Astrocytes are glial cells of the central nervous system and play a significant role in regulation of neuronal activity and maintenance of brain homeostasis. Unlike neurons, they are not electrically excitable but can respond to extracellular signaling molecules through the activation of surface metabotropic (G-protein coupled receptors) and ionotropic receptors, such as adrenergic and purinergic receptors. This leads to an increase in intracellular concentrations of secondary messengers – cAMP and Ca2+ – (i.e. cytosolic excitability) and the regulation of various cellular processes. When the central nervous system is activated, multiple different signaling molecules can be present in the extracellular space simultaneously activating various signaling pathways within individual cells. However, the impact of simultaneous activation of different receptors on cytosolic Ca2+- and/or cAMP excitability in astrocytes and cellular processes remains poorly understood. In this thesis, we aimed to investigate whether cAMP-excitability in astrocytes upon adrenergic receptor activation changes when purinergic receptors are simultaneously activated. To monitor cAMP-excitability in astrocytes, we employed a genetically encoded fluorescent biosensor, AKAR2, which allows indirect monitoring of cAMP activity via cAMP-dependent protein kinase A (PKA) activity using fluorescence microscopy and the FRET method.
The AKAR2 biosensor construct was introduced into rat cultured astrocytes through pDNA transfection. In individual astrocytes expressing AKAR2, we measured real-time changes in cAMP-dependent PKA activity using FRET microscopy after stimulating cells with agonists of adrenergic receptors (adrenaline (ADR)), purinergic receptors (ATP), or a combination of both. The changes in FRET signal over time were normalized. The final, cumulative, and velocity changes of the FRET signal were calculated for each stimulus and the data statistically compared between stimuli.
Stimulation of astrocytes with ADR triggered an increase in intracellular cAMP-dependent PKA activity, most likely due to activation of -adrenergic receptors. When astrocytes were stimulated with a combination of ADR and ATP, which simultaneously activated adrenergic and purinergic receptors, we observed a trend of higher values for the final, cumulative, and velocity changes of the FRET signal compared to cells stimulated with ADR alone, although these differences were not statistically significant. Stimulation with ATP did not significantly increase PKA activity compared to the control experiment. We conclude that the activation of adrenergic receptors with ADR has the primary effect on cAMP excitability in astrocytes, while the activation of purinergic receptors with ATP plays a modulatory role. When cells are co-stimulated with ADR and ATP, activation of purinergic receptors by ATP most likely contributes to the trend of increased cAMP-dependent PKA activity via activation of Ca2+-signalling pathway.
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