Different roles of cAMP/PKA and PKC signaling in regulating progesterone and PGE2 levels in immortalized rat granulosa cell cultures.

Follicular cells from various species secrete steroids and prostaglandins, which are crucial for reproduction, in response to gonadotropins. Here, we examined prostaglandin E2 (PGE2) secretion from immortalized rat granulosa cells derived from preovulaotry follicles expressing the rat follicle stimulating hormone receptor (denoted as FSHR cells) that produce progesterone in response to gonadotropins. The cells were stimulated with a) pregnant mare's serum gonadotropin (PMSG; a rat FSH receptor agonist), b) activators of the protein kinase A (PKA) pathway (forskolin and a cell permeable cAMP analog Dibutyryl-cAMP (DB-cAMP)) and c) protein kinase C (PKC) (12-O-tetradecanoylphorbol 13-acetate; TPA), alone and in combination for 24 h. Thereafter, PGE2 and progesterone levels in the culture media were determined. In accordance with previous studies, while PMSG and the PKA pathway activators induced progesterone accumulation in the media, TPA did not. In contrast, our data indicate that TPA, but neither PMSG, forskolin and DB-cAMP evoked PGE2 accumulation in the media. Western Blot analysis of cell lysate showed a drastic TPA induced increase of COX-2 levels, which was not seen with neither PMSG nor forskolin treatment. This association between the COX-2 and PGE2 levels suggests that the enzyme activity is the likely factor that determines the synthesis and levels of the prostaglandin in the culture media of the granulosa-derived cells. The addition of the PKA inhibitor H-89 to the FSHR cultures suppressed the gonadotropin and forskolin induction of progesterone secretion. Incubation in the presence of GF109203X (a PKC inhibitor) attenuated the TPA induced PGE2 accumulation in the culture media of the cells (a dose dependent reduction of 40-70%). In addition, while TPA inhibited the PMSG and forskolin induced-accumulation of progesterone in the media, the gonadotropin and forskolin inhibited the elevation of PGE2 levels evoked by TPA (a dose dependent decrease of 35-55%). These data suggest that cAMP/PKA and PKC signaling have opposite effects on PGE2 and progesterone synthesis in FSHR cells. We propose that this PKA and PKC interplay on progesterone and PGE2 may be advantageous for the coordination of these key mediators for successful ovulation and luteinization.

Celecoxib interferes to a limited extent with aspirin-mediated inhibition of platelets aggregation.

AIMS: The aim of the study was to analyze the interaction between celecoxib and low dose aspirin for COX-1 binding and its consequences on the aspirin-mediated antiplatelet effects. METHODS: We investigated ex vivo the interaction between celecoxib and aspirin for COX-1 binding and measured the resulting antiplatelet effects. We applied mechanism-based pharmacokinetic-pharmacodynamic (PKPD) modelling to analyze these data and to predict in vivo platelet aggregation for different doses and administration schedules of aspirin and celecoxib. RESULTS: The predictions of the PK-PD model were consistent with results from previous studies that investigated interaction between aspirin and celecoxib. The modelling results indicate that celecoxib can attenuate to a limited extent the in vivo antiplatelet effects of low dose aspirin. The extent of this interaction can be substantial (up to 15% increase in platelet aggregation by 200 mg day(-1) celecoxib when combined with low dose aspirin) during the first days of aspirin administration in patients who are already treated with celecoxib, and it cannot be prevented by separate administration of the interacting drugs. CONCLUSIONS: At the recommended therapeutic doses, celecoxib can attenuate to a limited extent the in vivo antiplatelet effects of low dose aspirin. Patients receiving a combination of low dose aspirin and the recommended doses of celecoxib were not identified to have increased risk of cardiovascular and cerebrovascular events due to competition between these drugs for COX-1 binding. Interaction between low dose aspirin and other COX-2 inhibitors and its clinical consequences requires further investigation.

Competition between low-dose aspirin and other NSAIDs for COX-1 binding and its clinical consequences for the drugs' antiplatelet effects.

