Developmental response to indomethacin: a comparison of isometric tension with PGE2 formation in the lamb ductus arteriosus

We studied the effects of oxygen and indomethacin on the isometric contractile response and the production of PGE2 in isolated rings of lamb ductus arteriosus from animals of different gestational ages (100 to 144 days; term is 150 days). Rings of ductus arteriosus from animals less than 110 days released significantly less PGE2 than did rings from animals greater than 120 days. The indomethacin-induced increase in muscle tension in relation to the decrease in endogenous PGE2 production in preparations from animals less than 110 days gestation was greater than in animals older than 120 days. These findings do not support the hypothesis that immature animals have a larger indomethacin-induced contraction due to an increased production of PGE2 earlier in gestation. They are, however, consistent with a decreased sensitivity to PGE2 in the more mature animals; they also support the hypothesis that the decreased effectiveness of indomethacin on the ductus arteriosus from later gestation animals reflects primarily a decrease in the sensitivity of the vessel to PGE2 during development.     

Ductus arteriosus: Developmental response to oxygen and indomethacin

It has been suggested that ineffective constriction in response to an increase in PO₂ is the primary cause for delayed closure of the ductus arteriosus in preterm infants. We studied the isometric contractile effects of increased PO₂ and indomethacin on isolated rings of lamb ductus arteriosus from animals of different gestational ages (87 to 147 days, term is 150 days). Rings from animals less than 110 days have a significantly smaller oxygen-induced contraction (2.53 ± .30 g/mm², n = 16) when compared with rings from animals near term (4.59 ± .69 g/mm², n = 9). Oxygen contracted rings from all gestational ages contract further upon addition of 1 μg/ml indomethacin. Rings from animals less than 110 days have a significantly larger indomethacin induced contraction (1.10 ± .17 g/mm², n = 16) than vessels near term (0.52 ± .12 g/mm², n = 9). Inhibition of prostaglandin production in rings less than 110 days results in a total combined oxygen and indomethacin induced tension that is not significantly different from the oxygen or oxygen and indomethacin induced tension developed in rings from animals near term. This is consistent with the hypothesis that, early during gestation, endogenous prostaglandins inhibit the vessel's ability to contract in response to oxygen. These observations are also consistent with the ability of indomethacin to constrict the patient ductus arteriosus in pre-term infants.     

Formation of prostacyclin (PGI2) by the ductus arteriosus of fetal lambs at different stages of gestation

Prostaglandins appear to play a role in maintaining patency of the ductus arteriosus during gestation. Prostacyclin (PGI₂) is the major product of prostaglandin biosynthesis in the lamb ductus arteriosus. This factor is both a vasodilator and a potent inhibitor of human platelet aggregation. We used inhibition of platelet aggregation as a sensitive bioassay to measure PGI₂ generation in rings of ductus arteriosus from fetal lambs. Mechanical manipulation accelerated the rate of PGI₂ released from the tissue 10 to 50 times. Tranylcypromine, an antagonist of prostacyclin synthetase, suppressed production of PGI₂ by rings of ductus arteriosus. Rings from immature animals (98–103 days gestation, term is 150 days) released significantly more PGI₂ (190 ± 28 ng/g wet weight/ 20 min, n=9) than did those from near term animals (136–146 days; 106 ± 23 ng/g wet weight/20 min, n=10). The capacity of the ductus arteriosus to generate more PGI₂ earlier in gestation is consistent with the observation that vessels from animals less than 110 days gestation have a significantly larger indomethacin induced contraction than do vessels near term.     

Formation of prostacyclin (PGI2) by the ductus arteriosus of fetal lambs at different stages of gestation.

Prostaglandins appear to play a role in maintaining patency of the ductus arteriosus during gestation. Prostacyclin (PGI2) is the major product of prostaglandin biosynthesis in the lamb ductus arteriosus. This factor is both a vasodilator and a potent inhibitor of human platelet aggregation. We used inhibition of platelet aggregation as a sensitive bioassay to measure PGI2 generation in rings of ductus arteriosus from fetal lambs. Mechanical manipulation accelerated the rate of PGI2 released from the tissue 10 to 50 times. Tranylcypromine, an antagonist of prostacyclin synthetase, suppressed production of PGI2 by rings of ductus arteriosus. Rings from immature animals (98-103 days gestation, term is 150 days) released significantly more PGI2 (190 +/- 28 ng/g wet weight/ 20 min, n = 9) than did those from near term animals (136-146 days; 106 +/- 23 ng/g wet weight/20 min, n = 10). The capacity of the ductus arteriosus to generate more PGI2 earlier in gestation is consistent with the observation that vessels from animals less than 110 days gestation have a significantly larger indomethacin induced contraction than do vessels near term.     

