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Program in Neuroscience, Department of Biological Science, Florida State University, Tallahassee, Florida 32306
Address all correspondence and requests for reprints to: Dr. Béla Kanyicska, Department of Biological Science, Biomedical Research Facility, Florida State University, Tallahassee, Florida 32306-4340. E-mail: bela{at}neuro.fsu.edu
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These data demonstrate that a large proportion of lactotrophs is capable of expressing and secreting ET-like peptides in biologically significant quantities. As low pituitary cell density in reverse hemolytic plaque assay minimizes cell to cell communications, these findings constitute direct proof of autocrine regulation of PRL secretion by ET-like peptides.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The evidence suggesting that ETs regulate pituitary hormone secretion is especially impressive. For instance, both ETs and their receptors are expressed in the pituitary gland and in the hypothalamic magnocellular nuclei (15, 16, 17, 18, 19). In addition, ET-like peptides have been detected in conditioned medium of long term pituitary cell culture (20). Moreover, it has been found that ET-like peptides are powerful modulators of hormone secretion from cultured pituitary cells, as they diminish PRL secretion and enhance LH, FSH, and TSH secretion (21, 22, 23, 24).
The source of ETs affecting hormone secretion from the anterior lobe of the pituitary gland in vivo has not yet been identified unequivocally. The magnocellular neurons of hypothalamic supraoptic and paraventricular nuclei have been considered potential sources of ET-like peptides (16, 21). It seemed conceivable that these neurons might provide ETs to the anterior lobe in a way similar to their delivery of oxytocin and vasopressin (25, 26, 27), specifically through their axon collaterals to the median eminence or by releasing ET in the posterior lobe from where it can reach the anterior lobe by either the long or short portal system, respectively. There is diminished enthusiasm for these possibilities, however, because ETs released by the magnocellular neurons will probably influence the blood flow through the portal vascularizations affecting nonselectively the entire neurohumoral input to the anterior lobe. Indeed, several experiments on pituitary (28) and nonpituitary tissues (14) indicated that ETs are produced locally. It is now thought that ETs, under physiological circumstances, do not reach their target cells through the general circulation (14). Taken together, these studies indicate strongly that ETs subserve a role as intercellular paracrine and/or autocrine messengers in the pituitary gland. However, no direct evidence for such a physiological role of ETs has yet been provided.
In the present study we sought to establish a role for ETs as autocrine regulators of PRL secretion. First, we applied immunocytochemical and reverse hemolytic plaque assay methods to demonstrate that lactotrophs are capable of expressing and releasing ET-like peptides. Second, we used a pharmacological blockade of ET receptors to investigate whether ETs, released by lactotrophs, can influence PRL secretion in an autocrine fashion. Previous studies found relatively high ET3 immunoreactivity in rat as well as human pituitary tissue (29, 30). Moreover, the release of ET1 and ET3 from long term pituitary culture is regulated differentially by insulin-like growth factors (20). These data demonstrated the presence of both ET1 and ET3 in the pituitary and indicated that ET3 might have a physiological role distinct from that of ET1. As we have previously found that ET1 is about 4 orders of magnitude more potent on lactotrophs than ET3 (31), we focused on ET1-like peptides in this study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunocytochemical detection of PRL and ET in sections of anterior
pituitary glands
An immunofluorescent double labeling technique was applied on
fixed pituitary tissue to demonstrate cellular colocalization of PRL
and ET1. Twenty-micron coronal cryostat sections were mounted on
gelatin-subbed glass slides and incubated with 10% normal horse serum
in PBS containing 0.4% (vol/vol) Triton X-100 and 0.1% (wt/vol)
sodium azide for 30 min at room temperature to prevent nonspecific
binding of antibodies. PRL immunoreactivity was visualized using a
rabbit polyclonal antiserum (NIH antirat PRL IC-5; 1:20,000) followed
by CY3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). ET-like immunoreactivity
was detected by applying monoclonal mouse anti-ET1 antibody (1:5,000;
MCE-6901-01, Peninsula Laboratories, Inc., Belmont, CA)
followed by CY2-conjugated donkey antimouse IgG (1:400; Jackson
ImmunoResearch Laboratories). Images were obtained with a Zeiss LSM 410
confocal laser scanning microscope (Zeiss, New York, NY) equipped with
two external (568 and 488 nm) argon-krypton lasers. Images were
acquired with a Plan-Neofluar 63/1.4 objective lens in a dual scan mode
with Z-sectioning (each optical section was 0.6 µm). The
colocalization was analyzed on the overlayed red and green images using
LSM 410 software (Zeiss) as previously described (33).\.
