Comparison of the pulmonary vascular response to prostaglandin A1, prostaglandin F2α and angiotensin II; Metabolism of PGA1 in lung

A rabbit lung preparation, perfused $\text{in vitro}$, was used to examine pulmonary metabolism of prostaglandin A₁ (PGA₁) and to compare the vasoconstrictor actions of PGA₁, prostaglandin F_2α (PGF_2α) and angiotensin II. PGF_2α caused significantly more, and angiotensin II significantly less, vasoconstriction than did an equimolar concentration of PGA₁. Of three likely PGA₁ metabolites only 15-keto-PGA₁ had any significant vasoconstrictor action. Furosemide and aminophylline (10^?3 M) reduced PGA₁, PGF_2α or angiotensin II-induced vasoconstruction. Diphloretin phosphate potentiated the vascular effect of angiotensin II. Furosemide (10^?3 M) and DPP (9.5 × 10^?6 M) significantly reduced pulmonary metabolism of PGA₁ while aminophylline (10^?3 M) had no effect on this process. Perfusion of the lungs with a hypoxic medium had no effect on PGA₁ metabolism.     

Prostaglandin inhibition of cartilage chondromucoprotein synthesis: Concept of “intrinsic activity”

We have recently reported that cartilage has two sites for prostaglandin (PG) action. One site (S₁) is stimulated by PGA₁, PGE₁ and PGF_1α and elevates tissue cyclic 3′5′adenosine monophosphate (cAMP). A second site (S₂) is activated by PGA₁ (but not PGE₁ or PGF_1α) and inhibits the synthesis of cartilage macromolecules. The present study is an investigation of the effects of PGB₁ on embryonic chicken cartilage chondromucoprotein synthesis in vitro. PGB₁ was found to inhibit chondromucoprotein synthesis with an apparent affinity for S₂ which was similar to that of PGA₁. The maximal inhibition produced by PGB₁ was, however, approximately one-half the maximal inhibition caused by PGA₁. Studies of the combined effects of PGB₁ and PGA₁ were consistent with the hypothesis that both classes of prostaglandins act at a common site (S₂) with about equal affinity but that PGB₁ has a lower intrinsic activity than PGA₁. Similar studies of the combined effects of PGE₁ or PGF_1α with PGA₁ indicate that neither PGE₁ nor PGF_1α binds significantly to S₂. An independent effect of PGB₁ to activate S₁ and elevate tissue cAMP was also found.     

A comparison of responses of guinea-pig isolated trachea to six prostaglandins

Effects of prostaglandins (PG) E₁, E₂, F_2α, A₁, A₂ nad B₂ were studied on guinea-pig isolated tracheal chains. PGF_2α, B₂ and A₂ produced contraction, PGE₁ and E₂ relaxation of the chain, but A₁ produced no response. 1) From the cumulative dose response curves, PGF_2α was more active in producing concentration than B₂ or A₂, though its effect was less than that of acetylcholine (ACh). PGE₁-induced relaxation was less than the response to isoproterenol. 2) PGE₁ and E₂ 1 μg/ml caused a 26.1 ± 3.83% (n=5) or a 9.5 ± 3.36 (n=6) decrease of ACh (1 μg/ml)-induced contraction respectively. The degree of relaxation produced by E₁ was greater than that by E₂ (P<0.01). 3) After five minutes preincubation with each of PGA₁, A₂, B₂ and F_2α in concentrations which did not produce any effect, ACh-induced contraction was augmented only after PGA₂ (P<0.05).     

