Importance of arachidonic acid metabolites in regulating ATP-induced calcium fluxes in thyroid FRTL-5 cells

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Importance of arachidonic acid metabolites in regutating ATP-induced calcium fluxes in thyroid FRTL-5 cells K. TC)RNQUIST, E. EKOKOSKI, L. FORSS and M. MATSSON

Department of Zoology,Division of Physiology, University of Helsinki and the Minerva Foundation Institute for Medical Research, Helsinki, Finland Abstract - Stimulating rat thyroid FRTL-5 ceils with the purinergic agonist ATP activates both the inositoi phosphate signal-transduction pathway and the phosphoiipase A2 pathway. in the present study we wanted to investigate the possible inter-relationships between these two systems during ATP-induced changes in intracellular free calcium ([Ca2+&). Pretreatment of Fura- loaded ceils with Cbromophenyiacyi, an inhibitor of phosphoiipaseA2, had no effect on the ATP-inducedentry of Ca2+but inhibited the release of sequestered Ca2+. Nordihydroguaiareticacid (NDGA), a iipoxygenase inhibitor, and 5,8,11,144cosatetraynoic acid (ETYA), an inhibitor of cytochrome P-450 enzymes, attenuated the ATP-evokedtransient increase in [Ca2”Ji.Furthermore,the capacitatfveentry of Ca2+was also attenuated in NDGA- and ETYA-treatedceils stimulatedwith ATP. Similar results were obtained using econazoie, an inhibitor of cytochrome P-450 enzymes. However, treatment of the ceils with indomethacin, a cyciooxygenase inhibitor, had no effect on the ATP-evoked response in [Ca2’]i. We also showed that stimulation of intact or permeabiiited FRTL-5 ceils with arachidonic acid released sequestered calcium. This calcium originated, at least in part, from an IPs sensitive calcium pool. in addition, arachidonic acid rapidly acidified the cytosol. The results suggest that metabolism of arachidonic acid by a non-cyciooxygenase pathway is of importance in supporting agonist-inducedcalcium fluxes evoked via stimulationof the inositoi phosphate pathway in FRTL-5 ceils. Furthermore,arachidonic acid per se may modify agonist-induced calcium fluxes in these cells.

In rat thyroid PRTL-5 cells, the purinergic agonist ATP and the admergic agonist noradmaliu activate phospholipase A2 (PLA2) with the concomitant release of arachidonic acid [l, 21. In addition, ATP and noradrenalin activate phospholipase C and the hydrolysis of phosphatidylinositol-45bisphosphate

(PIP2) to inositol 1,4,5-u&phosphate (IP3) and diacylglycerol [3-51. This activation of the PIP2-pathway rapidly releases sequestered Ca2’ and activates influx of extracellular Ca2’ [3-71. However, the possible involvement of PLA2 and arachidouic acid in the receptor-activated signal-transduction cascade 153

154

is presently obscure in FRTL-5 cells. Investigations in a variety of cells have suggested a role for arachidonic acid and/or its metabolites in regulating agonist-induced Ca2+ fluxes. These effects have been observed on both voltagesensitive Ca2’ channels I&9], as well as on voltageinsensitive Ca2’ channels [lO-121. These observations, and the fact that an increase in [Ca2+]lper se has been considered sufficient to activate phospholipase A2 activity [13, 141,prompted us to investigate the possible relationships between ATP-induced changes in ICa2+]iand PLA2 activity. The results show that in FRTLJ cells, non-cyclooxygenase metabolites of arachidonic acid have an important function in supporting both ATP-induced release of sequestered Cazt and the Ca2’ entry. Arachidonic acid per se increased [Ca2+]iby releasing sequestered Ca2’, apparently from an IP3 sensitive Ca2’ pool, and by activating influx of extracellular Ca2’. Furtbennore, arachidonic acid rapidly acidified the cytosol in FRTL-5 cells.

Materialsand methods Materials

CELL CALCIUM

hormones 1151 (insulin, 10 cLg/mI,transfenin, 5 ug/mk hydrocortisone, 10 nM, the tripeptide gly+ his-L-lys, 10 ng/mh TSH, 1 mu/ml; and somatostatin, 10 ng/ml). The cells were grown in a watersaturated atmosphere of 5% COZ 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 au experiment, with 2-3 changes of the culture medium. Fresh medium was always added 24 h prior to an experiment. [3H]-arachidonic acid release

The cells were incubated with [3H&irachidonicacid (0.1 cLci/ml)for 24 h [16] in 35 mm plastic dishes. The procedure is a modification of that described earlier [2]. After the incubation the cells were washed S-times (5 min intervals) with release buffer (in mM concentrations: NaCI, 134; KCl, 4.7; glucose, 5; CaCh, 2.0; MgSO4, 1.2; KH2PG4 1.2; NaHCO3,2.5; HEPES, 10; pH 7.2; and 0.1% bovine serum albumin). The cells were then incubated in 750 lrl of the release buffer for the times indicated with the appropriate agents. Then 600 u.l was removed and counted for radioactivity in a 1214 Rackbeta liquid scintillation spectrophotometer.