Does oxygen regulate prostaglandin-induced relaxation in the lamb ductus arteriosus?

It has been speculated that hypoxia might cause vasodilation of the ductus arteriosus by enhancing the relaxing action of endogenous prostaglandins. Using isolated rings of lamb ductus arteriosus, we measured immunoreactive PGE₂ released into the bath solution. We found that after a period of stabilization following suspension of the rings in low PO₂, only a negligible amount of PGE₂ was released by the rings (1.15 ± 0.52 pg PGE₂/mg wet weight per 45 min, n14, ±SEM). When rings were exposed to a high PO₂, significant amounts of PGE₂ were released (32.3 ± 12.6 pg PGE₂/mg wet weight per 45 min). These observations were supported by our findings that indomethacin had a negligible contractile effect (0.11 ± 0.09 g/mm², n=11) on rings equilibrated in a low PO₂, but caused a significant contraction (0.55 ± 0.12 g/mm², n=11) in rings incubated in a high PO₂. These findings do not support the hypothesis that low PO₂ increases PGE₂ production by the lamb ductus arteriosus. They are consistent with the hypothesis that endogenous PGE₂ inhibits the ability of the vessel to contract in response to oxygen. In addition (if these results can be extrapolated to the situation), the demonstration that the ductus arteriosus needs an oxygen tension greater than that present to produce effective amounts of PGE₂, strengthens the hypothesis that circulating levels of PGE₂ may be important in the prenatal maintenance of ductal patency.     

Studies on closure of the ductus arteriosus. XII. In utero effect of indomethacin and sodium salicylate in rats and rabbits

Administration of prostaglandin synthetase inhibitors to pregnant does and dams in late gestation was followed by $\text{in utero}$ contraction of the fetal ductus arteriosus when studied by the whole-body freezing method. In the rat this contraction was well established within 6 h and persisted up to 36 h following 15 mg/kg indomethacin p.o. No effect was observed in the 18 d rat fetus but fetuses at 20 d and 22 d of gestation responded significantly to indomethacin. Doses of indomethacin approaching clinical usage (2.5 mg/kg) also caused a positive response $\text{in utero}$. The rat was found to be sensitive also to sodium salicylate and in the rabbit both indomethacin and sodium salicylate were effective. Exposure $\text{in utero}$ to prostaglandin synthetase inhibitors with resulting contraction of the ductus may seriously disturb cardiac function in the fetus.     

Ductus arteriosus responses to prostaglandin E1 at high and low oxygen concentrations

It previously has been suggested that prostaglandin E₁ (PGE₁) relaxes the ductus arteriosus in a low but not in an elevated oxygen environment. However, in the experiments reported here PGE₁ relaxed rings on fetal lamb ductus arteriosus at both low (14 to 20 torr) and high (680 to 720 torr) oxygen tensions. The threshold concentration for PGE₁ was 10⁻¹⁰ M in either PO₂ and the ED50's of PGE₁ relaxation in high and low oxygen were 8.5 ± 3.4 × 10⁻¹⁰ M and 5.5 ± 0.7 × 10⁻¹⁰ M respectively. The magnitude of the relaxation was greater for the oxygen contracted ductus arteriosus than for that exposed to low oxygen. It is suggested that earlier reports of the lack of response of the ductus arteriosus to PGE₁ in a high oxygen environment following relaxation in a low oxygen environment may be related to loss of response of the ductus arteriosus to repeated doses of PGE₁ rather than to differences in PO₂. Prostaglandin E₁ therefore may play a significant role in the regulation of ductus arteriosus tone in the elevated oxygen environment of the newborn as well as the low oxygen environment of the fetus.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGFα

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE₂, PGF_2α, 6 keto PGF_1α (and its unstable precursor PGI₂). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂ and indomethacin. All the prostaglandins (except PGF_2α) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE₂, however, relaxed the vessel at concentrations below 10⁻⁸M. PGI₂ and 6 keto PGF_1α had approximately 0.001 and 0.0001 times the activity of PGE₂. Although PGE₂ has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE₂ might make it the most significant prostaglandin in regulating the patency of the vessel.     

