Prostaglandin F2α agonists induced enhancement in collagen1 expression is involved in the pathogenesis of the deepening of upper eyelid sulcus.

Previous our study reported that three-dimension (3D) cultures of human orbital fibroblasts (HOFs) replicated the etiology of deepening of the upper eyelid sulcus (DUES) caused by prostaglandin F2α analogues (PGF2α-ags). To examine this further, the effects of PGF2α-ags on HOFs were characterized by (1) lipid staining (2D; two-dimension, 3D), (2) comparison of the 3D organoid sizes of preadipocytes (DIF-) or adipocytes (DIF+) that had been treated with various concentrations of several PGF2α-ags, (3) physical stiffness (3D), and (4) the mRNA expression of adipogenic related genes, extracellular matrix (ECM), tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) (3D). PGF2α-ags caused a dramatic down-sizing of the 3D DIF+ organoids and this reduction was concentration dependent. The effects caused by PGF2α-ags were also observed in 3D preadipocytes. Micro-squeezer analysis clearly indicated that PGF2α-ags induced an increase in their physical solidity. The size of each organoid under several conditions was inversely correlated with the mRNA expression profile of collagen1 (COL1), TIMP2, and MMP2 and 9. These findings indicate that PGF2α-ags affect the expression of COL1, TIMP2, and MMP2 and 9 which, in turn, modulate the 3D ECM network within the organoids, thus resulting in their downsizing.

Prostaglandin F2α and EP2 agonists, and a ROCK inhibitor modulate the formation of 3D organoids of Grave's orbitopathy related human orbital fibroblasts.

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 µM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1ß (IL1ß) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1ß and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO.

Protection of 6-OHDA neurotoxicity by PGF2α through FP-ERK-Nrf2 signaling in SH-SY5Y cells.

6-Hydroxydopamine (6-OHDA) is a neurotoxin that destroy dopaminergic neurons and widely used to establish animal models of Parkinson's disease. Prostaglandins (PGs) are involved in various cellular processes, including the damage and repair of neuronal cells. However, the function of PGF2α in neuronal cells remains unclear. In this study, we investigated the effects of PGF2α against 6-OHDA-mediated toxicity in human neuroblastoma SH-SY5Y cells and elucidated its underlying molecular mechanism. When the cells were treated with 6-OHDA (50 µM) for 6 h, the expression levels of PGF2α synthetic enzymes; cyclooxygenase-2 and aldo-keto reductase 1C3 as PGF2α synthase were enhanced in an incubation-time-dependent manner. In addition, the production of PGF2α was increased in 6-OHDA-treated cells. Fluprostenol, a PGF2α receptor (FP) agonist (500 nM), suppressed 6-OHDA-induced cell death by decreasing the production of reactive oxygen species (ROS) and increasing the expression of the anti-oxidant genes. These fluprostenol-mediated effects were inhibited by co-treatment with AL8810, an FP receptor antagonist (1 µM) or transfection with FP siRNA (20 nM). Moreover, 6-OHDA-induced phosphorylation of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase family, was inhibited by co-incubation with AL8810. Furthermore, fluprostenol itself enhanced ERK phosphorylation and further elevated the 6-OHDA-induced phosphorylation of ERK. In addition, 6-OHDA induced nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), activating anti-oxidant gene expression, was repressed by co-culturing with AL8810. These results indicate that PGF2α suppressed 6-OHDA-induced neuronal cell death by enhancing anti-oxidant gene expression via the FP receptor-ERK-Nrf2 signaling. Thus, FP receptor is a potential target for inhibition of ROS-mediated neuronal cell death.

Prostaglandin F2α Agonists Negatively Modulate the Size of 3D Organoids from Primary Human Orbital Fibroblasts.

