Median Eminence Explants Research Articles

Tanycyte specific ablation of diacylglycerol lipase alpha stimulates the hypothalamic-pituitary-thyroid axis by decreasing the endocannabinoid mediated inhibition of TRH release.

In addition to the hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, a glial cell type, the tanycytes, also play a role in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis. Tanycytes modulate the feedback regulation of the axis by regulating the local thyroid hormone availability in the median eminence where the hypophysiotropic axons terminate. Recently, we showed that tanycytes produce diacylglycerol lipase alpha (DAGLα), the synthesizing enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG) that inhibits the release of TRH from the hypophysiotropic terminals in median eminence explants. To determine the importance of the endocannabinoid production of tanycytes, adult male Rax-CreERT2//DAGLαfl/fl mice were treated with tamoxifen to induce a tanycyte specific decrease of DAGLα expression (T-DAGLα KO). The effect of this genetic manipulation on the activity of the HPT axis was determined. Tanycyte specific decrease of DAGLα expression resulted in an approximately 2-fold increase of TSHβ mRNA level that was accompanied by increased levels of circulating free T4. The TRH mRNA level was, however, not influenced by the genetic manipulation. In addition to the effects on the HPT axis, the T-DAGLα KO mice showed increased fat mass ratio and decreased blood glucose levels. These data indicate that when endocannabinoid release of tanycytes is decreased, the disinhibition of the TRH release induces increased TSH synthesis and higher circulating T4levels. Thus it suggests that in wild-type mice, tanycytes exert a tonic inhibitory effect on the TRH release of hypophysiotropic axons. Furthermore, the endocannabinoid release of tanycytes also influences glucose homeostasis and fat deposition.

GnRH Neurons Directly Listen to the Periphery

GnRH Neurons Directly Listen to the Periphery

Role of Estradiol in the Dynamic Control of Tanycyte Plasticity Mediated by Vascular Endothelial Cells in the Median Eminence

In the ever-changing physiological context of the neuroendocrine brain, the mechanisms by which cellular events involving neurons, astroglia, and vascular cells are coordinated to bring forth the appropriate neuronal signaling is not yet known but is amenable to examination. In the median eminence of the hypothalamus, endothelial cells are key players in the plasticity of tanycytes (specialized astroglia) and neuroendocrine synapse efficacy. Here we report that estradiol acts on both purified endothelial cells and isolated tanycytes to trigger endothelial-to-glial communication that leads to a sudden and massive retraction of tanycyte processes. The blockade of endothelial nitric oxide synthase by in vitro adenoviral-mediated gene transfer of a dominant-negative form of endothelial nitric oxide synthase abrogates the estradiol-induced tanycyte plasticity mediated by endothelial cells. In parallel, increases in prostaglandin-E(2) (PGE(2)) due to changes in cyclooxygenase (COX)-1 and COX-2 expression induced by the exposure of tanycytes to estradiol promote acute tanycyte plasticity. We also demonstrate by electron microscopy that the administration of PGE(2) to median eminence explants induces rapid neuroglial plasticity at the neurovascular junction of neurons that release GnRH (the neuropeptide controlling reproduction). Conversely, preventing local PGE(2) synthesis in the median eminence of adult female rats with the COX inhibitor indomethacin impairs the ovarian cycle, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamo-hypophyseal portal system. Taken together, our findings show that estradiol controls the dialog between endothelial cells and astroglia to regulate neuroglial plasticity in the neuroendocrine brain.

