Evidence for a model of integrated inositol phospholipid pools implies an essential role for lipid transport in the maintenance of receptor-mediated phospholipase C activity in 1321N1 cells (original) (raw)
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Journal of Neurochemistry, 1988
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.
Journal of cellular …, 2010
Phosphoinositide specific phospholipase C (PI-PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI-PLC family display different expression and/or sub cellular distribution under non-physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI-PLCs compared to untreated cells. Special attention require PI-PLC beta3 and PI-PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U-73122 treatment. The meaning of these modifications is unclear, also because the use of this N-aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells.
A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization
European Journal of Biochemistry, 1989
A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 pmol . min-' . mg-l. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.
Neurochemical research, 2000
Intracellular compartmentation of inositol was demonstrated in primary cultures of mouse astrocytes, incubated in isotonic medium, by determination of efflux kinetics after "loading" with [3H]inositol. Three kinetically different compartments were delineated. The largest and most slowly exchanging compartment had a halflife of approximately 9 hr. This slow release leads to retention of a sizeable amount of pre-accumulated inositol in the tissue 24 hr after the onset of uptake inhibition, as confirmed by the observation that the inositol uptake inhibitor fucose caused a larger inhibition of unidirectional inositol uptake than of inositol pool size, measured as accumulated [3H]inositol after 24 hr of combined exposure to the inhibitor and the labeled isotope. Based upon the present observations and literature data, it is suggested that the large, slowly exchanging compartment is largely membrane-associated and participating in signaling via the phosphatidylinositide second m...
Biochemical Journal, 1997
The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4β-phorbol 12β-myristate 13α-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induce...
The Journal of Cell Biology, 1985
In PC12 cells, cultured in the presence of nerve growth factor to increase their complement of muscarinic receptors, treatment with carbachol induces muscarinic receptor-dependent rises in free cytosolic Ca2+ as well as hydrolysis of membrane phosphoinositides. Experiments were carried out to clarify the relationship between these two receptor-triggered events. In particular, since inositol-1,4,5-trisphosphate (the hydrophilic metabolite produced by the hydrolysis of phosphatidylinositol-4,5-bisphosphate) is believed to mediate intracellularly the release of Ca2+ from nonmitochondrial store(s), it was important to establish whether it can be generated at resting cytoplasmic concentration of Ca2+ (approximately 0.1 microM). Cells incubated in Ca2+-free medium were depleted of their cytoplasmic Ca2+ stores by pretreatment with ionomycin. When these cells were then treated with carbachol, their cytosolic concentration of Ca2+ remained at the resting level, whereas inositol-1,4,5-trisph...