Biomedical subjects

A Barnea

Publications and source records attributed to A Barnea.

At least 37 records · Page 2Linked to original sources

PubMed1996

Human fetal brain cells in aggregate culture: a model system to study regulatory processes of the developing human neuropeptide Y (NPY)-producing neuron.

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the brain and it has been implicated in a wide range of brain functions, including mentation. The aim of this study was to establish a culture system of human fetal brain cells expressing NPY in a regulated manner. The NPY production in response to forskolin and phorbol 12-myristate 13-acetate (PMA) was taken as a criterion for regulated expression of NPY. Aggregates were formed from dissociated cells derived from the cerebral hemispheres of human fetuses (12.5-19 weeks' gestation) by constant rotation and were maintained in serum-free medium. A 24 hr exposure to 10 microM forskolin + 20 nM PMA led to a 2-6-fold increase in NPY content of the cultures, most of which (80-90%) was secreted into the medium. The latter consisted of two substances differing in size: one corresponding in size to proNPY and the other to NPY. Thus, forskolin + PMA led to an increased production of NPY. Exposure to PMA alone led to an increase in NPY production comparable to that seen after forskolin + PMA and this effect of PMA was dose-dependent. In contrast, forskolin alone did not induce NPY production. Conditioned medium, derived from monolayer cultures enriched with human astrocytes, enhanced NPY production in response to forskolin+PMA in an age-dependent manner. The NPY production by aggregates derived from a 12.5 week-, 14-week- and 18-week-old fetus was enhanced 3-3.6-fold, 1.6-2-fold and 1.1-fold, respectively. Thus, expression of the NPY neurons in this culture system is a regulated process. The NPY production is enhanced markedly by activation of the protein kinase C pathway and by an astrocyte-derived soluble substance(s). Based on these results, we propose that this culture system can serve as a model for the study of regulatory processes of the human developing NPY neuron.

Astrocytes↗

PubMed1995

Brain-derived neurotrophic factor induces functional expression and phenotypic differentiation of cultured fetal neuropeptide Y-producing neurons.

A series of studies from our laboratory has established an aggregate culture system of fetal rat brain cells that can serve as a model for studying regulatory processes of the developing neuropeptide Y (NPY)-producing neurons. Using aggregate cultures derived from 17-day-old fetal rat cortex, we addressed these questions: 1) Does brain-derived neurotrophic factor (BDNF) stimulate NPY production, and if so, is stimulation a function of the developmental state of the cultured NPY neuron? 2) Does BDNF induce phenotypic differentiation of NPY neurons? BDNF led to an increase in NPY production and the accumulation of NPY-mRNA in a dose dependent manner. BDNF did not alter the stability of NPY-mRNA, judged by the disappearance rate of NPY-mRNA after blockade of RNA synthesis (estimated t1/2 was 6-8 hr). BDNF stimulation of NPY production was dependent on length of exposure to BDNF and on culture-age. A continuous 8-day exposure to BDNF resulted in a significantly higher level of NPY production than a pulse of 2 days (comparing BDNF exposure on days 0-8 vs. 6-8, or days 8-17 vs. 15-17). Moreover, older neurons (age 17 days) produced twice as much NPY as younger (age 8 days) neurons in response to a 2-day pulse of BDNF (50 ng/ml). BDNF was significantly more effective than NT-3 in inducing NPY production, and NGF was ineffective. Immunocytochemical analysis of 8-day NPY neurons revealed that a 2-day pulse of BDNF induced the appearance of an abundance of morphologically well-defined neurons bearing an elaborate network of neurites. This was in contrast to the control-treated NPY neurons, which were morphologically undefined. In summary, the age-dependent effect of BDNF on NPY production is consistent with induction of functional expression, rather than promotion of survival, of cultured NPY neurons. The neurotrophin specificity for stimulation of NPY production, and the lack of effect of BDNF on the stability of NPY-mRNA, implicate the TrkB receptor in mediating transcriptional activation of the NPY gene. Thus, BDNF exerts a dual effect on developing cultured NPY neurons: induction of functional expression, and phenotypic differentiation of immature neurons into mature neurite-bearing neurons.

