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Department of Physiology, Southern Illinois University School of Medicine (L.A.A.), Carbondale, Illinois 62901-6512; and the Department of Physiology, University of Kansas Medical Center (J.L.V.), Kansas City, Kansas 66160-7401
Address all correspondence and requests for reprints to: Dr. Lydia A. Arbogast, Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901-6512.
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Abstract |
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Introduction |
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There is increasing evidence that PRL receptors are localized in the hypothalamus, although the neuronal cell types that contain these receptors have not been elucidated. PRL-binding sites are found in hypothalamic tissue (14) and are detected in the arcuate nucleus and/or median eminence by autoradiographic methods (15, 16, 17). PRL receptor messenger RNA (mRNA) is detected in the hypothalamus by in situ hybridization techniques (18, 19, 20), with a pattern of expression in the arcuate nucleus anatomically similar to that in the TIDA neurons (20). To date, two isoforms of the PRL receptor, long and short, have been identified in normal rat tissues (21). Both long and short forms of the PRL receptor are expressed in hypothalamic tissue, as determined by reverse transcriptase-PCR (RT-PCR) (22, 23), although the long form of the receptor is the predominant form (23). A major goal of this study was to determine the existence of PRL receptors on dopaminergic neurons.
Although dopaminergic neurons are present in hypothalamic cell cultures, TH activity was not stimulated by PRL administration when the cells were cultured in a serum-free, chemically defined medium (24, 25). In contrast, hypothalamic dopaminergic neurons were activated by PRL under culture conditions that included horse serum and/or FBS (26, 27). The stimulatory effect of PRL was specific to dopaminergic neurons from hypothalamic, but not mesencephalic, tissue (27). However, it is not clear whether the presence of serum or different experimental conditions between laboratories contributed to differences in the responsiveness to PRL.
Mechanistic studies to understand PRL’s action on dopaminergic neurons are dependent on establishing an adequate in vitro model. The objectives for this study were 1) to assess the effects of medium composition on the ability of PRL to increase TH activity in vitro, 2) to establish time and concentration dependency for PRL’s action on TH activity, 3) to determine whether PRL receptors are colocalized with TH in hypothalamic dopaminergic neurons, and 4) to examine the PRL receptor isoform(s) expressed in the hypothalamic cell cultures.
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Materials and Methods |
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Fetal hypothalamic cell cultures
Fetal hypothalamic cells were cultured as described previously
(25), using a modification of the method described by Ahmed et
al. (28). Briefly, 3–5 five pregnant rats were anesthetized with
ether, and the fetuses were removed through a midline abdominal
incision under aseptic conditions. The medioventral hypothalamus was
excised to an approximate depth of 1 mm with fine dissecting scissors
using horizontal cuts posterior to the optic chiasm and anterior to the
mamillary bodies and vertical cuts halfway between the median eminence
and hypothalamic sulcus. The tissue was immediately placed in sterile
dispersion buffer without phenol red at 4–10 C. As described
previously (25), the hypothalamic cells were dispersed and resuspended
in a modified high glucose Dulbecco’s medium containing 2.5%
heat-inactivated FBS and 5% heat-inactivated horse serum. The medium
for all experiments was phenol red free. For some cultures, the medium
contained 5.4 mM potassium (K+) provided with
the commercial medium, whereas for other cultures, additional potassium
chloride was added for a final concentration of 25 mM
K+. The cells were plated on surfaces that had previously
been coated with poly-D-lysine (100 µg/ml). For
determination of TH activity, the cells were plated at a density of
150,000 cells/0.2 ml·well in 96-well plates. For RNA extraction, the
cells were plated at a density of 1 x 106 cells/1
ml·well in 24-well plates. For immunocytochemistry, the cells were
cultured at a density of 400,000 cells/0.4 ml·well in 8-well Lab-Tek
Permanox chamber slides (Life Science Products, Denver, CO). Eighteen
to 24 h after plating the cells, the medium was replaced with
serum-free, modified, high glucose Dulbecco’s medium supplemented with
ITS+ (1:100; Collaborative Biomedical Products, Bedford,
MA), 3.5 µM linolenic acid, and 10 µM
putrescine. For serum-containing medium, 5% heat-inactivated FBS was
also added. The K+ concentration was adjusted as indicated
for individual experiments. The medium was changed daily, and rat PRL
(rPRL; B-7, National Hormone and Pituitary Program) was included so
that the indicated treatment would be completed on day 10 in
vitro.