Hypothalamic Tumor Necrosis Factor-α Converting Enzyme Mediates Excitatory Amino Acid-Dependent Neuron-to-Glia Signaling in the Neuroendocrine Brain

Glial erbB1 receptors play a significant role in the hypothalamic control of female puberty. Activation of these receptors by transforming growth factor alpha (TGFalpha) results in production of prostaglandin E2, which then stimulates luteinizing hormone releasing hormone (LHRH) neurons to secrete LHRH, the neuropeptide controlling sexual development. Glutamatergic neurons set in motion this glia-to-neuron signaling pathway by transactivating erbB1 receptors via coactivation of AMPA receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Because the metalloproteinase tumor necrosis factor alpha converting enzyme (TACE) releases TGFalpha from its transmembrane precursor before TGFalpha can bind to erbB1 receptors, we sought to determine whether TACE is required for excitatory amino acids to activate the TGFalpha-erbB1 signaling module in hypothalamic astrocytes, and thus facilitate the advent of puberty. Coactivation of astrocytic AMPARs and mGluRs caused extracellular Ca2+ influx, a Ca2+/protein kinase C-dependent increase in TACE-like activity, and enhanced release of TGFalpha. Within the hypothalamus, TACE is most abundantly expressed in astrocytes of the median eminence (ME), and its enzymatic activity increases selectively in this region at the time of the first preovulatory surge of gonadotropins. ME explants respond to stimulation of AMPARs and mGluRs with LHRH release, and this response is prevented by blocking TACE activity. In vivo inhibition of TACE activity targeted to the ME delayed the age at first ovulation, indicating that ME-specific changes in TACE activity are required for the normal timing of puberty. These results suggest that TACE is a component of the neuron-to-glia signaling process used by glutamatergic neurons to control female sexual development.

Central Stimulatory Influence of Oxytocin on Preovulatory Gonadotropin-Releasing Hormone Requires More than the Median Eminence

The study was designed to determine whether the ability of central oxytocin (OT) to stimulate gonadotropin-releasing hormone (GnRH) on the afternoon of proestrus (PE) in the cycling female rat is mediated at the level of GnRH terminals within the median eminence (ME), or at higher hypothalamic levels where GnRH cell bodies and axons are located. Determining the location of this OT effect in vivo has proven difficult. Therefore, an in vitro system utilizing ME or basal hypothalamic (BH) explants containing GnRH terminals, or GnRH neurons including the cell bodies, axons and terminals, respectively, were harvested from regular cycling female rats at 15:00 h on PE or diestrus (DI). The explants were allowed to preincubate in Krebs Ringer Bicarbonate Buffer containing glucose, ascorbic acid, calcium, and a metalloprotease inhibitor (KRBG) and enriched with 95% O₂/5% CO₂ at 37°C until a stable baseline release of GnRH was achieved (30 min). The 0.05 level of probability was used as the minimum criterion of significance in all experiments. The ability of OT (10^–15–10^–9M) to stimulate the release of GnRH was determined in both ME and BH explants on PE and DI. The results demonstrated a sensitive, dose-dependent ability of OT to stimulate GnRH release from PE BH explants which was observed only in PE. Furthermore, OT failed to significantly stimulate GnRH release from ME explants on either PE or DI. The data indicate that the PE BH explant paradigm can be used to examine the manner and mechanisms by which OT influences GnRH release on the afternoon of PE. Furthermore, the results indicate for the first time that the stimulatory action of OT by itself on preovulatory GnRH release in cycling female rats is not mediated at the level of the GnRH terminals within the ME, but requires neuronal interactions and mechanisms within the BH explants.

Pulsatile Secretion of Gonadotropin-Releasing Hormone by Rat Hypothalamic Explants of GnRH Neurons without Cell Bodies

Gonadotropin-releasing hormone (GnRH) is typically secreted in a pulsatile manner. It is still unclear whether pulsatility depends on a GnRH pulse generator residing in the GnRH neurons or in other neurons. Since the cell bodies of GnRH neurons are located rostrally to the optic chiasm and the majority of GnRH terminals in the median eminence of the rat hypothalamus, we have compared GnRH secretion from individual preoptic, retrochiasmatic and median eminence explants using a static incubation system. GnRH is released from the three different types of explant in response to depolarization with veratridine or glutamate receptor stimulation using the agonist N-methyl-D-aspartate. Only the retrochiasmatic explants, however, show a characteristic pulsatile secretion of GnRH. The mean (+/- SD) interpulse interval is respectively 37 +/- 5, 25 +/- 4 and 12 +/- 1 min when the fractions are collected at 7.5-, 5.0- and 2.5-min intervals. The immunocytochemically stained GnRH cell bodies are normally distributed in the preoptic explants (n = 212-420) while only 3 GnRH cell bodies can be visualized in 7 retrochiasmatic explants. Semi-quantitative RT-PCR shows that GnRH mRNA is present in the retrochiasmatic explant in a ratio of about 1:600 relative to the preoptic explant. We conclude that pulsatile GnRH secretion occurs in the virtual absence of GnRH cell bodies but does not occur from GnRH terminals in the isolated median eminence. These data further indicate that a mechanism of GnRH pulsatility is located in the retrochiasmatic hypothalamus and involves neurons different from the GnRH neurons.

Glutamic acid induces luteinizing hormone releasing hormone release via alpha receptors

Glutamic acid (GA) and norepinephrine (NE) stimulate luteinizing hormone-releasing hormone (LHRH) release via release of nitric oxide (NO) from NOergic neurons in the arcuatemedian eminence region. To determine if GA releases LHRH via direct stimulation of NOergic neurons, or via stimulation of noradrenergic terminals, arcuate median eminence explants from male rats were incubated with various compounds, and the LHRH release into the medium was measured. GA-induced release of LHRH was completely blocked by phentolamine (1 μM), an alpha receptor blocker, which, by itself, had no effect on the release. Nitroprusside (NP), which spontaneously releases NO, more than doubled LHRH release. To determine if alpha receptors on the LHRH neuron are required for the action of NP, the tissue was incubated with phentolamine, plus NP. Phentolamine had no effect on the LHRH-releasing action of NP. The results are interpreted to mean that GA activates the release of NE from the noradrenergic terminals. This acts on alpha receptors on the NOergic neuron to produce the release of NO. This NO diffuses to the LHRH terminals and induces release of LHRH.

A putative role for extracellular ATP: Facilitation of 67copper uptake and of copper stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants

We have previously shown that extracellular copper stimulates the release of the luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA), that chelated copper (Cu) but not ionic Cu is the active form of the metal, and that there is a direct correlation between the ligand specificity for 67Cu uptake and Cu action. In this study, we examined the possibility that extracellular ATP can serve as a ligand facilitating Cu action on the LH-RH neuron. Hypothalamic slices or MEA explants of adult male rats were used. It was found that ATP facilitates 67Cu uptake by hypothalamic slices when Cu:ATP molar ratio was 1:2000 but not 1:2. Keeping the [Cu] constant (150 μM) and varying [ATP], ATP facilitation of Cu stimulation of LH-RH release from MEA explants was found to be a saturable function of [ATP]; maximal facilitation occurred with 2.5 mM ATP. When the nucleotide phosphate specificity for facilitation of Cu action was assessed, ADP, ATP, α,β-methylene-ATP (the non-hydrolyzable analogue of ATP) and GTP were equally effective, whereas AMP and adenosine were ineffective. These results indicate that extracellular ATP can facilitate Cu action on the LH-RH neuron and they are consistent with two mechanisms: (1) ATP facilitating Cu uptake and hence, Cu action and (2) ATP facilitating Cu action via an interaction with a purinergic receptor.

A rapid, Ca 2+-independent, onset of prostaglandin E 2 stimulation of the release of luteinizing hormone releasing hormone from copper-treated median eminence explants

Prostaglandin E 2 (PGE 2) is known to stimulate the release of luteinizing hormone releasing hormone (LHRH) from explants of the median eminence area (MEA). We have previously shown that a short preincubation of the MEA with copper markedly amplifies PGE 2 stimulation of LHRH release and that the Ca 2+-cAMP pathway is involved in this release process. In this study, we defined the kinetics of the onset of PGE 2 action and examined the Ca 2+ requirement for the onset and manifestation of PGE 2 action. MEA explants from immature female rats were incubated with copper (150 μM copper-histidine complex); thereafter, expiants were exposed to 10 μM PGE 2 for periods of 2−7 min with or without Ca 2+ (Ca 2+-free buffer containing 1 mM EGTA). A 2 min PGE 2 exposure was as effective as a 7 min PGE 2 exposure in stimulating LHRH release; the latter was manifested during the period between 2–7 min. When MEA were exposed to PGE 2 for 5 min, maximal rate of release was attained within this 5 min period. When MEA were exposed to PGE 2 for 2 min and then to forskolin (100 μM) for 5 min, there was no further increase in the rate of LHRH release (although exposure to forskolin alone maximally stimulated LHRH release). In the presence of EGTA (no Ca 2+), PGE 2 stimulation of LHRH release was abolished and this effect of EGTA was totally reversed by the addition of Ca 2+ 2 min after PGE 2 exposure and partially reversed (40%) by the addition of Ca 2+ 5 min after PGE 2 exposure in the presence of EGTA. In summary, (i) a 2 min exposure of copper-treated MEA expiants to PGE 2 suffices to maximally stimulate LHRH release presumably by maximally activating adenylate cyclase; (ii) maximal rates of LHRH release are attained within 3–5 min after PGE 2 exposure; (iii) there is a differential Ca 2+ requirement for the onset and for the manifestation of PGE 2 action: the initial 2 min interaction does not require extracellular Ca 2+; the subsequent events leading to LHRH release require Ca 2+ and the Ca 2+ requiring element(s) is short-lived (3–5 min).

Examination of a possible mechanism by which anisomycin suppresses pulsatile luteinizing hormone release in ovariectomized rats.

An initial study was performed to ascertain the effects of anisomycin, a reversible inhibitor of protein synthesis, on pulsatile luteinizing hormone (LH) release in adult, ovarietomized (OVX) rats. For this experiment, rats OVX 3-4 weeks earlier were fitted with indwelling atrial cannulae. On the next day (approximately 13.00 h), the rats received a subcutaneous injection of either 100 mg/kg body weight (BW) anisomycin or its saline vehicle. Administration of anisomycin significantly suppressed mean plasma LH levels, mean trough values, and both LH pulse frequency (saline: 6.3 pulses/3 h vs. anisomycin: 2.7 pulses/3 h) and amplitude. To determine whether anisomycin affected anterior pituitary LH responses to LH-releasing hormone (LHRH), a second experiment was performed in which saline- and anisomycin-treated OVX rats were given an intravenous injection of 10 ng/100 g BW LHRH 1.5 h later (14.30 h). Rats then were sacrificed and the anterior pituitary and brain removed. Whereas preinjection plasma LH levels were significantly lower in anisomycin-treated rats, they were significantly higher in anisomycin-treated rats 20 min after LHRH. Consequently, mean maximal increments and percent increments were significantly higher in anisomycin-treated rats. AP LH content and content of LHRH in the medial preoptic and suprachiasmatic nuclei were not influenced by anisomycin treatment. However, median eminence (ME) LHRH concentrations in anisomycin-treated rats were almost double the LHRH levels measured in control rats. A third study was conducted to assess the effects of anisomycin on basal and potassium (K+)-stimulated LHRH release from superfused ME explants.(ABSTRACT TRUNCATED AT 250 WORDS)

Atrial natriuretic factor inhibits luteinizing hormone secretion in the rat: evidence for a hypothalamic site of action.

