Mice deficient in inducible nitric oxide synthase (iNOS) were generated to test the idea that iNOS defends the host against infectious agents and tumor cells at the risk of contributing to tissue damage and shock. iNOS-/-mice failed to restrain the replication of Listeria monocytogenes in vivo or lymphoma cells in vitro. Bacterial endotoxic lipopolysaccharide (LPS) caused shock and death in anesthetized wild-type mice, but in iNOS-/-mice, the fall in central arterial blood pressure was markedly attenuated and early death averted. However, unanesthetized iNOS-/-mice suffered as much LPS-induced liver damage as wild type, and when primed with Propionobacterium acnes and challenged with LPS, they succumbed at the same rate as wild type. Thus, there exist both iNOS-dependent and iNOS-independent routes to LPS-induced hypotension and death.

The A 3 adenosine receptor (A3AR) is one of four receptor subtypes for adenosine and is expressed in a broad spectrum of tissues. In order to study the function of A3AR, a mouse line carrying a mutant A 3 allele was generated. Mice homozygous for targeted disruption of the A3AR gene, A3AR ؊/؊ , are fertile and visually and histologically indistinguishable from wild type mice. The lack of a functional receptor in the A3AR ؊/؊ mice was confirmed by molecular and pharmacological analyses. The absence of A3AR protein expression in the A3AR ؊/؊ mice was demonstrated by lack of N 6 -(4-amino-3-[ 125 I]iodobenzyl)adenosine binding to bone marrowderived mast cell membranes that were found to express high levels of A3AR in wild type mice. In A3AR ؊/؊ mice, the density of A 1 and A 2A adenosine receptor subtypes was the same as in A3AR ؉/؉ mice as determined by radioligand binding to brain membranes. Additionally, A 2B receptor transcript expression was not affected by ablation of the A3AR gene. A3AR ؊/؊ mice have basal heart rates and arterial blood pressures indistinguishable from A3AR ؉/؉ mice. Functionally, in contrast to wild type mice, adenosine and the A3AR-specific agonist, 2-chloro-N 6 -(3-iodobenzyl)-adenosine-5-N-methylcarboxamide (2-Cl-IB-MECA), elicit no potentiation of antigen-dependent degranulation of bone marrow-derived mast cells from A3AR ؊/؊ mice as measured by hexosaminidase release. Also, the ability of 2Cl-IB-MECA to inhibit lipopolysaccharide-induced tumor necrosis factor-␣ production in vivo was decreased in A3AR ؊/؊ mice in comparison to A3AR ؉/؉ mice. The A 2A adenosine receptor agonist, 2-p-(2-carboxyethyl)phenylamino)-5-Nethylcarboxamidoadenosine, produced inhibition of lipopolysaccharide-stimulated tumor necrosis factor-␣ production in both A3AR ؊/؊ and A3AR ؉/؉ mice. These results show that the inhibition in vivo can be mediated by multiple subtypes, specifically the A 3 and A 2A adenosine receptors, and A3AR activation plays an important role in both pro-and anti-inflammatory responses.Adenosine is a naturally occurring nucleoside that exhibits diverse and potent physiological responses in the central nervous system and the cardiovascular, renal, pulmonary, and immune systems. The actions of adenosine are mediated through G-protein-coupled receptors classified into four subtypes, A 1 , A 2A , A 2B , and A 3 , on the basis of their affinity order profiles for agonists and antagonists (1-3). The A 3 adenosine receptor (A3AR) 1 is the most recent subtype identified, since it had remained pharmacologically obscure until its gene was cloned in the early 1990s. The A3AR has been cloned from multiple species including rat (4, 5), human (6, 7), sheep (8), canine (9), and rabbit (10).The physiological importance of the A 3 adenosine receptor has not been established. A3AR is expressed in a broad spectrum of tissues. The most abundant expression in human is found in lung and aorta (7). The evaluation of the functional role of the A3AR has been hampered by the presence of multiple adenosine receptor s...

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Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility. In general, oral bioavailability of this class of compounds was found to be poor unless the imidazole was methylated on nitrogen. This work led to identification of 48, a potent (p38 MAP kinase inhibition IC50 0.24 nM) and selective p38 MAP kinase inhibitor which inhibits lipopolysaccharide-stimulated release of TNF-alpha from human blood with an IC50 2.2 nM, shows good oral bioavailability in rat and rhesus monkey, and demonstrates significant improvement in measures of disease progression in a rat adjuvant-induced arthritis model.

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)−/− mice that were also deficient in iNOS. ApoE−/− and iNOS−/− mice were cross-bred to produce apoE−/−/iNOS−/− mice and apoE−/−/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30–50% reduction in apoE−/−/iNOS−/− mice compared with apoE−/−/iNOS+/+ mice. Although the size of the lesions in apoE−/−/iNOS−/− mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45–70% reductions in both male and female apoE−/−/iNOS−/− mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.