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A Barnea

Publications and source records attributed to A Barnea.

At least 55 records · Page 3Linked to original sources
PubMed1990

Postovariectomy decline in the in vitro release of LH-releasing hormone is not manifested in aging female rats.

Aging of the female reproductive system is associated with a transition from a regular cyclic to an irregular cyclic and finally to an acyclic mode of function. We addressed the question: is the transition associated with a decline in the secretory response of the LHRH neuron to an external stimulus and with a change in steroid regulation of the secretory function of the LHRH neuron? Three groups of virgin female rats ranging from 2 to 7 months of age were used: intact, ovariectomized (8 days) and ovariectomized treated with estradiol-17 beta for 49-50 h; explants of the median eminence area (MEA) were incubated in vitro and LHRH release in response to the combined actions of copper and prostaglandin E2 was used as a criterion for secretory function. Based on vaginal cytology, young (2-3 months) females exhibited regular estrous cycles, the transition to irregular cycles manifested at 5 months of age (2/3 of animals) and to acyclicity at 7 months (all animals). In intact animals, there was a doubling in the magnitude of basal as well as stimulated LHRH release which paralleled the increase in MEA content of the peptide. By comparison to intact, stimulated LHRH release was markedly (greater than or equal to 50%; p less than 0.001) attenuated in 2- and 3-month-old ovariectomized rats but not in 5- and 7-month-old rats; thus, stimulated LHRH release at 5-7 months was approximately 2.5-fold greater than at 2-3 months of age. At each age group, estradiol treatment did not alter the magnitude of stimulated LHRH release. Ovariectomy with or without estradiol did not alter basal release nor the MEA content of LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1990

Expression of steroid metabolizing enzymes by aggregating fetal brain cells in culture: a model for developmental regulation of the progesterone 5 alpha-reductase pathway.

In this study, we established an in vitro model system for the study of developmental regulation of steroid enzyme expression in the perinatal brain. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation under constant rotation. On day 0, 3, 6, or 12 of culture, aggregates were incubated for 4 or 20 h with 3H-progesterone (P4) and the profile of 3H-steroids in the medium analyzed. Five major metabolites were formed from 3H-P4: 5 alpha-pregnan-3, 20-dione (DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP), and two unidentified polar substances designated A and B. Progressively with aggregate-age in culture, there was a decrease in the relative amounts of 3H-P4 recovered in the medium and a sequential increase in DHP, 3 beta-OH-DHP, 3 alpha-OH-DHP, A and B. Aggregates maintained in a chemically defined, serum-free medium metabolized P4 at an accelerated rate compared to those maintained in serum. An inhibitor of the enzyme 5 alpha-reductase completely inhibited P4 metabolism, indicating that 5 alpha-reduction is the primary step in this pathway. Thus, the aggregates express three key enzymes in P4 metabolism: 5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductases and the operation of this pathway is proposed: P4----DHP----3 alpha-OH-DHP + 3 beta-OH-DHP; A and B are derived from one or more of the latter three. Hence, this culture system can serve as a model to study regulatory processes in the developing steroidogenic brain.

3-Hydroxysteroid Dehydrogenases↗
PubMed1989

A rapid, Ca2+-independent, onset of prostaglandin E2 stimulation of the release of luteinizing hormone releasing hormone from copper-treated median eminence explants.

Prostaglandin E2 (PGE2) 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 PGE2 stimulation of LHRH release and that the Ca2+-cAMP pathway is involved in this release process. In this study, we defined the kinetics of the onset of PGE2 action and examined the Ca2+ requirement for the onset and manifestation of PGE2 action. MEA explants from immature female rats were incubated with copper (150 microM copper-histidine complex); thereafter, explants were exposed to 10 microM PGE2 for periods of 2-7 min with or without Ca2+ (Ca2+-free buffer containing 1 mM EGTA). A 2 min PGE2 exposure was as effective as a 7 min PGE2 exposure in stimulating LHRH release; the latter was manifested during the period between 2-7 min. When MEA were exposed to PGE2 for 5 min, maximal rate of release was attained within this 5 min period. When MEA were exposed to PGE2 for 2 min and then to forskolin (100 microM) 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 Ca2+), PGE2 stimulation of LHRH release was abolished and this effect of EGTA was totally reversed by the addition of Ca2+ 2 min after PGE2 exposure and partially reversed (40%) by the addition of Ca2+ 5 min after PGE2 exposure in the presence of EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Efficacy of ciprofloxacin against Leptospira interrogans serogroup icterohaemorrhagiae.

