Regulation of α1-adrenoceptor-linked phosphoinositide metabolism in cultured glia:: involvement of protein phosphatases and kinases

Noradrenaline-stimulated phosphomositide breakdown in cultured glia was found to be mediated by UIA-adrenoceptors. The alpha(1A)-selective agonist A61603 was as effective as noradrenaline in eliciting H-3-mositol phosphate (IP) accumulation but was approximately 50-fold more potent. In addition, the use of selective antagonists revealed a clear rank order of potency in the ability of these drugs to reverse the effect of noradrenaline on phosphomositide breakdown: RS17053 (alpha(1A)-selective)>>AH11110A (alpha(1B)-selective)>BMY7378 (alpha(1D)-selective). Pre-treatment of cultured glia with the protein phosphatase inhibitor okadaic acid resulted in a concentration- and time-dependent reduction in noradrenaline-evoked H-3-IP accumulation. This effect was mimicked by, but was not additive with, a phorbol ester, was reversed by protein kinase C (PKC) inhibitors and was not evident in cells which had been PKC depleted. The ability of cell extracts to dephosphorylate radiolabelled glycogen phosphorylase revealed the presence of the phosphatases PP1 and PP2A in almost equal abundance. Okadaic acid pre-treatment of intact cultures elicited a marked reduction in total phosphatase activity, particularly that mediated by PP2A. We also determined the effect of okadaic acid pre-treatment on PKC and cyclic AMP-dependent protein kinase (PKA) activities in these cells. PKC and PKA activities in cell extracts were assessed by determining the incorporation of P-32 into histone and kemptide, respectively. Okadaic acid elicited increases in both Ca2+-dependent and Ca2-/-- independent PKC activity; in addition, increases in both initial and total PKA activities were also recorded. The effect of okadaic acid on noradrenaline-stimulated H-3-IP accumulation were not, however, mimicked by either forskolin or 8-bromo-cyclic AMP, suggesting that this event is not regulated by PKA. Our data point to roles for both PKC and PP2A in the regulation of alpha(1A)-adrenoceptor-linked phosphomositide metabolism in cultured cortical glia. (C) 2003 Elsevier Science Inc. All rights reserved.