development of a novel, ins(1,4,5)p3-specific binding assay: its use to determine the intracellular...

Cellular Stgnalhng Vol 1, No 2, pp 147-156, 1989 0898-6568/89 $3 00+ 00 Pnnted m Great Britain Pergamon Press plc

DEVELOPMENT OF A NOVEL, INS(1,4,5)P3-SPECIFIC BINDING ASSAY. ITS USE TO DETERMINE THE INTRACELLULAR CONCENTRATION

OF INS(1,4,5)P 3 IN UNSTIMULATED AND VASOPRESSIN-STIMULATED RAT HEPATOCYTES

SUSAN PALMER, KELVIN T. HUGHES,* DENISE Y. LEE* and MICHAEL J. O. WAKELAM

Molecular Pharmacology Group, Institute of Biochemistry, University of Glasgow, G12 8QQ, U.K. and *Amersham International plc, Cardiff, Wales, U.K.

(Received 12 May 1988; and accepted 7 June 1988)

Abstract--The binding of [3H]Ins(l,4,5)P 3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites (Ks = 6.82 + / - 2 . 3 nM, Bm~x = 370+ / -38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e. Ins(2,4,5)P 3 was able to compete with [3H]Ins(l,4,5)P3 whereas Ins(1,3,4)P 3 was not. Ins(1,3,4,5)P4 showed a similar affinity for the receptor as Ins(2,4,5)P 3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [3H]Ins(1,4,5)P3-binding was independent of free Ca 2÷ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determine the basal and vasopressin- stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P 3 were calculated to be 0.22 +/-0.15/~M basal and 2.53 + / - 1.8/~M at peak stimulation. This assay provides a simple, specific and quantitatwe method for the measurement of Ins(l,4,5)P~ concentrations in the picomolar range.

Key words: Ins(1,4,5)P3; hepatocytes; vasopressin; binding assay.

I N T R O D U C T I O N

INOSITOL 1,4,5-trisphosphate (Ins(1,4,5)P3) is an intracellular second messenger produced upon phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) in response to CaZ+-mobilizing stimuli [reviewed in 1 and 2]. Ins(1,4,5)P 3 has been shown to elevate intracellular calcium concentrations by activating the release of Ca 2÷ from an intracellular store, probably the endo- plasmic reticulum [3]. It has been postulated that the mechanism of Ca z + release involves a specific intracellular receptor for Ins(1,4,5)P 3 [4]. Direct evidence for the presence of high affinity, intracellular InsP 3 binding sites has been obtained in a number of systems - - bovine adrenal cortex homogenate [5], permeabilised

Abbreviations: 2,3-DPG: 2,3-diphosphoglycerate; Ins(1,4,5)P3: inositol 1,4,5-trisphosphate; Ins(l,3,4)P3: inositol 1,3,4-triphosphate; Ins(1,3,4,5)P4: inositol 1,3,4,5- tetrakisphosphate; Ins(1,2cyc4,5)P3: inositol 1,2-cyclic 4,5- trisphosphate.

rabbit neutrophils [6, 7], permeabilised guinea pig hepatocytes [6], liver microsomal fraction [8, 9] and plasma membrane fraction [9], bovine adrenal cortex microsomes [9], bovine anterior pituitary microsomes [9, 10, 11], rat cerebellar membranes [12, 13, 14, 15], rat cerebral cortex membranes [16]. In addition, Willcocks et al. [14] have shown that the sites in rat cerebellum are stereospecific for Ins(1,4,5)P 3. More recently, the purification of an InsP~ binding protein, which may be the receptor from rat cerebella, has been reported [17].

Although the Ca 2 +-mobilizing role of Ins(l,4,5)P 3 is well documented [2, 3], few studies have been performed to determine the intracellular concentration of Ins(l,4,5)P 3. Measurement of Ins(1,4,5)P 3 mass has proved to be arduous and generally expensive. A number of techniques are available: alkaline phosphatase digestion of InsP 3 fraction obtained from Dowex-formate separation of inositol phosphates followed by gas chromatography [18];

