The effects of some natural prostaglandins on isolated human circular bronchial muscle.

PGE1 relaxed isolated human circular bronchial muscle over a wide concentration range as did isoprenaline. Surprisingly isoprenaline was more potent than PGE1. PGF2alpha weakly contracted this muscle preparation whereas histamine was more potent. PGE2, however, produced paradoxical results, relaxing some tissues and contracting others, always in a concentration-related manner irrespective of tissue tone. In preparations that contracted to PGE2, tachyphylaxis induced to PGF2alpha also applied to PGE2, but did not affect PGE1 relaxations of histamine contractions. These findings suggest that pge2 can stimulate either PGF2alpha or PGE1 receptors of isolated human bronchial muscle.     

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 1/20 of that of bradykinin (BK) on a weight basis. The activity of PGF₁ and PGF₂ was only 1/20 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 1/3–1/8 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 anti-histamine, pyrilamine (2.5 mg/kg, i.v.).     

Influence of dietary vitamin E on prostaglandin biosynthesis in rat blood

A vitamin E (-tocopherol) deficient diet stimulated prostaglandin biosynthesis in coagulating rat blood. Prostaglandins were extracted from serum, purified and bioassayed. The identity of prostaglandin E₂ was confirmed by gas chromatography-mass spectrometry. Withholding vitamin E from the diet caused a marked increase in PGE₂ and a lesser increase in PGF₂ production in serum. In rats maintained on diets containing different concentrations of vitamin E, serum concentrations of PGE₂ and PGF₂ were inversely related to serum concentrations of -tocopherol. These data suggest that in vivo -tocopherol inhibits the endogenous conversion of arachidonic acid into PGE₂ and PGF₂. The possibility that -tocopherol may inhibit the formation of endoperoxide intermediates of PGE₂ and PGF₂ biosynthesis and subsequent induction of platelet aggregation is discussed.     

Influence of prostaglandins on the lipid transfer between human high density and low density lipoproteins

Prostaglandin (PG) E₁ was demonstrated to stimulate the transfer of phosphatidylcholine and cholesterol esters from human high density lipoproteins (HDL₃) to low density lipoproteins (LDL). The enhancement effect of PGE₁, on the interlipoprotein lipid transfer was seen at low PG concentrations under conditions of spontaneous exchange as well as in the presence of lipoprotein-depleted plasma, or partly purified plasma lipid exchange protein. PGE₂ and PGF₂ showed no significant influence on the interlipoprotein lipid transfer. Evidence is presented suggesting that the PGE₁-induced stimulation of interlipoprotein lipid exchange results in enhancement of LCAT-catalyzed cholesterol esterification in plasma. It is proposed that the effect of PGE₁ is due to the previously described PGE₁-induced reorganization of the HDL surface [(1984) FEBS Lett. 173, 291-293] and that PG-lipoprotein interaction may be a factor regulating cholesterol homeostasis.     

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>PGF₂=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-PGF₂ 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-PGF₂ 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-P GE₂>16 phenoxy-17,18,19,20-tetranor-PGE₂=PGE₂=PGE₁=(15S)-15-methyl-PGE₂>PGF₂.     

Arachidonic acid metabolism in thrombocytes and vascular tissues of turkeys

Turkeys are hypertensive compared to mammals of similar size. In vitro synthesis of thrombocyte thromboxane B₂ (TxB₂), 12L-hydroxy-5, 8, 10 heptadecatrienoic acid (HHT), 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) and aortic prostaglandin (PG) production was studied in one to ten month old domestic white turkeys. Compared to normal human platelets, TxB₂ production was increased (55.4 vs. 31.4%) and HETE production was markedly reduced (6.5 vs. 34.6%) in control thrombocytes. Similar to human platelets in which cyclooxygenase inhibition with aspirin results in an increase in HETE production, block of the thrombocyte enzyme with aspirin doubled the production of HETE. In vitro conversion of radiolabeled arachidonic acid (AA) showed that the primary PG produced by turkey aorta was PGE₂. A 6-keto immunoreactive PG was present which comigrated with authentic 6-keto PGF₁, but failure of the aortic supernatant to inhibit adenosine diphosphate or AA induced platelet aggregation suggested that PGI₂ was not produced. The vasodepressor potency of PGE₁, PGE₂ and PGI₂ was altered in awake turkeys with PGE₁ and PGE₂ having five times the hypotensive effect as PGI₂. In addition, conversion of AA to PGE₂ by aorta in one month turkeys was greater (17.3 vs. 9.2%) than in ten month old turkeys. Systemic arterial pressure was increased in the ten month old turkeys (188 mmHg) compared to one month old turkeys (143 mmHg). Thus, both vascular AA metabolism and the vasodepressor potencies of PGE₂ and PGI₂ are altered and the activity of the lipoxygenase pathway in thrombocytes is limited in the turkey.     

