Control of astrocytic Ca signaling by nitric oxide-dependent S-nitrosylation of Ca homeostasis modulator 1 channels.

Background: Astrocytes Ca signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca signals in astrocytes. Importantly, astrocytes express the Ca-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca signals by triggering Ca influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex.

Results: Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca] and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca signaling.

Conclusions: Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca signaling.