Pulmonary uptake of PGE2 is inhibited by dipyridamole in rat isolated lungs

The metabolism of prostaglandin E₂ (PGE₂) is decreased by dipyridamole (20 μM) in rat isolated perfused lungs. The inhibition of the metabolism is reversible as the decreased metabolism returned to the control level when pulmonary infusion of dipyridamole was abolished. After pulmonary injection of ¹⁴C-PGE₂ (10 nmol) the radioactivity appeared more rapidly in the effluent when dipyridamole was infused into pulmonary circulation. Dipyridamole in vitro did not change the activity of 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) in the 100, 000 × g supernatant fraction of homogenized lungs. Thus, the decreased metabolism seems to be due to the inhibition of the uptake of PGE₂ into the lungs. When the rats were pretreated with dipyridamole in drinking water for one week the activity of 15-OH-PGDH in the 100, 000 × g supernatant fraction of the lungs was not changed significantly.     

Decrease of prostaglandin E2 receptor binding is accompanied by reduced antilipolytic effects of prostaglandin E2 in isolated rat adipocytes

The effect of treatment of isolated rat adipocytes with prostaglandin E2 (PGE2) on subsequent [3H]PGE2 binding was studied. In addition, the antilipolytic effects of was studied. In addition, the antilipolytic effects of PGE2, adenosine, and insulin were studied in control and PGE2-treated adipocytes. Treatment of adipocytes with PGE2 (1 microM) decreased the binding of [3H]PGE2 by 61% (from 11.0 to 4.6 fmol/10(6) cells, P less than 0.005). Scatchard analysis of the binding data demonstrated that the decrease of PGE2 receptor binding was due to a decrease in the apparent number of PGE2 receptors while the apparent receptor affinity was unaltered. Reduction of the PGE2 receptor binding was specifically regulated inasmuch as structurally related compounds such as PGF2 alpha and arachidonic acid had only minor effects on subsequent [3H]PGE2 receptor binding. Reduction of the available receptor number was associated with a significant decrease in the antilipolytic effect of PGE2 on the isoproterenol-stimulated lipolysis (P less than 0.05). The maximal antilipolytic effect of PGE2 was decreased by 45%. Desensitization of the biological effect of PGE2 (antilipolysis) was only partially specifically regulated inasmuch as the antilipolytic compound phenylisopropyladenosine also had reduced antilipolytic effect in PGE2-treated cells. However, the antilipolytic effect of insulin was similar in control and PGE2-treated cells. It was found that the PGE2-induced decrease of [3H]PGE2 receptor binding may be due to a very tight coupling between the PGE2 molecule and its specific receptor. This tight coupling may then represent an occupancy of the receptor rather than a true loss of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cigarette smoke ventilation decreases prostaglandin inactivation in rat and hamster lungs

The effects of cigarette smoke on the metabolism of exogenous PGE₂ and PGF_2α were investigated in isolated rat and hamster lungs. When isolated lungs from animals were ventilated with cigarette smoke during pulmonary infusion of 100 nmol of PGE₂ or PGF_2α, the amounts of the 15-keto-metabolites in the perfusion effluent were decreased. Pre-exposure of animals to cigarette smoke daily for 3 weeks did not change the metabolism of PGE₂ when the lungs were ventilated with air. Cigarette smoke ventilation of lungs from pre-exposed animals caused, however, a similar decrease in the metabolism of PGE₂ as in animals not previously exposed to smoke. After pulmonary injection of 10 nmol of ¹⁴C-PGE₂ the radioactivity appeared more rapidly in the effluent during cigarette smoke ventilation suggesting inhibition of the PGE₂ uptake mechanism. In rat lungs pulmonary vascular pressor responses to PGE₂ and PGF_2α were inhibited by smoke ventilation.     

