Modulation of Prolactin Secretion from Rat Anterior Pituitary Cells

0013-7227/90/1263-1435S02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 126, No. 3 Printed in U.S.A.

Interleukin-1-/? Modulation of Prolactin Secretion from Rat Anterior Pituitary Cells: Involvement of Adenylate Cyclase Activity and Calcium Mobilization* GENNARO SCHETTINI, TULLIO FLORIOt, OLIMPIA MEUCCIf, ELISA LANDOLFIt, MAURIZIO GRIMALDI, GAETANO LOMBARDI, GIUSEPPE SCALA, DENIS LEONG Institute of Pharmacology, Departments of Endocrinology (G.L.) and Biochemistry (G.S.), II School of Medicine, University of Naples, 80131 Naples, Italy; and the Department of Neuroscience and Physiology, University of Virginia (D.L.), Charlottesville, Virginia 22908

adenylate cyclase activity in both basal and VIP-stimulated conditions, while higher concentrations restored the enzymatic activity to the control value. ILl also caused a biphasic effect on the free intracellular calcium increase induced by maitotoxin, a calcium channel activator, being inhibitory at low and stimulatory at high concentrations. The effects of ILl on adenylate cyclase activity and calcium fluxes were reversed by preincubation of the monokine with its polyclonal antibody, thus confirming the specificity of the effects. In conclusion, our data show that ILl modulates PRL secretion by acting directly on pituitary cells through interaction with the adenylate cyclase-cAMP system and calcium flux. (Endocrinology 126: 1435-1441, 1990)

ABSTRACT. Recent findings indicate that interleukin-1/3 (IL1/3), a monokine secreted by stimulated macrophages and monocytes, modulates neuroendocrine functions in a manner similar to classical hormones. In this study we show that IL1 modulates PRL secretion, assessed by reverse hemolytic plaque assay, and describe the effect of the monokine on adenylate cyclase activity and calcium fluxes in rat normal pituitary cells. In basal and vasoactive intestinal peptide (VIP)-stimulated conditions, low doses of ILl reduced the mean plaque area, a direct index of PRL secretion without affecting the percentage of PRLsecreting cells. Similarly, low concentrations of ILl inhibited

I

NTERLEUKIN-1 (ILl) belongs to a group of proteins that are released by activated macrophages and monocytes during the acute phase of the immune response and that mediate important regulatory functions between leukocytes. This monokine is involved in a wide range of nonimmunological activities (1). Indeed, ILl induces thyroid cell (2), fibroblast (3), and astroglial (4) proliferation; modulates insulin secretion from isolated rat islets of Langerhans (5, 6); controls circulating hormones that cause fever (7); induces c-fos protooncogene expression of human endothelial cells (8); and increases the concentration of the c-myc protooncogene mRNA in the FRTL5 thyroid cell line (2). There appears to be a complete regulatory loop between the immune and neuroendocrine systems (9). Indeed, cells of the immune system are able to produce Received October 9, 1989. Address all correspondence and requests for reprints to: Prof. Gennaro Schettini, Institute of Pharmacology, II School of Medicine University of Naples, Via S. Pansini 5, 80131 Naples, Italy. * This work was supported by MPI 60% (1987) and MPI40% (1987) grants, and CNR Grant 87.00223.04 (1987; to Gennaro Schettini). t Recipient of Associazione Italiana Ricerea Canero (AIRC) fellowships.

neuroendocrine hormone mRNA, such as ACTH, GH, TSH, PRL, and they also possess receptors for all of these hormones (9, 10). On the other hand, ILl, whose receptors have been found in the brain (11), has recently been demonstrated to be involved in the control of the hypothalamic-hypophyseal-adrenal axis (12-14). In particular, Breder et al. (15) showed the existence of IL1/9immunoreactive fibers at the hypothalamic level, including the median eminence. This suggests that ILl may be released directly into the hypophyseal portal vessels, reach anterior pituitary cells, and affect hormonal secretion, probably interacting with ILl receptors identified in pituitary cell membranes (16). Indeed, ILl/3 has also been reported to stimulate GH, LH, TSH, and ACTH release from primary culture of dispersed normal rat anterior pituitary cells, while it inhibited PRL secretion (17). Moreover, the monokine has also been shown to directly stimulate POMC mRNA expression in normal rat anterior pituitary cells (18), and both ACTH (19) and /3-endorphin (20) secretion from the AtT20 mouse pituitary cell line. However, other researchers failed to observe that ILl exerted a direct effect on pituitary release, thus suggesting that its effect on pituitary hormone secretion is mediated via the hypothalamus (14, 21, 22).

