Effects of estrogens on choline-acetyltransferase immunoreactivity and GAP-43 mRNA in the forebrain of young and aging male rats

1. Previous work demonstrated that estradiol (E₂) treatment prevented the abnormal response to stress and the reduction of glucocorticoid receptors (GR) in hippocampus from aging male rats. The mechanisms originating these effects were unknown.2. In the present work, we investigated the E₂ effects on the cholinergic, growth-associated protein (GAP-43) expressing neurons of the medial septum (MS) and vertical limb of diagonal band of Broca (VDB). These areas project to the hippocampus, and may be involved in the mentioned E₂ effects in aging animals. Therefore, the response to E₂ of choline-acetyltransferase (ChAT) in neurons and cell processes and GAP-43 mRNA as a marker of neurite outgrowth was studied in young and old male rats.3. Young (3–4 months) and old (18–20 months) male Sprague-Dawley rats remained untreated or were implanted s.c. with a 14 mg pellet of E₂ benzoate during 6 weeks. We used immoucytochemistry to determine ChAT and isotopic in situ hybridization to analyze GAP-43 mRNA expression.4. Aging males showed a reduction in the number and length of ChAT-immunoreactive cell processes, but not in the number of positive neurons in MS and VDB. E₂ reverted both parameters in old rats to levels of young animals. Regarding basal levels of GAP-43 mRNA, they were similar in old and young animals, but E₂ treatment up-regulated GAP-43 mRNA expression in MS and VDB of old animals only.5. Our data suggest that prolonged E₂ treatment may affect hippocampal function of aging male rats by regulating in part the plasticity of cholinergic, GAP-43 expressing neurones of the basal forebrain. Without discarding a direct E₂ effect on the limbic tissue, effects on the cholinergic system may have a pronounced impact on the neuroendocrine and stress responses of the aging hippocampus.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.     

Norepinephrine alters the expression of genes involved in neuronal sprouting and differentiation: relevance for major depression and antidepressant mechanisms

Recent research into depression has focused on the involvement of long-term intracellular processes, leading to abnormal neuronal plasticity in brains of depressed patients, and reversed by antidepressant treatment. Given a suggested decrease in noradrenergic transmission in depression, and an antidepressant induced increase in norepinephrine (NE) level, a possible role for NE in mediating alterations in neuronal morphology and plasticity was examined. Human neuroblastoma SH-SY5Y cells treated with 10-5 m NE presented an elongated granule-rich cell-body and increased number of neurites, when compared with non-treated cells. Moreover, cell survival was enhanced in the presence of NE, while proliferation was inhibited. The above effects suggest a role for NE in cell differentiation. Indeed similar effects on cell survival and neurite outgrowth were induced in SH-SY5Y cells by retinoic acid (RA), an established differentiating agent. Finally, NE treatment resulted in a progressive decrease in the pluripotent marker Oct4 and an increase in the neuronal growth cone marker, growth-associated-protein 43 (GAP-43). Alongside these effects, NE-treated cells presented alterations in the expression of 44 genes as observed in a neurobiology cDNA microarray. Among the altered genes, an increase in the expression level of two neurite-outgrowth promoting genes, neural cell adhesion molecule L1 and laminin, was confirmed by RT-PCR. Taken together, the results support a role for NE in processes of synaptic connectivity, and may point to a role for this neurotransmitter in mediating the suggested neuronal plasticity in depression and in antidepressant treatment.     

Stimulation and conformational change of G<Subscript>o</Subscript>α induced by GAP-43

GAP-43 and G_o are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G_oα was highly purified from Escherichia coli cotransformed with pQE60 G_oα and pBB131 (NMT). GAP-43 stimulated GTPγS binding to G_oα and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that G_oα GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G_oα. In the presence of GAP-43, fluorescence spectrum of G_oα GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 ±0.22) ×10⁵ to (4.1±0.43) × 10⁵ (M⁻¹). However, no obvious changes of fluorescence spectra of G_oα GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G_oα GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.     

Expression pattern of diacylglycerol acyltransferase-1, an enzyme involved in triacylglycerol biosynthesis,in Arabidopsis thaliana

Triacylglycerol (TAG) is the major carbon storage reserve in oilseeds such as Arabidopsis. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyses the final step of the TAG synthesis pathway. Although TAG is mainly accumulated during seed development, and DGAT has presumably the highest activity in developing seeds, we show here that TAG synthesis is also actively taking place during germination and seedling development in Arabidopsis. The expression pattern of the DGAT1 gene was studied in transgenic plants containing the reporter gene -glucuronidase (GUS) fused with DNA sequences flanking the DGAT1coding region. GUS activity was not only detected in developing seeds and pollen, which normally accumulate storage TAG, but also in germinating seeds and seedlings. Western blots showed that DGAT1 protein is present in several tissues, though is most abundant in developing seeds. In seedlings, DGAT1 is expressed in shoot and root apical regions, correlating with rapid cell division and growth. The expression of GUS in seedlings was consistent with the results of RNA gel blot analyses, precursor feeding and DGAT assay. In addition, DGAT1gene expression is up-regulated by glucose and associated with glucose-induced changes in seedling development.     

Regulation of α-amylase activity in Amaranthus caudatus seeds by methyl jasmonate, gibberellin A3, benzyladenine and ethylene

Methyl jasmonate (JA-Me) at 10^–3 Minhibited Amaranthus caudatus seed germination anddecreased -amylase activity. Exogenous gibberellin A₃(GA₃) and ethylene, but not benzyladenine (BA), increased activity ofthe enzyme in the presence of JA-Me, with ethylene being the most effective. Theinhibitor of ethylene action, 2,5-norbornadiene (NBD) inhibited seed germinationand decreased -amylase activity. The inhibitory action of JA-Me onAmaranthus caudatus seed germination is associated with theinhibition of -amylase activity. Exogenous GA₃ and ethylenecontrol both -amylase activity and seed germination in the presence of JA-Me.     

Extracellular currents alter gap junction intercellular communication in synovial fibroblasts

We studied the effect of extremely low frequency (ELF) currents on gap junction intercellular communication (GJIC) mediated by connexin43 protein. Confluent monolayers of synovial fibroblasts (HIG-82) and neuroblastoma cells (5Y) were exposed in bath solution to 0-75 mA/m(2) (0-56 mV/m), 60 Hz. Single channel conductance, cell membrane current-voltage (I-V) curves, and Ca(2+) influx were measured using the nystatin single and double patch methods. The conductances of the closed and open states of the gap junction channel in HIG-82 cells were each significantly reduced (by 0.76 and 0.39 pA, respectively) in cells exposed to 20 mA/m(2). Current densities as low as 10 mA/m(2) significantly increased Ca(2+) influx in HIG-82 cells. No effects were seen in 5Y cells. The I-V curves of the plasma membranes of both types of cells were independent of 60 Hz electric fields and current densities, 0-75 mA/m(2), indicating that the effect of the 60 Hz fields on GJIC in HIG-82 cells was not mediated by a change in membrane potential. We conclude that ELF electric fields can alter GJIC in synovial cells via a mechanism that does not depend on changes in membrane potential, but may depend on Ca(2+) influx. The results open the possibility that GJIC mediated responses in synovial cells, such as for example, their secretory responses to proinflammatory cytokines, could be antagonized by the application of ELF electric fields.     

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.     

Microinjected anti-actin antibodies decrease gap junctional intercellular commmunication in cultured astrocytes

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.