Abstract
BackgroundThe properties of Ca2+ signaling mediated by purinergic receptors are intrinsically linked with functional activity of astrocytes. At present little is known concerning Ca2+-dependent purinergic responses in adult human astrocytes. This work has examined effects of purinergic stimulation to alter levels of intracellular Ca2+ in adult human astrocytes. Ca2+-sensitive spectrofluorometry was carried out to determine mobilization of intracellular Ca2+ following adenosine triphosphate (ATP) or 3′-O-(4-benzoyl)benzoyl-ATP (Bz-ATP) stimulation of adult human astrocytes. In some experiments pharmacological modulation of Ca2+ pathways was applied to help elucidate mechanisms of Ca2+ signaling. RT-PCR was also performed to confirm human astrocyte expression of specific purinoceptors which were indicated from imaging studies.ResultsThe endogenous P2 receptor agonist ATP (at 100 μM or 1 mM) applied in physiological saline solution (PSS) evoked a rapid increase of [Ca2+]i to a peak amplitude with the decay phase of response exhibiting two components. The two phases of decay consisted of an initial rapid component which was followed by a secondary slower component. In the presence of Ca2+-free solution, the secondary phase of decay was absent indicating this prolonged component was due to influx of Ca2+. This prolonged phase of decay was also attenuated with the store-operated channel (SOC) inhibitor gadolinium (at 2 μM) added to standard PSS, suggesting this component was mediated by SOC activation. These results are consistent with ATP activation of P2Y receptor (P2YR) in adult human astrocytes leading to respective rapid [Ca2+]i mobilization from intracellular stores followed by Ca2+ entry through SOC. An agonist for P2X7 receptor (P2X7R), BzATP induced a very different response compared with ATP whereby BzATP (at 300 μM) elicited a slowly rising increase in [Ca2+]i to a plateau level which was sustained in duration. The BzATP-induced increase in [Ca2+]i was not enhanced with lipopolysaccharide pre-treatment of cells as previously found for P2X7R mediated response in human microglia. RT-PCR analysis showed that adult human astrocytes in vitro constitutively express mRNA for P2Y1R, P2Y2R and P2X7R.ConclusionThese results suggest that activation of metabotropic P2YR (P2Y1R and/or P2Y2R) and ionotropic P2X7R could mediate purinergic responses in adult human astrocytes.
Highlights
The properties of Ca2+ signaling mediated by purinergic receptors are intrinsically linked with functional activity of astrocytes
adenosine triphosphate (ATP)-induced changes in [Ca2+]i We first confirmed that in excess of 99% cells in astrocyte culture were positive for glial fibrillar acidic protein (GFAP) under our culture conditions
The results from control experiments are consistent with the possibility that Ca2+ responses, induced by different concentrations of ATP in standard physiological saline solution (PSS), are mediated by a rapid release of intracellular Ca2+
Summary
The properties of Ca2+ signaling mediated by purinergic receptors are intrinsically linked with functional activity of astrocytes. Adenosine triphosphate (ATP) is one of the primary extracellular signaling molecules for astrocytes under both physiological and pathological conditions and evokes an astrocytic [Ca2+]i elevation through activation of P2 purinoceptors [1]. P2 purinoceptors are subdivided into two families consisting of metabotropic P2Y receptor (P2YR) and ionotropic P2X receptor (P2XR). In the former case subtypes of P2YR, such as P2Y1R and P2Y2R, are G-protein coupled and linked to inositol triphosphate-mediated release of Ca2+. Activation of purinergic receptors alters Ca2+-dependent pathways and intracellular levels of Ca2+ which in turn determine cellular functional responses to endogenous ligand, ATP. An alternative pathway for entry of Ca2+ from extracellular medium is provided by activation of family members of P2XR ionotropic channels
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