The presence of atrial natriuretic factor (ANF) immunoreactivity and receptors for ANF in the median eminence, hypothalamus, and anterior pituitary gland suggests a role for the peptide in the hypothalamic control of anterior pituitary function. In conscious ovariectomized female rats, transient elevation of plasma levels of ANF by volume loading, a stimulus known to release endogenous ANF from the heart, or by bolus iv injection of 0.1, 1.0, or 10 micrograms synthetic ANF failed to result in altered circulating levels of LH or GH. Constant iv infusion of ANF for 30 min, such that 2- to 3-fold elevations in plasma ANF were detected by RIA resulted, however, in significant inhibition of LH release in ovariectomized female rats (0.05 and 0.1 micrograms ANF/kg.min) and orchidectomized male rats (0.1 microgram ANF/kg.min). It was unlikely that this effect was exerted at the level of the anterior pituitary, since ANF failed to alter basal or LHRH-stimulated LH release from cultured anterior pituitary cells in vitro and since iv infusion of 0.1 microgram ANF/kg.min failed to alter pituitary responsiveness in vivo to a 10-ng bolus injection of LHRH. Significant inhibition of LH secretion was also observed after third cerebroventricular injection of 1.0 or 2.0 nmol ANF. As with iv infusion, central administration of ANF failed to significantly alter GH secretion. LHRH release from median eminence explants incubated in vitro in the presence of dopamine (60 or 120 microM) was inhibited by 10(-7) M ANF, suggesting a median eminence site of action of the peptide. Finally, an opiate involvement in the mechanism of ANF's action was suggested, since naloxone (0.5 mg, iv, followed by a 60-min infusion of an additional 1 mg) completely blocked the ability of ANF (0.1 or 0.5 microgram/kg.min, infused over the last 30 min of naloxone administration) to inhibit LH release. These data suggest that ANF can act centrally to alter the hypothalamic control of gonadotropin secretion, possibly by interacting with central dopaminergic and peptidergic systems. They further suggest actions of ANF within the brain unrelated to its previously described effects on fluid and electrolyte homeostasis.

Evidence for synergism between copper and prostaglandin E 2 in stimulating the release of gonadotropin-releasing hormone from median eminence explants: Na +/Cl − requirements

Copper (Cu) and PGE 2 are known to stimulate LHRH release from expiants of the median eminence area (MEA) by two mechanisms distinguishable by their Ca 2+ dependence. Moreover, exposure to Cu and PGE 2 results in an amplified release of LHRH which is partially Ca 2+ dependent, thus, resembling the release process stimulated by PGE 2 alone. We have shown that LHRH release stimulated by Cu alone is Na +/Cl − dependent. By defining the Na +/Cl − dependence of PGE 2- and Cu/PGE 2-stimulated release of LHRH, we wished to ascertain if there is synergism between Cu and PGE 2 actions. MEA of adult male rats were incubated for 5 min with 150 μM Cu and then for 15 min with 10 μM PGE 2 (Cu/PGE 21). Controls were incubated with Cu or PGE 2. LHRH release into the medium was evaluated by RIA. Substituting Cl − in the incubation buffer with the non-permeant anion, isethionate, did not alter PGE 2 stimulation of LHRH release, but it drastically inhibited Cu/PGE 2 stimulation of LHRH release, indicating that this process requires a permeant monovalent anion. PGE 2 and Cu/PGE 2 stimulation of LHRH release were both inhibited when Na + was substituted with Li +, or when 0.5 mM ouabain was included in the Na +-containing buffer; neither 10 μM tetrodotoxin (TTX) nor 100 μM amiloride were inhibitory. To ascertain if Na + is required for Cu uptake, we evaluated the uptake of 67Cu by MEA expiants and found that neither ouabain nor Li + inhibited uptake, indicating that the extracellular Na + and the activity of Na +/K + ATPase are required for the process of LHRH release. In summary, (i) Cu and PGE 2 stimulation of LHRH release are distinct processes, based on their Ca 2+ and Cl − requirements; and (ii) the process of Cu/PGE 2 stimulation of LHRH release exhibits the ionic requirements of both secretagogues. This is consistent with Cu and PGE 2 synergistically acting on the LHRH neuron, which results in amplification of the release processes initiated by each of the secretagogues.

In vivo and in vitro examination of an autoregulatory mechanism for luteinizing hormone-releasing hormone.