Animals↗

PubMed1995

Administration of systemic or local interleukin-2 enhances the anti-tumor effects of interleukin-12 gene therapy.

Interleukin-12 (IL-12) is a cytokine with a wide variety of immunoregulatory activities. These include stimulation of interferon-gamma production, cytolytic activity of natural killer (NK) cells and T-cell subsets, the development of cellular immunity, and induction of maturation of Th1 cells. IL-12 also has potent anti-tumor activity in vivo. In the present study the possibility of enhanced anti-tumor activity was examined using a combination of local IL-12 by cytokine gene therapy at the tumor site, combined with systemic or local IL-2 delivery. NIH 3T3 fibroblasts transfected with the genes for both subunits of IL-12, p35 and p40, were used as the source of IL-12 therapy producing 240 HLRU/10(6) cells/48 hr. In the first part of the study the effect of different regimens of systemic IL-2 delivery with local IL-12 administration on the size and growth rate of subcutaneous MCA-105 murine sarcoma was examined. Local IL-12 alone reduced the sizes of tumors after 32 days from 163 to 26.8 mm2 (P < 0.002). Adding the longer-acting polyethylene-glycol-modified IL-2 (PEG IL-2; 30,000 IU) for 5 days prevented the development of tumors in all treated mice compared to 1/3 mice treated with PEG IL-2 alone and 3/6 mice with IL-12, but this was a highly toxic therapy and most of the animals died. Administration of 60,000 IU of IL-2 on Days 1-5 postinoculation of tumor, delivered with IL-12 gene therapy, reduced the tumor growth rate compared to animals treated with IL-2 alone (P < 0.02) or IL-12 (0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1994

Seasonal recruitment of hippocampal neurons in adult free-ranging black-capped chickadees.

Neurogenesis and neuronal replacement in a population of wild free-ranging animals are described. Our subjects were adult black-capped chickadees (Parus atricapillus). Neuronal birth dates were determined by a single systemic injection of [3H]thymidine, followed by release of the bird and its recapture 6 or more weeks later. Newly formed neurons appeared in the hippocampal complex during all times of year, but with a marked peak in the fall (October). New neurons were also added to the hippocampal complex of captive chickadees, but at levels only half as high as seen in the wild birds. Neurons born at different times of the year lived for a few months and then disappeared. We suggest that the neurons added are part of a process of neuronal replacement and that they are important for the acquisition of new spatial memories, a need that is particularly acute in the fall. Hormonal and experiential factors may determine the rate of neuronal replacement.

Animals↗

PubMed1994

Role for glial cells in regulating the functional expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from dissociated fetal brain cells.

A series of studies from our laboratory have established an aggregate culture system of fetal rat brain cells expressing neuropeptide Y (NPY) which can serve as a model to study the role of glia-neuron paracrine interactions in the developmental expression of NPY neurons. In this system, NPY production increases progressively with culture-age and it is induced by forskolin (FOR) and phorbol 12-myristate 13-acetate (PMA). We addressed the following question: Is the functional expression of the NPY neurons impaired in the absence of glial cells (particularly astrocytes) and if so, can secretory products of aggregates composed of the full complement of brain cells (intact aggregates) restore the function of the impaired NPY neurons? Aggregates were generated from 17-day-old fetal rat cortex and maintained in serum-free medium for 13-15 days. Cytosine arabinoside (CA; doses of 0.5-8 microM) was added to the cultures on day 1 and the effectiveness in elimination of glial cells was verified on day 15 by measuring the incorporation of 3H thymidine into DNA and by immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Basal NPY production and FOR (10 microM) + PMA (20 nM) stimulated production of NPY on days 13-15 were taken as functional criteria. FOR + PMA induced approximately 2-fold increase in NPY production in control cultures (no CA). CA inhibited both basal and FOR + PMA induced production of NPY and DNA synthesis in a dose-dependent manner: at 6 microM CA, basal NPY production was reduced by about 50%, FOR + PMA stimulated production of NPY and DNA synthesis were completely inhibited, and astrocytes were essentially eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Absolute pitch--electrophysiological evidence.