Ciprofloxacin activity against Leptospira interrogans serogroup icterohaemorrhagiae was studied in vitro and in an animal model. The MBC of ciprofloxacin was 0.6 microgram/ml. Three of three Syrian hamsters died 8 to 9 days after intraperitoneal challenge with 10(6) leptospires. In contrast, five of six animals given ciprofloxacin 3 or 5 days after challenge survived.

Animals↗
PubMed1989

High-affinity uptake of 67Cu into a veratridine-releasable pool in brain tissue.

We have previously characterized two saturable, ligand-dependent processes for 67Cu uptake by hypothalamic slices: a high- and low-affinity process (22). In this study, we wished to ascertain if veratridine, a secretagogue that mimics a physiological release process, stimulates the release of newly taken up 67Cu and whether uptake of 67Cu into the releasable pool of copper is dependent on the process of 67Cu uptake. Hypothalamic or caudate slices from male rats were loaded for 30 min with 67Cu complexed to histidine (His) under conditions favoring high- or low-affinity uptake. First, we assessed the stability of the newly taken up 67Cu and found that, regardless of the mode of 67Cu entry into the tissue, greater than or equal to 85% of the 67Cu is retained in tissues incubated for 3 h in 67Cu-free buffer. Moreover, the 67Cu taken up by the high-affinity process was not displaced by 15-fold molar excess of nonradiolabeled Cu2+, histidine, albumin, or Zn2+, and only 20-30% of the 67Cu taken up by the low-affinity process was displaced by 10-fold excess Cu2+ or albumin. Next, we assessed veratridine stimulation of 67Cu release and found that 67Cu release occurred only from tissues loaded with the high- but not with the low-affinity process. This effect of veratridine was calcium dependent and was blocked by Tetrodotoxin, a specific blocker of the voltage-sensitive Na+ channel. In addition, we confirmed our earlier observation that a depolarizing concentration of K+ stimulates 67Cu release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Brain tissue accumulates 67copper by two ligand-dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process.

We characterized the mechanism of copper accumulation by the brain, using rat hypothalamic tissue slices incubated with 67Cu as a model system. Two ligand-dependent saturable processes were discerned: a high affinity, low capacity process and a low affinity, high capacity process. Vo versus [S] for the high affinity process was a hyperbolic function having an apparent Km and Vmax of 6 microM copper and 23 pmol/min/mg protein, respectively. Vo versus [S] for the low affinity process was a sigmoidal function having an "apparent Km" (So5) and maximal velocity at saturating [S] of 40 microM copper and 425 pmol/min/mg protein, respectively. The two processes were similar in that each exhibited: (a) a requirement for complexing of copper for optimal 67Cu accumulation; (b) a broad ligand specificity with respect to amino acids (histidine, cysteine, threonine, glycine) and peptides (Gly-His-Lys, glutathione) and ineffectiveness of albumin in serving as a facilitatory ligand; (c) a requirement for thermic but not metabolic energy. In spite of these similarities, a 50- or 1000-fold molar excess of ligand (histidine) inhibited 67Cu accumulation by the low affinity process by 60 and 85%, respectively, whereas excess histidine facilitated 67Cu accumulation by the high affinity process by 1.6-4-fold. These results are consistent with 1) a carrier-mediated facilitated diffusion, analogous to that of neutral amino acids, as a means of transporting complexed copper into brain tissue, and 2) the existence of two distinct carrier sites interacting in a positive cooperative manner: a high and a low affinity site.

Animals↗
PubMed1988

Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67copper.

The potential importance of copper (Cu) in neurosecretion can be inferred from the demonstration that extracellular Cu modulates the secretory function of peptidergic neurons (in vitro studies) and from the presence of high Cu concentrations in nerve terminals and secretory vesicles, primarily within the soluble matrix of the latter. We have previously hypothesized that vesicular Cu is released from neurons undergoing exocytosis and that such extracellular Cu plays an important modulatory role in the central nervous system. To test this Cu release hypothesis, rat hypothalami were incubated under in vitro conditions for 1 or 2 hr with 20 nM radiolabeled Cu (67Cu), and then 67Cu release was stimulated by a depolarizing concentration (60 mM) of K+. K+ markedly (P less than 0.001) stimulated 67Cu release in a Ca2+-dependent manner (stimulated release was 95 fmol/10 min/mg protein after 1 hr 67Cu loading and 160 after 2 hr). These amounts of released 67Cu account for about 10% of the total 67Cu taken up by the tissue. These results indicate that part of the 67Cu taken up by hypothalamic explants is directed into an intracellular compartment from where it can be released by a Ca2+-dependent mechanism, thus providing strong support to our hypothesis that release of copper is operative in situ in the brain.

Animals↗
PubMed1988

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)

Animals↗
PubMed1988

A correlation between the ligand specificity for 67copper uptake and for copper-prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from median eminence explants.

We have previously shown that copper (Cu) leads to a 3- to 4-fold amplification of prostaglandin E2 (PGE2) stimulation of LHRH release from explants of the median eminence area (MEA), that this amplification is a saturable function of [Cu], and that complexed Cu but not ionic Cu is the active form of the metal. This implicates a ligand-specific site in Cu action. In this study we address the following questions. Is there a ligand specificity for Cu amplification of PGE2 stimulation of LHRH release, and if so, does it correlate with the ligand specificity for Cu uptake? MEAs of 1-month-old female rats were incubated for 5 min with 150 microM Cu solution and then for 15 min with 10 microM PGE2 (Cu/PGE2); LHRH released into the medium was evaluated by RIA. To assess Cu uptake, MEAs were incubated with 100 microM67 Cu solution for 15 min, and 67Cu accumulation by the MEA was evaluated. The Cu was complexed to one of the following ligands: histamine, His, Cys, Thr, Gly, glutathione, Gly-His-Lys, or albumin. There was a high degree of correlation (r = 0.943) between the ligand specificity for Cu/PGE2 stimulation of LHRH release (Cu action) and 67Cu uptake. Complexation of ionic Cu with His facilitated Cu action and 67Cu uptake 3-fold each, and this was completely prevented by the inclusion of His in a 100-fold excess over the concentration of the Cu/His complex. Histamine, the amino acids, and the peptides facilitated Cu action and 67Cu uptake, whereas albumin did not do so. Of these facilitatory ligands, histamine and His were the most effective and Gly-His-Lys was the least effective. In summary, both 67Cu uptake and Cu action are ligand-dependent and ligand-specific; the Cu interactive sites have a common recognition for the Cu-ligand complex and for the ligand itself; and the ligand specificity for 67Cu uptake and for Cu action are highly correlated. These results are consistent with the ligand specificity for Cu uptake being the primary determinant of the ligand specificity for Cu/PGE2 stimulation of LHRH release.

Animals↗
PubMed1988

Progesterone augments copper-prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from explants of the median eminence of immature female rats: an estrogen-dependent process.

