ABSTRACT. Endothelin. (ET) peptides have recently been recognized as putative regulators of the endocrine system. Particularly in the gonadal system,. ET-3 ...
0013.7227/93/1322-0789$03.00/0 Endocrinology Copyright 0 1993 by The Endocrine
Vol. 132, No. 2 Printed in U.S.A.
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Endothelin-3 Stimulates Luteinizing Hormone-Releasing Hormone (LHRH) Secretion from LHRH Neurons by a Prostaglandin-Dependent Mechanism MARCEL0
MORETTO,
FRANCISCO
JO&
LOPEZ,
AND
ANDRfiS
NEGRO-VILAR
Reproductive Neuroendocrinology Section, Laboratory of Molecular and Integrative Neurosciences, Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
National
ABSTRACT Endothelin (ET) peptides have recently been recognized as putative regulators of the endocrine system. Particularly in the gonadal system, ET-3 stimulates LH secretion from anterior pituitary cells cultured in uitro. In these studies, we evaluate the actions of ET-3, the most abundant species of the ET family in the central nervous system, on LHRH release from arcuate nucleus-median eminence (AN-ME) fragments and an LHRH-secreting neuronal cell line (GTl cells) in uitro. ET-3 exhibited a stimulatory effect on LHRH secretion from AN-ME fragments and GTl cells incubated in a static system as well as in a dynamic perifusion paradigm. In all the systems used, the effects of ET-3 on LHRH secretion showed a dose dependency. The increase in LHRH secretion induced by ET-3 was accompanied by an increased secretion of prostaglandin E, (PGEZ), not only in the AN-ME incubations, but also in the GTl incubation and perifusion systems. Blockade
of arachidonic acid and/or PG synthesis significantly reduced the ET3-induced LHRH and concomitant PGE, release from both AN-ME fragments and GTl cells incubated in uitro. In AN-ME incubations, ET-3 effects were enhanced by potassium-induced depolarization. This suggests that activation of other transmitter system(s) may be needed for obtaining a physiological activation of the LHRH neuronal system. In summary, in these studies we provide evidence for a direct action of ET-3 on the LHRH neuronal system. This action is exerted directly on LHRH neurons either at the level of the nerve terminals, the perikaryon, or both. In addition, the effects of ET-3 on LHRH release require a functional arachidonic acid metabolic pathway, particularly involving PG synthesis, in order to obtain stimulation, indicating that PGs are involved in the intracellular events leading to ET-3-evoked LHRH secretion. (Endocrinology 132: 789-794, 1993)
E
hypothalamic neurons projecting to the median eminence (ME) (6) and the dense labeling for ET receptors on hypothalamic structures such as the ME (16) and other hypothalamic nuclei (17) suggest that, in addition to a pituitary site of action, the peptide could act at the hypothalamic level to modulate and/or regulate LHRH release. In this respect, it has recently been reported that ET-3 stimulates LHRH secretion from cultured fetal hypothalamic and immortalized neurons (18). In the present study, we explored in further detail the role of ET-3, the most abundant member of the ET family in the brain, in the control of LHRH secretion. First, experiments were designed to test whether ET-3 could release LHRH directly from the LHRH terminals, using arcuate nucleusmedian eminence fragments and an in vitro incubation system that we have characterized extensively (19-21). In addition, we also explored the role of endogenous arachidonic acid metabolites, particularly prostaglandin EZ (PGE*), in mediating the effects of ET-3 on LHRH release. Previous work from our laboratory has clearly documented that PGEZ is an essential intracellular mediator for the action of aminergic (19, 21) and peptidergic (20, 21) neurotransmitters on LHRH secretion. The results reported in this study show a stimulatory effect of ET-3 on LHRH release from nerve terminals in arcuate nucleus (AN)-ME fragments incubated in vitro as well as
NDOTHELINS (ETs) represent a family of peptides originally discovered in endothelial cells (l-3) from which three genes have been identified (1, 2, 4). In addition to the vascular location, ETs are expressed and receptors present in the central nervous system (1, 5-10). Regarding ET binding sites, two receptors have been identified, i.e. ETA and ETB, and originally defined as ET- 1 -selective and nonselective, depending on the relative affinity for the three endothelins. ETA receptors depict high selectivity for ET-1 and ET-2 (ll), whereas ETB does not discriminate between ETs (12). Earlier studies have indicated that ETs participate in the regulation of anterior pituitary function (6, 13-15). The observation that ET-3-immunoreactive neurons in the magnocellular subdivision of the paraventricular nucleus project to the median eminence (6) reinforces this notion by indicating a possible direct role of the peptide in the control of anterior pituitary hormone secretion. In this respect, ET-3 and ET-l have been reported to stimulate LH secretion from superfused anterior pituitary cells (13, 15) and to inhibit PRL release (13, 14). ETs, therefore, may represent an important regulator of reproductive and endocrine functions. Although intracerebroventricular administration of ET-3 does not appear to alter LH secretion (13), the presence of ET-3 in Received August 12, 1992. Address all correspondence and requests for reprints to: Dr. Francisco Jo& Lopez, Reproductive Neuroendocrinology Section, Laboratory of Molecular and Integrative Neurosciences, National Institute of Environmental Health Sciences, Building 101, MD C4-09, Research Triangle Park, North Carolina 27709.
from
an
LHRH
neuronal
cell
line
(GTl
neurons;
22).