Effects of arachidonic acid and prostaglandin F2 alpha in isolated rat lungs

Isolated rat lungs were ventilated and perfused by saline-Ficoll perfusate at a constant flow. The baseline perfusion pressure (PAP) correlated with the concentration of 6-keto-PGF1 alpha the stable metabolite of PGI2 (r = 0.83) and with the 6-keto-PGF1 alpha/TXB2 ratio (r = 0.82). A bolus of 10 micrograms exogenous arachidonic acid (AA) injected into the arterial cannula of the isolated lungs caused significant decrease in pulmonary vascular resistance (PVR) which was followed by a progressive increase of PVR and edema formation. Changes in perfusion pressure induced by AA injection also correlated with concentrations of the stable metabolites (6-keto-PGF1 alpha: r = -0.77, TxB2: -0.76), and their ratio: (6-keto-PGF1 alpha/TXB2: r = -0.73). Injection of 10 and 100 micrograms of PGF2 alpha into the pulmonary artery stimulated the dose-dependent production of TXB2 and 6-keto-PGF1 alpha. No significant correlations were found between the perfusion pressure (PAP) which was increased by the PGF2 alpha and the concentrations of the former stable metabolites. The results show that AA has a biphasic effect on the isolated lung vasculature even in low dose. The most potent vasoactive metabolites of cyclooxygenase, prostacyclin and thromboxane A2 influence substantially not only the basal but also the increased tone of the pulmonary vessels.     

Stimulation by prostaglandin E2 of glucagon and insulin release from isolated rat pancreas.

To ascertain whether prostaglandins (PG) may play a role in the secretion of glucagon and in an attempt to elucidate the conflicting observations on the effects of PG on insulin release, the isolated intact rat pancreas was perfused with solutions containing 1.1 x 10(-9) to 1.8 x 10(-5)m PGE2. In the presence of 5.6 mM glucose significant increments in portal venous effluent levels of glucagon and insulin were observed in response to minimal concentrations of 2.8 X 10(-8) and 1.4 X 10(-7) PGE2, respectively; a dose-response relationship was evident for both hormones at higher concentrations of PGE2. When administered over 60 seconds, 1.4 X 10(-6)M PGE2 resulted in a significant increase in glucagon levels within 24 seconds and in insulin within 48 seconds. Ten-minute perfusions of 1.4 X 10(-6)M PGE2 elicited biphasic release of both islet hormones; Phase I glucagon release preceded that of insulin. Both phases of the biphasic glucagon and insulin release which occurred in response to 15-minute perfusions of 10 mM arginine were augmented by PGE2. These observations indicate that PGE2 can evoke glucagon and insulin release at concentrations close to those observed by others in the extracts of rat pancreas. We conclude that PG may be involved in the regulation of secretion of glucagon and insulin and may mediate and/or modify the pancreatic islet hormone response to other secretagogues.     

Omega-hydroxylation of prostaglandin E1 in the isolated perfused lungs of pregnant rabbits

Cytochrome P450PG omega is induced in the rabbit lung in a gestational age-dependent manner and hydroxylates certain eicosanoids at their terminal, or omega (omega), carbon. This enzyme has been isolated from microsomal fractions and its activity has been characterized (Williams, D.E., et al., J. Biol. Chem. 259; 14600-14608, 1984). The experiments presented here examine the omega-hydroxylation activity of the intact lung during presentation of an eicosanoid substrate, prostaglandin E1 (PGE1), to the lung vasculature. Isolated, perfused lungs from three pregnant and four nonpregnant rabbits were injected with [3H]-PGE1. One-second fractions were collected from the perfusion effluent and were analyzed for metabolism of PGE1. Lungs isolated from pregnant rabbits metabolized PGE1 mainly to two polar derivatives, 20-hydroxy-PGE1 and 13,14-dihydro-15-keto-20-hydroxy-PGE1, whereas lungs from nonpregnant rabbits yielded mainly a relatively nonpolar metabolite, 13,14-dihydro-15-keto-PGE1. These metabolites were identified by coelution with standards that were generated enzymatically in vitro and whose structures were confirmed by gas chromatography/mass spectrometry (GC/MS).     

Microsomal prostaglandin E synthase-2 is not essential for in vivo prostaglandin E2 biosynthesis

Prostaglandin E₂ (PGE₂) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE₂ from the cyclooxygenase metabolite PGH₂ have been described. Here, we examine the contribution of one of these enzymes to PGE₂ production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE₂ levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE₂ synthase.     

