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 P2Y 2 nucleotide receptor (P2Y 2 R) 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 P2Y 2 R signaling has not been adequately explored. Hence, this study evaluated the role of caveolin-1 (Cav-1) in modulating P2Y 2 R subcellular distribution and signaling in human 1321N1 astrocytoma cells. Recombinant hP2Y 2 R expressed in 1321N1 cells and Cav-1 were found to co-fractionate in light-density membrane-raft fractions, colocalize via confocal microscopy, and co-immunoprecipitate. Raft localization was dependent on ATP stimulation and Cav-1 expression. This hP2Y 2 R/Cav-1 distribution and interaction was confirmed with various cell model systems differing in the expression of both P2Y 2 R 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 P2Y 2 R interaction with Cav-1 in membrane-raft caveolae of 1321N1 cells modulates receptor coupling to its downstream signaling machinery. Thus, P2Y 2 R/Cav-1 interactions represent a novel target for controlling P2Y 2 R function after CNS injury.Neurodegenerative conditions are among the leading causes of death and disability in the United States and have dramatically increased in incidence during the last decade (1, 2). The P2 receptors for extracellular nucleotides have emerged as key modulators of the pathophysiology of neurodegeneration (3-6). G protein-coupled P2Y 2 nucleotide receptors (P2Y 2 Rs) 3 have been identified in both neurons and glia as mediators of pro-inflammatory responses, neurotransmission, apoptosis, proliferation, and cell migration (3-5, 7, 8). In addition, the P2Y 2 Rs have also gained prominence, due to their association with some types of neoplasms, spinal cord injury, and the enhancement of neuronal differentiation (7, 9 -15). Further insight into the spatio-temporal organization of the P2Y 2 R and its signaling cascades in astrocytic cells is required to expand our knowledge of their role in neurodegenerative diseases. In this context, evidence suggests that receptors and associated signaling molecules are not randomly distributed in plasma membranes but are localized in specialized membrane microdomains, namely membrane rafts (MRs), such as caveolae (Cav) (16 -20). MRs are specialized membrane domains enriched in cholesterol and g...

Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007;Martínez et al., 2016). Changes in intracellular calcium concentration can be measured using the calcium sensitive fluorescent ratiometric dye fura-2 AM. This method is a high throughput way to measure agonist mediated calcium responses.

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A pharmacological characterization has been performed of the opioid receptor involved in modulation of phagocytosis in the protozoan ciliate Tetrahymena. Studies on inhibition of phagocytosis by mammalian prototypic opioid agonists revealed that morphine and beta-endorphin have the highest intrinsic activity, whereas all the other opioids tested can only be considered partial agonists. However, morphine (a mu-receptor agonist) is twice as potent as beta-endorphin (a delta-receptor agonist). Furthermore, the sensitivity for the opioid antagonist naloxone, determined in the presence of morphine and beta-endorphin, is very similar to the sensitivity exhibited by mammalian tissues rich in mu-opioid receptors. We suggest that the opioid receptor coupled to phagocytosis in Tetrahymena is mu-like in some of its pharmacological characteristics and may serve as a model system for studies on opioid receptor function and evolution.

SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer’s disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA’s expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA’s message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA’s mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD.