Mass Determination of Polyphosphoinositides and Inositol Triphosphate in Rat Adrenal Glomerulosa Cells with a Microspectrophotometric Method*

Abstract

A method is described for the assay of subnanogram amounts of phosphorus in phospholipids and organic phosphates. The formation of a complex with a high molar absorption coefficient at 600 nm when malachite green is added to phosphomolybdate at low pH and the adaptation of a microspectrophotometer to quantify the color in 10 .mu.l solution have made it possible for a dose-response curve from 0.1-1.2ng phosphorus to be developed. The method has been applied to the assasy of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns 4-P), phosphatidylinositol-4,5-diphosphate (PtdIns 4,5-P2), and inositol-1,4,5-triphosphate (Ins 1,4,5-P3) in rat adrenal glomerulosa cells after stimulation with angiotensin II (AII), K+, and ACTH for 0,2,4,6,8,10,12,15 and 60 sec. A control (nonstimulated) sample was incubated concomitantly for every time period. Nonstimulated cell levels (mean .+-. SEM; n = 216) were: PtdIns, 577 .+-. 6.4; PtdIns 4-P, 183 .+-. 3.1; PtdIns 4,5-P2, 59 .+-. 1.8; and Ins 1,4,5-P3, 94 .+-. 1.3 pmol/incubate. Maximum increase in levels of PtdIns, PtdIns 4-P, PtdIns 4,5-P2, and Ins 1,4,5-P3 above control values was obtained after 8 sec with AII (10-8 M) and after 6 sec with K+ (8.7 mM) stimulation. The values (picomoles per 2 .times. 105 cells incubate; n =4) were: PtdIns, 808 .+-. 28; PtdIns 4-P, 263 .+-. 20; PtdIns 4,5-P2, 112 .+-. 10; and Ins 1,4,5-P3, 136 .+-. 4 for AII stimulation, and PtdIns, 925 .+-. 76, PtdIns 4-P, 308 .+-. 11; PtdIns 4,5-P2 146 .+-. 28; and Ins 1,4,5-P3, 149 .+-. 5 for K+ stimulation. No increase above control levels could be found at any incubation time after ACTH stimulation. Thus both AII and K+ stimulate a short-lived increase in the mass of several elements of the phosphatidylinositol pathway. The discrepancy between these mass determinations and isotope study suggests that only some, but not all, pools are labeled by currently available techniques.