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Background: Astrocytes Ca 2+ 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 2+ signals in astrocytes. Importantly, astrocytes express the Ca 2+ -dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca 2+ signals by triggering Ca 2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca 2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca 2+ 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 2+ ] i 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 2+ 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 2+ signaling., (© 2024. The Author(s).)