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Endocrinology Vol. 138, No. 12 5374-5384
Copyright © 1997 by The Endocrine Society

ARTICLES

The Cortical Actin Cytoskeleton of Lactotropes as an Intracellular Target for the Control of Prolactin Secretion1

M. Eloísa Carbajal and María L. Vitale

Département d’Anatomie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4 Canada

Address all correspondence and requests for reprints to: María L. Vitale, Room P-808, Département d’Anatomie, Faculté de Médecine, Université de Montréal, Pavillon Principal, 2900 Boulevard Edouard-Montpetit, Montréal, Québec, H3T 1J4 Canada. E-mail: vitalem{at}ere.umontreal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of cortical actin filaments (F-actin) in the regulation of PRL secretion in cultured normal anterior pituitary cells. F-actin dynamics were evaluated by fluorescence microscopy, and PRL secretion from attached cells was measured by the reverse hemolytic plaque assay. F-actin localized to the periphery of lactotropes. PRL-releasing factors such as TRH, vasoactive intestinal peptide (VIP), and forskolin, or removal of the PRL-inhibiting factor dopamine (DA) from cultures chronically exposed to DA, caused fragmentation, i.e. focal disassembly of cortical F-actin. Basal, VIP-, and DA withdrawal-induced cortical F-actin disassembly were dependent on extracellular Ca2+ whereas TRH- and forskolin-induced disassembly were not. Short-term (5 min) treatment of cells with the F-actin-disrupting agent cytochalasin D (CD) enhanced basal PRL secretion but did not further stimulate TRH- or VIP-induced PRL secretion. The results support the existence of a causal link between F-actin disassembly and increased PRL secretion. On the other hand, exposure of cultures to DA decreased the percentage of cells showing cortical F-actin disassembly within minutes. Longer treatments (2–4 h) caused stabilization of cortical actin filaments as revealed by the protection vis-a-vis the depolymerizing effect of CD. The protective effect was specific for lactotropes and was evident with DA concentrations as low as 50 nM. Chronic exposure of the cells to DA blocked CD- and TRH-evoked actin disassembly and PRL secretion while VIP-induced effects were partially inhibited. Stabilization of F-actin with the marine sponge venom, jasplakinolide, also decreased basal and stimulated PRL secretion. In conclusion, our results suggest that, first, the cortical actin cytoskeleton of lactotropes is an integrator of the multiple factors regulating PRL secretion directly on the lactotrope, and second, the tonic inhibition of PRL secretion is mediated, at least in part, by DA-induced stabilization of cortical F-actin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPERIMENTAL evidence suggests that the cortical actin cytoskeleton is a physical constraint to regulated exocytosis (for a review see Ref.1). Disruption of actin filaments (F-actin) by depolymerizing drugs, such as the fungal toxins cytochalasins, enhances stimulated secretion in pancreatic ß-cells (2), cultured chromaffin cells (3), and permeabilized mast cells (4). Biochemical and fluorescence microscopy studies have shown that a rapid, transient, and reversible F-actin disassembly takes place in discrete zones of the cell cortex during hormone and neurotransmitter secretion (5, 6), and that areas of exocytosis correspond to cortical areas devoid of F-actin (7, 8). The participation of actin-regulatory proteins in stimulation-evoked actin reorganization is suggested by the finding that actin-binding proteins such as fodrin, an actin-anchoring protein (9), and scinderin, an actin-severing protein (7), redistribute and colocalize with cortical F-actin during stimulated secretion. Introduction of actin-monomer binding proteins into pancreatic acinar cells is followed by F-actin disassembly and by an increased amylase release (10). Similarly, introduction of recombinant scinderin into permeabilized platelets and chromaffin cells enhances disassembly of actin filaments and Ca2+-evoked secretion (11, 12). All these results strongly suggest F-actin plays an inhibitory role in secretion. In fact, disassembly of the cortical actin network in chromaffin cells facilitates access of secretory granules to the plasma membrane, enhancing the initial rate of exocytosis (13).

Anterior pituitary cells secrete their respective hormones by exocytosis. It has been shown in lactotropes and gonadotropes that a single depolarization causes an increase in intracellular Ca2+ ([Ca]i) that is too small to trigger exocytosis (14, 15). In contrast, in chromaffin cells, the same stimulus induces a sustained increase in [Ca]i and exocytosis (16). Depolarization-induced exocytosis is thought to be due to the entry of Ca2+ through voltage-gated Ca2+ channels. Only secretory vesicles near the channels will be affected by locally elevated [Ca]i and will undergo exocytosis. Therefore, secretory vesicles in anterior pituitary cells must be further away from the plasma membrane than in chromaffin cells. The cortical actin cytoskeleton may intervene in the segregation and movement of cortical vesicles in the anterior pituitary cells. Experimental evidence, although scarce, suggests that the actin cytoskeleton is involved in hormone secretion from the anterior pituitary (for a review see Ref.17). Morphological studies have shown that actin filaments are organized as a network underneath the plasma membrane of pituitary cells (18, 19) and that they either encompass or exclude the secretory granules from the subplasmalemmal space (19, 20). Studies performed in gonadotropin-secreting cell cultures revealed that migration of secretory granules toward the plasma membrane during cell stimulation required an assembled actin cytoskeleton (21). Reorganization of cytoskeletal actin in tumor-derived PRL- and GH-secreting cells during hormone secretion was also reported (22, 23). Furthermore, glucocorticoid-induced inhibition of ACTH secretion from a tumoral cell line is mediated, at least in part, by the cytoskeleton (24, 25).

The aim of the present work was to investigate the role of microfilaments in pituitary hormone secretion, specifically, PRL secretion. The PRL-secreting cell of the anterior pituitary presents characteristics that make it an interesting model to study the involvement of the cortical actin cytoskeleton in exocytosis. PRL secretion is under the control of inhibitory [PRL-inhibitory factors (PIFs) and PRL-releasing factors (PRFs)] (for a review see Ref.26). PRL release is tonically inhibited. Dopamine (DA) is considered to be the major PIF (26). Activation of the lactotrope-secretory activity involves both an increased influence of PRFs, such as TRH and vasoactive intestinal polypeptide (VIP) and a decreased dopaminergic tone (26). Here, we present evidence that the cortical actin cytoskeleton may act as a common target for PIFs and PRFs in their regulation of PRL secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Randomly cycling Sprague-Dawley female rats (Charles River, St. Constance, Québec) were used as a source of anterior pituitary cells. The pars distalis was dissected out free of pars intermedia and pars nervosa. Tissues from two to four rats were pooled, diced in small pieces, and dispersed into a single cell population by incubation with Mg2+/Ca2+-free Locke’s solution (154 mM NaCl, 2.6 mM KCl, 2.15 mM K2HPO4, 0.85 mM KH2PO4, 10 mM HEPES, 10 mM glucose; pH 7.2) containing 0.1% trypsin, 0.2% collagenase D, and 0.3% BSA for 30–35 min at 37 C. Digestion was stopped by addition of a volume of DMEM containing 0.2% of soybean trypsin inhibitor. Cells were recovered by centrifugation, rinsed with the DMEM, and resuspended in feeding medium (FM): DMEM supplemented with 2.5% FCS, 10% horse serum, antibiotics, and antifungi. Anterior pituitary cells were plated and cultured at 37 C in a 95% air-5% CO2 atmosphere. Under our experimental conditions, 99% of the cells recovered were anterior pituitary cells (stained positive for one anterior pituitary hormone), 35–40% of which were lactotropes (PRL-immunopositive cells).

Immunocytochemistry
Anterior pituitary cells were plated on poly-L-lysine-coated glass coverslips at a density of 2 x 105 cells per 35-mm Petri dish. Before any treatment, cells were allowed to recover for 24 h. Cells were then further incubated in FM, this time containing 100 µM ascorbic acid (AA) in the presence or in the absence of dopamine (DA) (several concentrations) for different periods according to the specific protocol. For DA treatments longer than 24 h, the media were removed after 24 h and replaced with fresh media containing 100 µM AA and the corresponding concentration of DA. After the treatments, dishes were removed from the incubator, and the experiments were started by rinsing the cells with regular Locke’s solution (Locke’s solution containing 1.2 mM MgCl2, 2.2 mM CaCl2, and 100 µM AA). In the case of cells preincubated with DA, the Locke’s solution also contained DA at the same concentration as the one used during the preincubation period. When studying the role of Ca2+, cells were rinsed three times with Ca-free Locke’s solution (1 mM EGTA). After rinsing, cells were challenged with different PRFs (TRH, VIP, DA withdrawal) or PIFs (DA) for increasing periods (0–600 sec) in the presence or in the absence of DA and/or Ca2+ depending on the specific protocol. Preparations were immediately fixed with 3.7% formaldehyde, permeabilized with acetone (3 min 50% acetone-3 min 100% acetone-3 min 50% acetone) and processed for fluorescence microscopy as previously described (27). Briefly, coverslips were thoroughly rinsed with PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) and incubated for 1 h at room temperature with 3% nonfat milk in PBS to block unspecific labeling. To study microfilament dynamics during stimulation/inhibition of PRL secretion, anterior pituitary cell cultures were double labeled for PRL and F-actin. After blockage, cells were incubated for 60 min at 37 C with rat PRL antiserum (1:1500 dilution in 1% nonfat milk in PBS) followed by a 60-min incubation at 37 C with fluorescein isothiocyanate conjugate (FITC)-antirabbit IgG (1:400 dilution in 1% nonfat milk in PBS). After these incubations, coverslips were washed with PBS and incubated for 45 min at room temperature in the dark with rhodamine-labeled phalloidin (1:200 dilution in PBS). Cells were observed with a Leitz Ortholux II fluorescent microscope equipped with an I-filter block for fluorescein and a M-filter block for rhodamine. To evaluate the state (assembled-disassembled) of the cortical actin network of lactotropes subjected to different treatments, the aspect (continuous-discontinuous) of the cortical rhodamine staining (F-actin) of fluorescein-positive cells (lactotropes) was recorded (7). This procedure, which was done without knowing the type of treatment applied to the cells (single blind design), was performed in 100 PRL-immunopositive cells per coverslip. Therefore, every final value for a given experimental condition is the result of the observation of not less than four coverslips (400 lactotropes) from two to three different experiments. Photographs were taken with T-MAX Kodak films (400 ASA).

Reverse hemolytic plaque assay
The reverse hemolytic plaque assay (RHPA) was performed as described previously (28, 29). After 2 days of culture in serum-supplemented medium, monodispersed pituitary cells were resuspended by brief trypsinization (0.05% trypsin, 1 min) followed by 5 min incubation with 0.1% soybean trypsin inhibitor and washed with DMEM-BSA. The pituitary cell suspension (1.5 x 106 cells/ml) was mixed with an equal volume of a 30% (vol/vol) protein A-conjugated ovine red blood cell suspension. Thirty-microliter aliquots were introduced into poly-L-lysine-coated Cunningham chambers by capillarity. Chambers were placed in a humidified container in a 95% air-5% CO2 incubator at 37 C for 1.5 h to allow the cells to attach. Next, chambers were rinsed with DMEM-BSA and filled with PRL antiserum (1:50, final dilution) supplemented or not with the appropriate drugs. Preparations were incubated for different periods; at the end of the incubation periods they were rinsed with DMEM-BSA to remove the excess of antibody and drugs. Plaques were developed by incubation for 30 min with guinea pig complement (1:40). Finally, cells were rinsed with DMEM-BSA, fixed with 0.5% paraformaldehyde for 24 h, and stained with toluidine blue to facilitate identification of pituitary cells. Controls included omission of either PRL antibody or the complement. To test the viability of pituitary cells subjected to different drug treatments, an additional slide for each treatment was filled with 0.2% trypan blue in PBS and incubated for 5 min at the end of the experiment. Each experimental condition was replicated in three separate slides per assay, and each experiment was repeated at least three times. One hundred cells per assay were examined. Plaques (zones of hemolysis) surrounding secreting cells were viewed with a Laborlux S Leitz microscope (Leica, Willowdale, Ontario, Canada) equipped with a camera (Javelin JTE 3462 RGB, Japan), and images were recorded with a VCR (SVO-9500 MD, SONY, Japan). Determination of the surface of each plaque was accomplished by using a video-based image-processing system Videoplan 2 (Carl Zeiss, Toronto, Ontario, Canada).

Statistical analysis
Uniformity between two samples of plaque area values was analyzed by the Kolmogorov-Smirnov test. Significance was determined at P < 0.05. Plaque area data were converted to a percentage of control. Differences between groups were statistically analyzed using Student’s t test.

Source of chemicals and antibodies
Enzymes for anterior pituitary cell dispersion were purchased from Boehringer-Mannheim Canada (Laval, Québec, Canada). Other materials for cell culture and guinea pig complement were from GIBCO Canada (GIBCO-Life Technologies, Burlington, Ontario, Canada). Antibiotics, soybean trypsin inhibitor, protein A (Staphylococcus aureus), poly-L-lysine, BSA (fraction V), VIP, TRH, dopamine, cytochalasin D, forskolin, and FITC-antirabbit IgG were purchased from Sigma Chemical Co (St. Louis, MO). PRL antibodies for immunocytochemistry and RHPA (NIDDK-anti-rPRL-IC-5 and anti-rPRL-S-9) were kindly provided by the NIH. Rhodamine-phalloidin and jasplakinolide were purchased from Molecular Probes (Eugene, OR). Stock solutions of TRH were prepared in Tris-buffered-saline; VIP was dissolved in 0.01% acetic acid. Dopamine solutions were prepared in 100 µM ascorbic acid. Forskolin, cytochalasin D, and jasplakinolide were prepared as stock solutions in DMSO; the final concentration of the solvent in the working dilutions was lower than 0.1%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cortical actin dynamics during stimulation or inhibition of PRL secretion
In cultured lactotropes (PRL-immunopositive cells) (Fig. 1Go, a and b), actin filaments were mainly distributed as a cortical network (Fig. 1Go, a' and b') that appeared either continuous (Fig. 1aGo') or fragmented (Fig. 1bGo'). Experimental evidence has shown that the continuity of the cortical rhodamine staining is associated with an assembled actin network, whereas fragmentation of the rhodamine-phalloidin staining was a signal of F-actin disassembly (5, 6, 30). This reorganization of cortical filaments is part of the secretory pathway in several cell types. To test whether this was also the case in lactotropes, we analyzed the effect of PRFs and PIFs on the dynamics, i.e. assembly/disassembly, of the cortical actin cytoskeleton of PRL-immunopositive cells. Anterior pituitary cells cultured for 48 h in DA-free medium were incubated with Locke’s solution alone (control) or containing either 100 nM TRH, or 500 nM VIP or 10 µM forskolin for increasing periods (15–600 sec). At the end of these incubations, cells were double labeled for PRL and F-actin, and the appearance of the cortical actin cytoskeleton of lactotropes was analyzed by fluorescence microscopy as described in Materials and Methods. The percentage of lactotropes displaying cortical actin disassembly in nontreated cells was 38 ± 6 (n = 400) and was not affected by further incubation with Locke’s solution (Fig. 2AGo, open circles). Instead, the presence of TRH in the incubation medium, an experimental condition leading to PRL secretion, caused a rapid and sharp increase in the percentage of cells showing cortical actin disassembly (Fig. 2AGo, filled triangles). The maximal percentage was reached 15 sec after the beginning of TRH stimulation. From then on, the percentage of lactotropes showing a fragmented cortical actin cytoskeleton slowly decreased to control levels. Stimulation of PRL secretion with VIP (Fig. 2AGo, filled diamonds) also caused an increase in lactotropes showing disassembly of cortical F-actin, although with a different kinetics than TRH. Upon VIP treatment, there was a slow increase in the number of cells with a discontinuous cortical F-actin ring. The maximal percentage was reached after a 90-sec incubation period with VIP. Values decreased thereafter, reaching control levels 300 sec after the initiation of VIP stimulation. Treatment of the cultures with forskolin, another substance that increases intracellular cAMP, also caused cortical actin disassembly (Fig. 2AGo, open squares). The maximal increase was observed after a 60-sec incubation period with forskolin. The kinetics of TRH- and VIP-induced cortical actin disassembly correlate with the time course of PRL secretion induced by these secretagogues, a fast response to TRH and a slow effect of VIP (31, 32). Forskolin was slower than TRH but faster that VIP. Disassembly of cortical actin evoked by TRH, VIP, or forskolin was a transient phenomenon, lasting not longer than 300 sec.



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  		Figure 1. Distribution of actin filaments in cultured lactotropes as revealed by fluorescence microscopy. Anterior pituitary cell cultures were sequentially stained with PRL antiserum and FITC-IgG (a-b) and rhodamine-phalloidin (a'-b') to evaluate the distribution of actin filaments in lactotropes. PRL staining was punctate, revealing the presence of secretory granules. F-actin was mainly localized at the lactotrope periphery. Lactotrope cortical actin filaments displayed two different patterns, a continuous cortical fluorescent ring (a', open arrowhead) or a fragmented cortical fluorescent ring (b', arrowheads). Bar, 5 µm.

 


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  		Figure 2. Time course study of lactotropes’ cortical actin disassembly induced by different secretagogues. Anterior pituitary cells incubated for 48 h in the absence of DA (A) were challenged for increasing periods (0–600 sec) with Locke’s solution alone (control) or containing 100 nM TRH, 500 nM VIP, or 10 µM forskolin. Cells pretreated with 500 nM DA for 48 h (B) were incubated with DA or with Locke’s solution (DA withdrawal (DAw)) in the presence or in the absence of 100 nM TRH, 500 nM VIP, or 10 µM forskolin for increasing periods (0–600 sec). After the treatments, cells were processed for double labeling using PRL antibodies and rhodamine-phalloidin. The percentage of lactotropes (PRL-immunopositive cells) displaying cortical actin disassembly (discontinuous rhodamine-fluorescent ring) was calculated for each experimental condition. Upon cell stimulation, there was an increase in the percentage of lactotropes having a disassembled cortical actin staining. Values shown are the mean ± SEM of a total of at least 400 lactotropes from three different cultures per experimental condition. *, P < 0.05: TRH, 15 sec, vs. control, 15 sec; VIP, 90 sec, vs. control, 90 sec; forskolin, 60 sec, vs. control, 60 sec; DAw, 30 sec, vs. DA, 30 sec; (DAw + TRH), 30 sec, vs. DA, 30 sec; (DAw + VIP), 120 sec, vs. DA, 120 sec; and (DAW + forskolin), 60 sec, vs. control, 60 sec.

 
PRL secretion is controlled by a complex interaction between PIFs and PRFs. Periods of increased PRL secretion are associated with both an increase in PRF concentrations and a decrease in PIF concentrations reaching the lactotrope. We investigated the possibility that the cortical actin cytoskeleton could be a common target for PRFs and PIFs to integrate their effects on PRL secretion. Cultures treated for 48 h with 500 nM DA were challenged with TRH or VIP for increasing periods (0–600 sec) in the presence or in the absence of 500 nM DA. Addition of TRH did not affect cortical actin disassembly in DA-inhibited cells (Fig. 2BGo, open circles). Stimulation of DA-treated cells with VIP seemingly increased the number of cells showing cortical actin disassembly after long incubation times (>2 min). However, due to the large dispersion of the experimental values, the percentages of lactotropes displaying cortical F-actin disassembly were not statistically different from those observed in cells treated with DA alone (not shown). Removal of DA from the medium caused a fast increase in the percentage of cells showing cortical actin disassembly (Fig. 2BGo, filled circles). The maximal percentage was reached 30 sec after DA withdrawal and decreased after that. Sixty seconds after DA withdrawal, the percentage of lactotropes displaying cortical actin disassembly was similar to the percentage obtained in cells incubated for 48 h in the absence of DA (compare with Fig. 2AGo, 0 sec). DA withdrawal-evoked cortical actin disassembly was not enhanced by the presence of 100 nM TRH in the incubation medium (Fig. 2BGo, filled triangles). On the contrary, in the presence of 500 nM VIP, the initial sharp increase in actin disassembly was replaced by a slow increase (Fig. 2BGo, filled diamonds), which reached DA withdrawal-evoked actin disassembly values 120 sec after the initiation of cell stimulation. Forskolin + DA withdrawal (Fig. 2BGo, open squares) induced an increase in the number of lactotropes showing cortical actin disassembly that was faster than VIP + DA withdrawal- but slower than TRH + DA withdrawal-induced actin disassembly.

To study the role of Ca2+ in cortical actin disassembly, cultures were challenged with different secretagogues in the presence or in the absence of extracellular Ca2+, and the state, i.e. assembled or disassembled, of the cortical actin cytoskeleton of PRL-immunopositive cells was analyzed by fluorescence microscopy. The time of exposure to each secretagogue was chosen from Fig. 2Go and was the one that induced the maximal increase in the percentage of lactotropes displaying cortical actin disassembly. The results depicted in Fig. 3Go show that basal, VIP-, and DA withdrawal-induced cortical actin disassembly were Ca2+-dependent events. DA withdrawal was more affected by the removal of external Ca2+ than the other experimental conditions. Neither TRH- nor forskolin-induced cortical actin disassembly was affected by removal of extracellular Ca2+.



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  		Figure 3. Effect of extracellular Ca2+ on basal and PRF-induced cortical actin disassembly. Anterior pituitary cells were grown for 48 h in the absence of DA (control, TRH, VIP, forskolin) or in the presence of 500 nM DA (DA withdrawal). After the incubation, cells were challenged for 30 sec with Locke’s solution (C), for 30 sec with 100 nM TRH, for 90 sec with 500 nM VIP, for 60 sec with 10 µM forskolin or for 30 sec with DA-free Locke’s solution (DAw). All these incubations were carried out in the presence or in the absence of Ca2+. Next, cells were processed for double labeling fluorescence microscopy, and the percentage of PRL-immunopositive cells showing cortical actin disassembly was calculated as explained in Materials and Methods. Data shown are the mean ± SEM of a total of 600 lactotropes from three different cultures (*, P < 0.05; **, P < 0.01).

 
Since our results show that incubation of lactotropes with PRFs caused disassembly of cortical actin, the next series of experiments were designed to test whether the state of lactotropes’cortical actin was also affected by experimental conditions leading to an inhibition of PRL secretion. Analysis of Fig. 2Go shows that the percentage of lactotropes displaying cortical actin disassembly was substantially reduced by exposure of cultures to 500 nM DA for 48 h (compare the percentage of lactotropes showing cortical F-actin disassembly at 0 sec in Fig. 2Go). To further study the effect of DA on the cortical actin cytoskeleton, anterior pituitary cells, cultured in the absence of exogenous DA, were treated for increasing periods (0–48 h) with 500 nM DA. At the end of each period, cells were processed for fluorescence microscopy, and the effect of the treatment on cortical F-actin was recorded. As depicted in Fig. 4Go, exposure of the cultures to DA slowly reduced the number of lactotropes showing cortical actin disassembly, suggesting that the presence of DA blocked disassembly of actin filaments. A 600-sec incubation period in DA containing medium significantly decreased (P < 0.05) the percentage of lactotropes showing cortical actin disassembly with respect to the nontreated cells (Fig. 4Go). A further decrease was observed 2–4 h later (Fig. 4Go). Four-hour exposure of the cultures to 100 nM, 1.0 µM, or 10 µM DA had a similar effect on cortical actin filaments. Comparison of data in Figs. 2Go and 4Go reveals that induction of actin filament disassembly by PRFs is faster than induction of actin filament assembly by DA.



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  		Figure 4. Effect of DA on cortical F-actin disassembly. Anterior pituitary cells cultured for 48–72 h in DA-free medium were incubated with 500 nM DA for increasing periods (0–48 h). Immediately after those periods, cells were processed for double labeling fluorescence microscopy to analyze the state (continuous = assembly or discontinuous = disassembly) of lactotropes’ cortical actin cytoskeleton. The percentage of lactotropes (PRL-immunopositive cells) having a disassembled cortical actin staining was calculated as explained in Materials and Methods. Exposure of cultures to DA caused a slow decrease in the percentage of lactotropes having a disassembled cortical actin staining. After a 600-sec incubation period with DA, there was a significant decrease in the percentage of lactotropes showing cortical actin disassembly (*, P < 0.05 vs. 600-sec control). Four hours later, the values were lower than at 600 sec (**, P < 0.05 600 sec vs. 4 h). Values shown are the mean ± SEM of a total of at least 400 lactotropes (two different cultures) per experimental condition.

