0013-7227/91/1286-2791$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 6 Printed in U.S.A.
Tumor Necrosis Factor-a Increases Release of Arachidonate and Prolactin from Rat Anterior Pituitary Cells KOJI KOIKE,* KENJI HIROTA, MASAHIDE OHMICHI, KOZO KADOWAKI, HIROMASA IKEGAMI, MASAAKI YAMAGUCHI, AKIRA MIYAKE, AND OSAMU TANIZAWA Department of Obstetrics and Gynecology, Osaka University Medical School, 1-1-50 Fukushima, Fukushimaku, Osaka 553, Japan
ABSTRACT. We investigated the effect of tumor necrosis factor-« (TNFa), a product of activated macrophages, on the release of arachidonate from dispersed anterior pituitary cells. Primary cultures of anterior pituitary cells from rats were preincubated with [3H]arachidonate to label their phospholipid-containing components. The cells were then washed and incubated with vehicle or test agents, and PRL release into the medium and [3H]arachidonate cleaved from phospholipid were measured. TNF« significantly increased the release of both PRL and [3H] arachidonate release in a time- and dose-dependent manner. Other cytokines, such as interleukin-lcv, interleukin-1/3, and yinterferon, had no effect on [3H]arachidonate release. To define the role of calcium in TNFa-induced arachidonate release, dis-
I
NFORMATION is rapidly accumulating which supports the existence of a close linkage between the neuroendocrine and immune systems. PRL and GH have recently been shown to stimulate immune functions both in vivo and in vitro (1, 2). Conversely, cytokines, the secretory products of macrophages, monocytes, and lymphocytes, stimulate anterior pituitary hormone release (3-6). Tumor necrosis factor-a (TNFa or cachectin) is a monokine produced by activated macrophages/monocytes which elicits various forms of biological activity. These include stimulation of collagenase activity and prostaglandin E2 (PGE2) production by synovial cells (7), promotion of angiogenesis (8), stimulation of plateletactivating activity in endothelial tissue (9), a cytotoxic effect on tumor cells (10, 11), and stimulation of proliferation of normal fibroblasts (12). TNFa also exerts powerful direct effects on the pituitary gland in vivo (13) and in vitro (14). Recently, we also found that TNFa stimulates the release of anterior pituitary hormones such as LH, FSH, PRL, and ACTH from cultured pituitary cells in vitro Received November 13, 1990. * To whom all correspondence and requests for reprints should be addressed.
persed pituitary cells weire incubated with low calcium medium, which decreased arachidonate release in response to TNFa. TNFa potentiated the release of [3H]arachidonate and PRL promoted by phospholipase-A2 and melittin, and markedly shifted the dose-response curve to the left. Inhibitors of phospholipase-A2, such as p-bromophenacyl bromide and quinacrine, had no effect on TNFa-induced [3H]arachidonate and PRL release. BW755C, an inhibitor of the conversion of arachidonate to its metabolites, decreased TNFa-induced PRL release, while indomethacin, a prostaglandin synthesis inhibitor, had no effect on TNFa-induced PRL release. These data indicate that arachidonate metabolites may be involved in the process of TNFainduced PRL release. (Endocrinology 128: 2791-2798, 1991)
(15). Little is known about the intracellular mechanism of TNFa-induced pituitary hormone release. TNFa receptors have recently been characterized in several cell lines (16,17), and postreceptor events such as promotion of extracellular calcium influx (18), activation of phospholipase-A2 (PLA2), and release and metabolism of arachidonic acid are implicated in other tissues (19-21). Exogenous arachidonate (22, 23), several arachidonate metabolites (22, 24), and pharmacological agents that increase the availability of endogenous arachidonate (25, 26) promote PRL release from anterior pituitary cells. In contrast, pharmacological agents that decrease arachidonate release or arachidonate metabolism decrease basal and secretagogue-induced PRL release (22, 25-27). Thus, the release of arachidonate from anterior pituitary phospholipids may be involved in the stimulus-secretion coupling of PRL release. In the present study we investigate whether TNFa modification of arachidonate release in dispersed anterior pituitary cells is temporally correlated with PRL release.
Materials and Methods Preparation of cultured pituitary cells Normal anterior pituitary cells from female Wistar rats (200250 g) were dispersed enzymatically, as described previously
2791 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:18 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1991 Voll28«No6
TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
2792
(28). The dispersed cells were seeded into Falcon 24-well plates (Oxnard, CA) at a density of 0.5 X 106 viable cells/well and allowed to attach for at least 4 days in a humidified 37 C atmosphere of 5% CO2 and 95% air before an experiment was performed.
were evaporated to dryness under a stream of nitrogen and stored at -20 C until assayed. cAMP assay was performed with the cAMP assay kit, using materials and protocols supplied by Amersham International pic (Amersham, Aylesbury, Buckinghamshire, United Kingdom).
Materials
Statistical analysis
Human recombinant TNFa (rTNFa) was a generous gift from Dainippon Pharmaceutical Co. (Tokyo, Japan). Recombinant human interleukin-la (IL-la), IL-1/3 (Wako Pure Chemical Industries Ltd., Osaka, Japan), 7-interferon (Shionogi Co., Ltd., Osaka, Japan), TNFa, PLA2 from Crotalus adematus (Sigma, St. Louis, MO), quinacrine (Sigma), 3-amino-l(s-trifluoromethylphenyl)2-pyrazoline hydrochloride (BW755c; a gift from Wellcome Research Laboratories, Bechenham, United Kingdom), and isobutylamethylxanthine (IBMX; Sigma) were dissolved directly in RPMI-1640 medium. Melittin (Sigma), p-bromophenacyl bromide (BPB; Sigma), and indomethacin (Sigma) were dissolved in 100% dimethylsulfoxide (Sigma) and then diluted with RPMI-1640 medium to the desired concentration. The maximum concentration of dimethylsulfoxide in the culture medium was 0.1%, and this did not affect PRL release from pituitary cells.