INTRODUCTION: NSAIDs are frequently used in modern medicine to inhibit the COX enzymes and induce analgesic, antipyretic, anti-inflammatory, and antiplatelet effects. Concomitant treatment with two or more NSAIDs can lead to their competition for binding and inhibition of the COX enzymes and altered time course of the pharmacological effects. AREAS COVERED: The competition between the low-dose aspirin and other NSAIDs for binding to COX-1 is described, including the recent findings on the differences in the interaction of NSAIDs with the individual COX-1 subunits, and the clinical consequences of this drug-drug interaction. The major pharmacokinetic (PK) and pharmacodynamic (PD) factors that govern the interaction of low-dose aspirin with other NSAIDs are explained, along with the approaches for prediction of the magnitude of this interaction using mechanism-based PK-PD modeling. EXPERT OPINION: Concomitant administration of other NSAIDs can diminish the antiplatelet effects of low-dose aspirin, increase the risk of thromboembolic effects (heart attacks and strokes), and lead to patient morbidity and mortality. The healthcare providers and the patients are seldom aware to this clinical problem and its consequences. Despite this drug interaction, low-dose aspirin possesses high clinical safety and it is not expected to be replaced by the recently approved drugs.

Down-regulation of cyclooxygenase-2 by the carboxyl tail of the angiotensin II type 1 receptor.

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or ß-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT1 in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT1 may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.

Prostaglandin receptor EP1-mediated differential degradation of cyclooxygenases involves a specific lysine residue.

The cyclooxygenase (COX) enzyme isoforms COX-1 and COX-2 catalyze the main step in the generation of prostanoids that mediate major physiological functions. Whereas COX-1 is a ubiquitously expressed stable protein, COX-2 is transiently upregulated in many pathologies and is often associated with a poor prognostic outcome. We have recently shown that an interaction of COX-2 with the prostaglandin EP1 receptor accelerates its degradation via a mechanism that augments its level of ubiquitination. Here we show that the sensitivity of both COX-1 and COX-2 to EP1 is altered upon modification of one lysine residue. A point mutation of lysine to-arginine in position 432 of COX-2 (K432R) yields an enzyme with decreased sensitivity to EP1 -mediated degradation. In contrast, insertion of a putative ubiquitination site into the corresponding position of COX-1 (H446K') yields an enzyme with higher levels of ubiquitination and reduced expression. Furthermore, compared to wild type COX-1, H446K' is significantly more sensitive to downregulation by EP1 . Together these data suggest that distinctive ubiquitination of COX-1 and COX-2 may be responsible for their different sensitivity to EP1 -mediated degradation.

Prostaglandin EP1 receptor down-regulates expression of cyclooxygenase-2 by facilitating its proteasomal degradation.

The enzyme cyclooxygenase-2 (COX-2) is rapidly and transiently up-regulated by a large variety of signals and implicated in pathologies such as inflammation and tumorigenesis. Although many signals cause COX-2 up-regulation, much less is known about mechanisms that actively down-regulate its expression. Here we show that the G protein-coupled receptor prostaglandin E(1) (EP(1)) reduces the expression of COX-2 in a concentration-dependent manner through a mechanism that does not require receptor activation. The reduction in COX-2 protein is not due to decreased protein synthesis and occurs because of enhancement of substrate-independent COX-2 proteolysis. Although EP(1) does not interfere with the entry of COX-2 into the endoplasmic reticulum-associated degradation cascade, it facilitates COX-2 ubiquitination through complex formation. Blockade of proteasomal activity results in degradation of the receptor and concomitant recovery in the expression of COX-2, suggesting that EP(1) may scaffold an unknown E3 ligase that ubiquitinates COX-2. These findings propose a new role for the EP(1) receptor in resolving inflammation through down-regulation of COX-2.

Coxibs interfere with the action of aspirin by binding tightly to one monomer of cyclooxygenase-1.

Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) through cyclooxygenase-2 (COX-2) pathways while thromboxane A(2) formed by platelets from AA via cyclooxygenase-1 (COX-1) mediates thrombosis. COX-1 and COX-2 are both targets of nonselective nonsteroidal antiinflammatory drugs (nsNSAIDs) including aspirin whereas COX-2 activity is preferentially blocked by COX-2 inhibitors called coxibs. COXs are homodimers composed of identical subunits, but we have shown that only one subunit is active at a time during catalysis; moreover, many nsNSAIDS bind to a single subunit of a COX dimer to inhibit the COX activity of the entire dimer. Here, we report the surprising observation that celecoxib and other coxibs bind tightly to a subunit of COX-1. Although celecoxib binding to one monomer of COX-1 does not affect the normal catalytic processing of AA by the second, partner subunit, celecoxib does interfere with the inhibition of COX-1 by aspirin in vitro. X-ray crystallographic results obtained with a celecoxib/COX-1 complex show how celecoxib can bind to one of the two available COX sites of the COX-1 dimer. Finally, we find that administration of celecoxib to dogs interferes with the ability of a low dose of aspirin to inhibit AA-induced ex vivo platelet aggregation. COX-2 inhibitors such as celecoxib are widely used for pain relief. Because coxibs exhibit cardiovascular side effects, they are often prescribed in combination with low-dose aspirin to prevent thrombosis. Our studies predict that the cardioprotective effect of low-dose aspirin on COX-1 may be blunted when taken with coxibs.

