Gilad Rimon

PGHS-2 inhibitors, NS-398 and DuP-697, attenuate the inhibition of PGHS-1 by aspirin and indomethacin without altering its activity.

Since the discovery of the inducible form of prostaglandin (PG) H synthase (PGHS), PGHS-2, considerable effort has been made to design selective inhibitors of this isozyme. N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) have been shown to interact reversibly with PGHS-1, while irreversibly inhibiting PGHS-2 in a time-dependent manner. In the present study we have tested the effects of DuP-697 and NS-398 on the activity of PGHS-1 and further explored the interactions between these agents and the inhibition of PGHS-1 by aspirin, indomethacin and ibuprofen. Three independent experimental systems, namely bovine aortic endothelial cells (BAEC), human fibroblasts and ram seminal vesicle microsomes were used to investigate the effects of DuP-697 and NS-398 on PGHS-1. The results show that DuP-697 and NS-398, at concentrations ranges which do not inhibit PGHS-1 activity, significantly attenuated the inhibition of PGHS-1 that was caused by aspirin and indomethacin. The same concentrations of DuP-697 and NS-398 did not affect the inhibition of PGHS-1 that was induced by the competitive reversible inhibitors ibuprofen and naproxen. Similar effects of DuP-697 and NS-393 were obtained with ram seminal vesicle microsomes. These results suggest that PGHS-2 inhibitors DuP-697 and NS-398 possibly interact with PGHS-1 at a site different from the enzymes catalytic site, thus causing attenuation of PGHS-1 inhibition by aspirin and indomethacin without altering PGHS-1 basal activity or the ibuprofen-induced inhibition.

Lateral mobility of phospholipids in the external and internal leaflets of normal and hereditary spherocytic human erythrocytes

The lateral diffusion coefficients (D) and the mobile fractions of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) and of membrane proteins labelled with fluorescein isothiocyanate, were measured by fluorescence photobleaching recovery on erythrocytes from healthy persons and from a hereditary spherocytosis patient. Measurements of lipid probe mobility were performed on ghosts labelled by NBD-PE exclusively at the external monolayer, or at both sides of the membrane. Our results indicate the following: (1) The mean values and the temperature dependence of D are different at the external and internal membrane leaflets. (2) In both normal and HS ghosts the mobile fraction of NBD-PE in the external monolayer does not depend significantly on temperature. On the other hand, the mobile fraction in the internal monolayer is reduced as the temperature is decreased. (3) At low temperatures, the mobile fraction of NBD-PE in the internal monolayer of spherocytic ghosts is significantly lower than the mobile fraction in the internal monolayer of normal ghosts. (4) No differences were observed between the mobilities of membrane proteins in normal and in spherocytic ghosts. However, differences were observed between the two cell populations in the temperature-dependence of the intrinsic fluorescence of unlabelled membrane proteins. The implications of these results for membrane phospholipid asymmetry and for cytoskeletal interactions with the internal lipid monolayer are discussed.

Prostaglandin H synthase-2 inhibitors interfere with prostaglandin H synthase-1 inhibition by nonsteroidal anti-inflammatory drugs.

Ram seminal vesicle microsomes, a rich source of prostaglandin H synthase-1, were incubated with 100 nM of the prostaglandin H synthase-2 inhibitors N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) and 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DuP-697) prior to exposure to the prostaglandin H synthase inhibitors aspirin, indomethacin, ibuprofen or naproxen. Activity of the enzyme was measured by following the conversion of arachidonic acid to prostaglandin E(2) and prostaglandin F2alpha. Although prostaglandin H synthase-1 activity was not altered by these concentrations of the prostaglandin H synthase-2 inhibitors, it was found that exposure to these agents prior to aspirin or indomethacin (irreversible prostaglandin H synthase inhibitors) significantly attenuated the inhibition obtained by the latter inhibitors. On the other hand, the same concentrations of the prostaglandin H synthase-2 inhibitors did not interfere with prostaglandin H synthase-1 inhibition that was induced by naproxen or ibuprofen (competitive prostaglandin H synthase inhibitors). Attenuation of the indomethacin inhibition of prostaglandin H synthase-1 by prostaglandin H synthase-2 inhibitors was observed only when the microsomes were pre-exposed to DuP-697 or NS-398 in the absence, but not in the presence, of arachidonic acid. The effect of DuP-697 was found to be irreversible, however, washing away the agent reversed the action of NS-398. Similar phenomena have been reported by us in bovine aortic endothelial cells and in human dermal fibroblasts. Attenuation of the inhibition by aspirin and indomethacin, without altering the enzymes basal activity or the inhibition induced by ibuprofen or naproxen may suggest the possibility that the prostaglandin H synthase-2 specific inhibitors DuP-697 and NS-398 affect prostaglandin H synthase-1 by interaction with a site different from the enzymes catalytic site.