Culture medium, serum and hormones needed for the cell cultnre were purchased from Gibco (Grand Island, NY, USA) and Sigma (St Louis, MO, USA). MeasurementOf[Ct.?]i Culture dishes were obtained from Falcon Plastic (Oxnard, CA, USA). ATP, nordihydroguaiaretic The medium was aspirated and the cells were then acid (NDGA), indomethacin, 4-bromophenacyl bro- harvested with buffered saline solution (BSS, conmide (CBPB), 5,8,11,14_eicosatetraynoicacid, ara- taining in mM concentrations; NaCl, 118; KCl, 4.6; chidonic acid, leukotrienes Bs, C4, D4, Es and nig- glucose, 10; CaClz, 1.0; HEPES, 20; pH 7.2) conericin were purchased from Sigma. Fun&YAM and taining 0.02% ElDA and 0.1% trypsin. After washbis-(carboxyethyl)-carboxyfluorescein/AM ing the cells 3-w by pelleting, the cells were in(BCEClVAM) were purchased from Molecular cubated with 1 pM Fun&YAM for 30 min at 37’C. Probes Inc. (Eugene, OR, USA). IP3 was purchased Following the loading period, the cells were washed from LC Services Corp (Wobum, MA, USA). All twice with BSS buffer, and incubated for at least 10 other chemicals used were of reagent grade. min at room temperature, and washed once again. The cells were added to a quartz cuvette, kept at Cell culture 37°C. and stirred throughout the experiment. Fluorescence was measured with a Hitachi F2000 FluoRat thyroid FRTL-5 cells were a generous gift of Dr rimeter. The excitation wavelengths were 340 and Leonard D. Kohn (NIH, Bethesda, MD, USA). The 380 urn, and emission was measured at 510 nm cells were grown in Coon’s modified Ham’s F 12 The signal was calibrated by addition of 1 mM medium, supplemented with 5% calf serum and six CaCh and digitonin to obtain Rmax. To obtain Rmin

ARACHIDONIC ACID(AA) METABOLISMAND CALCIUMENTRY extracellular Ca2’ was chelated with 5 mM EGTA and pH was elevated above 8.3 by the addition of T&base. [Ca2+]lwas calculated as described previously [17], using a computer program designed for the fluorimeter with a l&value of 224 nM for FuraCell permeabilization and measurement of pee ca2’ [Cd’]

The cells were grown and harvested as described above. The cells were then permeabilized as described recently for GH4CI cells [18, 191. After washing the cells twice by centrifugation (80 g, 5 ruin) in buffer A (containing in mM concentrations; KCl, 125; HEFES, 25; KHzPO42; EGTA, 0.25; and BSA, 1 mg/ml [PH 7.01). and an equilibration for 5 min at 37°C. the cells were permeabilized using 5 @f digitonin. The efficiency of the permeabilization was continuously assessed by trypan blue uptake. After 2-4 min incubation with digitonin, icecold solution B (identical to solution A, but contains

1200 -I

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ing no EGTA) was added, and the cells were washed twice by centrititgation (80 g, 5 min). Using this protocol, more than 95% of the cells were permeabilized. The permeabilized cells were incubated with 2 pM Fura-ZAM for 20 min at 37°C washed with solution B and kept on ice until used. Friar to an experiment, the cells were washed once, suspended in solution B and added to a quartz cuvette, kept at 37°C and stirred throughout the experiment The excitation and emission wavelengths, and the calibration procedure were identical to that used for the [Ca2+]iIIWWl~~ts. Measurementof intracellularpH (pHi)

The cells were grown and harvested as described above. The cells were then incubated for 35 min with 5 @I bis-(carboxyethyl)carboxyfluorescein/AM (BCECF-AM). pH1 was determined fluorometrlcally with a Hitachi F2000 spectrophotometer (Tokyo, Japan) using the excitation wavelengths 440 run and 500 nm and an emission wavelength of 530 MI. The calibration pro&rue was exactly as described previously [20]. Statistics

