Inhibitory action of fatty acids on calcium fluxes in thyroid FRTL-5 cells

ELSEVIER

Molecular and Cellular Endocrinology 103 (1994) 125-132

~ Molecularand Cellular Endocrinology

Inhibitory action of fatty acids on calcium fluxes in thyroid FRTL-5 cells E l i n a E k o k o s k i a,b, L i s b e t h F o r s s b, K i d T 6 m q u i s t a,b,* aDepartment of Zoology, Division of Physiology, University.of Helsinki, Helsinki, Finland bMinerva Foundation Institutefor Medical Research, Tukholmankatu 2, 00250 Helsinki, Finland

Received 11 February 1994; accepted 11 April 1994

Abstract

In the present study, we wanted to investigate the action of fatty acids on agonist-evoked changes in intracellular free calcium ([Ca2+]i) in thyroid FRTL-5 cells. Stimulating Fura 2 loaded cells with long chain unsaturated fatty acids increased [Ca2+]i in a dosedependent manner. This increase was in part dependent on extracellular calcium. Long chain saturated fatty acids and short chain fatty acids had no effects on [Ca2+]i per se. Pretreatment of the cells with long chain unsaturated fatty acids almost totally inhibited both the ATP- and thapsigargin-evoked release of sequestered calcium and the entry of extracellular calcium. Long chain saturated fatty acids also attenuated the ATP-evoked increase in [Ca2+]i, while short chain fatty acids had no effects on the ATP-evoked change in [Ca2+]i. The inhibitory effect of long chain unsaturated fatty acids on agonist-evoked changes in [Ca2+]i was not dependent on activation of protein kinase C, and was not due to an enhanced efflux of calcium. These fatty acids rapidly acidified the cytosol in the cells, which could, in part, explain the inhibitory effect of the long chain unsaturated fatty acids on agonist-evoked changes in [Ca2+]i. Addition of bovine serum albumin to the cells rapidly reversed the inhibitory effect of the fatty acids o n [Ca2+]i, and restored p H i. Thus, fatty acids could be potential modulators of calcium signaling in FRTL-5 cells, possibly by modulating calcium entry at the level of the plasma membrane. Keywords: Calcium entry; Fatty acids; Thyroid; Intracellular pH

1. Introduction

Activation of phospholipase A 2 and the production of arachidonic acid is an important event in regulating signal-transduction pathways in FRTL-5 cells (Burch et al., 1986; Okajima et al., 1989a; Tahara et al., 1991). Several other fatty acids may be liberated from the plasma membrane phospholipids by the action of phospholipase A 2 (Asaoka et al., 1992). Another important signal-transduction pathway in thyroid cells is mediated by activation of phospholipase C and the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (Mockel et al., 1987; Okajima et al., 1988, 1989b). Both these pathways interact to regulate the release of sequestered Ca 2÷ and the activation of influx of extracellular Ca 2÷ (Mockel et al., 1987; Okajima et al., 1988, 1989b; Rani et al., 1989; Ttirnquist, 1992b; T/Srnquist et al., 1994). Unsaturated fatty acids have been reported to either activate (Huang et al., 1992. Shimada and Somlyo, 1992) or inhibit (Shimada and Somlyo, 1992) voltage-operated * Corresponding author.

calcium channels. In some non-excitable cells, unsaturated fatty acids inhibited calcium entry, possibly via a direct inhibition of the entry pathway for calcium (Chow et al., 1990. Nordstr/3m et al., 1992). Another possibility is that the fatty acids enhance calcium extrusion, thus rapidly restoring the [Ca2+]i levels after stimulation (Breittmayer et al., 1993). Other studies have shown that unsaturated fatty acids enhance agonist-evoked calcium entry (Damron and Bond, 1993). In addition, unsaturated fatty acids have been shown to be potent activators of protein kinase C (see (Asaoka et al., 1992), and to inhibit Na÷/K÷ATPase (Oishi et al., 1990), two enzymes which may affect [Ca2+]i in cells. We have shown that PKC is of importance in regulating calcium entry in FRTL-5 cells (T6rnquist, 1993b). Since fatty acids, i.e. possible endogenous activators of PKC, may be produced during agonist-induced stimulation of the cells (Burch et al., 1986; Okajima et al., 1989a; Asaoka et al., 1992), we wanted to investigate the effects of these compounds on the regulation of calcium fluxes in FRTL-5 cells. Our results show, that several long chain unsaturated fatty acids potently attenuated both the agonist-evoked release of sequestered calcium, and the agonist-evoked calcium entry.

