Kinetic measurements of the release of prostaglandins from perfused rat lungs

Prostaglandins released from isolated, ventilated and perfused rat lungs were measured by a simple modification of the Vane technique using the rat stomach fundus as a continuous bioassay tissue. Exogeneously supplied arachidonic acid was converted mainly to PGF_2α which was determined by bioassay. A novel method for mixing a stream of inhibitors with the perfusate was used to determine PGF_2α in the presence of substrate amounts of arachidonic acid. Using this system the apparent K_m for PGF_2α production with arachidonic acid as the substrate was found to be 1.90 × 10⁻⁴M, while the K_i for aspirin was found to be 2.47 × 10⁻⁴M. These kinetic parameters are close to those reported for cell free systems and subcellular fractions suggesting that both substrate and inhibitor have ready access to the site of prostaglandin synthesis. The method appears to be generally useful to determine the effect of drugs and environmental factors on the release of prostaglandins by the lung.     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Effect of prostaglandin F2α on anterior pituitary function in man

Five healthy adult men received iv PGF_2α at dosages of 0.05, 0.20 and 2.0 μg/kg/min for 30 min. There were no significant changes in serum FSH, LH or TSH levels. Serum GH and cortisol levels were slightly increased at the highest dosage. These responses were associated with, and presumably a result of, stressful side effects. Thus, PGF_2α cannot be used as a provocative test of pituitary hormone reserve.Prostaglandins (PG's) have recently been implicated in the release of a number of hormones from the anterior pituitary gland. The stimulation of GH release by PG's of the E series from incubated rat pituitary slices has been demonstrated. $\text{In vivo}$ stimulation by PGE₁ of ACTH in rats and of GH release in man has also been shown.The present study was undertaken in order to examine the efficacy of iv administration of PGF_2α as a provocative test of anterior pituitary hormone reserve in man. The responses in circulating levels of gonadotropins, TSH, GH, and cortisol (as an index of ACTH) were measured.     

Paraplacental passage of 3H-prostaglandin F2

Prostaglandins F_2α permeates the live and denaturated whole fetal membranes as well as their isolated components chorion and amnion. The permeability constant in our vitro method is 1.3 × 10^?3 cm/min for PG. If 40 mg PG are instilled intra-amniotically at term — as used in therapeutical abortion — then 3.7 mg prostaglandin F_2α would pass per hour from the amniotic fluid through the fetal membranes into the decidua.     

Respiratory movements alter the generation of prostacyclin and thromboxane A2 in isolated rat lungs: The influence of arachidonic acid-pathway inhibitors on the ratio between pulmonary prostacyclin and thromboxane A2

The influence of hyperventilation on the spontaneous generation of prostacyclin and thromboxane A₂ by isolated rat lungs was studied. Both prostacyclin and thromboxane A₂, as measured by RIA of their stable end-products, 6-oxo-PGF_1α and TXB₂ respectively, were continuously released into the perfusate. However, the concentration of prostacyclin in the perfusate was higher than thromboxane A₂. Under normal ventilation at a rate 40–50 breaths/min, the ratio between these two compounds was 5:1. Increasing the rate of respiration to 100 breaths/min preferentially stimulated the release of prostacyclin. During hyperventilation, the ratio between 6-oxo-PGF_1α and TXB₂ was 12:1. Aspirin and indomethacin suppressed both basal and hyperventilation-stimulated release of prostacyclin and thromboxane A₂. Hydroperoxy-fatty acids and tranylcypromine inhibited only the release of prostacyclin but did not affect the generation of thromboxane A₂. Our findings confirm that the lung generates prostacyclin predominantly, and provide direct evidence that respiratory movements are involved in generation of pulmonary prostacyclin and thromboxane A₂.     

Progesterone and 20α-hydroxyprogesterone stimulate the in vitro release of GnRH by the isolated mediobasal hypothalamus

An $\text{in}$$\text{vitro}$ perifusion system was used to investigate the effect of progesterone (P₄) and 20α-hydroxyprogesterone (20α-OHP) on the release of GnRH from isolated hypothalamic tissue of the adult ovariectomized estradiol-17β primed rat. Pulse delivery of P₄ stimulated GnRH release not only from the isolated mediobasal hypothalamus-anterior hypothalamus-preoptic area (MBH-AHPOA) complex but also from the MBH alone. Release of GnRH from the MBH was also increased by 20α-OHP. These results suggest that the $\text{in}$$\text{vivo}$ increase of circulating P₄ or 20α-OHP associated with the initiation of the preovulatory LH surge may play a role in regulating the timing or amplitude of this surge by directly stimulating the release of GnRH from the MBH.     