People who have the ability to label or to produce notes without any reference are considered to possess Absolute Pitch (AP). Others, who need a reference in order to identify the notes, possess Relative Pitch (RP). The AP ability is assumed to reflect a unique, language-like representation of non-lexical musical notes in memory. The purpose of this study was to examine this assumption by comparing Event Related Potentials (ERP) of musicians with and without AP, to lexical and non-lexical representation of musical material. Subjects were eighteen young adult musicians. Seven were AP and eleven RP. Auditory stimuli, presented through earphones, were piano notes (non-lexical) or a voice saying the note's name (lexical). Visual stimuli, presented on a computer display were note symbols (non-lexical) or letters (lexical). Subjects performed a number of tasks, combining the two modalities (visual and auditory) and stimulus types (lexical and non-lexical), and reaction times (RT), performance accuracy and evoked potentials were recorded. The tasks forced the subjects to transfer mental representations of musical material from one mode to another. Our most important findings were the differences, between groups, in the scalp distribution of P300 amplitudes. We conclude that absolute pitch possessors use the same internal language as relative pitch possessors, when possible, but the distribution of the underlying brain activity is different between AP and RP subjects.

Acoustic Stimulation↗

PubMed1993

Morphological differentiation of neuropeptide Y neurons in aggregate cultures of dissociated fetal cortical cells: a model system for glia-neuron paracrine interactions.

The temporal changes in the morphological profiles of neuropeptide Y (NPY) neurons and their topographical relationship with glial cells (astrocytes) were characterized in aggregate cultures derived from fetal cortical tissue using immunocytochemical procedures. On day 6 of culture, structures labelled with NPY antibodies were small and uneven in size but many resembled neuronal cell bodies. On day 14, neuronal perikarya were well defined and several morphological types of NPY neurons could be distinguished most of which gave rise to beaded processes: unipolar or multipolar bitufted neurons whose processes branch in close proximity to the cell body; bipolar neurons; and multipolar neurons. On day 23, heavily punctate and asymmetrically labelled cell bodies were dispersed throughout the aggregate; neuronal processes were less conspicuous. At 14 and 23 days, cells expressing glial fibrillary acidic protein (GFAP) and neuronal specific enolase (NSE) were abundantly distributed throughout the aggregate. Using a double immunoreaction on 14-day-old aggregates revealed that GFAP+ cells and their processes were in close apposition to and engulfing the NPY neurons. Thus, dissociated fetal NPY neurons undergo morphological differentiation in culture along with astrocytes (GFAP+) and other neuronal cell types (NSE+). Based on the topographical association of astrocytes and neurons, particularly NPY neurons, we propose that the aggregate culture system can serve as a model to study the role of paracrine interactions in the regulation of the expression of NPY.

Animals↗

PubMed1993

An early and transient period of protein synthesis is required for induction of neuropeptide Y-mRNA by phorbol ester and forskolin in aggregate cultures of fetal brain cells.