0167-8140/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03315-Z

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E. Ekokoski et al. / Mol. Cell. Endocrinol. 103 (1994) 125-132

2. Materials and methods

2.1. Materials Culture medium, serum and hormones needed for the cell culture were purchased from Gibco (Grand Island, NY) and Sigma (St. Louis, MO). Culture dishes were obtained from Falcon Plastics (Oxnard, CA). ATP, arachidonic, oleic, linoleic, linolenic, valproic, heptanoic, myristic and palmitic acids, nigericin, staurosporin and bovine serum albumin (BSA, fatty acid free) were purchased from Sigma. Fura 2-AM and bis-(carboxyethyl)carboxyfluorescein-AM (BCECF-AM) were purchased from Molecular Probes, Inc. (Eugene, OR). Thapsigargin, and phorbol myristate acetate were purchased from LC Services Corp (Woburn, MA). All other chemicals used were of reagent grade.

2.4. Measurement of intracellular pH (pHi) The cells were grown and harvested as described above. The cells were then incubated for 35 min with 5/zM bis-(carboxyethyl)carboxyfluorescein-AM (BCECF-AM). pH i was determined fluorimetrically with a Hitachi F2000 fluorimeter (Tokyo, Japan) using the excitation wavelengths 440 nm and 500 nm and an emission wavelength of 530 nm. The calibration procedure was exactly as described previously (T6rnquist and Alinen, 1992).

2.5. Statistics The results are expressed as the mean + SEM. The figures show representative traces of each experiment. Statistical analysis was made using Student's t-test for paired observations. When three or more means were tested, analysis of variance was used.

2.2. Cell culture Rat thyroid FRTL-5 cells were a generous gift from Dr. Leonard D. Kohn (NIH, Bethesda, MD). The cells were grown in Coon's modified Ham's F 12 medium, supplemented with 5% calf serum and six hormones (Ambesi-Impiombato et al., 1980) (insulin, 10ktg/ml; transferrin, 5/zg/ml; hydrocortisone, 10 nM; the tripeptide gly-L-his-L-lys, 10 ng/ml; TSH, 0.3 mU/ml; somatostatin, 10 ng/ml). The cells were grown in a water-saturated atmosphere of 5% CO2 and 95% air at 37°C. Before an experiment, cells from one donor culture dish were harvested with a 0.25% trypsin solution and plated onto plastic 100-mm culture dishes• The cells were grown for 7-9 days before an experiment, with 2-3 changes of the culture medium. Fresh medium was always added 24 h prior to an experiment.

3. Results

3.1. Effects of fatty acids on [Ca2+]i in FRTL-5 cells Unsaturated fatty acids have been shown to stimulate the release of sequestered calcium in some cells types (Chow and Jondal, 1990a,b). Addition of 30/tM oleic ATP

