The Effects of Fatty Acids on Phosphoinositide Synthesis and myo ...

14 downloads 0 Views 792KB Size Report
depressant effect of arachidonate on phosphoinositide labeling ... creas, is the hydrolysis of phosphoinositides (PtdIns-4,5-P2, ..... a lipoxygenase metab~lite.~.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 36, Issue of December 25, pp. 17426-17431,1987 Printed in U.S.A.

The Effectsof Fatty Acids on Phosphoinositide Synthesisand myoInositol Accumulation in Exocrine Pancreas* (Received for publication, March 16, 1987)

Archana Chaudhry, SuzanneG . Laychock, and Ronald P. Rubin From the Department of Pharmacology, MedicalCollege of Virginia, Richmond, Virginia23298-0001

The effects of arachidonic acid (20:4) on phosphoi- which are putative cellular messengers. The diacylglycerol is nositide turnover were examined in rat pancreatic phosphorylated aciby ATP to form phosphatidic acid, which nar cells prelabeled with my~-[~H]inositol. Arachitogether with inositol contributes to the resynthesis of PtdIns donic acid (50 PM) increased the accumulation of myo- via CDP-diacylglycerol. The inositol utilized for the synthesis [3H]inositol, but not that of [3H]inositol monophos- of PtdIns is derived in part from the breakdown of inositol phate, [SH]inositol bisphosphate, or [3H]inositol tris- phosphates (3). A small portion (less than 10%) of the PtdIns phosphate. By contrast, 10 PM carbamoylcholine inis then phosphorylated to promote the synthesis of PtdIns-4creased the accumulation of all four compounds. A P and PtdIns-4,5,-Pz(5). combination of arachidonic acid plus carbamoylcholine In many secretory systems, including exocrine pancreas, caused a selective and marked accumulation of myo[SH]inositol, which was abolished by 10 mM LiCI. Ar- agonist-mediated breakdown of phosphoinositides, through achidonic acid (10-100 WM) produced a concentration- the activationof either phospholipase A, (6) or phospholipase dependent inhibitionof rny~-[~H]inositol incorporation C and diacylglycerol lipase (7), leads totheliberation of arachidonic acid, which may play an important role in the into phosphoinositides and markedly depressed carbamoylcholine-induced increases in my~-[~H]inositol secretory process (6, 8, 9). For example,arachidonicacid pancreas (8, IO), as well as incorporation into inositol phospholipids. Several evokes secretion from exocrine other secretory cells (11-16). While pancreatic secretagogues other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulate arachidonic acid metabolism (8, 17, 18), cyclooxystimulating my~-[~H]inositol accumulation and didgenase not and lipoxygenase inhibitors fail to block the secretory retard the incorporation of rny~-[~H]inositol into phos- response to receptor agonists (10,18,19). Thus, the exact role phoinositides. Thefactthat eicosapentaenoicacid of thearachidonic acidcascade in amylasesecretion has (20:5), but not arachidic acid (20:0), mimicked the remained elusive. depressant effect of arachidonate on phosphoinositide The stimulatory action of arachidonic acid and/or its melabeling suggeststhat the degree of unsaturation of the tabolites may be associated with its ability to activate phosfatty acid, rather than chain length, is important for pholipase C in intact cells (20-22) and cell-free preparations inhibition of phosphoinositide synthesis. The arachi(9,23-25). We have examined the effects of exogenous arachdonate-induced decrease in rny~-[~H]inositol incorpoidonate onphosphoinositide hydrolysis in isolated pancreatic ration was accompanied by a reduction in the steady acinar cells. The results reveal that, unlike Ca2+-mobilizing statelevel of [32P]phosphatidylinositol 4,5-bisphosreceptor agonists, arachidonic acid does not stimulate phosphate. The mass of arachidonic acid liberated in repholipase C-mediated phosphoinositide breakdown, but does sponse to carbamoylcholine was measured by gas chromatography-mass spectrometry,and the time course of interact with carbamoylcholine to enhance markedly the acstimulated arachidonate accumulation paralleled that cumulation of myo-inositol. This selective action of arachidonic acid appears toinvolve the inhibitionof the resynthesis of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pan- of inositol phospholipids that follows agonist-mediated inocreas, endogenous arachidonic acid serves as a nega- sitide breakdown. tive feedback regulatorof phosphoinositide turnover. EXPERIMENTALPROCEDURES

A biochemical response to Ca2+-mobilizing receptor agonists shared by many secretory cells, including exocrine pancreas, is the hydrolysis of phosphoinositides (PtdIns-4,5-P2, PtdIns-4-P, and PtdIns)’ (1-4). This phospholipase C-catalyzed reaction yields inositol phosphates and diacylglycerol, * This work was supported by Research Grant AM-28029 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The abbreviations used are: PtdIns-4,5-P2, phosphatidylinositol 4,5-bisphosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns, phosphatidylinositol; Ins-P, myo-inositol monophosphate; Ins-P,, myo-inositolbisphosphate; Ins-P3,myo-inositoltrisphosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

Materials-Collagenase was obtained from Boehringer Mannheim. ny~-[~H]Inositol (15 Ci/mM) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Essential amino acids (50 X concentrate) were purchased from Gibco. Hepes, carbamoylcholine, bovine serum albumin, ovalbumin, butylated hydroxytoluene, soybean trypsininhibitor, and eicosapentaenoic acid were obtained from Sigma. All other fatty acids were obtained fromNuCheck Prep (Elysian, MN). Unless otherwise indicated, the fatty acids were dissolved in either CH,OH or C2H60H, andimmediately prior to use were dried under a stream of N,, and resuspended in the incubation medium by sonication. All other drugs were dissolved in deionized HzO. Cell Preparation and Incubations-Pancreatic acinar cellswere prepared from male Sprague-Dawley rats (125-150 g) as described (26) and incubated ina Hepes-buffered Krebs-Henseleit medium containing in millimolar: NaCI, 98.3; KCl, 4.7; KH,PO,, 1.2; CaCI,, 1.3; MgS04, 1.2; NaHC03, 2.5; Hepes, 10; and dextrose, 11. The medium also contained soybean trypsin inhibitor (0.1 mg/ml) and essential amino acids, and was maintained at pH 7.4 under an

17426

Fatty Acids and Phosphoinositide atmosphere of 95% 0, and 5% CO,. To examine the effects of arachidonic acid, other fatty acids, and carbamoylcholine on cellular levels of myo-inositol and inositol phosphates, cells (50 mg of protein/ml) were suspended in medium containing 0.1% bovine serum albumin and incubated with [3H]inositol (100 Ci/ml) for 1 h at 37 “C with shaking (300 cpm). After incubation, the radiolabeled cells were isolated by layering the cells on top of 10 ml of medium containing 4% bovine serum albumin, and centrifuging at 50 x g for 5 min. The cells were then washed twice in bovine serum albumin-free medium and resuspended in the same albumin-free medium. A 250~~1 aliquot of cells (0.5-2 mg of cell protein/ml) was added to 250 ~1 of medium containing arachidonic acid, other fatty acids, and/or various drugs. Incubations were terminated after 30 min by the addition of 1 ml of cold 4.5% perchloric acid. To study effects of arachidonic acid, other fatty acids, and carbamoylcholine on myo-[3H]inositol incorporation into inositol phospholipids, acinar cells (0.5-2 mg) were incubated in 500 ~1 of albuminfree medium containing the test substance(s) and 3 &i of my~-[~H] inositol. Incorporation of myo-[3H]inositol into PtdIns was tarminated by the addition of 3 ml of CHCl.&H,OH (1:2, v/v), containing 0.1 mg/ml of butylatad hydroxytoluene. The direct effects of arachidonate on the metabolism of PtdIns4,5-P, were determined by exposing cells to [“‘PIP, for 60 min. This procedure labels the polyphosphoinositides (but not PtdIns) to equilibrium (2) and enables changes in radioactivity to reflect net changes in mass of these rapidly turning over phospholipids. Arachidonic acid was then added to some of the suspensions for 5 min, and aliquots were analyzed for PtdIns-4,5-P* as previously described (2). In experiments where the mass of unesterified arachidonic acid was measured, acinar cells (4-8 mg of cell protein) were incubated with or without carbamoylcholine in 8 ml of Kreb’s buffer containing 0.1% Pentex fatty acid-free bovine albumin (Miles Laboratories, Elkhart, IN). A 500~~1 aliquot of the cell suspension was withdrawn at various intervals and added to 3 ml of CHCl&H,OH (1:2, v/v), containing 0.1 mg/ml of butylated toluene to terminate the reaction. Analysis of Irwsitol Phospholipid Metabolism-For assay of myo[3H]inositol and [3H]inositol phosphates, perchloric acid extracts were centrifuged at 200 x g for 5 min and the pH of the supernatant adjusted to 8-9 with 0.5 M KOH, 9 mM NaZBIOP, 1.9 mM EDTA, 3.8 mM NaOH. Separation of myo-[3H]inositol, [3H]Ins-P, [3H]lns-Pz, and [3H]Ins-P3 was performed by anion exchange chromatography as described (27). Phospholipids were extracted from cells with CHClJ CH,OH/HCl as described previously (28, 29). The CHCL layer was dried, and the radioactivity quantitated by liquid scintillation spectrometry. In some cases, the extracted lipids were separated on silica type G plates as described by Weiss et al. (30). Greater than 90% of the radioactivity in the lipids comigrated with the PtdIns spot. In exocrine pancreas, PtdIns represents more than 90% of the total inositol phospholipids.’ Since PtdIns-4,5-Pz and PtdIns-4-P were not separated from PtdIns in this study, values are expressed as total inositol phospholipids. Measurement of Mass of Unesterified Arachidonic Acid-Arachidonic acid was extracted from acinar cell incubates as described above and separated from membrane phospholipids by elution from silicic acid columns using CHCl,/CH,OH (9:l) (31). Heneicosanoic acid (lo-100 ng) was added as an internal standard to each experimental sample prior to the extraction in order to determine the percent fatty acid recovery. Extracted fatty acids were methylated by reaction with ethereal diazomethane. The derivative was analyzed on a HewlettPackard carbowax column (25 mm length; inner diameter 0.2 mm) interfaced with a Hewlett-Packard gas chromatograph/mass spectrometer (model 5890). The separation of fatty acids was performed over a temperature range of 180-22O”C, at a helium flow rate of 1 ml/min. Quantitative analysis of fatty acids was achieved by selective ion monitoring for both arachidonic and heneicosanoic acids (m/e 55.2,67.2, and 79.2). Recovery of fatty acids averaged 60-70%. Statistical Analysis-Statistical analysis was determined using Student’s t test for paired comparisons. RESULTS