We have previously shown that both forskolin (F) and phorbol ester (P) induce neuropeptide Y (NPY) production by aggregate cultures formed from dissociated fetal rat brains. In this study, we addressed the question: Do F/P induce NPY-mRNA in aggregate cultures and if so, does induction require active protein kinases and on-going protein synthesis? On Northern blots, the NPY cDNA hybridized to a single species of about 0.8 kb. F and P each induced a time-dependent increase in NPY-mRNA relative abundance, and this was inhibited by staurosporine, an inhibitor of both protein kinase A and C. Cycloheximide (CHX) inhibited F/P induction of mRNA in a time-dependent manner. When aggregates were incubated with F+P for a total 12 h period, CHX added along with F+P (0 h) completely inhibited, CHX added 1.5 h after F+P partially inhibited, and CHX added 6 h after F+P did not inhibit the increase in NPY-mRNA. To rule out the possibility that this inhibitory profile reflects toxicity of CHX, blots were re-hybridized with a SRIF cRNA probe and, as expected for SRIF gene, a 12 h exposure to CHX did not inhibit F+P induction of SRIF-mRNA. Close inspection of the blots (derived from 1.5% agarose gels) suggested the presence in F/P-treated aggregates of 2 species NPY-mRNA [approximately 0.75 and approximately 0.85 kb, most likely differing in the length of poly(A) tail (Jamal et al., 1991)]; size-fractionation on a higher resolution gel (3% agarose) resolved the F/P induced mRNA into 2 distinct bands, while mRNA from untreated cultures migrated as a single band--the 0.75 kb.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Basic fibroblast growth factor selectively amplifies the functional state of neurons producing neuropeptide Y but not somatostatin in cultures of fetal brain cells: evidence for a cooperative interaction with insulin-like growth factor-I.

The role of basic fibroblast growth factor (bFGF) in regulating the functional state of neuropeptide Y (NPY) neurons in the brain was investigated, using aggregate cultures, derived from 17-day-old fetal rat cortex maintained for 16 days in serum-free medium, as a model. The criterion for the functional state was NPY production in response to a 24-h exposure to forskolin + phorbol 12-myristate 13-acetate (For + PMA). bFGF (0.1 nM) induced a approximately 2-fold increase in NPY production under basal conditions as well as after For + PMA (p < 0.001 vs control). To address the possibility that bFGF may interact with other growth factors, we assessed the effect of bFGF in the presence of long R3-insulin-like growth factor-I (l-IGF-I; 1 nM) and found that NPY production in response to For + PMA was even greater than with bFGF alone (2-fold; p < 0.001); even though l-IGF-I by itself was ineffective; suggesting that bFGF is the driving force of this amplification. To assess the selectivity of this process, we evaluated SRIF production in response to For + PMA and found that it was not amplified by bFGF, l-IGF-I, or bFGF + l-IGF-I. These results are consistent with bFGF selectively amplifying the functional state of the cAMP and protein kinase C (PKC) pathways leading to increased NPY-production, with cooperative interaction(s) between bFGF and IGF-I, and with a role for bFGF and IGF-I in the developmental expression/survival of the NPY neurons.

Animals↗

PubMed1992

Forskolin and phorbol ester stimulation of neuropeptide Y (NPY) production and secretion by aggregating fetal brain cells in culture: evidence for regulation of NPY biosynthesis at transcriptional and posttranscriptional levels.

Using fetal brain cells in culture, we have previously shown that activation of the cAMP pathway by forskolin induces the production and secretion of neuropeptide Y (NPY). In this study we wished to ascertain 1) if activation of the protein kinase C pathway induces NPY production and/or secretion and if there is synergism between the pathways, and 2) the role of protein/RNA synthesis and influx of extracellular calcium. Aggregates, formed from dissociated cells obtained from the hypothalamus-olfactory tubercle area of 17-day-old rat fetuses, were cultured in serum-free medium for 12 days. The NPY content of aggregates incubated for 24 h with solvent (control) was 4.4 ng/flask, and the medium content was 7.6 ng. Forskolin (10 microM) or phorbol 12-myristate 13-acetate (PMA; 20 nM) marginally affected aggregate content, but each increased medium content 2- to 3-fold; forskolin and PMA were additive. When cycloheximide (75 microM) was included along with forskolin, PMA, or forskolin plus PMA for a period of 10 h, the increase in NPY medium content was abolished. Actinomycin D (Act-D; 5 micrograms/ml) inhibited the response to each secretagogue in a time-dependent manner. When Act-D was included along with forskolin, PMA, or forskolin plus PMA for a total period of 12 or 24 h, the 12 h increase in content was not affected, whereas the 24 h increase was abolished. When the presence of Act-D was limited to 0-24, 6-24, or 12-24 h, and forskolin plus PMA were included for the entire 24-h period, the increase in NPY content was inhibited by 94%, 57%, and 12%, respectively. Verapamil (100 microM) totally inhibited the 24 h response to forskolin and partially (40-50%) inhibited the response to PMA or forskolin plus PMA. In none of these conditions was the inhibition of the increase in medium NPY content accompanied by an increase in aggregate content, nor was the NPY content of aggregate/medium of control cultures affected.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1991

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 microM) 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, alpha, beta-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.

Adenosine Triphosphate↗

PubMed1991

A potential mechanism for copper amplification of prostaglandin E2 action: attenuation of prostaglandin E2-induced efflux of cyclic AMP from median eminence explants.

It is known that prostaglandin E2 (PGE2) stimulation of LHRH release from the median eminence-arcuate nucleus (MEA) is mediated by the cAMP pathway, and a short pretreatment with copper markedly amplifies this release process. Because stimulation of cAMP accumulation is accompanied by cAMP efflux in many tissues, we considered the possibility that attenuation of cAMP efflux is one mechanism by which copper can enhance PGE2 action. When rat MEA explants were incubated in vitro, PGE2 induced a rapid (less than 2.5 min) and sustained (15 min) increase in cAMP efflux, the degree of which was a function of [PGE2]: by 5 min exposure to 10 microM PGEs2, efflux was 8-fold greater than the control (no PGE2) and it accounted for 12.4% of the total (tissue + medium) cAMP. Unlike the dramatic increase in cAMP efflux, PGE2 induced a moderate increase in cAMP content (49%) and in the incorporation of [3H] adenine into [3H] cAMP (78%); this increase was transient: it was evident after a 2.5 but not after a 5 min period of PGE2 exposure. Copper pretreatment did not alter this PGE2-induced increase in tissue cAMP content. In contrast, copper markedly inhibited (by 49%-66%) PGE2-induced cAMP efflux and this inhibition was noted regardless of the length of PGE2 exposure and PGE2 concentration. There was no evidence for hydrolysis of [3H]3',5'-cAMP included in the medium during the incubation with PGE2 with and without copper pretreatment. In summary, copper attenuated PGE2-induced cAMP efflux from MEA explants and this attenuation is not a consequence of a reduction in the availability of intracellular cAMP nor of hydrolysis of cAMP extracellularly.

Animals↗

PubMed1991

In vivo protective effect of lipopolysaccharide against Pseudomonas aeruginosa exotoxin A in mice.

Lipopolysaccharide (LPS) treatment of mice 1 to 5 days prior to administration of Pseudomonas aeruginosa exotoxin A (PA) induced full or partial protection against PA intoxication. The optimal LPS dose that induced resistance was 50 to 100 micrograms per mouse. Simultaneous administration of LPS and PA to mice, however, increased their sensitivity to PA two- to fourfold. Mice pretreated with LPS demonstrated a markedly enhanced clearance rate of 125I-labeled PA from peripheral blood, livers, and kidneys. In mice exposed to LPS and PA simultaneously, the rate of elimination of labeled PA was lower than that in control mice. While protein synthesis was inhibited significantly in livers and other organs of PA-exposed mice, in LPS-pretreated mice, PA-induced inhibition of protein synthesis was either diminished or totally prevented and elongation factor 2 (EF2) levels were normal. In mice treated only with LPS, enhanced protein synthesis and increased levels of EF2 were observed, suggesting that LPS protection against PA intoxication was perhaps a consequence of excessive amounts of EF2 induced by LPS.

ADP Ribose Transferases↗

PubMed1991

Regulated production and secretion of immunoreactive neuropeptide Y by aggregating fetal brain cells in cultures.