Prostaglandin E2 rather than lymphocyte-activating factor produced by activated human mononuclear cells stimulates increases in murine thymocyte cAMP

Culture supernatants (SUPS) of endotoxin (LPS)-activated human mononuclear cells (MNL) stimulated greater production of cAMP by thymocytes than by spleen cells of C3H/HeJ or nude ($\text{nu}\text{nu}$) mice. Similarly, the addition of prostaglandin E₂ (PGE₂) stimulated higher levels of cAMP in thymocytes and progressively lower levels in spleen cells from C3H/HeJ mice and $\text{nu}\text{nu}$ spleen cells, respectively. Partial purification on Bio-Gel P100 of the LPS-induced MNL SUPS yielded peaks of thymocyte proliferative activity characteristic of lymphocyte activation factor (LAF) but these fractions failed to stimulate cAMP levels in thymocytes. Moreover, MNL SUPS induced with LPS in the presence of indomethacin retained their LAF activity but no longer increased thymocyte cAMP levels. Radioimmunoassay of the SUPS for PGE₂ revealed significantly higher levels of PGE₂ in the media of those MNL cultures stimulated by LPS than when stimulated by phorbol myristic acetate, phytohemagglutin, or extracted cell wall fraction of Actinomyces viscosus. Thus, PGE₂ is produced by human MNL and may exert considerable immunoregulatory effects mediated by elevation of lymphocyte cAMP levels.     

Lamb ductus arteriosus: Effect of prostaglandin synthesis inhibitors on the muscle tone and the response to prostaglandin E2

The prostaglandin synthesis inhibitors, indomethacin and eicosa-5,8,11, 14-tetraynoic acid (ETA), have been tested on the isolated lamb ductus arteriosus at low and high PO₂ levels. Both compounds produced a gradual contraction of the hypoxic vessel, and at equal doses the effect of indomethacin was stronger. The maximal tension output of the hypoxic tissue under indomethacin was equal to that of the oxygen-contracted control. ETA- and indomethacin-treated preparations contracted further upon transfer from a low to a high oxygen environment, and the response under indomethacin exceeded significantly control values. Control preparations were relaxed markedly by PGE₂ in low oxygen but showed little or no response in high oxygen. In contrast, preparations pretreated with the inhibitors retained their sensitivity to PGE₂ during exposure to high oxygen. The data are consistent with the idea that E-type prostaglandins play a role in the regulation of the intrinsic tone of the ductus arteriosus during foetal life. It is also suggested that the sensitivity of ductal muscle to E-type prostaglandins is controlled by the rate of endogenous prostaglandin formation.     

Potentiation of bradykinin-induced vascular permeability increase by prostaglandin E2 and arachidonic acid in rabbit skin

The activity of prostaglandins (PG) in producing vascular permeability was quantitated by dye extraction method in skin of anaesthetized rabbits. PGE₁ and PGE₂ (0.01–10 μg) produced increase in vascular permeability. Activity was approximately equal to that of histamine (Hist) and $\text{1}\text{20}$ of that of bradykinin (BK) on a weight basis. The activity of PGF_1α and PGF_2α was only $\text{1}\text{20}$ of that of PGE₁ or PGE₂.In spite of the relatively low potency of PGE₁ and PGE₂ in the rabbit, near threshold doses (0.1 or 1 μg) of PGE₂ could potentiate permeability responses to bradykinin (0.1 μg) by 10 or 100-fold, respectively. Equivalent doses (0.1 or 1 μg) of histamine could not potentiate the bradykinin responses. Arachidonic acid (AA) at 1 μg, produced a 10-fold potentiation in the permeability response to bradykinin (0.1 μg). Pretreatment of the rabbits with indomethacin (20 mg/kg, i.p.) reduced the responses of BK (0.1 μg) + AA (1 μg) down to a similar magnitude of those seen with bradykinin alone. However, indomethacin did not block responses to either, BK alone, BK + PGE₂, or BK + Hist. Various doses (1, 10, 100 and 300 μg) of arachidonic acid alone also produced increase in cutaneous vascular permeability, although its potency was only $\text{1}\text{3}$–$\text{1}\text{8}$ of that of PGE₂. This activity of arachidonic acid was attributed in part to its bioconversion to PGE₂, since its activity was significantly reduced by the prostaglandin antagonist, diphloretin phosphate (DPP) (60 mg/kg, i.v.) and by indomethacin (20 mg/kg, i.p.), which blocks conversion of arachidonic acid to prostaglandins. Arachidonic acid may owe some of its permeability increaseing effects to histamine release, since its effects were also reduced by the antihistamine, pyrilamine (2.5 mg/kg, i.v.).     