Prostaglandin E2 can bimodally inhibit and stimulate the epididymal adipocyte adenylyl cyclase activity.

Measurements of prostaglandin E2 (PGE2)-induced adenylyl cyclase activity in membranes isolated from epididymal rat adipocytes revealed inhibition of cAMP production at low concentrations of PGE2 (less than 10 mM) and stimulation at higher concentrations. This biphasic effect of PGE2 was obtained when adenylyl cyclase was stimulated with GTP or NaF. In the presence of forskolin only the inhibitory phase by PGE2 was observed. Sulprostone, a PGE2 analogue, did not affect cAMP synthesis in the presence of either GTP or NaF; however, in the presence of forskolin, it inhibited cAMP production similarly to PGE2. Treatment of the membranes with cholera or pertussis toxin did not alter the biphasic effect of PGE2 on cAMP production. These findings raise the possibility that PGE2 acts through several receptor subtypes which are coupled to GTP binding proteins different from the classical Gi or Gs proteins.

Prostaglandin EP1 Receptor Down-regulates Expression of Cyclooxygenase-2 by Facilitating Its Proteasomal Degradation

Background: Little is known about cellular mechanisms that actively lower the levels of the proinflammatory enzyme COX-2. Results: An interaction between COX-2 and the prostaglandin receptor EP1 facilitates the degradation of COX-2 through the proteasome. Conclusion: EP1 negatively regulates COX-2 expression. Significance: Receptor-mediated regulation of COX-2 presents an important means of moderating inflammation and resolution of pathophysiological events. 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 E1 (EP1) 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 EP1 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 EP1 may scaffold an unknown E3 ligase that ubiquitinates COX-2. These findings propose a new role for the EP1 receptor in resolving inflammation through down-regulation of COX-2.

Modulation of endothelial prostaglandin synthesis by corticotropin releasing factor and antagonists.

Corticotropin releasing factor (CRF) is a hypothalamic hormone that also displays autocrine/paracrine roles at peripheral sites. High concentrations of CRF have been identified in endothelial cells and other inflammatory tissues. We investigated the effects of CRF and antagonists in the regulation of prostaglandin synthesis in bovine aortic endothelial cells, and also characterized the binding of CRF in these cells. Interleukin-1alpha increased prostacyclin (prostaglandin I2) synthesis in endothelial cells and this response to interleukin-1alpha was abolished by simultaneous exposure to CRF. The effect of CRF on interleukin-1alpha-induced prostaglandin synthesis was antagonised by the CRF receptor antagonist alpha-helical CRF-(9-41). In addition, this as well as another CRF receptor antagonist, namely [D-Phe12]CRF-(12-41), when applied alone at low concentrations inhibited the interleukin-1alpha-induced prostaglandin synthesis similarly to CRF, suggesting partial agonistic action. Binding of [125I]-labeled CRF in endothelial cells was saturable and fitted a two sites model. Kd for the higher-affinity class of receptors was 0.2 +/- 0.02 nM, and Bmax 0.79 +/- 0.095 fmol/mg protein. The lower-affinity class of receptors had a Kd of 1.77 +/- 0.14 microM and Bmax 0.97 +/- 0.12 fmol/mg protein. These findings suggest a direct role for CRF in the local regulation of inflammation.

