Caveolin-1 Regulates the P2Y2 Receptor Signaling in Human 1321N1 Astrocytoma Cells.

Damage to the CNS can cause a differential spatio-temporal release of multiple factors, such as nucleotides, ATP and UTP. The latter interact with neuronal and glial nucleotide receptors. The P2Y2 nucleotide receptor (P2Y2R) has gained prominence as a modulator of gliotic responses after CNS injury. Still, the molecular mechanisms underlying these responses in glia are not fully understood. Membrane-raft microdomains, such as caveolae, and their constituent caveolins, modulate receptor signaling in astrocytes; yet, their role in P2Y2R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y2R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y2R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, co-localize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y2R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y2R and Cav-1, and shRNA knockdown of Cav-1 expression. Furthermore, shRNA knockdown of Cav-1 expression decreased nucleotide-induced increases in the intracellular Ca(2+) concentration in 1321N1 and C6 glioma cells without altering TRAP-6 and carbachol Ca(2+) responses. In addition, Cav-1 shRNA knockdown also decreased AKT phosphorylation and altered the kinetics of ERK1/2 activation in 1321N1 cells. Our findings strongly suggest that P2Y2R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y2R/Cav-1 interactions represent a novel target for controlling P2Y2R function after CNS injury.

Profile of the average organ non-donor: can it be used predictively?

OBJECTIVE: To describe the profile of the average organ non-donor, compare it to that of the average donor, and identify characteristics that predict the likelihood that a given individual will be a non-donor. METHODS: The charts of 397 consenting potential organ donors of LifeLink of Puerto Rico from 2009 through 2011 were reviewed. Data regarding gender, age, BMI, the presence of diabetes, hypertension and/or kidney injury, death from cerebrovascular accident, and smoking were collected. RESULTS: Of the 397 charts reviewed, 283 were from donors, 96 were from non-donors, and 18 were excluded from the analysis. When compared to donors, non-donors were found more frequently to be 60 years old or older, diabetic, hypertensive, or obese; to have suffered from kidney injury, to have smoked and to have died of a cerebrovascular accident. On multivariate analysis, age, diabetes, kidney injury and smoking remained significant. However, after adjusting for age, only smoking and death from cerebrovascular accident remained statistically associated to non-donor status. CONCLUSION: Although being over 60 years old, having smoked and dying from a cerebrovascular accident were characteristics found significantly more frequently in non-donors, these characteristics were also present in some donors. Therefore, a careful evaluation of each potential donor is still mandatory to avoid the loss of transplantable organs.

Kinetic and structural studies on interactions between heparin or heparan sulfate and proteins of the hedgehog signaling pathway.

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and regulation of the tissue distribution of signaling molecules. In this study, we use surface plasmon resonance (SPR) to study interactions of HS and structurally related heparin with proteins in the Hedgehog signaling pathway. SPR analysis shows that heparin binds with different affinities to active fragments of the proteins Hedgehog (Hh), Interference Hedgehog (Ihog), Cam-related/Down-regulated by Oncogenes (CDO), and Sonic Hedgehog (Shh). Solution competition studies show that the minimum size of a heparin oligosaccharide capable of interacting with Ihog is larger than a tetrasaccharide and for interacting with Shh is larger than an octasaccharide. In comparison with heparin, Ihog and Shh exhibited a lower affinity for HS than for heparin, and CDO and Hh exhibit negligible binding to HS. This study clearly demonstrates Shh and Ihog are heparin and HS binding proteins and that both molecules preferentially bind heparin or HS having a high level of sulfation.