Purpose: To elucidate the molecular etiology of deepening of the upper eyelid sulcus (DUES) induced by prostaglandin (PG) analogs, a three-dimensional (3D) tissue culture system was employed using human orbital fibroblasts (HOFs). Methods: During adipogenesis, changes in HOF 3D organoid sizes, as well as their lipids stained by BODIPY and expression of the extracellular matrix (ECM) by immunolabeling and/or quantitative PCR, were studied in the presence or absence of either 100-nM bimatoprost acid or 100-nM prostaglandin F2α. Results: The size of the 3D organoids increased remarkably during adipogenesis, but such increases were significantly inhibited by the presence of PG analogs. Staining intensities by BODIPY and mRNA expression of peroxisome proliferator-activated receptor gamma were significantly increased upon adipogenesis but were not influenced by the presence of PG analogs. Unique changes in ECM expression observed with or without adipogenic differentiation were significantly modified by the presence of PG analogs. Conclusions: Our present study indicates that PG analogs have the potential to modulate the ECM network within HOF 3D organoids. Thus, a 3D tissue culture system may be a suitable strategy for understanding the disease etiology of DUES.

Prostaglandin F2α agonist-induced suppression of 3T3-L1 cell adipogenesis affects spatial formation of extra-cellular matrix.

To establish a deepening of the upper eyelid sulcus (DUES) model that can be induced by prostaglandin (PG) analogues, a three-dimension (3D) tissue culture was employed. Upon adipogenesis of the 3T3-L1 organoid, the effects of either Bimatoprost acid (BIM-A), or PGF2α were examined. During the adipogenesis, organoid size, lipid staining by BODIPY and expression of the extracellular matrix (ECM) by immunocytochemistry and/or quantitative PCR were employed. The size of the organoid increased remarkably during the adipogenesis, while such increases were significantly inhibited by the presence of PGF2α or BIM-A. BODIPY positive lipid-laden cells significantly increased during the adipogenesis, while in contrast they were greatly suppressed by the presence of PGF2α. Characteristic and spatial changes in ECM expressions observed upon adipogenesis were greatly modified by the presence of PGs. Our present study using a 3D tissue culture may be a suitable strategy toward understanding disease etiology of DUES.

The PGF2α agonists luprostiol and d-cloprostenol reliably induce luteolysis in luteal phase mares without evoking clinical side effects or a stress response.

In the present study we have evaluated a possible stress reaction in response to two different PGF2α analogs-luprostiol and D-cloprostenol--and their effects on estrous cycle characteristics. In a cross-over-design eight mares received in alternating order either luprostiol (Treatment LUP; 3.75 mg im), D-cloprostenol (Treatment CLO; 22.5µg im) or saline (Treatment CON; NaCl 0.9% 0.5ml im) on day 8 after ovulation. Injection of either LUP or CLO, but not of CON resulted in a significant decline of progesterone concentration in plasma to baseline concentrations within two days (time: p<0.001, treatment: p<0.01, time × treatment: p<0.05). The treatment to ovulation interval was significantly shorter in LUP and CLO than in CON cycles (LUP: 9.4 ± 0.4 d; CLO: 9.4 ± 1.3 d; CON: 16.1 ± 0.8 d; p<0.001). Injection of either LUP or CLO, but not of CON significantly increased salivary cortisol concentration (immediately before injection: CON 1.3 ± 0.2, LUP 1.4 ± 0.3, CLO 1.4 ± 0.3 ng/ml; 60 min after injection: CON 1.0 ± 0.3, LUP 8.0 ± 1.4, CLO 4.2 ± 0.7 ng/ml; time: p<0.01, treatment: p<0.001, time × treatment: p<0.001). Heart rate decreased over time (p<0.05) independent of treatment and no changes in heart rate variability were detected. Injection of the PGF2α analogs CLO and LUP reliably induced luteolysis and apart from a transient increase in salivary cortisol concentration no signs of a physiological stress response or apparent side effects occurred.

The induction of lipid peroxidation during the acute oxidative stress response induced by intratracheal instillation of fine crystalline silica particles in rats.