TH activity
TH activity was determined on day 10 of culture. The
hypothalamic cells were preincubated for 15 min in phenol red-free
Earles’ Balanced Salt Solution (Life Technologies, Grand Island, NY)
to which 20 µM tyrosine, 20 mM potassium
chloride, and rPRL, when indicated, were added. The preincubation
medium was removed, and 100 µl medium containing 100 µM
brocresine (4-bromo-hydroxybenzyloxyamine; gift from American Cyanamid
Co., Pearl River, NY), an aromatic amino acid decarboxylase inhibitor,
was added. The cells were then incubated for 60 min under a humidified
atmosphere of 90% air-10% carbon dioxide, with rPRL included as
indicated. At the end of the incubation period, the medium was removed,
acidified with 10 µl 1 N perchloric acid, and frozen
until samples were analyzed for medium
L-dihydroxyphenylalanine (DOPA) accumulation by HPLC with
electrochemical detection as described previously (6, 25).
Double labeled immunocytochemistry
On day 10 of culture, hypothalamic cells were fixed for 15 min
in 4% paraformaldehyde in PBS and washed twice in PBS for 5 min.
Nonspecific binding was blocked with 10% normal horse serum and 10%
normal donkey serum in PBS for 1 h. After rinsing three times with
PBS, the cells were incubated successively with avidin block and biotin
block (Vector Laboratories, Burlingame, CA) for 15 min and then rinsed
with PBS. The cells were incubated for 24 h at 4 C with mouse
monoclonal antibodies against the rPRL receptor (U-6; 5 µg/ml; gift
from Dr. Paul Kelly, INSERM, Paris, France) and/or a rabbit polyclonal
antiserum against rat TH (1:1250, East Acres Biologicals, Southbridge,
MA) diluted with 2% normal horse serum, 2% normal donkey serum, and
0.3% Triton X-100 in PBS. The PRL receptor antibody recognizes an
epitope on the extracellular dopamine distinct from the binding domain
(29) and, therefore, would recognize both the occupied and unoccupied
receptors of the long and short forms of the PRL receptor. Control
wells were incubated in the absence of antiserum or with mouse IgG1
.
In separate experiments, the fixed cells were incubated with PRL
receptor antibodies (U-6; 5 µg/ml) and either rabbit
antineuron-specific enolase (NSE; Incstar, Stillwater, MN) or rabbit
antiglial fibrillary acidic protein (GFAP; Incstar) at a concentration
supplied by the manufacturer. After rinsing six times with PBS at room
temperature, the cells were incubated for 1 h with a serum block
as described above, then for 1 h at room temperature with
biotinylated horse antimouse IgG (1:200; Vector Laboratories) and
fluorescein (dichlorotriazinylamino fluorescein)-conjugated donkey
antirabbit IgG (1:80; Jackson ImmunoResearch Laboratories, West Grove,
PA). After rinsing six times with PBS, the cells were incubated for
1 h at room temperature with lissamine rhodamine (lissamine
rhodamine B sulfonyl chloride)-conjugated strepavidin (22.5 µg/ml).
After rinsing six times with PBS, coverslips were mounted with
Fluoromount G. The immunostaining was visualized with a Nikon Optiphot
microscope (Nikon Corp., Melville, NY) using a mercury lamp with a blue
(492 nm) or green (570 nm) excitor filter block for fluorescein or
rhodamine, respectively.
PRL receptor RT-PCR
RNA was isolated by the method of Chomczynski and Sacchi (30) as
modified for the RNAzol B method (Tel-Test, Friendswood, TX).
Hypothalamic cells (2 x 106 cells) were lysed by
adding 0.8 ml RNAzol B reagent. RNA was extracted according to
manufacturer’s instructions and stored at -70 C. Complementary DNA
(cDNA) was synthesized from RNA by the SuperScript Preamplification
System for First Strand cDNA Synthesis (Life Technologies).
Oligonucleotide primers for the long and short forms of the PRL
receptor were synthesized by the Biotech Center at the University of
Kansas Medical Center and were identical to those used by Shirota
et al. (31). The primers for the short form of the receptor
recognized nucleotides at positions 624–653 and 924–953. The first
primer for the long form of the receptor was the same as the first
primer for the short form, whereas the second primer recognized
nucleotides at position 1014–1043 of the long form of the receptor.
These primers generate PCR products of 330 and 420 bp for the short and
long forms of the PRL receptor, respectively. These same primers have
been used for detection of PRL receptor mRNA in the rat hypothalamus
and pituitary (22, 23). cDNAs were amplified through 30 cycles using
Taq polymerase (22). After amplification, the samples were
electrophoresed on 1.5% agarose gels, and the PCR product was
visualized with ethidium bromide staining and compared to a known
standard for size determination. As negative controls, reactions were
performed with RNA that had not been reverse transcribed and with
samples lacking RNA in the RT reaction. Aliquots of the PCR products
were incubated for 1 h at 60 C with BsaBI to evaluate
digestion by an appropriate restriction endonuclease.
Statistical analysis
To normalize data, experimental values were adjusted to a
percentage of the control value for individual experiments. The control
value was the mean of 12 wells, and each experimental value was the
mean of triplicate wells for each hypothalamic culture. The results are
expressed as the mean ± SE of determinations from
four to nine different experiments. The percentage of immunoreactive
cells was quantified by counting the total number of cells and the
number of fluorescein- or rhodamine-labeled cells in 20 randomly
selected microscope fields under x400 magnification. The percentage of
labeled cells for each experiment was determined, and a final mean was
calculated form all eight experiments. Data were evaluated by ANOVA,
and multiple comparisons were made with Fisher’s least significant
procedures (32, 33).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). rPRL did not
alter TH activity in cells cultured with 5 mM
K+ in either defined or serum-containing medium (Fig. 1).
All subsequent experiments were performed with medium containing 25
mM K+ and 5% FBS.
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). The lowest
PRL concentration (1 ng/ml) did not cause a significant increase in TH
activity (Fig. 2). The time course for PRL’s action on dopaminergic
neurons was evaluated from 0–10 days. TH activity was 126%
(P = 0.06) of control levels after 12 h of rPRL
treatment (Fig. 3). TH activity was significantly
increased (P < 0.05) after 1 day of rPRL exposure and
remained elevated for 3 days. An additional increase in enzyme activity
was observed between 3 and 5 days of rPRL treatment, and TH activity
remained elevated after continuous exposure for 7 and 10 days.
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shows a number of TH-immunoreactive cells (fluorescein
label) that are also immunoreactive for PRL receptors (rhodamine label)
in both defined (Fig. 4, A and B) and serum-containing (Fig. 4, C and
D) media. The percentage of total cells that were immunopositive for TH
was 6.7 ± 0.6% (n = 8) or 6.8 ± 0.4% in defined or
serum-containing medium, respectively. Greater than 95% of
TH-containing cells were also immunopositive for PRL receptors.
However, a few TH-immunopositive cells did not show PRL receptor
immunostaining (Fig. 4A). PRL receptor immunostaining was also observed
in non-TH-containing cells. Indeed, 18.9 ± 2.3% or 24.2 ±
2.3% of the cells in defined or serum-containing medium were
immunopositive for PRL receptor. The TH-immunoreactive and PRL
receptor-immunoreactive cells were only subpopulations, as the majority
of cells were not labeled for either antigen. Similar results were
observed in wells treated with PRL (100 ng/ml) for 10 days (data not
shown). Both fluorescein and rhodamine staining were absent when the
primary antisera were omitted (Fig. 4, E and F, respectively) or when
mouse IgG1 was included as a control (data not shown).
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, A and B) and serum-containing medium (data not
shown). In contrast, there were no PRL receptors found in
GFAP-immunolabeled cells in either defined (Fig. 5, C and D) or
serum-containing medium (data not shown). There was no detectable
staining in control wells when the primary antiserum was omitted.
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, left panel).
Similarly, expression of the long form of the PRL receptor was also
evident in MBH tissue from adult ovariectomized rats. The mRNA for the
short form of the PRL receptor was not detectable or was only faintly
detectable in fetal hypothalamic cells with any treatment paradigm or
in adult MBH (Fig. 6, right panel). As a positive control,
both the long and short forms of the PRL receptor were expressed in
liver tissue from adult ovariectomized rats (Fig. 6). No PCR product
was detected when RT was omitted (Fig. 6) or when RNA was absent from
the RT reaction (data not shown). After digestion with
BsaBI, the 420-bp PCR product for the long form was cut into
the predicted 204- and 216-bp DNA fragments and the 330-bp PCR product
for the short form was cut into the predicted 204- and 126-bp DNA
fragments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The colocalization of PRL receptors and TH provides the first anatomical evidence for a direct effect of PRL on dopaminergic neurons. Indeed, immunostaining for the extracellular domain of the PRL receptor was observed in almost all of the TH immunoreactive neurons. However, as other, as yet unidentified, neuronal cells in the hypothalamic cultures were also immunopositive for PRL receptor, an indirect effect cannot be completely ruled out. Indeed, some neuropeptides alter TIDA neuronal activity in vivo (35, 36), and the neuronal activity of at least some of these is altered by PRL (7, 37). The cultured cells in this study were from the medioventral hypothalamus, which in the adult rat contains TIDA neurons (38). Thus, it is likely that dopaminergic cells in the fetal hypothalamic cell cultures correspond to TIDA neurons. Indeed, Beyer et al. (27) observed a PRL-induced increase in TH activity in cultured cells from the hypothalamus, but not in those from the midbrain. It is not clear whether the PRL receptor and TH colocalization observed in the fetal cultures is applicable to the adult rat. However, it is notable that PRL binding, PRL receptor immunostaining, and PRL receptor mRNA have been identified in the arcuate-median eminence region of the hypothalamus (15, 16, 17, 18, 19, 20). Although the expression of PRL receptor is widespread in the rat fetus and increases late in gestation (39), it is not known whether PRL receptor expression in the culture cells reflects the cell source or if expression is induced during the 10 days in vitro.
In addition to PRL receptor protein identified by immunocytochemistry, PRL receptor mRNA was identified in the hypothalamic cell cultures by RT-PCR. There are at least two PRL receptor isoforms, short and long, expressed in rat tissues (21). Although both forms of the PRL receptor are functional, there is differential regulation of expression of the two forms, but no functional variation is yet known. In the present study, only the long form was detected by RT-PCR in fetal hypothalamic cells and in adult MBH tissue of ovariectomized rats. No PCR product for the short form was detected in six different hypothalamic cultures or six different MBH samples, even though a strong signal for the short form was seen in liver tissues. These data indicate that, similar to the adult hypothalamus, the long form is the predominant form in the hypothalamic cell cultures, indicating that the cell cultures may be appropriate to examine the mechanism for PRL’s action on dopaminergic neurons. Both the long and short forms of the receptor have been identified by RT-PCR in the hypothalamus of estrogen-treated ovariectomized rats (22) and in cycling female rats (23) using the same primer pairs. However, 99% of PRL receptor mRNA in the hypothalamus encodes for the long form of the receptor in diestrous and proestrous rats (23). The inability to detect the short form of the PRL receptor in the present study may be due to the relative sensitivity of ethidium bromide staining or to the lack of estrogen or ovarian input. Indeed, ovariectomy decreases PRL binding in the hypothalamus, whereas estrogen reverses this decrease (14).
