Swelling-induced calcium signaling in wild-type and Mlc1-null astrocytes: paradoxical effects of pharmacological TRPV4 and Piezo1 inhibition.

INTRODUCTION: Astrocytes play a crucial role in brain ion and water homeostasis and continuously adapt their volume in response to osmotic challenges. This process is disturbed in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts (MLC). Intracellular calcium signaling in response to cell swelling has been implicated in astrocytic volume regulation. METHODS: We examined swelling-induced calcium signals in cultured astrocytes isolated from brains of wild-type and Mlc1-null mice, utilizing a microplate reader and Fura-2AM-based imaging. RESULTS: We showed that wild-type astrocytes respond to hypotonic shocks with a robust calcium signal proportional to the magnitude of the shock. Pharmacological experiments indicated that these responses reflect a combination of intracellular store-mediated calcium release and channel-mediated calcium influx. Mlc1-null astrocytes showed an increased baseline calcium level and a slower calcium response to large hypotonic shocks. Unexpectedly, pharmacological inhibition of mechanosensitive cation channels with the TRPV4 antagonist HC-067047 and the mechanosensitive channel inhibitor GsMTx4 did not reduce swelling-induced calcium signals but instead led to a paradoxical increase in amplitude and speed of calcium signals in wild-type and Mlc1-null astrocytes, with differences in the magnitude of alteration between the two genotypes. DISCUSSION: Together, our results suggest subtle alterations in calcium signaling that might affect the volume sensing machinery of Mlc1-null astrocytes, conforming with earlier studies that show mistuning of volume-regulated ion channels in MLC. The observed paradoxical effects are consistent with a role for TRPV4- and GsMTx4-sensitive pathways in setting basal responsiveness of astrocytes to osmotic stress, and argue against TRPV4 or Piezo1 being the main direct calcium entry routes during swelling. Overall, our findings highlight the complexity of astrocytic volume regulation in response to hypotonicity.