The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of κB-α isoform (IκB-α) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

Previous studies have suggested that the P2Z/P2X 7 purinergic receptor can participate in nucleotide-induced modulation of lipopolysaccharide (LPS) stimulated inflammatory mediator production. To test this hypothesis, we evaluated whether antagonism of the P2Z/P2X 7 receptor can influence LPS signaling and expression of the inducible form of nitric-oxide synthase (iNOS) in RAW 264.7 macrophages. In the present study, we demonstrate that pretreatment of RAW 264.7 macrophages with a P2Z/P2X 7 receptor antagonist, periodate oxidized adenosine 5-triphosphate (o-ATP), substantially inhibits LPS-stimulated NO production and iNOS expression without altering cell viability. This effect on LPS-induced iNOS expression is mimicked by a pyridoxal-phosphate-based antagonist (pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid) of the P2Z/P2X 7 purinergic receptor, indicating that these results are not unique to o-ATP. Additionally, o-ATP prevents cell death induced by P2Z/P2X 7 receptor agonists. To ascertain how P2Z/P2X 7 receptor antagonists influence LPS signaling, we evaluated the capacity of o-ATP to regulate LPSmediated activation of the transcription factor, nuclear factor-B, and the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2. These experiments reveal that pretreatment of RAW 264.7 cells with o-ATP attenuates the LPS stimulation of a nuclear factor-B-like binding activity. Moreover, the activation of ERK1 and ERK2 by LPS, but not by the phorbol ester, phorbol 12-myristate 13-acetate, is also blocked in RAW 264.7 cells by o-ATP pretreatment. In summary, these data suggest that the P2Z/P2X 7 receptor modulates LPS-induced macrophage activation as assessed by iNOS expression and NO production. This report implicates the P2Z/P2X 7 receptor in the control of protein kinase cascades and transcriptional processes, and these observations are likely to be important for the development of selective purinergic receptor antagonists for the treatment of septic shock.

Phylogenetic analysis of ca. 4500 base pairs of mitochondrial DNA sequence data reveals further genetic diversity in mouse lemurs (Microcebus) on the eastern and western coasts of Madagascar. Molecular data and phylogenetic analyses revise the previously monotypic species of eastern Madagascar, Microcebus rufus, with the description of 3 new species. Three additional Microcebus species are proposed in eastern Madagascar, along with another Microcebus species in western Madagascar. Correlating the molecular data with previously generated sequence data, we present a tentative pattern of distribution along the east coast. We show that the general distribution of Microcebus is based on a traditional eastern/western division. The preliminary model appears strongly influenced by both rivers and altitudinal differences acting independently as barriers.

Parthenogenesis has been documented in all major jawed vertebrate lineages except mammals and cartilaginous fishes (class Chondrichthyes: sharks, batoids and chimeras). Reports of captive female sharks giving birth despite being held in the extended absence of males have generally been ascribed to prior matings coupled with long-term sperm storage by the females. Here, we provide the first genetic evidence for chondrichthyan parthenogenesis, involving a hammerhead shark (Sphyrna tiburo). This finding also broadens the known occurrence of a specific type of asexual development (automictic parthenogenesis) among vertebrates, extending recently raised concerns about the potential negative effect of this type of facultative parthenogenesis on the genetic diversity of threatened vertebrate species.

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. Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-B signaling pathways in murine RAW 264.7 macrophages. Am J Physiol Cell Physiol 286: C923-C930, 2004. First published December 18, 2003; 10.1152/ajpcell.00417.2003.-Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X 7. In this regard, after exposure to bacterial LPS, P2X 7 activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X 7 has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-B, we tested the hypothesis that LPS and nucleotides regulate NF-B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X 7-selective ligand 2Ј-3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate (BzATP) results in the cooperative activation of NF-B DNA-binding activity and a sustained attenuation of levels of the NF-B inhibitory protein IB␣. Interestingly, a persistent reduction in IB␣ levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating IB␣ protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in IB␣ levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-B/ IB-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-B and Ras/MEK/ MAPK-associated pathways. nucleotide receptors; mitogen-activated protein kinases; nuclear factor-B; monocytes/macrophages; cytokines ALTHOUGH ATP IS KNOWN to be an important source of intracellular energy, numerous studies have revealed that high concentrations of ATP (5 mM) in the extracellular milieu following tissue damage or platelet degranulation can play a key role in the regulation of biological responses such as inflammation, platelet aggregation, and smooth muscle contraction (13,20,25,38). Nucleotides have been shown to greatly enhance the effects of bacterial LPS on macrophage and monocyte activation by augmenting the production of mediators such as nitric oxide (NO) and other free radicals, in addition to numerous cytokines, such as interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣ (26, 27, 39, 51, 55). The overproduction of these mediators, although important for the activation of the immune system and for bactericidal effects, can als...

Activation of the P2X7 receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg578, Lys579) within this motif are essential for LPS binding to P2X7 in vitro. Because P2X7 can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X7 is critical for receptor function and trafficking. In these studies we mutated Arg578 and Lys579 of P2X7, and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X7-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25°C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25°C the P2X7-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X7 C terminus controls receptor trafficking and modulates channel activity.

Macrophages express several lipopolysaccharide (LPS) binding proteins and are potently activated by LPS to produce inflammatory mediators. Recent studies have shown that receptors for exogenous nucleotides (P2X and P2Y purinergic receptors) can modulate macrophage production of TNF-alpha, IL-1beta and nitric oxide (NO) following LPS exposure. Macrophages and LPS-stimulated monocytes express elevated levels of P2Y1, P2Y2 and P2X7 mRNA, suggesting that both P2Y and P2X receptors can contribute to LPS-induced pathophysiology. In addition, oxidized-ATP treatment (which inhibits P2X7) of macrophages blocks LPS-induced NO production, NF-kappaB and ERK-1/2 activation. Also, an LPS-binding domain located in the P2X7 C-terminus appears important for receptor trafficking/function. Moreover, the purinergic receptor ligand 2-MeS-ATP attenuates LPS-induced cytokine and NO production in vivo and ex vivo. These data suggest that P2X7 and certain P2Ys are linked to LPS effects, although their relative contribution in vivo is unclear. Accordingly, we tested the capacity of several adenine nucleotides to modulate LPS-induced mortality in mice. We found that the P2X7-directed ligand BzATP was unable to prevent LPS-induced death, whereas 2-MeS-ATP and 2-Cl-ATP, which bind to multiple P2X and P2Y receptors were able to protect mice from LPS-induced death. These data suggest that the co-ordinate action of P2Y and P2X7 receptors are critical for controlling LPS responses in vivo and that agents directed against both receptor classes may provide the greatest therapeutic advantage.