循環持続症が起こることが知られている(Momma and Takao, 1987)。これらの疾患は新生児の生命の存続に関わり、重要である。また、生後一度収縮した動脈管が再開存する現象が知られているが、この再開存のメカニズムは不明であり、その解明は動脈管開存症の発生メカニズムの解明にもつながるものと考えられる。
　インドメタシンはシクロオキシゲナーゼ阻害という薬理作用から、動脈管開存症の治療に用いられているが、必ずしも成功率は高くない(Heyman et al., 1976; MaCarthy et al., 1987)。また、使用時期によっては逆効果を生じるという報告(Konishi et al., 1986)もある。
　また、アンギオテンシン変換酵素阻害薬であるマレイン酸エナラプリル(エナラプリル)は、降圧薬としてヒト妊婦によく使用されている(Johnston, 1984; Lund-Johansen, 1984)が、その妊婦の新生児において動脈管開存症の発現頻度が高いことが報告されている(Kreft-Jais et al., 1988; Pryde et al., 1993)。
　これらのことから本論文では、ラットを用いてインドメタシンやエナラプリル等の薬物が動脈管再開存に及ぼす影響を明らかにすることを目的とした。
　第1章においては、新生子を用いて、生後一度収縮閉鎖した動脈管の再開存について検討した。PGE_1とPGE_2(4、10μg/匹)を生後3時間の新生子に投与すると、動脈管の再開存が生じ、その作用はPGE_2の方が強い。PGE_2による動脈管の再開存は生後間もないほど強く、しだいに減退し、生後24時間後には消失することが示された。
　第2章においては、妊娠末期の母体ラットに非ステロイド系抗炎症薬を投与した時の新生子におけるPGE_2による動脈管の再開存に及ぼす影響を検討した。
　妊娠18日から20日の母体ラットにインドメタシン(1、3mg/kg)、アスピリン(100、300mg/kg)、イブプロフェン(10、30mg/kg)を3日間連続経口投与すると、得られた新生子においては、PGE_2(4μg/匹)による動脈管の再開存が著しく増強した。この再開存の増強作用は酸性の非ステロイド系抗炎症薬に共通すると考えられた。
　第3章においては、妊娠後期の母体にアンギオテンシン変換酵素阻害薬エナラプリルを投与した時の新生子におけるPGE_2による動脈管の再開存に及ぼす影響を検討した。
　妊娠14日から20日の母体にエナラプリル(0.1、1、10mg/kg)を7日間連続投与すると、新生子ではPGE_2による動脈管の再開存が著しく増強した。
　第4章においては、経胎盤的に非ステロイド系抗炎症薬(第2章)やアンギオテンシン変換酵素阻害薬(第3章)を投与すると、PGE_2による動脈管の再開存が著しく増強されるという現象が、どのようなメカニズムによって生じているかを明らかにするために、新生子の肺におけるPGE_2代謝に注目し、PGE_2の主要な代謝酵素である肺の15-ヒドロキシ-プロスタグランジンデヒドロゲナーゼ(PGDH)活性について検討した。
　母体にインドメタシン(3mg/kg)を投与した後に得た新生子の肺のPGDH活性は、対照群の約60％に低下していたので、このPGDH活性の低下がPGE_2代謝の遅延を引き起こし、インドメタシン投与後のPGE_2による動脈管の再開存の増強をもたらすものと考えられた。一方、母体にエナラプリル(10mg/kg)を投与しても、新生子の肺のPGDH活性は変化しなかったので、エナラプリル投与後のPGE_2による動脈管の再開存の増強には、新生子の肺のPGDH活性は関与していないものと考えられた。
　第5章においては、エナラプリルを母体に投与した時、動脈管中膜の平滑筋細胞の増殖とアポトーシスに及ぼす影響を検討した。
　細胞増殖とアポトーシスの指標としてそれぞれPCNA陽性細胞とTunel陽性細胞を観察した。胎齢19日の動脈管の平滑筋細胞の増殖は母体へのエナラプリル投与により約60%に低下した。
　動脈管のTunel陽性細胞は生後1日から動脈管の中央部には集団として、周辺部には円周状に散在して認められた。生後3日と5日には中央部のTunel陽性細胞の集団は消失し、血管壁全体に散在して認められた。電子顕微鏡により観察すると、Tunel陽性細胞はアポトーシス細胞に典型的なクロマチンの凝集像を示していた。母体にエナラプリルを投与すると、新生子の動脈管のTunel陽性細胞の出現パターンが遅延していた。
　以上の結果から、動脈管が一度収縮した後でも新生子においては、PGE_2とPGE_1により動脈管は再開存する。インドメタシンやエナラプリルは、経胎盤的に投与されると、新生子においてPGE_2による動脈管の再開存を著しく増強させる。その原因を調べたところ、インドメタシンを母体に投与すると、新生子の肺のPGDH活性が低下するので、肺におけるPGE_2代謝が遅延し、再開存作用が増強されるものと考えられた。しかし、エナラプリルにおいては、肺のPGDH活性は変化していなかったが、胎生期の動脈管の平滑筋の細胞増殖が抑制され、また、新生子の動脈管のアポトーシス細胞の出現パターンが遅延していた。これらのことから、平滑筋細胞の成熟化が抑制され、未熟型の動脈管の反応性を示した結果、PGE_2による動脈管の再開存が増強されたものと結論された。},
 title = {ラット動脈管の再開存に関する実験的研究},
 year = {}
}