Purinergic responses of calcium-dependent signaling pathways in cultured adult human astrocytes

Abstract

Doc number: 18

Abstract

Background: The 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[variant prime]-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.

Results: The 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.

Conclusion: These results suggest that activation of metabotropic P2YR (P2Y1R and/or P2Y2R) and ionotropic P2X7R could mediate purinergic responses in adult human astrocytes.