Measurement of Prostaglandins in the Cerebrospinal Fluid in Cat, Dog, and Man

Prostaglandins are involved in the modulation of various central functions (neurotransmitters and hypothalamic hormone release, thermoregulation, cerebro-vascular tone) and their levels increase in pathological situations [subarachnoid hemorrhage (SAH), stroke, convulsive disorders, etc.]. This study, using sensitive and specific antibodies, examined levels of four eicosanoids, Prostaglandins E₂ and F_2α(PGE₂, PGF_2α); and the metabolites of PGI₂, 6-keto-prostaglandin F_1α (6-keto-PGF1_α) and of thromboxane A₂, thromboxane B₂ (TxB₂), in the cerebrospinal fluid (CSF) obtained atraumatically from three species (human, canine, and feline). An assessment of the methodologic procedures (extraction and radioimmunoassay) was carried out. Human lumbar cerebrospinal fluid was shown to contain PGF_2α (15–44 pg/ml), 6-keto-PGF_1α (undetectable to 39 pg/ml), and TxB₂ (un-detectable to 28 pg/ml), whereas PGE₂ was undetectable (>18 pg) in all cases. In both animals species the eico-sanoid concentrations were 3-to 30-fold higher than humans for every prostaglandin examined. Although the prostaglandin profile for a given species remained constant (cat, PGE₂:6-keto-PGF_1α:TxB₂:PGF_2α; dog, TxB₂:PGE₂:6-keto-PGF_1α:PGF_2α), the absolute levels were found to be lower in the pentobarbital-anesthetized animals than in conscious cats. The correspondence of the prostaglandin profiles found in cerebrospinal fluid with the profiles reported in the literature in brain homogenates for the same species supports the hypothesis that cerebrospinal fluid levels of prostaglandins reflect the relative rates of synthesis in neural tissue.     

Prostaglandin removal and metabolism by isolated perfused rat lung

We have investigated the mechanism(s) involved in the removal of prostaglandins (PG) from the pulmonary circulation by the lung. Unidirectional fluxes of PG from the circulation into the lung are measured in an isolated perfused rat lung preparation. Evidence is presented which suggests that a transport system for PG exists in lung tissue. This transport system is responsible for the removal of some PG from the circulation by the lung. PGE₁ and PGF_2α are substrates for this system, whereas PGB₁, PGA₁, and 15-keto-PGF_2α are not. Since PGA₁ is a substrate for the intracellular PG dehydrogenase, the selectivity of the lung's metabolism system for circulating PG is probably due to the selectivity of the transport system for PG. It is shown that the percentage of the pulmonary arterial concentration (C_A) of PGE₁ or PGF_2α that is metabolized on passage through the pulmonary circulation decreases rapidly as C_A increases. When the lungs were perfused with PGE₁ (PGF_2α), the metabolites detected in the venous effluent were 15-keto-PGE₁ (PGF_2α) and 15-keto-13,14-dihydro-PGE₁ (PGF_2α). The time course pattern of the appearance of metabolites in the venous effluent after the initiation of a constant C_A, and the relative concentrations of the metabolites in the venous effluent, were examined as a function of C_A.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Prostaglandin stimulation of ovarian ornithine decarboxylase in vitro.

Prostaglandins E₁ and E₂ caused a 5–10 fold stimulation of ornithine decarboxylase activity in granulosa cells isolated from porcine ovarian follicles. The minimally effective concentration of prostaglandin E₂ was 10 ng/ml and the plateau of activity was reached at 500 ng/ml. Prostaglandin F_2α was ineffective. 1-Methyl,3-isobutyl-xanthine, a phosphodiesterase inhibitor, potentiated the effect of both submaximal and maximal effective doses of prostaglandin E₂, suggesting that the effect of prostaglandin E₂ is mediated by cAMP. The effect of prostaglandin E₂ was similar to that of luteinizing hormone and a cAMP analogue, 8-Bromo-cAMP.     

Prostaglandin synthesis in isolated glomeruli

Prostaglandins are thought to play an important role in the local regulation of glomerular blood flow and in the release of renin from the juxtaglomerular apparatus. We therefore examined prostaglandin synthesis by isolated rat glomeruli. Isolated glomeruli were either prelabeled with [14_C] arachidonic acid or were incubated with [14_C] arachidonic acid for the entire experimental incubation in Krebs buffer. Prostaglandin synthesis was determined by thin layer radiochromatography of acid extracts of the supernatant solutions. Indomethacin inhibitable synthesis of small amounts of 6-keto-PGF_1α, the metabolite of prostacyclin(PGI₂,) and larger amounts of PGF_2α, and PGE₂, and possibly thromboxane B₂ (TXB₂) by isolated glomeruli could be demonstrated with either prelabeling or direct incubation. These findings support the hypothesis that prostaglandins are produced within the glomerulus where they may affect local glomerular blood flow and function.     