Interaction between prostaglandin E2 and l-cis-diltiazem, a specific blocker of cyclic nucleotide gated channels in bovine aortic endothelial cells.

Prostaglandins are known to transduce their signals via 7 transmembrane prostanoid receptors, which typically signal through coupling to G proteins and downstream second messenger molecules and protein kinase activation. Recently we have shown that cyclic nucleotides affect prostaglandins binding to bovine aortic endothelial cells independent of protein kinases. Here we show that incubation of bovine aortic endothelial cells with permeable analogs of cAMP or cGMP leads to a rapid and reversible reduction in PGE(2) binding to the cells. Since cyclic nucleotides are known modulators of cyclic nucleotide gated channels, we examined the effect of a specific cyclic nucleotide gated channel blocker l-cis-diltiazem on prostaglandin E(2) (PGE(2)) binding to bovine aortic endothelial cells. L-cis-diltiazem is shown to displace PGE(2) binding to bovine aortic endothelial cells in a dose dependent manner. In addition the effect of PGE(2) and l-cis-diltiazem on thapsigargin induced calcium elevation in the cells was compared. Both agents reduced in bovine aortic endothelial cells the thapsigargin induced calcium elevation by about half. PGE(2) also retarded the time course of the response to thapsigargin. Simultaneous treatment of the cells with both PGE(2) and l-cis-diltiazem did not yield an inhibitory effect beyond that observed with l-cis-diltiazem alone. Together our data point at the cyclic nucleotide gated channels as a feasible candidate for association with the PGE(2) binding site in bovine aortic endothelial cells.

Partnering between monomers of cyclooxygenase-2 homodimers.

Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2 convert arachidonic acid to prostaglandin H2 in the committed step of prostanoid biosynthesis. These enzymes are pharmacological targets of nonsteroidal antiinflammatory drugs and cyclooxygenase (COX) 2 inhibitors. Although PGHSs function as homodimers and each monomer has its own COX and peroxidase active sites, the question of whether there is cross-talk between monomers has remained unresolved. Here we describe two heterodimers in which a native subunit of human PGHS-2 has been coupled to a subunit having a defect within the COX active site at some distance from the dimer interface. Native/G533A PGHS-2, a heterodimer with a COX-inactive subunit, had the same specific COX activity as the native homodimer. Native/R120Q PGHS-2, a heterodimer in which both subunits can oxygenate arachidonic acid but in which the R120Q subunit cannot bind the COX inhibitor flurbiprofen, was inhibited by flurbiprofen to about the same extent as native PGHS-2. These results imply that native PGHS-2 exhibits half-of-sites reactivity. Isothermal titration calorimetry established that only one monomer of the native PGHS-2 homodimer binds flurbiprofen tightly. In short, binding of ligand to the COX site of one monomer alters its companion monomer so that it is unable to bind substrate or inhibitor. We conclude that PGHS monomers comprising a dimer, although identical in the resting enzyme, differ from one another during catalysis. The nonfunctioning subunit may provide structural support enabling its partner monomer to catalyze the COX reaction. This subunit complementarity may prove to be characteristic of other dimeric enzymes having tightly associated monomers.

Herpes simplex virus thymidine kinase gene transduction enhances tumor growth rate and cyclooxygenase-2 expression in murine colon cancer cells.