The concentration dependence and time response for PRL’s action on TH activity in the hypothalamic cell cultures was very similar to the in vivo effect of PRL on TIDA neuronal activity. The effective concentrations of PRL were 10–1000 ng/ml, which would correspond to basal circulating levels (10 ng/ml) and the range of circulating PRL levels (100–1000 ng/ml) during the PRL surges associated with reproduction. The PRL concentration (1 ng/ml), similar to bromocriptine-suppressed circulating PRL levels, did not significantly alter TH activity. A significant increase in TH activity was observed between 12 and 24 h of PRL exposure. This is very similar to the time course required for in vivo PRL treatment to increase TIDA neuronal activity (2). Indeed, Johnston et al. (40) reported that protein synthesis was required early during the delay period for PRL’s action, as cycloheximide administered during the first 4 h after PRL administration prevented the increase in TH activity.
It was somewhat surprising that PRL receptor expression could not be correlated with the responsiveness of dopaminergic neurons to PRL stimulation. Dopaminergic cells cultured in a serum-free, chemically defined medium expressed PRL receptors, but did not respond to PRL treatment. The confirms previous data from our laboratory (25) and from that of Porter and colleagues (24) that there was no significant increase in TH activity in response to PRL treatment with cells in defined medium. All cultures were started in serum-containing medium for 18–24 h, but this brief exposure did not impart PRL responsiveness to the cells if they were subsequently cultured in defined medium for 9 days. In contrast, inclusion of FBS in the maintenance medium for the entire 10 days conferred PRL responsive to the dopaminergic neurons. Indeed, Sarkar (26) and Beyer et al. (27) previously reported an increase in TH activity with cells cultured in serum-containing medium. Thus, it appears that a serum factor contributes to the ability of cultured dopaminergic neurons to respond to PRL by a mechanism other than altered PRL receptor expression. However, we cannot rule out the possibility that other cell types were responsive to PRL in the cultures, even with defined medium.
Understanding of the signal transduction pathways for the PRL receptor has increased remarkably during the last few years, and these are currently being intensely investigated in a number of cellular models (21, 41, 42, 43, 44, 45, 46, 47). It is not clear whether components of the signal transduction pathways are altered in the dopaminergic neurons during conditions of nonresponsiveness vs. responsiveness to PRL, but this hypothesis requires further investigation. PRL-responsive and -nonresponsive states are not unique to the cell culture system. There are in vivo precedents for TIDA neurons to become unresponsive to PRL stimulation during normal physiological changes and in response to hormonal manipulations. TIDA neurons do not respond to PRL treatment during midlactation (12, 13) or in estrogen-treated ovariectomized rats (48). This is somewhat paradoxical because estrogen stimulates PRL receptor mRNA in peripheral tissues (49) and increases PRL binding in the hypothalamus (14). Moreover, PRL receptor mRNA levels are greatest in the mammary gland during lactation (50). Although a role for decreased PRL receptor expression cannot be entirely dismissed without further investigation, these data do not support the idea that decreased PRL receptor expression contributes to the unresponsive state of TIDA neurons to PRL stimulation in vivo.
In conclusion, the colocalization of PRL receptor and TH supports a direct effect of PRL on dopaminergic neurons, but an indirect effect resulting from PRL’s action on another neuronal cell type(s) cannot be ruled out. Identification of the other PRL receptor-containing neurons could contribute to our understanding of PRL’s action in hypothalamic cells. The lack of correlation between PRL receptor expression in dopaminergic neurons and degree of responsiveness to PRL stimulation raises significant questions. Investigation of the intracellular events that occur within the dopaminergic neurons in response to PRL stimulation and which of these events are compromised in the nonresponsive state would aid in our understanding of PRL’s action.
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Acknowledgments |
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Footnotes |
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Received October 14, 1996.
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