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Department of Human Anatomy and Genetics, University of Oxford, Oxford, United Kingdom OX1 3QX
Address all correspondence and requests for reprints to: Prof. John F. Morris, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, United Kingdom OX1 3QX. E-mail: john.morris{at}anat.ox.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric
acidA chloride channel blocker picrotoxin (100
µM). T-BSA (0.1 nM to 1 µM)
for 5 or 20 min also caused an increased release of immunoreactive PRL
into the medium compared with control incubations. T and T-BSA did not
stimulate exocytosis from gonadotrophs or cause LH release. In
conclusion, we report for the first time a rapid, nongenomic effect of
T on PRL secretion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acidA(GABAA) receptors (4). The
circulating plasma steroids, estradiol (5, 6), corticosterone (7), and
testosterone (T) (8, 9), have all been shown to exert rapid effects on
the electrical activity of hypothalamic and hippocampal neurons. Many
similar, rapid, nongenomic effects of steroids have been reported in
other tissues. For example, progesterone stimulates oocyte maturation
(10) and induction of the acrosome reaction in sperm (11) and inhibits
smooth muscle contractility (12). In rodents two main subtypes of lactotroph can be characterized morphologically by electron microscopy (13). Type 1 cells contain large polymorphic electron-dense secretory granules, whereas type 2 cells contain numerous smaller electron-dense granules. In male rodents the two types of lactotroph are present in approximately equal proportions. T is the major circulating steroid in male rodents. In the anterior pituitary T is known to exert genomic inhibitory effects on PRL synthesis via classical cytoplasmic receptor binding and regulation of DNA transcription (14). However, little is known regarding rapid, nongenomic actions of any steroid in the anterior pituitary and the mechanism(s) by which these might occur. Steroids conjugated to BSA are impermeant to the plasma membrane and therefore cannot access cytoplasmic receptors. They have been used increasingly as tools to investigate nongenomic, cell surface effects of steroids (15, 16).
As part of an investigation of the varied effects of steroids in the male pituitary, we have, therefore, investigated whether T and the conjugate T-BSA can exert a rapid, nongenomic action on the release of PRL by use of a combination of tannic acid and electron microscopic analysis and RIA. Tannic acid was used to capture the cores of granules as they are exocytosed (17), thereby enabling the cellular sites of exocytosis to be localized and quantitated by electron microscopy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Secretion of PRL in vitro by anterior pituitary segments
Identification of secreting cells. Anterior pituitary glands
were cut into 4 roughly equal segments. The segments were distributed
randomly (1 segment/well) in the wells of 24-well tissue culture plates
(Costar, Cambridge, MA) and incubated at 37 C for 90 min
in a humidified atmosphere saturated with 95%
O2-5% CO2 in 1 ml
incubation medium [1% (vol/vol) aprotinin (Bayer Corp.,
Saffron Waldon, UK) and 1% (vol/vol) penicillin/streptomycin
(Sigma) in oxygenated Earle’s Balanced Salt solution
(EBSS; phenol red free; Sigma, Dorset, UK, pH 7.4]. The
segments were then transferred to fresh incubation medium to which had
been added 1.2 mM tannic acid (BDH, Poole, UK) and T (1 or
100 nM), T-BSA (1 or 100 nM), or 28
mM K+ (positive control) and
incubated for an additional 5 or 20 min. To eliminate the possibility
of nonspecific steroid membrane effects or effects of the BSA conjugate
on hormone release, a range of other steroids at equal concentrations
were subsequently tested, namely 17
- and 17ß-estradiol,
corticosterone, progesterone, and progesterone-BSA. In separate
experiments tissue was equilibrated for 15 min before steroid contact
with nifedipine (100 nM), thapsigargin (150
nM), retinal (10 µM), picrotoxin (100
µM), or dopamine (500 nM) which were then
included in the medium with T-BSA for the remainder of the experiment.
At the end of the final incubation, tissue was immersion fixed in
glutaraldehyde (2.5% in 0.1 M phosphate buffer, pH 7.2)
for 1 h and then processed into Spurr’s resin.