Differential production of prostaglandins D2 and E2 and of unusual prostanoid by chick spinal cord and meninges in vitro

In order to specify the source of locally synthesized prostaglandin (PG) E₂ which is able to saturate the large class of low affinity PGE₂ receptors in chick spinal cord, bioconversion of [1-¹⁴C]arachidonic acid into prostanoids was studied in homogenates of chick spinal cord and meninges first without addition of exogenous glutathione (GSH). Homogenates of spinal cord produced ¹⁴C-labeled PGE₂, PGD₂ and PGF₂. Homogenates of meninges accumulated much larger amounts of [¹⁴C]PGE₂ than spinal cord and surprisingly a ¹⁴C-labeled arachidonate metabolite referred to as compound Y. Compound Y generation, which was inhibited by indomethacin and enhanced by esculetin, was therefore mediated through the cyclooxygenase pathway. The fact that no labeled compound Y was detected in homogenates incubated with [³H]PGD₂ or [³H]PGE₂ indicated that compound Y was not degradation product of PG_s. Secondly, after addition of exogenous GSH, ¹⁴C-labeled compound Y was totally converted into [¹⁴C]PGE₂. The compound Y which is converted into PGF_s after a strong reduction with NaBH₄ and into PGE₂ after a mild reduction with GSH-hemin system or SnCl₂ was therefore assumed to be a 15 hydroperoxy-PGE₂ (15 HP-PGE₂). These results suggest that PGE₂ can be synthesized in meninges either by the classical isomerization of PGH₂ or by isomerization of PGG₂ followed by a GSH-sensitive reaction.     

Effect of prostaglandins on protein, RNA, DNA and collagen synthesis in experimental wounds

The effect of exogenous prostaglandins E₁, E₂ and F₂ (PGE₁, PGE₂ and PGF₂) on ³H-leucine, ³H-uridine, ³H-thymidine and ³H-proline incorporation in experimental cutaneous wounds has been studied in rats.

Prostaglandins E₁ and E₂ markedly stimulate the incorporation of these tritiated precursors, into protein, RNA, DNA and collagen synthesis, whereas F₂ inhibits it. All tested prostaglandins exhibit their maximum effect within the first hours following administration. Most active is PGE₁. These observations indicate that application of prostaglandins significantly stimulate incorporation with protein, RNA, DNA and collagen synthesis in the skin of wounded rats and thus, may play a role in epidermal cell growth and division as well as in scar-forming tissue.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

Angiotensin II and norepinephrine after indomethacin in isolated perfused canine kidneys. Tachyphylaxis vs. modulator effect of prostaglandins

High levels of radioimmunoassayable PGE₂ were measured in the perfusate of isolated kidneys. Indomethacin inhibited PGE₂ release in this system. Small reductions in the pressor effects of norepinephrine (NE) were associated with increasing perfusate levels of PGE₂; a large increase in the pressor effect of NE followed additions of indomethacin and reductions in perfusate PGE₂ levels. A marked reduction in pressor responsiveness to angiotensin II (AII) was measured in the isolated kidney which could not be prevented or reversed by indomethacin. It is believed that tachyphylaxis was responsible for the marked reduction in pressor responsiveness to AII and that this is independent of alterations in prostaglandin metabolism. However prostaglandins appeared to modulate the pressor effects of AII as they did NE in the isolated perfused kidney.     

Human isolated bronchial muscle preparations from asthmatic patients: effects of indomethacin and contractile agonists

Airway reactivity to histamine was determined in a group of non-asthmatic and asthmatic patients prior to thoracotomy. The latter group was more reactive to histamine provocation than the former (PC40: 28.40 +/- 6.27 mg/ml and 1.15 +/- 0.19 mg/ml, respectively). Subsequent to the surgical intervention, isolated human bronchial muscle preparations were obtained from both groups (15 non-asthmatic and 5 asthmatic subjects). Histamine concentration-effect curves were generated both in the absence and in the presence of indomethacin (1.7 microM; 30 min). Neither the basal tone nor the histamine response and sensitivity of the preparations were altered by the antiinflammatory drug. In bronchial preparations from one asthmatic subject, indomethacin significantly reduced the prostaglandin production during histamine contraction. Prostaglandin E2 and F2 alpha contracted isolated human bronchial muscle preparations from these asthmatic individuals. These data suggest that endogenous prostaglandins may not regulate the contractile response to histamine in vitro.     