Crystalline silica (SiO2) is an important material for industry but is considered potentially carcinogenic. Inhalation of a crystalline SiO2 aerosol may contribute to serious lung diseases. Crystalline SiO2 particles are commonly used as a positive control in toxicity assays of particulate materials (e.g. nanoparticles). Crystalline SiO2 induces oxidative stress resulting in lipid peroxidation, but the acute oxidative stress response in the lung is not well understood. Lipid peroxidation during the acute stage of oxidative stress after instillation of crystalline SiO2 into rats was examined by bronchoalveolar lavage fluid (BALF) analysis. The levels of 8-iso-prostaglandin F2α and hydroxyoctadecadienoic acid (HODE) in the BALF were measured using liquid chromatography coupled to quadrupole mass spectrometry. The concentration of the antioxidant protein heme oxygenase-1 (HO-1) in the BALF was determined using enzyme-linked immunosorbent assay. Intratracheal instillation of crystalline SiO2 increased the level of HODE and HO-1 in BALF at 24 h after administration. The levels of HODE and HO-1 returned to baseline at 72 h after instillation. Lactate dehydrogenase leakage was observed only after 1 h instillation. These results suggest that the contribution of oxidative stress to the pulmonary toxicity of crystalline SiO2 is minimal in the early acute stage after exposure.

Evaluation of the Effect of Latanoprostene Bunod Ophthalmic Solution, 0.024% in Lowering Intraocular Pressure over 24 h in Healthy Japanese Subjects.

INTRODUCTION: Latanoprostene bunod is a novel nitric oxide (NO)-donating prostaglandin F2α receptor agonist in clinical development for the reduction of intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension. We evaluated the effect of latanoprostene bunod 0.024% instilled once daily (QD) on lowering IOP over a 24-h period in healthy Japanese subjects following 14 days of treatment. METHODS: This was a single-arm, single-center, open-label clinical study of 24 healthy Japanese male volunteers. A baseline IOP profile was established in both eyes in the sitting position at 8 PM, 10 PM, 12 AM, 2 AM, 4 AM, 8 AM, 10 AM, 12 PM, and 4 PM using a Goldmann applanation tonometer. Subjects subsequently instilled latanoprostene bunod 0.024% QD at 8 PM for 14 days in both eyes. The absolute and change from baseline in sitting IOP was assessed on day 14. RESULTS: The mean (SD) age of the subjects was 26.8 (6.3) years, and mean (SD) baseline IOP was 13.6 (1.3) mmHg in the study eye. Latanoprostene bunod 0.024% instilled QD for 14 days reduced IOP at all the evaluated time points (P < 0.001) with a mean (SD) 24-h reduction of 3.6 (0.8) mmHg or 27% from the baseline in the study eye. Peak and trough IOP lowering occurred at 8 AM and 8 PM (12 and 24 h following instillation) with a mean reduction of 4.2 (1.8) mmHg, or 30%, and 2.8 (2.2) mmHg, or 20%, respectively. Punctate keratitis and ocular hyperemia, both mild in severity, were the most common adverse events. CONCLUSION: Latanoprostene bunod ophthalmic solution 0.024%, dosed QD for 14 days, significantly lowered mean IOP in healthy Japanese subjects during the entire 24-h period. Studies of latanoprostene bunod in patients diagnosed with normal tension glaucoma are warranted. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01895985. FUNDING: Bausch & Lomb, Inc.

Effects of CP-900691, a novel peroxisome proliferator-activated receptor α, agonist on diabetic nephropathy in the BTBR ob/ob mouse.

