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Department of Physiology and Biophysics, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064
Address all correspondence and requests for reprints to: Celia D. Sladek, Ph.D., Department of Physiology and Biophysics, Finch University of Health Sciences/The Chicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064. E-mail: sladekc{at}mis.finchcms.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence for modulation of these osmotic responses by gonadal steroids has been obtained from in vivo studies. In early studies, contradictory results were obtained relative to the effect of castration, hormone replacement, gender, and stage of the estrous cycle on plasma VP concentration (11, 12, 13, 14, 15, 16, 17, 18). More recent studies examined the effects of gonadal steroids on osmotic stimulation of VP mRNA and reported that gonadectomy prevented the increase in hypothalamic VP mRNA in response to increased osmolality in both males and females, and administration of exogenous testosterone or the testosterone metabolite, dihydrotestosterone (DHT), but not estradiol, restored the osmotic response of VP mRNA in male rats (19, 20).
To determine whether these effects of the gonadal steroids reflect direct actions on the hypothalamo-neurohypophyseal system (HNS) as opposed to actions on other organ systems that might secondarily impact this system, the effect of gonadal steroids was evaluated in vitro using HNS explants. In previous studies, HNS explants have been shown to respond to increases in the osmolality of the culture medium with both an increase in VP release and an increase in VP mRNA content (21, 22). In the current experiments, HNS explants were first used to determine whether castration would alter hyperosmolar-induced VP release in vitro. Second, HNS explants were used to study the effects of testosterone and its metabolites, estradiol and DHT, on VP and OT release and VP mRNA content. All three steroid hormones were found to inhibit the increase in VP and OT release in response to an increase in osmolality. Therefore, additional experiments were performed to determine whether estradiol and DHT would have an effect on another mode of VP stimulation, exposure to potassium chloride (KCl). KCl increases VP release by directly depolarizing the neural lobe of the pituitary, thus enabling us to determine whether estradiol and DHT are acting directly on the nerve terminals. Finally, to determine whether the inhibition of osmotically stimulated VP and OT release is a genomic or nongenomic event, similar perifusion studies were performed using estradiol or DHT conjugated with BSA. Genomic mechanisms of steroids involve DNA binding and regulation of gene transcription. This requires the hormone to diffuse across the cell membrane and bind to intracellular hormone receptors (23). Nongenomic effects of steroids involve actions at the plasma membrane (24, 25) and are extracellular events. As BSA is a large molecule, the bound hormone is unable to cross the cell membrane, thus limiting its actions to the extracellular environment (26, 27).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After decapitation, HNS explants were prepared as described previously (28). The brain and pituitary were removed from the skull using a caudal approach with the pituitary stalk still intact. Under a dissecting microscope, the anterior pituitary was removed, and a hexagonal block of tissue was dissected by cutting rostral to the optic chiasm, lateral to either side of the median eminence, and undercutting at a depth of 1–2 mm. These explants included the magnocellular neurons of the supraoptic nucleus with their axonal projections extending through the median eminence and terminating in the neural lobe. The organum vasculosum of the lamina terminalis (OVLT) was contained in the explant as well.
Perifusion conditions
The HNS explants were then placed in individual closed chambers
(500 µl), which were positioned in a multiple microchamber unit
(Endotronics, Minneapolis, MN) and perifused with F-12 nutrient mixture
supplemented with 20% FCS, 1 mg/ml glucose, 50 µU/ml penicillin, 50
µg/ml streptomycin, and 1 x 10-4 M
bacitracin. Bacitracin was added to prevent hormone degradation (29).
The final osmolality of the basal medium was approximately 300
mosmol/kg H2O. The medium was warmed to 37 C and aerated
with 95% O2-5% CO2 before placement of the
HNS explants in the chamber and for the duration of the experiment. Six
explants were perifused simultaneously at a rate of approximately 2.4
ml/h. For 10 h, effluent from each chamber was collected at 20-min
intervals with a fraction collector housed in a refrigerator (4 C).
