Christine Morrow

Astrocyte glutamate receptor activation promotes inositol phospholipid turnover and calcium flux

Astrocyte-enriched cultures prepared from the neonatal rat cortex were prelabelled with either [3H]myoinositol or 45Ca2+ and then exposed to various excitatory amino acids. This resulted in an increase in both the breakdown of membrane inositol phospholipids and Ca2+ flux with the following rank order of efficacy: quisqualate greater than or equal to glutamate (Glu) greater than kainate much greater than N-methyl-D-aspartate. Experiments performed with the Ca2+ ionophore A23187 and in the absence of medium Ca2+ suggested that Glu-evoked 45Ca2+ efflux was primarily the result of an increased influx of extracellular Ca2+. However, Glu-stimulated inositol lipid metabolism was found to be only partially dependent on extracellular Ca2+. The quisqualate-preferring receptor antagonist gamma-glutamylaminomethylsulphonic acid was found to be effective in reversing both Glu-evoked inositol lipid breakdown and Ca2+ flux. The results presented are suggestive of some form of interaction between Glu receptors coupled to inositol lipid turnover and Ca2+ channel opening in astrocytes.

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 45Ca2+ 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 α‐adrenoceptors also caused PPI breakdown and 45Ca2+ 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.

Activation of Muscarinic and of α1‐Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Abstract: Astrocyte‐enriched cultures prepared from the newborn rat cortex incorporated [3H]myo‐inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose‐dependent and blocked by specific α1‐adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+ ‐free medium, but was abolished when EGTA was also present in the Ca2+ ‐free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo‐inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.

Receptor-mediated inositol phospholipid hydrolysis in astrocytes.

Astrocyte-enriched cultures of the neonatal rat cortex were incubated for 24 h with [3H]inositol to prelabel the membrane inositol phospholipids. Exposure of the cultures to either noradrenaline or carbachol in the presence of Li+ produced a time- and dose-dependent accumulation of intracellular [3H]inositol phosphates. The separation of the individual inositol phosphates formed in response to receptor stimulation revealed that the major 3H-metabolite accumulated under these conditions was inositol monophosphate but that at least some of this was due to the initial formation of inositol trisphosphate. The use of selective receptor antagonists showed that noradrenaline- and carbachol-induced [3H]inositol phosphate accumulation was the result of the activation of alpha 1-adrenoceptors and muscarinic acetylcholine (probably of the M1 subtype) receptors respectively. Agonist-evoked [3H]inositol phosphate accumulation were found to be additive but the simultaneous addition of agonists and the Ca2+ ionophore A23187, which also promoted inositol phospholipid hydrolysis, was not. Agonist-induced [3H]inositol phosphate accumulation was only partially dependent on extracellular Ca2+, whilst that elicited by A23187 was entirely Ca2+-dependent. The results suggest that alpha 1-adrenoceptors and muscarinic acetylcholine receptors in these cultures are present either on the same cells and linked to separate inositol lipid pools or associated with different subpopulations of astrocytes in these cultures. Moreover, inositol lipids other than phosphatidylinositol 4,5-bisphosphate may be hydrolysed in response to agonist stimulation.

Effects of Neurotransmitters on Astrocyte Glycogen Stores In Vitro

Abstract: We have used receptor binding assays to determine the presence of three neurotransmitter receptors in a crude membrane fraction derived from neonatal rat cortical astrocyte cultures and subsequently determined the effects of transmitter receptor activation on astrocyte glycogen content in vitro. β‐Adrenergic (KD= 88 pM; Bmax= 51 fmol/mg of protein), serotonin (KD= 70 nM; Bmax= 44 pmol/mg of protein), and muscarinic cholinergic receptors (KD= 79 pM; Bmax= 44 fmol/mg of protein) were found to be present on astrocyte membranes using [3H]dihydroalprenolol, [3H]serotonin, and [3H]quinuclidinyl benzilate, respectively, as ligands. Astrocyte cultures exposed to noradrenaline but not specific α‐ and β‐receptor agonists contained 33% less glycogen than controls. Neither serotonin nor carbachol caused alterations in astrocyte glycogen content under normal conditions. Reserpine‐treated cultures, however, responded to serotonin with a 28% decrease in glycogen content and contained higher levels of glycogen than non‐reserpine‐treated controls (a 55% increase). These results show that both noradrenaline and serotonin can evoke astrocyte glycogenolysis and that noradrenergic control of glycogen metabolism is probably exerted through both α‐ and /S‐receptors. Neurotransmitter control of astrocyte glycogen turnover may represent a form of neuron‐astrocyte signalling in addition to that provided by changes in external potassium concentration.

Phorbol ester stimulates proliferation of astrocytes in primary culture

Near-confluent primary cultures of astrocytes from the neonatal rat cerebral cortex were transferred to low serum (0.1%) growth medium for 24 h before a single addition of phorbol-12-myristate-13-acetate (0.01-100 ng X ml-1), a phorbol ester which mimics diacylglycerol activation of protein kinase C. After 48 h the cultures were pulsed with [methyl-3H]thymidine. Cultures exposed to phorbol ester exhibited dose-dependent increases in thymidine incorporation which were reversed by amiloride.

