Abstract
In the rat pancreatoma cell line, AR4-2J, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin. Two other isomers were identified, (1,4,5,6) and a fifth isomer which was either (1,2,3,4) or (1,2,3,6), which have not previously been detected in any cell type. To study the metabolic interrelationships between these compounds and inositol 1,3,4,5,6-pentakisphosphate in the intact cell, their turnover was assessed under different protocols of [3H]myo-inositol labeling; the inositol phosphates were labeled to near steady state or under conditions where either rapidly or slowly turning over inositol polyphosphates were preferentially labeled. The relative specific radioactivities of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,6-tetrakisphosphate were very similar in bombesin-stimulated cells, consistent with the pathway for the conversion of inositol 1,4,5-trisphosphate to the other three inositol polyphosphates. Compared with these inositol phosphates, the turnover of inositol 1,3,4,5,6-pentakisphosphate was slow. An accumulation of radioactivity into inositol 1,3,4,5,6-pentakisphosphate was observed only under labeling conditions where its relative specific radioactivity was substantially below that of inositol 1,3,4,6-tetrakisphosphate. This indicated that the precursor for de novo synthesis of inositol 1,3,4,5,6-pentakisphosphate was inositol 1,3,4,6-tetrakisphosphate. Bombesin stimulated the net breakdown of inositol 1,3,4,5,6-pentakisphosphate and increased the level of inositol 3,4,5,6-tetrakisphosphate; the relative specific radioactivities of these two compounds were similar under all conditions. These data led to the novel proposal that inositol 3,4,5,6-tetrakisphosphate is the product of inositol 1,3,4,5,6-pentakisphosphate breakdown. This reaction was apparently stimulated by a regulated change in the enzyme(s) which interconvert inositol 1,3,4,5,6-pentakisphosphate and inositol 3,4,5,6-tetrakisphosphate.
Highlights
In the rat pancreatoma cell line, AR4-ZJ, three inositol tetrakisphosphate isomers were identified, (1,3,4,6), (1,3,4,5), (3,4,5,6), which were increased during activation of phospholipase C by bombesin
We have examined the effects of agonist activation on inositol tetrakis- and pentakisphosphate metabolism in the rat pancreatoma cell line, AR4-2J
Our findings indicate that agonist activation causes an accumulation of three inositol tetrakisphosphates in the AR4-2J cells
Summary
The inositol phosphates and lipids are abbreviated according to the “Chilton Co&en&on”. Example, (1,4,5)IP3 for D-myo-inositol (1,4,5)trisphosphate (34) as, for and PIP*. For phosphatidylinositol (4,5)bisphosphate; HEPES, 4-(2-hydroxyethyl)-l-piperazieethanesulfonic acid; MOPS, 3-(N-morpholino)-. Propanesulfonic acid); HPLC, high performance liquid chromatography. (1,3,4,6)IP4 are derived from lipase C product, (1,4,5)IPs. (3,4,5,6)IP4, pendent pathway involving (1 , 3 , 4 f 5 , 6)IPs. the metabolism Surprisingly, appears to agonist-induced of the phosphothe third inositol arise by an indebreakdown of EXPERIMENTAL
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