147

148 S. PALMER et. al

h.p.l.c, separation of inositol phosphates with on line alkaline phosphatase digestion and inor- ganic phosphate analysis [19]; paper electro- phoresis of [32p]inositol phosphates and determination of the specific activity of ATP and Ptdlns(4,5)P 2 [and therefore Ins(1,4,5)P3] [20, 21]; h.p.l.c, separation of inositoi phosphates, acid hydrolysis and mass spectrometry [22] and alkaline phosphatase digestion of InsP 3 fraction from Dowex-formate separation of inositol phosphates followed by enzymatic fluorimetric assay of inositol [23]. Furthermore, unless h.p.l.c, separation of inositol phosphates is used, any quantitation of InsP 3 will be of both lns(1,4,5)P 3 and Ins(1,3,4)P 3. Therefore, there is a need for an inexpensive, simple and specific assay for Ins(l,4,5)P 3. This need might be met by the development of a radioligand binding assay using a microsomal preparat ion which demon- strates Ins(1,4,5)P3-specific binding.

The data presented here illustrates the specificity of [3H]Ins(1,4,5)P3 binding in a crude microsomal preparation of bovine adrenal cortex. This subcellular fraction has been used to quantitate Ins(1,4,5)P 3 in acid extracts of rat hepatocytes and the intracellular concentration of Ins(1,4,5)P 3 calculated.

M A T E R I A L S A N D M E T H O D S

Materials

[3H]Ins(1,4,5)P3 (specific activity 20-70Ci/mmol), 3H20 and [U-~4C]sucrose (specific activity 350Ci/ mmol) were supplied by Amersham International. Ins(l,3,4)P~ and Ins(2,4,5)P 3 were the generous gift of Dr. R. F. Irvine (A.F.R.C., Cambridge, U.K.). Other inositol phosphates, all of which were >95% pure, were obtained from Amersham International. Colla- genase (150U/mg, CLS 2) was purchased from Worthington Biochemical Corporation (New Jersey, U.S.A.). Silicone oils (API00 and AR20) were obtained from Wacker Chemicals Ltd. (Walton-on- Thames, Surrey, U.K.). All other chemicals were of the highest grade commercially available.

(20raM) with DTT (1 mM) and the homogenate centrifuged at 5000 g for 15 min. The supernatant was centrifuged at 35 000g for 20 min, the resulting pellet resuspended in the homogenisation buffer and centrifuged again at 35 000g for 20 min. The final pellet was resuspended in homogenisation buffer at a protein concentration of approx. 20-40 mg/ml.

Preparation of inositol phosphate extracts from hepatocytes

Isolated hepatocytes were prepared by collagenase digestion of the livers from male Wistar rats (200- 240 g) as described by Elliot et al. [24]. Cells were incubated at a density of approx. 60-70 mg dry wt/ml in bicarbonate buffered saline [25] containing 2.5 mM CaCI 2, 10mM glucose and bovine serum albumin (Fraction V, 2%, w/v). Aliquots (120pl) of the cell suspension were distributed into plastic vials and incubated at 37°C with either buffer or vasopressin (230 nM). Incubations were terminated by addition of 25 pl HCIO 4 (10%, v/v) at the appropriate time and the samples stood on ice. Samples were centrifuged (12000g, 2rain, 4°C) and the supernatants neutralised with approx. 10pl KOH (I.5M) containing 60 mM HEPES in the presence of Universal Indicator [26].

Estimation of intracellular volume of hepatocytes

The mtracellular volume of hepatocytes was estimated as described by Shears and Kirk [27]. Hepato- cyte suspensions were incubated in bulk with 3H20 (0.2#Ci/ml) and [U-"C]sucrose (0.4pCi/ml) for 15min. Samples (120/d) of cell suspension were placed on 500/d of silicone oil (d= 1.045; a 1:1 mixture of API00 and AR20) which was layered above 100/A of HC104 (10%, v/v) in 1.5ml plastic centrifuge tubes. These samples were centrifuged at 12 000 g for 20 s. Radioactivity of the upper and lower phases was determined. [U-~4C]sucrose of the lower phase was assumed to represent contamination by the extracellular medium. 3H20 of the lower phase, after correction for contamination by the extracellular medium, was assumed to be intracellular. It has been estimated that 87% of intracellular water is cytosolic [28]. Cytosolic water was calculated to be 1.92+/ -0.38 ml/mg dry wt (n =4 from 6 hepatocyte preparations).

