Biochimica et Biophysica Acta, 1014 (1989) 239-246 Elsevier

239

BBAMCR 12592 Review

Mass measurement of inositol phosphates

Susan Palmer and Michael J.O. Wakelam Molecular Pharmacology Group, Department of Biochemistry, University of Glasgow, Glasgow ( U.K.)

(Received 9 June 1989) (Revised manuscript received 1 September 1989)

Key words: Inositol phosphate; Mass spectrometry; NMR

Contents

I. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

If. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

III. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

IV. Methods of inositoi phosphate measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

V. Use of radioactive inositol lipid precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

VI. Optical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

VII. Gas chromatography, mass spectrometry, nuclear magnetic resonance, fast atom bombardment . . . 242

VIII.Analytical methods specific for Ins(1,4,5)P3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

!. Summary

This review summarises the methods available for the mass measurement of inositol phosphates, i.e., use of radioactive inositol lipid precursors, optical techniques, gas chromatography, mass spectrometry, nuclear mag- netic resonance, fast atom bombardment and assays specific for Ins(1,4,5)P3. Examples of the use of each method, its sensitivity, advantages and drawbacks are given.

Abbreviations: InsP, inositol phosphate, PtdInsP, phosphatidyl- inositol phosphate; DAG, diacylglycerol; DPG, 2,3-diphospho- glycerate.

Correspondence: M.J.O. Wakelam, Molecular Pharmacology Group, Department of Biochemistry, University of Glasgow, Glasgow (312 8QQ, U.K.

If. Introduction

A range of hormones, neurotransmitters and growth factors stimulate cellular events via the generation of second messenger molecules derived from inositol phos- pholipids [1]. Binding of the agonist to its specific cell surface receptor results in the activation of phospho- inositidase C by a process which involves an as yet uncharacterised guanine nucleotide binding regulatory protein (G-protein), Gp. Hydrolysis of PtdIns(4,5)P2 generates two second messengers: inositol 1,4,5-tris- phosphate (Ins(1,4,5)P3) and sn-l,2-diacylglycerol (DAG). DAG, the physiological activator of protein kinase C, is metabolised to either phosphatidic acid or monoacylglycerol by the actions of diacylglycerol kinase and diacylglycerol lipase, respectively. Mass measure- ment of DAG is achieved by enzymatic means [2], but will not be considered further in this article.

Ins(1,4,5)P3 stimulates the release of intracellular stores of Ca 2+ by a mechanism which involves specific

0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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intracellular receptors. Removal of Ins(1,4,5)P3 is by both phosphorylation to Ins(1,3,4,5)P4 and dephospho- rylation to Ins(1,4)P2, catalysed by Ins(1,4,5)P3 3-kinase and Ins(1,4,5)P3 5-phosphatase, respectively [1]. It has been proposed that lns(1,3,4,5)P4 also has a second messenger function, in stimulating Ca 2 + entry into cells [3]. Dephosphorylation of ins(1,3,4,5)P4, by an enzyme which may be the same as that acting upon Ins(1,4,5)P3, generates lns(1,3,4)P3, an inositol trisphosphate which does not appear to have a signalling role under physio- logical conditions. Inositol phosphates that may not be involved in receptor mediated signal transduction have also been identified in cells. The pathways involved in the metabolism of these phosphates appear to lead to the generation of inositol penta- and hexakisphos- phates, the functions of which, within cells, remain unclear. The increasingly complex metabolism of in- ositol phosphates has recently been reviewed elsewhere [41.

It has been demonstrated that cells can also contain cyclic inositol phosphates [5], although this is not the case for all cell types [6]. The physiological role of the cyclic inositol phosphates is unclear since, despite earlier claims, it is apparent that Ins(l:2-cyclic,4,5)P3 has a low affinity for the InsP3 receptor linked to Ca 2+ homeostasis [7,8]. However, when measuring inositol phosphate levels in cells, these compounds cannot be ignored, since the acid treatment, frequently used to terminate cell incubations, will result in the hydrolysis of the cyclic bond and thus lead to spurious estimations of individual inositol phosphates.