500 - A

100o

OA

B

ATP 50O

250

=

0

.-'n

2.3. Measurement of[Ca2+] i The medium was aspirated and the cells were then harvested with buffer buffered saline solution (BSS, containing in millimolar concentrations: NaC1, 118; KC1, 4.6; glucose, 10; CaC12, 1.0; HEPES, 20; pH 7.2) containing 0.02% EDTA and 0.1% trypsin. After washing the cells three times by pelleting, the ceils were incubated with 1/zM Fura 2-AM for 30 min at 37°C. Following the loading period, the cells were washed twice with BSS buffer, and incubated for at least 10 min at room temperature, and washed once again. The cells were added to a quartz cuvette, kept at 37°C, and stirred throughout the experiment. Fluorescence was measured with a Hitachi F2000 fluorimeter. The excitation wavelengths were 340 and 380 nm, and emission was measured at 510 nm. The signal was calibrated by addition of 1 mM CaC12 and digitonin to obtain Rmax. To obtain Rmin extracellular Ca 2+ was chelated with 5 mM EGTA and pH was elevated above 8.3 by the addition of Tris-base. [Ca2+]i was calculated as described previously (Gryenkiewicz et al., 1985), using a computer program designed for the fluorimeter with a Kd-value of 224 nM for Fura 2.

ATP

()A •

;'50

•

,i

0

,ooo

E

Goo

400

,'l

200

o

"0--

......

o

,

........

1 Otelc

,

lO ac*d

........

,

loo

(~,M)

Fig. l. Action of oleic acid on [Ca2+]i in FRTL-5 cells. The traces denote: Fura 2-loaded cells stimulated with 30/~M oleic acid (OA) and then with 100/~M ATP (A), or with 100/~M ATP only (B) in a calcium-containing buffer. (C) The cells were stimulated with 30,uM (OA) and then with 100/zM ATP, or with 100/zM ATP only (D) in a calcium-free buffer. The horizontal bar denotes 1 min. (E) shows the dose-dependent inhibitory effect of OA on the ATP-evokedincrease in [Ca2+]i in a calcium-containingbuffer (ll), or in a calcium-freebuffer (El). Each point gives the mean+ SEM of 4-7 separate determinations.

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E. Ekokoski et al. / Mol. Cell. Endocrinol. 103 (1994) 125-132

Table 1 The effects of several fatty acids on [Ca2+]i and on the ATP-evoked increase in [Ca2+]i Compound

A[Ca2+]i (nM) FA

Control Oleic acid Arachidonic acid Linoleic acid Linolenic acid Palmitic acid Myristic acid Heptanoic acid Valproic acid

63 + 86 + 68 + 62 + -

ATP 6 15 12 19

758 + 43 68 + 9a 68 + 31a 56 + 9a 84 + 50a 412 + 52a 291 + 79a 716 _+72 602 + 42

The cells were harvested and loaded with Fura 2 as described in Section 2. The cells were then stimulated with the appropriate fatty acid (FA, final concentration 30/~M), and after 3-4 min, the cells were stimulated with 100/~M ATP. Each number gives the mean + SE of 3-9 separate determinations. The dashes after fatty acids indicate that no effect was observed. aSignificantly different (P < 0.05) from the ATP-evoked increase in [Ca2+]i in control cells. acid to FRTL-5 cells rapidly increased [Ca2+] i both in calcium-containing buffer (63 + 6 nM, Fig. 1), and in nominally calcium-free buffer containing 100/~M E G T A (25 + 2 nM, Fig. 1) . The effect of oleic acid was dosedependent in both buffers (data not shown). Although the increase in [Ca2+]i evoked by oleic acid appeared to depend on extracellular calcium, the change in [Ca2+]i was not inhibited by the calcium-channel inhibitor S K F 96365 (103 + 20 nM, data not shown). The effect of oleic acid on [Ca2+]i was not due to a non-specific permeabilization of the plasma membrane, as evaluated using trypan blue, measurement of the membrane potential with the fluorescent dye bisoxonol, and by 45Ca2+-flux studies (data not shown). Several other unsaturated fatty acids also increased [Ca2+] i in FRTL-5 cells (Table 1).

pared with control cells (25 + 2 nM, P < 0.05, data not shown). Several of the other fatty acids tested attenuated the ATP-evoked increase in [Ca2+]i (Table 1). The above results suggested that oleic acid and ATP, at least in part, mobilized calcium from the same intracellular stores. W e then tested whether oleic acid, like ATP, induced capacitative calcium entry. Stimulating the cells with oleic acid in a calcium-free buffer, and then adding calcium (final concentration 1 mM) to the cells, increased [Ca2+]i by 73 + 2 nM (Fig. 2). If the cells were stimulated with both oleic acid and ATP, the increase in [Ca2+] i was 81 + 17 nM (data not shown). In cells stimulated with ATP only, addition of calcium increased [Ca2+]i transiently by 1 5 0 + 13 nM, which then stabilized 1 0 2 + 13 nM above the prestimulatory [Ca2+]i -level (Fig. 2). If oleic acid was added to the cells after ATP, but prior to Ca 2÷, the increase in [Ca2+] i in response to the addition of calcium was 52 + 8 nM (P < 0.05, Fig. 2). The effect of oleic acid was not due to an enhanced efflux of calcium, as oleic acid did not enhance the ATP-evoked efflux of 45Ca2+ from FRTL-5 cells (data not shown). Furthermore, if barium was added to the cells after stimulation with ATP and addition of oleic acid, the entry of barium was smaller in cells treated with oleic acid, compared with control cells (Fig. 3). As barium rapidly enters the cells through calcium channels in a manner similar to that of calcium, but cannot be extruded by the Ca2÷ATPase, the results suggested that oleic acid inhibited the entry of barium. W e have shown that thapsigargin is able to activate capacitative calcium entry in FRTL-5 cells (Trrnquist, 1993b). Addition of 2/~M thapsigargin to cells stimulated with 30/~M oleic acid resulted in a small, transient inA

B

250

125E , -. -S

,J

0

3.2. Effect o f fatty acids on the ATP- and thapsigarginevoked increase in [Ca2+]i in FRTL-5 cells In some cell types, fatty acids have a modulatory effect on calcium entry (Huang et al., 1992; Shimada and Somlyo, 1992). In FRTL-5 cells stimulated first with 30/~M oleic acid, and then with 100/~M ATP, the ATP-evoked increase in [Ca2+] i was almost totally attenuated (Fig. 1). A similar result was obtained when the experiment was repeated in a nominally calcium-free buffer containing 1 0 0 / t M E G T A (Fig. 1). The inhibitory effect of oleic acid on the A T P - e v o k e d increase in [Ca2+]i was dosedependent (Fig. 1). Addition of oleic acid after stimulating the cells with A T P resulted in an increase in [Ca2+]i similar to that in control cells (63 + 6 n M and 5 7 + 10 nM, respectively, data not shown). In a calcium-free buffer, addition of oleic acid after A T P resulted in a slightly attenuated response in [Ca2+] i (16 + 2 nM), com-

500 - C

ATP •

O• A C a• 2÷

D

A•T P

C•a 2÷

250

~

I

I 1 " ~

j

~ ' ~ ' ~ ~'¸~ ~

_fI'.... , F.....

Fig. 2. Inhibitory action of oleic acid on capacitative calcium entry in FRTL-5 cells. Fura 2 loaded cells were in a calcium free buffer containing 100/.tM EGTA. In (A), the cells were stimulated with oleic acid (OA, final concentration 30/~M), and then calcium (final concentration 1 mM) was added. In (B), the cells were stimulated with calcium only. In (C), the cells were stimulated first with ATP (final concentration 100/~M), and then with OA, and then calcium was added to the cell suspension. In (D), the cells were stimulated with ATP only, and then calcium was added. The horizontal bar denotes 1 min.

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A ATP

Ba 2+

B

'I'

ATP

OA Ba 2+

V'

•

•

50

25

Fig. 3. Inhibitory action of oleic acid on barium entry in FRTL-5 cells. Fura 2 loaded cells were in a calcium free buffer containing 100/aM EGTA. In (A), the cells were stimulated first with ATP (final concentration 100/aM), and then barium (final concentration 1 mM) was added to the cells. In (B), the cells were stimulated first with ATP, and then with oleic acid (OA, final concentration 30/aM), and then barium was added to the cell suspension. The ordinate is given in arbitrary units. The horizontal bar denotes 1 min.