Measuring PRL secretion from individual lactrotrophs by reverse
hemolytic plaque assay (RHPA)
Cunningham chambers were constructed, and the hemolytic
plaque assay was carried out as described previously (34, 35). Briefly,
pituitary cells mixed with protein A-coupled ovine red blood cells were
plated on poly-L-lysine-coated microscope slides (1 ml 2%
ovine red blood cells contained approximately 5 x 104
pituitary cells). After 1-h equilibration in a humidified
CO2 incubator, the cells were washed with approximately 400
µl DMEM-BSA (Life Technologies, Gaithersburg, MD)
containing 1 mg/ml BSA (fraction V, Sigma Chemical Co.,
St. Louis, MO). Subsequently, the chambers were filled with DMEM-BSA
containing antirat PRL antiserum (1:50; provided by G. M. Nagy)
and different concentrations of ET receptor antagonists or other test
materials (listed below). Chambers filled with DMEM-BSA containing only
the PRL antiserum served as controls. Each experiment contained three
to five replicates of the same treatment. After 4 h of incubation,
hemolytic plaques were developed by applying guinea pig complement for
30 min (1:50; Life Technologies). The complement’s action
was stopped by infusing PBS containing 4% formaldehyde and 0.1%
glutaraldehyde into the chambers. After 1 h at room temperature,
the chambers were kept at 4 C overnight. The following day, the
fixative was removed with a wash of PBS, and the slides were stored in
PBS at 4 C until ICC. Compounds tested in PRL RHPA were the selective
ETA receptor antagonists BQ123,
cyclo(D-Asp-Pro-D-Val-Leu-D-Trp),
and BQ610,
(N,N-hexamethylene)carbamoyl-Leu-D-Trp(CHO)-D-Trp
(36, 37); the selective ETB receptor antagonist BQ788,
N-cis-2,6-dimethylpiperidinocarbonyl-L-
-methyl-Leu-D-1-methoxycarbonyl-Trp-D-Nle
(38); and ET-converting enzyme inhibitory peptide (ECi),
[D-Val22]big ET1-(16–38), purchased from
Peninsula Laboratories. ET1, the most potent
ETA receptor agonist (31), was obtained from Sigma Chemical Co.\.
Characterization of ET-secreting lactotrophs by ET specific
RHPA
The hemolytic plaque assay for ET was performed essentially as
described above, except that the anti-PRL antiserum was replaced by an
anti-ET antiserum. During the development of the ET plaque assay,
several anti-ET antisera from varying sources were tested in incubation
for 3, 6, 12, 18, and 24 h in concentrations of 1:25, 1:50, 1:100,
1:200, 1:400, and 1:800. We found two polyclonal rabbit anti-ET1
antisera (IHC-6901 and IHC-6910, both from Peninsula Laboratories) suitable for RHPA in 1:25 to 1:50 concentrations,
whereas the applications of monoclonal anti-ET1 antibodies (MCE-6901-01
from Peninsula Laboratories and MET-1 from
Accurate Chemical & Scientific Instruments Corp.,
Westbury, NY) did not result in hemolytic plaques (Table 1
). The earliest plaque formation was
detected after 6 h of incubation. The incidence of plaque-forming
pituitary cells increased up to 18 h, but extending the incubation
time to 24 h did not result in a further increase in the number of
plaques.\.
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Data collection and analysis
Plaques were examined using a Microphot-FXA microscope
(Nikon) equipped with a x4 phase contrast objective (used
for measuring PRL plaques) and a x20 DIC objective (used for ET1
plaque measurements). Plaques were always verified by visual inspection
before measurements were made, and only plaques with a single pituitary
cell in the center surrounded by red blood cell "ghosts" were
accepted. Numerical values of each plaque area (square microns) were
obtained individually by ImagePro Plus software and transported to a
Microsoft Corp. (Redmond, WA) Excel spreadsheet.
For statistical analysis, one-way ANOVA followed by Dunnett’s multiple
comparison test were applied using the Prism 2.01 program from
GraphPad Software, Inc. (San Diego, CA), where
P < 0.05 was considered the threshold of significance.
For further analysis of cell populations, logarithmically binned data
were analyzed by the Pstat program within the pClamp6 software package
(version 6.03, Axon Instrument Co., Foster City, CA). Each dataset,
representing different treatment groups, was fitted with Gaussian
curves using the Levenberg-Marquart least squares method. The first,
second, and third orders of fitted Gaussian curves within each groups
were compared statistically using Pstat. The higher order was accepted
only if it resulted in a significantly improved fit (signified by
F > 3.0).
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Results |
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). All
of the antisera capable of recognizing ET1 gave positive staining on
pituitary sections (Table 1
). The staining for ET-like immunoreactivity
was completely eliminated when the antisera were preabsorbed with 10
µg/ml ET1 or when the primary antiserum was omitted from the staining
protocol (Table 1). The monoclonal anti-ET1 antibodies (either
MCE-6901-01 or MET-1) and the polyclonal antiserum specific for ET1
(IHC-6901) showed similar staining patterns and complete colocalization
(data not shown).
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). On fixed tissue sections, we estimated
that approximately 5% of the lactotrophs were stained positive for
ET1. This appraisal of the incidence of ET-expressing lactotrophs is
comparable with data obtained previously from dispersed pituitary cells
plated on microscope slides (39).
Individual lactotrophs secrete an ET1-like substance(s)
By using protein A-coupled sheep red blood cells in conjunction
with rabbit anti-ET antisera that recognize ET1 (either IHC-6901 or
IHC-6910, see Table 1), we were able to detect hemolytic plaque
formations around PRL-immuno-positive cells (Fig. 2), thus demonstrating that lactotrophs
are capable of releasing an ET1-like immunoreactive substance(s).
To compare ET release from lactotrophs of male and female origins,
plaque assays were run in parallel sessions with cells obtained from
male or female animals. After an incubation of 24 h in the
presence of ET1-specific antiserum (IHC-6901), many ET plaque-forming
lactotrophs were detected in both cases. After enumerating lactotrophs
based on their PRL immunoreactivity, we found that 24.6% of
lactotrophs from males (1221 of 4963) and 43.9% of lactotrophs from
females (825 of 1879) released an ET1-like substance(s) in a quantity
sufficient to initiate hemolytic plaque formation. The mean plaque area
was significantly greater in the case of females: 1299 ± 40
vs. 758 ± 16 µm2 (Fig. 3A). The relative frequency of
logarithmically binned plaque sizes can be fitted best by a simple
Gaussian function (Fig. 3B), suggesting a homogeneous population of
ET-secreting lactotrophs. It is noteworthy that a sizable proportion of
the ET plaque-forming cell population (56% in males and 34% in
females) was not immunopositive for PRL. This observation indicates
that ET-releasing capacity is not exclusive to lactotrophs, and ETs are
probably used for intercellular communication by other pituitary cell
types as well.
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). Thus endogenous ET-like peptides
released by lactotrophs are indeed capable of effecting PRL secretion.
