Elsevier

Neuroscience

Volume 148, Issue 4, 21 September 2007, Pages 876-892
Neuroscience

Cellular neuroscience
Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes

https://doi.org/10.1016/j.neuroscience.2007.06.039Get rights and content

Abstract

Cell–cell communication in astroglial syncytia is mediated by intracellular Ca2+ ([Ca2+]i) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca2+]i elevation through Ca2+ 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 Ca2+-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 (4αPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4αPDD and hypotonic challenge promoted [Ca2+]i elevation mediated by influx of extracellular Ca2+. 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.

Section snippets

Astrocyte culture

The experiments were performed according to the Italian and international laws on protection of laboratory animals, with the approval of a local bioethical committee and under the supervision of a veterinary commission for animal care and comfort of the University of Bologna. Every effort was made to minimize the number of animals used and their sufferings.

Primary cultures of pure cortical rat astrocytes were prepared as previously described (Ferroni et al., 1995). Briefly, neonatal cerebral

TRPV4 transcript and protein are expressed in cultured rat cortical astrocytes

In order to assess the expression of TRPV4, we first performed RT-PCR analysis of mRNA extracted from primary cultures of rat neocortical astrocytes. The presence of TRPV4 transcripts was determined by using two different sets of primers. Fig. 1A shows that a faint, specific PCR product of ∼500 bp, corresponding to that expected for TRPV4, was detected in cDNA synthesized from total RNA. Re-amplification of the first PCR product with nested-TRPV4 primers gave rise to a stronger signal of

Discussion

The main finding of this study is the demonstration that astroglial cells in vitro and in situ possess TRPV4 channel proteins. We show that hypotonicity and exposure to the phorbol derivative 4αPDD, two stimuli that activate TRPV4, promote [Ca2+]i responses in cultured cortical astrocytes. Furthermore, 4αPDD elicits a cationic conductance with biophysical and pharmacological features identical to those of recombinant human TRPV4. The immunohistochemical analysis reveals that TRPV4 is

Acknowledgments

We are grateful to Dr. Antonio Ferrer Montiel (University Miguel Hernandez, Alicante, Spain) for supplying the human transient receptor potential channel (hTRPV4) hTRPV4/pEGFP-N1 and the Alexa Fluor 488–conjugated wheat germ agglutinin. We thank Alessia Minardi for preparation and maintenance of primary cultures of cortical astrocytes. The skilful technical assistance of Miss Lucia Dipietrangelo and Dr. Fulvio Celsi (confocal microscopy) is gratefully acknowledged. A special thanks to Bjørg

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