Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl(-) currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on d-[(3)H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated d-[(3)H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (>or=80% inhibition) by intracellular Ca(2+) chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control d-[(3)H]aspartate release activated by the substantial hyposmotic swelling showed little (<or=25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca(2+)-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca(2+)/calmodulin-independent signaling mechanism. Ca(2+)-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication.