Galanin reduces PDBu-induced protein phosphorylation in rat ventral hippocampus.

The effect of galanin (GAL) on basal and phorbol-12,13-dibutyrate (PDBu) induced protein phosphorylation in rat ventral hippocampal miniprisms was investigated. GAL (0.5, 1 and 2 microM) inhibited PDBu stimulation in a concentration-dependent manner without altering basal protein phosphorylation. This inhibitory effect was prevented by the GAL antagonist galantide. GAL did not affect either the activity of protein kinase C (PKC) from rat brain or basal phosphorylation in ventral hippocampal hippogenates, suggesting that it did not directly modulate PKC activity. Depolarization of miniprisms from ventral hippocampi by 18 mM K+ prevented the effect of GAL on PDBu-induced phosphorylation. The results indicate that GAL indirectly regulates neuronal protein phosphorylation by a GAL receptor-mediated action.     

A study on co-localization of FSH and its receptor in rat hippocampus

It has been known that GnRH, LH and their receptors exist in hippocampal neurons. However, whether FSH and its receptor also exist in hippocampal neurons remained unknown yet. In situ hybridization, double-labeled immunofluorescence stain and double-labeled immunohistochemistry stain in adjacent sections were used in our research to study the distribution, co-localization of FSH and its receptor and co-localization of FSH and GnRH receptor in rat hippocampus. The result found that pyramidal neurons from CA1 to CA4 region and granule neurons in dentate gyrus could express FSH and its receptor, majority of hippocampal neurons co-expressed FSH and its receptor, FSH and GnRH receptor. These suggested that hippocampal neurons not only express FSH but also act as FSH target cells. FSH may regulate the function of hippocampal neurons by ways of paracrine or autocrine. At the same time, GnRH may regulate the function of FSH neuron in hippocampus through GnRH receptor.     

The NOTCH receptor and its ligands

An intricate interplay of signalling pathways dictates the acquisition of specific cell fates during development. The NOTCH receptor is the central element in a cell-interaction mechanism that controls the fate of a very broad spectrum of precursor cells. Conservation across species implies that signalling through this receptor is a tool frequently used by metazoans to modulate the fate of precursor cells. This article describes recent advances in the genetic and molecular dissection of this developmentally fundamental pathway that have provided new insights into the mechanism by which extracellular signals act through the NOTCH receptor to determine or alter cellular fate.     

Galanin receptor activation attenuates norepinephrine release from rat spinal cord slices

Galanin and norepinephrine both act on sensory neurons in the rat spinal cord dorsal horn. We looked for the effects of galanin on the basal and electrically-evoked release of [³H]norepinephrine from slices of the dorsal spinal cord. 0.1 to 1.0 μM reduced the basal efflux by maximal 10 % and the stimulation-evoked release in a concentration-related manner by maximal 27 %. In the presence of 0.1 μM galanin receptor antagonist (M-15) reduction of basal efflux persisted but significant effects on the stimulation-evoked release were no longer observed. The antagonist, given alone, was without effect on the release. Thus galanin can reduce the stimulation-evoked norepinephrine release from spinal cord dorsal horn nerve terminals by an action on galanin receptors, however, the release seems not to be tonically inhibited by galanin.     

大鼠海马神经元内11β-HSD1和GR的共存及其意义

本研究旨在探讨糖皮质激素代谢酶-11β-羟基类固醇脱氢酶Ⅰ型（11β-HSD1）和糖皮质激素受体（GR）在大鼠海马神经元内的共同分布及其意义。用免疫细胞化学方法研究显示,海马神经元内不仅存在11β-HSD1免疫反应物质,还存在GR免疫反应物质,而且11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLC）方法研究表明,地塞米松（DEX）可以促进11β-HSD1与GR共存于同一个海马神经元内,用Western印迹杂交和薄层层析（TLG）方法研究表明,地塞米松（DEX）可以促进11β-HSD1蛋白表达及其酶的活性,利用11β-HSD1基因启动子区序列构建的以CAT酶为报告基因的pBLCAT6质粒转染PC12细胞,证实DEX能够促进CAT酶的表达。以上糖皮质激素的作用均可为GR受体阻断剂RU38486所阻断,结果提示;糖皮质激素（GC）与GR结合后,可以作用于与其共存的11β-HSD1基因启动子区,使11β-HSD1表达增加,从而使更多的GC代谢产物转化为有活性的GC,此机制可能与保证GC在海马神经元内与亲和力较低的GR结合有关。     

Differential effects of haloperidol and olanzapine on the expression of erythropoietin and its receptor in rat hippocampus and striatum

Compared with first-generation antipsychotics (FGAs), second-generation antipsychotics (SGAs) seem to be neuroprotective and trigger neuroplasticity. Because neuroplasticity is regulated by a variety of neurotrophic factors we studied differential effects of haloperidol (HAL, a FGA) and olanzapine (OLZ, a SGA) on temporal expression of erythropoietin (EPO), a potent neuroprotective factor and its receptor (EPOr) in rat brain. Rats (8-10/group) were treated with HAL or OLZ for 14 days (HAL-14 or OLZ-14) or 45 days (HAL-45 or OLZ-45). Animals were killed by decapitation or by perfusion to collect brains for immunoblotting and immunohistochemical analysis respectively. In hippocampus, the levels of both EPO and EPOr were significantly increased in HAL-14 (p < 0.001) and OLZ-14 (p < 0.001) groups. Their levels decreased in HAL-45 compared with levels in HAL-14 (EPO, p < 0.001; EPOr, p < 0.05), whereas the levels were further increased (EPO, p < 0.05) in OLZ-45 compared with OLZ-14. In striatum, the levels of both EPO and EPOr were unchanged in HAL-14 and EPO levels significantly decreased in HAL-45 (p < 0.05), whereas their levels were significantly increased in OLZ-14 and OLZ-45 compared with the vehicle-treated control (p < 0.001). Both EPO and EPOr were primarily expressed by neurons and endothelial cells. These data suggest that SGAs such as OLZ may have neuroprotective effects through expression of EPO that may be clinically relevant for long-term safe and beneficial management of psychotic patients.     

Differential regulation of ciliary neurotrophic factor and its receptor in the rat hippocampus in response to kainic acid-induced excitotoxicity

We analyzed the changes in expression of ciliary neurotrophic factor (CNTF) and its receptor, ligand-binding subunit a (CNTFRa), in the hippocampus following intraperitoneal administration of a convulsant dose of kainic acid (KA). Immunohistochemistry and immunoblotting showed that CNTF levels rose dramatically between day 1 and day 10, and that the CNTF was located in reactive astrocytes. In contrast, upregulation of CNTFRalpha mRNA, occurred in neurons as well as astrocytes. A rapid, and short-lived (3 h-2 d) increase in CNTFRalpha was also observed in the more resistant granule cells and CA2 pyramidal neurons. The increase in astrocytes was detected by day 1 and was sustained for more than 5 d. These results show that CNTF and CNTFRalpha are differentially regulated in hippocampal neurons and reactive astrocytes following KA injection, indicating that these proteins may be involved in the regulation of astrocyte and neuronal degenerative responses.     

Galanin coexists with pituitary hormones in the fetal rat

Immunocytochemical analysis of endocrine cells of the developing rat pituitary has shown clearly that, starting from E16, the galanin-immunoreactive cells are heterogeneous, the neuropeptide being present in cells containing the majority of pituitary hormones. A proportion of galanin-containing corticotropes, starting from about 17% at E16 reached about 29% at E21, while the galanin-containing lactotrope increase in the same period was from about 13% to 34%. Galanin-containing thyrotropes ranged from about 11% at E16 to about 13% at E21, while the proportion of galanin-containing luteotropes increased consistently from about 17% at E19 to about 27% at E21. Only the trend of galanin-containing somatotropes was inverted, falling from about 26% to 13%. We conclude that the multiple coexistence of galanin with most of the pituitary hormones during fetal development is a further example of plasticity of endocrine pituitary cells, and that galanin may have a role in cytodifferentiation.     

Galanin and its analogues: A structure-activity relationship studies in rat isolated gastric smooth muscles

Summary Galanin (GAL), a 29-amino-acid-residue neuropeptide, modulates gastric smooth muscles activity by interacting with specific receptors. However due to the lack of specific antagonists in the gastrointestinal (GI) tract the actual level of GAL involvement in GI motility remains largely unknown. In our studies we have performed structure-activity relationship studies of two porcine galanin fragments, two chimeric galanin analogues and several 15-amino-acid-residue galanin analogues modified in positions 2, 3, 4, 6, 8 or 14, investigating their contractile action on rat isolated gastric fundus strips, employed as in vitro assay of peptides activity. Thus we intended to characterize the molecular domains of GAL responsible for binding and activation of GAL receptors in rat gastric smooth muscle cells. The data acquired in the course of our structure-activity relationship studies suggest that both N-and C-terminal fragment of GAL molecule contribute towards the affinity and activity of GAL gastric smooth muscle cell receptors. Moreover, we concluded that positions 2, 3, 4, 6, 8 and 14 in the amino acid sequence of GAL may play important roles in binding and activation of GAL receptors in rat gastric smooth muscle cells. Abbreviations: The symbols of the amino acids, peptides and their derivatives are in accordance with the 1983 Recommendations of the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Eur. J. Biochem. 138, 9 (1984)). Other symbols     

Galanin and its analogues: A structure-activity relationship studies in rat isolated gastric smooth muscles

Galanin (GAL), a 29-amino-acid-residue neuropeptide, modulatesgastric smooth muscles activity by interacting with specific receptors. However due to the lack of specific antagonists in thegastrointestinal (GI) tract the actual level of GAL involvement in GI motility remains largely unknown. In our studies we have performed structure-activity relationship studies of two porcinegalanin fragments, two chimeric galanin analogues and several 15-amino-acid-residue galanin analogues modified in positions 2, 3, 4, 6, 8 or 14, investigating their contractile action on rat isolated gastric fundus strips, employed as in vitro assay of peptides activity. Thus we intended to characterize the moleculardomains of GAL responsible for binding and activation of GAL receptors in rat gastric smooth muscle cells. The data acquired in the course of our structure-activity relationship studies suggest that both N- and C-terminal fragment of GAL molecule contribute towards the affinity and activity of GAL gastric smooth muscle cell receptors. Moreover, we concluded that positions 2, 3, 4, 6, 8 and 14 in the amino acid sequence of GAL may play important roles in binding and activation of GAL receptors in rat gastric smooth muscle cells.     

Galanin inhibits pancreatic amylase secretion in the pentobarbital-anesthetized rat.

The potent inhibitory effect of galanin on basal and pentagastrin-stimulated gastric acid secretion in vivo, and the presence of galanin-containing nerves in gastrointestinal tract and pancreas, suggested that this peptide may regulate the exocrine secretion of the GI system. Male rats were anesthetized with pentobarbital and the dose-dependent inhibitory effects of galanin on basal and stimulated pancreatic protein and amylase secretions were investigated in separate experiments. Galanin was administered intravenously in the following doses: 3, 6, 10, 15 and 20 micrograms/kg/h (0.93, 1.86, 3.1, 4.65 and 6.2 nmol/kg/h), and pancreatic secretions measured. The maximal effective dose of galanin (3.1 nmol/kg/h) on basal pancreatic secretions was found, and was used for evaluating the inhibitory effect of galanin on pancreatic protein and amylase secretions stimulated by bombesin, secretin and cholecystokinin. Galanin potently inhibited basal, bombesin-, secretin- and cholecystokinin-stimulated pancreatic protein and amylase secretion. Inhibitory effect of galanin was dose-dependent and biphasic.     

Dynamic increases in AMPA receptor phosphorylation in the rat hippocampus in response to amphetamine

Increasing evidence supports the critical role of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) glutamate receptors in psychostimulant action. These receptors are regulated via a phosphorylation‐dependent mechanism in their trafficking, distribution, and function. The hippocampus is a brain structure important for learning and memory and is emerging as a critical site for processing psychostimulant effects. To determine whether the hippocampal pool of AMPA receptors is regulated by stimulants, we investigated and characterized the impact of amphetamine (AMPH) on phosphorylation of AMPA receptors in the adult rat hippocampus in vivo. We found that AMPH markedly increased phosphorylation of AMPA receptor GluA1 subunits at serine 845 (S845) in the hippocampus. The effect of AMPH was dose dependent. A single dose of AMPH induced a rapid and transient increase in S845 phosphorylation. Among different hippocampal subfields, AMPH primarily elevated S845 phosphorylation in the Cornu Ammonis area 1 and dentate gyrus. In contrast to S845, serine 831 phosphorylation of GluA1 and serine 880 phosphorylation of GluA2 were not altered by AMPH. In addition, surface expression of hippocampal GluA1 was up‐regulated, while the amount of intracellular GluA1 fraction was concurrently reduced in response to AMPH. GluA2 protein levels in either the surface or intracellular pool were insensitive to AMPH. These data demonstrate that the AMPA receptor in the hippocampus is sensitive to dopamine stimulation. Acute AMPH administration induces dose‐, time‐, site‐, and subunit‐dependent phosphorylation of AMPA receptors and facilitates surface trafficking of GluA1 AMPA receptors in hippocampal neurons in vivo.

     

Aspirin increases NMDA receptor subunit 2A concentrations in rat hippocampus

The N-methyl-d-aspartate receptor (NMDAR), a heteromeric protein, is a glutamate receptor that has three classes of subunits: NR1, NR2, and NR3. It has been reported that these receptors are involved in synaptogenesis, synaptic plasticity, and many other processes in the central nervous system. The aim of this study is to investigate the efficacy of aspirin on hippocampal NMDARs. Sixteen rats were studied in two groups, with eight animals in each group. The first group was the control group, and the second one was the aspirin-given group. Aspirin (acetylsalicylic acid) was administered orally to the rats (200 mg/kg). Tissue samples were obtained after 3 h. The brain was removed, and both hippocampi were dissected out for evaluation. It was found that acute doses of aspirin caused increases on the levels of NMDAR 2A (NR2A) receptors and malondialdehyde (MDA), the end product of lipid peroxidation. Production was significantly increased in the aspirin-given group. We know that MDA is a marker for free radical-mediated tissue damage. In conclusion, lipid peroxidation, caused by acute doses of aspirin may lead to excitotoxicity effects by a hippocampal NR2A-mediated mechanism.     