The synthesis and biological activities of some 12-Aza-prostaglandin analogues

12-Aza-prostaglandin (PG) analogues containing the pyrrolidine-2,4-dione ring system have been synthesized from ,2-disubstituted glycine esters via cyclisation of their -ethoxycarbonylacetyl derivatives. 5-(6-Carboxyhexyl)-1-octylpyrrolidine-2,4-dione (5) had little or no PG-like activity on superfused intestinal or vascular smooth muscle preparations but it selectively antagonised smooth muscle responses to PGE₂, PGE₁, PGF_2α and PGA₂in vitro. At a concentration of 10⁻⁵ g/ml it reduced responses of the rat stomach strip to PGE₂ by over 80% but did not affect responses of this tissue to acetylcholine, 5-hydroxytryptamine (5-HT) or bradykinin. Polyphloretin phosphate (PPP), the known PG antagonist, had a similar effect at the same concentration (10⁻⁵ g/ml).5-(6-Carboxyhexyl)-1-(3-hydroxyoctyl)pyrrolidine-2,4-dione (12) had the same profile of activity on superfused smooth muscle preparations as PGE₂ or PGA₂. On intravenous injection into anaesthetised rats it caused dose-dependent falls in arterial blood pressure with associated tachycardias, which is typical of the response to PGE₂. The smooth muscle activity of (12) was not reduced by passage through isolated perfused guinea-pig lungs nor was its potency as a vasodepressor increased when given intra-arterially to rats. These results suggest that, unlike PGE₂, this analogue is not removed by the pulmonary circulation.     

Uptake and metabolism of prostaglandins by isolated perfused lung: Species comparison and the role of plasma protein binding

We have investigated the uptake and subsequent metabolism of the prostaglandins (PGs) PGE₁, PGA₁, and PGB₁ by rat, guinea pig and rabbit isolated perfused lungs (IPL). Significant species differences were not observed in the uptake or metabolism of any PG on passage through the IPL. However, differences in the uptake of PGA₁ and PGB₁ and in the metabolism of PGA₁ were observed with a given species when the composition of the perfusion medium was varied. The IPL removed minimal amounts (<20% of the supply rate) of PGA₁ and PGB₁ from the circulation when the perfusate contained 4.5% bovine serum albumin (BSA). In the absence of BSA, however, both PGA₁ and PGB₁ were substantially removed from circulation (～53% of the supply rate) and PGA₁ was also metabolized. The composition of the perfusate had no effect on the uptake and metabolism of PGE₁ which was always taken up and metabolized to a greater extent than was PGA₁ and PGB₁. Thus, the apparent species differences previously reported for the pulmonary biotransformation of PGA can result from differences in the perfusion medium used. Our data suggest that both plasma protein binding and a transport system play important roles in determining the selectivity of the uptake of PGs by the lung.     

The effect of cigarette smoke on the metabolism of arachidonic acid in isolated hamster lungs

The effects of cigarette smoke on the metabolism of exogenous arachidonic acid (AA) were investigated in isolated hamster lungs. Arachidonate was injected into the pulmonary circulation and the metabolites were analysed from the nonrecirculating perfusion effluent by thin layer chromatography. After the pulmonary injection of 66 nmol of ¹⁴C-AA about 20 % of the injected radioactivity appreated in the perfusion effluent mostly as metabolites in six minutes. When isolated lungs were ventilated with cigarette smoke during the perfusion, the amounts of PGF_2α, PGE₂ and two unidentified metabolite groups increased in the lung effluent. In two other experimental series hamsters were exposed to cigarette smoke before the lung perfusion either once for 30 min or during one hour daily for ten consecutive days. Neither pre-exposures caused any changes in the amounts of arachidonate metabolites in the lung effluent.     

Direct actions of prostaglandins E1, A1, and F2α on myocardial contraction

The inotropic and chronotropic actions of prostaglandin (PG) types PGE₁, PGA₁, and PGF_2α were studied in isolated guinea pig right and left atria, and papillary muscles; rabbit atria; and toad ventricular strips in order to more completely define the cardiac contractile properties of PG. All three prostaglandins, in muscle bath concentrations of 10μg/ml, exerted positive inotropic and chronotropic actions on guinea pig atrium. These contractile effects were persistent after removal of PG from the muscle bath and appeared to limit the relative response to a subsequent dose of PG. The inotropic action of PGE₁ was present over a wide range of bath calcium concentrations (1.1 to 4.4 mM/L). Beta adrenergic receptor blockade, histamine blockade, and pretreatment with reserpine failed to significantly affect the inotropic actions of PG. Norepinephrine and histamine produced more potent inotropic and chronotropic effects on guinea pig atria than did PG and these contractile effects did not exhibit persistence or tachyphylaxis. The prostaglandins did not significantly affect dose response curves for norepinephrine inotropic and chronotropic actions. The prostaglandins had no effect on the force or frequency of contraction in rabbit atria. PGE₁ exerted a positive inotropic effect on toad ventricular myocardium whereas PGA₁ had no effect and PGF_2α had a negative inotropic action.     

Prostaglandin A1 inhibition of chondrosarcoma growth

The effects of various prostaglandins (PG) on the in vitro synthesis of macromolecules by two transplantable chondrosarcomas were studied. Prostaglandin A₁ markedly inhibited the incorporation of radioactive precursors into chondromucoprotein, total protein, RNA and DNA of both a well differentiated rat chondrosarcoma and a poorly differentiated murine chondrosarcoma. PGE₁ and PGF_1α had no effects on the synthesis of macromolecules by either tumor. PGA₁ inhibitory effects occurred over a dose range of 1 to 25 μg/ml. PGA₁ had no effect on the synthesis of macromolecules by liver. The data indicate that malignant transformation of cartilage cells does not alter their responsiveness to PGA₁.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Influences of prostaglandins on electrophoretic mobility and aggregation of rabbit platelets

Influences of prostaglandin(PG)s on electrophoretic mobilities and aggregation of rabbit platelets were studied. The PGs studied (PGI₂, PGE₁, PGD₂, PGE₂, PGF_2α, PGA₂ and PGA₁) had no effect on platelet electrophoretic mobility. However, both PGE₁ and PGI₂ in 0.3 and 3.0 μM inhibited ADP-induced aggregation and ADP-induced decrease in the mobility. PGD₂ in 0.3 and 3.0, and PGE₂ in 30 μM inhibited the aggregation but did not depress the ADP-induced decrease in the mobility. PGF_2α, PGA₂ and PGA₁ had no effect on the decrease in electrophoretic mobility and on the aggregation caused by ADP.     

Effect of uterine luminal perfusion of PGF2α and PGE2 on uterine vasomotor response and PGF2α output in cyclic cattle

Six cyclic Holstein dairy cows were anesthetized on days 12–14 post-oestrus. Reproductive tract was exposed by midventral incision, and the ovarian (utero-ovarian) vein and facial artery cannulated. Oviduct was ligated, and a catheter (affluent) introduced into the tip of the uterine horn. The uterine horn was ligated above the uterine body, a second catheter (effluent) introduced into the uterine lumen, and an electromagnetic blood flow transducer placed around the uterine artery. On the day following surgery, the uterine horn was infused constantly for 9 h with PGF_2α dissolved in PBS (0.7 ml/min, 177 ng/ml). During periods 1 and 3 (first 3 h and last 3 h, respectively) only PGF_2α was perfused; during period 2 (between 3 h and 6 h) 101tgμg/ml of PGE₂ were added to the perfusate together with PGF_2α. Uterine venous and peripheral blood samples were collected simultaneously every 15 min, and uterine blood flow recorded continuously. Least-square means for PGF measured in uterine venous drainage for periods 1, 2 and 3 were 315 ± 26, 557 ± 24 and 511 ± 26 pg/ml, respectively (P < 0.05). Uterine blood flow values were 52 ± 5, 67 ± 4 and 61 ± 4 ml/min for periods 1, 2 and 3 (P < 0.08), respectively.Results do not support the hypothesis that the antiluteolytic effect of PGE₂ is associated with a suppression of uterine PGF_2α release into the circulation. Greater release of PGF to the circulation in period 2 (addition of PGE₂) is probably the result of the vasodilatory effect of PGE₂ on uterine endometrial vasculature.     

The metabolism of arachidonic acid in isolated perfused fetal and neonatal rabbit lungs

The developmental pattern of fetal and neonatal rabbit lungs to metabolize arachidonic acid (AA) to different cyclo-oxygenase products was studied in isolated rabbit lungs, which were perfused with Krebs bicarbonate buffer. ¹⁴C-AA (66 nmol) was injected into the pulmonary circulation and the nonrecirculating perfusion effluent was collected for four minutes. About ten per cent of the injected radioactivity was found in the 0–4 min perfusion effluent. The metabolites of AA in the effluent were analyzed by thin layer chromatography. The major metabolites of AA were PGE₂ and its 15-keto-derivates, but also PGF_2α and its 15-keto-derivates, TXB₂ and 6-keto-PGF_1α were found in the effluent. The most drastic developmental change was the increase in the amount of 15-keto-metabolites of PGE₂ from late fetal period to the lungs of one day old rabbits (1.8 fold increase between birth and first postnatal day). Smaller changes were detected in the amounts of other cyclo-oxygenase products.     