In each experiment, all chambers were perifused with basal osmolality medium (300 mosmol/kg H2O) for the first 4 h to allow stabilization of hormone release. After this equilibration period, explants were either perifused with basal osmolality medium for the remaining 6 h, exposed to a ramp increase in osmolality or exposed to 25 mM KCl. For the explants receiving the osmotic ramp, the osmolality of the medium was increased at a rate of 5 mosmol/kg H2O·h by increasing the sodium chloride concentration. This was accomplished by the use of the Endotronics Automated Perifusion System (APS 10, Endotronics, Coon Rapids, MN) that allowed manipulation of the rate of rise in osmolality. In previous experiments, HNS explants were shown to respond similarly to increases in osmolality achieved with either NaCl or mannitol (22). At the end of the 10-h perifusion period, the VP concentration in the collected fractions was determined by RIA, the osmolality was monitored by microvapor pressure osmometry (Wescor, Buffalo, NY), and all explants were frozen in liquid nitrogen for subsequent RNA extraction.
In the steroid-treated groups, immediately after dissection and throughout the perifusion period, explants were continuously maintained in medium supplemented with testosterone (3 ng/ml), 17ß-estradiol (50 pg/ml), or DHT (3 ng/ml; Sigma Chemical Co., St. Louis, MO) dissolved in ethanol or 3-carboxymethyloxime:BSA-estradiol (200 nM) or 3-carboxymethyloxime:BSA-DHT (10 µM; Steraloids, Wilton, NH) dissolved in 50 mM Tris at pH 8.5. The steroids were covalently bound to the BSA molecules, and concentrations were used such that they were equal to the molar concentrations of the respective nonconjugated steroids. BSA-estradiol contained 30 mol steroid:1 mol BSA, whereas BSA-DHT contained 35 mol steroid:1 mol BSA. In all experiments, comparable amounts of ethanol or Tris were added to the medium perifusing the nonsteroid-treated explants. The impact of the addition of steroids on the osmolality of the medium was not a concern, because the calculated increase of 3.6 nosmol/liter is 6 orders of magnitude below either detectable or physiologically relevant changes in osmolality. Similarly, the impact of the addition of BSA in the experiments with the conjugated steroids would be minimal due to the presence of 20% FBS in the medium. Both total and free testosterone were measured by RIA in the testosterone-supplemented medium as well as in the nonsupplemented medium. These values were compared to the plasma testosterone levels measured in trunk blood collected from the sham and castrated rats.
RIAs
VP and OT.
VP and OT concentrations in the perifusate
fractions were determined by RIA as previously described (30, 31). The
antisera used were generated in conjunction with Arnel Products
(Brooklyn, NY) and were used at a final dilution of 1:100,000. The VP
assay buffer was 0.1 M PBS (pH 7.6) with 1 mg/ml BSA and 1
mg/ml sodium azide. The OT assay buffer was 0.05 M PBS (pH
7.6) with 0.5 mg/ml BSA and 1 mg/ml sodium azide. In both assays, 100-
and 50-µl aliquots of effluent medium from each 20-min fraction were
brought to 400 µl with buffer and assayed. The standards and samples
were incubated for 72 h at 4 C in the presence of the antiserum
and tracer (5,000 cpm [125I]arginine VP or 2,500 cpm
[125I]; New England Nuclear, Boston, MA). Antibody-bound
VP or OT was separated from free hormone with dextran-coated charcoal
and counted. All samples from a given experiment were assayed
simultaneously. The minimum sensitivity was 1.0 pg/tube for VP and 0.5
pg/tube for OT.
Testosterone.
Both total and free testosterone double
antibody assay kits were obtained (Diagnostic Systems Laboratories,
Webster, TX) and used to assay plasma testosterone in duplicate from
the trunk blood of decapitated castrated and noncastrated rats as well
as from the medium used in the perifusion experiments. The minimum
sensitivity of the assays were 0.1 ng/ml and 0.25 pg/ml for total and
free testosterone, respectively.
RNA extraction and quantification
Explants were frozen in liquid nitrogen after each perifusion
experiment. Total RNA was extracted from each explant using Tri-Reagent
(Molecular Research Center, Cincinnati, OH) and assayed in the
ribonuclease (RNase) protection assay using a 32P-labeled
full-length VP complementary RNA probe (680 bp) synthesized (Promega
Riboprotein System) from the pGEM 4-AVP8C construct provided by T. G.