Stimulation of prostaglandin E2 biosynthesis by estradiol in rat kidneys

The effect of estradiol administration on renal prostaglandin (PG) E2 biosynthetic activity in rats was studied. A specific radioimmunoassay for PGE2 was developed and applied in the quantitation of PGE2 biosynthesis in kidney. Conversion of exogenous arachidonic acid into PGE2 by renal microsomal fraction was assayed. Formation of PGE2 was linear in fashion up to 5 min incubation at 37 degrees C, and linear in fashion up to 3.5 mg of microsome used as enzyme source. The renal biosynthesis of PGE2 was significantly increased by estradiol treatment.     

Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E?

Variations in the spontaneous contractile activity during 6 hours following isolation of uterine horns from proestrus, metestrus and spayed rats, were explored. In estrus and metestrus preparations the contractions declined during 60 min and between 180--200 min a progressive spontaneous recovery (abolished by indomethacin) was observed up to 360 min. Uteri from proestrus and spayed animals exhibited a continuous depression without recovery during the whole experimental period. At 60 min, uterine horns from estrus animals (which showed a marked contractile decrement) released to the suspending medium significantly less prostaglandin E-like material than at 360 min, i.e. when contractions had almost completely recovered. No modification in the amount of prostaglandin F-like material was detected accompanying these spontaneous contractile variations. In the spayed group at 60 min of functioning (i.e. when the contractile impairment was significantly smaller than at a later time) the release of PGE was greater than at 360 min. These findings suggest a possible control of rat uterine contractions by PGE, rather than by PGF.     

Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E?

Variations in the spontaneous contractile activity during 6 hours following isolation of uterine horns from proestrus, metestrus and spayed rats, were explored. In estrus and metestrus preparations the contractions declined during 60 min and between 180–200 min a progressive spontaneous recovery (abolished by indomethacin) was observed up to 360 min. Uteri from proestrus and spayed animals exhibited a continuous depression without recovery during the whole experimental period. At 60 min, uterine horns from estrus animals (which showed a marked contractile decrement) released to the suspending medium significantly less prostaglandin E-like material than at 360 min, i.e. when contractions had almost completely recovered. No modification in the amount of prostaglandin F-like material was detected accompanying these spontaneous contractile variations. In the spayed group at 60 min of functioning (i.e. when the contractile impairment was significantly smaller than at a later time) the release of PGE was greater than at 360 min. These findings suggest a possible control of rat uterine contractions by PGE, rather than by PGF.     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

Mechanism of action of aldosterone release by prostaglandin E in isolated rat adrenal glomerulosa cells

The effect of prostaglandin E (PGE) on aldosterone release and the mechanism of action of PGE in mediating the release of aldosterone were studied using isolated rat glomerulosa cells. PGE1 stimulated aldosterone release in a dose-dependent fashion at concentrations between 10(-8) and 10(-6) M and caused approximately a two-fold increase over the basal aldosterone level at 10(-6) M. A significant and dose-dependent increase in cAMP production was also produced by PGE1 at concentrations greater than 10(-8) M. Aldosterone release induced by 10(-7) M or 10(-6) M PGE2 was significantly reduced by a competitive receptor blocking PG-antagonist, SC 19220 (10(-7) M), but not affected by (Sar1, Ileu8)-angiotensin-II (A-II), a competitive inhibitor of A-II. PGE-stimulated aldosterone release was almost completely abolished by depleting the extracellular Ca2+ by EGTA, or by verapamil, a Ca2+-channel blocker or W-7, a calmodulin inhibitor. These findings suggest that PGE stimulates aldosterone release through the membrane receptor binding and activation of adenylate cyclase and that Ca2+-calmodulin system plays an essential role in mediating the steroidogenic action of PGE in the adrenal glomerulosa cells. However, the physiological significance of PGE in the regulation of aldosterone secretion remains to be elucidated.     