Data are expressed as nanograms of PRL per well, femtomoles of cAMP per well, or disintegrations per min of [3H] arachidonate released/well. Each experiment was repeated two or more times to ascertain the reliability of the results. In this study each point is presented as the mean ± SEM. All data were subjected to analysis of variance, and differences between groups were assessed using the multiple range test of Duncan. P < 0.05 was considered to represent a significant statistical difference.
Arachidonate release On the day of an experiment, the cells were preincubated for 2 h with [3H]arachidonate (0.25 AiCi/ml; 100 Ci/mmol; New England Nuclear, Boston, MA) to incorporate the [3H]arachidonate into esterified lipids. The cells were then washed four times with 1.5 ml RPMI-1640 medium (Handai Biken, Osaka, Japan) containing 0.25% BSA (Sigma) without [3H]arachidonate. In selected experiments the cells were incubated for 30 min with selected agents (BW755c, quinacrine, BPB, low calcium medium, and indomethacin) in the final wash buffer to allow these agents to exert their effects before the cells were exposed to TNFa. The cells were then incubated for 30 min in vehicle, TNFa, and/or test agents. A 100-/xl aliquot of medium was removed for PRL RIA, and 900 n\ medium were extracted with ethyl acetate and analyzed for arachidonate by TLC (29). The solvent system employed to separate arachidonate from the PGs was isooctane-ethyl acetate-water-acetic acid (5:11:10:2). Typical Rf values were 0.85, 0.6, and 0.33 for arachidonate, PGE2, and PGF2n, respectively.
Results 3
Effect of TNFa on I H]arachidonate and PRL release The dose responses of TNFa-induced release of both [3H]arachidonate and PRL are illustrated in Fig. 1. Incubation for 30 min with TNFa caused dose-dependent stimulation of [3H]arachidonate and PRL release. As shown in Fig. 2, 3 X 10"9 M TNFa significantly (P < 0.01) increased the release of both [3H]arachidonate and PRL within 30 sec of incubation, and this effect continued throughout the next 30 min of incubation. IL-1/3 (0.2-2000 pg/ml) had no effect on [3H] arachidonate release, while IL-1/3 caused an additive effect on
-1600
800-
•I 600-
1
-1400 I-1200 i 6
400-
1000
=
800
|
PRL RIA PRL concentrations were determined with the protocol and reagents provided by the NIDDK Rat Pituitary Hormone Distribution Program. The results are expressed in terms of NIDDK rat PRL RP-2. The intra- and interassay coefficients of variance were less than 6% and 10%, respectively. Assay for pituitary cAMP The cells were preincubated in serum-free medium with 0.25 mM IBMX to inhibit phosphodiesterase activity. The medium was then removed and replaced with fresh medium containing IBMX and TNFa for 30 min. At the termination of the study, the medium was quickly removed, and the cyclic nucleotides were extracted by the rapid addition of 65% ethanol. Samples
600 200-
en
400 200
Vehicle
3 x 10" 3x 10i: 3 x 10s 3 x 10( 3xiO"! TNF-a (M)
FIG. 1. Effects of TNFa on [3H]arachidonate and PRL release from cultured pituitary cells. [3H]Arachidonate and PRL release were significantly (P < 0.01) increased in a concentration-dependent manner by 3 x 1011 M or more of TNFa. Incubation time was 30 min. Each point represents the mean ± SE of results for six wells.
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TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
Effect of TNFa on PLA2-induced release o arachidonate and PRL
600-
As previously reported, PLA2 significantly increased PRL (22) and [3H]arachidonate release in a concentration-dependent manner (Fig. 4). TNFa at a concentration of 1.5 X 10"8 M caused a cumulative effect on the release of [3H] arachidonate promoted by PLA2 and markedly shifted the curve for arachidonate release to the left. Similarly, the effect of TNFa (1.5 x 10 8 M) on PLA2 (100 mU/ml)-induced PRL release was additive (Fig. 4).
500 J
400-
1 o
300
2793
g> 200 100-
Effect of TNFa on melittin-induced release of ^H] arachidonate and PRL
0 600
Melittin, a PLA2 activator, is known to stimulate the release of both [3H]arachidonate and PRL (23, 26). TNFa (1.5 X 10"8 M) enhanced this effect and caused a shift in the concentration-response curve for melittininduced release of both [3H]arachidonate and PRL (Fig. 5). The EC5o of arachidonate release was 0.5 yuM with melittin treatment and 0.82 /xM with melittin and 1.5 x 10 8 M TNFa treatment. The EC50 of PRL release was 0.9 yuM with melittin and 1.1 /xM with melittin and TNFa treatment.
500 4001
300 -
PC
200100-
10
20
30
Time (min)
FIG. 2. Time courses of effects of TNFa (3 x 109 M) on [3H]arachidonate release (bottom) and PRL release (top) from cultured pituitary cells. TNFa was added at time zero. TNFa at all time points significantly (P < 0.01) increased [3H]arachidonate and PRL releases. Each point represents the mean ± SE of results for four wells.
PRL release stimulated by TNFa. Similarly, other cytokines, such as IL-la (2-2000 pg/ml) and 7-interferon (1-1000 U/ml), had no effect on basal and TNFa-stimulated [3H]arachidonate release, while these cytokines also caused an additive effect on TNFa-induced PRL release (Table 1). TNFa, even at 3 x 10 7 M, had no effect on cell viability, as determined by trypan blue exclusion, and the cells remained adherent to the wells. Effect of low calcium medium on TNFoc-induced arachidonate release Pituitary cells were preincubated for 30 min in low calcium medium before the start of the 30-min incubation with TNFa (3 X 109-3 X 10"8 M). The low calcium medium had little effect on basal [3H]arachidonate release, but significantly (P < 0.01) decreased TNFainduced arachidonate release (Fig. 3). The low calcium medium also decreased (P < 0.01) TNFa-induced PRL release (data not shown).
Effect of inhibition of PLA2 activity on PLA2-, melittin-, and TNFa-induced release of I3H]arachidonate and PRL Anterior pituitary cells were preincubated for 30 min with BPB, an inhibitor of PLA2 (30), and then incubated for 30 min with vehicle, PLA2, melittin, and/or BPB. BPB (0.5 and 5 MM) blocked the induction of [3H]arachidonate release by PLA2 (200 mU/ml; P < 0.01) and 1 ixM melittin (P < 0.01; Fig. 6). BPB (5 ^M) also abolished PLA2- and melittin-induced PRL release (P < 0.01). On the other hand, BPB (0.5 and 5 ^M) had no effect on TNFa (1.5 x 10"8 M)-induced [3H]arachidonate or PRL release (Fig. 7). Furthermore, pituitary cells were preincubated for 30 min with quinacrine (10 and 50 /xM), another inhibitor of PLA2 (31), and then incubated for 30 min with vehicle, TNFa (1.5 X 10'8 M) and/or quinacrine. Quinacrine also had no effect on basal and TNFainduced [3H]arachidonate or PRL release (Fig. 8). Effects of BW755c and indomethacin on TNFa-induced PRL release Pituitary cells were exposed to BW755C or indomethacin for 30 min before the start of the final 30-min incubation period. BW755c (250 IXM), an inhibitor of both the cyclooxygenase and lipoxygenase pathways of arachidonate metabolism (31), abolished TNFa (30 nM)induced PRL release. In contrast, 50 juM indomethacin, a potent inhibitor of only the cyclooxygenase pathway
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TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
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Endo • 1991 Voll28«No6
TABLE 1. Effects of IL-la, IL-1/3, and 7-interferon on TNFa (1.5 X 10 8 M)-induced [3H]arachidonate and PRL release from cultured pituitary cells
Treatment
[3H]Arachidonate (dpm/well)
Cone.
Vehicle
PRL (ng/well)
-TNFa
-l-TNFa
-TNFa
+TNFa
278 ± 21
492 ± 25°
198 ± 16
393 ± 16a 569 ± 526 ± 563 ± 537 ±
17 12 15 18°
30*'c 79* 27*'c 43*'c
IL-la
2 pg/ml 20 pg/ml 200 pg/ml 2000 pg/ml
272 ± 20 260 ± 9 285 ± 20 304 ± 13
523 ± 26* 504 ± 18* 501 ± 33* 522 ± 34*
247 ± 216 ± 230 ± 294 ±
IL-1/3
0.2 pg/ml 2 pg/ml 20 pg/ml 200 pg/ml 2000 pg/ml
320 ± 312 ± 279 ± 356 ± 306 ±
14 20 11 16 23
487 ± 42* 516 ± 28* 498 ± 24* 474 ± 30* 507 ± 45*
183 ± 23 244 ± 41 296 ± 12° 268 ± 14° 321 ± 14°
511 ± 49* 477 ± 41* 504 ± 25*'d 553 ± 64* 643 ± 23*lC
7-Interferon
lU/ml 10 U/ml 100 U/ml 1000 U/ml
300 ± 295 ± 308 ± 284 ±
17 13 28 10
432 ± 28* 425 ± 23* 411 ± 42* 454 ± 17*
179 ± 24 176 ± 41 173 ± 12 326 ± 19°
446 ± 45* 405 ± 47* 427 ± 21* 547 ± 23*-c
Incubation time was 30 min. Each point represents the mean ± SE of results for four wells. P < 0.01 vs. vehicle. *P < 0.01 vs. corresponding concentration of IL-la, IL-1/3, or 7-interferon. c P < 0.01 us. TNFa alone.
0
(31), had no effect on TNFa-induced PRL release (Fig. 9). Effect of TNFa on intracellular cAMP accumulation Thirty-minute incubation with TNFa (3 X 10"n-3 X 10"7 M) had no effect on pituitary cAMP levels (data not shown). Discussion This is the first report to demonstrate that TNFa increases the release of arachidonate from rat anterior pituitary cells. In the present study we also demonstrated that TNFa potentiated PLA2- and melittin-induced arachidonate release, and that inhibition of lipoxygenase pathways abolished TNFa-induced PRL release, while inhibitors of PLA2 had no effect on TNFa-induced arachidonate or PRL release. Recently, the amino acid sequence of the TNFa receptor was determined from a cDNA clone (32), and such postreceptor events of TNFa as the activation of PLA2 and the release and metabolism of arachidonate have been implicated in other tissues (19-21). The data in this study support the hypothesis that arachidonate metabolism may be related to the process of TNFa-induced PRL release. Specifically, it was found that the release of arachidonate occurred within 30 sec of TNFa stimulation, the earliest time measured, and that the amount of arachi-
donate release correlated with the amount of PRL release in the time-course and dose-response experiments. Since other cytokines, such as IL-la, IL-1/3, IL-6, (our unpublished observations), and 7-interferon failed to stimulate arachidonate release, these actions may be specific to TNFa. It has been reported that IL-1 increases arachidonate metabolites in a variety of cells (33, 34) and also that IL1 and 7-interferon are synergistic with TNFa in their biological effects (34, 35). It is noteworthy that none of these cytokines had any effect on TNFa-induced arachidonate release, but potentiated the TNFa-induced release of PRL. These results suggest that the mechanism of action of TNFa on PRL release is different from that of other cytokines. It is of interest that a low calcium medium greatly inhibited TNFa-induced arachidonate and PRL release. We also observed that calcium channel blockers, such as cobalt and verapamil, decreased basal and TNFa-induced PRL release (submitted for publication). These results may imply that extracellular calcium is necessary for TNFa-induced arachidonate and PRL release, but it must be remembered that calcium and arachidonate may interact at several levels during the PRL secretion process. For example, 5-hydroxyeicosatetraenoic acid stimulated PRL release from rat anterior pituitary cells appears to be calcium dependent (24), and arachidonate has been demonstrated to mobilize intracellular calcium in GH3 cells (36). Therefore, calcium and arachidonate
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TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL 2mM CaJ
800-
2795
4000
TNF-a (1.5X1O-'3M)>
1500-
3000
600T3
.9 1000
o 2000-
3 o -a
X''
500
1000-
400-
i E a.
Low C a 2 +
•o
Vehicle O.I
O.5
1
Melittin (vM)
Vehicle O.I
0.5 1 Melittin ( M M )
FIG. 5. Effects of TNFa on melittin-induced [3H]arachidonate release (left) and PRL release (right) from cultured anterior pituitary cells. TNFa significantly (P < 0.01) enhanced both [3H]arachidonate and PRL release induced by melittin. Incubation time was 30 min. Each point represents the mean ± SE of results for four wells.
200-
Vehicle
3X10'9
1,5 x 10'8 3 x 10'8
TNF-a (M) FIG. 3. Effect of low calcium medium on TNFa-induced [3H]arachidonate release from cultured anterior pituitary cells. Low calcium medium decreased (P < 0.01) [3H]arachidonate release induced by TNFa (3 X lO9-3 x 10"8 M). Cells were preincubated for 30 min in low calcium medium before start of the 30-min incubation. Each point represents the mean ± SE of results for four wells.
700-
Vehicle PLA2 (mU/ml)
FIG. 4. Effects of TNFa (1.5 x 10 8 M) on PLA2-induced [3H]arachidonate release (left) and PRL release (right) from cultured anterior pituitary cells. TNFa significantly potentiated PLA2-induced [3H]arachidonate and PRL release. Incubation time was 30 min. Each point represents the mean ± SE of results for four wells. *, P < 0.05 us. TNFa; **, P < 0.01 vs. TNFa; t, P < 0.05 us. vehicle; ft, P < 0.01 us. vehicle.
metabolisms may interact in a complex manner at multiple sites in the PRL release process. TNFa has been reported to induce PLA2 activity and increase the synthesis of arachidonate metabolites in
many cell types (20, 21, 33, 34). It has also been reported that TNFa induces PLA activity and synthesis of a PLA2-activating protein in endothelial cells (19). Therefore, we have speculated that the activation of PLA2 is involved in TNFa-induced arachidonate release from rat anterior pituitary cells. As previously reported (22, 26), PLA2 and melittin, stimulators of endogenous arachidonate release, increase the release of both arachidonate and PRL, and such inhibitors of PLA2 as BPB and quinacrine blocked release. However, these inhibitors had no effect on TNFa-induced arachidonate or PRL release, indicating that PLA2 was not involved in this process. Furthermore, we found additive effects of TNFa on PLA2- and melittin-induced arachidonate and PRL release. These data also suggest that the mechanism of TNFa induction of arachidonate release is different from that of PLA2 and melittin induction. The enzyme responsible for the release of arachidonate from cellular phospholipids has not been determined conclusively. It has been suggested that free arachidonate is released by the sequential action of phosphoinositide-specific PLC, followed by diacylglycerol lipase and/or direct deacylation of phospholipids by PLA2. Earlier reports (19, 20) have suggested that PLA2 is largely responsible for TNFa-induced release of arachidonate from several cell types, although it has been reported that TNFa does not increase PLA2 release from rabbit chondrocytes (37) and that PLA2 activity and arachidonate metabolite release are not coordinately regulated in rabbit chondrocytes (38). Therefore, it is possible that TNFa-induced arachidonate release may be mediated by some other mechanisms, such as a diacylglycerol-specific lipase (39) or an acyl coenzyme-A
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2796
TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
Endo• 1991 Vol 128 • No 6
900-
|
FIG. 6. Effects of BPB on PLA2- and melittin-induced [3H]arachidonate release (left) and PRL release (right) from cultured anterior pituitary cells. BPB significantly (P < 0.01) blocked both PLA2- and melittin-induced [3H]arachidonate and PRL release. Cells were preincubated for 30 min with BPB and then incubated for 30 min with vehicle, PLA2, melittin, and/or BPB. Each point represents the mean ± SE of results for four wells.
| Vehicle PLA2 (200mU/ml)
800-
melittin _
I
700-
is
600
a
500
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Vol. 128, No. 6 Printed in U.S.A.