% P,

155

900

V

600

The results are expressed as the mean f SE. The figures show representative traces of each experiment. Statistical analysis was made using Student’s t-test for paired observations. When 3 or more means were tested, analysis of variance was used. Results

300 ATP-inducedrelease of [3H]-arachidonicacid m

0 Ng. 1 Effect of ATP on [3H]-mchitkmic acid release in FRTL-5 cells. [3H]-machidonicacid-labelled cells wem incubatedin buffer (filled rectangle). or buffer containing 100 @l ATP for 9 min (hatched rectangle), and the released [%I-arechidonic acid was measured. Bach bar is the mtan f SE of triplicatedeterminations. ATP significantly (P < 0.05) increased the release of [‘HI-arachidonicacid.

Stimulating [%#arachidonic acid pretreated FRTL5cellswith100pMATFfor9minincreased(P< 0.05) the release of [3H]-arachidonicacid (Fig. 1). Action of inhibitionof arachidonic acid metabolism on ATP-inducedchanges in [Cd+]t

In control c$ls stimulation with 100 p&l ATF in[CP]i by 503 + 27 nM (n = 13; Fii. 2).

creased

156

CELL CALCIUM

BPB) [123to FRTL-5 cells had no significant effect ,. In these cells stimulation with on resting [Ca2+l* 100 pM ATP increased [Ca2+]iby 600 f 31 nM (n = 6) in a Ca2+-containingbuffer and by 173 + 15 nM (P < 0.05) in a Ca2’ -free buffer (Fig. 2). Pretreatment of the cells with the lipoxygenase inhibitor nordihydroguaiaxeticacid, NDGA, (30 pM for 8-10 min 1221) almost totally abolished the ATP-evoked response in [Ca2+]i(40 Z!Z 16 nM and 8 ? 4 nM. n = 6, P < 0.05, in Ca2+-containingand Ca2+-freebuffer, respectively; Fig. 2). Pretreatment with econazole or 5,8,11,14-eicosatetraynoic acid (ETYA), which are known to inhibit cytochrome P450 pathway enzymes [22 , also attenuated the AT-evoked response in [Caa ‘Ii: 284 + 14 nM and 157 + 21 r&f, respectively (P < 0.05, n = 5-9, Fig. 2) in Ca2+-containingbuffer. In a Ca2’-free buffer, econazole had no effect on the ATP-induced increase in [Ca2+]i(274 + 41 nM, n = 5, Fig. 2). while pretreatment with ETYA decreased the response (41 + 12 nM, n = 6, Fig. 2). However, pretreatment with the cyclooxygenase inhibitor indomethacin (30 pM for 10 min; [22]) had no sisyificant effect on the ATP response in either a Ca -containing or a Ca2+-freebuffer (513 f 20 nM, n = 4, and 275 + 35 nM. n = 4, respectively). A summary of all the results is shown in Figure 3. The effect of NDGA and econazole on the ATP-evoked changes in [Ca2+]iwas dose-dependent (not shown).

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Fura-

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buffer (upper traces), or in nominally 100 ph4 EGTA (lower traces),

with 100 pM ATP.

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right): control cells, after a 5 min incubation

with 30 p.M 4-BPB,

after a 10 min incubation

with 30 @l NDGA,

after a 10 min incubation

with 10 @l econazole.

30 ph4 ETYA, or The horizontal

bar denotes 1 min.

The increase in [Ca2+]iis dependent both on the release of sequestered Ca2+and on influx of extracellular Ca2’ [7,21]. In a Ca2+-freebuffer the increase in [Ca2+]iwas 280 f 12 nM. Addition of 30 p.M of tbe PLA2 inhibitor Cbromophenylacyl bromide (4600

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Fig. 3

Summary

of the effects of compounds

4-BPB

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arachidonic

Fun-2

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loaded cells were treated as in Figure 1, and the effects of 30 pM QBPB. NDGA. ElYA, ewnazole. were compared with the ATP-induced increase in [Ca’+]i in control cells in Ca” containing buffer (filled nctangles) cells.

Ca”-free

buffer containing

0.05 compared

100 ph4 EGTA (hatched rectangles).

with control cells.

or indomethacin or in nominally

Each bar gives the mean f SE of 6-13 separate experiments.