crease in [Ca2÷]i (49 + 14 nM, Fig. 3). In control cells, thapsigargin induced a biphasic change in [Ca2+]i (167 + 7 nM, and 111 + 11 nM, for the initial increase in [Ca2+]i and the new plateau level, respectively) (Fig. 4). Furthermore, if oleic acid was added to the cells after thapsigargin, [Ca2÷]i immediately decreased (Fig. 4). In a calciumfree buffer, thapsigargin increased [Ca2÷]i by 108 + 5 nM in control cells. Addition of calcium to these cells resulted in a biphasic increase in [Ca2÷]i: a transient increase (354 + 16 nM), followed by a plateau phase in [Ca2+]i (224 + 13 nM above the prestimulatory [Ca2+]i level). In cells treated with oleic acid in a calcium-free buffer, the thapsigargin-induced increase in [Ca2+]i was only 48 + 13 nM (P<0.05). When calcium was added to these cells, [Ca2+]i increased by 89 + 9 nM (P < 0.05) (Fig. 4). If oleic acid was added to the cells after thapsigargin, but prior to Ca 2÷, the ~ncrease in [Ca2÷]i was 140 + 48 nM, and then stabilized 69 + 17 nM above the prestimulatory [Ca2+]i-level (P < 0.05) (Fig. 4). Taken together, the above results suggested that oleic acid apparently, has a dual effect on [Ca2+]i in FRTL-5 cells: a release of sequestered calcium and an inhibition of calcium entry.

506

"A OA

3.3. Mechanism of action of oleic acid on [Ca2+]i in FRTL-5 cells Fatty acids have been shown to activate PKC in several cell types (Asaoka et al., 1992), and activation of PKC inhibited calcium entry in FRTL-5 cells (T6rnquist 1993a,b). However, inhibition of PKC with staurosporin, or downregulation of PKC (200 nM phorbol myristate acetate for 24 h) did not affect the change in [Ca2÷]i evoked by oleic acid (data not shown). Furthermore, addition of ATP to oleic acid treated cells in which PKC was inactivated, did not restore the ATP-evoked change in [Ca2+]i to control levels (data not shown). Thus, the effect of oleic acid on [Ca2+]i and the inhibition of the ATP-evoked [Ca2+]i transient was apparently not dependent on the activation of PKC. Oleic acid has been shown to inhibit Na÷/K÷-ATPase (Oishi et al., 1990). Inhibition of this enzyme could in= crease [Ca2+]i indirectly due to Na÷-Ca 2÷ exchange (T6rnquist, 1992a). Addition of oleic acid to cells incubated in a Na÷-free choline buffer increased [Ca2+]i by 60 + 2 nM, which was not different from that seen in control cells (63 + 6 nM) (Fig 5). However, the overall

B Tg

C Tg

Tg

OA

250

cJ

500

D OA

E Tg

Ca 2+

F Tg

OA Ca 2÷

Tg

Ca 2 .

f~,

250

Fig. 4. Action of oleic acid and thapsigargin on [Ca2+]i in FRTL-5 cells. Upper traces: (A) Fura 2 loaded cells were stimulated with oleic acid (OA, final concentration 30/aM) and thapsigargin (Tg, final concentration 2/aM) in a calcium buffer. (B) The cells were stimulated first with thapsigargin, and then oleic acid was added. (C) Control cells stimulated with Tg only. Lower traces: (D) Fura 2 loaded cells were stimulated with oleic acid (OA, final concentration 30/aM) and thapsigargin (Tg, final concentration 2/zM) in a nominally calcium-free buffer containing 100/aM EGTA, and then calcium (final concentration 1 mM) was added. (E) The cells were stimulated first with thapsigargin, and then oleic acid was added prior to the addition of calcium. (F) Control cells stimulated with Tg only. The horizontal bar denotes 1 min.

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E. Ekokoski et al. / Mol. Cell. Endocrinol. 103 (1994) 125-132 500

1000

OA %

250

ATP

•

/!

