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Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes Benfenati V; Amiry-Moghaddam M; Caprini M; Mylonakou MN; Rapisarda C; Ottersen OP; Ferroni SNeuroscience 2007[Sep]; 148 (4): 876-92Cell-cell communication in astroglial syncytia is mediated by intracellular Ca(2+) ([Ca(2+)](i)) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca(2+)](i) elevation through Ca(2+) influx, the molecular identity of the channel protein underlying this response is still elusive. Here we report that primary cultured cortical astrocytes express the transient receptor potential vanilloid-related channel 4 (TRPV 4), a Ca(2+)-permeable cation channel gated by a variety of stimuli, including cell swelling. Immunoblot and confocal microscopy analyses confirmed the presence of the channel protein and its localization in the plasma membrane. TRPV4 was functional because the selective TRPV4 agonist 4-alpha-phorbol 12,13-didecanoate (4alphaPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4alphaPDD and hypotonic challenge promoted [Ca(2+)](i) elevation mediated by influx of extracellular Ca(2+). This effect was abolished by low micromolar concentration of the TRPV4 inhibitor Ruthenium Red. Immunofluorescence and immunogold electron microscopy of rat brain revealed that TRPV4 was enriched in astrocytic processes of the superficial layers of the neocortex and in astrocyte end feet facing pia and blood vessels. Collectively, these data indicate that cultured cortical astroglia express functional TRPV4 channels. They also demonstrate that TRPV4 is particularly abundant in astrocytic membranes at the interface between brain and extracerebral liquid spaces. Consistent with its roles in other tissues, these results support the view that TRPV4 might participate in astroglial osmosensation and thus play a key role in brain volume homeostasis.|Animals[MESH]|Animals, Newborn[MESH]|Astrocytes/cytology/*physiology[MESH]|Calcium/metabolism[MESH]|Cell Membrane/metabolism[MESH]|Cells, Cultured[MESH]|Chlorocebus aethiops[MESH]|Dose-Response Relationship, Drug[MESH]|Electric Stimulation/methods[MESH]|Gene Expression/*physiology[MESH]|Glial Fibrillary Acidic Protein/metabolism[MESH]|Green Fluorescent Proteins/metabolism[MESH]|Membrane Potentials/drug effects/physiology/radiation effects[MESH]|Microscopy, Immunoelectron/methods[MESH]|Occipital Lobe/*cytology/metabolism/ultrastructure[MESH]|Patch-Clamp Techniques[MESH]|Phorbols/pharmacology[MESH]|Rats[MESH]|Reverse Transcriptase Polymerase Chain Reaction/methods[MESH]|Ruthenium Red/pharmacology[MESH]|TRPV Cation Channels/genetics/*metabolism[MESH]|Transfection/methods[MESH] |
