Analysis of molecular heterogeneity of prolactin in human systemic lupus erythematosus.

Hyperprolactinemia without clinical manifestations has been reported in some patients with systemic lupus erythematosus (SLE) because an increase of prolactin (PRL) is produced due to the BIG/BIG molecular variant (molecular variant < 150 kD). This research project aimed to determine levels of PRL: its bioactive form, the little nonglycosylated form (NGPRL) and variants with decreased bioactivity such as the BIG/BIG and the little glycosylated (GPRL), in 29 women and five men with SLE. PRL was assayed by IRMA with a kit from Immunotech Laboratory, the BIG/BIG form by precipitation with polyethyleneglycol 6000, and the NGPRL and GPRL by chromatography on Concanavalin-A- Sepharose. Increased PRL was detected in seven patients (20.6%) of whom three had increased BIG/BIG, six had increased GPRL and only four had increased NGPRL. The three cases with increased BIG/BIG were contrasted by chromatography on Sephadex G-100. No increased PRL or any of the other variants assayed were found in men. Results were similar when PRL was evaluated in the same blood samples by a different IRMA (DPC Laboratory). The etiology of the hyperprolactinemia in some of these patients is unknown, but their lack of symptoms (galactorrhea or amenorrhea) could be due to the BIG/BIG forms and basically to the glycosylation of the hormone. As for the relation between PRL and SLE activity, we found that hyperprolactinemic patients were younger, had a shorter history of illness, although it was not statistically significant, and a higher SLEDAI score. This would indicate a relation between hyperprolactinemia and lupus activity. The patients with increased BIG/BIG form also had a very active illness at the time of the study.

Hyperprolactinemia in asymptomatic patients is related to high molecular weight posttranslational variants or glycosylated forms.

Circulating human Prolactin (PRL) exists in different variants related to posttranslational modifications, dimerization or association with other serum proteins. Compared to monomeric prolactin these variants usually have little or no biologic activity and include BigBig (BB PRL), Big (B PRL), and Glycosylated forms (G PRL). The aim of the present study was to assess levels of BB PRL, B PRL, little PRL (L PRL) and G PRL in hyperprolactinemic patients with no menstrual alterations or galactorrhea. L PRL, B PRL, and BB PRL were identified by gel filtration chromatography on Sephadex G-100; G PRL and NG PRL were identified by chromatography on Concanavalin A Sepharose. PRL was measured by IRMA DPC. Eleven women, aged 22-50 yrs, were studied for: breast dysplasia (1), controlled hypothyroidism (3), dysmenorrhea (3), microadenoma follow-up (2), and gynecological control (2). Pituitary MRI was normal in all but one patient, who had a microadenoma discovered by Magnetic Resonance Imaging. Six patients had normal L PRL levels, and their hyper PRL was due to excess BPRL or BB PRL. Five patients had increased L PRL levels, but excess G PRL. Patients harboring molecular PRL variants do not present the symptoms typical of the hyperprolactinemic syndrome. Furthermore in patients with clinically controlled prolactinomas the presence of PRL variants should be ruled out to avoid an unnecessary increase of dopamine agonist dosage.

Analysis of the hypothalamic-pituitary-ovary axis in the neonatally-androgenized female rat.

The administration of testosterone propionate (TP) in the female rat at the neonatal age has been used for several yr as a model to study anovulation during adulthood. The present work was designed in order to see whether some neuroendocrine parameters vary with age in this animal model. Hypothalamic LHRH content and LH-FSH anterior pituitary (AP) content and plasma levels were evaluated in samples taken from both neonatally-androgenized and littermate control female rats at different ages (15 to 100 days old). Additionally, we have studied pulsatile LH-FSH released in plasma and in vivo AP response to LHRH in both neonatally-androgenized and control female rats during adulthood. The results indicate that the neonatal TP treatment did not induce any change in hypothalamic LHRH content over development. Neonatally androgenized rats have decreased both LH-FSH AP content and plasma levels at the infantile age (15-day old). LH-FSH AP content remained reduced in samples taken up to the 30th day of age. Plasma LH-FSH levels on the day 30 of age were similar in both groups. TP-treated rats studied on the 100th day of age had: a) an altered pulsatile rhythm of gonadotropin release in plasma due to the decreased LH-FSH trough and average mean values, and to the diminished FSH peak amplitude values, as well as an increased LH:FSH ratio; and b) an impaired in vivo LHRH-induced LH-FSH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for synergism between copper and prostaglandin E2 in stimulating the release of gonadotropin-releasing hormone from median eminence explants: Na+/Cl- requirements.