•

500

0

--

Fig. 5. Effect of oleic acid on [Ca2+]iin FRTL-5 cells in Na+-free buffer. Fura 2 loaded cells were incubated in a Na+-containingbuffer (A) or a Na+free buffer (Na+ exchanged to equimolar concentrations of choline (B)), and were stimulated with oleic acid (OA, final concentration 30ktM), and ATP (final concentration 100~M). (C) Control cells in Na+-free buffer stimulated with ATP only. The horizontal bar denotes 1 min. change in [Ca2+] i was different from that seen in control cells. The ATP-evoked increase in [Ca2+]i was enhanced (197 + 16 nM, P < 0.05), which is not unexpected if extrusion of calcium through Na÷-Ca 2÷ exchange is prevented. Stimulating control cells in a Na+-free buffer with ATP increased [Ca2÷] i by 461 + 20 nM (Fig. 5). Recent investigations have suggested that fatty acids could enhance the extrusion of calcium from cells (Breittmayer et al., 1993). However, we were not able to observe an enhanced extrusion of 45Ca2+ after stimulation of the cells with ATP and oleic acid (or arachidonic acid, data not shown). It has been suggested that fatty acids could directly inhibit the calcium-entry pathway, and that this effect could be reversed by BSA (Chow et al., 1990; Chow and Jondal, 1990b). The results in Fig. 6 show that addition of BSA (final concentration 0.2%), almost totally restored the ATP-evoked increase in [Ca2+]i (534 + 28 nM). Furthermore, addition of BSA to cells stimulated first with thapsigargin, and then with oleic acid, rapidly restored the plateau phase of the thapsigargin-evoked increase in [Ca2+] i (Fig. 6). 3.4. E f f e c t s o f o l e i c a c i d on p H i in F R T L - 5 cells

We have recently observed that arachidonic acid rapidly acidifies the cytosol in FRTL-5 cells (T6rnquist et al., 1994). Similar results have been observed with other fatty acids (Nordstr6m et al., 1992). It was thus of'interest to investigate whether oleic acid had a similar effect on pHi. Addition of oleic acid to FRTL-5 cells rapidly acidified the cytosol in a dose-dependent manner (Fig. 7). Fur-

1000

500

A

OA

BSA

ATP

B

thermore, addition of BSA (final concentration 0.2%) rapidly restored the intracellular pH (Fig. 7). To test whether the oleic acid-induced change in calcium was due to the observed acidification, we acidified the cells with the K+-H÷ ionophor nigericin, using a dose (0.3/~g/ml) which acidified the cytosol to roughly the same extent as 30ktM oleic acid (Fig. 8). This dose of nigericin had a very modest effect on [Ca2+]i. Stimulating nigericin-treated cells with 100 ktM ATP increased [Ca2+] i by 556 + 44 nM, compared with 758 + 43 nM (P < 0.05) in control cells (Fig. 8). Thus, part of the inhibitory effect of oleic acid (and other fatty acids) on the ATP-evoked increase in [Ca2+]i may be due to the observed acidification of the cytosol.

4. Discussion In the present report, we show that several fatty acids significantly impair both the agonist-evoked release of sequestered calcium, and the capacitative calcium entry in FRTL-5 cells. Our results suggest that the long-chain unsaturated fatty acids tested (oleic, arachidonic, linoleic, and linolenic acids), are potent inhibitors of calcium fluxes. Saturated long-chain fatty acids (palmitic and myristic acids) did not induce any changes in [Ca2+]i per se, but their inhibitory effect on agonist-evoked increase in [Ca2+]i was significant. The short-chain fatty acids tested (heptanoic and valproic acids) did not modulate [Ca2+]i in FRTL-5 cells. The long-chain fatty acids tested all increased [Ca2+]i in a manner dependent on extracellular calcium, although C 500

Tg

OA BSA

250

C,J

r

Fig. 6. Bovine serum albumin reverses the effect of oleic acid on [Ca2+]iin FRTL-5 cells. (A) Fura 2-loaded cells were stimulated with 30/~M oleic acid (OA) and then bovine serum albumin (BSA, final concentration 0.2%) was added prior to stimulating the cells with 100/tM ATP. (B) Stimulation of control cells with ATP. (C) Stimulation of cells with thapsigargin (Tg, final concentration 2/tM), and the addition of oleic acid and BSA. The horizontal bar denotes 1 min.