Piperidine-based peroxisome proliferator-activated receptor-α agonists are agents that are efficacious in improving lipid, glycemic, and inflammatory indicators in diabetes and obesity. This study sought to determine whether CP-900691 ((S)-3-[3-(1-carboxy-1-methyl-ethoxy)-phenyl]-piperidine-1-carboxylic acid 4-trifluoromethyl-benzyl ester; CP), a member of this novel class of agents, by decreasing plasma triglycerides, could prevent diabetic nephropathy in the Black and Tan, BRachyuric (BTBR) ob/ob mouse model of type 2 diabetes mellitus. Four-week old female BTBR WT and BTBR ob/ob mice received either regular chow or one containing CP (3 mg/kg per day) for 14 weeks. CP elevated plasma high-density lipoprotein, albuminuria, and urinary excretion of 8-epi PGF(2α), a product of the nonenzymatic metabolism of arachidonic acid and whose production is elevated in oxidative stress, in BTBR WT mice. In BTBR ob/ob mice, CP reduced plasma triglycerides and non-esterified fatty acids, fasting blood glucose, body weight, and plasma interleukin-6, while concomitantly improving insulin resistance. Despite these beneficial metabolic effects, CP had no effect on elevated plasma insulin, 8-epi PGF(2α) excretion, and albuminuria, and surprisingly, did not ameliorate the development of diabetic nephropathy, having no effect on the accumulation of renal macrophages, glomerular hypertrophy, and increased mesangial matrix expansion. In addition, CP did not increase plasma high-density lipoprotein in BTBR ob/ob mice, while paradoxically increasing total cholesterol levels. These findings indicate that 8-epi PGF(2α), possibly along with hyperinsulinemia and inflammatory and dysfunctional lipoproteins, is integral to the development of diabetic nephropathy and should be considered as a potential target of therapy in the treatment of diabetic nephropathy.

Depressed levels of prostaglandin F2α in mice lacking Akr1b7 increase basal adiposity and predispose to diet-induced obesity.

Negative regulators of white adipose tissue (WAT) expansion are poorly documented in vivo. Prostaglandin F(2α) (PGF(2α)) is a potent antiadipogenic factor in cultured preadipocytes, but evidence for its involvement in physiological context is lacking. We previously reported that Akr1b7, an aldo-keto reductase enriched in adipose stromal vascular fraction but absent from mature adipocytes, has antiadipogenic properties possibly supported by PGF(2α) synthase activity. To test whether lack of Akr1b7 could influence WAT homeostasis in vivo, we generated Akr1b7(-/-) mice in 129/Sv background. Akr1b7(-/-) mice displayed excessive basal adiposity resulting from adipocyte hyperplasia/hypertrophy and exhibited greater sensitivity to diet-induced obesity. Following adipose enlargement and irrespective of the diet, they developed liver steatosis and progressive insulin resistance. Akr1b7 loss was associated with decreased PGF(2α) WAT contents. Cloprostenol (PGF(2α) agonist) administration to Akr1b7(-/-) mice normalized WAT expansion by affecting both de novo adipocyte differentiation and size. Treatment of 3T3-L1 adipocytes and Akr1b7(-/-) mice with cloprostenol suggested that decreased adipocyte size resulted from inhibition of lipogenic gene expression. Hence, Akr1b7 is a major regulator of WAT development through at least two PGF(2α)-dependent mechanisms: inhibition of adipogenesis and lipogenesis. These findings provide molecular rationale to explore the status of aldo-keto reductases in dysregulations of adipose tissue homeostasis.

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models.

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 µg) and 0.12% (36 µg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 µg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.

Involvement of prostaglandin F2α in preeclamptic human umbilical vein vasospasm: a role of prostaglandin F and thromboxane A2 receptors.

OBJECTIVE: Preeclampsia is characterized by hypertension and proteinuria developing after 20 weeks of gestation. Increased vasoconstriction can be one of the major underlying pathophysiological event in this syndrome. We examined the role of vasoconstrictor prostanoid, prostaglandin F2α (PGF2α) in preeclamptic and normotensive human umbilical veins. METHODS: Umbilical veins were set up in organ bath. The concentration-response curves of PGF2α (endogenous agonist of prostaglandin F receptor) and fluprostenol (prostaglandin F receptor selective agonist) were determined in normal and preeclamptic veins either in the absence or presence of BAY u3405 (thromboxane A2 receptor selective antagonist). PGF2α and its major metabolite concentrations were measured by enzyme immunoassay kit. The expression of vasoconstrictor prostanoid receptors was determined by western blot. RESULTS: The concentration-dependent contractions to PGF2α and fluprostenol were significantly increased in umbilical vein preparations derived from preeclamptic women compared with those of normotensives. Increased reactivity was related with enhanced sensitivity to these spasmogens in preeclamptic veins. BAY u3405 (10 µmol/l) did not modify the responsiveness to PGF2α in normal umbilical veins whereas moderately reduced the contractions in preeclamptic preparations. Serum concentrations of PGF2α and its major metabolite, 13,14-dihydro-15-keto-PGF2α, were comparable between preeclamptics and normotensives whereas the metabolite concentration was elevated in umbilical cord serum of preeclamptics. 13,14-dihydro-15-keto-PGF2α, release was also increased in umbilical vein preparations of preeclamptic women. An increased prostaglandin F receptor protein expression was determined whereas EP3 and thromboxane A2 protein expressions were unchanged in preeclamptic umbilical veins. CONCLUSION: Prostaglandin F and thromboxane A2 receptors activation by PGF2α could be involved in umbilical vasospasm observed in preeclampsia.