Copper (Cu) and PGE2 are known to stimulate LHRH release from explants of the median eminence area (MEA) by two mechanisms distinguishable by their Ca2+ dependence. Moreover, exposure to Cu and PGE2 results in an amplified release of LHRH which is partially Ca2+ dependent, thus, resembling the release process stimulated by PGE2 alone. We have shown that LHRH release stimulated by Cu alone is Na+/Cl- dependent. By defining the Na+/Cl- dependence of PGE2- and Cu/PGE2-stimulated release of LHRH, we wished to ascertain if there is synergism between Cu and PGE2 actions. MEA of adult male rats were incubated for 5 min with 150 microM Cu and then for 15 min with 10 microM PGE2 (Cu/PGE2). Controls were incubated with Cu or PGE2. LHRH release into the medium was evaluated by RIA. Substituting Cl- in the incubation buffer with the non-permeant anion, isethionate, did not alter PGE2 stimulation of LHRH release, but it drastically inhibited Cu/PGE2 stimulation of LHRH release, indicating that this process requires a permeant monovalent anion. PGE2 and Cu/PGE2 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 microM tetrodotoxin (TTX) nor 100 microM amiloride were inhibitory. To ascertain if Na+ is required for Cu uptake, we evaluated the uptake of 67Cu by MEA explants 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Amplification of prostaglandin E2 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) E2 stimulation of the release of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA). 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 CuCl2 (ionic) or copper complexed to histidine (CuHis) at a concentration of 200 microM each and then for 15 min in the presence of 10 microM PGE2. It was found that chelated copper but not ionic amplified PGE2 action, and that the magnitude of PGE2 stimulation of LH-RH release was 3-4-fold in copper-treated than untreated tissue. Moreover, PGE2-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 microM each) and then for 15 min with 10 microM PGE2. Controls were incubated with metal(II) complex or PGE2. Of these complexes, only CuHis and to a lesser extent NiHis stimulated LH-RH release. However, CuHis was the only complex that amplified PGE2 stimulation of LH-RH release. Thus, amplification is specific for copper. The finding that chelated copper but not ionic copper amplifies PGE2 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.(ABSTRACT TRUNCATED AT 250 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.

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)

Copper stimulation of LHRH release from median eminence explants. I. A divalent metal specific process that does not require extracellular calcium.

We have previously shown that chelated copper stimulates the release of LHRH from explants of the median eminence area (MEA) incubated under in vitro conditions, and that this stimulation involves a ligand specific interaction. To further elucidate the mechanism of action of copper, we addressed two questions: (a) What is the divalent metal [metal(II)] specificity for this release process? (b) Is the stimulation of LHRH release by CuHis dependent on influx of extracellular calcium? MEA obtained from adult male rats were incubated for 15 min with one of the following divalent metals Cu, Ni, Fe, Zn, Cd or Mn (each complexed to histidine at an equimolar ratio; 100 microM) and then in the absence of metal for an additional period of 30 min. We found that CuHis and to a lesser extent NiHis stimulated LHRH release, and that FeHis, ZnHis, CdHis or MnHis did not do so. In addition, MEA were incubated for 15 min with CuHis in the presence or absence of CaCl2. Under these two conditions, the temporal pattern and magnitude of CuHis-stimulated LHRH release were identical, indicating that extracellular calcium is not required for copper action. Since, in this series of metals, Cu2+ and Ni2+ are the most potent oxidizing agents, our finding strongly supports our previous proposition that an oxidation reaction is involved in the process of copper stimulation of LHRH release. It has yet to be elucidated whether copper action is totally independent of an increase in intracellular calcium or whether copper leads to LHRH release via mobilization of calcium from intracellular stores.

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)

A role for extracellular copper in modulating prostaglandin E2 (PGE2) action: facilitation of PGE2 stimulation of the release of gonadotropin-releasing hormone (LHRH) from median eminence explants.

Copper is present in high concentrations in axonal terminals of hypothalamic neurons (1). We have previously postulated that copper is released from axonal terminals of hypothalamic neurons (2). In this study, we addressed the question: Does extracellular copper facilitate PGE2 action, specifically, the stimulation of LHRH release? The median eminence area (MEA) of male rats was incubated under in vitro conditions, and LHRH release into the medium was quantified by RIA. When MEA were incubated for 5 min with 100 microM copper and then for 15 min with 50 microM PGE2 (Cu/PGE2), Cu/PGE2-stimulated release of LHRH was significantly (P less than 0.001) greater (2 times) than the sum of copper- and PGE2-stimulated release. When MEA were first incubated for 15 min with PGE2 and then for 5 min with copper (PGE2/Cu), PGE2/Cu-stimulated release equalled the sum of PGE2- and copper-stimulated release of LHRH. Thus, copper facilitates PGE2 stimulation of LHRH release from the MEA, and the characteristics of this facilitation are consistent with an enhancement of PGE2 binding to its receptor. These results support the proposition that extracellular copper can serve as a modulator of PGE2 action.

A ligand-specific action of chelated copper on hypothalamic neurons: stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants.

We have previously shown that chelated copper stimulates the release of luteinizing hormone-releasing hormone (LHRH) from isolated hypothalamic granules. In this study, we wished to ascertain if chelated copper acts on hypothalamic neurons to stimulate LHRH release and, if so, what is the ligand specificity of this interaction. An in vitro system of explants of the median eminence area (MEA) was established and characterized. MEA explants were exposed for 15 min to 50 microM copper, and then they were incubated for 75 min in copper-free medium. Copper led to a transient increase in the rate of LHRH release; the maximal rate was attained 15 min after transfer of the MEA to copper-free medium. In addition, we found that copper complexed to histidine (Cu-His), but not ionic copper, stimulated LHRH release, the magnitude of which was dependent on the dose of Cu-His. The chelator specificity for Cu complex action was such that Cu-His stimulated LHRH release 4.9-fold and Cu-Cys stimulated release 2.5-fold, whereas neither Cu-Thr, Cu-Gly-His-Lys, Cu-bovine serum albumin, nor ceruloplasmin stimulated LHRH release. Based on these results and those of others indicating that the concentration of copper in hypothalamic axonal terminals is 1-2 orders of magnitude greater than plasma, we propose that copper released in the vicinity of the LHRH neurons interacts with specific sites on the LHRH axonal terminals, which leads to release of the peptide.