Assay of secreted PRL and LH. The experiments in which hormone released into the medium was assayed followed the protocol described above, except that no tannic acid was added to the incubation medium. Medium from the final incubation was collected and stored in aliquots (300 µl; 20 C) for subsequent measurement of immunoreactive (ir-) PRL and ir-LH by RIA. Pituitary segments were weighed on a torsion balance and discarded.
Preparation and incubation of dispersed anterior pituitary
cells
Suspensions of dissociated anterior pituitary cells were
prepared as described previously (18). Briefly, anterior pituitary
cells were dissociated by incubation (1 h, 37 C) with collagenase
(0.2%, wt/vol; Roche Molecular Biochemicals, Sussex, UK)
and deoxyribonuclease in EBSS enriched with BSA (0.4%;
Sigma); the dispersion was aided by gentle trituration (30
sec every 10 min). The resulting cell suspension was centrifuged
(300 x g, 10 min), the pellet was resuspended in 5 ml
BSA-enriched EBSS, and the suspension was filtered through 20-µm
nylon mesh to remove any large clumps of debris. The filtrate was then
centrifuged (300 x g, 10 min), and the pellet was
resuspended in 5 ml incubation medium. The cells were examined at the
light microscope level to verify the effectiveness of the dispersion
and counted using a hemocytometer. Cell viability was assessed by the
trypan blue exclusion test and was always found to be more than
95%.
The cells were plated at a density of 2.5 x 105 cells/ml·well in 24-well cell culture plates (Costar, Cambridge, MA) and incubated for 90 min at 37 C in a humidified atmosphere saturated with 95% O2-5% CO2. They were then challenged for 5 min with T, T-BSA (1 pM to 1 µM), or 28 mM K+; controls were incubated in an equal volume of incubation medium alone. After centrifugation (600 x g, 4 C, 10 min), the supernatant fluid was harvested and assayed for ir-PRL. In some experiments, 1.2 mM tannic acid was also added with incubation medium, and the pituitary cells were retained for quantitative electron microscopy.
Quantitative electron microscopy
The anterior pituitary segments and isolated cells were prepared
for electron microscopy using standard methods. Briefly, segments were
postfixed in osmium tetroxide (1%, wt/vol, in 0.1 M sodium
phosphate buffer) contrasted with uranyl acetate (2%, wt/vol, in
distilled water), dehydrated through increasing concentrations of
ethanol (70–100%), and embedded in Spurr’s resin (Agar Scientific (UK), Stansted, UK). Ultrathin sections (50–80 nm)
were viewed with a JEM-1010 transmission microscope (JEOL USA, Inc., Peabody, MA). Cut sections were collected once a full
block face was presented; thus, sections were relatively superficial.
Immunogold labeling for PRL was performed to aid cell identification;
rabbit antirat PRL (National Hormone and Pituitary Program,
Gaithersburg, MD) was used at a dilution of 1:5000. For control
sections, the primary antibody was omitted and replaced with 0.1
M sodium phosphate buffer containing 1% (wt/vol) egg
albumin. Endocrine cells in sections taken systematically from
different depths of the embedded tissue were identified on the basis of
their secretory granule populations (shape, electron density, size, and
distribution), organelle structures, nucleus size, and chromatin
characteristics (13) and by immunogold labeling (19). Cells from
individual samples were always identified and counted on four to eight
randomized grids according to a systematic random procedure
(20).
RIA of PRL and LH
PRL was determined in duplicate by RIA using a primary antibody
of defined specificity raised in rabbits against rat PRL, with
synthetic PRL as a reference preparation and
[125I]PRL as tracer (all reagents generously
supplied by the National Hormone and Pituitary Program). The assay
sensitivity was 0.5 ng/ml, and the inter- and intraassay coefficients
of variation were 10% and 4%, respectively. Dilution curves of test
samples were parallel that of the standard PRL preparation. LH was
determined by RIA using a primary antibody raised in rabbits against
rat LH, rat LH reference preparation, and rat
[125I]LH as tracer (reagents also supplied by
the National Hormone and Pituitary Program). The assay sensitivity was
1 ng/ml, and the inter- and intraassay coefficients of variation were
11% and 8%, respectively.