Expression and functional evidence of the prostaglandin F2alpha receptor mediating contraction in human umbilical vein.

Our purposes were to perform the pharmacological characterization of PGF(2alpha) receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF(2alpha), and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC(50)) order was PGF(2alpha) (6.01+/-0.05)>latanoprost free acid (5.65+/-0.07)=bimatoprost free acid (5.59+/-0.08). The contractile effects of PGF(2alpha) and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (pK(B)) of AL-8810 vs. PGF(2alpha) (5.93+/-0.05) and vs. latanoprost free acid (6.40+/-0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF(2alpha) in this tissue.

Regulation of Nur77 gene expression by prostanoids in cementoblastic cells.

OBJECTIVE: The inflammatory cytokine interleukin-1 (IL-1) decreases mineralisation by immortalized mouse-derived cementoblastic cells (OC-CM cells), whilst various prostanoids, including fluprostenol (flup) increase it. Subtraction hybridisation conducted on flup minus IL-1-treated OC-CM cells revealed that one of the primary response genes preferentially induced by flup is the transcription factor Nur77. The objective of this study was to examine the signal transduction cascades regulating prostanoid induction of Nur77 gene expression in OC-CM cells. METHODS: Confluent OC-CM cells were treated with prostaglandin E(2) (PGE(2)), prostaglandin F(2alpha) (PGF(2alpha)), specific activators of the various EP prostanoid receptors and of the FP prostanoid receptor, and direct activators/inhibitors of the cyclic AMP-protein kinase A (PKA), protein kinase C (PKC) and intracellular calcium pathways. Nur77 gene expression was examined by mRNA extraction and Northern blot analysis. RESULTS: PGE(2) and PGF(2alpha) treatment of OC-CM cells significantly increased Nur77 mRNA expression in a time- and dose-dependent fashion. Both the EP1 prostanoid receptor-specific activator 16,16-dimethyl-PGE(2) and the FP prostanoid receptor-specific activator flup significantly increased Nur77 gene expression by OC-CM cells as compared to vehicle-treated controls. Increase in Nur77 gene expression was also observed when direct activators of the PKA, PKC and intracellular calcium pathways were used to treat OC-CM cells. Direct inhibition of the PKA, PKC and intracellular calcium pathways abrogated Nur77 gene expression induced by OC-CM cell treatment with PGE(2) and PGF(2alpha). CONCLUSION: Nur77 is a primary gene expressed by OC-CM cells and its induction appears to be mediated by the PKA, PKC and intracellular calcium pathways. Nur77 may affect expression of downstream target genes in OC-CM cells and partially regulate cementoblast cell function.

PGF2alpha-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70S6k, and PTEN.

Prostaglandin F2alpha (PGF2alpha) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2alpha-induced VSMC hypertrophy we examined the ability of the PGF2alpha analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3beta (GSK-3beta), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3beta, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.

Progesterone metabolism in bovine endometrial cells and the effect of metabolites on the responsiveness of the cells to OT-stimulation of PGF2alpha.

