John W. Regan

Enzymes and Receptors of Prostaglandin Pathways with Arachidonic Acid-derived Versus Eicosapentaenoic Acid-derived Substrates and Products

Dietary fish oil containing ω3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the ω6 fatty acid arachidonic acid (AA) and the ω3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA3 is about equipotent to TxA2 at the TPα receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of ω3 fatty acids such as EPA.

Prostaglandin E2 Induced Functional Expression of Early Growth Response Factor-1 by EP4, but Not EP2, Prostanoid Receptors via the Phosphatidylinositol 3-Kinase and Extracellular Signal-regulated Kinases

Prostaglandin E2(PGE2) mediates its physiological effects by interactions with a subfamily of G-protein-coupled receptors known as EP receptors. These receptors consist of four primary subtypes named EP1, EP2, EP3, and EP4. The EP2 and EP4 subtypes are known to couple to Gαs and stimulate intracellular cyclic 3,5- adenosine monophosphate formation, whereas the EP1 and EP3 receptors are known to couple to Gαq and Gαi, respectively. Recently we found that EP2 and EP4 receptors can activate T-cell factor signaling; however, EP2 receptors did this primarily through a cAMP-dependent protein kinase-dependent pathway, whereas EP4 receptors primarily utilized a phosphatidylinositol 3-kinase (PI3K)-dependent pathway (Fujino, H., West, K. A., and Regan, J. W. (2002) J. Biol. Chem. 277, 2614–2619). We now report that PGE2 stimulation of EP4 receptors, but not EP2 receptors, leads to phosphorylation of the extracellular signal-regulated kinases (ERKs) through a PI3K-dependent mechanism. Furthermore, this activation of PI3K/ERK signaling by the EP4 receptors induces the functional expression of early growth response factor-1 (EGR-1). Under the same conditions induction of EGR-1 protein expression was not observed following PGE2 stimulation of EP2 receptors. These findings point to important differences in the signaling potential of the EP2 and EP4 receptors, which could be significant with respect to the potential involvement of EP4 receptors in inflammation and cancer.

Forskolin stimulation of water and cation permeability in aquaporin 1 water channels

Aquaporin1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3′,5′-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin1. The cationic permeability of the aquaporin1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

Cloning of a Carboxyl-terminal Isoform of the Prostanoid FP Receptor

An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FPB receptor, is identical to the original isoform, the FPA, throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FPA, whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FPB terminates after only one amino acid. The FPA isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FPB isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FPA or the FPB receptor cDNAs, prostaglandin F2α stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FPB as compared with cells transfected with the FPA isoform. Using the polymerase chain reaction, mRNA encoding the FPB isoform was identified in the ovine corpus luteum.

High affinity renal [3H]flunitrazepam binding: Characterization, localization, and alteration in hypertension

Abstract The binding of [ 3 H]flunitrazepam was studied in membranes prepared from the kidney and cerebral cortex of unilaterally nephrectomized rats made hypertensive by simultaneous deoxycorticosterone acetate (DOCA) and NaCl administration. A significant 35–43% increase in the number of [ 3 H]flunitrazepam binding sites (Bmax) was found in the renal membranes prepared from the hypertensive rats; there was no change in the density of binding sites in the membranes obtained from the cerebral cortex. The K d of [ 3 H]flunitrazepam binding did not change either in the renal or in the cerebral membranes (∼ 12 nM in the kidney and ∼2.0 nM in the brain). Drug specificity studies with renal membranes showed that the inhibition of [ 3 H]flunitrazepam binding by various benzodiazepines did not jibe with their pharmacologic potency as anxiolytic agents. An intrarenal distribution of specific [ 3 H]flunitrazepam binding was found in the bovine kidney; specific binding was greatest in the outer cortex and virtually absent in the medulla, the minor calyx and the renal artery. The evidence that the renal benzodiazepine binding site is of high affinity, is specific, has a unique distribution, and is regulated during hypertension suggests that it may be associated with an important pathophysiologic structure.