Consistent with the previously established ETA receptor
dominance in lactotrophs (31), the ETB-specific antagonist,
BQ788, was ineffective in changing PRL secretion (Fig. 4). When
endogenous ET1 formation was impaired by ET convertase enzyme
inhibitory peptide [D-Val22]big ET1-(16–38)
(42), the average PRL plaque size was increased by 56.9 ± 14.1%
(Fig. 4). Exogenous ET1 at a concentration of 10-8
M inhibited PRL secretion by 30.5% (Fig. 4), indicating
that the responsiveness of lactotrophs to ETs in a plaque assay
preparation is comparable to that of lactotrophs in monolayer cultures
(24, 31). The inhibitory effect of ET1 at a higher
concentration(10-6 M) was diminished (Fig. 4), suggesting that under RHPA conditions, lactotrophs possess similar
biphasic biological responsiveness to the ETs described previously
using different culture conditions (28, 43, 44). The average number of
PRL plaques in the control group was 212 ± 27 plaques/slide
(n = 5), constituting approximately 60% of the total number of
lactrotrophs determined by ICC. Masking the effects of endogenous ETs
by either ETA antagonists or an ET convertase inhibitory
peptide did not affect the overall incidence of plaque-forming
lactotrophs.
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). However, there were salient
differences between dose-response curves of BQ123 on lactotrophs
obtained from female vs. male rats (Fig. 5). In lactotrophs
obtained from diestrous females, the effect of BQ123 on PRL secretion
followed a bell-shaped dose-response curve, reaching a maximum at
10-7 M (155.1 ± 14.5%), whereas at
higher concentrations, the effect of BQ123 on PRL secretion gradually
diminished (Fig. 5A). In lactotrophs obtained from males, the
BQ123-induced increase in PRL secretion followed a sigmoid
dose-response curve with an estimated half-maximum concentration of
1.34 x 10-8 M BQ123. The maximum
increase in PRL secretion was 242.0 ± 17.4% of that in the
untreated groups (Fig. 5B).
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). After retransformation of the maxima
of the fitted Gaussian functions, estimates for the mean plaque area of
the two populations were 1,180 and 6,531 µm2 (female) and
946 and 4,852 µm2 (male). In the presence of
10-7 M BQ123, the smaller plaque-forming cell
population disappeared, and populations in both cases could be fitted
best with a first order Gaussian function, where estimated maxima were
5,956 µm2 (female) and 9,862 µm2 (male). At
higher BQ123 concentrations (10-6 or 10-5
M), the smaller plaque-forming population (with a maximum
at 1,762 µm2) reappeared among female lactotrophs (Fig. 6), resulting in a decrease in the overall secretory activity as
reflected by the descending phase of the bell-shaped dose-response
curve presented in Fig. 5A. In males, treatment with higher
concentrations of BQ123 also revealed two population of lactotrophs
(Fig. 6). In this case, however, increased maxima of the large
plaque-forming group (15,452 µm2) offset the reappearance
of the smaller plaque-forming group (4,045 µm2),
resulting in a small overall increase in PRL secretion, as reflected by
the saturation phase of the dose-response curve shown in Fig. 5B.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have not identified the immunoreactive ETs unequivocally as ET1 in these studies. However, based upon the results with antisera having differential cross-reactivities for ET1, ET2, and ET3, we have reason to believe that the staining observed in pituitary sections is due to the presence of ET1 and/or its precursors. We can be less certain of the identity of the released ET-like material detected by the RHPA, although a similar assumption that the released substance is ET1 seems justified. The relatively low incidence of double labeled lactotrophs can be related to the fact that the steady state ET1 concentration in the pituitary gland is very low (29). The low tissue concentration of ETs is not surprising considering that the half-maximal concentration of ET1 affecting PRL secretion is around 10-15 M (31). Therefore, assuming an autocrine or paracrine mode of action, very little ET1 would be needed to act at such short distances from its source.