Preparation of binding protein

Bovine adrenal glands were removed from animals and stored at - 20°C prior to the date of preparation of the binding protein. The cortex was dissected from the adrenal glands, homogenised in NaHCO 3

Radioligand binding studies

Aliquots of the bovine adrenal cortex microsomal fraction, containing 500-1000 mg protein, were incubated in 25 mM Tris (pH 9), 1 mM EDTA, 5mM NaHCO3, 0.25mM DTT, 1 mg/ml bovine serum

Mass measurement of Ins(1,4,5)P 3 149

albumin (Fraction V). Incubations were performed, unless otherwise stated, for 15 min on ice in a final volume of 100#1 with [aH]Ins(1,4,5)P3 (approx. 3000 cpm = 52 fmol = 0.52 nM) and an aliquot of the hepatocyte inositol phosphate extract or inositol phosphate standard as appropriate. Non-specific binding was determined in the presence of 942 nM Ins(1,4,5)P 3. Incubations were terminated by centrifugation (12000g, 3min, 40C) and removal of the supernatant. Particulate bound radioactivity was analysed by liquid scintillation spectrometry.

Specific binding in the absence of unlabelled Ins(1,4,5)P 3 varied between 25 and 40% of total radioactivity in the incubation and was generally in excess of 1000 cpm. Non-specific binding was approx- imately 5-10% of total radioactivity per incubation.

Under the conditions specified, there was no signifi- cant breakdown of [3H]Ins(l,4,5)P3 (evaluated by h.p.l.c.) during the period of the experiment (data not shown).

C a 2 + studies

When the influence of Ca 2+ on [3H]Ins(1,4,5)p~ binding was investigated, aliquots of the binding protein preparation were centrifuged (12 000 g, 3 min, 4°C), washed with the appropriate Ca 2÷ buffer and centrifuged again before resuspension in the appropriate Ca 2÷ buffer. The Ca 2÷ buffers prepared were similar to the homogenisation buffer except that EGTA and Ca 2÷ were added to achieve the required free Ca 2+ concentration. Free Ca 2+ concentrations were calculated using a computer program, written on the basis of previously published programmes for hand-held calculators [29, 30].

R E S U L T S

Character&ation of binding protein

Kinetic studies showed that specific binding of 0.52 nM [3H]Ins(1,4,5)P3 to the microsomal prepara t ion at 0°C was half maximal at approx. 90 s, reached a plateau by 10 min and remained constant for up to 30 min (Fig. 1). Addi t ion o f I # M unlabelled Ins(l ,4,5)P 3 resulted in the rapid displacement o f bound ligand, with half time o f approx. 6 min, indicating a dynamic l igand-receptor interaction.

Tracer binding was significantly reduced in the presence o f 0.5 nM Ins(1,4,5)P 3 and almost completely inhibited by 6 2 . 5 n M Ins(l ,4,5)P 3 (Fig. 2). Scatchard analysis o f specific [3H]Insl,4,5P3 binding data [Fig. 2 (inset)] was

TABLE 1. INFLUENCE OF FREE C a 2+ CONCENTRATION

ON [3H[lns(1,4,5)P3_ BINDING TO ADRENOCORTICAL MICROSOMES

% of max. specific [Free Ca 2÷] (M) [3H]Ins(1,4,5)P~ bound

0 100 + / - 13 5 × 10 -tt 86 + / - 2 5 x 10 L0 98 + / - 4 5 × 10 9 91 + / - 1 5 × 10 -s 80 + / - 7 5 × 10 -7 85 + / - 3

The adrenocortical microsomal preparation was centrifuged, washed and resuspended in the appropriate Ca 2+ buffer before adddition to the reaction mixture which was Ca -'+ free. Binding of 0.52 nM [3H]Ins(l,4,5)P3 was determined as described in Materials and Methods. Results are means + / - S.D. (n = 3) for a single representative experiment.

consistent with a single popula t ion o f binding sites with apparent dissociation constant (KD) 6.82 + / - - 2.3 nM and binding capacity (Bm,~) o f 3 7 0 + / - 38 fmol /mg protein (combined data f rom 7 determinations using 3 batches o f binding protein). These data are similar to that o f Guillemette et al. [9] a l though the prepara t ion used in the experiments presented here exhibited a greater binding capacity. The ECs0 of Ins(1,4,5)P 3 binding was estimated to be 5 , 9 + / - 0.9 nM. This value is similar to that observed in other systems: bovine adrenocort ical microsomes - - 5 n M [5] and 1 .3nM [9]; rat liver microsomes - - 8 nM [8] and 1.9 nM [9]; bovine anterior pituitary microsomes - - 3.5 nM [l l] and l . l nM [9, 10]; rat cerebellar membranes - - 4 0 n M [12, 13], 2 3 n M [14] and 6 6 n M [15] and cerebral cortical microsomes - - 20 nM [16].