Sherman, Georgetown University (Washington DC). The radiolabeled probe
was purified by nucleic acid purification cartridges (New England
Nuclear Research Products). As the probe hybridizes to the full-length
VP mRNA as well as the neurophysin-coding portion of OT mRNA that is
homologous between VP and OT, both VP and OT mRNA could be measured
simultaneously in the same assay (Fig. 1
). Total RNA from each explant
was used in the hybridization.
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The complementary RNA-mRNA pellet was washed and reconstituted in 8
µl 0.5 x Tris-borate-EDTA electrophoresis buffer. The fragments
were fractionated on a 1.3% agarose gel with 0.5 x
Tris-borate-EDTA for 55 min at 70 mA. The gel was dried, and direct
contact autoradiography was performed by exposing the gel to Kodak
X-Omat XAR5 film (Eastman Kodak, Rochester, NY) for 24–48 h (Fig. 1
). VP and OT mRNA were quantitated by determining
counts per min in cut gel fragments containing the VP and OT mRNA
bands. Quality control between assays was evaluated by including
standard preparations of 0, 50, 100, 500, and 1000 pg sense VP mRNA
generated from the cAVP8c plasmid with each experimental group of
explants as well as control preparations of mRNA from hydrated and
dehydrated rats and mRNA extracted from a nonperifused explant.
Statistical analysis
In the perifusion studies, basal hormone release was calculated
for each explant as the mean hormone release during the fourth hour of
the equilibration period (i.e. immediately before either the
osmotic ramp was started or KCl was added). VP and OT release is
expressed as a percentage of this basal value, and the basal release
for each group is expressed as picograms per ml. Results are expressed
as the mean ± SEM. After log10
transformation of the data, statistical significance was tested by
two-way ANOVA with repeated measures followed by simple main effect
analysis. In some cases, when these analyses indicated statistical
significance, subsequent ANOVA and post-hoc mean comparisons
by Newman-Keuls test were performed to establish specific group
differences at individual time points. The level of significance was
set at P < 0.05.
VP and OT mRNA content were analyzed by normalizing the counts per min in the appropriate band to one of the quality control standards included in each gel to control for variations between hybridization assays. Results are expressed as the mean ± SEM. Statistical analysis was performed as described above without the repeated measures. The level of significance was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, both the sham explants and the
castrated explants had a significant increase in VP release in response
to the increase in osmolality compared to their basal groups. The
osmotic responses did not significantly differ from each other.
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Effect of testosterone on VP and OT
To determine whether the reason why similar responses were
obtained in the explants from castrated and noncastrated rats was due
to the absence of testosterone in the perifusion medium, the effect of
adding testosterone to the medium was evaluated. Explants from intact,
unoperated rats were either maintained in testosterone (3
ng/ml)-supplemented or nonsupplemented medium throughout the entire
experiment. Explants were perifused with basal medium throughout or
were exposed to a ramp increase in osmolality during the last 6 h
of the perifusion. Hormone release was measured by RIA, and mRNA was
quantitated by the RNase protection assay. As shown in Fig. 3A, testosterone completely inhibited the osmotically
induced increase in VP release in response to the osmotic stimulus. In
addition, the increase in VP mRNA normally observed with increased
osmolality was not present in the testosterone-supplemented explants
(Fig. 3B). Testosterone had the same effects on osmotically stimulated
OT release and mRNA content (Fig. 4). As depicted in
Fig. 5, total and free testosterone levels found in the
medium were similar to the concentration measured in trunk blood
collected from rats used as explant donors.
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) and mRNA content (Fig. 6B).
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). However, in contrast to the effect of
estradiol, although the osmotic stimulation of hormone release was
attenuated, the osmotic stimulation of VP mRNA remained intact in the
presence of DHT (Fig. 7B).
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).