Tumor Necrosis Factor-a Increases Release of Arachidonate and Prolactin from Rat Anterior Pituitary Cells KOJI KOIKE,* KENJI HIROTA, MASAHIDE OHMICHI, KOZO KADOWAKI, HIROMASA IKEGAMI, MASAAKI YAMAGUCHI, AKIRA MIYAKE, AND OSAMU TANIZAWA Department of Obstetrics and Gynecology, Osaka University Medical School, 1-1-50 Fukushima, Fukushimaku, Osaka 553, Japan
ABSTRACT. We investigated the effect of tumor necrosis factor-« (TNFa), a product of activated macrophages, on the release of arachidonate from dispersed anterior pituitary cells. Primary cultures of anterior pituitary cells from rats were preincubated with [3H]arachidonate to label their phospholipid-containing components. The cells were then washed and incubated with vehicle or test agents, and PRL release into the medium and [3H]arachidonate cleaved from phospholipid were measured. TNF« significantly increased the release of both PRL and [3H] arachidonate release in a time- and dose-dependent manner. Other cytokines, such as interleukin-lcv, interleukin-1/3, and yinterferon, had no effect on [3H]arachidonate release. To define the role of calcium in TNFa-induced arachidonate release, dis-
I
NFORMATION is rapidly accumulating which supports the existence of a close linkage between the neuroendocrine and immune systems. PRL and GH have recently been shown to stimulate immune functions both in vivo and in vitro (1, 2). Conversely, cytokines, the secretory products of macrophages, monocytes, and lymphocytes, stimulate anterior pituitary hormone release (3-6). Tumor necrosis factor-a (TNFa or cachectin) is a monokine produced by activated macrophages/monocytes which elicits various forms of biological activity. These include stimulation of collagenase activity and prostaglandin E2 (PGE2) production by synovial cells (7), promotion of angiogenesis (8), stimulation of plateletactivating activity in endothelial tissue (9), a cytotoxic effect on tumor cells (10, 11), and stimulation of proliferation of normal fibroblasts (12). TNFa also exerts powerful direct effects on the pituitary gland in vivo (13) and in vitro (14). Recently, we also found that TNFa stimulates the release of anterior pituitary hormones such as LH, FSH, PRL, and ACTH from cultured pituitary cells in vitro Received November 13, 1990. * To whom all correspondence and requests for reprints should be addressed.
persed pituitary cells weire incubated with low calcium medium, which decreased arachidonate release in response to TNFa. TNFa potentiated the release of [3H]arachidonate and PRL promoted by phospholipase-A2 and melittin, and markedly shifted the dose-response curve to the left. Inhibitors of phospholipase-A2, such as p-bromophenacyl bromide and quinacrine, had no effect on TNFa-induced [3H]arachidonate and PRL release. BW755C, an inhibitor of the conversion of arachidonate to its metabolites, decreased TNFa-induced PRL release, while indomethacin, a prostaglandin synthesis inhibitor, had no effect on TNFa-induced PRL release. These data indicate that arachidonate metabolites may be involved in the process of TNFainduced PRL release. (Endocrinology 128: 2791-2798, 1991)
(15). Little is known about the intracellular mechanism of TNFa-induced pituitary hormone release. TNFa receptors have recently been characterized in several cell lines (16,17), and postreceptor events such as promotion of extracellular calcium influx (18), activation of phospholipase-A2 (PLA2), and release and metabolism of arachidonic acid are implicated in other tissues (19-21). Exogenous arachidonate (22, 23), several arachidonate metabolites (22, 24), and pharmacological agents that increase the availability of endogenous arachidonate (25, 26) promote PRL release from anterior pituitary cells. In contrast, pharmacological agents that decrease arachidonate release or arachidonate metabolism decrease basal and secretagogue-induced PRL release (22, 25-27). Thus, the release of arachidonate from anterior pituitary phospholipids may be involved in the stimulus-secretion coupling of PRL release. In the present study we investigate whether TNFa modification of arachidonate release in dispersed anterior pituitary cells is temporally correlated with PRL release.
Materials and Methods Preparation of cultured pituitary cells Normal anterior pituitary cells from female Wistar rats (200250 g) were dispersed enzymatically, as described previously
2791 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 16:18 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1991 Voll28«No6
TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
2792
(28). The dispersed cells were seeded into Falcon 24-well plates (Oxnard, CA) at a density of 0.5 X 106 viable cells/well and allowed to attach for at least 4 days in a humidified 37 C atmosphere of 5% CO2 and 95% air before an experiment was performed.
were evaporated to dryness under a stream of nitrogen and stored at -20 C until assayed. cAMP assay was performed with the cAMP assay kit, using materials and protocols supplied by Amersham International pic (Amersham, Aylesbury, Buckinghamshire, United Kingdom).
Materials
Statistical analysis
Human recombinant TNFa (rTNFa) was a generous gift from Dainippon Pharmaceutical Co. (Tokyo, Japan). Recombinant human interleukin-la (IL-la), IL-1/3 (Wako Pure Chemical Industries Ltd., Osaka, Japan), 7-interferon (Shionogi Co., Ltd., Osaka, Japan), TNFa, PLA2 from Crotalus adematus (Sigma, St. Louis, MO), quinacrine (Sigma), 3-amino-l(s-trifluoromethylphenyl)2-pyrazoline hydrochloride (BW755c; a gift from Wellcome Research Laboratories, Bechenham, United Kingdom), and isobutylamethylxanthine (IBMX; Sigma) were dissolved directly in RPMI-1640 medium. Melittin (Sigma), p-bromophenacyl bromide (BPB; Sigma), and indomethacin (Sigma) were dissolved in 100% dimethylsulfoxide (Sigma) and then diluted with RPMI-1640 medium to the desired concentration. The maximum concentration of dimethylsulfoxide in the culture medium was 0.1%, and this did not affect PRL release from pituitary cells.