Recent evidence has been presented that demonstrates the existence of ultrashort feedback circuits for a number of neuropeptides in the central nervous system. The present investigation was undertaken to examine the possible existence of an autoregulatory mechanism for LHRH. In the first experiment, long term ovariectomized rats bearing lateral ventricular and atrial cannulae received intracerebroventricular (icv) injections of saline or LHRH every hour from 1200-1500 h. Blood was collected at 10-min intervals from 1200-1500 h to assess the effects of icv LHRH on pulsatile LH and FSH release. After the 1500 h collection, LHRH was injected iv and blood was collected to determine the effects of icv LHRH on pituitary responsiveness to LHRH. Central injections of 0.1 pg and 1 ng LHRH, but not 10 pg LHRH, significantly suppressed mean LH levels, trough LH levels, and LH pulse frequency compared to those in saline-treated control rats. The amplitude of LH pulses was not significantly affected by any dose of LHRH. In contrast to LH, multiple icv injections of LHRH failed to alter pulsatile FSH release. Pituitary LH and FSH responses to iv LHRH injection were not suppressed by icv LHRH. In a second experiment, hourly icv injections of 1 ng LHRH into proestrous rats markedly suppressed preovulatory LH release only during the middle to latter phases of the surge. In a final study using an in vitro superfusion system, addition of a LHRH agonist to the superfusion medium at a concentration that does not cross-react in the LHRH RIA suppressed basal and K+-stimulated LHRH release from medial basal hypothalamic fragments, but not from median eminence explants. These results support the existence and operation of an autoregulatory mechanism for LHRH in the central nervous system which may participate in the control of episodic LHRH release in ovariectomized rats and preovulatory LHRH release in proestrous rats. Seemingly, ultrashort-loop negative feedback regulation of LHRH requires the presence of structures other than nerve terminals in the MBH (i.e. cell bodies).

Amplification of prostaglandin E 2 stimulation of luteinizing hormone-releasing hormone release from median eminence explants: a metal(II)-specific effect of chelated copper

We have previously demonstrated that extracellular copper amplifies prostaglandin (PG) E 2 stimulation of the release of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area(MEA) 1. Two questions were addressed: what is the active form of copper and the metal(II) specificity for copper action? MEA explants were incubated for 5 min in the presence of CuCl 2 (ionic) or copper complexed to histidine (CuHis) at a concentration of 200 μM each and then for 15 min in the presence of 10 μM PGE 2. It was found that chelated copper but not ionic amplified PGE 2 action, and that the magnitude of PGE 2 stimulation of LH-RH release was 3–4 fold in copper-treated than untreated tissue. Moreover, PGE 2-stimulated release was directly related to the dose of CuHis. To test the metal specificity, MEA explants were incubated for 5 min with one of the following metal(II) complexes: CuHis, NiHis, FeHis, ZnHis, CdHis, MnHis, or BaHis (200 μM each) and then for 15 min with 10 μM PGE 2. Controls were incubated with metal(II) complex or PGE 2. Of these complexes, only CuHis and to a lesser extent NiHis stimulated LH-RH release. However, CuHis was the only complex that amplified PGE 2 stimulation of LH-RH release. Thus, amplification is specific for copper. The finding that chelated copper but not ionic copper amplifies PGE 2 is suggestive that the copper-interactive sites on the LH-RH neurons are not exposed to the extracellular space but that they are either embedded in the plasma membrane, facing the intracellular space, or in the cytoplasm. Such an orientation is consistent with copper modulation of the functional state of a postreceptor component involved in the cascade of events initiated by PGE 2 and leading to LH-RH release.

Evidence that a short-lived effect of copper leads to amplification of prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from median eminence explants.