 
DA-mediated stabilization of cortical actin filaments
The aforementioned results demonstrate that experimental conditions evoking PRL secretion were accompanied by disassembly of lactotrope’s cortical actin network, while inhibition of PRL secretion by DA was associated with decreased cortical actin disassembly. We hypothesized then, that the inhibitory effect of DA on PRL secretion could be due, at least in part, to a DA-induced stabilization of the cortical cytoskeleton. To test this hypothesis, we investigated whether cortical actin filaments of DA-treated lactotropes had a higher resistance to the depolymerizing effect of cytochalasin D (CD) than cells not exposed to DA. Five-minute exposure of anterior pituitary cell cultures to 1 µM CD caused fragmentation of cortical actin filaments and the appearance of fluorescent aggregates in the cytoplasm of most lactotropes (Fig. 5aGo'). Lactotropes cultured for 48 h in a 500 nM DA-supplemented medium showed an almost intact cortical actin staining in spite of the presence of CD (Fig. 5bGo').



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  		Figure 5. Depolymerizing effect of CD on lactotrope actin cytoskeleton and evidence for a protective effect of DA. Anterior pituitary cultures were incubated in the absence (a-a') or in the presence (b-b') of 500 nM DA for 48 h. Next, they were treated for 5 min with 1 µM CD in the absence (a-a') or in the presence (b-b') of DA. At the end of those periods, preparations were processed for fluorescence microscopy to evaluate the effect of CD and DA on lactotrope cortical actin cytoskeleton. CD treatment caused disruption of actin filaments; aggregates of rhodamine-fluorescent material could be seen in the cell periphery and interior (a', open arrowheads). Exposure of the cultures to 500 nM DA for 48 h partially protected cortical actin filaments to CD-induced depolymerization (b', arrowhead). Bar, 5 µm.

 
Time course studies indicate that a fast increase in the number of lactotropes showing signs of actin filament disruption occurred upon CD treatment (Fig. 6AGo, open circles). Cortical actin filaments in lactotropes cultured in DA-free medium appeared more fragile than those in nonlactotropes present in the same preparations. Indeed, a 10-min incubation period with CD caused disruption of cortical actin in 85 ± 3% PRL-immunonegative cells, whereas almost 100% PRL-immunopositive cells were already affected (Fig. 6Go, A and A', open circles). Exposure of anterior pituitary cell cultures to 500 nM DA for 48 h significantly reduced the depolymerizing effect of CD on cortical actin filaments. The protective effect, i.e. cells showing a continuous cortical actin staining in spite of CD (Fig. 5bGo'), was maximal during short-term treatments (5–10 min) of cultures with CD and diminished thereafter (Fig. 6AGo, filled circles). There was no protective effect of DA on cortical F-actin in PRL-immunonegative anterior pituitary cells (Fig. 6AGo', filled circles). The results suggest that in lactotropes, DA stabilized cortical actin filaments whereas the absence of DA made the filaments more susceptible to disassembly.



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  		Figure 6. A and A', Effect of exposure of cultures to DA on CD-induced actin filament disruption. Anterior pituitary cells were cultured for 48 h in medium supplemented (filled circles) or not (open circles) with 500 nM DA. Disruption of actin filaments was induced by treatment of the cell cultures with 1 µM CD for increasing periods (0–30 min). In the case of cells previously exposed to DA, the incubation medium also contained 500 nM DA. Immediately after the treatment, cells were double labeled for PRL and F-actin, and the disruption of the cortical F-actin staining in PRL-immunopositive cells (A) and in PRL-immunonegative cells (A') was analyzed. Each value shown is the mean ± SEM of the percentage of discontinuous cortical rhodamine fluorescent ring of four coverslips containing cells from three experiments. Statistical analysis: **, P < 0.01 5, 10, and 15 min CD treatment of cells preincubated with DA vs. 5, 10, and 15 min CD treatment in cells not incubated with DA, respectively; *, P < 0.05 30 min CD treatment of cells preincubated with DA vs. 30 min CD treatment of cells not incubated with DA. B, Time course studies on the development of DA-induced protective effect on cortical actin filaments. Cultures were exposed to 500 nM DA for different periods (0–48 h); next they were treated with 1 µM CD for 5 min. After the incubation with CD, preparations were processed for PRL and F-actin fluorescent labeling. The percentage of cells displaying cortical actin disassembly was calculated for each experimental condition. Values shown are the mean ± SEM of percentage data from four coverslips (400 lactotropes) from two different cell cultures. (*, P < 0.05 2 h vs. 0 h; **, P < 0.05 4 h vs. 2 h and P < 0.01 vs. 0 h). C, Dose response study on the protective effect of DA against CD-depolymerizing action. Anterior pituitary cells were cultured for 48 h in DA-free medium (control (-)) or with medium supplemented with increasing concentrations of DA (from 50 nM to 5 µM). After the treatment, cells were challenged with 1 µM CD for 5 min, and the state of cortical actin filaments (continuous/discontinuous) of lactotropes was analyzed by fluorescence microscopy as explained above. Data shown are the mean ± SEM of the percentages of cortical actin disassembly of four to five coverslips (400–500 lactotropes) per experimental condition (*, P < 0.02 vs. (-); **, P < 0.05 vs. 0.05 µM DA).

 
The next series of experiments were designed to find out the minimum exposure time required for DA to stabilize lactotropes’ actin filaments. Two-day-old anterior pituitary cells were incubated in FM supplemented or not with 500 nM DA for 0, 2, 4, 12, 24, or 48 h at 37 C. Cells were next treated with Locke’s solution containing 1 µM CD for 5 min at room temperature. Cultures were processed for double labeling with PRL antibodies and rhodamine-phalloidin, and the protective effect of DA on CD-induced actin disruption was evaluated as explained above. DA-induced stabilization of microfilaments was already apparent upon 2 h of exposure of the cultures to DA-supplemented medium and was maximal upon 4 h (Fig. 6BGo). We also performed a dose-response study on the protective effect of DA treatment against depolymerization of cortical microfilaments (Fig. 6CGo). Anterior pituitary cells were treated for 48 h with different concentrations of DA (50 nM to 5 µM) and next incubated with 1 µM CD for 5 min. Evaluation of the state of cortical actin filaments by fluorescence microscopy demonstrated that exposure of lactotropes to DA concentrations as low as 50 nM protected cortical actin filaments against the depolymerizing action of CD (Fig. 6CGo). Interestingly, concentrations of DA higher than 100 nM did not further increase the percentage of lactotropes that were protected against depolymerization, suggesting that there was a subpopulation of lactotropes (~20% of lactotropes) that was refractory to DA.

Involvement of actin disassembly in PRL secretion
To investigate the existence of a causal link between the disassembly of cortical actin filaments and the lactotrope-secretory response, we studied the effect of CD on basal and stimulated PRL secretion. RHPA was chosen to measure PRL secretion. In this method, hormone secretion is observed as areas of hemolysis surrounding cells. The surface of the plaque of hemolysis is directly related to the amount of hormone secreted from the cell during a specific period (29). RHPA enabled us to evaluate the effect of actin-disrupting or -stabilizing substances on PRL release under the same experimental conditions the studies on actin disassembly were carried out (seeCortical actin dynamics during stimulation or inhibition of PRL secretion and DA-mediated stabilization of cortical actin filaments): in single, attached lactotropes in a mixed population of anterior pituitary cells.

Lactotropes spontaneously secrete PRL in the absence of hypothalamic control. Figure 7Go shows a typical profile of basal PRL secretion. Basal release of PRL increased linearly in a time-related fashion (Fig. 7Go, open circles). Exposure of cultures to the depolymerizing agent CD (1 µM) had a biphasic effect on PRL secretion (Fig. 7Go, filled circles). During the first 5 min, PRL secretion from CD-treated cultures was higher than control levels; at 10 min there was not significant differences between treated and control preparations, whereas longer exposure times to CD caused a progressive decline of PRL secretion. Viability of pituitary cells was affected by CD. After 5, 10, 15, 20, and 30 min incubation with 1 µM CD, cell viability measured by trypan blue exclusion was 92, 89, 86, 82, and 76%, respectively. The experimental data indicate that short-term (5 min) incubations with CD enhanced basal PRL secretion, whereas longer incubations seemed to have deleterious effects on the cells.



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  		Figure 7. Effect of CD on basal PRL secretion from anterior pituitary cell cultures. Cultures were incubated for increasing periods (5–15 min) in medium containing (filled circles) or not (open circles) 1 µM CD. PRL secretion was measured by RHPA. The surface of zones of hemolysis surrounding the cells (PRL secretion) was determined with the aid of a Videoplan image analyzer. Data shown are from a representative experiment. Values are the mean plaque area (µm2) ± SEM calculated from the area of hemolysis of 100–150 cells per experimental condition. The same experiment was performed four times with similar results. P < 0.05 CD, 5 min, vs. control, 5 min, and CD, 15 min, vs. control, 15 min.

 
Since the only known effect of CD is to depolymerize actin filaments, we speculated that actin polymerization facilitated the secretory response in CD-treated cells. Moreover, our fluorescence microscopy experiments showed that PRFs induced cortical actin disassembly. The experimental evidence suggests that PRF-induced actin disassembly is part of the mechanism involved in PRL secretion. Accordingly, we tested whether TRH and VIP were still able to stimulate PRL secretion in cells with a disrupted actin cytoskeleton. Dispersed pituitary cells were incubated with 100 nM TRH or 500 nM VIP in the presence or in the absence of 1 µM CD. Five-minute incubation with CD enhanced PRL secretion (Fig. 8Go A). The increase was due to a larger secretion of PRL by single lactotropes but also to an increase in the number of lactotropes secreting PRL (41% over control values). Treatment of cultures with 100 nM TRH for 5 min stimulated PRL to a similar extent as CD (Fig. 8AGo); however, TRH could not further increase PRL secretion in the presence of CD (Fig. 8AGo). VIP alone caused a 36% increase in PRL secretion, but cells exposed simultaneously to CD and VIP did not show a higher secretory response than cells incubated with VIP alone (Fig. 8AGo). Removal of tonic DA inhibition, also a powerful stimulatory signal for PRL secretion, did not cause an additional release of PRL in the presence of CD (Fig. 8BGo). The presence of neither TRH nor VIP enhanced PRL secretion induced by DA withdrawal or DA withdrawal plus CD (Fig. 8BGo). Interestingly, CD, TRH, VIP, DA withdrawal, and any combination of those compounds induced the release of similar amounts of PRL during a 5-min incubation period (Fig. 8Go). Moreover, none of the physiological PRFs potentiated the effect of CD on PRL secretion. Taken together, the results suggest that CD, TRH, VIP, and DA withdrawal elicited the exocytosis of a pool of secretory granules by inducing a common mechanism, which could be, perhaps, the disassembly of cortical actin filaments.



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  		Figure 8. Effect of CD on the ability of TRH, VIP, and/or DA withdrawal to stimulate PRL secretion. A, Anterior pituitary cells were treated for 5 min with DMEM alone (control) or containing either 1 µM CD, 100 nM TRH, or 500 nM VIP, the latter two also in the presence or absence of 1 µM CD. B, Cells chronically exposed to 500 nM DA (48 h) were incubated for 5 min with buffer containing DA (DA) or with DA-free buffer (DAw) alone or containing 100 nM TRH (DAw + TRH) or 500 nM VIP (DAw + VIP), each condition also in the presence or absence of 1 µM CD. In panels A and B, PRL secretion was evaluated by the RHPA and expressed as a percentage of basal release (control = 100%). Each bar represents the mean ± SEM of the percentage of plaque areas of 100–150 cells from three distinct experiments. *, P < 0.05 vs. control.

 
Involvement of the actin cytoskeleton in DA-mediated inhibition of PRL secretion
An interesting possibility is that the cortical cytoskeleton may mediate inhibition of secretion especially in cells such as the lactotropes where the secretory activity is tonically inhibited. Our fluorescence microscopy experiments indicate that exposure of lactotropes to DA stabilized the cortical actin cytoskeleton against depolymerization. Exposure of cultures to 500 nM DA for 48 h reduced basal PRL secretion by 27 ± 3%. Lower DA concentrations such as 50 nm and 100 nm induced 12 ± 3% and 16 ± 5% inhibition, respectively. DA treatment completely blocked the stimulatory effect of CD and TRH on PRL secretion (Fig. 9AGo). The presence of CD in the incubation medium slightly overcame DA inhibition on TRH-induced PRL secretion (Fig. 9AGo). VIP-induced PRL secretion was partially inhibited (Fig. 9AGo) by long-term exposure to DA (Fig. 9AGo). CD did not affect VIP-evoked PRL secretion in DA-treated cells (Fig. 9AGo).



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  		Figure 9. Role of the actin cytoskeleton on the inhibition of PRL secretion. Anterior pituitary cell cultures were exposed for 48 h to 500 nM DA (A) or for 5 min to 5 µM jasplakinolide (B). They were next treated for 5 min with different secretagogues in the presence of DA or jasplakinolide (Jasp). As controls, cultures not exposed to DA or jasplakinolide were incubated with CD, TRH, or VIP for 5 min. PRL secretion was assessed by RHPA and expressed as a percentage of basal release (control = 100%). Results are the mean ± SEM of percentages of plaque area of 100–150 cells from three independent experiments. Panel A: *, P < 0.05 vs. control; **, P < 0.05 CD vs. (DA + CD); 0, P < 0.05 (DA + TRH) vs. (DA + TRH + CD). Panel B: *, P < 0.05 CD or PRF vs. CD or PRH + Jasp.

 
To find out whether stabilization of actin filaments indeed blocked basal and induced PRL secretion, anterior pituitary cells were treated with jasplakinolide, a membrane-permeant peptide isolated from the marine sponge Jaspis sp. (33) that stabilizes actin filaments (34). Jasplakinolide compete with phalloidin for binding sites on actin filaments (34). Under our experimental conditions, 5-min incubation with 5 µM jasplakinolide was enough for the compound to enter the cells as revealed by the lack of actin filament staining by rhodamine-phalloidin but not by actin antibodies. Jasplakinolide treatment inhibited CD-induced actin disruption. Cell viability after 5 min treatment with 5 µM jasplakinolide was 95%. Figure 9BGo shows a histogram of the effect of jasplakinolide on PRL secretion. Jasplakinolide by itself decreased by 20% basal PRL secretion. Stabilization of actin filaments by jasplakinolide completely inhibited the stimulatory effect of CD, TRH, and VIP on PRL release (Fig. 9BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results suggest the cortical actin cytoskeleton of normal cultured lactotropes is involved in the control of PRL secretion. Cortical actin filaments were locally disassembled during stimulation of PRL secretion, and inhibition of actin disassembly inhibited basal and stimulated PRL release. Moreover, here we present evidence that the lactotrope cortical actin network seemed to mediate, at least partly, the tonic inhibition exerted by DA on PRL secretion.

The secretory granules of anterior pituitary cells are linked to each other and to the surrounding cytoskeleton (19). Some granules are next to the plasma membrane; others localize beneath the peripheral microfilaments or are trapped in this network (19, 20). Anterior pituitary secretory granules already docked at the plasma membrane may constitute a "release-ready" pool of granules, and those trapped in the network or lying behind the microfilaments may constitute a "reserve" pool of granules characterized by a slow rate of release as it has been shown in chromaffin cells (35) and melanotropes (36). Hormone secretion proceeds at a slower pace than neurotransmitter secretion (14), and most anterior pituitary secretory granules are excluded from the subplasmalemmal region (20). Therefore, local dismantling of the cortical actin network may be necessary for the excluded granules to reach the plasma membrane for exocytosis. Experimental evidence indicates that fragmentation of F-actin staining correlates with F-actin disassembly, while the continuity of the cortical F-actin staining correlates with actin filament assembly (5, 6). Our present fluorescence microscopy studies show that stimulation of lactotropes with PRFs led to a transient increase in the percentage of lactotropes displaying cortical actin disassembly. Transient disassembly of cortical actin filaments have also been described in synaptosomes (37), chromaffin cells (5, 6, 38), clonal PRL secreting cells (22), and pancreatic acinar cells (10). Other intracellular events occurring during stimulation of PRL secretion, such as elevation of [Ca]i (39), hydrolysis of phosphoinositides, and translocation of protein kinase C (40, 41, 42), are also transient. In the absence of tonic hypothalamic inhibition, lactotropes have a high rate of basal (nonstimulated) PRL secretion (26). Under such an experimental condition, i.e. cells incubated in DA-free medium for 24–48 h, we found peripheral actin filaments partially disassembled in 39 ± 4% of lactotropes examined (n = 400). This percentage correlates with the 43% of lactotropes having spontaneous Ca2+ action potentials (active lactotropes) recently reported by Ho et al. (43). The correlation suggests that active lactotropes may possess a disassembled cortical actin network, again indicating a possible association between actin disassembly and secretory activity.

The occurrence of cortical actin disassembly was independent of the second messenger pathway used by the different secretagogues tested, suggesting that different intracellular routes may converge at later steps, one of them being the cortical cytoskeleton. The maximal percentage of cells displaying actin disassembly observed after addition of TRH or removal of DA occurred during the first minutes of treatment when the increase in intracellular Ca2+ has been shown to take place (39, 43, 44). VIP caused a slower increase in the percentage of cells displaying cortical actin disassembly, which is consistent with the slow rise in intracellular cAMP induced by VIP (41). It has been previously shown in chromaffin cells that evoked actin disassembly is a Ca2+-dependent event (7), although other intracellular pathways, such as activation of protein kinase C (38), are also able to induce actin disassembly. Here, we observed that evoked actin disassembly was affected by removal of extracellular Ca2+ only when it was induced by VIP or DA withdrawal or under basal conditions. These results are in agreement with the experimental observation that, in the absence of Ca2+, lactotropes have spontaneous Ca2+ transients (43) and that DA withdrawal also induces the entry of extracellular Ca2+ (43). Moreover, DA withdrawal-induced PRL secretion is inhibited by blocking the entry of Ca2+ (43). The role of Ca2+ in VIP-induced PRL secretion is more controversial. In pituitary tumor cells, VIP induces Ca2+ transients (41) that are dependent on extracellular Ca2+ (45). Extracellular Ca2+ has been also shown to be necessary for VIP-induced PRL secretion (41). However, Pizzi et al. (46) observed that omission of Ca2+ from the incubation medium did not affect the ability of VIP to release PRL, although extracellular Ca2+ enhanced VIP-induced PRL secretion. Our observation that removal of external Ca2+ decreased, but did not abolish, VIP-induced increase in the number of cells showing cortical actin disassembly is in agreement with the report of Pizzi et al (46) that external Ca2+ enhances VIP effects on lactotropes. Furthermore, stimulation of adenylate cyclase by forskolin induced disassembly of cortical actin that was independent of extracellular Ca2+. Interestingly, it has been recently demonstrated that cAMP controls the movement of secretory granules in pancreatic ß-cells (47). Therefore, it is possible that VIP-induced actin disassembly involves both signaling pathways. TRH-induced actin disassembly was independent of extracellular Ca2+. Stimulation of lactotropes with TRH induces hydrolysis of phosphatidylinositol followed by an initial and transient release of Ca2+ from intracellular stores (48, 49), which is responsible for the initial phase of TRH-induced PRL secretion (39, 50). The results suggest that release of Ca2+ from intracellular stores mediates TRH-induced cortical actin disassembly.