IlL Sample preparation

The choice of extraction procedure is a critical con- sideration in the analysis of inositol phosphates, espe- cially if mass measurements are to be performed. As a general rule, it is advisable that recovery measurements are made with appropriate radiolabelled standards be- fore a particular method is adopted. Termination of incubations with neutral chloroform/methanol [9] is suitable for lnsP, lnsP2 and InsP3 (> 85~ recovery), although the recovery of higher inositol phosphates may be reduced [10]. Therefore, acidic extraction procedures are preferable. The acids used are either perchloric or trichloroacetic acid. Removal of perchloric acid can be achieved by precipitation of potassium perchlorate on addition of KOH [11], whereas trichloroacetic acid is removed by repeated washing with water-saturated di- ethyl ether [12]. The use of Freon and tri-n-octylamine for neutralisation [13] has recently become popular, although poor recoveries of Ins(1,4,5)P3 have been re- ported for this method [14]. When low concentrations of inositol phosphates are present in a sample, their re- coveries can be poor. However, when radiolabelling is used for inositol phosphate analysis, Wreggett et al. [10]

propose the use of a phytate hydrolysate to increase bulk and improve recoveries. The measurement of cyclic phosphates requires greater care since the cyclic bond is acid sensitive. A phenol-based extraction procedure has been shown to yield good recoveries of InsP, InsP2, Ins/'3 and InsP4 (> 95%) and to preserve the cyclic moiety of radiolabeUed cyclic inositol phosphate stan- dards [6,15].

IV. Methods of inositol phosphate measurement

The choice of technique adopted for the determina- tion of inositol phosphate mass is dictated by: (i) the inositol phosphates of interest; (ii) the availability of material for analysis; (iii) capital outlay and running costs. Several techniques can be applied to all inositol phosphates, whereas others apply only to a certain few. Generally, the more inositol phosphates of interest, the more involved the analysis becomes. However, mass measurement of Ins(1,4,5)P3, the inositol trisphosphiite isomer currently of most interest, is probably the sim- plest to achieve.

For the majority of studies, the resolution of inositol phosphate isomers is required since mass determination of bulk InsP, InsP2, Ins/'3, etc. is of limited use (e.g., Refs. 16 and 17). A variety of inositol polyphosphate isomers occur in vivo including at least two of InsP 3 (Ins(1,4,S)P3 and Ins(1,3,4)P 3, [18]), three of InsP4 (Ins(1,3,4,S)P4, Ins(1,3,4,6)P4, Ins(3,4,5,6)P4, [12,19-21]) and several isomers of lnsP2 and InsP. A variety of HPLC procedures are now available for the separation of these isomers [22] and HPLC is usually an integral part of inositol phosphate mass measurement.

V. Use of radioactive inositol lipid precursors

Labelling of cellular inositol lipids with [32p]p~ or [3H]inositol has often been the method used to measure agonist-stimulated changes in polyphosphoinositides and inositol phosphates, respectively [22].

Inositol lipids can be labelled to a constant specific activity, i.e., their specific activities remain unchanged during the experimental period, by preincubation of cells in the presence of the radiolabel followed by incubation in its absence. However, metabolically dis- tinct pools of inositol lipids have been proposed to exist [23,24] and the distribution of label between agonist-sensitive and -insensitive pools is unclear. Therefore, it is incorrect to assume that, under these conditions, the specific activity of inositol lipids is equivalent to that of inositol phosphates and, thus, inositol phosphate mass cannot be calculated. Neverthe- less, labelling of cellular inositol lipids to isotopic equi- librium, when the specific activities of all inositol-con-

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taining compounds are identical, can be used. This procedure was originally used for 32p-labelling of plate- lets [25], but is particularly suited to cells in culture since incubation with [3H]inositol for several days may be required [26-28].

Dangelmaier et al. [25] incubated freshly isolated platelets with [32p]p i for 2 h, at which time, the specific activities of the monoester phosphate groups of Ptdlns- (4,5)P 2 and [¥-32p]ATP were identical. Neutralised per- chloric acid extracts of inositol phosphates were treated with 2,3-diphosphoglycerate phosphatase prior to reso- lution of InsP3 by two-dimensional paper electrophore- sis. InsP 3 mass, calculated from the associated radioac- tivity, was determined to be 1-4 pmol/10 s cells (ap- prox. 1/zM) and 10-30 pmol/10 s cells (approx. 20/zM) in resting and thrombin-stimulated platelets, respec- tively. More recently, the inositol phosphates have been separated by HPLC [29]. The basal concentration of Ins(1,4,5)P3 was below the detection limit of the system but, the maximal concentration was estimated to reach 2/zM (approx. 10% of total InsP3, the majority being Ins(1,3,4)P3). 32p-labelled contaminants were identified as ATP, inorganic pyrophosphate and 2,3-DPG by their specific removal using charcoal [22], inorganic pyro- phosphatase and 2,3-DPG phosphatase, respectively.