1435

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ILl MODULATION OF PRL SECRETION

Two forms of ILl, a and 0, have been characterized as products of two different genes (1). Although ILl a and ILl/? share only a 26% homology in their amino acid sequences, they produce similar immunological responses by interacting through the same cell receptor (23, 24). The nature of the biochemical events after ILl receptor activation remains to be clarified. Recently, it has been shown that ILl stimulated cAMP production in lymphocytes (25) and caused diacylglycerol production through a mechanism not involving phosphatidylinositol breakdown (26). Moreover, we have shown that ILl affects adenylate cyclase activity and calcium fluxes in 235-1 clonal pituitary cells (27, 28). The aim of the present study was to clarify, by means of reverse hemolytic plaque assay (RHPA), the effect of ILl/3 on PRL secretion from a single anterior pituitary cell and to characterize the transducing mechanisms by which ILl exerts its action in normal rat anterior pituitary cells. Materials and Methods Primary cultures of anterior pituitary cells Male Wistar rats (200-250 g) were decapitated, and the anterior pituitary glands were quickly removed under sterile conditions. The tissue was cut into cubic millimeter fragments and enzymatically dispersed by serial incubations with trypsin (Worthington, Freehold, NJ; 2.5 mg/ml; 15 min at 37 C) and pancreatin (2 mg/ml; 12 min at 37 C); the fragments of tissue were further dispersed by mechanical trituration through plastic pipette tips in a few milliliters of minimum essential medium Eagle for suspension cultures (Gibco, Grand Island, NY). Cell viability was greater than 95%, as measured by the trypan blue exclusion test. The cells were plated in petri dishes with RPMI1640 culture medium containing 7.5% horse serum, 2.5% fetal calf serum, and antibiotics [100 IU/ml penicillin (Flow, Rockville, MD), 100 Mg/ml streptomycin sulfate (Flow), 15 Mg/ml gentamicin (Flow), and 0.5 Mg/ml amphotericin-B (Flow)] in a humidified atmosphere (5% CO2-95% air) at 37 C for 2-3 days before experiments (29). RHPA RHPA was performed according to the method described by Leong (30). Briefly, after trypsin dispersion, 1 ml pituitary cells (~ 500,000 cells) was mixed with a 1-ml suspension of 12% protein-A-coated ovine red blood cells (Sclavo, Siena, Italy), and the mixture was introduced into Cunningham chambers. One hour later each chamber was incubated with medium (RPMI-1640, 0.1% BSA, trypsin inhibitor, and antibiotics), with test substances [ILl and/or vasoactive intestinal peptide (VIP)] at the appropriate concentrations, and with antiserum (PRL-Ab diluted 1:100) for 2 h, followed by incubation with medium plus guinea pig complement (diluted 1:15) for 40-50 min. Plated cells were fixed by 1% glutaraldehyde in ice-cold RPMI-1640. After staining, pituitary cells were viewed and counted with the aid of a Leitz Diavert microscope (Leitz,

