ATP‐Evoked Ca²⁺ Mobilisation and Prostanoid Release from Astrocytes: P₂‐Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Abstract

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.

Structural Basis of Agonist-induced Desensitization and Sequestration of the P2Y2 Nucleotide Receptor

“…The number of HA-tagged wild-type P2Y 2 receptors in 1321N1 cells was estimated by comparison with the fluorescence intensity due to HA-tagged ␤ 2 -adrenergic receptors (500 fmol/mg of total protein) in HEK-293 cells (20). The ␤ 2 -adrenergic receptor density was determined to be 32,667 Ϯ 6692 receptors/cell compared with 22,767 Ϯ 9753 receptors/cell for the wild-type P2Y 2 receptor.…”

“…It is possible that, during our experiments, too much UTP and too few 1321N1 cells were present to demonstrate significant hydrolysis of nucleotide. P2Y 2 Receptor Sequestration Is Reversible-The reversibility of sequestration of the wild-type P2Y 2 receptor was determined after preincubating cells for 10 min with 1 mM UTP. Following incubation of the cells for various times in the absence of agonist, cell-surface receptor expression was determined.…”

“…Recombinant P2Y 2 Receptor Expression Levels in 1321N1 Cells-In the absence of an established method to detect P2Y 2 receptors on the cell surface, all receptor constructs were Nterminally tagged with an epitope of the HA antigen from influenza virus. The fluorescence intensities of cells expressing the epitope-tagged P2Y 2 receptor constructs were determined by flow cytometry, and the resulting scans were superimposed (Fig.…”

“…Multiple P2Y subtypes have been classified pharmacologically and molecularly and are predominantly linked to activation of phospholipase C and increased levels of inositol 1,4,5-trisphosphate and diacylglycerol, leading to elevations in the intracellular free calcium concentration ([Ca 2ϩ ] i ) and the activation of protein kinase C (PKC) (1)(2)(3)(4). The P2Y 2 nucleotide receptor subtype (formerly the P 2U receptor) is distinguished pharmacologically from the other known mammalian P2Y receptor subtypes by the equal potency and efficacy of the naturally occurring agonists ATP and UTP.…”

Astrocytes in Alzheimer's Disease

“…40 -42 Subsequent in vitro experiments have demonstrated that glial cells express the very same diverse variety of neurotransmitter receptors and ion channels as do neurons, [43][44][45][46][47][48][49][50][51][52][53][54][55] thus raising the question of the role for neuroglia in information processing in the brain. Further experiments have found that the expression pattern of neurotransmitter receptors in situ is very much restricted by the immediate neurotransmitter environment; as a consequence glial cells are properly endowed to sense the neurotransmitters released in their territorial domains.…”