We have previously shown that extracellular copper (Cu) amplifies prostaglandin E2 (PGE2) stimulation of LHRH release from explants of the median eminence area (MEA) of adult male rats, and that amplification is a post-PGE2 receptor event involving the adenylate cyclase system. We addressed the question: Is the process of Cu-amplified PGE2 stimulation of LHRH release regulated by ovarian steroids and, if so, is the regulatory steroid estrogen and/or progesterone? Immature female rats (30-32 days old) were ovariectomized and 6 days later treated with a combination of these steroids: E, sc implant of 17 beta-estradiol in a Silastic capsule for 3 days; EEi, E plus im injection of 17 beta-estradiol (2 micrograms/rat) 24 h before killing; and P, either im injection of 0.08 mg/kg progesterone 1 h before killing or 10(-9) M P included in the incubation buffer starting 1 h before Cu/PGE2 exposure. Controls were treated with the vehicle. MEAs were incubated for 5 min with 150 microM Cu, for 15 min with 10 microM PGE2, and then for 45 min with buffer (Cu/PGE2); LHRH release into the medium was measured by RIA. Cu/PGE2-stimulated LHRH release from MEA of intact rats was about 10 times greater than basal release. Ovariectomy led to a 50% reduction in Cu/PGE2-stimulated release [sham, 20.6 +/- 2.0 (mean +/- SE); ovariectomized, 9.4 +/- 1.8], pg/30 mm/MEA; and neither E, EEi, nor P significantly altered this response. In contrast, administration of P to either E- or EEi-primed rats augmented Cu/PGE2 stimulation of LHRH release 3.5-fold (E vs. EP or EEi vs. EEiP); however, P did not augment stimulation of LHRH release by Cu alone or PGE2 alone. Also, inclusion of P in the incubation buffer was as effective as in vivo P in augmenting Cu/PGE2 stimulation of LHRH release from the MEA of EEi-primed rats. On the other hand, in vitro P by itself did not alter LHRH release. These effects of P on the response of the MEA to Cu/PGE2 were not accompanied by a significant increase in the MEA content of LHRH. The process of Cu/PGE2 stimulation of LHRH release is regulated by ovarian steroids, so that ovariectomy leads to a marked reduction of the response of the MEA to Cu/PGE2, and P augments this response in an estrogen-dependent manner. Moreover, it is the secretory process elicited by the combined effects of Cu and PGE2 that is augmented by P.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

The maturational process of gonadotropin-releasing hormone neurons in the male rat: a role for the adrenal gland in the increase in secretory function.

The aim of this study was to define the maturational process of the LHRH neuron in the male rat and the role of the adrenal and testis. Maturing (7-week-old) and adult (12-week-old) male rats were adrenalectomized (ADX), castrated (TX), or ADX and TX; controls were sham operated. Two weeks later, LHRH release from explants of the median eminence area (MEA), MEA LHRH content, and serum testosterone (T) were determined. MEA were incubated for 5 min with 150 microM copper and then for 15 min with 10 microM prostaglandin E2 (Cu/PGE2). LHRH release in response to Cu/PGE2 increased 3-fold between 7 and 9 weeks of age [from 10.4 +/- 1.8 to 29.2 +/- 2.1 pg/15 min.MEA (mean +/- SE)], and there was no further increase thereafter. ADX, TX, or ADX plus TX of 7-week-old rats prevented the increase in Cu/PGE2-stimulated release. In contrast, ADX of 12-week-old rats did not alter LHRH release, whereas TX or ADX plus TX drastically reduced it (75%). The MEA content increased gradually between 7 and 14 weeks of age (from 2.0 +/- 0.2 to 3.0 +/- 0.3 ng). ADX of either 7- or 12-week-old rats did not alter LHRH content, whereas TX or ADX plus TX reduced it to 1.3 ng in both age groups. Serum T increased 6.7-fold between 7 and 9 weeks of age (from 1.06 +/- 0.42 to 6.70 +/- 0.57 ng/ml), and there was no further increase thereafter. ADX of 7-week-old rats did not alter serum T, whereas ADX of 12-week-old rats reduced serum T to 0.62 +/- 0.15 ng/ml. Serum T was undetectable in TX or ADX plus TX rats of both age groups. In summary, maturation of the male reproductive system is associated with a moderate increase in LHRH content of the median eminence and marked increases in the secretory function of the LHRH neuron and in serum T; the testis is essential for these maturational increases. In addition to the testis, the adrenal is necessary for the maturational increase in the secretory function of the LHRH neuron, but not for the increase in LHRH content or that in serum T. It is proposed that dissociable mechanisms regulate LHRH secretion and content in the maturing male, and that T and a substance of adrenal origin (steroid?) are required for the maturational increase in secretory function of the LHRH neuron.