Morgan et al. [26] cultured anterior pituitary cells for 3 days in the presence of both [32PIP i and [3H]inositol. The specific activity of [32p]ATP was determined and assumed to be identical to that of [32p]Ptdlns(4,5)P2. The specific activity of inositol, and thus inositol phos- phate mass, was calculated using the 32p/3H ratio of inositol lipids. However, some of the assumptions made may have led to an underestimation of inositol phos- phate mass. For example, Ins(1,4,5)P3 levels were esti- mated to be 56 nM after 30 rain stimulation with gonadotrophin-releasing hormone, which is consider- ably less than estimates from other cells (see below).

Horstman et al. [27] and Michell et al. [28] have used [3H]inositol to label inositol lipids and their metabolites to isotopic equilibrium in AR42J pancreatoma cells and HL60 cells, respectively. Inositol phosphates were re- solved by HPLC and their mass was calculated directly from the associated radioactivity. For example, intra- cellular concentrations of Ins(1,4,5)P3 obtained in un- stimulated and agonist-stimulated cells were 2 #M and 25 #M, respectively, for AR42J cells and 0.4 #M and 1.6 #M, respectively, for HL60 cells.

However, equilibrium labelling experiments are not without their problems. Stewart et al. [30], using a human T-lymphocyte cell line, observed that [3H]in- ositol continued to be incorporated into InsP5 when the specific activity of inositol lipids was constant. Since the metabolism of ImPs remains unclear, the possibility arises that this observation may have been a conse- quence of receptor-independent synthesis of InsP5 and its precursors. Indeed, receptor stimulation has been

shown to be without effect upon InsP5 and InsP6 levels [311.

VI. Optical techniques

A number of simple, optical techniques of variable sensitivities are now available for the mass measurement of inositol phosphates. These include microspectropho- tometric and enzymatic/fluorimetric assays and a novel metal-dye detection method.

A microspectrophotometric assay, developed by Un- derwood et al. [32], involves the reaction of phos- phomolybdate with malachite green to generate a com- plex with a high molar extinction coefficient at 600 nm. A dose response curve of 0.1-1.2 ng phosphorus was determined by adaptation of a microspectrophotometer to quantify the colour in 10 #l of solution. InsP3, which co-chromatographed with [3Hllns(1,4,5)P a on thin-layer chromatography using polyethyleneimine cellulose plates, was found to be 90 pmol/2.5.105 in resting adrenal glomerulosa cells and a maximum, at 6 s, of 130 pmol/2.5.105 cells and 150 pmol/2.5.105 cells on stimulation with angiotensin II and high K +, respec- tively. No interference of up to 1000 ng of ATP or GTP was found. This method, although only used to measure inositol lipids and InsP3 by these workers, should be able to be applied to all inositol phosphates. Analysis of inositol phosphate isomers using this method, however, will necessitate a phosphate-free HPLC methodology.

Meek [33] described the HPLC separation of inositol polyphosphates on a Pharmacia Mono Q HR 5/5 an- ion-exchange column eluted with increasing concentra- tions of sulphate ions. Inositol phosphate mass was determined by on line alkaline phosphatase treatment and phosphate analysis, calibrated with nanomolar amounts of inositol polyphosphate standards. This method requires a minimum of 0.1 g of tissue per sample, the removal of nucleotides prior to HPLC and detects nanomolar qaantities of inositol phosphates. However~ Mayr [34] suggests that this technique is unreliable due to strong interference of incompletely dephosphorylated higher inositol polyphosphate iso- mers (InsPx, x >t 4) with the molybdate complex. In addition, incomplete dephosphorylation of inositol polyphosphates by alkaline phosphatase will result in their underestimation.