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and 10A). The effects of the steroid
conjugates on VP mRNA content also duplicated the effects of the
unbound steroids on VP mRNA content. BSA-estradiol significantly
inhibited the osmotically induced increase in VP mRNA (Fig. 9B). This
inhibition was not seen with BSA-DHT (Fig. 10B). The effects of the
conjugated hormones on OT mRNA content were similar to their effects on
VP mRNA. There was no significant difference in the OT mRNA content of
osmotically stimulated control and BSA-DHT explants. Although not
reaching statistical significance (P = 0.08), there was
a tendency for the OT mRNA in the BSA-estradiol explants to be lower
than that in the control explants as was previously observed for VP
mRNA (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Due to the absence of testosterone in our perifusion medium, the explants from both the sham and gonadectomized rats were exposed to "castrated" conditions during the 10 h of perifusion. This led to the hypothesis that the VP response to an increase in osmolality was actually attenuated in the explants from both castrated and intact rats. Therefore, similar perifusion studies were performed to determine whether the addition of testosterone to the medium affected VP release and/or VP mRNA content. Based on the study by Crowley et al. (19), a more robust VP response to increased osmolality in the testosterone-treated explants was expected. Surprisingly, testosterone completely inhibited the increase in both VP and OT release and mRNA in response to the osmotic stimulus. This did not appear to be a pharmacological effect due to a hyperphysiological concentration of free testosterone in the medium, as the concentration present in the medium was similar to that found in the plasma of rats used as explant donors. Also, since the basal rate of VP release was not significantly different in the absence and presence of testosterone, the inhibitory effect of the steroid probably did not reflect an effect on cell volume that inhibited osmoreceptor function.
As testosterone can be aromatized to estradiol or metabolized to DHT in the brain, testosterone’s inhibitory effect on hormone release may be mediated by either estrogenic or androgenic mechanisms. DHT binds to androgen receptors with a stronger affinity than testosterone and is androgen specific, as it cannot be further aromatized to estradiol. Therefore, the effects of estradiol and DHT on osmotically stimulated hormone release and mRNA were compared. Both estradiol and DHT inhibited osmotically stimulated VP and OT release, but the effects of these hormones on VP mRNA diverged. In the case of estradiol, both VP release and VP mRNA response to osmotic stimulation were inhibited. However, in the case of DHT, although secretion was inhibited, the osmotically induced rise in VP mRNA content was still present. This is an important observation, because it suggests that hormone release and mRNA content may be regulated independently. It is also important because it suggests that the inhibition of osmotic responses by estradiol and DHT may be through different mechanisms. Given the potential for both genomic and nongenomic actions of estradiol and DHT and the potential that these steroids might act either directly on the VP and OT neurons or on other neurons in the osmoregulatory pathway, both parallel and divergent effects are quite possible.
Steroid receptors have not been located in the magno-cellular neurons (33, 34, 35). Therefore, genomic effects of estradiol and DHT directly on the VP and OT neurons is unlikely. However, genomic effects may be possible elsewhere in the osmotic circuitry, for example, on osmosensitive neurons in the OVLT region or other neurons involved in transmitting osmotic information from the OVLT region to the supraoptic nucleus (3, 36). Autoradiographic studies have demonstrated binding of both estrogens (37) and androgens (38) to neuronal membranes in the supraoptic nucleus. Hence, these gonadal steroids may also be inhibiting hormone release and message through nongenomic actions directly on the magnocellular neurons or on other neurons in the osmotic circuit.
As DHT inhibited the release of VP but not the increase in mRNA content, KCl was used to evaluate whether DHT was exerting its effects at the neural lobe by inhibiting depolarization-secretion coupling in the nerve terminals. KCl did induce a large increase in hormone release, but this increase was not affected by the addition of either estradiol or DHT to the medium, indicating that neither estradiol nor DHT interrupts depolarization-secretion coupling. Therefore, their actions must interrupt mechanisms before this step in hormone release, e.g., action potential generation from the cell body or afferent signals that either transmit or influence hormone secretion in response to osmotic stimulation.
