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. PGF₂ 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 PGF₂ also applied to PGE₂, but did not affect PGE₁ relaxations or histamine contractions. These findings suggest that PGE₂ can stimulate either PGF₂ or PGE₁ receptors of isolated human bronchial muscle.     

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.     

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.     

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.     

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.     

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.     

Interactions of Malondialdehyde and 4-Hydroxynonenal with Rat Liver Plasma Membranes and their Effect on Binding of Prostaglandin E2 by Specific Receptors

We investigated effect of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydrox-ynonenal (HNE) on prostaglandin (PG) E₂ receptors of liver plasma membranes. The modification of the membranes by MDA diminished PGE₂ binding, decreasing receptor affinity for PGE₂ and receptor density whereas HNE increased PGE₂ binding, enhanced receptor density but did not changed receptor affinity. ESR study showed the decrease of the whole membrane fluidity after modification by MDA whereas HNE lowered membrane fluidity only in the internal zone of lipid bilayer and increased it in the surface area. The possible effects of membrane changes caused by MDA and HNE on PGE₂ receptor parameters are discussed.     

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.).     

Hemichannels Formed by Connexin 43 Play an Important Role in the Release of Prostaglandin E2 by Osteocytes in Response to Mechanical Strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E₂ (PGE₂) release, and intercellular coupling, and PGE₂ is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE₂ in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE₂ per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE₂ release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE₂ increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE₂ in response to FFSS.     

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.     

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.     

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.     

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.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

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.     

Chronobiological Studies on the Hypotensive Effect of Prostaglandin E2 and Arachidonic Acid in the Rat

The present study was designed to determine whether biological rhythm variations could be detected in the hypotensive action of prostaglandin E₂ (PGE₂) and arachidonic acid (AA) in normal rats. Doses of 1.0 μg kg^-1 of PGE₂ or 0.5 mg kg^-1 of AA were administered to pentobarbital-anesthetized rats at 6 times of the day. Maximal reduction of systolic and diastolic blood pressures was obtained when PGE₂ or AA were administered to rats between 0930 and 1200. The lowest falls in blood pressure were found when the same doses of the two substances were injected between 0300 and 0500. Mechanisms to explain these circadian variations are suggested.     

Oxidant Stress and Glomerular Prostanoid Production: Influence of Angiotensin Converting Enzyme Inhibition

1. The effect of H₂O₂ (4.7 × 10^-9 4.7 × 10^-3M) on prostanoid production by isolated glomeruli from normotensive (WKY) and, spontaneously hypertensive rats (SHR) has been studied.

2. Oxidant stress significantly increased synthesis of prostaglandin E₂(PGE₂), I₂(PGI₂)and thromboxane A₂ (TxA₂) by glomeruli from both strains whereas the ratio (PGE₂ + PGI₂)/TxA₂ increased in only SHR.

3. Pre-incubation of glomeruli with the angiotensin converting enzyme inhibitors captopril or lisinopril, had virtually no effect on H₂O₂-induced synthesis of individual prostanoids nor on the ratio (PGE₂ + PGI₂)/TA₂ by glomeruli from either WKY or SHR.

4. The findings suggest that H₂O₂-induced changes in glomerular function may be mediated, in part, by PGs but fail to support the suggestion that the ability of ACEI to protect glomeruli from H₂O₂-induced damage is determined by PGs.     

Effects of dietary saturated, n-6 and n-3 polyunsaturated fats on blood pressure and prostaglandins outflow from perfused mesenteric vascular bed in rats

Effects of the dietary administration of saturated fat and of n-6 and n-3 polyunsaturates on blood pressure, prostaglandin metabolism in small vessels, tissue fatty acid distribution and urinary PGE₂ excretion were compared. Rats were divided into three groups. Diets contained 10% hydrogenated cocunut oil (HCO), 10% safflower oil (SFO) or 10% cod liver oil (CLO) added to a basic fat free diet for 10 weeks. Systolic blood pressure was increased in the CLO group animals. Urinary PGE₂ excretion was decreased in the HCO and CLO groups as compared to that in the SFO group animals. PGE₂, 6-keto-PGF₁ and thromboxane (Tx) B₂ outflow from isolated perfused mesenteric arterial beds were extremely decreased in the CLO group animals, and to a lesser extent in the HCO group as compared to the SFO animals. In the tissue phospholipid, 20:3n−9/20:4n−6 ratios were increased in the HCO group indicating essential fatty acid deficiency, and n-6 and n-3 polyunsaturates were elevated in the SFO and the CLO group animals respectively. Arachidonic acid concentration was highest in the SFO group, while there was no significant differences between the HCO and the CLO group. These results suggest that dietary fatty acid manipulation effects urinary PGE₂ excretion and PGI₂, PGE₂ and TxA₂ synthesis in mesenteric arterial beds and also changes the tissue fatty acid distribution. Furthermore, n-3 polyunsaturates caused an extreme reduction of 2-series PGs synthesis in small resistance vessels.