In contrast to the data o f Worley et al. [13] using cerebellar membranes , binding o f [3H]Ins(l,4,5)P3 to the adrenocort ical microsomes was independent o f free Ca 2 + concentration in the range o f 5 × 10 - I I to 5 × l 0 ×7 M (Table 1).

The results o f compet i t ion studies performed using 0.52 nM [3H]Ins(1,4,5)P3 and a range of inositol po lyphosphates are illustrated in Fig. 3. These experiments demonst ra ted a clear specificity o f the binding site for Ins(1,4,5)P 3.

g~

~ E = " c .

100 -

80 ¸

6O

40

20

J

1 0 2 0 3 '0 4 0

150 S. PALMER et al.

Time (rain)

FIG. 1. Kinetics of [3H]Ins(1,4,5)P3 association with and dissociation from adrenocortical microsomes. Binding to adrenocortical microsomes (O) was initiated by the addition of 0.52 nM [3H]Ins(I,4,5)P3 to the reaction mixture. For dissociation experiments (©), Ins(l,4,5)P 3 (to a final concentration of I#M) was added to the reaction mixture after a 15 minute preincubation in the presence of [3H]Ins(l,4,5)P~. Incubations were terminated by centrifugation at 12 000g for 15 s and the supernatant removed imme- diately. Specific binding is expressed as a percentage of total specific binding at 30 min. Results, expressed as mean + / - S.D. (n = 6), are combined data from 3 experiments.

lns(l,3,4,5)P 4, a naturally occurring inositol tetrakisphosphate, was able to compete for the binding site although the ECs0 of Ins(l,3,4,5)P 4 was approx. 18.6 fold greater than that of Ins(l,4,5)P 3. Ins(2,4,5)P 3, a synthetic, Ca z+- mobilizing inositol trisphosphate, exhibited a similar affinity for the binding site as Ins(l,3,4,5)P 4. In addition to this, Ins(1,3,4)P 3 at concentrations up to 5 #M was without effect on binding of 0 .52nM [3H]Ins(1,4,5)P3. These results demonstrate that the vicinal phospate groups at the 4 and 5-positions of the inositol ring are required for binding. Other inositol phosphates and molecules found intracellularly which bear a structural similarity to inositol phosphates also show limited competition with

[3H]Ins(1,4,5)P3 binding. The EC50 values obtained are presented in Table 2. Preliminary evidence suggests that Ins(1,2cyc4,5)P 3 is able to show limited competit ion for the Ins(1,4,5)P 3 binding site (ECs0 approx. 23-fold greater than that of Ins(1,4,5)P3, data not shown). In addition, acid treatment of Ins(1,2cyc4,5)P 3 [incubation with an equal volume of 10% (w/v) HCIO 4 for 1 h, then neutralisation with KOH/HEPES] resulted in increased competit ion with [3H]Ins(l,4,5)P 3, presumably due to the production of the more competitive Ins(l,4,5)P 3.

The adrenocortical microsomal preparation, therefore, exhibited a single population of high affinity, Ins(l,4,5)P3-specific binding sites which were independent of Ca 2 + concentrations in the

Mass measurement of Ins(l,4,5)P 3 151

0

10-

08 ̧

06

04

02

O0

0 5 "

04'

03"

100 200 300 /rag protein)

. . . . . . . . i . . . . . . . . i . . . . . . . . i

1 10 100

log [Ins( 1,4,5)P8]

FIG. 2. Specific binding of [3H]Ins(1,4,5)P3 as a func- tion of increasing concentration of ligand. Adreno- cortical microsomes were incubated on ice with 0.52 nM labelled ligand and increasing concentrations of unlabelled ligand as described in Materials and Methods. Inset: Scratchard analysis of the binding data. Results, expressed as mean + / - S.D. (n = 3), are from a single, typical experiment.

i

400

physiological range. The preparation is therefore suitable for use as a binding protein for the determination of lns(1,4,5)P 3 concentrations in cell extracts.

