P2Y receptor regulation of cultured rat cerebral cortical cells: calcium responses and mRNA expression in neurons and glia

Abstract

1 We have investigated increases in cytosolic Ca 2+ in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron-free glial cultures. 2 In both cultures, the agonist-response profile was 2-methylthioADP(2MeSADP)42-methylthioATP(2MeSATP)4ADP4ATP4adenosine 5 0 -O-(3-thiotriphosphate), consistent with a P2Y 1 receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. 3 Suramin, pyridoxal-phosphate-6-azophenyl-2 0 ,4 0 -disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA 2 values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. . This further indicated that the 2MeSADP response was not in the neurons. 6 Reverse transcriptase -polymerase chain reaction studies detected transcripts for P2Y 1 , P2Y 4 and P2Y 6 in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y 2 transcripts were not detected in the embryonic cortex. 7 Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca 2+ in glial cells and presynaptic sites on neurons.

The Effects of pH on Beta-Endorphin and Morphine Inhibition of Calcium Transients in Dorsal Root Ganglion Neurons

“…22,30,62 Although DRG neurons respond to high-K ϩ with a rapid increase in intracellular Ca 2ϩ , glia in these cultures do not; therefore this stimulus can also be used as a marker for neuronal cells. 6,17,40 We found that high-K ϩ (40 mmol/L)-evoked depolarization of DRG neurons initiates a reversible and reproducible increase in intracellular Ca 2ϩ (Fig 4D). Pilot experiments showed an attenuation of high-K ϩ -evoked Ca 2ϩ responses at pH e 6.1 and pH e 6.5, consistent with reports in the literature, 15,26 and we therefore chose to assess morphine inhibition at pH e 7.6, pH e 7.2, and pH e 6.8.…”

ATP Induces Synaptic Gene Expressions in Cortical Neurons: Transduction and Transcription Control via P2Y1 Receptors

“…The P2Y 1 R is well established to be in the family of G q/11 -linked receptors and thus is expected to signal upon agonist activation primarily via phospholipase C, inositol trisphosphate, and mobilization of Ca 2ϩ from intracellular stores (Abbracchio et al, 2006). With rat cortex mixed-cell cultures, Bennett et al (2003) found that only 9 to 15% of the neurons mobilized Ca 2ϩ in response to P2Y 1 R agonists (but all glia did so) in testing at an earlier stage than ours (E14 cortex, cultured only for 8 days, and with ϳ50% glia present). Therefore, our cortex cultures, being more mature and with glial suppression, were compared here in this response.…”

“…This extends the previous findings on P2Y 1 R mRNA in the cortex. Thus, Moore et al (2001) found it is strongly expressed in total extracts of the adult human cortex, and Bennett et al (2003) showed that it is abundant in extracts of E14 rat cortex and in mixed-cell cultures derived from it, and in glia cultured alone. Specifically, in the multiprocess-bearing astrocytes freshly isolated from the rat cortex or hippocampus, 10 to 30% of the cells have been found to contain P2Y 1 R mRNA and to show P2Y 1 R functional responses, with this proportion increasing to ϳ90% of astrocytes after 2 weeks in culture (Zhu and Kimelberg, 2001).…”

P2Y1 receptor signaling enhances neuroprotection by astrocytes against oxidative stress via IL‐6 release in hippocampal cultures

“…sion molecularly with selective siRNAs, strongly supporting our notion that P2Y 1 R mediates the protective effect of ATP. Several studies have shown that the expression level of P2Y 1 R in astrocytes changes under different culture conditions and is different in different tissues of origin (Bennett et al, 2003;Cheung et al, 2003;Pearce and Langley, 1994;Kimelberg, 2001, 2004). However, ATP could also prevent the cell death of astrocytes via activation of P2Y 1 R in hippocampal cultures, as well as in forebrain astrocyte cultures.…”