Binding of prostacyclin by plasma glycoproteins

The binding of prostacyclin (PGI₂) to plasma proteins and the resulting increase in PGI₂ stability was investigated. Using gel filtration to separate bound and free PGI₂, we have found that Cohn Fraction VI can bind PGI₂, and retard its hydrolysis to 6-keto-PGF₁ (6KPGF₁). The biological activity of the bound PGI₂ correlated well with the quantity of bound PGI₂, measured as 6KPGF₁ by RIA. Fraction VI bound a greater percentage of PGI₂ than the other eicosanoids tested (i.e., PGI₂ > TXB₂ > LTB₄ > PGE₁ > PGF₂). The PGI₂ binding activity of Fraction VI was lost after neuraminidase treatment. Our data suggest that Fraction VI glycoproteins may play an important role in the binding and stabilization of PGI₂ by plasma proteins.     

Fibroblastic regulation of osteoblast function by prostaglandins

The effects of osteogenic inhibitory factors secreted by human periodontal ligament fibroblasts were studied in rat bone marrow stromal cell cultures. Serum-free conditioned medium from cultures of fibroblasts strongly depressed formation of mineralized tissue by bone marrow cell cultures. The inhibitory activity was reduced by treatment of fibroblast cultures with indomethacin or by pretreatment of conditioned medium with specific antibodies to prostaglandins (PGs) E₂ and F₂. Passage of conditioned medium over octadecyl columns enriched PGs four-fold and significantly increased inhibitory activity. Inhibition of mineralization was replicated by treatment of bone-cell cultures with PGs B₂, D₂, E₂, and I₂ at concentrations of 350 ng/ml to 350 pg/ml. All combinations of these agents were inhibitory but PGE₂ and PGF₂ exhibited the greatest inhibition at low concentrations (350 pg/ml). These experiments indicate that fibroblasts secrete PGs which can inhibit bone formation, and this may be one mechanism whereby fibroblasts can modulate osteogenesis at the interfaces of soft and mineralizing connective tissues.     

Relaxation of isolated human pulmonary muscle preparations with prostacyclin (PGI2) and its analogs

The effects of PGI₂ and two analogs Iloprost and ZK 96480 were examined on isolated human pulmonary muscle preparations. High concentrations of these agents reduced the basal tone in all types of preparations. In addition, they relaxed tissues which had been maximally contracted with histamine (50 μM). PGI₂ was more potent on pulmonary arterial muscle preparations (pD₂ value : 6.33, n = 3) than on bronchial muscles. The relaxations induced by PGI₂ in bronchial preparations were quite variable, that is, some tissues relaxed while others did not. The analogs also relaxed arterial preparations and the pD₂ values were approximately the same (Iloprost : 7.42, n = 4 and ZK 96480 : 7.48, n = 4). The isolated human pulmonary vascular preparations were approximately 10-fold more sensitive to the analogs than bronchial muscle preparations. In bronchial tissues we noted that the PGI₂ relaxant effect was spontaneously reversed with time, an activity not observed with both analogs. A pretreatment of the bronchial tissues with indomethacin (1.7 μM) did not reduce the variations observed with PGI₂ nor modify the transient relaxation observed with this agent. These data demonstrate that vascular tissues from the human lung are considerably more sensitive to these relaxant agonists than bronchial preparations.     

Biphasic effect of sodium fluoride and guanyl nucleotides on binding to prostaglandin E2 receptors in rat epididymal adipocyte membranes

Both NaCl and NaF promoted PGE₂ binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the ‘salt effect’ of sodium on PGE₂ binding, the effects of Mg²⁺ and guanyl nucleotides on PGE₂ binding in the presence of NaCl or NaF were compared. Mg²⁺ decreased PGE₂ binding; high NaF concentration abolished this inhibition, while increased NaCl concentratipns did not affect the Mg²⁺ inhibition. In the presence of Mg²⁺ the effects of NaCl and NaF were additive. The enhancement of PGE₂ binding by fluoride, unlike sodium, was dependent on the presence of Mg²⁺. Induction of the membranes with GDPβS, Gpp(NH)p, GTP or GTPγS increased PGE, binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE₂ binding at low NaF concentrations and inhibition of PGE₂ binding at higjh NaF concentrations. No changes in the stimulatory action of NaCl on PGE₂ binding were observed in the simulatenous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE₂ binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE₂ binding. The implications of these findings are discussed.     