Measurement of Ins ( 1,4,5 ) P3 concen tra tion in cell extracts

Using this assay system, we have investigated the concentration of Ins(1,4,5)P 3 in neutralised acid extracts of unstimulated and vasopressin- stimulated hepatocytes. This cell type was cho- sen as it has already been extensively studied with regard to inositol phosphate metabolism and Ca2+-mobilization by Ins(1,4,5)P 3. There- fore, estimates of Ins(1,4,5)P 3 concentration made using this assay system can be compared

directly with estimates previously made using other techniques and related to physiological effects.

The addition of neutralised acid extracts prepared in the absence of hepatocytes to the radioligand binding assay had no effect on the radioactivity associated with the binding protein (Table 3). Therefore, changes in particulate bound radioactivity on assay of hepatocyte extracts represent real changes in Ins(1,4,5)P 3 concentration. Indeed, addition of a known amount of Ins(1,4,5)P 3 to an HCIO 4 terminated hepatocyte sample followed by K O H / H E P E S neutralisation and assay for Ins(l,4,5)P 3 gave 8 8 o recovery (n= 3 from a single experiment).

Stimulation of hepatocytes with vasopressin resulted in a rapid increase in Ins(l,4,5)P 3 con-


g ==

100 '

80 "

60 ¸

40 ¸

20

0 - 10

0 [ ] 0 17

i l i i

- 9 - 8 - 7 - 6

log [inositol phosphate]

FIG. 3. Competition by inositol polyphosphates for pH]Ins(l,4,5)P 3 binding sites. [3H]Ins(1,4,5)P3-binding to adrenocortical microsomcs was determined in the presence and absence of the inositol polyphosphates indicated: O, Ins(1,4,5)P3; ©, Ins(l,3,4,5)P4; / ins(2,4,5)P3 [~, Ins(1,3,4)p3" Results are means (n = 3-6) for combined data of 4 experiments. S.D. values (which did not exceed 10%) have been omitted for clarity.

i

- 5

tent, approx. 6 fold above basal at 3 s (see Fig. 4). Ins(1,4,5)P 3 content peaked at 5-15s before falling towards basal levels. However, Ins(1,4,5)P 3 content remained significantly ele- vated after 60 s stimulation at approx. 3 fold of basal (see Fig. 4). Using the value determined for the intracellular volume of hepatocytes (see Methods), basal concentrations of Ins(1,4,5)P 3 were calculated to be 0 . 2 2 + / - 0 . 1 5 / ~ M (n = 18, combined data from 6 hepatocyte preparations). Whilst some variation in basal concentrations of Ins(l,4,5)P 3 was observed between hepatocyte preparations, unstimulated Ins(1,4,5)P 3 concentrations were generally in the order of 0.2/~M. Peak stimulation of Ins(1,4,5)P 3 concentration was between 6 and 10 fold that of unstimulated cells (intracellular concentration calculated to be 2.53 + / - 1.8/aM, n = 12, combined data from 4 hepatocyte preparations). Acid extraction of inositol phosphates hydrolyses the cyclic moiety

of any cyclic inositol phosphates that are present whereas preservation of the cyclic moiety requires neutral extraction [31, 32, 33, 34]. How- ever, there is no evidence for cyclic inositol phosphates in hepatocytes and it has been shown that Ins(1,2cyc4,5)P 3 is not present in all tissues [35].

DISCUSSION

The characteristics of [3H]Ins(1,4,5)P3 binding to the adrenocortical microsomal fraction presented in this paper are generally similar to those observed by Guillemette et al. [9]. [3H]Ins(l,4,5)P3 binding is rapid, reversible and saturable and there is a single population of receptor sites. This study provides the first evidence that the 'InsP 3 receptor' of adrenocortical microsomes exhibits positional specificity.

