Actions of prostaglandins E1 and F2alpha on isolated intrapulmonary vascular smooth muscle.

The effects of PGE1 and PGF2alpha were studied on isolated strips of intrapulmonary arteries and veins from dog, sheep, swine and man. PGF2alpha contracted human arterial strips in a dose-dependent fashion, relaxed slightly sheep arteries and had no effect on dog arteries. Canine, sheep and human venous strips were contracted by PGF2alpha. PGE1 relaxed slightly both veins and arteries from dog and sheep. Human arteries usually contracted slightly and human veins usually relaxed slightly to PGE1. In a limited number of experiments, swine arteries and veins failed to respond to PGF2alpha or PGE1. All the vascular strips contracted well when exposed to NE. These results suggest that the responses of intrapulmonary vessels to PGF2alpha and PGE1 are species-dependent. PGF2alpha generally exhibits a contractile action, especially on veins. PGE1 usually relaxes intrapulmonary vessels. With regard to vessels from man, PGF2alpha is a powerful stimulant while PGE1 produces only small, variable effects.     

Synthesis of thromboxane B2 and 6-keto-prostaglandin F1 alpha by bovine gastric mucosal and muscle microsomes

Bovine gastric mucosal and muscle microsomes synthesize prostaglandins and thromboxane b/ (TXB2) from aratchidonic acid (AA). TXB2 and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) were the majro products synthesized by pylorus, body, and cardiac region of the gastric mucosa. Gastric muscle mainly synthesized 6-keto-PGF1 alpha. TXB2 and 6-keto-PGF1 alpha synthesis occurs at an appreciable rate from endogenous precursors but more rapidly with added arachidonate. Prostaglandins E2, F2 alpha and D2 were synthesized in smaller amounts under the conditions studied.     

Rat osteogenic sarcoma cells: comparison of the effects of prostaglandins E1, E2, I2 (prostacyclin), 6-keto F1alpha and thromboxane B2 on cyclic AMP production and adenylate cyclase activity.

The effects of prostaglandins (PG's) E₁, E₂, I₂ (prostacyclin), 6-keto F_1α and thromboxane (Tx) B₂ were compared in freshly isolated cells from a rat osteogenic sarcoma and in membranes from cultured cells of the same tumour. Cyclic AMP production was measured in cells and adenylate cyclase activity was measured in cell membranes. In both systems PGI₂ was less potent than either of the PGE's, and both TxB₂ and 6-keto PGF_1α were only weak agonists. These effects on bone-derived cells suggest that PGI₂ is unlikely to be a potent bone resorbing agent. Resitance experiments suggested that all the PG's share the same receptor site which appears distinct from the site of action of parathyroid hormone.     

Specific receptors for thromboxane A2 in cultured vascular smooth muscle cells of rat aorta

The specific binding site for thromboxane A2 (TXA2) was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. [3H]SQ29,548, a potent and selective TXA2 receptor antagonist, displayed high-affinity and specificity, as well as saturable and displaceable binding to rat VSMC in culture. Scatchard analysis of equilibrium binding at 24 degrees C revealed a single class of binding sites with a Kd of 1.7 nM and a Bmax of 8.0 fmol/10(6) cells. A series of TXA2 receptor antagonists completely suppressed [3H]SQ29,548 binding to rat VSMC, and the rank order of their inhibitory potencies (Ki) correlated well with the potencies for suppression of the U46619-induced contraction of rat thoracic aorta. These results suggest that specific binding sites for [3H]SQ29,548 represent the TXA2 receptor in rat VSMC.     

Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta

Prostaglandin synthesis in aortic smooth muscle cells originating from healthy an atherosclerotic rabbits was studied by incubating [14C]arachidonic acid with intact confluent cells and cell homogenates. In spite of a reduced 6-keto prostaglandin F1 alpha formation, no potentiating effect on the prostaglandin E2 generation occurred. Indeed, both cyclooxygenase and prostaglandin I2 synthetase activities appear to be reduced. These results suggest that an impaired arachidonic acid utilisation in aortic smooth muscle cells may be involved in the course of the atherosclerotic process.     

