Regulation Of Ca²⁺ Mobilization On Cultured Cortical Astrocytes And Synaptic Plasticity On The Hippocampus By SKF83959

Background: Recent evidences indicate the existence of a putative novel phosphatidylinositol (PI)-linked D1 dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase Cβ. Astrocytes form the largest population of non-excitable cells in mammalian central nervous system (CNS) and play an important role in CNS. To gain further insight into this newly-appreciated receptor in brain, The present study explored the effect of a selective PI-linked D1 dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca2+]i) and neurotransmitter release in cultured rat prefrontal cortical astrocytes. Methods: The expression of D1 dopamine receptor in cultured rat prefrontal cortical astrocytes was evaluated by immunofluorescence, RT-PCR, western blotting. The effects of SKF83959 on [Ca2+]i and neurotransmitter release were measured with calcium imaging and HPLC study. Results: D1 dopamine receptor was expressed in rat prefrontal cortical astrocytes. SKF83959 caused a transient dose-dependent increase in [Ca2+]i. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic/endoplasmic reticulum (ER) completely blocked this potentiation induced by SKF83959. In Ca2+-free solution, SKF83959 was able to induce a significant increase of [Ca2+]i, although the magnitude of elevation was less than that in the presence of extracellular Ca2+. Voltage-gated calcium channels (VGCC) blocker verapamil slightly reduced the SKF83959-induced [Ca2+]i increase. Receptor-operated calcium channels (ROCC) antagonist D-AP5 and CNQX attenuated the SKF83959-induced elevation of [Ca2+]i. Meanwhile, capacitative Ca2+ entry (CCE) contributed to SKF83959-induced elevation of [Ca2+]i. Furthermore, the inhibitor of phospholipase C (PLC) U73122 and inositol-1,4,5- trisphosphate(IP3) receptor antagonist 2-APB completely inhibited this potentiation. However, pCPT-cAMP, a membrane permeable cAMP analog had no effect. SKF83959-stimulated increase in [Ca2+]i was coincided with an increase in glutamate and GABA release. SKF83959-induced neurotransmitter release was inhibited by D1 receptor antagonist SCH23390 and chelating of intracellular Ca2+ with BAPTA-AM. Conclusion: These results suggested that activation of D1 receptor by SKF83959 mediated a dose-dependent mobilization of [Ca2+]i and subsequent release of glutamate and GABA from cultured rat prefrontal cortical astrocytes.Part II SKF83959 facilitates long-term depression in rat hippocampal CA1 synapsesBackground: Recent work has demonstrated that a phosphatidylinositol (PI)-linked D1 dopamine receptor selective agonist SKF83959 mediated phosphatidylinositol hydrolysis via activation of phospholipase Cβin brain. Specific contributions of SKF83959 to synaptic plasticity have not been well elucidated. So the present study examined the roles of SKF83959 in low frequency stimulation (LFS)-induced long-term depression (LTD) at Schaffer collateral-CA1 synapses in rat hippocampal slices. Methods: The roles of SKF83959 in LFS-induced LTD at Schaffer collateral-CA1 synapses were measured with field potential study. The expression of Ng, CaMKII, CaN was determined by western blotting. Results: SKF83959 stimulation significantly depressed field excitatory postsynaptic potentials (fEPSP) in a dose-dependent manner and facilitated the induction of LTD by LFS. NMDA receptor antagonist D-AP5 partially blocked SKF83959-facilitated LTD. PLC inhibitor U73122 and IP3 receptor antagonist 2-APB completely prevented SKF83959-facilitated LTD. However, H-89, a membrane permeable PKA inhibitor had no effect. Calcium chelator, BAPTA-AM prevented SKF83959-facilitated LTD. Furthermore, SKF83959-facilitated LTD was significantly depressed in the presence of calcineurin (CaN, PP2B) inhibitors cyclosporin A (CsA) and associated with a persistent increase in the expression of calcineurin A. Conclusion: SKF83959 facilitated LFS induced-LTD via PLC signal pathway and needed the activation of NMDA receptor and the elevation of [Ca2+]i. The activation of CaN was involved in SKF83959-facilitated LTD.