Mass measurement of Ins(1,4,5)P 3 153

TABLE 2. INHIBITION OF SPECIFIC [3H]Ins(l,4,5)P3_ BINDING TO ADRENOCORTICAL M1CROSOMES BY INOSITOL

PHOSPHATES, ATP, GTP AND 2,3-I~ISPHOSPHO- GLYCERATE

[3H]Ins(l,4,5)P3 binding to adrenocortical microsomes

Competitor ECs0 (M)

Ins(l,4,5)P~ 5.9 x 10 -9* Ins(2,4,5)P 3 1.2 x 10 -7* Ins(l,3,4,5)P 4 1.1 × 10 -7* Ins(l,3,4)P 3 No displacement at 5.0 x 10 -6* Inositol No displacement at 2.5 x 10-4~

Ins(l)P 6.4 x 10 5t Ins(l,2cyc)P No displacement at 2.5. + 10 -4"I" Ins(4)P 3.5 x 10 5t Ins(l,4)P 2 2.5 × 10-5"} . Ins(2,4)P 2 1.9 × 10-4t InsP 5 7.3 x 10-6t InsP 6 1.3 × 10-st ATP 30% displacement at 2.5 × 10-4t GTP 20% displacement at 2.5 × 10-4t 2,3-DPG 4.0 x 10-4t

*Results are means (n=3-6) of combined data from 4 separate experiments.

eResults are estimates from a single experiment (n = 2), typical of 3.

Ins(2,4,5)P 3 displayed a significantly lower affinity than Ins(1,4,5)P 3 whereas Ins(1,3,4)P 3 was inactive at the site. This is in agreement with the data of Willcocks et al. [14] using rat cerebellum. Fur the rmore , in cont ras t to the da ta o f Guille- met te et al. [9], the p repa ra t ion exhibits a relatively low affinity for Ins ( l ,4 )P 2, Ecs0 approx . 2.5 x 10 -5 M which is 250-fold higher than that determined by Gui l lemet te et al. [9]. The identity and funct ion of the ' r ecep tor ' remain unknov~n. However , the binding site is distinct f rom

Ins(1,4,5)3 3-kinase (which is p redominan t ly sol- uble [36] and for which Ins(2,4,5)P 3 is a p o o r substrate [37]) and Ins( l ,4 ,5) 3 5-phosphatase (which exhibits a K m for Ins( l ,4 ,5)P 3 of 17/zM in adrenal cortex [9]). The high affinity and specificity of the binding site suggest that it m a y be the ' receptor ' p roposed to couple Ins(1,4,5)3 to Ca 2+ release f rom intracellular stores [4]. To suppor t this, the relative affinity for Ins(2,4,5)P 3 binding and its ability to mobil ize Ca 2+ are similar [3, 4, 6]. In addit ion, Ins( l ,4 ,5)P 3- induced Ca 2+ mobi l iza t ion has been demon- strated in an adrenocor t ica l mic rosomal p repa ra t ion [9]. In agreement with the da ta o f Willcocks et al. [14] using rat cerebellum, Ins( l ,3 ,4)P 3 is unable to compete with [3H]Ins(1,4,5)3 for the binding site whereas it is a weak Ca 2 +-mobil izing agent in Swiss 3T3 cells [38]. In addit ion, Ins( l ,3 ,4,5)P 4 is able to compete with [3H]Ins(1,4,5)P 3 for the receptor but is unable to mobil ize Ca 2+ f rom permeabi l ised Swiss 3T3 cells [38]. Fur the rmore , the high affinity of the Ins( l ,4 ,5)P 3 binding shown here and in other studies (cited earlier) contras ts with the relatively lower potency o f Ins( l ,4 ,5)P 3 for mobil iz ing Ca 2 + [ECs0 0.1-3.0 pM-reviewed in 3]. However , the exper imental condi t ions necessary for the two assays differ considerably. The identity and funct ion o f the ' InsP 3 receptor ' requires fur ther investigation.

