Astrocytes, the most numerous glial cells, are now recognized as active and indispensable participants in the functioning of the central nervous system. Their roles are diverse, ranging from providing metabolic support to neurons and participating in synaptic transmission, to complex intracellular signaling involving calcium and other secondary messengers. Recent studies have also identified astrocytes as intracranial baroreceptors that detect changes in cerebral blood flow via mechanosensitive channels such as TRPV4 and Piezo1, thereby contributing to the regulation of cerebral perfusion. We investigated the impact of these channels’ activation on the concentrations of the metabolite lactate and the key signaling molecules, cAMP and Ca²⁺, aiming to explore the potential connection between mechanical sensing, metabolism, and cellular signaling. To monitor dynamic changes in individual cells in vivo, we employed fluorescence microscopy methods. Using fluorescence resonance energy transfer (FRET) and the genetically encoded nanosensors Laconic and Epac1-camps, we measured intracellular lactate and cAMP concentrations. Intracellular calcium dynamics were monitored using fluorescence microscopy with the highly sensitive calcium dye Calbryte 520 AM. Our results demonstrate that the TRPV4 channel antagonist HC067047 increases mechanically induced intracellular changes in lactate but does not influence cAMP concentrations. The Piezo1 channel antagonist Dooku1 influenced transient changes in intracellular calcium levels, whereas the TRPV4 channel antagonist RN-1734 exhibited no detectable effect. Notably, we also showed that the TRPV4 channel antagonist HC-067047 influences the morphology of astrocytes and causes an increase in the basal concentration of cAMP. The results indicate that caution is needed when using these antagonists, as their acute application can have non-specific effects.
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