Adrenal Glands↗
PubMed1987

Effects of partial hepatectomy on the immune responses in mice.

Experiments were performed to investigate the immune responses occurring as a result of partial hepatectomy (HEP) in mice. On Day 12 mice subjected to HEP showed a twofold rise in serum levels of IgG when compared with sham-operated (ShO) controls. The effects of HEP on specific antibody production following a single immunization with sheep red blood cells (SRBCs) were investigated. An early appearance of direct (IgM) splenic plaque-forming cells (PFCs) and significantly elevated indirect (IgG) PFCs were found in HEP mice. Elevated, early-appearing mercaptoethanol-resistant (IgG) hemagglutinating antibodies were also demonstrated in the sera of HEP mice. In addition to these findings our study showed that humoral and cell-mediated responses are affected by HEP in opposing fashion. Partial hepatectomy performed immediately after skin grafting suppressed a first set allograft rejection in mice. Furthermore, inhibited delayed-type hypersensitivity (DTH) response against SRBCs, as evaluated by a footpad weight assay, was demonstrated in HEP mice compared with the ShO controls. When SRBC-primed mice were partially hepatectomized 15 days later, they responded in a manner typical of the secondary immune response, showing an increased production of indirect (IgG) splenic PFCs. A similar anamnestic response was observed in mice sensitized with leptospiral antigen 50 days prior to HEP or exposed to carbon tetrachloride (CCl4) hepatotoxin. The mice responded by elevated serum IgG-specific antibodies as measured by solid-phase enzyme-linked immunosorbent assay (ELISA). Since HEP induced in mice immunological disturbances similar to those associated with liver disease, it is suggested that partial hepatectomy creating liver deficiency followed by regeneration may be a useful experimental model to study the immune status of various forms of hepatic damage.

Animals↗
PubMed1987

Copper amplification of prostaglandin E2 stimulation of the release of luteinizing hormone-releasing hormone is a postreceptor event.

We have shown that copper amplifies prostaglandin E2 (PGE2) stimulation of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA) and that this process is calcium-dependent. Since a Ca-cAMP pathway has been implicated in PGE2 action on the LH-RH neuron, in this study we wished to ascertain if copper exerts its effect on the PGE2 receptor or on a postreceptor component involved in PGE2 action. MEA of adult male rats were incubated for 5 min with 200 microM Cu/histidine (CuCl2 mixed with L-histidine at an equimolar ratio) and then incubated for 15 min either with 10 microM PGE2 (Cu/PGE2), 100 microM forskolin (Cu/forskolin), or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (Cu/cAMP). Controls were incubated without Cu/histidine or with Cu/histidine alone. Basal release of LH-RH was 4.6 +/- 0.45 pg/15 min per MEA (mean +/- SEM). Net stimulated release during the 15-min exposure to PGE2, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate was 3.6 +/- 0.52, 3.1 +/- 0.39, and 1.6 +/- 0.42 pg/15 min per MEA, respectively. Net stimulated release after exposure to Cu/PGE2, Cu/forskolin, or Cu/cAMP was 12.7 +/- 2.2, 9.9 +/- 1.46, and 1.4 +/- 1.9 pg/15 min per MEA, respectively, indicating that copper amplifies the action of PGE2 and forskolin but not cAMP action. When MEA were exposed to a mixture of PGE2 and forskolin for 15 min, the effects of these two secretagogues on LH-RH release were not additive, regardless of whether the MEA were pretreated with Cu/histidine. In contrast to PGE2 and forskolin, copper did not amplify K+ stimulation of LH-RH release and, moreover, when Cu/histidine-treated MEA were exposed to a mixture of PGE2 and 30 mM K+, the effects of these two secretagogues were additive. These results are supportive of the proposition that PGE2 stimulation of LH-RH release is mediated by the Ca-cAMP pathway and that copper amplification of PGE2 action is a postreceptor event.

1-Methyl-3-isobutylxanthine↗
PubMed1986

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)

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

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)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