Endo• 1990 Voll26-No3

Rockleigh, NJ). The number of plaque-forming cells was expressed as a percentage of all cells (randomly counting 200 cells/slide). Plaque area was measured by a videoplan (IBAS system-CONTRON). Adenylate cyclase (AC) assay The AC assay was performed according to the method described by Salomon et al. (31) with modifications (32). After being dissected the pituitaries were homogenized in a glass potter fitted with Teflon pestle in a buffer containing 20 mM Tris (pH 7.5; 4 C), 0.32 M sucrose, 5 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, and 25 Mg/ml leupeptin (1:20, wt/vol). The homogenate was centrifuged at 400 X g for 5 min. The pellet was discarded, and the supernatant was centrifuged at 14,000 X g. The supernatant was discarded, and the pellet was resuspended in a buffer containing 20 mM Tris, 6 mM MgCl2, 1.2 mM EGTA, 3 mM dithiothreitol, 0.25 M sucrose, and 25 Mg/ml leupeptin. The reaction mixture contained 53 mM HEPES (pH 7.4; 30 C), 0.3 mM EGTA, 1 mM dithiothreitol, 5 mM MgCl2, 0.05 mM cAMP, 0.05 mM 3-isobutylmethylxanthine, 10 nM GTP, 0.5 mM ATP, 5 mM creatine phosphate, 50 U/ml creatine phosphokinase, 0.1 mg/ml BSA, and 0.5 mM [a-32P]ATP (2550 cpm/pmol; Amersham, Arlington Heights, IL). The final reaction volume was 100 MI divided into 50 MI reaction mixture containing [32P]ATP, 25 MI test substances, and 25 MI membrane preparation. The reaction was started by the addition of 25 MI membrane to 75 pd reaction mixture and test substances, and was carried out for 10 min at 30 C. The reaction was stopped by the addition of 100 M! of a solution containing 2% sodium dodecyl sulfate, 20 mM ATP, and 6.25 mM cAMP (pH 7.5). [3H]cAMP (15,000 cpm; Amersham) was added to the stop to monitor recovery from chromatography. Proteins were determined according to Bradford's method (33), using a solution reagent purchased from Bio-Rad (Milan, Italy). Measurement of intracellular free calcium concentrations The cytosolic free calcium was measured by the quin-2 technique (34). Normal anterior pituitary cells were detached from petri dishes by a rapid treatment with 0.25% trypsin and then resuspended in 20 ml serum-free RPMI-1640. For quin-2 loading the cells were prepared as previously described (35) in serum-free RPMI-1640 containing 0.5% BSA. Aliquots of 20 mM quin-2 acetoxymethylester (Amersham) in dimethylsulfoxide were used to obtain a final concentration of 50 fiM. After incubation with the fluorescent probe the cells were washed in serum-free RPMI-1640 and finally resuspended in the same medium at a density of 3 million/ml. Before fluorescence determination, each aliquot of cells was centrifuged and resuspended in 2 ml Hanks' Balanced Salt Solution plus 1 mM MgCl2,2 mM CaCl2, 0.2 mg/ml BSA, and 0.35 mg/ml NaH2CO3. The cell suspension was then placed in a quartz cuvette in a Perkin-Elmer spectrophotofluorimeter (Norwalk, CT) and continuously gently stirred. Cytosolic calcium concentrations were calculated with the calibration procedure by Tsien et al. (34).

IL1 MODULATION OF PRL SECRETION

1437

Chemicals Human recombinant ILl/3 (1,000,000 IU/ml), kindly provided by Dr. A. Shaw (Glaxo Institute, Geneva, Switzerland, and murine ILla (10,000 IU/ml) were used for the experiments. During ILl/3 treatment cells were incubated in growth medium in the presence of appropriate IL1/3 concentrations. Dilutions of ILl were made in sterile distilled water just before use. Sheep pOlyclonal anti-ILl/3 antibody was also obtained from Dr. A. Shaw. It was specific for human recombinant ILl/3 and was able to neutralize 100 pg ILl at a dilution of 1:100. In our experiments the anti-ILl/3 antibody was used at a 1:100 titer and was added to cells together with ILl/3 after a 40-min preincubation with the monokine. Maitotoxin (MTX), kindly provided by Prof. T. Yasumoto, was prepared as previously described (35) and added directly to cell suspension. MTX was dissolved in 10% ethanol-90% distilled water as a stock solution of 0.1 mg/ml, and then diluted to the required concentrations.

BASAL

VIP 1HM

4O

3O

20-

Statistics Experiments were performed in triplicate or quadruplicate and repeated at least three times. Statistical analysis was performed by means of analysis of variance, followed by the Newman-Keuls test. P < 0.05 was considered statistically significant.