In
addition, evidence for the involvement of arachidonic acid metabolites and, in particular, PGs as important intracellular 789
790 mediators sented.
ET-3-STIMULATED of the action
of ET-3
Materials
on LHRH
neurons
is pre-
and Methods
Animals Adult Sprague-Dawley male rats (CD-Charles River, Raleigh, NC) were used to provide tissue for the AN-ME incubations. Animals were kept under a controlled environment consisting of a 12 h-light-12-h darkness schedule and temperature of 22 C. Purina rat chow (Ralston Purina, St. Louis, MO) and water were provided ad libitum.
Reagents Indomethacin and ET-3 were (St. Louis, MO) and Peninsula 5,8,11,14-Eicosatetraynoic acid (Plymouth Meeting, PA).
AN-ME
purchased from Sigma Chemical Co. Labs (Belmont, CA), respectively. (ETYA) was obtained from Biomol
incubations
AN-ME fragments were rapidly dissected after animal decapitation and the explants incubated in a static system as described (20, 23). Fragments were incubated in Krebs-Ringer-Bicarbonate-Glucose buffer (KRB) gassed with 95% 02-5% COz containing different doses of rat ET3 for 30.min periods. In some experiments, medium containing high potassium concentration (28 mM) was used. The osmolarity of this medium was maintained by reducing sodium concentrations, Rat ET-3 was initially dissolved in 0.1 N acetic acid and further diluted to the final concentrations with KRB. The amount of acetic acid present in the medium did not affect basal LHRH secretion and was approximately 100 nM or smaller.
LHRH
SECRETION
Endo. Voll32.
perifused simultaneously at approximately 0.1 ml/min with KRB gassed with 95% 02-5% CO1 at 37 C. Fractions were collected at 5-min intervals. Flow in each chamber was measured by the dilution principle using a i2’I-marker as tracer in 6-10 fractions collected during the preincubation period (60 min). In order to reduce interchamber variability, the flow values were used for the final calculations, expressing, therefore, LHRH output as picograms of hormone secreted in a 5-min fraction.
RIAs LHRH concentrations from in vitro incubations and perifusion experiments were measured by RIA using the anti-LHRH sera FMS-FJL 123a. This antiserum was raised in sheep using LHRH (Peninsula Labs) coupled to bovine thyroglobulin as immunogen. Coupling of hapten to the carrier was performed by using carbodiimide, as previously described (26). The molar ratio hapten:carrier was 1:lOO. The titer of the antiserum when used in RIA was l:lOO,OOO. FMS-FJL 12-3a antiserum recognizes the C-terminal portion of LHRH. Changes as little as deamidation of the last amino acid dramatically reduced the ability of the antiserum to recognize the molecule, since free acid LHRH cross-reacted less than 0.03% with the native molecule. Cross-reactivity with several LHRH analogs was as follows, LHRH, 100%; Trp7,Leus-LHRH, 0.0616%; Tyr’,Leu5,Glu6,Trp7,Lys8-LHRH, 0.3186%; DAla6-LHRH, 4.4080%; DArg6,Trp7,Leu*-LHRH-NHethylamide, less than 0.0048%; DTrp6LHRH-NHethylamide, 0.0915%; Gln*-LHRH, 9.7051%; Des-NH>LHRH, 0.0294%; Des-Gly’r’-LHRH-NHethylamide, 0.0039%; DTrp’,Des-Gly”-LHRH, less than 0.0047%; DAla6,Des-Gly”-LHRHNHethylamide, less than 0.0044%; DLys6-LHRH, 2.6557%; and His5,Trp’,Tyr8-LHRH, less than 0.0046%. The assay was performed under equilibrium conditions. In brief, 150 ~1 sample were used, and this vol brought up to 200 ~1 with assay buffer, antibody 100 ~1 (l:lOO,OOO initial dilution), and tracer (iodinated LHRH; 10,000 cpm) 100 ~1. Standard curves contained 150 ~1 KRB in order to provide similar incubation conditions to those of the samples. After overnight incubation at 4 C, bound and free LHRH were separated by adding-l ml ice-cold ethanol. Thereafter, the assav was centrifuged (2,500 rum at 4 C for 30 min), the supernatant discarded, and the pellet counted in an automatic y-counter. The sensitivity of the assay oscillated between 0.25-0.5 pg/ tube, and the intra- and interassay variabilities were always smaller than 12%. ET-3 did not displace LHRH tracer when tested in the assays at the concentrations used in the experiments, PGE2 was measured with a commercial kit from DuPont/New England Nuclear (Boston, MA). The sensitivity of the assay was less than 0.25 pg/tube; the inter- and intraassay variations were smaller than 10%. Cross-reactivity of the PGEz antiserum was as follows: 3.7% with PGE,; less than 0.4% with 13,14-dehydro-15-keto-PGEZ, PGF,, thromboxane Bl, and l’GF*,. _I
Static incubation
of
immortalized
LHRH
neurons