*P <

157

ARACHIDONIC ACID (AA) METABOLISM AND CALCIUM ENTRY

Addition of Ca2’ to cells in Ca2+-free buffer stimulated with ATP resulted in a rapid increase in [Ca2+]i by 184 k 13 nM (n = 8), which then stabilized at 132 f 13 nM above the prestimulatory [Ca2+]i level (Fig 4). In cells pretreated with 30 pM Q-BPB the initial increase in [Ca2’]i was 161 + 21 nM (n = 5) and the plateau level stabilized 113 + 18 nM above prestimulatory level (not shown). When the effects of pretreating the cells with 30 J.IM NDGA, 10 pM econazole or 30 pM ETYA were investigated, addition of Ca2’ increased [Ca2+]i by 32 k 2 nM, 51 zk 16 nM and 41 ?r:8 nM (n = 5-8; P < 0.05 for all the tested compounds; Fig. 4). Surprisingly, indomethacin attenuated the increase in [Ca2+]i (138 It 6 nM and 78 f 9 nM, n = 4, for the initial and the plateau phase of [Ca2+]i, respectively, Fig. 4). ATP v 250 5:

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Fig. 4 Effects of compounds affecting amchidonic acid metabolism on [Ca”]t after addition of Ca2’ to cells stimulated with ATP in FRTL-S cells. Upper trace: Fura- loaded cells were stimulated with 100 @I ATP in nominally Ca”-free buffer containing 100 @I EGTA. After termination of the msponse, Ca2+ (Enal cone 1 mM) was added. Lower figure: After stimulating the cells with 100 pM ATP in nominally Ca2’-free buffer containing 100 pM EGTA, and addition of Ca” (fmal corm 1 mM). the initial increase in [Ca2+]t (filled rectangles) and the elevated steady-state of [Ca’+]t (hatched rectangles) in response to added Ca2+ was measured in control cells, after a 5 min incubation with 30 pM 4-BPB, 30 pM NDGA, 30 @I ETYA. 10 pM econazole, or after a 10 min incubation with 30 @I indomethacin. Each bar gives the. mean f SE of 5-9 separate determinations. *P < 0.05 compared with control cells.

Fig. 5 Inhibitory effect of NDGA and 5,8,11,14-eicosat&aynoic acid on Ca2+entry in FRTL-S cells stimulated with ATP. Fum-2 loaded cells were stimulated with 100 pM ATP in nomhtally Ca2’-free buffer containing 100 pM EGTA. Upper trace. Afta termination of the response the cells were incubated for 5 mitt and then Ca” (final concentration 1 mM) was added. Middle trace. After termination of the response the cells were incubated with NDGA for 5 mm and then Ca2’ (fmal concentmtion I mM) was added. Lower trace. After termination of the reapouae the cells were incubated with 5,8,11,14-eicosatetraynoic acid (ETYA) for 5 min and then Ca2’ (final concentration 1 r&f) was added. The horizontal bar denotes 1 min.

In the next experiments, the cells were stimulated with ATP in a Ca2’-free buffer. After incubat-

ing the cells for 9 miu, Ca2+was added, which increased [Ca2+]iby 153 f 13 nM (n = 6, Pig. 5). If 30 p.M mA or NDGA were added prior to Ca2’ the increase in [Ca2’]i was attenuated (88 f 11 nM, and 112 f 8 uM, respectively, P < 0.05, Fig. 5). Thus, of the inhibitors of the PLA2-arachidonicacid pathway tested, 4-BPB inhibited the release of sequestered Ca2+, whereas NDGA and ETYA inhibited both the release and the entry of Ca2’, aud econazole inhibited entry of Ca2’ only.

158

CEU CALCIUM

500

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ATP v

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Fig. 6 Action of arachidonicacid and ATF on [Ca*‘]i in FRTL-5 cells. Fura- loaded cells wm incubatedin Ca*‘-containingbtier (upper traces) or in nominally Ca”-free buffer containing 100 @I EGTA (lower traces). The cells wem stimulate-dwith 30 pM arachidonic acid (AA, left traces), simultaneously with arachidonic acid and 100 ph4 ATP (AA + ATP, (middle traces), or with ATF (right traces). The horizontalbar denotes 1 min.

