Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation.

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear.

Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli.

Sigma receptors once considered as a class of opioid receptors are now regarded as unique orphan receptors, distinguished by the ability to bind various pharmacological agents such as the progesterone (steroid), haloperidol (anti-psychotic), and drugs of abuse such as cocaine and methamphetamine. The sigma-1 receptor is a 223 amino acid protein, proposed to have two transmembrane segments. We have developed a scheme for the purification of the guinea pig sigma-1 receptor following overexpression in Escherichia coli as a maltose binding protein (MBP) fusion and extraction with Triton X-100.

Human P2X7 pore function predicts allele linkage disequilibrium.

Background: Innate immune response amplification is achieved by leukocyte expression of the purinergic nucleotide receptor P2X7, an extracellular nucleotide-gated pore. Previously, low P2X7 pore activity in whole blood was associated with loss-of-function genotypes in correlation with a decreased ratio of lipopolysaccharide-stimulated tumor necrosis factor-alpha to interleukin-10, of relevance to a variety of infectious and inflammatory disorders. We hypothesized that evaluation of participants with discordance between the P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype.

Detection of human P2X7 nucleotide receptor polymorphisms by a novel monocyte pore assay predictive of alterations in lipopolysaccharide-induced cytokine production.

The nucleotide receptor P2X(7) is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X(7) gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X(7) pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 +/- 4; AC, 42 +/- 4; and CC, 6 +/- 1-fold stimulation).

A novel assay to detect nucleotide receptor P2X7 genetic polymorphisms influencing numerous innate immune functions.

The importance of accessory signaling pathways amplifying endotoxin responses has recently been highlighted by genetic studies describing LPS-hyporesponsive individuals despite carrying the common allele for TLR4. The nucleotide receptor P2X7 modulates the production of numerous LPS-stimulated inflammatory mediators. We have recently described the largest phenotypic screen known for genetic polymorphisms associated with the nucleotide receptor P2X7, a global regulator of leukocyte function.

The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho.

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems.

Purinergic receptor regulation of LPS-induced signaling and pathophysiology.

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.

A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production.

Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia. Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities. Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types.