GTl cells (22) were grown on poly-L-lysine-precoated (40 rg/ml; Sigma Chemical Co) tissue culture flasks (Nunc Inc., Naperville, IL) for 3-4 days in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Grand Island, NY) supplemented with 5% horse serum (GIBCO, lot 3Op5516), 5% fetal calf serum (Hyclone Inc., Logan, UT; lot 11151068), and 1% penicillin/streptomycin (GIBCO, lot 10133222) as described (22), with slight modifications. After reaching confluence, cells were detached from the flasks using a collagenase/dispase (0.4% collagenase and 0.2% dispase in HEI’ES buffer containing 0.25% BSA, 0.8% NaCl, 0.072% KCl, 0.01% Na2P04H, 0.6% HEPES, 0.2% dextrose, and 0.001% DNase) solution (2-4 ml/flask) for 15 min, gently mixed, centrifuged at 800 rpm for 5 min, the supernatant discarded, the pellet resuspended in neuraminidase (0.0008% neuraminidase, 0.02% EDTA in HEPES buffer) solution (2 ml/flask) for 5-10 min, and gently mixed until a single cell suspension was obtained. The cells were centrifuged, the supernatant discarded, and the cell pellet resuspended in DMEM supplemented as described above. Thereafter, cells were plated on polyL-lysine-coated six-well dishes (Nunc) in a final vol of 2.5 ml/well (approximately lo6 cells per well). After growing to confluence (3-4 days), the cells were washed three times with DMEM, preincubated for 30-60 min, and then exposed to the different treatments for 30-min periods. After the incubation time, 1 ml of the medium was collected for RIA measurements, the remaining medium discarded, and cells were exposed to 0.1% trichloroacetic acid in 95% ethanol for 10 min, followed by scraping off the cells for protein measurement by the method of Lowry (24).
Perifusion
of
immortalized
GTl cells were cultured Cytodex-3 beads (Pharmacia described (25). After 3-4 days, the final cell-matrix vol was
LHRH
1993 No 2
v
~
Data analysis and statistics Results are expressed as means f SEM. Dose-response curves were fitted to the four-parameter logistic equation using the algorithm Allfit (27). The parameters obtained after the fitting were used to plot the dose-response curves. In the perifusion experiments, the dose dependency was evaluated by using the ratio of the area under the secretory curve and the area of the three samples previous to the stimuli multiplied by four in order to obtain an estimate of basal release during a 60.min period. Statistical evaluation of the data was performed using the SAS package (SAS Institute, Cary, NC). Statistically significant differences were evaluated using one-way analysis of variance on the raw, the logarithmically transformed or on the ranked (Kruskal-Wallis’s test) data depending on normality and variance homogeneity criteria (28). As a posteriori test, Dunnett’s or Scheffe’s test was used either in the raw, the logarithmically transformed data, or on the ranks, when appropriate. Results were considered statistically significant if in any of the abovementioned tests a F < 0.05 was obtained.
neurons
as indicated above and then grown on LKB Biotechnology, Piscataway, NJ), as cells were loaded in l-ml plastic syringes, adjusted to 0.2 ml, and 8 chambers were
Results ET-3 stimulated LHRH release from AN-ME fragments incubated in vitro in a dose-dependent manner (Fig. 1, upper
ET-3STIMULATED panel). When compared with the control group, doses from 10 nM to 1 PM induced a statistically significant elevation in LHRH release. The PGE2 response followed a similar doserelated profile when compared with that of LHRH (Fig. 1, lower panel), suggesting that this intracellular messenger might be involved in ET-3-induced LHRH release. To further evaluate the participation of PGs in ET-3induced LHRH secretion from AN-ME fragments, a cyclooxygenase blocker, indomethacin, was used at 10 PM. ET-3 (1 PM) elicited an approximate 2.5-fold increase in both LHRH and PGE2 release into the medium (Fig. 2, upper and lower panels, respectively). Indomethacin treatment induced a slight but statistically significant reduction in LHRH release (Fig. 2, upper panel). As expected, PGE;! release was undetectable in the medium of fragments treated with the blocker (Fig. 2, lower panel). Interestingly, indomethacin completely blocked the ability of ET-3 to stimulate LHRH secretion and 12
10
6
6
* ** ii’
SECRETION 2
15
791 ,
*
I
1
* < CONT
1 PM ET-3
10 PM INDO
* ET-3 + INDO
FIG. 2. Indomethacin (INDO; 10 KM) blocks the ET-3 (1 PM)-induced LHRH (upper panel) and PGE, (lower panel) release from AN-ME fragments incubated in uitro. Asterisks denote statistically significant differences US. the control group (CONT) by Kruskal-Wallis’s test followed by the nonparametric Dunnett’s test.