Basal PRL secretion was not only stimulated by TRH and VIP but by CD as well. Exposure of cultures to TRH, VIP, or CD for the same incubation periods induced the release of similar amounts of PRL. TRH- and VIP-induced stimulation of PRL secretion were not additive to CD-induced secretion, in spite of the different second messenger systems used by TRH and VIP. Furthermore, jasplakinolide, an actin filament-stabilizing agent not only inhibited CD-induced PRL secretion but also TRH- and VIP-evoked PRL release. Since 1) the only known effect of CD is to depolymerize actin filaments (51, 52); 2) TRH and VIP also induced cortical actin disassembly; and 3) CD-, TRH-, and VIP-induced PRL release are blocked by stabilization of actin filaments, it is tempting to speculate that actin disassembly is part of the mechanisms underlying PRL secretion. In the absence of hypothalamic inhibition, actin depolymerization was sufficient to induce PRL secretion. A similar result was observed in pancreatic acinar cells (10); however, actin disassembly alone does not to induce catecholamine secretion in adrenal chromaffin cells (38). The discrepancy between the results may indicate a difference in the mechanism underlying fast secretion (neurotransmitters, mostly docked secretory vesicles) and slow secretion (hormones, mostly segregated secretory granules). However, as suggested by Muallem et al. (10), the fact that actin disassembly alone induces secretion may also indicate that some hormone- secreting cells release their respective hormones in a constitutive way and that the role of the cortical actin cytoskeleton is to be a barrier to constitutive exocytosis. This may be the case in lactotropes that are characterized by a high rate of basal secretion when freed from the tonic hypothalamic inhibition.

It is known that the balance between PIFs and PRFs determines the extent of PRL secretion (26, 53). PIFs and PRFs probably affect intracellular targets involved in the control of PRL secretion, i.e. the cortical actin cytoskeleton, in opposite ways, i.e. stabilization/disassembly, to inhibit/stimulate PRL secretion. Addition of DA to cells grown in DA-free medium decreased both the percentage of lactotropes with a partially disassembled cortical actin network and basal PRL secretion. Moreover, chronic exposure of cultures to DA blocked TRH-induced actin disassembly and PRL secretion, and CD partly overcame the inhibition. A complete different picture was observed when actin disassembly and PRL secretion were induced by VIP. The inhibitory effect of DA on VIP-induced actin disassembly was not obvious due to the large dispersion of the percentages of cells showing actin disassembly for each experimental condition. Chronic exposure of lactotropes to DA slightly decreased VIP-evoked PRL secretion. It is known that VIP-induced PRL secretion is delayed (31, 49). Moreover, it has been recently shown that VIP stimulates PRL secretion in an indirect way, by inducing secretion of galanin from a subpopulation of anterior pituitary cells (54). VIP-sensitive anterior pituitary cells are not affected by DA (54). Therefore, we should expect a delay in the onset of VIP-induced actin disassembly and a large dispersion in the percentages of lactotropes that displaying actin disassembly. In fact, forskolin, which also activates adenylate cyclase, was faster that VIP in inducing cortical actin disassembly.

In cells cultured in the absence of PRL-inhibitory factors, addition of DA slowly reduced cortical actin disassembly within minutes. When cells were exposed to DA for longer periods (at least 2–4 h), an increase in filaments’ stability vis-a-vis the depolymerizing effect of CD was noticed. The stabilizing effect of DA was exclusive to lactotropes and was observed with concentrations of DA as low as 50 nM. In cultures not treated with DA, lactotrope microfilaments were more sensitive to the deleterious action of CD than microfilaments of PRL-immunonegative anterior pituitary cells. Stabilization of actin filaments by glucorticoids was observed in an ACTH- secreting clonal cell line and suggested to mediate glucocorticoid-induced inhibition of ACTH secretion (24, 25). Our experiments performed in normal cultured lactotropes indicate that DA-induced stabilization of the cortical actin cytoskeleton may be implicated in the mechanism by which DA tonically inhibits PRL release. Indeed, exposure of the cells to DA for 48 h blocked the stimulatory effect of actin depolymerization on PRL secretion, suggesting that DA-induced stabilization of the cortical actin filaments is a barrier to constitutive PRL secretion. The stabilization of cortical actin filaments by DA was a slower process than the inhibition of cortical actin filament disassembly also by DA. Moreover, DA-induced inhibition of actin disassembly was slower than secretagogue-evoked disassembly of the same microfilaments. The relocalization of the endoplasmic reticulum of lactotropes, also suggested to be a physical barrier to PRL secretion, has been shown to take place within 2 min (55, 56). Nicotine-induced actin disassembly in chromaffin cells is also faster than actin reassembly (7). Therefore, intracellular signals involved in actin network disassembly during secretion might be faster than intracellular mechanisms necessary to induce polymerization and reassembly of actin filaments during inhibition of secretion.

In conclusion, the present work suggests the cortical actin cytoskeleton is an intracellular integrator of the multiple signals involved in the control of PRL secretion at the level of the lactotrope. The stabilization of the lactotrope’s cortical actin cytoskeleton may be part of the intracellular mechanism by which DA tonically inhibits basal PRL release.


   Acknowledgments
 
The authors are grateful to Dr. R.-M. Pelletier for helpful and critical comments during the preparation of the manuscript, to Dr. M. Bendayan for the use of the fluorescence microscope and the image processing system, and to Dr. I. Londoño for her advice in the use of the Videoplan. The technical assistance of Mrs. F. Dionne in the preparation of the anterior pituitary cell cultures is gratefully acknowledged. The authors would like to thank the National Hormone and Pituitary Program of the NIDDKD for the gift of PRL antibodies and M. Luc Parent from Sainte Justine Hospital (Montréal, Quebec, Canada) for the sheep blood.


   Footnotes
 
1 This work was funded by Grants MT-12879 from the Medical Research Council of Canada and CAFIR-U.Montréal (to M.L.V.). M.L.V. is supported by a scholarship from Fonds de la recherche en santé du Québec. Back

Received April 30, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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