Comparative Effects of Arachidonic Acid, Carbamoylcholine, and Various Fatty Acids on myo-pH]Inositol and pH]Inositol Phosphate Levels in Acinar Cells-In previous studies, we showed that stimulation of myo-[3H]inositol prelabeled pan’ A. Chaudhry

and R. P. Rubin,

unpublished

data.

17427

Turnover

creatic acini with carbamoylcholine and other Ca’+-mobilizing receptor agonists causes the accumulation of [3H]Ins-P3, [3H] Ins-Pz, and [3H]Ins-P (32,33). Table I shows that arachidonic acid alone does not alter the levels of the three inositol phosphates. However, arachidonic acid enhanced the accumulation of myo-[3H]inositol from a mean control value of 4.8 to 6.3% and 8.2% at concentrations of 50 and 100 PM, respectively. This action of arachidonic acid was not a consequence of a detergent effect on the acinar cell, for the following reasons: 1) it was not accompanied by the release of lactate dehydrogenase (data not shown); 2) arachidonic acid did not render acinar cells permeable to trypan blue (data not shown). To determine further whether arachidonate compromised cellular integrity and/or viability, in one experiment we examined the permeability of acinar cells to [Ylmannitol, a low molecular weight compound that is normally not taken up by cells. Cells were incubated for 30 min with 50-100 FM arachidonate, 0.2 &i of [i4C]mannitol (2 mCi/mmol), and 1.25 &i of 3H20. The cells were pelleted and radioactivity measured to determine the extracellular space ([Wlmannitol) and the total space (3Hz0). Arachidonate did not alter the W3H ratio (0.2), implying that arachidonate did not increase acinar cell permeability. Carbamoylcholine stimulated the accumulation of myo-[3H] inositol (Fig. 1A), as well as the [3H]inositol phosphates (Fig. 1, B, C, and D), although the relative increase in [3H]Ins-P, accumulation (Fig. 1B) was much greater than the relative increase in myo-[3H]inositol accumulation (Fig. 1A). Arachidonic acid did not significantly affect carbamoylcholine-induced accumulation of the three inositol phosphates (Fig. 1, B, C, and D). However, the combination of arachidonate and carbamoylcholine resulted in myo-[3H]inositol levels (7.2% of total [3H]inositides) that were significantly greater than the predicted sum of responses of the two agents given alone (3.0%) (Fig. lA). The effect of an equivalent concentration (50 fiM) of four other fatty acids on the accumulation of myo-[3H]inositol is shown in Fig. 2. A modest, but significant, increase in cellular myo-[3H]inositol levels was observed with the saturated and mono-, di-, and trienoic species of fatty acid. However, unlike arachidonic acid, these fatty acids elicited an additive, rather than a synergistic, response with carbamoylcholine (Fig. 2). Inhibition of Arachidonate-induced myo-pH]Znositol Accumulation by LXX--In order to elucidate the biochemical mechanism involved in the action of arachidonate, the effect of LiCl on myo-[3H]inositol levels was next investigated. Li’ acts as an inhibitor of inositol phosphatases to promote the cellular accumulation of inositol phosphates in various tissues, including exocrine pancreas (3,33-35). As a consequence of this action, there is a reduction in cellular inositol levels, which leads subsequently to the impairment of the resynthesis TABLE

