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Regulation of α1-adrenoceptor linked phosphoinositide breakdown in cultured glia: role of protein phosphatases

Assari, Tracy Louise; (2002) Regulation of α1-adrenoceptor linked phosphoinositide breakdown in cultured glia: role of protein phosphatases. Doctoral thesis (Ph.D.), University College London (United Kingdom). Green open access

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Abstract

This study attempts to identify the protein phosphatases and kinases involved in regulating receptor-mediated phosphoinositide metabolism in astrocytes derived from newborn rat cerebral cortex and maintained in culture. Noradrenaline produced a dose and time dependent accumulation of [3H] inositol phosphates which was mimicked by A61603 and blocked by RS17053 indicating the involvement of α1A-adrenoceptors in this response. Okadaic acid inhibited noradrenaline- evoked [3H] inositol phosphate accumulation in a time (t½, 5 min) and concentration (IC 50, 0.3μM) dependent manner; an effect which was reversed by the non-selective protein kinase inhibitor staurosporine. This suggests that protein phosphatase inhibition in these cells allows a kinase-mediated down regulation of receptor activity. A myristoylated protein kinase C inhibitor, at 10μM, reversed the effect of okadaic acid by some 60% whilst the cAMP-dependent protein kinase inhibitor, at the same concentration was without an effect. The involvement of protein kinase C in this response was further continued with the use of a protein kinase C activator, phorbol 12-myristate 13-acetate which inhibited noradrenaline-stimulated [3H] inositol phosphate accumulation (IC50 3nM) but was not additive with okadaic acid. The inhibitory effect of okadaic acid on noradrenaline-evoked [3H] inositol phosphate accumulation was overcome by long termphorbol ester treatment, thereby down regulating protein kinase activity in these cells. Pre-treatment of cultures with okadaic acid produced a loss of phosphatase activity and an increase in protein kinase C activity in these cultures. The effect of noradrenaline was found to be largely dependent upon extracellular Ca 2+. Ca2+ uptake into glial cells stimulated by α1-adrenoceptor activation was blocked in cultures pre-treated with a phosphatase inhibitor (okadaic acid) or kinase activator suggesting that receptor-coupled Ca 2+ influx is regulated by phosphorylation. Ca2+ release was also stimulated by α1-adrenoceptor activation but potentiated in cultures pre-treated with okadaic acid. Results thus point towards a role for a protein kinase in the mechanism by which receptors are coupled to and regulate phosphoinositide metabolism in glial cultures. The identity of particular protein phosphatases and protein kinases involved in this aspect of glial cell function are hereby a subject for investigation.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Regulation of α1-adrenoceptor linked phosphoinositide breakdown in cultured glia: role of protein phosphatases
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: (UMI)AAI10104870; Health and environmental sciences; Adrenoceptor; Alpha;1-adrenoceptor; Breakdown; Cultured; Glia; Phosphatases; Phosphoinositide; Protein
URI: https://discovery.ucl.ac.uk/id/eprint/10104841
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