Action o arachidonic acid on ATP-inducedchanges 2f in [Ca ]i in FRTL-5 cells

Effects of arachidonic acid permeabilizedFRlLS cells

Addition of 30 @I arachidonic acid to Furaloaded cells rapidly increased [Ca2+]1by 86 zk9 nM (n = 13; Fig. 6). Part of this increase in [Ca2+]lwas dependent on influx of extracellular Ca2’ as the increase was only 53 zk6 nM (n = 10; P < 0.05) in a Ca2+-freebuffer (Fig. 6). The results suggest that arachidonic acid (or a metabolite of arachidonic acid) may release sequestered Ca2’ in FPTL-5 cells and that this release resulted in activation of entry of extracellular Ca2+. Addition of ATP to cells stimulated with arachidonic acid resulted in a very blunted increase in [Ca2+]iin cells in Ca2’ containing buffer (24 f 8 nM, n = 7; P < 0.05). In a Ca2’tree buffer almost no response in [Ca2+]iwas observed (10 f 6 nIvI, n = 7; P < 0.05; Fig. 6). Furthermore, addition of arachidonic acid and ATP simultaueousl increased [Ca2+]1by 286 f 24 nM (n = 6) in a CaXt-containing buffer, and by 125 rt 11 nM (n = 5) in a Ca2’-free buffer (Fig. 6). Both these responses were of lower ma uitude (P < 0.05) compared to the response in [Ca5t ]i obtained with ATP only.

Stimulating permeabilized cells with 10 pM arachidonic acid rapidly increased [Ca2’] (Fig. 7). The cells still responded to 10 p.M lP3 added afkr arachidonic acid (Fig. 7). However, arachidonic acid go

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Fig. 7 Action of arachidonic acid and W on [Ca*‘] in permeabilized FRTL-5 cek Fura- loaded permeabilized cells were: (upperQUCC) stimulated first with 10 @I arachidtic acid (AA) and then with 10 ph4 Ip3, (lower trace) stimulated fmt repeatedlywith 10 @l lF$ and then with 10 pM arachidonicacid (AA). The horizontalbar denotes 1 min.

ARACHIDONIC

ACID (AA) METABOLISM

159

AND CALCIUM ENTRY

released [Ca2+]at least in part from an BYsensitive Ca*+ pool, because when arachidonic acid was added to cells repeatedly stimulated with 10 pM IP3, only a modest change in [Ca*+] was observed (Fig. 7). Affect of arachidonic acid on intracellular pH in FRTLJ cells

Addition of arachidonic acid to FRTLJ cells rapidly acidified the cytosol (Fig. 8). The increase in [Ca2+]iobserved after addition of arachidonic acid may thus in part be due to the acidification of the cytosol, because acidification of the cells with the I?‘/-H+ ionophore nigericin also increased [Ca2+]iin FRTL-5 cells (Fig. 8). However, the effects of nigericin (0.3 pg/ml) on [Ca2+]iand pHi were much slower in onset than that observed with arachidonic acid. Addition of a higher dose of nigericin (1 pg/ml) rapidly acidified the cytosol and increased [Ca2+]i(Fig. 8).

Discussion

The findings in this report suggest that the metabolism of arachidonic acid may be of im rtance in the regulation of agonist-induced Ca2pofiuxes in FRTL-5 cells, and that arachidonic acid per se is mobilizing sequestered Ca*’ in these cells. Our study further suggests that especially non-cycloox-

ygenase metabolites of arachidonic acid appear to be important in maintaining Ca*’ fluxes: preincubation with the lipoxygenase inhibitor NDGA, or the cytochrome P-450 enzyme inhibitors ecouazole and ETYA [22] decreased the ATP-induced changes in [Ca*+]i, while the cyclooxygenase-inhibitor indomethacin had no effects. Non-cyclooxygenase metabolites of arachidonic acid appear to have diverse effects on calcium fluxes in different cell types. These metabolites regulate Ca* entry in response to thrombin 1111and EGF 1121. Furthermore, these metabolites have been suggested to release sequestered Ca2+or to enhance agonist-induced release of Ca2’ in endothelial cells [ill, platelets [23], pituitaty cells [24], and mesengial cells [25]. However, 14,15-cis-epoxyeicosatrienoic acid, a cytochromc P-450 metabolite, inhibited both the thapsigargin and the thrombin-induced entry of Ca2+in platelets [26]. Furthermore, AA per se has been reported to enhance Ca*’ currents in myocytes [8] and in pituitary cells 191and to mobilize sequestered Ca*’ in permeabilixed pancreatic islet cells [27] and to deplete Ca2’ stores in pituitary cells [28]. NDGA, ETYA and CBPB, but not econazole, decreased the ATP-evoked release of sequestered Ca2’ in FRTL5 cells. This observation suggests that some metabolite of arachidonic acid may be important for the release of sequestered Ca2’, although a nonspecific effect of these compounds cannot be excluded. Another possibility is that they interfere

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pg/ml; right traces) and the changes in [Ck*+]t (upper traces)or pHi (lower traces) were measured. in the right trace.