10

C O.OIpM 1 pM

Results We evaluated the effect of human recombinant IL1/3 on PRL secretion from single normal rat anterior pituitary cells. In particular we analyzed 1) the number of plaque-forming cells, which represent the percentage of PRL-secreting cells; 2) the mean area of plaques, which is proportional to the amount of hormone secreted; and 3) the frequency distribution of plaque mean area. ILl/3 (0.01 and 1 pM) did not significantly affect the percentage of PRL-secreting cells under both basal and VIP (1 HM)-stimulated conditions (Fig. 1). Conversely, ILl/3 (0.01 pM) reduced plaque mean area under basal conditions, while it was ineffective at the highest concentration tested (1 pM; Fig. 2). VIP (1 /XM) significantly stimulated PRL secretion, as shown by the increase in the plaque mean area (+44%; p < 0.01). Both ILl concentrations tested (0.01 and 1 pM) caused a reduction of VIP-stimulated PRL release (p < 0.01; Fig. 2). Moreover, the analysis of the frequency distribution of plaque mean area showed that ILl significantly (p < 0.05) reduced the shift to large plaques induced by VIP, thus confirming the cytokine inhibitory effect on PRL secretion [control, 61% of large plaques; VIP (1 /*M), 75%; VIP plus ILl (1 pM), 60%]. To assess the possible postreceptor mechanisms coupled with ILl/3 receptor activation, we studied the effect of ILljS on both anterior pituitary AC activity and free intracellular calcium concentrations. IL1/3 inhibited basal AC activity in normal anterior

I L1.

C 001 pM 1pM

ILin

FIG. 1. Effect of IL1/3 on the percentage of PRL-secreting cells under

basal and VIP-stimulated conditions. pituitary cells showing a biphasic pattern of response (Fig. 3). Low IL1/3 concentrations (0.01 and 0.1 pM) caused a significant reduction of the enzyme activity, while high concentrations of the monokine (from 1 pM to 10 nM) were ineffective in modulating AC activity. Similarly, ILl/? (from 0.1-10 pM) significantly inhibited VIP (1 JUM) -stimulated AC activity, being maximally effective at 1 pM (-30% of VIP-stimulated AC activity). Also under VIP-stimulated conditions high ILl/3 concentrations (100 pM and 10 nM) did not affect AC activity. Similar results were obtained using murine ILla. ILla (1 pM) inhibited VIP-stimulated AC activity (p < 0.01), while in basal conditions we only observed an inhibitory trend of enzyme activity (at 0.01 pM; Table 1). The specificity of ILl/? effects was tested by polyclonal anti-ILl/3 antibody (titer, 1:100), which blocked ILl/3 inhibition of AC activity in both basal and VIP-stimulated conditions (Fig. 4). On the contrary, we did not observe any modulation of the DA (10 ixM) inhibition of AC activity (data not shown). The effect of ILl/3 on free cytosolic calcium concentrations in normal anterior pituitary cells in suspension was also studied. In basal conditions neither low (0.01 and 1 pM) nor high (1 and 10 nM) ILl/? concentrations modified

IL1 MODULATION OF PRL SECRETION

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Endo* 1990 Vol 126« No 3

• VIP

VIP

BASAL.

• BASAL

280O 50-

2600:2400< 2200-

40-

UJ

2000 180030-

160fr 1400-

120a

20-

C

001 pM 1pM IL1

c

C.CipM

M

FIG. 2. Effect of IL1/3 on plaque mean area of PRL-secreting cells under basal and VIP-stimulated conditions. *, P < 0.05; **, P < 0.01 (vs. respective control values).

