Mazal Rubin

Inhibition of Cyclooxygenase 2 by Nimesulide Decreases Prostaglandin E2 Formation But Does Not Alter Brain Edema or Clinical Recovery After Closed Head Injury in Rats

Recently, the enzyme cyclooxygenase (COX) has been recognized to exist as constitutive (COX-1) and inducible isoforms (COX-2). In previous studies, drugs that were inhibitors of both COX-1 and COX-2 failed to decrease brain edema formation or improve Neurological Severity Score (NSS) after closed head trauma (CHT), although some did decrease prostaglandin-E2 (PGE2) formation. The present study examined whether or not a specific inhibitor of COX-2 (nimesulide) exerts a beneficial effect after CHT in rats. Halothane-anesthetized rats (n = 8 in each group) were randomly assigned to one of four groups: surgery, no CHT, no drug (group 1); surgery, no CHT, nimesulide 30 mg/kg intraperitoneally (IP) (group 2); surgery, CHT, no drug (group 3); and surgery, CHT, nimesulide 30 mg/kg IP (group 4). NSS was determined at 1 and 24 h, and brain tissue PGE2 concentration and water content were determined after killing at 24 h. Treatment with nimesulide did not improve NSS (NSS at 24 h = 11+/-6 [median +/- range] in group 3 and 12+/-4 in group 4) or edema formation (brain water content at 24 h = 84.3+/-1.8% [mean +/- SD] in group 3 and 83.8+/-1.9% in group 4). However, nimesulide did decrease cortical and hypothalamic PGE2 formation by 41% and 47%, respectively during the first hour of incubation after brain tissue sampling. The authors conclude that although nimesulide does reduce tissue PGE2 formation, it does not exert a beneficial effect on brain tissue edema or functional activity after CHT in rats.

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.

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.

Effects of Nimesulide, a Selective Cyclooxygenase-2 Inhibitor, on Cardiovascular Alterations in Endotoxemia

Prostanoids and cytokines are known to play a pivotal role in the mechanisms leading to endotoxin-induced cardiovascular failure. We investigated the effect of nimesulide (NIM), a selective cyclooxygenase-2 (COX-2) inhibitor, on the cardiovascular alterations occurring during endotoxemia, and on prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in endotoxemic rats. NIM significantly reduced endotoxin-induced elevation of plasma and myocardial levels of TNF-α, but not those of IL-1β. Searching for the mechanism underlying the anti-TNF-α effect of NIM, it was found that the drug reduced nuclear factor kappa B activation through diminished nuclear levels of p-65 accompanied by a protective effect against the cardiovascular alterations and mortality seen during endotoxemia. In addition, the inhibitory effect of NIM on endotoxin-induced elevation in plasma and hypothalamic levels of PGE2 was noteworthy, and this may suggest that the large amounts of PGE2 observed during endotoxemia are mainly produced via COX-2.

Carrier-mediated uptake of Levofloxacin by BeWo cells, a human trophoblast cell line

ObjectivePlacental transfer of Levofloxacin (LF), a broad spectrum fluoroquinolone antibiotic, and its inhibition was investigated in BeWo cells, a human trophoblast cell line.MethodsThe experiments of LF uptake by BeWo cells were performed after preincubation and in the presence of the P-glycoprotein inhibitors (Cyclosporin A, Verapamil and Quercetin), the organic anion/cation transporter inhibitor (Cimetidine) and the MCT substrates (lactic acid and salicylic acid).ResultsP-glycoprotein inhibitors increased the uptake of LF by BeWo cells. The increase in LF accumulation by Cyclosporin A, Verapamil and Quercetin was by 30, 90 and 80%, respectively. Cimetidine, the organic cation inhibitor, increased the transport of LF by 48%. Lactic acid and salicylic acid, the MCT substrates, initially decreased the accumulation of LF by 30% and subsequently increased the uptake of LF by 500 and 53%, respectively.ConclusionsThe uptake of LF by human trophoblast cells is mediated by multiple transporters as well as passive diffusion.

Channel modulators affect PGE2 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.

Effects of closed head trauma and lipopolysaccharide on body temperature, brain tissue water content, and PGE2 production in rats.

Closed head trauma (CHT) increases brain tissue prostaglandin E2 (PGE2) concentration, and that increase is associated with cerebral edema formation and worsening of the neurologic severity score (NSS). Injection of the bacterial endotoxin lipopolysacharride (LPS) increases cerebral and hypothalamic PGE2, and the hypothalamic increase is associated with increased body temperature. The present study determined (a) whether LPS-induced increase of PGE2 causes brain edema or worsens NSS and (b) whether CHT increases hypothalamic PGE2 and thereby increases body temperature. Halothane-anesthetized rats were divided into four groups: group 1 = surgery with no CHT and no LPS (n = 8); group 2 = surgery with LPS and no CHT (n = 8); group 3 = surgery with CHT and no LPS (n = 8); and group 4 = surgery with CHT plus LPS (n = 8). NSS was determined at 1 and 24 h after injury, and brain tissue PGE2 and edema were determined when animals were killed 24 h after injury. As compared with group 1, LPS alone, but not CHT or CHT plus LPS, increased rectal temperature. CHT and CHT plus LPS, but not LPS alone increased brain water content and worsened NSS. LPS, CHT, and CHT plus LPS all increased hypothalamic and cerebral PGE2 production. We conclude that although LPS and CHT increased PGE2 levels, LPS alone did not affect neurologic status or brain edema, CHT did not increase rectal temperature, and addition of LPS to CHT did not aggravate the sequelae of CHT.