Drugs
The following were used. T
3-(O-carboxy-methyl)oxime:BSA (20–30 mol steroid/mol BSA),
T 3-(O-carboxy-methyl)oxime:BSA-fluorescein isothiocyanate
conjugate, progesterone 3-(O-carboxy-methyl)oxime:BSA
(15–45 mol steroid/mol BSA), and BSA-fluorescein isothiocyanate (all
from Sigma) were initially dissolved in EBSS adjusted to
pH 9 with 1 M NaOH and diluted in EBSS, pH 7.4.
T, 17- and 17ß-estradiol, progesterone, and corticosterone (all
from Sigma) were initially dissolved in a small amount of
ethanol and subsequently diluted with EBSS; the final concentration of
ethanol never exceeded 0.1%, and appropriate controls were included in
each experiment. Nifedipine, picrotoxin, thapsigargin, retinal,
dopamine (all from Sigma) were dissolved and diluted in
EBSS immediately before use. All solutions were adjusted to pH 7.4
before use.
Statistical analysis
Quantitation of secretion by electron microscopy was analyzed by
the Mann-Whitney U test. Preliminary analysis of RIA by the
Shapiro-Wilks test showed that the data were normally distributed.
Subsequent analysis was performed using ANOVA with post-hoc
comparisons made using Duncan’s multiple range test. Differences were
considered significant at P < 0.05. As the basal rate
of ir-PRL and ir-LH release varied between experiments, statistical
analyses were made within experiments only. Each of the studies shown
was repeated at least three times (for specific details, see figure
legends), and in all instances a similar data profile was seen.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and LH (Fig. 3A). T-BSA
(10-10–10-6
M) for 5 or 20 min significantly (P <
0.05) stimulated ir-PRL at all concentrations tested (Fig. 1A). By
contrast, T-BSA exerted no detectable effect on the amount of ir-LH
released (Fig. 3A).
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) stimulated the release of PRL only from
the small granule type 2 lactotrophs. T-BSA (1 and 100
nM) for 5 or 20 min in vitro caused a
significant (P < 0.01) increase in the mean number of
exocytotic events observed per releasing type 2 lactotroph (Fig. 2A). The proportion of secreting type 2
lactotrophs also increased (1 nM T-BSA, 56
± 2%; 100 nM, 50 ± 3%; P
< 0.01 vs. control, 23 ± 1%). Nearly all (85 ± 4%) of
the exocytosis occurred at the vascular borders of the cells. In
contrast, no effect of the steroid was observed either on the number of
type 1 lactotrophs releasing PRL-containing granules (1
nM T-BSA, 23 ± 1%; 100
nM, 20 ± 2%; P =
vs. control, 22 ± 1%) or on the number of exocytoses
from these cells (Fig. 2B). Also, no significant difference compared
with EBSS controls was observed with either dose of steroid on the
number of exocytoses occurring from gonadotrophs (Fig. 3B). Although a significant
(P < 0.05) increase in PRL release in response to 28
mM K+ between 5 and 20 min
was detected, the exocytotic responses to T-BSA treatment for 5 or 20
min did not differ, nor did the responses to the three concentrations
of T-BSA (0.1 nM, 10 nM,
and 1 µM) tested (Fig. 1A).
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-estradiol, progesterone, progesterone-BSA, or corticosterone were
all without effect (Fig. 4).
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), as was the proportion of responsive
type 2 lactotrophs (from 56 ± 2%, control, to 23 ± 1%
with 1 nM T-BSA and 9.0 ± 0.4% with 100
nM T-BSA, respectively; P < 0.05). By
contrast, none of the treatments designed to test the involvement of
extracellular Ca2+ (Fig. 5B), L-type
Ca2+ channels (nifedipine; Fig. 5C),
intracellular Ca2+ release (thapsigargin; Fig. 5D), or protein kinase C (retinal; Fig. 5E) had a detectable effect on
the T-induced exocytosis. Furthermore, treatment of anterior pituitary
segments with the GABAA channel inhibitor
picrotoxin (Fig. 5F) had no significant effect on either the number of
secreting type 2 lactotrophs or the amount of exocytosis induced from
these cells in response to either concentration of T-BSA (1 and 100
nM). Parallel studies revealed similar profiles
of responses in pituitary tissue from male mice to nonconjugated T at
the same concentrations (data not shown).