A number of workers have employed an enzymatic/ fluorimetric assay to measure inositol phosphate mass. The method is based on that of MacGregor and Matschinsky [35] for the chemical analysis of myo-in- ositol. Briefly, myo.inositol is oxidised by NAD-depen- dent myo-inositol dehydrogenase coupled to reoxidation of NADH with oxaloacetate and malate dehydrogenase. The resultant malate is measured fluorimetrically. A macroassay (for tissue samples of 0.5 mg or more) and a microassay (for samples of as little as 25 ng dry weight)

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are described, of which the sensitivity of the latter compensates for the somewhat protracted analytical procedure. The assay is suitable for the determination of 0.2 pmol to 8 nmol of myo-inositol.

Inositol phosphate mass measurements have been made by the introduction of a minor modification, i.e., incubation of desalted inositol phosphate fractions from anion-exchange resin columns with alkaline phos- phatase prior to the myo-inositol assay [36]. The re- covery of inositol phosphates during the desalting pro- cedure varied between 75 and 90% and was assessed using radiolabelled standards. Trichloroacetic acid ex- tracts of primary cultured rabbit papillary-collecting tubule cells were found to contain 0.16 + 0.05 pmol//~g protein and 0.34 + 0.08 pmol//~g protein of InsP3 in unstimulated and bradykinin-stimulated cells, respec- tively [36]. Estimation of the intracellular volume was used to calculate the intraceilular concentration of InsP3 to be 7.5/~M basal and 16 pM following bradyldnin stimulation. Similar results were obtained using freshly isolated rabbit papillary-tubule collected cells [17].

Similarly, Rogers and Hammerman [371 determined Ins(1,4,5)P3 mass (isolated by HPLC [33]) in canine renal proximal tubular cells. IGF-II treatment resulted in the elevation of Ins(1,4,5)P3 from approx. 0.5 nmol/mg protein to a maximum of approx. 1.55 nmol/mg protein at 15 s.

The enzymatic/fluorimetric assay of myo-inositol can be subject to interference from detergents and salt. However, Laursen et al. [38] have developed a rapid, simple method for the removal of interfering substances with close to 100% recovery of inositol. Briefly, deter- gent was removed when samples were passed through Sep-Pak CIs cartridges and washed with water. The combined eluants were desalted by slow percolation through Ambolite MB-3 deionizing columns and the eluant freeze-dried. This procedure was found to be effective in the removal of Triton X-100, NADH and malate.

Preliminary studies by Kargacin et al. [39] suggest that a novel HPLC analysis of fluorescent derivatives of inositol and inositol phosphates may prove useful. To date, up to 60 nmo] of inositol and Ins(2)P have been reacted with isatoic anhydride in the presence of tri- ethanolamine and quantitatively recovered from HPLC. However, the reactivity of isatoic anhydride with InsPx (x = 2-5) has not yet been investigated. This fnethod is not suitable for phytate which lacks the necessary hy- droxyl groups for derivatization. Quantitative analytical methods for phytate include HPLC on a ~Bondapak C!8 column with refractive index measurement of the column eluate (directly proportional to the quantity of phytate up to 120/tg) and a less sensitive colorimetric method involving precipitation of phytate with FeCI3, acid hydrolysis of the phosphate groups and analysis of phosphate using ammonium molybdate [40].

Metal-dye detection, developed by Mayr [34], in- volves the competition of inositol phosphates with a cation-specific dye (4-(2-pyridylazo)resorcinol, PAR) for a tervalent transition metal cation, e.g., yttrium ions. Inositol phosphate standards and perchloric acid cells extracts (neutrarised, treated with charcoal to remove nucleotides and desalted) were resolved on a Pharmacia Mono Q column, eluted with an upward-concave gradi- ent by mixing 0.2 and 0.4 M HCI containing 9 or 18/tM and 14 or 28 /~M yttrium, respectively. The column eluate was mixed with PAR (buffered with tri- ethanolamine at pH 8.4) and absorbance at 546 nm measured. At this pH, phosphorylated compounds be- come very strong polycation complexing agents, i.e., yttrium ions preferentially bind to inositol polyphos- phates and the absorbance decreases. The peak area was demonstrated to be proportional to the chemical amount of inositol polyphosphates up to I nmol. The magnitude of the peaks obtained varied between inositol polyphos- phates, i.e., the peak area obtained with a fixed con- centration of Ins(1,3,4,5)P4 > Ins(1,4,5)P a > Ins(1,4)P2, etc. such that sensitivity is greatest for the higher in- ositol polyphosphates.