The aim of this study was to establish a culture system of fetal brain cells that could serve as a model for the study of the developmental regulation of the neuropeptide Y (NPY) neuron. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation in serum-free medium under constant rotation. Aggregate formation was complete within 24-48 h, and cultures were maintained for up to 23 days. The content of immunoreactive (IR) NPY in the medium and in the aggregates increased progressively with time in culture and at each time point, the medium contained 5- to 10-fold more NPY-IR. A 48-hour exposure to forskolin resulted in a 2-fold increase in the accumulation of NPY-IR in the aggregates and in the medium, indicating that both production and secretion of NPY are regulated by the cAMP intracellular pathway. Sephadex gel filtration revealed the presence of proNPY- and NPY-size substances. The ratio of NPY- to proNPY-size substances increased progressively with age of the aggregates as well as in tissues obtained from perinatal rats of comparable age. Thus, production and secretion of NPY-IR in the cultured aggregates are regulated processes and hence, this culture system can serve as a model to study regulatory processes in the developing NPY neuron.

Animals↗

PubMed1991

Dexamethasone-induced accumulation of neuropeptide-Y by aggregating fetal brain cells in culture: a process dependent on the developmental age of the aggregates.

Neuropeptide-Y (NPY) and glucocorticoid receptors are coexpressed in many neurons in the brain. We addressed the question: Do glucocorticoids regulate the accumulation and/or secretion of immunoreactive (IR) NPY by fetal rat brain cells in culture, and if so, is the effect developmental stage dependent? Aggregates, formed from dissociated cells obtained from the hypothalamus-olfactory tubercle of 17-day-old fetuses, were cultured in serum-free medium for 23 days. On day 23, the aggregate NPY content was 6 ng/flask, and secretion (last 2 days) was approximately 12 ng/24 h. Exposure to dexamethasone (Dex; 20 nM) between days 0-23 led to a 1.9-fold increase in the aggregate content of NPY, whereas NPY secretion was not altered. When Dex exposure was limited to days 12-23, 16-23, 19-23, or 21-23, only a 12- to 23-day exposure induced NPY accumulation, and it was as effective as a 0- to 23-day exposure. The Dex-induced increase in NPY content was evident after a lag period of 4 days or more. When Dex exposure occurred on days 0-12, the aggregate NPY content on day 12 or 23 was not altered. None of these treatments altered the aggregate/medium content of immunoreactive somatostatin (SRIF) or the response to a 48-h exposure to forskolin (10 microM). Dex induction of NPY accumulation was a saturable function of the Dex concentration (maximal at 20 nM), and it was completely inhibited by RU486, a glucocorticoid/progesterone receptor antagonist; neither progesterone, 17 beta-estradiol, nor testosterone altered aggregate/medium NPY contents. Protein/DNA contents of the aggregates were either unaffected or slightly reduced by Dex. Thus, 1) Dex stimulates the accumulation of immunoreactive NPY, but not SRIF, by cultured fetal brain cells; 2) this effect requires a continuous 8-12 days of exposure to Dex during a late developmental stage in culture; 3) Dex does not potentiate or attenuate forskolin action on the NPY neuron; and 4) Dex action appears to be mediated by the glucocorticoid receptor. These results are consistent with glucocorticoid induction of production and/or decreased intracellular degradation of NPY, and with glucocorticoids regulating the NPY neuron in the perinatal brain in a developmental age-dependent manner.

Animals↗

PubMed1990

The ligand specificity for uptake of complexed copper-67 by brain hypothalamic tissue is a function of copper concentration and copper:ligand molar ratio.