As binding of both estradiol and DHT to supraoptic neurons was reported (37, 38), the possibility that the inhibition of osmotic responses by estradiol and DHT reflected nongenomic actions was further evaluated in experiments with the BSA-conjugated steroids. When estradiol and DHT are conjugated to the large molecule, albumin, they are unable to cross the cell membrane, thus preventing activation of cytoplasmic receptors and subsequent genomic actions (26, 27). The conjugated forms of both estradiol and DHT duplicated the actions of the nonconjugated steroids. This evidence supports the hypothesis that the inhibitory actions of these steroids reflect nongenomic actions. It is unlikely that these effects are due either to the addition of BSA to the medium or to enzymatic degradation of the BSA:steroid conjugates, because the culture medium already contains substantial amounts of BSA contributed by the 20% FBS, and the medium contains bacitracin, which prevents enzymatic degradation of VP and OT (29).
There has been increasing evidence for nongenomic actions of steroids
in the central nervous system (25). There is evidence for binding sites
for androgens, estrogens, and progestins on brain membranes (39),
including the supraoptic nucleus (37, 38) as mentioned previously.
These steroids could act directly at the VP neuron to alter ion
permeability. For example, estrogen was shown to directly inhibit the
release of GnRH by opening potassium channels on the neurons (40). They
might also exert direct nongenomic effects by altering the efficacy of
other neurotransmitters that are important in VP release
(i.e. 
-aminobutyric acid and glutamate). Excitatory amino
acids are important regulators of excitatory transmission in the
central nervous system, and it has been reported that androgens alter
N-methyl-D-aspartic acid receptor-mediated
responses in hippocampal neurons (41, 42). This could be a potential
mechanism for the inhibition of VP by DHT.
Androgens and estrogens could also inhibit VP secretion indirectly by
affecting the release of other neurotransmitters that influence the
activity of the magnocellular neurons. Lagrange et al. (40)
also demonstrated that GnRH is secondarily inhibited by estradiol
modulation of µ-opioids from projection neurons. Both estrogens (35)
and androgens (43, 44, 45) have been reported to increase -aminobutyric
acidergic tone in the preoptic area of the hypothalamus and in the
median eminence. It is thought that this may be a potential mechanism
for the inhibition of LHRH by gonadal steroids.
The finding that both estradiol and DHT inhibited osmotically stimulated VP release raises the concern that this may be a nonspecific action of steroids in the explant. This concern is mitigated by the fact that estradiol and DHT had distinct effects on osmotic stimulation of VP mRNA with estradiol, but not DHT, inhibiting the osmotically induced increase in VP mRNA. Previous experiments evaluating the effect of corticosterone on VP release by HNS explants (46) also provide evidence of the specificity of these effects. Although corticosterone also inhibited osmotically stimulated VP release, this effect was reversed by RU-486, the glucocorticoid antagonist, suggesting that the effect is mediated by the glucocorticoid receptor through either genomic or nongenomic mechanisms (46).
In conclusion, this study provides evidence implicating nongenomic actions of estradiol and DHT in the osmotic regulation of VP and OT. The physiological significance of these findings remains to be determined, because in vivo, VP and OT release is responsive to osmotic stimulation in the presence of circulating estrogen and/or testosterone. A possible explanation for this discrepancy is that in vivo there are multiple pathways providing osmoregulatory input to the VP and OT neurons (36), whereas in the explants only the osmoreceptive elements in the OVLT region and supraoptic nuclei are included. Furthermore, in vivo, the HNS receives a multitude of afferent pathways carrying information about other physiological parameters, e.g. blood pressure, blood volume, etc., and this information contributes to the regulation of neurohypophyseal hormone release in response to dehydration and other disturbances in fluid and electrolyte balance. Thus, the HNS explant model enables us to evaluate hypothalamic osmotic control mechanisms in isolation from the multitude of afferent pathways carrying information about other physiological parameters, and thereby provides the opportunity to elucidate influences of gonadal steroids on these specific regulatory mechanisms.
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Acknowledgments |
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Footnotes |
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Received December 23, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid turnover: effect of castration on
-aminobutyric acid turnover in microdissected brain regions of the
male rat. J Neurochem 60:2254–2264[CrossRef][Medline]
-aminobutyric acidergic
neurones. Endocrinology 143:165–174
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