Differential effects of prostaglandins and arachidonic acid on gastric circulation and oxygen consumption

Experiments were carried out on anesthetized dogs to compare the effects of prostaglandin E₂ (PGE₂), prostacyclin (PGI₂) and arachidonic acid (AA) administered intraarterially on gastric blood flow and oxygen consumption during constant arterial pressure perfusion and constant flow perfusion of the stomach. Both PGE₂ and PGI₂ increased total blood flow and oxygen consumption both in the resting stomach and following histamine stimulation although the effects of PGE₂ on the oxygen consumption in stimulated stomach were not statistically significant. On the contrary, AA decreased both gastric blood flow and oxygen consumption in the histamine stimulated stomach. To determine if these compounds can influence gastric oxygen consumption independently of their effects on blood flow, the experiments with constant flow perfusion were performed. Both PGE₂ and PGI₂ decreased both the perfusion pressure and oxygen consumption in the resting as well as in the histamine-stimulated stomach whereas AA increased perfusion pressure and decreased oxygen consumption during histamine administration. Effects of AA were blocked by indomethacin suggesting that not AA itself but some of its metabolites, most likely thromboxanes were responsible for the hemodynamic and metabolic changes resulting from the contraction of gastric arterioles and precapillary sphincters. On the contrary, both PGE₂ and PGI₂ caused gastric hyperemia and an increase in oxygen consumption in the resting stomach, but decreased the latter parameter in the stimulated stomach, most probably as a result of secretory inhibition overcoming direct vascular effects of these compounds.     

The effect of prostaglandin F2β on expiratory flow rates

Prostaglandin F_2β (PGF_2β), a stereoisomer of F₂ was administered by ultrasonic nebulization to eight patients with bronchial asthma and four normal subjects in increasing doses up to a 200 μg maximum dose. Maximum expiratory flow (MEF) and forced vital capacity (FVC) were analyzed at 5, 15, 30, 60 and 120 minutes after administration of aerosol.

All expiratory flow rates were reduced after 5 minutes. Some increase in terminal flow rates was observed after 60 minutes. We conclude that PGF_2β is not an effective bronchodilator at this dose level.     

Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F₂ (PGF₂) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10⁶ cells, and PGF₂ increased from a normal value of 31 pgrams to 482 pgrams/10⁶ cells. In CLL lymphocytes, levels of PGE and PGF₂ were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF₂ fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3′:5′-adenosine monophosphate (c-AMP) level was constantly low, and the initial high level of PGF₂ fluctuated in relation to similar oscillations of cyclic 3′:5′-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF₂, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

The prostaglandins of articular cartilage I. Correlates of prostaglandin activity in a chondrocyte culture system

Suspensions of aggregated chondrocytes display active prostaglandin (PG) production. Radioimmunoassay of culture media and thin layer chromatographic analysis suggests that PGE₂ is the primary PG synthesized. In order of decreasing concentration, the following PG were tentatively identified; PGE> PGI> PGA + PGB PGF₁₊₂ > T×B. An inverse logarithmic relationship was identified between PG synthesis and cells cultured at densities of 1.5 to 7.5 × 10⁶ cells/ml. Little or no change in the PG distribution profile was seen at these high cell densities. Maximum PG synthesis was attained after 36 hours of incubation with persistence of high synthetic levels up to 48 hours. PGE₂ production measured at various post-isolation intervals indicated an initial high rate of synthesis during the first 4 hours which decreased with time up to 24 hours. Cartilage explant organ cultures demonstrated a similar level of PG synthesis suggesting minimal effect of matrix on cellular PG production. Indomethacin (5 μg/ml) inhibited PG synthesis by 70% within 4 hours and 85% after 24 hours of exposure. Arachidonic acid supplementation (10 μM) stimulated PG synthesis by 300%.     

Actions of endothelin-1 on prostaglandin production by gestational tissues

Endothelin-1 (10⁻¹¹M-10⁻⁷M) was incubated with human umbilical vein endothelial cells and cells derived from amnion and decidua and prostaglandin production was determined. The rates of biosynthesis of 6-keto-prostaglandin F₁ (6-keto-PGF₁) and prostaglandin E₂ (PGE₂) by endothelial cells were increased significantly by treatment with endothelin-1. Amnion cell PGE₂ production was reduced significantly by endothelin-1 treatment whereas decidual PGE₂ and prostaglandin F₂ production was unaffected by this treatment. Thus, it is possible that endothelins may play a part in the regulation of uteroplacental hemodynamics and the mechanisms of parturition.