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells

We have compared the production of prostaglandins in fibroblast-like cells and endothelial cells in culture. Of the fibroblasts studied $\text{10T}\text{1}\text{2}$, SHE, BP6T and KD produce significant amounts of PGI₂, PGE₂ and PGF_2F2 under optimal culture conditions, but only 3T3 and BHK produce TxA₂ in addition to PGI₂. The adult bovine aortic endothelial cells (ABAE) and fetal bovine heart endothelium (FBHE) synthesise PGI₂ but not TxA₂, either from endogenous or exogenous substrates. Both cultured endothelial cells and fibroblasts apparently lack 15-hydroxyprostaglandin dehydrogenase pathway and the ability to convert 6-Keto PGF_1α into 6-Keto PGE₁. PGI₂ production by ABAE was 3–5 times that of FBHE, about twice that of SHE cells and 6–8 times that of $\text{10T}\text{1}\text{2}$ or BP6T cells. Supernatants or media obtained from these cells inhibited aggregation of human platelet-rich plasma, a known biological effect of PGI₂. This effect was abolished when cell monolayers were preincubated with indomethacin or tranylcypromine. RIA and chromatographic data of 6-Keto PGF_1α from these experiments confirmed that the inhibition of platelet aggregation was due to the formation of PGI₂. The production of all prostanoids by endothelial cells or fibroflasts was significantly higher during the exponential phase of growth as compared to confluent monolayers. We propose that fibroblasts $\text{10T}\text{1}\text{2}$ or SHE can serve as useful experimental models for the study of metabolism and transport of PGI₂ and/or TxA₂ in cells of nonendothelial nature.     

Norepinephrine-induced down regulation of alpha 1 adrenergic receptors in cultured rabbit aorta smooth muscle cells

Drug-induced refractoriness of alpha-adrenergic receptor-mediated vasoconstriction may be a clinically important phenomenon. We have investigated the possible molecular mechanisms underlying this phenomenon in cultured vascular smooth muscle cells derived from the rabbit aorta. alpha 1-Adrenergic receptors were identified in membranes prepared from these cells by [125I]HEAT binding. The radioligand bound to a high affinity site (Kd = 140 pM) in a saturable fashion (202 fmol/mg protein). Adrenergic agonists and antagonists competed for binding of [125I]HEAT with the expected order of potency for an alpha 1-receptor, (-)epinephrine greater than or equal to (-) norepinephrine greater than (+)epinephrine greater than isoproterenol and prazosin greater than phentolamine greater than yohimbine. Exposure of cells for 26 hours to 10 microM norepinephrine resulted in a 70% decrease in the number of alpha 1-receptors as measured by [125I]HEAT binding without any significant change in the affinity of the receptor for the ligand. When the alpha-receptors were blocked with 10 microM phentolamine the loss of receptors induced by norepinephrine was completely prevented. Similar down-regulation of the [125I]HEAT binding sites was observed when the alpha 1-agonist phenylephrine was used instead of norepinephrine. It is concluded that alpha-agonists induce down-regulation of aortic smooth muscle alpha 1-receptors. This reduction of alpha-receptors could be important in the mechanisms by which vascular smooth muscle develops refractoriness to alpha-adrenergic stimulation.     

Thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin F2 alpha production by contracting pregnant rate uteri in vitro

While prostaglandin production by uterine tissue has been shown to be involved in the contractile mechanism of this tissue, less attention has focused upon the involvement of other prostanoids. We have simultaneously measured in vitro isometric contractility of pregnant rat uteri with the release of prostaglandin F2 alpha (PGF), 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and thromboxane B2 (TXB2) into the bathing medium under various conditions. Frequency of uterine contractions and integrated contractile force (ICF) increased from 15 days of gestation and peaked at the time of parturition. Activity was generally greatest during the first 15 min of incubation except during parturition and on Day 1 postpartum when the uterine segment remained active for 1 h experimental period. Indomethacin (INDO) significantly reduced contractile activity regardless of gestational stage. PGF, TXB2, and 6-k-PGF1 alpha increased with gestational age, peaking at the time of parturition. Production was greatest during the first 15 min of incubation and INDO inhibited production of each prostanoid regardless of gestational stage. Imidazole (100 micrograms/ml) inhibited TXB2 production without affecting PGF or 6-k-PGF1 alpha levels. Frequency of contraction and ICF were not affected by imidazole treatment despite TXB2 reduction. These data demonstrate that the in vitro uterus from pregnant rats is capable of producing prostanoids other than prostaglandins and their production generally parallels uterine contractile activity. Thus, the possibility that these prostanoids are involved in physiologic changes during parturition warrants further investigation.     

Effect of prostaglandins A1, A2, B1, B2, E2 and F2 alpha on human umbilical cord vessels

Human umbilical blood vessels have the ability to close spontaneously following delivery at term. It has been suggested that prostaglandins may have a possible physiological role in its closure. This study investigates the effects of 6 naturally occurring prostaglandins (A1, A2, B1, B2, E2, F2a) on the umbilical blood vessels. Umbilical cords were collected from cases of normal spontaneous vaginal deliveries and cesarian section at term. A total of 41 strips of umbilical arteries and 26 strips of umbilical veins from 24 cords were used. A 4-point bioassay method was used to compare the potency of prostaglandins A1, A2, B1 and F2a with PGE2. The effect of Polyphloretin Phosphate (PPP) on prostaglandin-induced contractions was studied on umbilical artery strips from 12 cords. The 6 prostaglandins exerted a stimulant effect on the isolated strips of human umbilical arteries. Prostaglandin B2 was the most potent compound on the umbilical vein, followed by PGA2. PPP in the concentration range of 10 to 40 mcg/ml completely eliminated the responses of PGE2, F2a, A1, A2, and B1. Responses to PGB2 were considerably but not completely abolished. PPP (up to 40 mcg/ml) did not affect contractions induced by 5-hydroxytryptamine, suggesting the presence of discrete receptor sites in the blood vessels for different pharmacologically active compounds. This is the first report of the constrictor effect of PGA and PGB compounds. These naturally occuring prostaglandins with high potencies (compared with other prostaglandins and other vasoactive substances) may play a role in spontaneous closure of umbilical vessels. PGE1, E2, F1 and F2a are found in umbilical blood vessels obtained at term.     

Effect of prostaglandins E1, E2 and F2alpha on neutrophil aggregation.

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E1, E2 and F2alpha alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E1, E2 and F2alpha were 10(-7), 10(-6) and 10(-5)M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell-conditioned medium.

We have previously shown (Berrou et al., J. Cell. Phys., 137:430-438, 1988) that porcine endothelial cell-conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. 1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]-sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. 2) Whereas ECCM stimulation of [35S]-methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine-labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. 3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine-labeled proteoglycans 7 protein cores of high apparent molecular mass (550-200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]-serine incorporation in response to ECCM (51%, as measured by Sepharose CL-6B chromatography). 4) Finally, incorporation of [35S]-sulfate into chondroitinase-generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL-6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that 1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and 2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.     