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. The objective of this work was to characterize P4 metabolism by endometrial cells in vitro and determine if metabolites were able to modify prostaglandin secretion in response to oxytocin (OT). Endometrial epithelial and stromal cells were incubated with 3H-P4 or 3H-pregnenolone (P5) for 6 or 24 h. Metabolites in the medium were separated by HPLC. The results showed that P4 and P5 were converted to two major polar metabolites and a less polar metabolite that was identified as 5alpha- or 5beta-pregnanedione by LC/MS. Progesterone metabolism was similar in both stromal and epithelial cells. To determine if 5alpha- or 5beta-pregnanedione were able to modify PGF(2)alpha synthesis, cells were cultured with P4, 5alpha- or 5beta-pregnanedione (100 ng ml(-1)) for 48 h and then each group of cells was incubated for a further 4-6 h with or without OT (200 ng ml(-1)). Results showed that only P4 caused significant (P<0.001) increase in basal, but not OT-stimulated, PGF(2)alpha synthesis. OT binding assays showed no significant effect of progesterone or its metabolites on OTR concentration. In conclusion, bovine endometrial cells are able to metabolize pregnenolone and progesterone but neither 5alpha- nor 5beta-pregnanedione altered prostaglandin synthesis or OTR number in endometrial epithelial cells. These data suggest that 5-pregnanediones do not play a role in the regulation OT-stimulated PGF(2)alpha secretion during the bovine estrous cycle.

Intracellular calcium mobilization as a target for the spasmolytic action of scopoletin.

The coumarin scopoletin was isolated in a pure form from the roots of Brunfelsia hopeana Benth. (Solanaceae). In isolated rat aortic rings, scopoletin (26-520 microM) inhibited to approximately the same extent the contractions induced by a variety of substances, including phenylephrine, potassium chloride, serotonin and PGF(2) (alpha). The effect of the coumarin on phenylephrine-induced contractions was not affected by endothelium removal or NO-synthase blockade by L-NAME (100 microM). Scopoletin (78 - 590 microM) antagonized in a concentration-dependent manner (IC(50) = 300 +/- 20 microM, n = 5), transient contractions in Ca(2+)-free media induced by noradrenaline, but not those induced by caffeine. Also, scopoletin did not interfere with the refilling of noradrenaline-sensitive intracellular calcium stores. It is suggested that the non-specific spasmolytic action of scopoletin can be attributed, at least in part, to its ability to inhibit the intracellular calcium mobilization from the noradrenaline-sensitive stores.

Formation of an F2-isoprostane in vascular smooth muscle cells by elevated glucose and growth factors.

Recently a series of non-cyclooxygenase-derived prostanoids were identified in vivo in humans and in animal models of free radical injury as products of free radical-catalyzed peroxidation of arachidonic acid. One of these, an F2-isoprostane, 8-epiprostaglandin F2 alpha (8-epi-PGF 2 alpha), is a potent renal vasoconstrictor and can increase vascular smooth muscle cell (VSMC) DNA synthesis. In the present study we have evaluated whether F2-isoprostanes play a role in diabetic vascular dysfunction by studying the formation of 8-epi-PGF2 alpha in porcine VSMC (PVSMC) cultured under hyperglycemic conditions. 8-Epi-PGF2 alpha levels were quantitated by a specific enzyme immunoassay. We also examined whether certain VSMC growth factors, such as angiotensin II, platelet-derived growth factor, and transforming growth factor-beta, could also regulate the formation of 8-epi-PGF2 alpha. We observed that PVSMC cultured under high glucose (HG) conditions produced significantly higher amounts of 8-epi-PGF2 alpha compared with normal glucose (NG) conditions (3.7 +/- 0.13 ng/10(6) cells in HG vs. 2.9 +/- 0.2 ng/10(6) cells in NG, P < 0.05). Furthermore, all three growth factors tested evoked significant dose-dependent formation of 8-epi-PGF2 alpha (ranging from 125 to 220% of control). These results suggest that 8-epi-PGF2 alpha formation, as a result of hyperglycemia or due to growth factor action, may lead to increased VSMC growth and contribute to the complications of diabetes and cardiovascular disease.