We have previously shown that copper (Cu) amplifies prostaglandin E2 (PGE2) stimulation of LHRH release from explants of the median eminence area (MEA) incubated under in vitro conditions. In this study, we have carried out an extensive comparative study of the kinetics of the process of PGE2 stimulation and C alpha-amplified PGE2 (Cu/PGE2) stimulation of LHRH release. MEA explains obtained from adult male rats were incubated under in vitro conditions, and LHRH released into the medium was assayed by RIA. Kinetic parameters were established by varying the time of exposure of the MEA to Cu or PGE2 and the dose of Cu or PGE2. Cu action requires a minimal 5-min period of exposure of the MEA, is rapidly manifested (less than 5 min after transfer of Cu-free medium), and is reversible (half-life, 10-15 min). Cu amplification of PGE2 action is a saturable function of the concentration of both Cu and PGE2; half-saturation and saturation were achieved with 120 and 200 microM copper, respectively, and with 0.6 and 10 microM PGE2, respectively. A 5-min exposure of Cu-treated MEA to PGE2 has the following time course of PGE2 action: the maximal rate of LHRH release is attained within 5 min; release is maintained at the maximal rate for another 5 min and then declines. Importantly, this decline is not prevented by a longer (15-min) exposure of the MEA to PGE2, indicating desensitization to PGE2 action. When MEA treated with Cu (5 min) and PGE2 (15 min) is allowed to recover for 45 min in buffer, the tissue regains its responsiveness to PGE2. All of the kinetic parameters of the process induced by PGE2 alone are similar to those described above for Cu/PGE2, except that the magnitude of the maximal rate of PGE2-stimulated release is 4- to 6-fold lower and that release is maintained at this rate for about 45 min. We also examined the PG structure specificity for stimulation of LHRH release and found that PGD2 by itself or after Cu pretreatment is much less effective than PGE2 in stimulating LHRH release. In summary, Cu amplification of PGE2 stimulation of LHRH release from LHRH neuronal terminals (i.e. MEA explants) involves rapid activation of a short-lived component.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular calcium is required for copper-amplified prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from median eminence explants.

Prostaglandin E2 (PGE2) is known to stimulate the release of LHRH from hypothalamic tissue incubated under in vitro conditions. We have previously shown that a short preincubation of explants of the median eminence area with copper(II) [Cu(II)] complexed to histidine (CuHis) markedly amplifies PGE2 stimulation of LHRH release. In this study, we wished to ascertain if Cu-amplified PGE2 stimulation of LHRH release is dependent on influx of extracellular calcium (Ca) and, if so, whether it is the copper and/or PGE2 action that is Ca dependent. Median eminence area explants were incubated for 5 min with 200 microM CuHis, then for 15 min with 10 microM PGE2, and finally for 45 min in buffer (Cu/PGE2). Controls were incubated with CuHis or PGE2. In the presence of Ca (1.8 mM), Cu/PGE2 stimulation of LHRH release reached a peak value within 15 min of exposure to PGE2 (accelerated phase), after which the rate of release declined, with a half-life of about 20 min (decelerated phase). In the absence of Ca, Cu/PGE2 stimulation of LHRH release was inhibited by 70% during the accelerated phase, and it was completely inhibited during the decelerated phase. When incubation was carried out in the presence of Ca and 100 microM verapamil, Cu/PGE2 stimulation of LHRH release was similar to that seen in the absence of extracellular Ca. When Ca was omitted from the incubation medium during the exposure to CuHis but was included during and after the exposure to PGE2, the response to PGE2 was fully restored. In addition, we evaluated the extracellular Ca requirement for stimulation of LHRH release by PGE2 alone (without pretreatment with CuHis) and found that release was inhibited by 65% in the absence of Ca. These results indicate that the process of PGE2 stimulation of LHRH release is partially dependent on extracellular Ca, that the process of Cu-amplified PGE2 stimulation of release is highly dependent on influx of extracellular Ca, and that PGE2 action, but not Cu action, is dependent on Ca influx. Since cAMP has been implicated in Ca2+-dependent secretion in other cells, we propose that Cu amplifies the functional state of a postreceptor component involved in the Ca-cAMP secretory pathway stimulated by PGE2.

Copper stimulation of LHRH release from median eminence explants. II. A process dependent on chloride transport.