Metabolism of [14C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.     

A radioimmunoassay for 13,14-dihydro-15-keto prostaglandin F2α with chromatography and internal recovery standard

Antibodies to the 13,14-dihydro-15-keto metabolite of prostaglandin F_2α(PGF_2αM) were raised in sheep using a bovine thyroglobulin conjugate of PGF_2αM. Labeled 13,14-dihydro-15-keto prostaglandin A₂ (PGA₂M), 13,14-dihydro-15-keto prostaglandin E₂ (PGE₂M) and PGF_2αM were prepared from their corresponding high specific activity parent prostaglandins with swine kidney homogenate and purified using reverse phase liquid-liquid partition chromatography. A rapid method of column chromatography for use prior to radioimmunnoassay was developed.Mathematical corrections for the effect of recovery tracer on the logit/log transformation are presented with a new approach to expression of radioimmunoassay cross reactions allowing continuous expression of the variation of these cross reactions with displacement. These mathematical approaches are widely applicable to other radioimmunoassay systems and transformations.The assay was used for measurement in groups of human volunteers: males, females, women at delivery and paired venous umbilical cord bloods. A correlation between venous cord and maternal peripheral PGF_2αM levels is demonstrated with a gradient from the cord plasma to maternal plasma.     

Prostaglandin actions in established insect cell lines

Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals mediating physiological functions. We reported that PGs influence protein expression in insect cell lines, which prompted the question: do PGs influence cell proliferation or viability in insect cell lines? Here, we report on the outcomes of experiments designed to address the question in cell lines from three insect orders: Hemiptera (squash bug, Anasa tristis, BCIRL-AtE-CLG15A), Coleoptera (red flour beetle, Tribolium castaneum, BCIRL-TcA-CLG1), and Lepidoptera (tobacco budworm, Heliothis virescens, BCIRL-HvAM1). Treating the insect cell lines with PGA₁, PGA₂, or PGD₂ led to dose-dependent reductions in cell numbers. All three cell lines were sensitive to PGA₁ and PGA₂ (IC₅₀s = 9.9 to 26.9 μM) and were less sensitive to PGD₂ (IC₅₀s = 31.6 to 104.7 μM). PG treatments also led to cell death at higher concentrations, as seen in mammalian cell lines. PGE₁, PGE₂, and PGF_2α treatments did not influence AtE-CLG15A or HvAM1 cell numbers at lower concentrations, but led to dose-related reductions in TcA-CLG1 cells at higher concentrations. Similar treatments with pharmaceutical inhibitors of PG biosynthesis also led to reduced cell numbers: MAFP (inhibits phospholipase A₂), indomethacin (inhibits PG biosynthesis), and esculetin (inhibits lipoxygenase). Because these pharmaceuticals are used to relieve inflammation and other medical issues in human medicine, they are not toxic to animal cells. We infer PGs are necessary in optimal quantities for ongoing homeostatic functions in established cell lines; in quantities outside the optimal concentrations, PGs are deleterious.     

Prostaglandin-induced enhancement of the in vitro anamnestic response

The ability of various prostaglandins (PGs) to affect the $\text{in vitro}$ anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA₁, PGE₂ and PGF_2α), PGE₁ was found to have a stimulatory effect, whereas PGA₁, PGE₂ and PGF_2α were ineffective in stimulating or inhibiting the $\text{in vitro}$ anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE₁-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 μg of PGE₁ were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Metabolism of prostaglandin A. I. The kidney cortex as a major site of PGA2 degradation