Transduction of tumor cells with herpes simplex virus thymidine kinase (HSV-tk) gene and subsequent treatment with the prodrug ganciclovir (GCV) is the most common system utilized to date for "suicide" gene therapy of cancer. In the current report, we show that HSV-tk gene transduction enhances tumor growth rate of murine colon cancer cells, that are implanted subcutaneously in syngeneic mice, and enhances cyclooxygenase-2 (COX-2) protein expression and prostaglandin E(2) (PGE(2)) release in vitro and in vivo. It is further shown that the observed phenomenon is related to the presence of the HSV-tk sequence insert in the retroviral vector used for HSV-tk gene delivery. Transduction of murine colon cancer cells with control vector, carrying the neomycin-resistance gene alone, failed to increase tumor growth rate and COX-2 protein expression or PGE(2) production. On the contrary, it even decreased tumor growth, COX-2 protein expression and PGE(2.) The growth rate of HSV-tk-transduced murine tumors was significantly reduced by treatment with the selective COX-2 inhibitor nimesulide. Additionally, we demonstrate herein that both enhanced growth rate of HSV-tk-transduced murine tumors and increased levels of PGE(2) in HSV-tk-transduced cells persist upon the development of GCV resistance. Taken together, these results provide a better understanding of the direct effect of HSV-tk gene transduction on tumor cell biology and target tumor development.

On the interaction of specific prostaglandin H synthase-2 inhibitors with prostaglandin H synthase-1.

Previous studies with both intact cells and ram seminal vesicles microsomes have shown that the specific PGHS-2 inhibitors NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide) and DuP-697 (5-bromo-2[4-fluorophenyl]-3-[4-methylsulfonylphenyl]-thiophene) attenuate the inhibition of PGHS-1 caused by aspirin and indomethacin. This effect occurs at concentrations of PGHS-2 inhibitors that do not inhibit the cyclooxygenase activity of PGHS-1. Here we study the effect of NS-398 and ibuprofen, a nonspecific inhibitor, on the indomethacin-induced inhibition of purified PGHS-1 and compare this effect with that observed with microsomal enzyme. Dissociation constants are obtained for the interaction of NS-398 with the purified and microsomal PGHS-1 using curve fitting of experimental data on the interaction of indomethacin with the enzyme. The dissociation constants for ibuprofen and NS-398 for interaction with PGHS-1 are similar. This finding indicates that specific PGHS-2 inhibitors are similar to ibuprofen in their ability to compete with aspirin, an irreversible time-dependent inhibitor of PGHS-1 often used for prevention of spontaneous thrombosis. Importantly, the concentrations at which PGHS-2 inhibitors attenuate the inhibition induced by aspirin and indomethacin are well below those required to cause inhibition of PGHS-1. Our results suggest that arachidonic acid not only competes with PGHS-2 inhibitors for binding to the cyclooxygenase site of PGHS-1 but it also reduces the affinities of PGHS-1 for these inhibitors by an additional, as yet unresolved mechanism.

Channel modulators affect PGE(2) binding to bovine aortic endothelial cells.

PGE(2), PGF(2alpha) and the thromboxane agonist U-46619 bind to bovine aortic endothelial cells and compete on the same binding site with similar affinity. In addition, binding remains unaffected by prolonged exposure to the ligand. These characteristics differ significantly from those of any known G-coupled prostaglandin receptor. Binding of PGE(2) to the cells is reduced in the presence of the cyclic nucleotides cGMP and cAMP, and is unaffected by protein kinase inhibitors. Removal of permeable cyclic nucleotides from the cell medium results in a fast and complete restoration of PGE(2) binding to the cells, suggesting that both cyclic nucleotides reduce PGE(2) binding by a reversible interaction with the prostaglandin-binding site, without the involvement of second messenger-activated protein kinases. Our data further show that binding of prostaglandins to bovine aortic endothelial cells is sensitive to heavy metals and to activators and blockers of calcium, ATP-sensitive K(+) and chloride channels. Nickel, a specific cyclic nucleotide-gated (CNG) channel activator, decreases PGE(2) binding and so do the CNG channel activators Rp-8-Br-PET-cGMPS and Sp-8-Br-PET-cGMPS. On the other hand, the calcium channel blockers pimozide, diltiazem as well as LY-83,583, a guanylate cyclase inhibitor, which were reported to block CNG channels, enhance PGE(2) binding. The sensitivity of PGE(2) binding to selective CNG channel modifying agents, as well as the rapid and reversible interaction with cyclic nucleotides, may suggest that the common low-affinity prostanoid-binding site on bovine aortic endothelial cells is associated with a molecular entity, which possess several properties of a CNG channel.