intracellular calcium levels, measured by quin-2 fluorescent probe (data not shown). Conversely, ILl/? was able to influence the cytosolic calcium increase induced by the calcium channel activator MTX (29). Indeed, ILl/? (1 pM) significantly inhibited MTX-stimulated calcium fluxes (—22.3%), while nanomolar concentration of the cytokine caused a marked potentiation of the MTX effect (+300%; Fig. 5). The ILl modulation of MTX-induced calcium fluxes was also present when the cells were treated with the monokine 48 h before the experiment. However, in these conditions, the inhibition induced by ILl/? (1 pM) was more evident than after acute treatment (—40% us. —22.3%), while the stimulatory effect of the high concentration of IL1/3 was unaffected by prolonged exposure to the monokine (fig. 6). As shown in Fig. 7, in presence of its specific polyclonal antibody (titer, 1:100) ILl/? is unable to affect MTX stimulation of calcium fluxes. Discussion Although the relationship between ILl and stress hormones, via activation of the hypothalamic-hypophysealadrenal axis, seems now well accepted (13, 14), studies on ILl modulation of other anterior pituitary hormone

-15

-14

1

-13

-12

10

-9

I LIB ( L o g M )

'

FIG. 3. Dose-response curve of ILl/3 modulation of AC activity under basal and VIP-stimulated conditions. CO, P < 0.01 vs. basal value; **, P < 0.01 vs. respective control (C) value. TABLE 1. Modulation of AC activity by murine ILla pmol cAMP/mg protein •min

Basal VIP (1 MM)

Control

ILla (0.01 pM)

ILla (lpM)

93.4 ± 3.1 145.2 ± 12.2°

70.28 ± 6.7 116.57 ± 3.9

86.49 ±7.3 88.39 ±6.2"

°P< 0.01 vs. basal value, b P< 0.01 vs. respective control value. secretion are extremely controversial. In particular, as far as PRL secretion is concerned, ILl has been reported to stimulate, inhibit, or be ineffective on hormone release (17, 22, 36). Moreover, it has not been clarified whether the ILl effect is due to the direct action of the cytokine at pituitary level (17) or is mediated via the hypothalamus (21). Our findings show that ILl is able to modulate PRL release by a direct action on the pituitary gland. Indeed, by means of RHPA, a new technique able to evaluate hormone secretion at the single cell level, we found that ILl inhibits PRL secretion under both basal and VIPstimulated conditions. Moreover, the effect of the mon-


1439

MTX

(5x1O" 9 M)_

10-

8

6 UJ

u

tr U C

ILiP Ab ILiB-Ab QOIpM ' BASAL

?

4

1pM -VIP1]

FIG. 4. Reversal of the inhibitory effect of IL1/3 on AC activity under basal and VIP-stimulated conditions by means of specific anti-ILl polyclonal antibody (Ab). Data are expressed as a percentage of the respective control values. Control values were 35.56 ± 2.3 (basal; C) and 54.16 ± 4.7 (VIP) pmol cAMP produced/mg protein/min. **, P < 0.01 vs. respective control value.

okine on PRL secretion we found, occurs for very low ILl concentrations (0.01 and 1 pM). These findings are in line with the report of Tracey and De Souza (16), which identified ILl receptors on pituitary membranes, and with the report of Breder et al. (15), which demonstrated the presence of immunoreactivity for ILl in fibers of various hypothalamic areas, including the median eminence. Thus, ILl could be secreted from median eminence directly into the hypophyseal portal vessels, reach the anterior pituitary, and modulate hypophyseal hormone secretion. Furthermore, by means of RHPA we can exclude any cell to cell interaction in secretory studies, therefore avoiding, for instance, the paracrine inhibitory influence of ACTH on PRL release. This observation confirms that ILl directly inhibits PRL secretion from lactotropes without excluding, however, that ACTH may participate in vivo in ILl inhibition of PRL release. In light of these results we can hypothesize that since the immunopermissive role of PRL is well known, the decrease in PRL secretion induced by ILl may serve as a mechanism to limit the acute responses otherwise induced by the monokine. Our studies on second messenger systems confirm these functional data. Indeed, the in vitro addition of ILl to pituitary membranes caused a significant inhibition of the AC activity in basal and VIP-stimulated conditions for concentrations equimolar (0.01 and 1 pM) to that which inhibits PRL secretion. The specificity of this

2

1pM 1nM 10nM FIG. 5. IL1/3 modulation of the increase in free cytosolic calcium concentration induced by MTX under acute conditions. The basal intracellular calcium concentration was 257 ± 14 nM. ILl (1 pM) inhibits the MTX-induced calcium rise by 22.3%, while 1 and 10 nM ILl enhance MTX stimulation to 30% and 300%, respectively. *, P < 0.05; **, P < 0.01 [vs. control (C) value].

