Rho-family GTPases modulate Ca(2+) -dependent ATP release from astrocytes.

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G(q)/phophospholipase C (PLC)/Ca(2+) mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca(2+) mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G(q)-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca(2+)-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.

Defining the expression pattern of the LGI1 gene in BAC transgenic mice.

The LGI1 gene has been implicated in the development of epilepsy and the invasion phenotype of glial cells. Controversy over the specific tissue expression pattern of this gene has stemmed from conflicting reports generated using immunohistochemistry and the polymerase chain reaction. LGI1 is one of a four-member family of secreted proteins with high homology and here we demonstrate, using GFP-tagged constructs from the four LGI1family members, that commonly used antibodies against LGI1 cross-react with different family members. With the uncertainty surrounding the use of commercially available antibodies to truly establish the expression pattern of LGI1, we generated transgenic mice carrying the LGI1-containing BAC, RP23-127G7, which had been modified to express the GFP reporter gene under the control of the endogenous regulatory elements required for LGI1 expression. Three founder mice were generated, and immunohistochemistry was used to determine the tissue-specific pattern of expression. In the brain, distinct regions of glial and neuronal cell expression were identified, as well as the choriod plexus, which is largely pia-derived. In addition, strong expression levels were identified in glandular regions of the prostate, individual tubules in the kidney, sympathetic ganglia in the kidney, sebaceous glands in the skin, the islets of Langerhans, the endometrium, and the ovary and testes. All other major organs analyzed were negative. The pattern of reporter gene expression was identical in three individual founder mice, arguing against a position effect altering expression profile due to the integration site of the BAC.

Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation.

1 Transient accumulation of extracellular ATP reflects both release of ATP from intracellular stores and altered rates of ATP metabolism by ecto-enzymes. Ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) and ecto-nucleotide pyrophosphatases (eNPPs) degrade ATP, while ecto-nucleotide diphosphokinases (eNDPKs) synthesize ATP from ambient ADP. 2 Although the methylene ATP analogs betagamma-meATP and alphabeta-meATP are widely used as metabolically stable tools for the analysis of purinergic signaling, their specific effects on eNTPDase, eNPP, and eNDPK activities have not been defined. This study compared the actions of these analogs on extracellular ATP metabolism by human 1321N1 astrocytes, rat PC12 pheochomocytoma cells, and rat C6 glioma cells. 3 Both analogs significantly reduced clearance of extracellular ATP by 1321N1 cells that express both eNTPDases and eNPPs, as well as by C6 cells that exclusively express eNPPs. In contrast, both analogs were much less efficacious in inhibiting ATP clearance by PC12 cells that predominantly express eNTPDases. Betagamma-meATP, but not alphabeta-meATP, was effectively hydrolyzed by the 1321N1 and C6 cells; PC12 cells did not significantly degrade this analog. 4 Alphabeta-meATP, but not betagamma-meATP, acted as a substrate for purified yeast NDPK to generate ATP via trans-phosphorylation of ADP. alphabeta-meATP also acted as substrate for the eNDPK activities expressed by 1321N1, PC12, and C6 cells and thereby induced extracellular ATP accumulation in the presence of ambient or exogenously added ADP. 5 These results indicate that methylene ATP analogs exert complex and cell-specific effects on extracellular ATP metabolism that can significantly modify interpretation of studies that use these reagents as probes of purinergic signal transduction in intact tissues.

Colocalization of ATP release sites and ecto-ATPase activity at the extracellular surface of human astrocytes.

Extracellular ATP and other nucleotides function as autocrine and paracrine signaling factors in many tissues. Recent studies suggest that P2 nucleotide receptors and ecto-nucleotidases compete for a limited pool of endogenously released nucleotides within cell surface microenvironments that are functionally segregated from the bulk extracellular compartment. To test this hypothesis, we have used luciferase-based methods to continuously record extracellular ATP levels in monolayers of human 1321N1 astrocytoma cells under resting conditions, during stimulation of Ca2+-mobilizing receptors for thrombin or acetylcholine, and during mechanical stimulation by hypotonic stress. Soluble luciferase was utilized as an indicator of ATP levels within the bulk extracellular compartment, whereas a chimeric protein A-luciferase, adsorbed to antibodies against a glycosylphosphatidylinositol-anchored plasma membrane protein, was used as a spatially localized probe of ATP levels at the immediate extracellular surface. Significant accumulation of ATP in the bulk extracellular compartment, under either resting (1-2 nm ATP) or stimulated (10-80 nm ATP) conditions, was observed only when endogenous ecto-ATPase activity was pharmacologically inhibited by the poorly metabolizable analog, betagamma-methylene ATP. In contrast, accumulation of submicromolar ATP in the cell surface microenvironment was readily measured even in the absence of ecto-ATPase inhibition suggesting that the spatially colocalized luciferase could effectively compete with endogenous ecto-ATPases for released ATP. Other experiments revealed a critical role for elevated cytosolic [Ca2+] in the ATP release mechanism triggered by thrombin or muscarinic receptors but not in basal ATP release or release stimulated by hypotonic stress. These observations suggest that ATP release sites are colocalized with ecto-ATPases at the astrocyte cell surface. This colocalization may act to spatially restrict the actions of released ATP as a paracrine or autocrine mediator of cell-to-cell signaling.