We concluded from the immunocytochemical data that lactotrophs are capable of producing ET-like peptides, although only a small proportion of the cells have the cellular level of ETs sufficiently high to be detectable by immunocytochemical methods. However, these observations should not detract from considering endogenous ETs as important regulatory peptides within the pituitary gland, as the number of immunocytochemically identified ET-positive cells probably underrepresents the true number of lactrotrophs that are capable of expressing and releasing ET-like peptides. Indeed, by using the ET-specific RHPA method, we found that the number of lactotrophs in which ET release was detectable by hemolytic plaque formation vastly outnumbered the number of ET-positive lactotrophs revealed by ICC. The discrepancy in the data obtained with the two methodological approaches can be resolved by considering that the incidence of ET-positive cells is determined by the intracellular ET contents at the moment of fixation, whereas in the plaque assay the incidence of plaque-forming cells depends on the rate of ET-like peptide secretion and the length of the incubation.
The RHPA was first developed to measure antigen secretion at the single cell level (46) and was adapted later for detection and measurement of hormone secretion from individual pituitary cells in culture (34, 47). This method has became indispensable in studying hormone secretion at the single cell level, and its applications provided important insights concerning heterogeneity and population dynamics of different hormone-producing phenotypes in the pituitary gland (48, 49, 50, 51, 52, 53, 54). However, attempts to develop a hemolytic plaque assay for small peptides (e.g. vasoactive intestinal polypeptide) have been unsuccessful to date. Rather serendipitously, we found two polyclonal anti-ET antisera that, in the presence of ET-like antigen and guinea pig complement, are capable of inducing hemolytic plaque formation. The observed ET plaques compared with PRL plaques are rather small, but easily detectable. As the presence of ET receptors has already been detected in lactotrophs (19, 31), demonstration of the release of ETs from individual lactotrophs immediately suggested the possibility of autocrine regulation of PRL secretion by ET-like peptides. It is not yet known how the synthesis, storage, and secretion of ETs are regulated in the lactotrophs or whether the regulation of ET release from the lactotrophs parallels that of PRL secretion. Similarly to that of vascular endothelial cells (14), the regulation of ET synthesis and release is probably coupled in the lactotrophs as well; hence, ET would only be produced in substantial amounts if there is a demand for ETs, presented by a heretofore unidentified stimulus. This scenario could explain why only a small amount of ET1 is actually stored intracellularly.
Interestingly, lactotrophs obtained from cycling female rats showed signs of more vigorous ET secretion than lactotrophs obtained from males. The physiological significance of these observations is not clear at present. It is tempting to speculate, however, that ETs play a role in setting the responsiveness of lactotrophs to hypothalamic PRL-releasing factors. As the action of ETs on PRL secretion is predominantly inhibitory, a higher incidence of ET-secreting lactotrophs in cycling female rats seems compatible with the idea that the overall responsiveness to PRL secretagogues is lower in females than in males (except perhaps a short time window preceding the proestrous PRL surge).
Although our data strongly support a role for ET in autocrine regulation of PRL secretion, they do not rule out other possibilities. As lactotrophs constitute a majority of the cells in the anterior lobe of the pituitary gland, and many of the lactotrophs actively secrete ETs, our data imply that a paracrine type of interaction among lactotrophs through ETs may also exist. In addition, the fact that ET plaque formations were detected among nonlactotrophs leaves open the possibility of an ET-mediated paracrine type of communication between lactotrophs and other, heretofore unidentified, cellular phenotypes.
It has been recognized earlier that the RHPA offers an ideal approach to investigate autocrine regulation, because by applying this method, the secretory activity of a single cell can be assessed quantitatively and without interference from neighboring cells (55, 56, 57). In the present study, we have found that pharmacological blockade of ETA receptors increased PRL secretion. The plausible interpretation of the observed effects with the ETA receptor antagonists is that these compounds relieve the cells from inhibition exerted by their own secreted ETs. Similarly to ETA antagonism, inhibition of ET biosynthesis increased PRL secretion. The importance of the latter result is that it supports our interpretation that the effects of ETA antagonists on PRL secretion are indeed caused by antagonizing endogenously released ET, rather than result from their effects on ETA receptors as inverse agonists (58). Taken together, these experiments provided evidence that endogenous ETs modulate PRL secretion in an autocrine manner. In addition, as exogenous ET1 could significantly lower PRL secretion, the presumed autocrine inhibition of PRL secretion by endogenous ETs is probably not at its possible maximum. This latter observation could at least in part account for the relatively modest overall increases in PRL secretion induced by ETA antagonist applications.
