Clinical estimation of corneal endothelial pump function. (57/6807)

PURPOSE: To develop a technique to estimate the corneal endothelial pump rate in human subjects. METHODS: Corneal hydration control is thought to be maintained by a pump-leak mechanism whereby the leak of solutes and fluid across the endothelial barrier into the stroma is, in the steady state, exactly balanced by the pumping of solutes and passive fluid transfer across the endothelium to the aqueous humor. Overall corneal hydration control can be measured from the rate at which the swollen cornea thins (deswells), and a measure of the leak can be obtained simultaneously from the endothelial permeability to fluorescein. From the pump-leak hypothesis, the deswelling rate is directly proportional to the pump rate and inversely proportional to the leak rate. The relative endothelial pump rate can be estimated as the product of the normalized deswelling rate and the normalized endothelial permeability. This procedure was used to obtain the relative endothelial pump rate in 41 patients with diabetes mellitus, 12 patients with long-term corneal transplants, 20 long-term wearers of contact lenses, and 19 normal volunteer subjects after the short-term administration of topical dorzolamide. RESULTS: The relative endothelial pump rate did not differ significantly from that of control subjects in diabetics, in contact lens wearers, and after dorzolamide administration, but was markedly decreased in the patients with corneal transplants, despite a reduction in permeability (reduced leak). CONCLUSIONS: This method allows the estimation of both the barrier and pump arms of corneal endothelial function and should be useful in the investigation of causes and mechanisms of functional endothelial insufficiency.  (+info)

Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomes. (58/6807)

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.  (+info)

Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action. (59/6807)

Macrophage migration inhibitory factor (MIF) is an important pro-inflammatory mediator with the unique ability to counter-regulate the inhibitory effects of glucocorticoids on immune cell activation. MIF is released from cells in response to glucocorticoids, certain pro-inflammatory stimuli, and mitogens and acts to regulate glucocorticoid action on the ensuing inflammatory response. To gain insight into the molecular mechanism of MIF action, we have examined the role of MIF in the proliferation and intracellular signaling events of the well characterized, NIH/3T3 fibroblast cell line. Both endogenously secreted and exogenously added MIFs stimulate the proliferation of NIH/3T3 cells, and this response is associated with the activation of the p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinases (MAP). The MIF-induced activation of these kinases was sustained for a period of at least 24 h and was dependent upon protein kinase A activity. We further show that MIF regulates cytosolic phospholipase A2 activity via a protein kinase A and ERK dependent pathway and that the glucocorticoid suppression of cytokine-induced cytoplasmic phospholipase A2 activity and arachidonic acid release can be reversed by the addition of recombinant MIF. These studies indicate that the sustained activation of p44/p42 MAP kinase and subsequent arachidonate release by cytoplasmic phospholipase A2 are important features of the immunoregulatory and intracellular signaling events initiated by MIF and provide the first insight into the mechanisms that underlie the pro-proliferative and inflammatory properties of this mediator.  (+info)

Relationship of nimesulide safety to its pharmacokinetics: assessment of adverse reactions. (60/6807)

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most widely used drugs and their use is frequently associated with severe or even serious adverse events, which increase morbidity and mortality. The increase of toxic effects, primarily of the digestive system, due to treatment with NSAIDs, underlines a need for safer NSAIDs. Nimesulide (4-nitro-2-phenoxymethanesulphonanilide) is a chemically unique anti-inflammatory agent in that it has a higher pKa (6.5) than conventional acidic NSAIDs and it is one of the newer class of NSAIDs that are selective for cyclooxygenase-2. Nimesulide also has additional activities, among them effects on production/action of oxy-radicals and other components of neutrophil activation that may contribute to the spectrum of its anti-inflammatory activity as well as potentially reducing the likelihood of gastrointestinal ulcerogenicity. An analysis was performed of the safety data of nimesulide collected in clinical studies and from those reported spontaneously worldwide in the post-marketing phase. The results show that nimesulide is associated with a relatively low occurrence of adverse drug reactions especially in the gastrointestinal tract while those in the liver are within or below the general incidence with other NSAIDs.  (+info)

Effect of nimesulide on glucocorticoid receptor activity in human synovial fibroblasts. (61/6807)

