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The synthesis and the pharmacological activity of a series of 1,5-diarylimidazoles developed as potent and selective cyclooxygenase-2 (COX-2) inhibitors are described. The new compounds were evaluated both in vitro (COX-1 and COX-2 inhibition in human whole blood) and in vivo (carrageenan-induced paw edema, air-pouch, and hyperalgesia tests). Modification of all the positions of two regioisomeric imidazole cores led to the identification of 4-[4-chloro-5-(3-fluoro-4-methoxyphenyl)imidazol-1-yl]benzenesulfonamide (UR-8880, 51f) as the best candidate, which is now undergoing Phase I clinical trials.

The mechanism that promotes transition from the ATP-to the ADP-liganded state of ATP-sensitive K ؉ (K ATP ) channels and consequent channel opening in a cytosolic environment of high ATP concentration has yet to be understood. A mechanism examined here that could reverse the ATP-inhibited state is based on the action of adenylate kinase to catalyze phosphoryl transfer between ATP and AMP, resulting in transformation of ATP into ADP. In membrane patches excised from guinea pig cardiomyocytes, AMP alone did not affect channel behavior but increased the open probability of ATP-inhibited K ATP channels. This required MgCl 2 and a hydrolyzable form of ATP and was prevented by P 1 ,P 5 -di-adenosine-5 -pentaphosphate, an inhibitor of adenylate kinase. The single channel amplitude and kinetics of channel openings induced by the ADP-generating substrates of adenylate kinase, AMP and MgATP, were indistinguishable from the biophysical properties of the K ATP channel exhibited after addition of MgADP. In whole cell voltage-clamped cardiomyocytes, introduction of exogenous adenylate kinase along with millimolar MgATP and AMP induced a K ؉ current that was suppressed by a sulfonylurea blocker of K ATP channels. Enriched sarcolemmal membrane preparations were found to possess ATP⅐AMP phosphotransferase activity with properties attributable to an extramitochondrial isoform of adenylate kinase. These results indicate that adenylate kinase is a naturally occurring component of sarcolemmal membranes that could provide dynamic governance of K ATP channel opening through its phosphoryl transfer catalytic action in the microenvironment of the channel.ATP-sensitive K ϩ (K ATP ) 1 channels are involved in signaling networks that transduce cellular metabolic events into membrane potential changes and have been implicated in glucoseinduced insulin secretion in pancreatic ␤ cells or ischemiaassociated action potential shortening in heart muscle (1-5). Although the defining property of K ATP channels is their inhibition by intracellular ATP (5), which is readily demonstrable in excised membrane patches, as is the effect of ADP to reverse this ATP-inhibited state (1, 2, 6 -9), the mechanism by which opening of this channel is governed in situ has not been elucidated. The major question still to be resolved is how transition from the ATP-to the ADP-liganded state is accomplished.The common assumption that changes in the cytosolic concentrations of adenine nucleotides are the sole determinant of K ATP channel opening has been contested (2, 4, 6, 10 -13). In cardiac cells, the ATP concentration (ϳ5-10 mM) exceeds by over 100-fold the IC 50 value for K ATP channel closure (12, 13). This translates into a requirement for a change of two orders of magnitude in the intracellular ATP concentration, which is incompatible with cell viability, to achieve a mass action-induced change in the state of ATP-liganding and channel opening.Considering that altered concentrations of cytosolic ATP and/or ADP are not readily detectable nor correlated with predi...

The efficacy with which sulfonylurea drugs inhibit cardiac ATP-sensitive K+ (KATP) channels is reduced during metabolic compromise and cellular contracture. Disruption of the actin microfilament network, which occurs under similar conditions, reduces the sensitivity of the channel toward intracellular ATP. To investigate whether a disrupter of actin microfilaments could also affect the responsiveness of the KATP channel to sulfonylurea drugs, single-channel currents were measured in the inside-out configuration of excised patches from guinea pig ventricular myocytes. Treatment of the internal side of patches with deoxyribonuclease (DNase) I (100 micrograms/ml), which forms complexes with G actin and prevents actin filament formation, antagonized sulfonylurea-induced inhibition of KATP channels that was coupled with a loss of sensitivity to ATP. The apparent dissociation constant and Hill coefficient for the inhibitory effect of glyburide, a prototype sulfonylurea, on KATP-channel opening were, respectively, 0.13 microM and 0.95 before and 2.7 microM and 0.98 after DNase treatment. DNase did not alter intraburst kinetic properties of the channel. When DNase was denatured or coincubated with purified actin (200 micrograms/ml), it no longer decreased glyburide-induced channel inhibition. This suggests that sulfonylurea-inhibitory gating of cardiac KATP channels may also be regulated through a mechanism involving subsarcolemmal actin microfilament networks.

