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