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Notes: Abstract: The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5-trisphosphate [Ins(l,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second-messenger inositol 1,3,4,5-tetra-kisphosphate [Ins(l,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4-trisphosphate [Ins(l,3,4)P3], eightfold; Ins(l,3,4,5)P4, 6.4-fold; Ins(l,4,5)P3, 75%; inositol 1,4-bisphosphate [Ins(1,4)P2, 0%; inositol 3,4-bisphosphate, 100%; inositol 1-phosphate/inositol 3-phosphate, 30%; and inositol 4-phosphate, 40%. [3H]Inositol 1,3-bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin-stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(l,4,5)P3 is metabolized primarily by Ins(1,4,5)P3-3’ kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 nM), [3H]Ins(l,3,4,5)P4 and [3H]Ins(l,3,4)P3 levels were increased, even at 1 μM serotonin. Ketanserin (7 μM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 μM) also increased the levels of [3H]Ins(l,4,5)P3, [3H]Ins(l,3,4,5)P4, and [3H]Ins(l,3,4)P3, but the profile of these increases was different. The quisqualic acid-stimulated formation of inositol phosphate isomers was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that it was not due to the ionotropic properties of the quisqualate receptor. Similar results were obtained on stimulation of the labeled slices with glutamate, but the magnitudes were less. The data show that in the guinea pig cerebellum, stimulation of the serotonin2 and metabotropic quisqualic acid receptors leads to the initial formation of Ins(l,4,5)P3, but its subsequent metabolism varies, presumably owing to two kinds of receptors, localized on different cell types in the cerebellum with varying levels of inositol phosphate-metabolizing enzymes.