Quantitative Changes in Inositol 1,4,5-Trisphosphate in ...

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The role of myo-inositol 1,4,5-trisphosphate (1ns-1,4,5-P3)l as a second ... lular receptor and also by its ability to mobilize ATP-depend- ent intracellular calcium ...
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JOURNAL OF BIOLOGICAL CHEMISTRY

Vol . 261, No. 33, Issue of November 25, pp. 15644-15647 1986 Printed in S. A.

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(0 1986 by The American Society of Biological Chemists, Inc

Quantitative Changes in Inositol 1,4,5-Trisphosphatein Chemoattractant-stimulatedNeutrophils* (Received for publication, July 18, 1986)

Peter G. Bradford and Ronald P. Rubin$ From the Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5tri~[~~P]phosphate binding and to stimulate release of sequestered stores of 46Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tri~[~~P]phosphate binding and to release 46Ca2+correlated wellwith the ISH] inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [“H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucylphenylalanine, the inositol 1,4,5-trisphosphatecontent of the extract increased from 0.05 to 0.55 pmol/106 cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 p ~ These . studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ fromintracellular stores.

amount of Ins-1,4,5-P3produced by cells has not been established. The mass of Ins-1,4,5-P3 formed in stimulated cells has been estimated based upon specific activity measurements of the polyphosphoinositide substrate pool and assessmentof the total radioactivity in released the Ins-P3 (8).A more direct massmeasurement of aninositolpolyphosphatefraction based upon quantitationby capillary gas chromatography has been reported in thrombin-stimulated platelets(9). However, this fraction contained both isomers of Ins-P3 (Ins-1,4,5-P3 and Ins-1,3,4-P3), as well as Ins-1,3,4,5-P,; and so, the reported value represents the sum total of the three inositol polyphosphates. Inthisstudy, we have utilizedtwobiochemical assays specific for Ins-1,4,5-P3 to determine the Ins-1,4,5-P3 content inextracts derivedfrom stimulatedneutrophils.TheIns1,4,5-P3 in the extracts was assayed both by its ability to compete with the binding of In~-1,4,5[~~P]P, tointracelits lular receptor and also by its ability to mobilize ATP-dependent intracellular calcium stores. Both assays yielded quantitatively similar results. In addition to quantitating the mass of Ins-1,4,5-P3, this study provides the first demonstration in rabbit neutrophils that chemoattractant stimulation causes the rapid formation of Ins-1,3,4-P3 and Ins-1,3,4,5-P4. Evidence will be presented that these novel inositol polyphosphates do not possess biological activity comparable to that exhibited by Ins-1,4,5-P3. EXPERIMENTALPROCEDURES

Preparation of Neutrophil Extracts-Peritoneal neutrophils from New Zealand Whiterabbits were collected by heparinized saline The role of myo-inositol 1,4,5-trisphosphate (1ns-1,4,5-P3)l lavage 10-12 h after intraperitoneal injection of molluscan glycogen, as a second messenger of Ca2+-mobilizing hormones is now as described previously (3). Cells (>95% neutrophils) were washed well-established (1, 2). In the neutrophil and the promyelo- and resuspended (2 X 10s/ml) in a modified Hanks’ balanced salts cytic HL-60 cell, Ins-P, is rapidly formed following chemo- solution containing, in millimolar: NaC1, 124; KCI,4; Na,HPO,, 0.64; attractant receptor stimulation(3-5). Moreover, when added KH,PO,, 0.66; NaHC03, 15; Hepes, 10; MgCI,, 0.2; CaCI,, 0.5; glucose, to permeabilized neutrophils, Ins-1,4,5-P3 stimulates 45Ca2’ 5.6 (pH 7.4); and bovine serum albumin, 0.05%. Neutrophils (6-8 X 10’) were labeled with [3H]inositol (15 Ci/mmol, 0.1 mCi/ml) for 3-4 release from cellular stores (6, 7). h in an agitator water bath under an atmosphere of 95% 0,and 5% PermeabiIized neutrophils are sensitive to submicromolar CO,. The cells were washed and resuspended in fresh buffer. Aliquots concentrations of Ins-1,4,5-P3 (7); however, theabsolute of cells (-10’ in 200 pl) were added to buffer (100 p l ) containing Met-Leu-Phe to give a final concentration of 10 nM. The reaction * This work was supported in part by Grants HL-07357 and AM- was stopped at selected times (5-300 s) by the addition of 200 p1 of 28029 fromthe National Institutes of Health. The costs of publication ice-cold 25% trichloroacetic acid. Alternatively, reactions were conof this article were defrayed in part by the payment of page charges. ducted for 15 s with varying concentrations of met-Leu-Phe and This article must therefore be hereby marked “aduertisement” in terminated by acid extraction. After 20 min on ice, the samples were microcentrifuged for 2 min. The supernatants, which contained the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence and reprint requests should be ad- inositol phosphates, werewashed four times with 1.5 volumes of dressed Dept. of Pharmacology, P. 0. Box 524, MCVStation, Medical diethyl ether in order to remove the trichloroacetic acid and then neutralized (pH 7.0-7.4) with dilute NH,OH and stored at -70 “C College of Virginia, Richmond, VA 23298. The abbreviations used are: Ins-1,4,5-P3, myo-inositol 1,4,5-tris- until analyzed (