Regulation of a common, low-affinity binding site for primary prostanoids on bovine aortic endothelial cells

Bovine aortic endothelial cells contain a prostaglandin site which binds with similar low-affinity PGE2, PGF2alpha and the thromboxane agonist U-46619. Treatment of the cells with agents that increase the level of cellular cAMP such as forskolin, a direct activator of adenylate cyclase or IBMX, a phosphodiesterase inhibitor, decreased the binding of PGE2 to the cells. Addition of dibutyryl cAMP to intact cells caused a quick reduction in PGE2 binding with a half time of less than 2 min. The reduction in PGE2 binding was completely reversible after removing the dibutyryl cAMP. The reduction in PGE2 binding after addition of dibutyryl cAMP to the intact cells was also observed after a mechanical disruption of the cells or after permeabilization with digitonin. Incubation of the cells with myristoylated PKI(14-22) amide, a specific protein kinase A inhibitor, resulted in partial suppression of the reduction of PGE2 binding by dibutyryl cAMP. Pretreatment of intact cells for 24 h with 10(-6) M PGE2 or a PKC activator did not reduce the specific binding of [3H]-PGE2. These results suggest that PKA, but not PKC, is involved in a fast reversible regulation of the common prostanoid receptor on bovine endothelial cells.

Corticotropin releasing factor modulates interleukin-1-induced prostaglandin synthesis in fibroblasts: receptor binding and effects of antagonists

Corticotropin releasing factor (CRF) is a predominant regulator of the neuroendocrine, autonomic and behavioral responses to stress. In addition, numerous studies support autocrine/paracrine roles for this peptide at peripheral sites. CRF and CRF binding sites have been identified in different regions of the central nervous system as well as in the heart, spleen, adrenal and testis, and high levels of CRF were detected in inflamed fibroblasts. However, the precise physiological or pathophysiological role of peripheral CRF cannot yet be discerned. Here we show that CRF, through interaction with specific membrane receptors, blocks the interleukin-1alpha (IL-1alpha)-stimulated prostaglandin (PG) synthesis in fibroblasts. Binding of [125I]-labeled CRF in fibroblasts was saturable and fitted a two sites model. K(D) for the higher-affinity class of receptors was 20+/-2.2 pM, and Bmax 1.95+/-0.22 fmol/mg protein. For the lower-affinity class of receptors K(D) was 160+/-17 nM, and Bmax 2.38+/-0.27 fmol/mg protein. CRF blocked the effect of IL-1alpha on PGE2 synthesis, and this was antagonised by D-PheCRF12-41. In addition, the CRF receptor antagonists alpha helical CRF9-41 and D-PheCRF12-41 at high concentrations inhibited the IL-1alpha-induced PG synthesis similarly to CRF, suggesting partial agonistic action. Taken together, these results suggest a modulatory role of CRF in inflammation.

The involvement of nuclear factor-kappa B in cyclooxygenase-2 overexpression in murine colon cancer cells transduced with herpes simplex virus thymidine kinase gene

We have previously reported that transduction of murine colon cancer cells (MC38) with herpes simplex virus thymidine kinase (HSV-tk) gene results in a significant enhancement of tumor growth rate in vivo and overexpression of cyclooxygenase-2 (COX-2). Our current study aimed to investigate the involvement of nuclear factor-kappa B (NF-κB), a pivotal transcriptional regulator of COX-2, in the upregulation of COX-2 expression by HSV-tk. It was found that HSV-tk gene transduction of MC38 cells results in significantly enhanced NF-κB activity, increased phosphorylation and degradation of inhibitor-kappa Bα (IκBα) and enhanced translocation of NF-κB to the nucleus. Treatment of HSV-tk-transduced MC38 cells with sulfasalazine, a potent NF-κB inhibitor, led to dose-dependent inhibition of NF-κB activity, IκB phosphorylation and nuclear translocation of NF-κB, accompanied by significantly decreased COX-2 expression and reduced release of prostaglandin E2. Transient transfection experiments with COX-2 promoter constructs fused to luciferase reporter gene revealed that mutation in NF-κB-responsive element of COX-2 promoter significantly reduced promoter activity in HSV-tk-transduced MC38 and COS-7 cells, whereas it had no effect on promoter activity in the respective wild-type cells. At last, it was found that HSV-tk gene transduction causes significant enhancement of NF-κB activity and COX-2 expression in two additional tumor cell lines, 9L and T24. These findings suggest that HSV-tk gene transduction results in NF-κB pathway activation, which is essential for COX-2 overexpression by HSV-tk.

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.