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BCECF-loaded &IS

Ca*~-c.ontaining buffer and stimulated with amchidonic acid (30 pM, AA; lefi traces) or n&ricin Note the different pHicalibration

i

W-

incubated in

(0.3 p&m& Nig; middle traces; or 1 The horizontal bar denotes 1 min.

160

with the production of IP3. Despite substantial effort, we have not been able to measure the ATP-induced production of IP3 on a physiologically relevant time scale. The possible effects of NDGA or ETYA on the IP3 production is thus still unknown. In glomemlar mesangial cells, NDGA pretreatment did decrease the AVP-induced release of sequestered Ca2+,without any effects on the AVP-induced production of IP3 [25]. An important observation was, though, that NDGA and ETYA substantially inhibited Ca2+ entry when added after ATP (see Fig. 5). We also showed that addition of arachidonic acid to FRTL-5 cells released sequestered Ca2’ and stimulated entry of Ca2’. Although arachidonic acid apparently released Ca2’ at least in part from an IP3 pool, some other mechanism of action is probably also present The increase in [Ca2+]lcould be due to release of Ca2’ from the mitochondria [28]. Arachidonic acid is, however, probably not directly affecting the production of IP3 [27]. Another interesting possibility is that the arachidonic acid-induced increase in [Ca2+]lis the result of an acidification of the c osol. Recent investigaIcf tions have indicated that H may compete with Ca2’ for common intracellular buffer sites [29]. The suggestion is also strengthened by the observations that the effect of arachidonic acid was not abolished in a Na+-free buffer (thus excluding an effect mediated via Nat/-H+ exchange combined with Nat/-Ca2’ exchange (data not shown), or in the presence of the Cazt-entry blocker SKF 96365 (data not shown), and by the observation that the Kt/Ht ionophore nigericin, which acidifies the cytosol in FRTLJ cells 1201 also increased [Ca2+]* Studies with FRTL-5 cel& have shown that agonists like ATP and noradrenalin, which are known to release sequestered Ca2+via the IP3 pathway [3-51 and activate Ca2+influx [7, 211 are also potent activators of PLAZ activity and arachidonic acid release [ 1, 21. It is theoretically possible that activation of arachidonic acid production and metabolism may produce a metabolite which is the actual mediator of Ca2+entry in FRTL-5 cells. In P19 839 murine carcinoma cells leukotriene B4, a metabolite of arachidonic acid, has been shown to mediate the EGF-induced Ca2’ entry [12]. However, addition of leukotrienes B4, C4, D4, or E4 did not affect

CJXL CALCIUM

[Ca2+]i per se or the ATP-induced increase in [Ca2+]l(data not shown). Furthermore, activation of arachidonic acid release requires influx of extracellular Ca2’ 113,301, suggesting that arachidonic acid metabolites may participate in maintaining Ca2+ entry rather than in activating Ca2+entry. Furthermore: in FRTLJ cells stimulated with ATP in a Ca2’-free buffer, a Ca2’ entry pathway is activated which remains ‘open’ for long periods after termination of the stimulus [7, 31, 321. Addition of Ca2’ to these cells resulted in a rapid influx of Ca2’ (capacitative Ca2’ entry, see [33]). This influx of Ca2’, initiated several minutes after termination of the stimulus, was shown to be sufftcient to activate arachidonic acid release (manuscript submitted), and was potently attenuated by non-cyclooxygenase inhibitors (the present report). Thus, several different regulatory mechanisms must robably interact to coordinate that gating of the CaR entry pathway. In conclusion, the results in the present study show that the ATP-induced entry of Ca2’ in FRTL5 cells appears to be dependent on non-cyclooxygenase metabolites of arachidonic acid. In addition, arachidonic acid per se may modify agonist-induced release of sequestered Ca2’ at least in part by emptying lP3-sensitiveCa2+pools in these cells. Acknowledgements This study was supportedby the Sip-id Juselius Foundation and the Academy of Finland.

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ARACHIDONIC ACID (AA) METABOLISM AND CALCIUM ENTRY adenosine mceptor enhances alpha- 1-adrenergic receptor-mediated activatiop of phoapholipase C and Ca*’ mobilization jn a prtussir toxin-sensitive manner in FRTL-5 thyroid cells. FEEIS Lett., 248, 145149.

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Received : 21 April 1993 Revised : 12 July 1993 Accepted : 20 July 1993

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