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7.5

pH i

A

(Damron and Bond, 1993). The increase in [Ca2+]i observed with oleic acid in our study was, however, not inhibited by SKF 96365, an inhibitor of agonist-evoked calcium entry via a presently uncharacterized type of calcium channels (Merritt et al., 1990). We have not been able to demonstrate VOCC in FRTL-5 cells. Quite opposite results were obtained in rabbit intestinal smooth muscle cells (Shimada and Somlyo, 1992), and neurons (Keyser and Alger, 1990), where VOCCs were inhibited by fatty acids. An inhibitory effect of fatty acids on agonist-mediated Calcium entry has also been observed in Tcells (Chow et al., 1990; Nordstr/Sm et al., 1992). Our results suggest that the fatty acids inhibited an entry pathway for calcium in FRTL-5 cells. In addition to inhibiting calcium entry in FRTL-5 cells, the fatty acids appeared to inhibit the release of sequestered calcium. A similar finding has been observed in some cells (Chow and Jondal, 1990b), but not in other cells (Breittmayer et al., 1993). Several explanations may account for our observation. The most likely explanation is that although the increase in [Ca2+]i in response to the tested fatty acids was slow in onset and of small magnitude, it was sufficient to deplete the stores. Thus, no or only very little sequestered calcium was available for a subsequent stimulation. The fatty acids could also impair the receptor-induced production of IP 3 (Richieri and Kleinfeld, 1989). However, the calcium-mobilizing effect of thapsigargin was also attenuated, suggesting that the fatty acids had a direct effect on the calcium stores. Finally, the fatty acid-induced acidification of the cytosol could hamper the release of sequestered calcium. At least the IP3-induced release of sequestered calcium is pHdependent (Guillemette and Segui, 1988). Several reports have shown that fatty acids are potent activators of PKC (Asaoka et al., 1992). In FRTL-5 cells, activation of PKC has a potent inhibitory effect on both the ATP- and the thapsigargin-evoked changes in [Ca2÷] i (Trrnquist, 1993a,b). We were not able to abolish the increase in [Ca2÷] i induced by oleic acid by inhibiting

OA

7.1 6.9 6.7

0.25

B 0.20 0.15

A PHi

{

0.10

t'

0.05 0.00

........

i

........

i

1

10

Oleic

C

acid

OA

7.5 _

........

100

(.uM)

BSA

•

•

phi

Fig. 7. Action of oleic acid on pHi in FRTL-5 cells. BCECF loaded cells were stimulated with oleic acid (OA, final concentration 30/zM (A)). The horizontal bar denotes 1 min. (B) Dose-dependent effect of oleic acid on pHi in FRTL-5 cells. Each point gives the mean + SEM of 4-6 determinations. (C) The cells were stimulated with OA and then BSA (final concentration 0.2%) was added to the cells. a part of the increase in [Ca2+]i was apparently due to mobilization of calcium from intracellular stores. Fatty acids have been shown to enhance the activity of voltage dependent calcium channels (VOCC) in some cell types (Huang et al., 1992; Shimada and Somlyo, 1992), and to enhance agonist-evoked calcium entry in other cells A

B

OA •

7.5

N •

6.7 1000 ~.

"%'~%""~ C

D OA

ATP

N •

ATP

•

•

500

Fig. 8. Comparison of the effects of oleic acid and nigericin on pH and [Ca2+]iin FRTL-5 cells. Upper traces: BCECF loaded cells were stimulated with oleic acid (OA, final concentration 30/zM (A)) or nigericin (N, final concentration 0.3 Fg/ml (B)) Lower traces: Fura 2 loaded cells were stimulated first with oleic acid (C) or nigericin (D) and then with ATP (final concentration 100/zM). (E) Control cells stimulated with ATP only. The horizontal bar denotes 1 min.

