RADIO-LABEL AND MASS DETERMINATIONS OF INOSITOL 1,3,4,5-TETRAKISPHOSPHATE FORMATION IN RAT CEREBRAL CORTICAL SLICES - DIFFERENTIAL-EFFECTS OF MYOINOSITOL

To investigate the effects of increasing concentrations of myo-inosito l (inositol) on receptor stimulated [H-3]inositol polyphosphate format ion in the absence of lithium, slices of rat cerebral cortex were incu bated with various concentrations of [H-3]inositol (1 to 30 muM). Carb achol stimulated formation of [H-3]inositol trisphosphate (InsP3) and [H-3]inositol 1,3,4,5-tetrakisphosphate {Ins(1,3,4,5)P4} increased sev eral fold when the inositol concentration was increased reaching a pla teau at approximately 12 muM inositol. Time course studies revealed th at in the presence of low concentrations of inositol (1 muM), [H-3]Ins P, and [H-3]Ins(1,3,4,5)P4 formation in response to carbachol stimulat ion increased slowly over a 10 to 20 min time period, whereas in the p resence of 4 and 12 muM inositol, carbachol stimulated [H-3]InsP3 and [H-3]Ins(1,3,4,5)P, formation was rapid and essentially complete withi n 3 to 5 min after carbachol addition. Although the carbachol dose res ponse in 12 muM inositol had a much greater maximal efficacy, there wa s no change in potency. Similar to the effects of carbachol on [H-3]In s(1,3,4,5)P4 formation from prelabeled phosphoinositides, muscarinic r eceptor stimulation increased Ins(1,3,4,5)P4 mass formation by seven f old. Furthermore, Li+ (8 mM) completely inhibited carbachol stimulated increases in Ins(1,3,4,5)P, mass formation. In contrast to the effect s of increasing inositol on carbachol stimulated formation of radiolab eled inositol phosphates, increasing inositol had no effect upon mass formation of Ins(1,3,4,5)P4. These results show that when measuring in ositol polyphosphate formation by the radiolabeling technique in the a bsence of Li+, increasing the inositol concentration greatly increases the stimulated component of [H-3]InsP3 and [H-3]Ins(1,3,4,5)P4 format ion. However, this inositol induced increase in agonist stimulated Ins (1,3,4,5)P4 formation is not reflected as an increase in mass formatio n.