The effects of thyroxine and methimazole treatment on the synthesis of prostacyclin (PGI2) in the rat

The effects of thyroxine (T4) and methimazole administration on plasma prostacyclin (PGI₂) levels $\text{in vivo}$ and on PGI₂ release by aortic rings incubated $\text{in vitro}$ were investigated in rats. Male rats were given single injection of T4 (200 μg/100 g body wt) ip every 24 h for either 3, 7 or 14 days for hyperthyroid rats. For hypothyroid rats, a group of rats were given methimazole (0.01 % in drinking water) for 14 days. PGI₂ concentrations were determined in plasma and also in the medium in which aortic rings were incubated. PGI₂ was measured as 6-keto-PGF1α by RIA. Plasma PGI₂ levels in T₄-treated groups were found to be significantly higher than those of control animals. Aortic rings obtained from rats given single injection of T₄ for 7 and 14 days showed significant increases in release of PGI₂ into the incubation medium. In contrast, rats given methimazole for 14 days showed a significant decrease in the production of PGI₂ by aortic rings without any significant changes in plasma levels. Direct addition of T₄ into the incubation medium did not cause any significant changes in PGI₂ release by aortic rings obtained from control rats.These results suggest the regulatory role of thyroid hormone in PGI₂ synthesis $\text{in vivo}$.     

Effects of prostaglandin PGE2 on the synthesis of placental proteins and human placental lactogen (HPL)

The biosynthesis of placental proteins and placental lactogen (HPL) was studied $\text{in vitro}$ in 10–12 week, 16–18 week and term human placenta in the presence and absence of PGE_2α. The highest ¹⁴C-leucine incorporation was detected in 10 to 12 weeks old placentas. Addition of PGE_2α to the induction medium depressed the rate of incorporation of ¹⁴C-leucine into placental proteins on a dose dependent manner. Placentas most sensitive to this action of PGE_2α were those obtained at 18 weeks gestation followed by placentas at term. $\text{In vivo}$ application of PGE_2α for tharapeutic induction of abortions resulted in the marked inhibition of placental protein synthesis $\text{in vitro}$.     

The measurement of human endometrial prostaglandin production a comparison of two in vitro methods

Two $\text{in vitro}$ methods for measuring human endometrial prostaglandin production were compared. Endometrial samples from eight patients were incubated over eight hours by a perifusion and a superfusion technique. The collected fractions were assayed by radioimmunoassay for PGE₂ and PGF_2α.There was no significant difference between the perifusion and superfusion methods for the pattern and amount of PGE₂ and PGF₂ production with time. Significantly higher production levels of PGE₂ and PGF_2α were found in secretory phase endometria than in proliferative phase endometria. Histological examination of the tissue specimens by light and electron microscopy showed that both methods caused gross tissue damage after eight hours experimentation. The superfusion method produced more morphological damage than the perifusion method. However, no tissue damage could be detected after one hour of incubation with either method.Over an eight hour period neither the perifusion nor the superfusion technique appears to be a good indicator of $\text{in vivo}$ endometrial prostaglandin production. Either reflect the $\text{in vitro}$ situation.     

Studies on closure of the ductus arteriosus. X. in vivo effect of prostaglandin

The effect of prostaglandins F_2∝, E₁ and of 7-oxa-13-prostynoic acid on the newborn rat and rabbit ductus can be studied using the whole-body freezing technique. PGF_2∝ and PGE₁ were able to re-open the closing ductus arteriosus in adequately oxygenated animals. PGF_2∝ administration was accompanied by a strong physical reaction in the rat but less in the rabbit. PGF₁ had sedative effects in both animals. A prostaglandin antagonist, 7-oxa-13-prostynoic acid had no effect on normal ductal closure nor did it counteract the effects of PGF_2∝ and PGE₁. The role of prostaglandins in homeostasis during the fetal and newborn period may be to modify ductal tone.     