6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP2 receptor antagonist

On studying the interaction of various ligands with the pharmacologically defined, recombinant human EP2 receptor (Regan et al., Mol Pharmacol 46: 213-220, 1994), we discovered that the putative EP1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) also has affinity for the human EP2 receptor. Moreover, AH 6809 behaved as an EP2 receptor antagonist and inhibited prostaglandin E2 (PGE2)-stimulated increases in cyclic AMP. These findings have significant implications for studies that employ AH 6809 to determine the pharmacological basis of PGE2-induced responses in human cells and tissues.

Activation of FP Prostanoid Receptor Isoforms Leads to Rho-mediated Changes in Cell Morphology and in the Cell Cytoskeleton

Prostaglandin F2α(PGF2α) exerts its biological effects by binding to and activating FP prostanoid receptors. These receptors, which include two isoforms, the FPA and FPB, have been cloned from a number of species and are members of the superfamily of G-protein-coupled receptors. Previous studies have shown that the activation of FP receptors leads to phosphatidylinositol hydrolysis, intracellular calcium release, and activation of protein kinase C. Here, we demonstrate that PGF2α treatment of 293-EBNA (Epstein-Barr nuclear antigen) cells that have been stably transfected with either the FPA or FPB receptor isoforms leads to changes in cell morphology and in the cell cytoskeleton. Specifically, cells treated with PGF2α show retraction of filopodia and become rounded, and actin stress fibers are formed. Pretreatment of the cells with bisindolylmaleimide I, a protein kinase C inhibitor, has no effect on the PGF2α-induced changes in cell morphology, although it does block the effects of phorbol myristate acetate on cell morphology. On the other hand, the PGF2α-induced changes in cell morphology and formation of actin stress fibers can be blocked by pretreatment of the cells with C3 exoenzyme, a specific inhibitor of the small G-protein, Rho. Consistent with FP receptor induced formation of actin stress fibers and focal adhesions, FPA receptor activation also leads to rapid (within two minutes) tyrosine phosphorylation of p125 focal adhesion kinase (FAK) which can be blocked by pretreating the cells with C3 exoenzyme. Taken together, these results suggest that the FP receptor isoforms are coupled to at least two second messenger pathways, one pathway associated with protein kinase C activation, and the other with activation of Rho.

Molecular cloning and expression of human EP3 receptors: evidence of three variants with differing carboxyl termini.

1 The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. 2 Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3′;untranslated sequence. 3 The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none. 4 Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg−1 protein of [3H]‐prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E. 5 PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3′ untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.

EP 4 prostanoid receptor coupling to a pertussis toxin- sensitive inhibitory G protein

The EP2 and EP4 prostanoid receptor subtypes are G-protein-coupled receptors for prostaglandin E2 (PGE2). Both receptor subtypes are known to couple to the stimulatory guanine nucleotide binding protein (Gαs) and, after stimulation with PGE2, can increase the formation of intracellular cAMP. In addition, PGE2 stimulation of the EP4 receptor can activate phosphatidylinositol 3-kinase (PI3K) leading to phosphorylation of the extracellular signal-regulated kinases (ERKs) and induction of early growth response factor-1 (EGR-1) (J Biol Chem 278: 12151–12156, 2003). We now report that the PGE2-mediated phosphorylation of the ERKs and induction of EGR-1 can be blocked by pretreatment of EP4-expressing cells with pertussis toxin (PTX). Furthermore, pretreatment with PTX increased the amount of PGE2-stimulated intracellular cAMP formation in EP4-expressing cells but not in EP2-expressing cells. These data indicate that the EP4 prostanoid receptor subtype, but not the EP2, couples to a PTX-sensitive inhibitory G-protein (Gαi) that can inhibit cAMP-dependent signaling and activate PI3K/ERK-dependent signaling.

Prostanoid receptors and phosphatidylinositol 3-kinase: a pathway to cancer?

Abstract The significance of receptor heterogeneity has, in many cases, been unclear, particularly in the case of closely related receptor subtypes that are activated by the same endogenous ligands and appear to signal through the same second messenger pathways. In this article, recent studies of the EP and FP prostanoid receptors are reviewed, showing that receptor subtypes previously thought to activate the same signaling pathways, in fact, differ through novel interactions with phosphatidylinositol 3-kinase and activation of nuclear signaling pathways. These findings might be applicable to other families of G-protein-coupled receptors and have implications in cancer and other diseases.