thromboxane-a2 and sulprostone

1. The influence of endogenously produced prostanoids on active transepithelial Ca2+ transport and cAMP formation was investigated in immunodissected rabbit kidney connecting and cortical collecting tubule cells grown to confluency on permeable supports. 2. The cyclo-oxygenase inhibitor indomethacin dose-dependently (IC50 = 18 nM) reduced the net apical-to-basolateral Ca2+ transport by 57%. Inhibition was reversed in medium obtained from monolayers incubated in the absence of indomethacin. 3. HPLC analysis following incubation with 14C-labelled arachidonic acid revealed the presence of a wide variety of radiolabelled prostanoids in both the apical and basolateral media. These findings are compatible with the endogenous production and subsequent release of stimulatory prostanoids. 4. The inhibitory action of indomethacin was reversed by the addition of the prostanoids PGE1, PGE2 and PGA2, but not PGD2, PGF2 alpha, the stable PGI2 analogue cicaprost or the thromboxane A2 mimetic U-46619. PGE2 stimulated transepithelial Ca2+ transport dose dependently (EC50 = 3 nM), irrespective of the compartment of which it was added. The stimulatory effect of PGE2 was paralleled by increased cAMP formation, suggesting the apical and basolateral presence of stimulatory prostanoid receptors EP2 and/or EP4. 5. Sulprostone, an analogue selective for EP1 and EP3 receptors, inhibited transepithelial Ca2+ transport in indomethacin-treated monolayers only when applied basolaterally, suggesting the exclusive presence of inhibitory EP receptors on the basolateral membrane. 6. The percentage by which parathyroid hormone and arginine vasopressin increased both transepithelial Ca2+ transport and cAMP formation was dramatically increased in indomethacin-inhibited cells as compared with control cells, demonstrating that indomethacin unmasks the actions of these hormones to their full extent.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arachidonic Acid; Arginine Vasopressin; Biological Transport; Calcium; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclic AMP; Cyclooxygenase Inhibitors; Dinoprostone; Eicosanoids; Epoprostenol; Indomethacin; Kidney Tubules; Models, Biological; Parathyroid Hormone; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Rabbits; Receptors, Prostaglandin; Thromboxane A2; Virulence Factors, Bordetella

[3H]PGE2 and [3H]PGF2 alpha were shown to bind with similar binding capacity and dissociation constants to bovine aorta endothelial cells. The similarity in the binding parameters suggests that both agonists may bind to the same binding site. Displacement of [3H]PGE2 performed with PGE2, PGF2 alpha or U-46619, a thromboxane agonist, shows that all three prostanoids displaced the bound [3H]PGE2 with comparable potency (IC50 = 10(-7) M). These results indicated that the three different prostanoids, which serve as specific agonists to different prostanoid receptors, also compete for the same binding site in bovine endothelial cells with similar affinity. Comparison of the displacement of [3H]PGE2 or [3H]PGF2 alpha by a number of prostaglandin agonists and antagonists further supports the notion that the natural prostanoids bind with similar affinities to the same binding site. Thus, sulprostone, an EP1/EP3 agonist, displaced bound [3H]PGE2 and [3H]PGF2 alpha with IC50 of about 10(-7) M. On the other hand, thromboxane antagonists (BAY u-3405 and GR-32191B), EP1 specific antagonist (SC-19220) EP1/DP antagonist (AH-6809) and iloprost, a stable prostacyclin agonist, failed to displace bound [3H]PGE2 or [3H]PGF2 alpha at a concentration range of 10(-9)-10(-6) M. Gradual increase of sodium fluoride (NaF), a general activator of G binding proteins, or incubation of permeabilized cells with GTP gamma S resulted in a decrease in [3H]PGE2 binding, suggesting that the binding site represents a low-affinity common prostanoid receptor which, similar to other prostanoid receptors, is probably coupled with G binding proteins.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Binding Sites; Biphenyl Compounds; Carbazoles; Cattle; Cells, Cultured; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Endothelium, Vascular; Epoprostenol; Heptanoic Acids; Iloprost; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Sulfonamides; Thromboxane A2; Thromboxanes; Xanthenes; Xanthones

We wished to determine whether any evidence indicates that the ductus arteriosus has prostanoid receptors coupled to contractile pathways and whether the sensitivity of the ductus to the dilator effect of prostaglandin E2 (PGE2) was inhibited by other prostanoids. Rings of ductus arteriosus were isolated from fetal New Zealand White rabbits (28 days of gestation) and mounted in vitro. In the presence of 1 microM indomethacin, the vessel was relaxed with either 300 nM forskolin or 10 nM PGE2, and cumulative concentration-contraction response curves to several synthetic prostanoids were obtained with or without a receptor antagonist when available. The vessel was also precontracted with 1 microM indomethacin and 25 mM K+ in 13-14.5 kPa O2, and cumulative concentration-relaxation response curves to PGE2 were obtained with and without addition of prostanoids. In 300 nM forskolin, both U46619 and sulprostone caused concentration-dependent contractions of the ductus in the nanomolar range (EC50 values, i.e., the interpolated molar concentration of the drug causing 50% of its own eventual maximum response of 33 and 42 nM, respectively). Responses to GR63799X and PGF2 alpha were complicated by the fact that these agonists caused relaxation at high concentrations (> or = 30 nM). The response to U46619 was shifted to the right by the thromboxane receptor antagonist EP 092. In 10 nM PGE2, U46619, sulprostone, and GR63799X elicited similar contractile responses, whereas PGF2 alpha had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Analysis of Variance; Animals; Colforsin; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Ductus Arteriosus; Female; In Vitro Techniques; Indomethacin; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Pregnancy; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E, Synthetic; Rabbits; Receptors, Prostaglandin; Thromboxane A2; Thromboxanes; Vasoconstrictor Agents

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Cyclic AMP; Dinoprostone; Dose-Response Relationship, Drug; Histamine; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Receptors, Prostaglandin E; Saphenous Vein; Swine; Thromboxane A2; Vasoconstrictor Agents; Venae Cavae