Up- and down-regulation of membrane receptors as possible mechanisms related to the antiulcer actions of milk in rat gastric mucosa

The effects of a cow's milk diet on receptor activity and histamine metabolism in gastric glands and mucosa isolated from adult rats were examined. The milk diet was associated with (1) a decreased mobilization of H₂ receptors by histamine and (2) an increased mobilization of PGE₂ (prostaglandin E₂) receptors in mucous cells (cytoprotective effect) and parietal cells (antiacid effect). These changes are not observed for the receptors reducing pentagastrin- and histamine-induced gastric acid secretion (pancreatic/enteroglucagons, somatostatin) and stimulating mucus, bicarbonate and pepsin secretions in the rat (secretin). Cimetidine produced a parallel displacement of the histamine dose-response curve, suggesting competitive inhibition between this classical H₂ receptor antagonist and histamine in the two experimental groups. Prostaglandins and other components in milk such as EGF (epidermal growth factor) and somatostatin might therefore protect gastric mucosa by a differential control of PGE₂ and histamine H₂ receptor activity eitherdirectly (PGE₂ in milk) orindirectly (inhibition of endogeneous histamine synthesis/release and stimulation of PGE-I synthesis/release).     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10⁻⁶M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10⁻⁶M, while a lower concentration (1 × 10⁻⁷M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Solubilized myocardial adenylate cyclase: activation by prostaglandins

Prostaglandins E₁, E₂, F_2α, and F_2α activated solubilized myocardial adenylate cyclase from guinea pigs and cats. The activation did not require the presence of added phospholipids in contrast to stimulation of the solubilized enzyme by catecholamines, glucagon, and histamine. The data may provide insight into the mechanism and cellular site of action of the prostaglandins.     

Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE₄ release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 μM) prior to anti- IgE challenge inhibited the 6-keto prostaglandin F_1α (6-keto PGF_1α) release and enhanced (36%) the quantities of LTE₄ detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 μM) and the IC₅₀ values for both inhibitors were similar (0.13 μM). BAY x1005 (1 μM) produced the same percent of inhibition of anti-IgE-induced LTE₄ release in the presence or absence of indomethacin. BAY x1005 (1 μM) did not alter the 6-keto PGF_1α release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.     

Synthesis and release of prostaglandins by rat brain synaptosomal fractions.

Abstract— Particulate fractions from rat brain homogenate containing the synaptosomes synthesize and release prostaglandins F and E on aerobic incubation. The prostaglandin of the F-typc released could be further identified as proslaglandin F_2α using specific radioimmunoassays for prostaglandins F_1α, and F_2α-. The metabolite 13,14-dihydro-15-keto-prostaglandin F_2α could not be detected. The amount of prostaglandins released is dependent on incubation time and temperature as well as pH and osmolarity of the incubation medium. Total brain homogenate released more prostaglandins than purified synaptosomes per mg protein, indicating that synaptosomes are probably not a main source of prostaglandins when compared with other subcellular brain fractions. While prostaglandin synthesis was only moderately increased by the addition of the precursor fatty acid arachidonic acid, anti-inflammatory drugs like indomethacin, high concentrations of some local anaesthetics and Δ¹-tetrahydrocannabinol inhibited prostaglandin release. The neurotransmitters noradrenaline, dopamine and 5-hydroxytryptamine did not influence prostaglandin release from the synaptosomal rat brain fractions.     

Tetrandrine,a calcium antagonist of Chinese herbal origin,interacts with vascular muscle α1-adrenoceptor

The effects of tetrandrine (TET) on the contractile responses of rat aortic rings and perfused rat mesenteric arteries to phenylephrine (PE) were investigated. TET inhibited the maximal contraction to PE in a concentration-dependent manner. TET significantly inhibited the transient contraction in Ca²⁺-free medium presumably due to release of intracellular Ca²⁺ after activation of α₁-adrenoceptors. However, it caused a stronger inhibition of the sustained contraction in Ca²⁺-containing medium presumably the result of Ca²⁺ influx. TET has no inhibitory effect on caffeine-induced transient contraction. Radioligand receptor binding study using isolated dog aortic muscle membranes indicated that TET inhibited the binding of ³H-prazosin in a competitive manner, hence showing that TET interacted directly with the α₁-adrenoceptors. Thus, TET affected PE-induced aortic contractions by multiple mechanisms, inhibiting interaction of PE with α₁-adrenoceptors and interfering with PE-induced responses involving both Ca²⁺ entry and release.     

Detection of hydrogen peroxide production in the isolated rat lung using Amplex red

Abstract

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H₂O₂) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H₂O₂ using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR’s highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O₂) concentration of ventilation gas on lung rate of H₂O₂ release into the perfusate. Baseline lung rate of H₂O₂ release was 8.45?±?0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O₂ in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O₂-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H₂O₂, and that most of acute hyperoxia-enhanced lung rate of H₂O₂ release is from nonmitochondrial rather than mitochondrial sources.     

Oxytocin-induced release of prostaglandin-like substance in isolated rat uterus

Isolated rat uterine horns were incubated in van Dyke-Hastings solution containing 1.0 mU/ml of oxytocin (Pitocin) at 30°C for 60 to 90 min. The uterine strips were found to remain strongly contracted throughout the incubation period and release into the bathing fluif a prostaglandin-like activity which was detectable by bioassays on the isolated rat uterine and stomach preparations. It was also found that ethyl acetate used in the extraction procedure had a potent inhibitory effect on the responses of the rat uterine and stomach strips to oxytocin and PGF_2α.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.