This metal-dye detection system is compatible with the anions chloride, formate and acetate and the cations trimethylammonium, Tris, triethanolamine, imidazole and ammonium. However, sulphate and phosphate are not suitable, since these anions form strong complexes with tervalent metal ions. Therefore, cells (approx. 10 mg of tissue is needed) should be incubated in the absence of sulphate and in the presence of low con- centrations of phosphate, EDTA and polyvalent ca- tions. The interference caused by bisphosphorylated sugars, pyrophosphate and 2,3-diphosphoglycerate was overcome by increasing the length of the Mono Q column, upon which the resolution of inositol polyphos- phate isomers was particularly good.

VII. Gas chromatography, mass spectrometry, nuclear magnetic resonance, fast atom bombardment

These techniques, which involve considerable capital outlay, have been used both individually and in combi- nation to make mass measurements of inositol and, more recently, inositol phosphates.

Gas chromatography has been used to quantitate inositol in mammalian tissues [41-43], inositol mono- phosphate isomers in rat brain [44] and inositol tris- phosphate (after complete dephosphorylation) in hu- man platelets [16,45]. Briefly, inositol or inositol phos- phate extracts were prepared, desalted, dephospho- rylated in the case of InsP3, and converted to trimethyl- silyl or pertrimethylsilyl derivatives. Samples were ap- pried to a gas chromatography column in the presence of a known amount of chiro-inositol standard and quantitated by flame ionization and integration of the

detected peaks in comparison with standard curves. Trimethylsilyl ether dimethylphosphate derivatives have also been used to separate enantiomers of myo-ino.~itol 1-phosphate [46,471. Only milligram quantities of tissue are required az~d, under ideal conditions, 0.1 pmol of inositol monophosphate can be detected.

Similarly, gas chromatography has been used in con- junction with mass spectroscopy to quantitate inositol monophosphate isomers [481, Ins(1,4)P2 and Ins(1,4,5)P 3 [47,491. The polar trimethylsilyl derivatives of inositol polyphosphates tend to adsorb onto metal surfaces. Hence, the detection limits for trimethylsilyl-InsP2 and tdmethylsilyl-InsP3 are 0.2 nmol and 1 nmol, respec- tively, before adsorptive losses become significant.

Portilla et al. [50] investigated the bradykinin-in- duced production of Ins(1,4,5)P3 in MDCK cells. In- ositol phosphates, extracted using chloroform/ methanol, were subjected to HPLC and the Ins(1,4,5)Pa fraction was isolated, lyophilized and dephosphorylated by acid hydrolysis before conversion to hexatri- fluoroacetyl derivatives. Bradykinin caused an increase in the mass of Ins(1,4,5)P3 from basal levels of 152 pmol/mg protein to 537 pmol/mg protein by 10 s stimulation.

More recently, fast atom bombardment (FAB) mass spectrometry has been used to identify inositol phos- phates [49]. This technique was used to demonstrate that cyclic inositol phosphates were produced on clea- vage of polyphosphoinositides by phospholipase C [51]. However, this technique can not be used to distinguish between isomers since analysis of four isomers of in- ositol pentakisphosphate resulted in four identical spec- tra [49]. The limit of detection for this system is approx. 10 nmol.

alp-nuclear magnetic resonance (NMR) has been used to quantitate inositol pentakis- and hexakisphosphates in mammalian tissues [52]. Inositol phosphates were extracted from approx. 5 g of tissue using chloroform/ methanol and high salt. After the phases were split, the aqueous supernatant was subjected to gel filtration on Sephadex G-50 and G-10 columns. The eluate was lyophilized and redissolved in 1)20 prior to 3~p-NMR analysis. Peaks were identified by 31P-NMR analysis of InsP5 and InsP6 standards. InsP5 and InsP6 were found to be present at concentrations of at least 5-15/~M in all of the samples examined.

Vlli. Analytical methods specific for lns(l,4,5)P 3

All of the aforementioned techniques can be used to make mass measurements of Ins(1,4,5)Pa if it is first resolved from all other inositol tdsphosphate isomers. However, there are two methods which specifically quantitate Ins(1,4,5)P3. Therefore, if measurements of Ins(1,4,5)P3 only are required, it is more practical to use one of the techniques described below.