Data are expressed as nanograms of PRL per well, femtomoles of cAMP per well, or disintegrations per min of [3H] arachidonate released/well. Each experiment was repeated two or more times to ascertain the reliability of the results. In this study each point is presented as the mean ± SEM. All data were subjected to analysis of variance, and differences between groups were assessed using the multiple range test of Duncan. P < 0.05 was considered to represent a significant statistical difference.
Arachidonate release On the day of an experiment, the cells were preincubated for 2 h with [3H]arachidonate (0.25 AiCi/ml; 100 Ci/mmol; New England Nuclear, Boston, MA) to incorporate the [3H]arachidonate into esterified lipids. The cells were then washed four times with 1.5 ml RPMI-1640 medium (Handai Biken, Osaka, Japan) containing 0.25% BSA (Sigma) without [3H]arachidonate. In selected experiments the cells were incubated for 30 min with selected agents (BW755c, quinacrine, BPB, low calcium medium, and indomethacin) in the final wash buffer to allow these agents to exert their effects before the cells were exposed to TNFa. The cells were then incubated for 30 min in vehicle, TNFa, and/or test agents. A 100-/xl aliquot of medium was removed for PRL RIA, and 900 n\ medium were extracted with ethyl acetate and analyzed for arachidonate by TLC (29). The solvent system employed to separate arachidonate from the PGs was isooctane-ethyl acetate-water-acetic acid (5:11:10:2). Typical Rf values were 0.85, 0.6, and 0.33 for arachidonate, PGE2, and PGF2n, respectively.
Results 3
Effect of TNFa on I H]arachidonate and PRL release The dose responses of TNFa-induced release of both [3H]arachidonate and PRL are illustrated in Fig. 1. Incubation for 30 min with TNFa caused dose-dependent stimulation of [3H]arachidonate and PRL release. As shown in Fig. 2, 3 X 10"9 M TNFa significantly (P < 0.01) increased the release of both [3H]arachidonate and PRL within 30 sec of incubation, and this effect continued throughout the next 30 min of incubation. IL-1/3 (0.2-2000 pg/ml) had no effect on [3H] arachidonate release, while IL-1/3 caused an additive effect on
-1600
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-1400 I-1200 i 6
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PRL RIA PRL concentrations were determined with the protocol and reagents provided by the NIDDK Rat Pituitary Hormone Distribution Program. The results are expressed in terms of NIDDK rat PRL RP-2. The intra- and interassay coefficients of variance were less than 6% and 10%, respectively. Assay for pituitary cAMP The cells were preincubated in serum-free medium with 0.25 mM IBMX to inhibit phosphodiesterase activity. The medium was then removed and replaced with fresh medium containing IBMX and TNFa for 30 min. At the termination of the study, the medium was quickly removed, and the cyclic nucleotides were extracted by the rapid addition of 65% ethanol. Samples
600 200-
en
400 200
Vehicle
3 x 10" 3x 10i: 3 x 10s 3 x 10( 3xiO"! TNF-a (M)
FIG. 1. Effects of TNFa on [3H]arachidonate and PRL release from cultured pituitary cells. [3H]Arachidonate and PRL release were significantly (P < 0.01) increased in a concentration-dependent manner by 3 x 1011 M or more of TNFa. Incubation time was 30 min. Each point represents the mean ± SE of results for six wells.
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TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
Effect of TNFa on PLA2-induced release o arachidonate and PRL
600-
As previously reported, PLA2 significantly increased PRL (22) and [3H]arachidonate release in a concentration-dependent manner (Fig. 4). TNFa at a concentration of 1.5 X 10"8 M caused a cumulative effect on the release of [3H] arachidonate promoted by PLA2 and markedly shifted the curve for arachidonate release to the left. Similarly, the effect of TNFa (1.5 x 10 8 M) on PLA2 (100 mU/ml)-induced PRL release was additive (Fig. 4).
500 J
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1 o
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Effect of TNFa on melittin-induced release of ^H] arachidonate and PRL
0 600
Melittin, a PLA2 activator, is known to stimulate the release of both [3H]arachidonate and PRL (23, 26). TNFa (1.5 X 10"8 M) enhanced this effect and caused a shift in the concentration-response curve for melittininduced release of both [3H]arachidonate and PRL (Fig. 5). The EC5o of arachidonate release was 0.5 yuM with melittin treatment and 0.82 /xM with melittin and 1.5 x 10 8 M TNFa treatment. The EC50 of PRL release was 0.9 yuM with melittin and 1.1 /xM with melittin and TNFa treatment.
500 4001
300 -
PC
200100-
10
20
30
Time (min)
FIG. 2. Time courses of effects of TNFa (3 x 109 M) on [3H]arachidonate release (bottom) and PRL release (top) from cultured pituitary cells. TNFa was added at time zero. TNFa at all time points significantly (P < 0.01) increased [3H]arachidonate and PRL releases. Each point represents the mean ± SE of results for four wells.