E. Ekokoski et al. / Mol. Cell. Endocrinol. 103 (1994) 125-132

PKC, nor was the ATP-evoked increase in [Ca2+]i restored in these cells. However, we cannot exclude the possibility that some PKC-isoenzymes were not inhibited by staurosporin or downregulated by phorbol esters. Oleic acid is known to selectively regulate PKC isoenzymes (Khan et al., 1993). Furthermore, oleic acid had no effect on cell viability, excluding the possibility that the increase in [Ca2+]i was the result of a non-specific permeabilization of the plasma membrane. We also tested whether oleic acid or arachidonic acid could modify the membrane potential, but neither compound had any effects on the membrane potential (data not shown). Thus, the effects of the fatty acids were probably not due to modulation of K÷-channel activity (see Ordway et al., 1989), or to a permeabilization of the cells. In addition, we have obtained a very strong release of sequestered calcium after stimulating permeabilized cells with ATP (T6rnquist, unpublished observations). Furthermore, we could not detect any effects of oleic acid (or arachidonic acid) on the extrusion of 45Ca2+ from FRTL-5 cells, excluding an enhancing effect of oleic acid on the plasma membrane Ca2+-ATPase, as has been suggested to occur in other cells (Breittmayer et al., 1993). Our results with barium, a cation which cannot be transported by the Ca2+-ATPase, but readily permeates calcium channels, also suggested that the fatty acids did not enhance the extrusion of calcium in FRTL-5 cells. Oleic acid has been shown to inhibit Na+/K+-ATPase in rat brain (Oishi et al., 1990). If this occurred in FRTL-5 cells, [Ca2+]i could increase due to enhanced Na+-Ca 2+ exchange. In a Na+-free buffer solution, oleic acid increased [Ca2+]i, but the shape of the change in [Ca2+]i was different from that seen in a Na+-containing buffer. However, part of the oleic acid-evoked increase in [Ca2+]i could be the result of the acidification-induced activation of Na+-H + exchange, followed by enhanced Na+-Ca 2+ exchange. Bovine serum albumin (BSA) has been shown to potently bind fatty acids (Purdon and Rao, 1989), and to reverse or to abolish the effects of fatty acids on calcium fluxes (Chow et al., 1990; Nordstr6m et al., 1991; Breittmayer et al., 1993). We observed a similar effect in our study, supporting the suggestion that the effect of oleic acid (and arachidonic acid) on calcium entry could occur at the level of the plasma membrane. Furthermore, the fact that BSA reversed the effect of the fatty acids suggested that the effect of the fatty acids was not due to a disruption of the cell membrane. Addition of BSA also rapidly restored the pH i of the cells. Studies with model membranes have suggested that the acidification of the cytosol with fatty acids was due to a rapid movement of un-ionized fatty acids to the inner leaflet of the membrane, followed by a release of protons from the fatty acid molecules (Kamp and Hamilton, 1992). Addition of BSA rapidly removed the fatty acids, thus leading to a rapid increase in pH i. Presently, we do

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not know whether this 'flip-flop' of fatty acids occurs in FRTL-5 cells, or whether the fatty acids are transported over the membrane with the aid of transport proteins (Storch et al., 1981). In conclusion, we have shown that several fatty acids potently modulated calcium fluxes in FRTL-5 cells, apparently by inhibiting calcium entry and by releasing sequestered calcium. Several fatty acids are liberated from the plasma membrane phospholipids by the action of phospholipase A 2 during the activation of signaltransduction pathways in many cells (see Asaoka et al., 1992). The fatty acids produced may be important modulators of the overall calcium homeostasis, affecting calcium-dependent processes in FRTL-5 cells.

Acknowledgements This study was supported by the Sigrid Juselius Foundation and the Academy of Finland.

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