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Tarver et al. [53] used partially purified rat brain Ins(1,4,5)P 3 3-kinase and [y-a2p]ATP to phosphorylate Ins(l,4,5)P 3 to [32p]Ins(1,3,4,5)P4. Tracer [3H]Ins(1,4, 5)P 3 was included to determine the percentage conver- sion (85-90% after 2 h at 37°C). Less than 270 of Ins(1,3,4)P 3 was converted to InsP4 under these condi- tions. Incubations were quenched in boiling water for 5 rain and excess [y-32p]ATP and. 2,3-di[a2p]phos- phoglycerate was removed enzymatically using apyrase and phosphoglycerate mutase. [32p]Ins(1,3,4,5)P4 was isolated on DEAE-Sephacel and lfigh voltage paper electrophoresis. The original mass of Ins(1,4,5)P3 was calculated from the radioactivity in [32p]Ins(1,3,4,5)P4, the recovery of [3HlIns(1,4,5)P3 tracer and the specific activity of [y-32p]ATP. The assay is quantitative (linear with respect to Ins(1,4,5)P3 up to 50 pmol), sensitive to 1 pmol and specific.

This assay was used in conujunction with estimation of intracellular volume to measure intraceUular Ins(1,4,5)P 3 concentration in human platelets (0.2 #M basal, 1/.tM at 10 s stimulation with 5 U/ml of throm- bin). In addition, the mass of Ins(1,3,4)P a was calcu- lated by subtracting the mass of Ins(1,4,5)Pa (de- termined by the above assay after acid extraction) from the total mass of InsP 3 determined by gas chromato- graphy [16,45]. Ins(1,3,4)P a was found to comprise more than 9070 of the total InsP 3 mass formed at 10 s stimulation with thrombin. An estimation of Ins(l:2- cyclic,4,5)P3 was also made by incubation of neutral and acid-treated neutral extracts with Ins(1,4,5)P a 3- kinase. The increase in Ins(1,4,5)P 3 detected in the acid-treated samples was assumed to reflect Ins(l:2- cyclic,4,5)P3 mass.

Alternatively, Ins(1,4,5)P 3 mass can be determined using an Ins(1,4,5)P3-specific binding assay [54,55]. The pioneers of this method, Bradford and Rubin [56], competed [ 32 P]Ins(1,4,5)/'3 with Ins(I,4,5) Pa of extracts prepared from unstimulated and fMetLeuPhe-stimu- la~ed neutrophiis for the intracellular Ins(1,4,5)P3 re- ceptor of saponin-permeabilised neutrophils. Intracellu- lar Ins(1,4,5)P 3 concentrations were found to be 0.05 and 0.55 pmol/10 6 cells in control and stimulated in- cubations, respectively (0.1 /~M and 1.1 #M). The Ins(1,4,5)Pa-specific binding assay involves the competi- tion of high specific activity [3H]Ins(1,4,5)P3 with Ins(1,4,5)P3 for Ins(1,4,5)P3-specific binding sites in adrenocortical microsomes. Palmer et al. [54] incubated increasing concentrations of Ins(1,4,5)P3 (up to 100 nM) with a fixed amount of [aH]Ins(1,4,5)Pa and the binding protein at 0°C for 15 min. Samples were centrifuged and the radioactivity associated with the pellet was determined. Nonspecific binding was de- termined in the presence of 1/tM Ins(1,4,5)P3. A typical binding curve is illustrated in Fig. 1. Competition stud- ies, using a range of inositol polyphosphates, demon- strated a clear specificity of the binding site for

244

100,

6 0

40,

201

0 . . . . . . . . i . . . . . . . . i . . . . . . . . i

0,1 I 10 100 Ens(1,4,fb) ~ (nM)

Fig. 1. Specific binding of [3H]Ins(1,4,5)P3 to bovine adrenocortical microsomes, Binding protein was incubated for 15 rain on ice with approx. 100 fmol [3H]lns(1,4,5)P3 and increasing concentrations of unlabelled Ins(1,4,5)P3. Samples were centrifuged (12000 X g, 3 rain, 4°C), the supematant was removed, the pellet was dissolved in scintillation fluid and its radioactivity was determined. Non-specific binding was determined in the presence of 1 ItM unlabelled lns(1,4,5)P3. Results are means :t: S.D. (n = 6) for combined data of

three experiments.