PRL release stimulated by TNFa. Similarly, other cytokines, such as IL-la (2-2000 pg/ml) and 7-interferon (1-1000 U/ml), had no effect on basal and TNFa-stimulated [3H]arachidonate release, while these cytokines also caused an additive effect on TNFa-induced PRL release (Table 1). TNFa, even at 3 x 10 7 M, had no effect on cell viability, as determined by trypan blue exclusion, and the cells remained adherent to the wells. Effect of low calcium medium on TNFoc-induced arachidonate release Pituitary cells were preincubated for 30 min in low calcium medium before the start of the 30-min incubation with TNFa (3 X 109-3 X 10"8 M). The low calcium medium had little effect on basal [3H]arachidonate release, but significantly (P < 0.01) decreased TNFainduced arachidonate release (Fig. 3). The low calcium medium also decreased (P < 0.01) TNFa-induced PRL release (data not shown).
Effect of inhibition of PLA2 activity on PLA2-, melittin-, and TNFa-induced release of I3H]arachidonate and PRL Anterior pituitary cells were preincubated for 30 min with BPB, an inhibitor of PLA2 (30), and then incubated for 30 min with vehicle, PLA2, melittin, and/or BPB. BPB (0.5 and 5 MM) blocked the induction of [3H]arachidonate release by PLA2 (200 mU/ml; P < 0.01) and 1 ixM melittin (P < 0.01; Fig. 6). BPB (5 ^M) also abolished PLA2- and melittin-induced PRL release (P < 0.01). On the other hand, BPB (0.5 and 5 ^M) had no effect on TNFa (1.5 x 10"8 M)-induced [3H]arachidonate or PRL release (Fig. 7). Furthermore, pituitary cells were preincubated for 30 min with quinacrine (10 and 50 /xM), another inhibitor of PLA2 (31), and then incubated for 30 min with vehicle, TNFa (1.5 X 10'8 M) and/or quinacrine. Quinacrine also had no effect on basal and TNFainduced [3H]arachidonate or PRL release (Fig. 8). Effects of BW755c and indomethacin on TNFa-induced PRL release Pituitary cells were exposed to BW755C or indomethacin for 30 min before the start of the final 30-min incubation period. BW755c (250 IXM), an inhibitor of both the cyclooxygenase and lipoxygenase pathways of arachidonate metabolism (31), abolished TNFa (30 nM)induced PRL release. In contrast, 50 juM indomethacin, a potent inhibitor of only the cyclooxygenase pathway
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TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
2794
Endo • 1991 Voll28«No6
TABLE 1. Effects of IL-la, IL-1/3, and 7-interferon on TNFa (1.5 X 10 8 M)-induced [3H]arachidonate and PRL release from cultured pituitary cells
Treatment
[3H]Arachidonate (dpm/well)
Cone.
Vehicle
PRL (ng/well)
-TNFa
-l-TNFa
-TNFa
+TNFa
278 ± 21
492 ± 25°
198 ± 16
393 ± 16a 569 ± 526 ± 563 ± 537 ±
17 12 15 18°
30*'c 79* 27*'c 43*'c
IL-la
2 pg/ml 20 pg/ml 200 pg/ml 2000 pg/ml
272 ± 20 260 ± 9 285 ± 20 304 ± 13
523 ± 26* 504 ± 18* 501 ± 33* 522 ± 34*
247 ± 216 ± 230 ± 294 ±
IL-1/3
0.2 pg/ml 2 pg/ml 20 pg/ml 200 pg/ml 2000 pg/ml
320 ± 312 ± 279 ± 356 ± 306 ±
14 20 11 16 23
487 ± 42* 516 ± 28* 498 ± 24* 474 ± 30* 507 ± 45*
183 ± 23 244 ± 41 296 ± 12° 268 ± 14° 321 ± 14°
511 ± 49* 477 ± 41* 504 ± 25*'d 553 ± 64* 643 ± 23*lC
7-Interferon
lU/ml 10 U/ml 100 U/ml 1000 U/ml
300 ± 295 ± 308 ± 284 ±
17 13 28 10
432 ± 28* 425 ± 23* 411 ± 42* 454 ± 17*
179 ± 24 176 ± 41 173 ± 12 326 ± 19°
446 ± 45* 405 ± 47* 427 ± 21* 547 ± 23*-c
Incubation time was 30 min. Each point represents the mean ± SE of results for four wells. P < 0.01 vs. vehicle. *P < 0.01 vs. corresponding concentration of IL-la, IL-1/3, or 7-interferon. c P < 0.01 us. TNFa alone.