It has been previously shown that complexation of Cu2+ is essential for effective uptake of Cu2+ by brain tissues and that 67Cu complexed to His is taken up by a high affinity and a low affinity saturable process (Hartter, D. E., and Barnea, A. (1988) J. Biol. Chem. 263, 799-805). Using rat hypothalamic tissue slices, we defined the ligand specificity for these two uptake processes. The effectiveness of stereoisomers or methyl (Me) derivatives of His in facilitating 67Cu uptake by the high affinity process was in this decreasing order: L-His = D-His = Me-3-N-His greater than Me-ester-His greater than Me-alpha-N-His greater than or equal to Me-1-N-His. By the low affinity process it was: L-His = D-His = Me-3-N-His = Me-ester-His = Me-alpha-N-His greater than Me-1-N-His. When facilitation of 67Cu uptake by 14 different amino acids was evaluated using copper:ligand (Cu:L) ratios of 1:2,000 (high affinity process) or 1:2 (low affinity process), His stood out as the most effective. However, when [Cu2+] was 0.1 microM and the Cu:L ratio was increased from 1:2,000 to 1:20,000, Ala, Gly, Lys, Ser, or Thr was each as effective as His; when [Cu2+] was 10 microM and the Cu:L ratio was increased from 1:2 to 1:2,000, Gln, Glu, Gly, Lys, or Ser was each superior to His in facilitating 67Cu uptake. Moreover, by comparison to 67Cu uptake at a Cu:L ratio of 1:2, increasing the ratio attenuated (His) or enhanced (Gln, Glu, Gly, Lys, Ser) 67Cu uptake. These results indicate that 1) coordination of Cu2+ with the 1-N-imidazole and the alpha-amino (but not with the carboxyl) is essential for His facilitation of 67Cu uptake, and 2) the amino acid specificity for uptake of complexed Cu2+ is a function of both [Cu2+] and the molar ratio of copper to amino acid. These results are consistent with coordination of Cu2+ with at least three nitrogens being a primary factor facilitating copper uptake by brain tissue.

Amino Acids↗

PubMed1990

Uptake of 67copper complexed to 3H-histidine by brain hypothalamic slices: evidence that dissociation of the complex is not the only factor determining 67copper uptake.

It was previously shown that complexation of 67Cu with His facilitates 67Cu uptake by hypothalamic slices and that His, in a concentration that is 1000-fold greater than Cu(His)2, inhibits 67Cu uptake (D. E. Hartter and A. Barnea, J. Biol. Chem. 263, 799-805 (1988)). We addressed the question: Does dissociation of the Cu(His)2 complex occur during the process of Cu2+ uptake and if so, is dissociation the only factor determining uptake? Rat hypothalamic slices were incubated with 67Cu(3H-His)2 and the kinetic profiles of 67Cu and 3H-His uptake were evaluated. 67Cu uptake was linear for up to 60 min, Vo vs S [0.1-160 microM Cu(His)2] was sigmoidal, Lineweaver-Burk plot was non-linear, Scatchard plot was bell-shaped, and Hill plot had multiple slopes. In contrast, 3H-His uptake was linear for up to 30 min, Vo vs S was biphasic, Lineweaver-Burk plot was linear, Scatchard plot was biphasic, and Hill plot had a single slope. Keeping [67Cu] constant and increasing [3H-His] resulted in a dose-dependent inhibition of 67Cu uptake which was not accompanied by an inhibition of 3H-His uptake. Substituting His in the complex with Phe or Lys resulted in a marked shift to the right in Vo vs S for 67Cu uptake and at S less than 40 microM, only His facilitated 67Cu uptake relative to ionic 67Cu2+. However, Vo vs S for 3H-His, 3H-Phe, and 3H-Lys uptake were superimposeable, indicating comparable dissociation of the complexes. In summary, we demonstrate that, although complexation of Cu2+ is essential for 67Cu uptake by hypothalamic tissue, 67Cu and 3H-His are taken up by distinct processes, which implies dissociation of the complex at the level of the membrane. Moreover, even though dissociation occurs, it is not the only factor that determines Cu2+ uptake by the hypothalamic tissue. It is suggested that the physicochemical properties of the Cu complex is an important factor determining Cu uptake by brain tissue.

Animals↗

PubMed1990

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain.

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.

Animals↗