lns(1,4,5)P3 (Table I). ATP, a weak competitor for the Ins(1,4,5)P3 binding site (Ki > 0.25.10 -3 M), is pre- sent in millimolar quantities in all cells. However, the final concentration of ATP present on addition of a cell extract to the lns(1,4,5)P3 binding assay is less than 50 I~M, at which concentration there is no displacement of Ins(1,4,5)P 3. In addition, it is unlikely that competing cellular inositol phosphates attain concentrations rela- tive to Ins(1,4,5)P3 to interfere with the binding assay. Therefore, inositol phosphate extracts of cells can be

TABLE I

Competition for lns(l,4,5)P 3 binding sites in bovine adrenocorticai mi- crosomes by inositol polyphosphates

Competitor ICs0 (nM)

Ins(1,4,5)P3 5.9+ 0.9 a Ins(2,4,5)P3 120 + 3.0 a Ins(l:2-cyclic,4,5)P 3 124 +16 a Ins(1,3,4,5)P4 110 + 8 a Ins(1,3,4)P 3 No displacement at 5-10 -e M a ATP 30~ displacement at 0.25-10 4 M b

a Results are means (n -- 3-6) of combined data from four separate experiments.

b Results are estimates from a single experiment (n--2) , typical of three.

assayed directly for Ins(1,4,5)P3 mass. However, the direct addition of tri-n-octylamine-Freon neutralized cell extract (see Section III) results in denaturation of the binding protein. The sensitivity of the assay is such that levels of Ins(1,4,5)P 3 as low as 0.2 pmol can be de- tected. As with the 3-kinase assay, Ins(1 : 2-~yclic,4,5)P3 mass can also be determined by analysis of neutral and acid-treated neutral cell extracts.

The lns(1,4,5)P3-specific binding assay has been used successfully by a number of workers. Palmer et al. [54] determined the intracellular concentration of Ins(l,4, 5)P 3 in rat hepatocytes to be 0.2 + 0.15 ltM basal and 2.53 + 1.8 ltM at peak stimulation with vasopressin (10 to 15 s). Similarly, this assay has been used to measure lns(1,4,5)P3 mass in rat cerebral cortical slices (18.8 + 2.6 pmol/mg protein basal, 31.3 + 2.3 pmol/mg pro- tein, 10 s high K + [57]), NIH-3T3 fibroblasts and NG108-15 neuroblastoma-glioma hybrid cells (8.1 + 1.1 pmol/106 cells and 11.5 :[: 0.8 pmol/106 cells basal,

TABLE II

Techniques available for the mass measurement of inositol phosphates

Technique Inositol phosphates measured Sensitivity Re£

Radiolabelled precursors InsPl_ 6 Limited by extent of incorporation and 25-28 resolution of inositol phosphate isomers

Megal-,.,...,.~ection InsP1-6 Minimum of 100 pmol inositol phosphate 34 Fast atom bombardment (FAB) InsPl_ 6 Minimum of 4-20 nmol inositol phosphate 49-51

cyclic inositol phosphates HPLC/alkaline phosphatase InsPl_ 4 Minimum of 0.1 g of tissue 33

and phosphate analysis GC-MS

HPLC of fluorescent derivatives 31P.NMR HPLC/RI measurements Gas chromatography

Enzymatic/fluorimetric assay of inositol

Microspectmphotometry Ins(l,4,5)P 3 3-kinase lns(l,4,5)P3-specific binding assay

lnsPi.3 Minimum of 0.1 pmol InsP, 0.2 nmol 47-49 lnsP 2, I nmol InsP 3 50

lns(2)P Minimum of 5 nmol inositol phosphate 39 InsPs and InsP 6 Minimum of 500 mg tissue 52 InsP6 Minimum of 10 ~g phytate 40 InsP Minimum of 0.1 pmol inositol phosphate 44 insP3 Minimum of 10 pmol inositol phosphate 16,45 InsP3 Minimum of 0.2 pmol inositol phosphate 35,36

Ins(1,4,5) P3 Minimum Ins(l,4,5)P3 Minimum Ins(l,4,5) P3 Minimum

of 0.1 ng phosphorus 32 of 1 pmol inositol phosphate 53 of 0.2 pmol inositol phosphate 54-56

TABLE III

Summary of Ins(l,4,5)P 3 mass measurements obtained to date

245

Tissue Agonist Ins(1,4,5)P3 content Ref.