The high affinity and specificity o f the ' Ins(1,4,5)P 3 receptor ' m a k e it ideal for use in a radiol igand binding assay to determine Ins(1,4,5)P 3 concent ra t ions in cell extracts. There is no evidence to suggest that the relative concent ra t ions of inositol phospha tes other than Ins(1,4,5)P 3 which would be needed to influence

TABLE 3. INFLUENCE OF CELL EXTRACT ON [3H]INs(I,4,5)P~-BINDING

Specific binding of [3H]Ins(1,4,5)P 3 at concentration of Ins(l,4,5)P 3 specified (cpm)

Addition 0 nM 3.1 nM 25 nM

H20 1070 703 216 25pl neutralised acid extract 1039 711 212

[~H]Ins(1,4,5)P3-binding to adrenocortical microsomes was determined in the presence of 25 pl H20 or neutralised acid extract (prepared in the absence of hepatocytes), a fixed amount of [3H]Ins(1,4,5)P 3 and three concentrations of unlabelled Ins(l,4,5)P 3. Results are means (n = 2) from a single experiment, typical of 3.

800 ,


6 0 0 •

400 -

2 0 0 •

i i i

2 0 4 0 6 0

Time(sec)

FIG. 4. Timecourse of Ins(1,4,5)P 3 production in vasopressin-stimulated rat hepatocytes. Hepatocytes were incubated with vasopressin (230nM) for the times indicated. Incubations were terminated with ice cold perchloric acid, centrifuged and the resulting supernatant neutralised with KOH/HEPES. An aliquot of each neutralised supernatant was incubated with adrenocortical microsomes and [3H]Ins(1,4,5)P3 in parallel with incubations containing no cell extract but increasing concentrations of unlabelled Ins(1,4,5)Pv The Ins(l,4,5)P3 concentration of each hepatocyte extract was read off the standard curve. Results, expressed as means + / - S.D. (n=6) are from a single, typical experiment.

this radioligand binding assay can occur in cell extracts. The sensitivity of the assay described is such that levels of Ins(l,4,5)P 3 as low as 0.2 pmol can be detected.

The use of the assay has been shown by the determination of the time course of vasopressin- stimulated Ins(1,4,5)P 3 production in rat hepatocytes. The characteristics of the response, i.e. initiation, magnitude and duration, are identical to those observed using h.p.l.c, separation of [3H]Ins(1,4,5)P3 in guinea pig hepatocytes [39, 40] and rat hepatocytes (Palmer, unpublished data).

Previously, it has only been possible to determine Ins(l,4,5)P 3 mass by laborious and expen-

sive techniques. Alternatively, rough estimates of Ins(1,4,5)P a concentrations have been made based on the PtdIns(4,5)P 2 content of cells and using isotopic techniques where the problem of pool equilibration makes accurate estimates impossible. Using the latter technique, Thomas et al. [41] calculated that the increase in InsP 3 (mixed isomers) concentration on stimulation was 10pM in the absence of Li ÷ and 30pM in the presence of Li +. Similarly, intracellular concentrations of InsP 3 in Li+-treated rat hepatocytes are calculated to be 9 #M basal and 23 #M on stimulation [26]. Resolution of Ins(1,4,5)P 3 on h.p.l.c, yields figures of 5.56/~M basal and 37.9 pM stimulated for guinea pig hepatocytes

Mass measurement of Ins(1,4,5)P~ 155

(calculated from the data of Burgess et al. [39]). In the present study, the intracellular concentrations of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes were calculated to be approx. 0.2 and 2.5 #M respectively. Not only are these concentrations an order of magnitude lower than those calculated using isotopic techniques but they are much closer to what might reasonably be expected in view of the fact that the ECs0 for Ins(1,4,5)P3-induced Ca 2 + release in permeabilised guinea pig hepatocytes is 0.1/aM [42]. Similar values have been calculated for Ins(1,4,5)P 3 concentration in neutrophils by competing [32p]Ins(l,4,5)P3 with cellular Ins(1,4,5)P 3 with cellular Ins(1,4,5)P 3 in permeabilised cells [7] and in platelets by h.p.l.c. separation, two-dimensional paper chromatography and specific activity determination [21].

The development of this radioligand binding assay should enable the rapid and accurate measurement of Ins(1,4,5)P 3 concentrations without the use of h.p.l.c, and other expensive, time-consuming techniques.

Acknowledgements--Parts of this work were sup- ported by grants from the Cancer Research Cam- paign and the Medical Research Council.

R E F E R E N C E S

I. Berridge M. J. (1984) Biochem. J. 220, 345-360. 2. Downes C. P. and Michell R. H. (1985)

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development of a novel, ins(1,4,5)p3-specific binding assay: its use to determine the intracellular...

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