Characteristics of Phorbol Ester- and Agonist-Induced Down-Regulation of Astrocyte Receptors Coupled to Inositol Phospholipid Metabolism

We have examined some of the characteristics of phorbol ester‐ and agonist‐induced down‐regulation of astrocyte receptors coupled to phosphoinositide metabolism. Our results show that preincubation of [3H]inositol‐labelled astrocyte cultures with phorbol 12‐myristate 13‐acetate (PMA) resulted in a time‐ (t1/2, 1–2 min) and concentration‐dependent (IC50, 1 nM) decrease in the accumulation of [3H]inositol phosphates (IP) evoked by muscarinic receptor stimulation. Much longer (30–40 min) preincubation periods with higher concentrations (IC50, 600 μM) were required to elicit the same effect with the receptor agonist carbachol. Following preincubation, agonist‐stimulated [3H]IP accumulation recovered with time; in both cases pretreatment levels of inositol lipid metabolism were attained within 2 days. Both phorbol ester and agonist pre‐treatments were also effective in reversing the carbachol‐evoked mobilisation of 45Ca2+ in these cells. However, their effects on phosphoinositide metabolism were found not to be additive. Although neither pretreatment affected the incorporation of [3H]inositol into phosphoinositides, both resulted in a loss of membrane muscarinic receptors as assessed by [3H]N‐methylscopolamine binding. In washed membranes prepared from [3H]inositol‐labelled cultures, the guanine nucleotide analogue, guanosine 5′‐O‐thiotri‐phosphate (GTP‐γ‐S), caused a dose‐dependent increase in [3H]IP formation. This response was enhanced when carbachol was also included in the incubation medium, although the agonist alone was without effect. Pretreatment with either PMA or carbachol had no effect on GTP‐γ‐S‐stimulated [3H]IP accumulation but did reduce the ability of carbachol to augment this response. Similar findings were obtained when membranes were exposed directly to PMA. Phorbol ester pretreatment was also effective in reversing the increase in [3H]IP accumulation and 45Ca2+ mobilisation evoked by noradrenaline. However, following preincubation with carbachol there was no loss of nor‐adrenaline‐stimulated phosphoinositide breakdown although its ability to mobilise 45Ca2+ was blocked.

Effects of Extracellular Potassium on Glycogen Stores of Astrocytes In Vitro

Abstract: Astrocyte‐enriched and meningeal cell cultures of the rat cerebral cortex were prepared, and their glycogen content was measured after 10–90 min under control (2.5 mM) concentrations of potassium after prefeeding with 20 mM glucose. No net change in glycogen level was noted in either culture over this period. Cell cultures were then exposed to increased concentrations of potassium (5, 10, and 15 mM), and their glycogen content was measured after 10–90 min. Both types of cell culture showed complex and variable changes in glycogen content. In general, increased potassium concentrations caused astrocyte glycogen stores to be reduced at physiological increases of potassium levels (from 2.5 to 5 mM and above), although a period of resynthesis was evident at all potassium concentrations. Meningeal cell glycogen levels were highly variable and only affected by high (10 and 15 mM) levels of potassium. These results are discussed with respect to the theory that changes in the external potassium concentration caused by neuronal activity might act as a signal controlling astrocyte glycogen stores.

Phorbol ester stimulation of prostanoid synthesis by cultured astrocytes.

The role of protein kinase C in mediating the synthesis and release of various prostanoids (prostaglandins E2, I2, F2 alpha and thromboxane A2) from astroglial cells derived from neonatal rat cerebrum and maintained in primary culture was investigated using phorbol ester. Phorbol myristate acetate stimulated the release of arachidonic acid from prelabelled cells and all 4 prostanoids in a dose-dependent manner (EC50 = 300 nM). This effect was inhibited by the protein kinase inhibitor 1-(-5-isoquinolinylsulfonyl)-2-methylpiperizine (IC50 = 25 microM) and the phospholipase A2 inhibitor, mepacrine (IC50 = 5 microM). In addition, the stimulatory effect of the phorbol ester was not apparent in cells which had been depleted specifically of protein kinase C. In the presence of the calcium ionophore A23187, phorbol ester-stimulated prostanoid release was enhanced. In the absence of extracellular calcium, there was no prostanoid-stimulation by phorbol ester, but the calcium channel blocker verapamil did not mimic this effect. We conclude that stimulation of protein kinase C by phorbol ester elicits prostanoid synthesis and release by a process that involves calcium influx and the activation of phospholipase A2.

Phosphatidic acid promotes phosphoinositide metabolism and DNA synthesis in cultured cortical astrocytes

The addition of exogenous phosphatidic acid (PA) to cultured cortical astrocytes prelabelled with [3H]inositol resulted in the accumulation of intracellular [3H]inositol phosphates (IP) in a concentration-dependent (EC50 = 20 microM) manner. Analysis of the individual IPs formed following a PA challenge revealed a rapid but transient generation of [3H]inositol trisphosphate (IP3) indicating the involvement of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown in this response a fact which was confirmed when the recovery of radiolabel in membrane phosphoinositides was assessed. PAs ability to stimulate IP3 accumulation was found to be dependent upon its acyl-chain length. Dioleoyl-PA (C18:1) was equally as effective as PA from egg yolk lecithin in this respect whilst dipalmitoyl-PA (C16:0) was less so and dimyristoyl-PA (C14:0) and dilauroyl-PA (C12:0) were without effect. In subconfluent, serum-deprived cultures, PA was found to increase DNA synthesis following a 48 h exposure period. This effect was observed over the same concentration range used to measure phosphoinositide breakdown and was found to be mediated by the activation of protein kinase C. As with its effect on phosphoinositide metabolism, PAs ability to promote DNA synthesis was correlated with its acyl-chain length. These data show that PA is capable of stimulating both phosphoinositide metabolism and DNA synthesis in cultured astrocytes possibly via the activation of specific membrane receptors. However, the precise relationship between these events remains to be elucidated.