Human platelets None 1-4 pmol/10 s cells (1 ~M) a 25 Thrombin 10-30 pmol/10 s cells (20 ~M) a 25 Thrombin 2.0/~ M 29 None 0.2/tM 53 Thrombin 1.0/t M 53

Rat anterior pituitary cells GnRH 56 nM 26 Rat salivary gland None 99 pmol/mg protein 33 Rat brain None 497 pmol/mg protein 33 Rat cerebral cortex None 18.8 pmol/mg protein 57

High K + 31.3 pmol/mg protein 57 Rat adrenal glomerulosa cells None 360 pmol/106 cells a 32

AngII 520 pmol/106 cells a 32 High K + 600 pmol/106 cells a 32

Rat papillary collecting tubule cells None 160 pmol/mg protein (7.5 ~t M) 36 Bradykinin 340 pmol/mg protein (16/x M) 17

Rat hepatocytes None 0.22/tM 54 Vasopressin 2.53 ~tM 54

Rabbit neutrophils None 0.05 pmol/10 6 cells (0.1 ~tM) 56 fMetLeuPhe 0.55 pmol/106 cells (1.1 ~tM) 56

Canine renal proximal tubular cells None 500 pmol/mg protein 37 IGF-II 1550 pmoi/mg protein 37

MDCK cells None 152 pmoi/mg protein 50 Bradykinin 537 pmol/mg protein 50

AR42J pancreatoma cells None 2/xM 27 Substance P 25/tM 27

HL60 cells None 0.4 laM 28 fMetLeuPhe 1.6/~M 28

NG108-15 (neuroblastoma x glioma hybrid) cells None 11.5 pmol/106 cells 58 Bradykinin 115 pmol/106 cells 58

NIH3T3 fibroblasts None 8.1 pmol/106 cells 58 Bradykinin 27 pmol/106 cells 58

Human fibroblasts None 300 pmol/106 cells 59 Bradykinin 2660 pmoi/106 cells 59

a Mixed isomers of inositol trisphosphate.

respectively; approx. 27 pmol/106 cells and 115 pmol/106 cells, respectively, at 10 s stimulation with bradykinin [58]) and in fibroblasts from progressive systemic sclerotic patients [59].

A second Ins(1,4,5)P3-specific binding assay, using rat cerebella, has recently been described [60]. The sensitivities of the two Ins(1,4,5)P3 binding assays are the same although the rat cerebellum preparation pos- sesses a higher density of binding sites and a greater specifity for Ins(1,4,5)P3. However, as detailed for the assay employing bovine adrenocortical microsomes, er- rors in the estimation of Ins(1,4,5)P 3 concentration due to competition by other inositol polyphosphates and ATP are insignificant. Therefore, the ready availability of bovine adrenal glands may make this method prefer- able.

Ins(1,3,4,5)P4-specific binding sites have been identi- fied in HL-60 membranes [61] and rat cerebella mem- branes [62]. Therefore, the development of an Ins(1,3,4,5)P4-specific binding assay is a likely possibil- ity.

A summary of methods available for the mass mea- surement of inositol phosphates is provided in Table II. The maximum sensitivities of the assays are as given by the respective authors. In addition, Table Ill details the studies, to date, where mass measurements of Ins(1,4,5)P3, Ins(1 : 2-cyclic,4,5)P3 and InsP3 have been made, albeit by a variety of techniques. Analysis of the data suggests that there may be considerable differences in the basal Ins(1,4,5)P3 content of different tissues, e.g., rat brain 497 pmol/mg protein, rat salivary gland 99 pmol/mg protein [33]; NG108-15 (neuroblastoma x glioma hybrid) cells 11.5 pmol/10 ° cells, NIH3T3 fibroblast~ ~,.1 pmol/106 cells [58], human fibroblasts 300 pmol/106 cells [59]. Such differences do ilot neces- sarily correlate with the use of different assay tech- niques. However, the relevance of these observations in terms of a second messenger role for Ins(1A,5)P3 re- mains unclear and will require further investigation.

Mass measurements of inositol phosphates are essen- tial for the elucidation of signal transduction via in- ositol phospholipids. Analysis of inositol phosphate

246

mass in a wide variety of cell types should now be possible, given the diversity of techniques available.

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