Fibroblasts from human synovial membranes were cultured with nimesulide, naproxen or dexamethasone. Nimesulide, but not naproxen, showed effects on the glucocorticoid system that may contribute importantly to its anti-inflammatory activity. Nimesulide at therapeutically relevant concentrations induced the intracellular phosphorylation and activation of glucocorticoid receptors, and activated their binding to the target genes. Naproxen or dexamethasone markedly reduced the number of glucocorticoid receptor binding sites, in contrast to nimesulide, which had no significant effect.  (+info)

Molecular model of the interaction between nimesulide and human cyclooxygenase-2. (62/6807)

The cyclooxygenase-2 (COX-2) isoenzyme is a key target for COX-2-selective non-steroidal anti-inflammatory drugs (NSAIDs). An important difference in binding of nimesulide compared with non-selective NSAIDs appears to involve the amino acid at position 523 of the enzyme. Replacement of valine with isoleucine at this position provides access to a binding site that is larger in COX-2 than in COX-1. Nimesulide appears to exploit this enlarged binding site for establishing a number of favourable contacts with the enzyme that lead to selective inhibition of COX-2. We made these conclusions from a three-dimensional molecular model of the active site of human COX-2, constructed using the X-ray coordinates of COX-1 from sheep seminal vesicles and COX-2 from mouse fibroblasts as templates, with the aid of sequence alignment methods and molecular modelling techniques. The resulting model was refined, and the active site was probed for regions of steric and electrostatic complementarity for ligand binding. Docking studies were then undertaken with many different nimesulide conformers, a family of which could establish very favourable interactions with the NSAID binding site of human COX-2 by exploiting the extra space made available by the isoleucine/valine replacement. The stability of the resulting complexes was studied by simulating molecular dynamics.  (+info)

The in vivo assessment of nimesulide cyclooxygenase-2 selectivity. (63/6807)

In man, nimesulide selectively inhibits cyclooxygenase-2 (COX-2) with little effect on haemostatic function or gastric prostaglandin formation. It causes significantly less gastrointestinal injury than naproxen, but has anti-inflammatory efficacy similar to that of naproxen and other currently available non-steroidal anti-inflammatory drugs. Naproxen suppressed arachidonic-acid-mediated platelet aggregation, reduced serum thromboxane B2 levels by 98% throughout the treatment period and reduced gastric mucosal prostaglandins (PGE2 and 6-keto-PGF1alpha) production by an average of 80%. This contrasts with nimesulide: platelet aggregation was not significantly affected, thromboxane B2 levels were only 29% lower and the gastric mucosal prostaglandins were inhibited in the order of approximately 20%. During the treatment period, both nimesulide and naproxen significantly inhibited COX-2-dependent PGE2 synthesis in the whole blood; however, naproxen had a lesser effect than nimesulide.  (+info)

Gastrointestinal toxicity of non-steroidal anti-inflammatory drugs: the effect of nimesulide compared with naproxen on the human gastrointestinal tract. (64/6807)

This overview includes theories and evaluation of non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal toxicity. Factors in damage include microvascular aspects, neutrophil recruitment, mucosal prostaglandins, gastrointestinal secretions and bacteria. We have proposed an extensive simplified framework that includes an important local initiating effect which may involve NSAID accumulation, interaction with surface phospholipids, events that alter cellular ATP, and local/systemic effects of cyclooxygenase (COX) inhibition. COX-2-selective drugs are desirable not only because they spare COX-1 and so avoid gastrointestinal toxicity, but also because COX-2-selective agents are only weakly acidic and therefore avoid substantial accumulation in the gastric mucosa. Short-term endoscopy studies of NSAIDs are important initially to evaluate human gastroduodenal tolerability. They show that injury increases with the amount of NSAIDs even though the lowest therapeutic doses inhibit gastric COX almost completely, and that the more-acidic NSAIDs tend to cause greater gastric damage. Long-term endoscopy studies involve NSAID ingestion for at least 3 months. A main question is the extent to which the ulcers seen cause symptoms, substantial bleeding and/or perforation. Measurement of serious outcomes is thought by many to be the best assessment of gastrointestinal safety, but studies find marked variations even with the same drug. Damage to the small intestine by NSAIDs is even more frequent than to the upper gastrointestinal tract, but is difficult to evaluate. Conventional acidic NSAIDs increase the permeability of human small intestine, probably by a non-prostaglandin mechanism, but nimesulide does not do so, possibly because of its very weak acidity.  (+info)