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 microliters solution have made it possible for a dose-response curve from 0.1-1.2 ng phosphorus to be developed. The method has been applied to the assay 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 X 10(5) cell 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.

Vascular smooth muscle cells from the Milan hypertensive rat exhibit decreased functional angiotensin II receptors.

“…23 In these experiments, we confirmed previous observations that Ang II effect peaked at 15 seconds. In subsequent experiments, VSM monolayers were grown in 35 mm dishes and labeled by adding 30 ^tCi/ml [ 3 H]myoinositol for 24-48 hours as previously described.…”

Endothelium‐dependent and BRL 34915‐induced vasodilatation in rat isolated perfused mesenteric arteries: role of G‐proteins, K+ and calcium channels

“…Even though these three agents are known to require Ca2+ for their vasoconstrictor action, they differ remarkably in their dependence on extracellular Ca2+ for this effect; AVP-and KCl-induced vasoconstriction was abolished in the absence of extracellular calcium, while NA-induced vasoconstriction persisted (present study, data not shown). NA, which acts on a1-adrenoceptors (Lynch et al, 1985), and AVP on V1-receptors (Fox et al, 1987;Nabika et al, 1985) and KCl, which acts directly on smooth muscle cells (Underwood et al, 1988) have all been shown to be capable of inducing the breakdown of polyphosphoinositides to myoinositol trisphosphate and diacylglycerol. Therefore, the differential effects of pertussis toxin on these Ca2+-mobilizing processes activated by these agents suggest that either the events associated with each of the agents are coupled to different G-proteins or only a,-receptors, but not V1-receptors or voltage-dependent calcium channels are coupled to PTX-sensitive G-proteins in rat mesenteric arteries.…”

The Physiologic Concentration of Inositol 1,4,5-Trisphosphate in the Oocytes of Xenopus laevis

“…The dissociation constant of the IP 3 receptor suggests that [IP 3 ] in an unstimulated cell is in the picomolar range and certainly not greater than a few tens of nanomolar. However, most experimental measurements on unstimulated cells place [IP 3 ] well above its dissociation constant for the IP 3 receptor, generally in the micromolar range (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). These discrepant predictions have yet to be resolved.…”

“…These discrepant predictions have yet to be resolved. A similarly wide range of predicted [IP 3 ] exists for the maximal concentration attainable in cells (100 nM to 100 M) (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The purpose of this investigation was to establish the range of [IP 3 ] in a cell, the Xenopus oocyte, before and after application of a physiologic stimulus.…”