Supercritical fluid-aerosolized vitamin E pretreatment decreases leak in isolated oxidant-perfused rat lungs

Hybertson, Brooks M., Roger P. Kitlowski, Eric K. Jepson,and John E. Repine. Supercritical fluid-aerosolized vitamin Epretreatment decreases leak in isolated oxidant-perfused rat lungs.J. Appl. Physiol. 84(1): 263-268, 1998.We hypothesized that direct pulmonary administration ofsupercritical fluid-aerosolized (SFA) vitamin E would decrease acuteoxidative lung injury. We previously reported that rapid expansion ofsupercritical CO₂ formedrespirable particles of vitamin E and that administering SFA vitamin Eto rats increased lung vitamin E levels and decreased neutrophil-mediated lung leak. In the present investigation, we foundthat pretreatment with SFA vitamin E protected isolated rat lungsagainst the oxidant-induced lung leak caused by perfusion with xanthineoxidase (XO) and purine, an enzyme system that generates superoxideanion () and hydrogenperoxide. SFA vitamin E droplets were 0.7-3 µm in diameter, andinhalation of the airborne droplets for 30 min deposited ~55 µg ofvitamin E in rat lungs. Isolated rat lungs perfused with XO (0.02 U/ml) and purine (10 mM) gained more weight (1.75 ± 0.12 g,n = 8), retained more Ficoll(11.5 ± 1.2 mg/left lung,n = 7), and accumulated more Ficoll intheir lung lavages (700 ± 146 µg/ml,n = 8) than control lungs [0.25 ± 0.06 g (n = 10), 6.2 ± 1.2 mg/left lung (n = 9), and 141 ± 31 µg/ml (n = 8), respectively,P < 0.05]. In contrast,isolated lungs from rats that were pretreated with SFA vitamin E haddecreased (P < 0.05) weight gains(0.32 ± 0.06 g, n = 7), Ficollretentions (3.3 ± 1.1 mg/left lung,n = 7), and lung lavage Ficollconcentrations (91 ± 26 µg/ml,n = 6) after perfusion with XO andpurine compared with isolated lungs from control rats perfused with XOand purine. This protective effect was not observed in rat lungs givensham treatments (CO₂ alone orvitamin E acetate aerosolized with supercriticalCO₂). Our results suggest thatdirect pulmonary supplementation of vitamin E decreases susceptibilityto vascular leakage caused by XO-derived oxidants.

     

Release of prostaglandin E2 and thromboxane B2 by mixed isolated human lung cells

Human lung specimens were minced and treated for 30 min with collagenase (1 mg ml-1) and DNase (0.1 mg ml-1) to obtain a suspension of viable (approximately 80%) and metabolically active lung cells (5 x 10(6) cells per gram of tissue). Treatment of these mixed lung cells with bradykinin (1.25 x 10(-6) to 1 x 10(-5) M) and f-Met-Leu-Phe (f-MLP; 1 x 10(-8) to 5 x 10(-6) M) did not stimulate to a substantial extent the release of prostaglandins and thromboxanes (measured with novel Enzyme Immunoassays). The only concentration of PAF that stimulated significantly the release of icosanoids from lung cells was 5 x 10(-7) M. Phorbol myristate (PMA; 5 x 10(-8) to 2 x 10(-6) M) and ionophore a-21387 (2.5 x 10(-6) to 2 x 10(-5) M) strongly stimulated the release of prostaglandins and thromboxanes by dispersed human lung cells. These findings support previous observations showing that human lungs have the enzymes necessary for the synthesis and release of prostaglandins and thromboxanes but stimulation of the release of these mediators is not obtained with the hormonal stimuli that are active in guinea pigs. Studies in progress will purify the cell populations and characterize the cells responsible for the release of these icosanoids.     

Stimulation of CFU-f formation by prostaglandin E2 is mediated in part by its degradation product, prostaglandin A2

Prostaglandins (PG) of the E series are known to rapidly undergo non-enzymatic dehydration in culture medium containing serum albumin to produce the cyclopentenone PGs of the A series. We investigated the actions of PGA1 and A2 in the in vitro calcifying fibroblastic-colony forming unit assay which can partially mimic the in vivo anabolic effects of PGE2. It was found that PGA1 and A2 both stimulated colony formation in a dose-dependent manner with a maximum at 10(-6) M and to a similar degree to PGE2. In contrast to PGE2, PGA1 and PGA2 both caused an inhibition of cAMP accumulation. Furthermore, the addition of protein kinase A inhibitors, H8 and H89, had no significant effect on the stimulation of colony number by PGE2. These data suggest that (a) the bone anabolic effects of PGE1 and E2 are, in part at least, mediated by their dehydration products PGA1 and A2 and (b) that they are mediated via pathways not necessarily involving the cAMP/protein kinase A cascade.     

Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.