Studies of inositol phosphate export from neuronal tissue in vitro

Recent in vivo microdialysis studies have demonstrated the presence of extracellular levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] that can be increased in a concentration-dependent manner by muscarinic receptor activation. The aim of the present study was to determine whether extracellular levels of Ins(1,4,5)P3 could be measured in vitro. Despite rapid increases in internal Ins(1,4,5)P3 levels after stimulation with 1 mM carbachol, there was no change in external levels in both rat brain cortical slices and human neuroblastoma SH-SY5Y cells. Suprafusion of myo-[3H]inositol-prelabelled hippocampal slices with 1 mM carbachol caused an increase in 3H-inositol phosphates over basal levels in the perfusate after 10 min, reaching a peak (223 +/- 56% of basal) 20 min after suprafusion with carbachol was started. This response to carbachol was potentiated in the presence of 30 mM K+. Analysis of the individual 3H-inositol phosphates in the perfusate revealed that levels of [3H]inositol monophosphate, [3H]inositol bisphosphate, [3H]inositol trisphosphate, and [3H]inositol tetrakisphosphate were all significantly increased. A similar increase in extracellular 3H-inositol phosphates was demonstrated in SH-SY5Y cells incubated with 1 mM carbachol for 30 min. This response was again enhanced by 30 mM K+, although the intracellular response was not potentiated. Possible roles for extracellular inositol phosphates are discussed.