Fetal bovine serum contains biologically available ATP.

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300-1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells.

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies.

The Purinergic Landscape of Type 2 Diabetes Mellitus.

Adenosine triphosphate (ATP) is the key energy intermediate of cellular metabolic processes and a ubiquitous extracellular messenger. As an extracellular messenger, ATP acts at plasma membrane P2 receptors (P2Rs). The levels of extracellular ATP (eATP) are set by both passive and active release mechanisms and degradation processes. Under physiological conditions, eATP concentration is in the low nanomolar range but can rise to tens or even hundreds of micromoles/L at inflammatory sites. A dysregulated eATP homeostasis is a pathogenic factor in several chronic inflammatory diseases, including type 2 diabetes mellitus (T2DM). T2DM is characterized by peripheral insulin resistance and impairment of insulin production from pancreatic ß-cells in a landscape of systemic inflammation. Although various hypoglycemic drugs are currently available, an effective treatment for T2DM and its complications is not available. However, counteracting systemic inflammation is anticipated to be beneficial. The postulated eATP increase in T2DM is understood to be a driver of inflammation via P2X7 receptor (P2X7R) activation and the release of inflammatory cytokines. Furthermore, P2X7R stimulation is thought to trigger apoptosis of pancreatic ß-cells, thus further aggravating hyperglycemia. Targeting eATP and the P2X7R might be an appealing novel approach to T2DM therapy.