The interactions between indomethacin and cytotoxic drugs in mice bearing B-16 melanomas

We have compared the effects of indomethacin alone (100 μg/mouse/day) with those of indomethacin plus adriamycin, 5-FU, nitrogen mustard, thioTEPA, and vincristine on B-16 tumor cell proliferation in vivo. As we have previously descriged, after four days of treatment with indomethacin, subcutaneous tumors were slightly smaller and lighter in weight, but contained $\text{more}$ melanoma cells. Addition of indomethacin to cytotoxic regimens resulted in either no change or a decrease in the effectiveness of the chemotherapy. In previous studies we demonstrated that treatment of tumor-bearing mice with a long-acting synthetic analogue of PGE₂ (di-M-PGE₂) stimulated the synthesis of endogenous prostaglandins. In order to evaluate if these endogenously synthesized prostaglandins were responsible for the inhibition of B-16 growth in vivo, mice were treated with di-M-PGE₂ or di-M-PGE₂ plus indomethacin. Addition of indomethacin did not alter the tumor inhibitory effects of di-M-PGE₂.     

Effect of gestational age on pulmonary metabolism of prostaglandin E1 & E2

The fetus and prematurely delivered newborn lamb have high concentrations of circulating PGE₂ that may play a hormonal role, particularly in maintaining the patency of the ductus arteriosus. We studied the ability of the isolated, perfused lung from immature (100 ± 150 days) lamb fetuses to metabolize PGE₂ as a function of PGE₂ concentration in the perfusate. After an intra-arterial infusion of ³H-PGE₂ and ¹⁴C-inulin (to act as a marker of extracellular space), the bulk of the ¹⁴C-inulin was rapidly cleared through the isolated lung and the majority of the ³H activity appeared after the ¹⁴C activity had fallen to negligible values. The ³H activity that was retained longer in the lung was primarily associated with the 15-keto prostaglandin E₂ and 15-keto-13,14 dihydro prostaglandin E₂ metabolites. Lungs from immature fetal lambs metabolized 25% less PGE₂ than did lungs from animals near term. This is consistent with our prior observation that premature lambs have decreased plasma clearance rates (in vivo) and elevated circulating concentrations of PGE₂ when compared with term newborn lambs.     

Relative biological activity of certain prostaglandins and their enantiomers

A series of mirror image ($\text{ent}$) forms of prostaglandins F₂ and E₂ have been compared for potency in a hamster antifertility test. In the PGF₂ series, $\text{ent}$-compounds surveyed had less potency than corresponding natural structures. For the PGE₂ series, 11α-(15S)-ent-PGE₂ methyl ester was 10-fold more potent than PGE₂. Altering the C-9 hydroxy configuration in the PGF₂ series from the natural α to β decreased potency dramatically for compounds tested.     

Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells

We have compared the production of prostaglandins in fibroblast-like cells and endothelial cells in culture. Of the fibroblasts studied $\text{10T}\text{1}\text{2}$, SHE, BP6T and KD produce significant amounts of PGI₂, PGE₂ and PGF_2F2 under optimal culture conditions, but only 3T3 and BHK produce TxA₂ in addition to PGI₂. The adult bovine aortic endothelial cells (ABAE) and fetal bovine heart endothelium (FBHE) synthesise PGI₂ but not TxA₂, either from endogenous or exogenous substrates. Both cultured endothelial cells and fibroblasts apparently lack 15-hydroxyprostaglandin dehydrogenase pathway and the ability to convert 6-Keto PGF_1α into 6-Keto PGE₁. PGI₂ production by ABAE was 3–5 times that of FBHE, about twice that of SHE cells and 6–8 times that of $\text{10T}\text{1}\text{2}$ or BP6T cells. Supernatants or media obtained from these cells inhibited aggregation of human platelet-rich plasma, a known biological effect of PGI₂. This effect was abolished when cell monolayers were preincubated with indomethacin or tranylcypromine. RIA and chromatographic data of 6-Keto PGF_1α from these experiments confirmed that the inhibition of platelet aggregation was due to the formation of PGI₂. The production of all prostanoids by endothelial cells or fibroflasts was significantly higher during the exponential phase of growth as compared to confluent monolayers. We propose that fibroblasts $\text{10T}\text{1}\text{2}$ or SHE can serve as useful experimental models for the study of metabolism and transport of PGI₂ and/or TxA₂ in cells of nonendothelial nature.