Metabolism of glycosaminoglycans in cultured smooth muscle cells from pig aorta

Arterial wall smooth muscle cells, originating from the inner layer (media) of pig aortas, were grown in culture. The synthesis and secretion of proteoglycans by these cells were investigated. These cells were incubated in the presence of [³⁵S] sulfate or [¹⁴C] glucosamine and these precursors incorporation into glycosaminoglycans was followed.Proteoglycans synthesized by media cells exhibit different glycosaminoglycan distribution patterns according to their localization. The glycosaminoglycan components are largely confined to the medium (80 per cent) and exhibit a distribution pattern that ressembles closely that found in pig aorta tissue. In comparison with the extracellular and intracellular pools, the pericellular pool (trypsin released material) contains proportionally more heparan sulfate.Isotopic chase experiments demonstrated that glycosaminoglycans leave the intracellular and pericellular compartments with initial half-lives of 7 – 8 h and 13 – 14 h, respectively.About half of the labelled glycosaminoglycans was released into the medium, in an apparently undegraded form, while the rest was degraded.The production of proteoglycans is not affected by modifying the exogenous concentration of hyaluronic acid or chondroitin sulfate present in the culture medium. The synthesis of proteoglycans, but not their secretion is inhibited with cytochalasin-B, a microfilament modifying agent. The secretion of proteoglycans and also — in part — their synthesis is inhibited by antimicrotubular agents: colchicine and vinblastine, with observed intracellular accumulation of proteoglycans.These data suggest that, in arterial cells, the intracellular movement of proteoglycans during the secretory process is mediated by microtubular elements.In conclusion, our results provide evidence for the responsiveness of cultured mediacytes to antimicrotubular and antimicrofilamentar drugs, the utilization of which allows modification in the metabolism and secretion of arterial proteoglycans.     

Effects of 60Co radiation on synthesis of prostaglandins F2 alpha, E, and thromboxane B2 in lung airways of guinea pigs

At 1 hr to 14 days after total-body exposure of guinea pigs to 3.0 Gy 60Co, changes were detected in prostaglandin concentrations in bronchial airway tissues. At 3 hr postexposure, tissue levels of PGE were significantly elevated, while at 48 hr transiently elevated levels of PGF2 alpha were observed. By 72 hr, levels returned to control values. Airway synthesis of thromboxane B2 in irradiated animals did not differ from that in controls. Also assessed were the capacities of bronchial airway preparations to respond to H-1 receptor stimulation by the exogenous addition of histamine or transmembrane divalent cation transport stimulation with ionophore. Tissues from irradiated animals demonstrated alterations in the amount and type of prostaglandins generated, varying with time postirradiation.     

Interleukin-1 beta induces the production of an L-arginine-derived relaxing factor from cultured smooth muscle cells from rat aorta.

The effect of interleukin-1 beta on the production of non-prostanoid vasoactive factors by cultured rat aortic smooth muscle cells was investigated. Under bioassay conditions, the perfusate from a column of confluent cells grown on beads and treated with interleukin-1 beta (1 ng/ml for 18 to 24 hr) abolished the contraction of a canine coronary ring without endothelium contracted by phenylephrine (1 microM), while the perfusate from control cells had no effect. The relaxing activity of the perfusate was observed when transit times were increased from 1 sec to 5 min. Nitro L-arginine (100 microM) reversed the relaxations and L-arginine stereoselectively restored the relaxations. Interleukin-1 beta (1 ng/ml) evoked a time-dependent accumulation of cyclic GMP but not cyclic AMP in cultured smooth muscle cells. The transfer of fresh or stored (-70 degrees C) conditioned culture medium from interleukin-1 beta-treated cells but not from control cells, to cultured smooth muscle cells stimulated the production of cyclic GMP. These observations demonstrate that interleukin-1 beta induces the production of transferable factor which relaxes vascular smooth muscle and stimulates the production of cyclic GMP.     

Effects of prostaglandins E1, E2, and F2alpha on the growth of leukaemia cells in culture.

Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine, uridine and leucine. At concentrations of 50 and 25 mug per ml, there was significant inhibition of thymidine and uridine incorporation, but not of leucine, PGF2alpha showed significant inhibition of thymidine and uridine incorporation but not leucine incorporation, in all 3 concentrations studied (100, 50, and 25 mug/ml). The ability of the cells to form colonies in soft agar was significantly inhibited by PGE1 and PGE2 at concentrations as low as 1-8 mug/ml. For F2alpha, however, a concentration as high as 56mug/ml was required to show inhibitory effect, but at 1-8 mug/ml it was found to be stimulatory.