We have previously shown that chelated copper stimulates LHRH release from explants of the median eminence area (MEA). Characteristics of this release process are: ligand and metal specificity, the involvement of a limited number of copper interactive sites, and a lack of dependence on extracellular calcium. Since chloride transport is essential for exocytosis of peptides and biogenic amines, we wished to ascertain if chloride transport is essential for the process of CuHis-stimulated release of LHRH. MEA explants were incubated for 15 min with 100 microM CuHis (phase I) and then for 15 min in copper-free medium (phase II) and LHRH released into the medium was evaluated by RIA. In the presence of 136 mM Cl, CuHis stimulated the release of LHRH from a basal level of 5 +/- 0.4 pg/15 min per MEA to 17 +/- 0.9 pg during phase I and to 30 +/- 1.2 pg during phase II. In the absence of Cl-, the CuHis-stimulated release of LHRH during phases I and II was inhibited by 80 and 90%, respectively. In the presence of 136 mM Cl- and the anion transport inhibitor SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) the stimulated release was completely inhibited in both phases. When the selectivity of this release process for monovalent anions was tested, the effectiveness of the anions in supporting CuHis-stimulated LHRH release was in this decreasing order: Cl- greater than Br- greater than SCN- = acetate greater than I- = isethionate.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper stimulation of LHRH release from median eminence explants. III. A process dependent on extracellular sodium.

Copper, complexed to histidine (CuHis), stimulates LHRH release from explants of the median eminence area (MEA). To gain further understanding of the mechanism of copper action, in this study, we assessed the Na+ and energy requirements for CuHis stimulation of LHRH release. MEA explants, obtained from adult male rats, were incubated at 37 degrees C for 15 min with 100 microM CuHis and then for 45 min in CuHis-free medium (Krebs-Ringer-phosphate buffer, pH 7.4). LHRH released into the medium was evaluated by RIA. When the incubation buffer contained 143 mM Na+, CuHis stimulated the release of LHRH from a basal level of 17.2 +/- 1.26 (mean +/- SEM, n = 7) to 74.5 +/- 6.2 pg/60 min per MEA. When [Na+] was reduced to 16 mM Na+ (by substituting with Li+), CuHis-stimulated LHRH release was inhibited by 80% (p less than 0.001); indicating a requirement for Na+. In addition, we found that CuHis-stimulated LHRH release was a saturable function of Na+ concentration; saturation achieved with about 100 mM Na+. To assess the requirement for Na+ transport, we evaluated the effect of 1 mM ouabain, 10 microM tetrodotoxin (TTX), or 100 microM amiloride on CuHis stimulation of LHRH release. Ouabain inhibited CuHis stimulation of LHRH release by 80%, whereas TTX and amiloride were ineffective. In addition, we observed that CuHis did not stimulate LHRH release when incubation was carried out at 4 degrees C or at 37 degrees C in the presence of 5 mM KCN.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Castration and Maturational Age of Male Rats on the Process of Copper-Stimulated Release of Luteinizing Hormone Releasing Hormone from Median Eminence Explants: Evidence that Androgens Increase the Affinity of the Copper-Interactive Sites for Copper

We have previously shown that chelated copper stimulates the release of luteinizing hormone releasing hormone (LHRH) from explants of the median eminence area (MEA) incubated under in vitro conditions and that this stimulation involves a ligand-specific interaction. In this study, we addressed the question: do testicular steroids regulate the secretory response of LHRH neurons to copper? MEA, obtained from immature, mature, immature castrated and sham-operated rats, were incubated in the presence of various concentrations of copper for 15 min and then in the absence of copper for an additional period of 30 min. We noted that after a lag period of 5 min of incubation, the rate of LHRH release increased in a linear fashion for a period of 15 min. In addition, the rate of LHRH release as well as the rate at which LHRH release was accelerated were saturable functions of the concentration of copper. When incubation was carried out in the presence of a nonsaturating concentration of copper (50 microM), the fractional amount (percent of the total MEA content) of LHRH released from the MEA of castrated rats was significantly (p less than 0.001) lower than that from sham-operated rats; stimulated release being 0.9% and 1.4%, respectively. Similarly, copper-stimulated release from the MEA of immature rats was lower than that from the MEA of mature rats. However, when incubation was carried out in the presence of saturating concentrations of copper (100 or 200 microM), the percentage of stimulated release from the MEA of castrated rats was similar to that of sham-operated rats and significantly lower than that of mature and sham-operated rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium messenger system: an integrated view.

Calcium messenger system: an integrated view.