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demonstrates that a similar data
profile was observed when comparing the effects of T and T-BSA in the
isolated anterior pituitary cell preparation to those measured in
anterior pituitary segments. T and T-BSA (1 pM to 1
µM) produced significant (P < 0.01)
concentration-dependent increases in ir-PRL release within 5 min (Fig. 6A). No significant difference was detected between responses to T or
T-BSA at matching concentrations. Electron microscopic analysis of
tannic acid-captured exocytosis confirmed that the source of PRL
released was the type 2 and not type 1 lactotrophs (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxy-4-pregnen-20-one, which is readily converted from and to
progesterone, has been shown selectively and rapidly (within minutes)
to suppress GnRH-induced FSH release (21, 22). Rapid actions of
estradiol to stimulate PRL secretion from GH3
clonal pituitary cells (23) have been reported, and we have
demonstrated similar actions of estradiol on PRL release from type 2
lactotrophs in male rat pituitary glands (24). In the present study,
tannic acid capture of granule exocytosis and quantitative electron
microscopy have demonstrated that physiological concentrations of T
(10-10–10-6
M) can elicit significant PRL release from type 2
lactotrophs within 5 min. In addition, T can recruit nonreleasing type
2 lactotrophs into the secreting population of cells. These effects
were detected in both in vitro anterior pituitary
preparations tested: anterior pituitary segments in which the intimate
cell-cell contacts and three-dimensional tissue arrangements are
intact, and isolated anterior pituitary cells where these arrangements
are lost but diffusion of test ligands to target sites might be
expected to be more efficient. These data suggest that 1) in the
segment preparation, adequate diffusion of steroids and steroid-BSA
conjugates was achieved within the 5-min treatment periods; and 2) it
is likely that T acts directly on type 2 lactotrophs and not on
adjacent cells to regulate the release of a local intrapituitary
paracrine regulator of PRL secretion. Neither type 1 lactotrophs nor
gonadotrophs were observed to respond to T. It was perhaps not
surprising that no effect of T on gonadotrophs was demonstrated, as any
effect might well be an inhibition of stimulated release, which we did
not test. T via conversion to the metabolite dihydrotestosterone is
known to regulate GnRH-induced Ca2+ signaling
directly in the pituitary by a genomic mechanism (25).
The rapid effects of T on PRL release from type 2 lactotrophs appear to
be specific because only this population of cells was affected by
exposure to the steroid. If the effects of T reported here were due to
general membrane depolarization or alterations in membrane fluidity,
then a more general stimulation of anterior pituitary secretion would
have been expected. The lack of effect on type 1 lactotrophs of T
cannot be attributed to a lack of affect of tannic acid on these cells,
because K+-stimulated release was readily
detectable. Furthermore, the range of other steroids tested, except for
17ß-estradiol, was without effect on PRL release. As the effects of
T-BSA were not mimicked by those of progesterone-BSA, the possibility
that the BSA conjugate may be mediating a membrane effect is unlikely.
Furthermore, the effects of nonconjugated T were indistinguishable from
those of T conjugated to BSA in both anterior pituitary in
vitro preparations tested. Therefore, it would appear likely that
1) the stimulatory action of T is exerted at the cell surface; and 2)
the effect is not mediated via intracellular conversion of T to either
estrogen (by aromatase) or 5-dihydrotestosterone (by 5-reductase)
in lactotrophs, although conversion by other cell types cannot be
excluded. In the PRL-secreting cell line GH3 B6,
estradiol rapidly stimulates PRL release by acting both directly, to
cause a sustained train in action potentials (23), and indirectly (26),
via reversal of dopamine inhibition. The reason why T exerts its
nongenomic secretagogue effect specifically on type 2 lactotrophs could
be that only type 2 lactotrophs express putative surface receptors for
T, and this possibility is currently under investigation in our
laboratory. A single class of estradiol-specific thermolabile binding
sites on pituitary membranes from female rats has previously been
characterized (27). The absence of a difference in the amount of PRL
released in response to 5- or 20-min exposure to T or T-BSA might
suggest that tannic acid is acting in some way to prevent either
membrane recycling of putative T-binding proteins, which hinders
resensitization of these proteins, or that, once used, sites of
exocytosis are blocked by the captured cores. However, this would not
appear to be the case, as no significant difference was found between
the PRL responses to T-BSA measured at each time point by RIA in the
absence of tannic acid, whereas a difference was found in response to
the depolarizing stimulus of high molarity
K+.