0
(31), had no effect on TNFa-induced PRL release (Fig. 9). Effect of TNFa on intracellular cAMP accumulation Thirty-minute incubation with TNFa (3 X 10"n-3 X 10"7 M) had no effect on pituitary cAMP levels (data not shown). Discussion This is the first report to demonstrate that TNFa increases the release of arachidonate from rat anterior pituitary cells. In the present study we also demonstrated that TNFa potentiated PLA2- and melittin-induced arachidonate release, and that inhibition of lipoxygenase pathways abolished TNFa-induced PRL release, while inhibitors of PLA2 had no effect on TNFa-induced arachidonate or PRL release. Recently, the amino acid sequence of the TNFa receptor was determined from a cDNA clone (32), and such postreceptor events of TNFa as the activation of PLA2 and the release and metabolism of arachidonate have been implicated in other tissues (19-21). The data in this study support the hypothesis that arachidonate metabolism may be related to the process of TNFa-induced PRL release. Specifically, it was found that the release of arachidonate occurred within 30 sec of TNFa stimulation, the earliest time measured, and that the amount of arachi-
donate release correlated with the amount of PRL release in the time-course and dose-response experiments. Since other cytokines, such as IL-la, IL-1/3, IL-6, (our unpublished observations), and 7-interferon failed to stimulate arachidonate release, these actions may be specific to TNFa. It has been reported that IL-1 increases arachidonate metabolites in a variety of cells (33, 34) and also that IL1 and 7-interferon are synergistic with TNFa in their biological effects (34, 35). It is noteworthy that none of these cytokines had any effect on TNFa-induced arachidonate release, but potentiated the TNFa-induced release of PRL. These results suggest that the mechanism of action of TNFa on PRL release is different from that of other cytokines. It is of interest that a low calcium medium greatly inhibited TNFa-induced arachidonate and PRL release. We also observed that calcium channel blockers, such as cobalt and verapamil, decreased basal and TNFa-induced PRL release (submitted for publication). These results may imply that extracellular calcium is necessary for TNFa-induced arachidonate and PRL release, but it must be remembered that calcium and arachidonate may interact at several levels during the PRL secretion process. For example, 5-hydroxyeicosatetraenoic acid stimulated PRL release from rat anterior pituitary cells appears to be calcium dependent (24), and arachidonate has been demonstrated to mobilize intracellular calcium in GH3 cells (36). Therefore, calcium and arachidonate
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TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL 2mM CaJ
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TNF-a (1.5X1O-'3M)>
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o 2000-
3 o -a
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i E a.
Low C a 2 +
•o
Vehicle O.I
O.5
1
Melittin (vM)
Vehicle O.I
0.5 1 Melittin ( M M )
FIG. 5. Effects of TNFa on melittin-induced [3H]arachidonate release (left) and PRL release (right) from cultured anterior pituitary cells. TNFa significantly (P < 0.01) enhanced both [3H]arachidonate and PRL release induced by melittin. Incubation time was 30 min. Each point represents the mean ± SE of results for four wells.
200-
Vehicle
3X10'9
1,5 x 10'8 3 x 10'8
TNF-a (M) FIG. 3. Effect of low calcium medium on TNFa-induced [3H]arachidonate release from cultured anterior pituitary cells. Low calcium medium decreased (P < 0.01) [3H]arachidonate release induced by TNFa (3 X lO9-3 x 10"8 M). Cells were preincubated for 30 min in low calcium medium before start of the 30-min incubation. Each point represents the mean ± SE of results for four wells.
700-
Vehicle PLA2 (mU/ml)
FIG. 4. Effects of TNFa (1.5 x 10 8 M) on PLA2-induced [3H]arachidonate release (left) and PRL release (right) from cultured anterior pituitary cells. TNFa significantly potentiated PLA2-induced [3H]arachidonate and PRL release. Incubation time was 30 min. Each point represents the mean ± SE of results for four wells. *, P < 0.05 us. TNFa; **, P < 0.01 vs. TNFa; t, P < 0.05 us. vehicle; ft, P < 0.01 us. vehicle.
metabolisms may interact in a complex manner at multiple sites in the PRL release process. TNFa has been reported to induce PLA2 activity and increase the synthesis of arachidonate metabolites in
many cell types (20, 21, 33, 34). It has also been reported that TNFa induces PLA activity and synthesis of a PLA2-activating protein in endothelial cells (19). Therefore, we have speculated that the activation of PLA2 is involved in TNFa-induced arachidonate release from rat anterior pituitary cells. As previously reported (22, 26), PLA2 and melittin, stimulators of endogenous arachidonate release, increase the release of both arachidonate and PRL, and such inhibitors of PLA2 as BPB and quinacrine blocked release. However, these inhibitors had no effect on TNFa-induced arachidonate or PRL release, indicating that PLA2 was not involved in this process. Furthermore, we found additive effects of TNFa on PLA2- and melittin-induced arachidonate and PRL release. These data also suggest that the mechanism of TNFa induction of arachidonate release is different from that of PLA2 and melittin induction. The enzyme responsible for the release of arachidonate from cellular phospholipids has not been determined conclusively. It has been suggested that free arachidonate is released by the sequential action of phosphoinositide-specific PLC, followed by diacylglycerol lipase and/or direct deacylation of phospholipids by PLA2. Earlier reports (19, 20) have suggested that PLA2 is largely responsible for TNFa-induced release of arachidonate from several cell types, although it has been reported that TNFa does not increase PLA2 release from rabbit chondrocytes (37) and that PLA2 activity and arachidonate metabolite release are not coordinately regulated in rabbit chondrocytes (38). Therefore, it is possible that TNFa-induced arachidonate release may be mediated by some other mechanisms, such as a diacylglycerol-specific lipase (39) or an acyl coenzyme-A
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2796
TNFa INCREASES RELEASE OF BOTH ARACHIDONATE AND PRL
Endo• 1991 Vol 128 • No 6
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FIG. 6. Effects of BPB on PLA2- and melittin-induced [3H]arachidonate release (left) and PRL release (right) from cultured anterior pituitary cells. BPB significantly (P < 0.01) blocked both PLA2- and melittin-induced [3H]arachidonate and PRL release. Cells were preincubated for 30 min with BPB and then incubated for 30 min with vehicle, PLA2, melittin, and/or BPB. Each point represents the mean ± SE of results for four wells.
| Vehicle PLA2 (200mU/ml)
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