The antihypertensive and natriuretic prostaglandin A₂ (medullin) has been isolated and identified in rabbit renal papilla. Since PGA₂, unlike prostaglandin F₂ (PGE₂) and prostaglandin F_2α (PGF_2α), is not metabolized by the lung, studies were undertaken to determine if the site of PGA₂ metabolism is in the renal cortex where its primary vasodilatory and natriuretic effects occur. In $\text{in vitro}$ experiments, homogenates of renal cortex and outer medulla were incubated with ³H-PGA₂ (0.2 μc, 20 μg) at 37°C for 30 minutes. A metabolite(s) less polar than ³H-PGA₂ was observed following silicic acid chromatography of acidic lipid extracts of cortical, but not outer medullary homogenates. In $\text{in vivo}$ studies, ³H-PGA₂ (2 μc, 50 μg) was injected into the renal artery of the rabbit and blood withdrawn from the ipsilateral renal vein. At least three less polar major metabolites of PGA₂ were observed in the plasma within 15 seconds following injection. No appreciable ³H-PGA₂ was observed in venous plasma 30 seconds following injection of ³H-PGA₂. In contrast to plasma, the major urinary metabolites were more polar than PGA₂. The present study reveals that PGA₂ is almost completely metabolized in a single passage through the rabbit kidney suggesting this organ is a major site of PGA₂ metabolism.     

Prostaglandin E1 specific binding in rhesus myometrium

Myometrial low speed supernatant prepared from non-pregnant rhesus uteri was incubated with ³H-Prostaglandin (PG)E₁ with or without addition of unlabelled prostaglandins. The uptake of ³H-PGE₁ was inhibited in a dose dependent fashion by PGE₂>PGE₁>PGA₁>PGF_2α=PGA₁>PGB₁=PGB₂≥PGD₂. PGE₁ metabolites inhibited ³H-PGE₁ binding in the following order: 13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁=15-keto-PGE₁. The specific binding of ³H-PGE₁ and ³H-PGF_2α was similarly affected by the temperature and time of incubation. Equilibrium binding constants determined using rhesus uteri obtained during the luteal phase of the menstrual cycle indicate the presence of high affinity PGE₁ binding sites with an average (n=3) apparent dissociation constant of 2.2 × 10⁻⁹M and a lower affinity PGE₁ binding site with a K_d ≅ 1 × 10⁻⁸M. No high affinity — low capacity ³H-PGF_2α sites could be demonstrated.Relative uterine stimulating potencies of some natural prostaglandins and prostaglandin analogs tested after acute intravenous administration in mid-pregnant rhesus monkeys corresponded with the PGE₁ binding inhibition of the respective compound. The uterine stimulating potencies of the prostaglandin analogs tested were: (15S)-15-methyl-PGE₂=16,16-dimethyl-PGE₂>17-phenyl-18,19,20-trinor-PGE₂>16 phenoxy-17,18,19,20-tetranor-PGF_2α=PGE₂=PGE₁=(15S)-15-methyl-PGF_2α>PGF_2α.     

Prostaglandin binding in human serum follows the polarity rule

Human serum binds PGA₁ > PGE₁ > PGF_2α. This is in inverse order of their polarity. Approximately 90% of PGA₁ is bound. By analogy with steroid and iodothyronine metabolism, it is likely that the tighter binding of PGA₁ accounts for its relatively slow clearance.     

Effect of oestradiol 17 β on prostaglandin biosynthesis by the uterus of ovariectomized rat and guinea pig

$\text{In vitro}$ prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 β, administered subsutaneously, was measured by R.I.A. of PGF_2α and PGE₂. Incubations with [1-¹⁴C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF_1α and in decreasing order of magniture, PGF_2α and PGE₂. In guinea pig PGF_2ga was the main product. Ovariectomy in rats completely changed the pattern of synthesized prostanoids: PGI₂ production was doubled when compared to cycling rats and PGE₂ increased 10 fold. PGF_2α walues were similar to the mean value measured during the cycle. OE₂ treatment almost completely inhibited PGI₂ synthesis and reduced PGE₂ by half. Total PG synthesis in OE₂ treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF_sα synthesis was slightly stimulated. In the guinea pig OE₂ treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF_2α was always the main metabolite. In conclusion OE₂ regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxyhenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.