Androgens have previously been shown to induce rapid increases in intracellular Ca2+ in cardiac myocytes (28), mouse kidney cells (29), and human prostate cancer cells (30). These effects of T appear to be initiated through hormone-specific membrane androgen-binding sites, although these have yet to be characterized (28, 31). Our experiments, however, using Ca2+-free EBSS, nifedipine, and thapsigargin, demonstrated that influx of extracellular Ca2+ through L-type voltage-gated Ca2+ channels and release of intracellular Ca2+ stores are not prerequisites for the T-induced effects on type 2 lactotrophs. Similarly, we have previously shown that the effect of estradiol on the exocytosis of oxytocin and vasopressin from the dendrites of magnocellular neurosecretory neurons is not affected by the absence of Ca2+ in the bathing medium (32). Studies by others have also indicated that secretion from certain cell types, for example intact neutrophils (33), platelets (34), and a component of insulin release from pancreatic islets (35), is not dependent on a rise in intracellular Ca2+. It is possible that T may cause local release of intracellular Ca2+ at the site of exocytosis, but this would be very difficult to demonstrate or inhibit by established methods (36).
The GABAA-chloride channel receptor complex is well established as the target for a number of nongenomic actions of steroids on neuronal excitability (4). GABAA receptor subunits have been localized in the anterior pituitary (37, 38), and functional studies have demonstrated that GABA and GABAA receptor agonists exert biphasic effects on PRL release: transient stimulation followed by prolonged sustained inhibition (38). Therefore, it appeared possible that T-BSA induced PRL release by potentiation of the GABAA receptor-mediated transient stimulation. However, blockade of GABAA receptor chloride channels with picrotoxin had no influence on T-BSA-induced PRL release, so modulation of GABAA receptor complex function is unlikely to be involved in the secretagogue effect of T on type 2 lactotrophs. Furthermore, because retinal did not influence induced PRL release from type 2 lactotrophs, signaling through protein kinase C does not appear to be involved.
Importantly, it should be noted that acute changes in steroid milieu to which the pituitary tissue was subjected in vitro would not occur in vivo. The effects of T on type 2 lactotrophs were inhibited by dopamine to amounts not significantly different from control levels, and dopamine is known to have differential effects on lactotroph subtypes (39, 40, 41). This suggests that under conditions of normal inhibitory dopaminergic tone in vivo no such nongenomic secretagogue effect of T would be apparent. However, it is likely that circulating steroids would play an active role to modulate the activity of the population of type 2 lactotrophs in relation to normal PRL pulsatility when dopamine tone is removed. The physiological relevance to male reproduction is not immediately obvious. Stimulation of lactotrophs is known to inhibit gonadotropins (42). Therefore, T, by facilitating PRL release, may contribute to the negative feedback of T on gonadotropin release.
In conclusion, we report for the first time a rapid, nongenomic effect of T on PRL secretion. The determination of the signaling pathways and mechanisms activated by T and the detection and characterization of putative pituitary plasma membrane T-binding sites are the focus of ongoing investigation.
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Acknowledgments |
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Footnotes |
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Received December 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxy-4-pregnen-20-one involves cytosolic calcium. Endocrinology 134:377–382-Amino butyric acid
receptor function in rat anterior pituitary: evidence for control of
prolactin release. Endocrinology 105:754–759This article has been cited by other articles:
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, 5-min
exposure to steroid; 
, 20-min exposure to steroid. Values are the
mean ± SEM (n = 6).The data are typical of those
from three replicate experiments. **, P < 0.01
vs. corresponding basal control. 
,
P < 0.05, 20-min vs. 5-min exposure to 28
mM K+ (by ANOVA plus
Duncan’s multiple range test). B, Electron micrograph showing
exocytosis from a type 2 lactotroph in response to T-BSA (1
nM for 5 min). Exocytoses were either single
(arrowheads) or multiple (arrow). Magnification,
x12,000.