The pharmacological data in this study, in agreement with those from previous reports (22, 23, 28), indicated that the effect of endogenous ETs on PRL secretion is predominantly inhibitory in nature. However, the bell-shaped PRL dose-response curve derived from administration of the ETA antagonist BQ-123 to lactotrophs obtained from female rats also suggests that the autoregulation of PRL secretion by endogenous ETs can be both stimulatory and inhibitory in nature. We propose that the observed bell-shaped dose-response curve with the ETA antagonist BQ123 resulted from differential sensitivities of the inhibitory and the stimulatory effects of the endogenous ETs toward ETA antagonism. Under different culture conditions, unusual dose-response curves of the effect of ET1 on PRL secretion have been observed, indicating the complexity of ET’s action on PRL secretion (31, 41, 43). However, using monolayer cultures where cell to cell interactions probably persist, the observed biphasic effects of ETs on PRL secretion could not be interpreted unequivocally (31). Our present data indicate that the lactotrophs themselves can perceive the effect of ETs either as stimulatory or inhibitory; therefore, the potentially bidirectional responsiveness to ETs is inherent to lactotrophs and does not require cell to cell interactions.
Analysis of the frequency distribution of different PRL plaque sizes revealed that in terms of PRL-releasing activity, the population of lactotrophs is not homogeneous. In agreement with previous observations (34, 48, 49, 50, 51, 52, 53, 57), under basal conditions, two populations of lactotrophs were apparent, one with relatively low and the other with higher PRL-releasing activity. The treatment with ETA receptor antagonist dose dependently modified the size distribution pattern of lactotrophs. At lower antagonist concentrations, the small plaque-forming population shifted toward the large plaque-forming group, and together they formed an apparently uniform population. At higher concentrations of the ETA antagonist, the relatively smaller plaque-forming population reappeared. The apparent redistribution of lactotrophs between different subpopulations can be interpreted by assuming that the inhibitory and stimulatory effects of ETs have differential sensitivities toward the ETA antagonist. At low antagonist concentrations inhibitory influences will be blocked, whereas stimulatory effects are still in place. At higher antagonist concentrations, at least in a certain proportion of lactotrophs, the stimulatory influence of the endogenous ETs will be blocked; hence, the smaller plaque-forming subpopulation reappears. A sequential application of PRL plaque assay under basal conditions as well as in the presence of low and high concentrations of an ETA antagonist would be necessary to establish whether the same individual lactotroph can respond in both ways to endogenous ETs or whether the stimulation and inhibition by ETs affect two different subpopulations. It seems likely that the cellular responsiveness to ETs and the size of the ET-responsive subpopulation of lactotrophs depend upon the physiological status of the animal. Preliminary data suggest that ovarian steroids might play a decisive role in that respect (39).
Taken together, the most important finding of these studies is that lactotrophs are capable of synthesizing and releasing ETs. By showing that PRL secretion, measured at the single cell level, increased upon ETA receptor antagonist application, we provided direct evidence that autocrine regulation by ETs is indeed operational in lactotrophs. In addition to vasoactive intestinal polypeptide, which was the first putative autocrine stimulator of PRL secretion described (56, 59), it has been recently reported that two other peptides with established PRL-releasing capacity, angiotensin II (60) and galanin (61), perform similar regulatory functions. ET is the first regulatory peptide identified that is capable of strong autocrine inhibition of PRL secretion. However, further data derived from in vivo experiments are required to assess the relative contribution of the autocrine regulation to the overall control of PRL secretion under varying physiological conditions.
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Acknowledgments |
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Footnotes |
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Received July 28, 1998.
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References |
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