Effect

I

of arachidonic

acid (AA) on myo-[3H]inositol and [3H]inositol phosphate levels Acinar cells prelabeled with myo-[3H]inositol were exposed to arachidonic acid for 30 min and the accumulation of myo-[3H]inositol and [3H]inositol phosphates was determined. Results are expressed as percent of total phospho-[3H]inositide radioactivity, which averaged 126,573 cpm. Each value is the mean k S.E. of 3-10 independent experiments. AA

[3H]Ins

[3H]Ins-P

[3H]Ins-P,

[3H]Ins-P3

% total phospho-[3HlinositidecEes

Mf 0

50 100

4.80 + 0.88 6.29 + 1.30” 8.18 + 1.96”

‘p < 0.05 compared

1.51 + 0.27 1.67 + 0.32 1.86 + 1.15 to paired

untreated

0.09 f 0.01 0.09 f 0.01 0.09 + 0.02 samples.

0.06 + 0.01 0.05 + 0.01 0.06 zk 0.02

AcidsFatty

17428

and Phosphoinositide Turnover

of phosphoinositides (36-38). Fig. 3A shows that LiCl reduced the accumulation of my~-[~H]inositol elicited by a combination of arachidonic acid and carbamoylcholine from 6.2 to 0.9% (Fig. 3A). By contrast, in Li+-pretreated cells, [3H]InsP3 levels after exposure to arachidonate plus carbamoylcholine were comparable to those observed after exposure to carbamoylcholine alone (Fig. 3B). Fig. 3 also reaffirms the

6-

0.3-

4-

0.2-

E

2-

0.1-

f

0-

8 a

.s

A

Li+

[ 3H] Inositol

19 ['HI Ins-P3

I

r1

0-

0.6-

0.6-

2

1

-

0.4-

0.4

0.2- AA

CCh 0.2-

.,

ability of Li+ to retard inositol phosphate degradation in pancreatic acinar cells (32, 35) by showing that Li' reduced the accumulation of my~-[~H]inositol elicited by carbamoylcholine to below basal values, while [3H]Ins-P3levels were markedly enhanced. Effects of Arachidonic Acid and Other Fatty Acids on myoPHJInositol Incorporation into Inositol Phospholipids-Fig. 4 illustrates the concentration-dependent reduction in phosphoinositide labeling produced by arachidonic acid in acinar cells concomitantly exposed to myo- [3H]inositol.This reduction was significant at 25 p~ arachidonic acid (18%)and was maximal at a concentration of 100 p M (67%).

n-

AA

AA

AA

+ CCh

nd AA

CCh A t

"

AA

+ h

CC

CCh A$

CCh

CCh

FIG. 1. Effects of arachidonic acid andcarbamoylcholine on the levels of my~-[~H]inositol and [sH]inositol phosphates in for pancreatic acinar cells. Cells prelabeled with my~-[~H]inositol 1h were exposed to arachidonic acid ( A A ; 50 p ~ in) the presence or absence of carbamoylcholine (CCh; 10 p M ) for 30 min. Drug-induced accumulation of myo-[3H]inositoland [3H]inositol phosphates is expressed as percent total ph~spho-[~H]inositides minus the radioactivity in untreated control samples. Radioactivity in each control fraction was: A, myo-['Hlinositol, 4.6 f 0.9;B, ['H]Ins-P3, 0.07 & 0.01; C, ['HIIns-P, 1.7 f 0.3; and D, [3H]Ins-P2,0.09 f 0.01 percent of total. Total phospho-['Hlinositide radioactivity averaged 116,896 cpm. Each value is the mean f S.E. for five to eight independent experiments. Asterisk, significantly different from the sum of arachidonate and carbamoylcholine responses, as determined by the Student t test for paired comparisons (p < 0.05).

FIG. 3. Effect of LiCl on the accumulation of myo-["H]inositol and [SH]Ins-P~ in acinar cells exposed to arachidonic acid and carbamoylcholine. Cells preloaded with my~-[~H]inositol were ) carbamoylexposed for 30 min to arachidonic acid ( A A ; 50 p ~ and ) or in combination, in the presence or choline (CCh; 10 p ~ alone absence of LiCl (10 mM). Drug-induced accumulation of m ~ o - [ ~ H ] inositol ( A ) and 13H]Ins-P3( B ) is shown after subtracting the total radioactivity in untreated control samples. In each control fraction, radioactivities of my~-[~H]inositol and [3H]Ins-P3were 5.2 f 1.3 and which 0.06 f 0.01%, respectively, of total ph~spho-[~H]inositides, averaged 94,356 cpm. Li+ did not alter theamount of radioactivity in either control fraction. Each value is the mean f S.E. of three to four similarly independent experiments. Asterisk, p