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Diarrhea induced by current, demonstrating that inhibition of Cl؊ flux reflected selective disruption of ligand stimulation of GCC rather than the chloride channel itself. Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing the utility of this pathway to elucidate the mechanisms regulating ST-dependent guanylyl cyclase signaling and intestinal fluid homeostasis. In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for enterotoxigenic diarrhea.Guanylyl cyclase C (GCC), 1 the receptor for Escherichia coli heat-stable enterotoxin (ST a ) expressed in intestinal mucosa cells, is a member of the receptor guanylyl cyclase family that possesses receptor and catalytic domains on a single transmembrane protein (1, 2). Occupancy by ST a of the extracellular receptor domain induces catalytic conversion of intracellular GTP to cyclic GMP (cGMP), resulting in sequential alterations in epithelial cell chloride flux, electrolyte and fluid secretion, and diarrhea (3-7). Interventions that specifically interrupt the ST a -induced GCC-mediated signal sequence have not been defined. In cell-free systems, GCC is allosterically inhibited by 2-substituted adenine nucleotides (8, 9). Yet, the impermeance of intact cells to phosphorylated nucleotides and the absence of endogenous 2-substituted nucleotides has precluded the disruption of ST a -induced signaling in intestinal cells through this inhibitory pathway. However, intestinal cells express transporters, which carry 2-substituted nucleosides into the cytosol, and adenosine kinase, which catalyzes conversion of 2-substituted nucleosides into 2-substituted nucleotides (10). The present studies examine whether that mechanism can be exploited to interrupt transmembrane signaling and alterations in chloride flux induced by ST a in intact intestinal epithelial cells. EXPERIMENTAL PROCEDURESCyclic GMP Accumulation in Intact Cells-Caco 2 cells, well differentiated human colon carcinoma cells, were seeded in 24-well plates, allowed to reach confluence, and grown for an additional 14 -21 days to ensure differentiation of these cells into colonic enterocytes. HEK293 cells, human embryonic kidney cells expressing recombinant GCC, were seeded in 24-well plates, allowed to reach confluence, and used for assays at least 5 days after seeding (1, 11). Cells were incubated in OPTI-MEM serum-free media (Life Technologies, Inc.) (0.5 ml/well) containing indicated concentrations of the test substances for the given period of time. Cells were washed three times with OPTI-MEM, then incubated in OPTI-MEM (0.2 ml/well) containing 0.12 mM isobutylmethylxanthine to inhibit endogenous phosphodiesterases for 10 min. ST a was added to a final concentration of 0.5 M for 10 min. Trichloroacetic acid (0.2 ml of 12% solution) was added to the wells to lyse the cells and terminate the reaction. Well contents were collected and centrifuged 15 min in a micro...

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EditorialLas enfermedades cardiovasculares: un problema de salud pública y un reto global Las enfermedades cardiovasculares son responsables de la mayor parte de las muertes en el mundo. De acuerdo con el Informe del Estado Global en Salud de la Organización Mundial de la Salud (OMS), publicado en abril de 2011 (1), las enfermedades crónicas no transmisibles fueron la causa de, aproximadamente, el 63 % (36 millones) del total (57 millones) de muertes ocurridas en el mundo en el año 2008. De las cuatro principales enfermedades crónicas no transmisibles -las enfermedades cardiovasculares, el cáncer, la diabetes y las enfermedades respiratorias crónicas-las cardiovasculares fueron las causantes del 29,82 % (17 millones) de las muertes (1).La OMS estimó la mortalidad y la prevalencia de enfermedades crónicas en cada estado miembro. Resulta alarmante que, en todos los continentes, con la excepción de África, las muertes por enfermedades no transmisibles superan las de las enfermedades transmisibles, maternas y perinatales, nutricionales y causas combinadas (1,2).En los países desarrollados, por ejemplo, los Estados Unidos, más de 2.200 personas mueren de enfermedades cardiovasculares cada año y éstas son la causa de una de cada 2,9 muertes; además, en promedio, cada día muere una persona cada 39 segundos en este país (1-4). En los países europeos se observa una tendencia similar a la de los norteamericanos. Para los hombres europeos, por ejemplo, las muertes por las enfermedades crónicas no transmisibles son 13 veces más altas que otras causas combinadas, y para los hombres en la región occidental del Pacífico, son ocho veces mayor (1-3).La muerte por enfermedades infecciosas ha disminuido en la mayoría de los países en las últimas décadas. A medida que los países más pobres mejoren sus condiciones de vida, es probable que sus sistemas de salud mejoren y que se reduzcan los niveles de enfermedades infecciosas y parasitarias. A pesar de la prevalencia de las enfermedades transmisibles (como el VIH/sida, la malaria y la tuberculosis), las enfermedades cardiovasculares siguen en aumento en el número de muertes y cobran más "años de vida perdidos" en los países en desarrollo, porque afectan no sólo a personas mayores, sino también a personas jóvenes (4,5).