The role of inositol 1,4,5-trisphosphate 5-phosphatase in inositol signaling in the CNS of larval Manduca sexta

Production of inositol 1,4,5-trisphosphate (IP3) in cells results in the mo bilization of intracellular calcium. Therefore, the dynamics of IP3 metabol ism is important for calcium dependent processes in cells, This report inve stigates the coupling of mAChRs to the inositol lipid pathway in the CNS of the larval Manduca sexta. Stimulation of intact abdominal ganglia prelabel ed with [3H] inositol using a muscarinic agonist, oxotremorine-M (oxo-M), i ncreased total inositol phosphate levels in a dose dependent manner (EC50=4 .23 mu M). These inositol phosphates consisted primarily of inositol 1,4-bi sphosphate (IP2) and inositol monophosphate (IPI). Similarly, when nerve co rd homogenates were provided with [3H]-phosphatidylinositol 4,5-bisphosphat e ([3H]-PIP2) (10-13 mu M) the predominant products were IP3 and IPI. In co ntrast, incubation of purified membranes with 1 mM oxo-M in the presence of 100 mu M GTP gamma S and [3H]-PIP2 increased IP3 levels, suggesting that t he direct activation of phospholipase C (PLC) by mAChRs occurs in a membran e delimited process. Together, these results suggest that in the intact ner ve cord and in crude homogenates, a cytosolic 5-phosphatase quickly metabol izes IP3 to produce to IP2 and IPI. This enzyme was kinetically characteriz ed using IP3 (Km=43.7 mu M, Vmax=864 pmoles/min/mg) and IP4 (Km=0.93 mu M, Vmax=300pmoles/min/mg) as substrates. The enzyme activity can be potently i nhibited by two IP thiol compounds; IP3S3 (1,4,6) and IP3S3 (2,3,5, that sh ow complex binding kinetics (Hill numbers<1) and can distinguish different forms of the 5-phosphatase in purified membranes. These two inhibitors coul d be very useful tools to determine the role of the inositol lipid pathway in neuroexcitability. (C) 1999 Elsevier Science Ltd, All rights reserved.