Astrocytes are a heterogeneous group of glial cells with many functions that maintain homeostasis of central nervous system (CNS). Among other things, astrocytes provide metabolic support to neurons and regulate synaptic signal transmission. In addition to roles in physiological processes, they are also involved in the response to pathological alterations in the CNS. Unlike neurons, astrocytes are electrically silent, however, they display intracellular excitability, the central part of which are the second messengers cyclic adenosine-3',5'-monophosphate (cAMP) and Ca2+. These two regulate a wide range of cellular processes related to astrocyte metabolism, morphology and intercellular communication. Cytosolic levels of Ca2+ and cAMP are dynamically regulated by extracellular stimuli via G-protein coupled receptors (GPCRs) that are present on the cell surface and, upon activation, cause the dissociation of trimeric G-proteins into subunits. The latter trigger the release of Ca2+ from the endoplasmic reticulum and stimulate or inhibit production of cAMP through interaction with adenylate cyclase.
Latrophilins are classified as adhesive GPCRs. In mammalian cells one can find type 1, 2 and 3 latrophilins, which modulate second messengers in various ways – they increase cytosolic Ca2+ concentration and decrease or increase cytosolic cAMP levels. Latrophilins are also present in the CNS and may be involved in excitatory synapse formation, axonal attraction, and neuronal migration. Their activity in the context of astrocyte function is still poorly investigated, so the aim of the thesis was to determine which signaling pathways are triggered in astrocytes after stimulation with endogenous latrophilin ligand. In isolated rat astrocytes, changes in the intracellular levels of cAMP and Ca2+ were measured by real-time fluorescence microscopy, and the ectodomain of mouse teneurin 2 (mTen2) was used as a stimulus. Prior to intracellular Ca2+ recordings, astrocytes were stained with the Ca2+-indicator dye Fluo-4 AM. To monitor intracellular cAMP levels cells were transfected with Förster Resonance Energy Transfer (FRET)-based cAMP nanosensor Epac1-camps. Our results suggest that stimulation of astrocytes with mTen2 results in a statistically significant decrease in intracellular Ca2+ levels, whereas intracellular cAMP levels are unaffected. In addition, pre-stimulation of cells with mTen2 reduced the increase in intracellular Ca2+ concentration triggered by adrenergic GPCR agonist noradrenaline compared to control non pre-stimulated cells. Our results suggest that mTen2 reduces noradrenaline-mediated Ca2+-excitability of astrocytes.
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