effect was demonstrated by coincubation of the monokine with the anti-ILl polyclonal antibody, which completely abolished the inhibitory ILl effect on AC activity. Similar but less pronounced results were obtained with murine ILla, with evidence of a common biological activity of ILla: and IL1/3 and their capacity to bind to the same receptor (23, 24). These data are in agreement with our previous report on the 235-1 PRL-secreting pituitary cell line (27) and with the study of Calkins et al. on Leydig cells (37), reporting an inhibitory action of ILl on AC activity and cAMP formation, respectively. Although ILl did not modify basal free intracellular calcium levels, low concentrations of the monokine caused a reduction of the calcium fluxes elicited by MTX. This marine toxin is known to increase intracellular calcium levels in many cell types, probably acting through the voltage-sensitive calcium channels of the L-type (29, 35, 38, 39), and to induce PRL release from normal anterior pituitary cells


1440 MTX (5x1O-9M)

5• •

3 Q

2 1-

C

IL,p1pM ( 4 8 hr t r e a t . )

FIG. 6. Effect of 48-h treatment with ILl/3 on the MTX-induced free intracellular calcium rise. Basal free cytosolic calcium levels were: control (C), 250 ± 14 nM; IL (1 pM) 243 ± 32 nM; and IL (1 nM), 218 ± 20 nM. The percent ILl (1 pM) inhibition of the MTX effect was 40%; ILl (1 nM) stimulation of the MTX-induced increase in calcium levels was 33%. *, P < 0.05; **, P < 0.01 [vs. control (C) value]. 6-

MTX

(5XX>~9M)

Ui

Endo • 1990 Vol 126 • No 3

fibroblasts. Alternatively, since a growing bulk of evidence shows that cAMP-dependent phosphorylation is able to promote the opening of calcium channels during depolarization (41), it is possible to infer that a prolonged reduction of cAMP formation induced by the monokine may participate in the inhibitory effect of ILl on calcium conductance. As far as the stimulatory effect of 1 nM ILl is concerned, on the basis of the existence of two different subpopulations of ILl/? receptors, with high and low affinities for the ligand (1), it cannot be completely ruled out that low affinity binding sites coupled in a stimulatory way with the calcium channels exist. However, since ILl has been reported to exert cytotoxic effects (42), it is possible that the increases in free intracellular calcium levels elicited by high concentrations of the monokine could be determined by the additive toxicity of ILl and MTX. In conclusion, our data show that IL1/3 modulates PRL secretion through a direct action at the pituitary level. However, the coexistence of an indirect modulation of pituitary hormone release via the hypothalamus, as previously reported (21, 22), cannot be excluded. Moreover, our findings suggest that ILl modulation of the ACcAMP system and calcium fluxes seems to be responsible for the inhibitory effect of the monokine on PRL release at the pituitary level.

< Ui

a. u ? 4

Acknowledgments T

We are grateful to Prof. T. Yasumoto for providing us with MTX and to Dr. A. Shaw (Glaxo Institute, Geneva, Switzerland) for providing us with ILl/3 and anti-ILl antibody.

References 2-

FIG. 7. Reversal of IL1/3 (1 pM) inhibition of the MTX-induced cytosolic calcium rise after coincubation of the monokine with its specific polyclonal antibody (Ab). **, P < 0.01 [vs. control (C) value]; -(rtr, P < 0.01 vs. ILl alone.

(29). This effect was more pronounced, with ILl incubating with normal anterior pituitary cells for 48 h. Also, ILl modulation of calcium fluxes was demonstrated to be specific, since it was reversed by adding the anti-ILl polyclonal antibody. The higher sensitivity of normal anterior pituitary cells to ILl inhibition of MTX-induced calcium fluxes after 48-h incubation with the monokine could be ascribed to the ability of the monokine to stimulate its own receptor expression after 48 h of treatment, as reported by Akahoshi et al. (40) in human

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