Evaluation of quaternized and neutral muscarinic receptor ligands in normal and DES-treated rat

The localization of quaternized muscarinic receptor (mAChR) antagonists, [¹¹C]methyl tropanyl benzilate ([¹¹C]MTRB) and [¹¹C]methyl quinuclidinyl benzilate ([¹¹C]MQNB), in rat pituitary was compared to that of [¹¹C]tropanyl benzilate ([¹¹C]TRB), a neutral antagonist. The quaternized ligands localize via a mAChR-mediated mechanism as shown by 60% reduction in radioactivity concentrations in the presence of QNB. [¹¹C]TRB appears to localize primarily by a non-mAChR specific mechanism. Induction of pituitary prolactinomas by diethylstilbestrol resulted in a reduction of [¹¹C]MTRB pituitary localization compared to normals. Elevated serum prolactin levels due to prolactinoma presence had no measurable effect on myocardial [¹¹C]MTRB uptake or on K_d values. B_max values for myocardial mAChR were similar for controls and for DES exposure of 10 weeks.     

Changes in phosphorylation of the NMDA receptor in the rat hippocampus induced by status epilepticus

Systemic administration of pilocarpine preceded by lithium induces status epilepticus (SE) that results in neurodegeneration and may lead to the development of spontaneous recurrent seizures. We investigated the effect of Li/pilocarpine-induced SE on phosphorylation of the NMDA receptor in rat hippocampus. Phosphorylation of NR1 by PKC on Ser890 was decreased to 45% of control values immediately following 1 h of SE. During the first 3 h following the termination of SE, phosphorylation of Ser890 increased 4-fold before declining to control values by 24 h. Phosphorylation of NR1 by PKA was also depressed relative to controls immediately following SE and transiently increased above control values upon the termination of SE. SE was accompanied by a general increase in tyrosine phosphorylation of hippocampal proteins that lasted for several hours following the termination of seizures. Tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDAR increased 3-4-fold over control values during SE, continued to increase during the first hour following SE and then declined to control levels by 24 h. SE resulted in the activation of Src and Pyk2 associated with the postsynaptic apparatus, suggesting a role for these enzymes in the SE-induced increase in tyrosine phosphorylation. Changes in phosphorylation of the NMDA receptor may play a role in the pathophysiological consequences of SE.     

Structure-function relationship between the human chemokine receptor CXCR3 and its ligands

I-TAC, IP10, and Mig are interferon-gamma inducible CXC chemokines that share the same G-protein-coupled receptor CXCR3, which is preferentially expressed on Th1 lymphocytes. We have explored the structure-function relationship of the CXCR3 ligands, in particular of I-TAC, which has highest affinity for CXCR3 and is the most potent agonist. A potent antagonist for CXCR3 was obtained by NH(2)-terminal truncation of I-TAC. I-TAC (4-73), which lacks the first three residues, has no agonistic activity but competes for the binding of I-TAC to CXCR3-bearing cells and inhibits migration and Ca(2+) changes in such cells in response to stimulation with I-TAC, IP10, and Mig. It does also not induce internalization of CXCR3, which is in support of the lack of agonistic effects. Hybrid chemokines between I-TAC and IP10 were used to identify regions responsible for the higher activity of I-TAC. I-TAC-like IP10 analogs are obtained by substituting the NH(2) terminus (residues 1-8) or N-loop region (residues 12-17) of IP10 with those of I-TAC, suggesting that the differences in function of the CXCR3 ligands can be assigned to distinct regions and that these regions are interchangeable. Structure-activity studies with Mig showed that the extended basic COOH-terminal region, which is not present in I-TAC and IP10, is important for binding and activity.     

Galanin receptor in the rat pancreatic beta cell line Rin m 5F. Molecular characterization by chemical cross-linking

125I-Galanin was cross-linked to receptor in Rin m 5F cell membranes using the bifunctional reagent disuccinimidyl tartarate. Regardless of the presence of reducing agents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cross-linked galanin-receptor complexes revealed the presence of a radioactive band at Mr 57,000. Excess unlabeled galanin completely inhibited the labeling of the band while other regulatory peptides had no effect. Labeling of the Mr 57,000 complex was abolished by galanin concentration from 10(-9) to 10(-6) M (IC50 = 5 X 10(-9) M). Initial incubation with 125I-galanin in the presence of increasing concentrations of guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) (10(-7) to 10(-4) M) also inhibited the labeling of the Mr 57,000 complex. Moreover, pretreatment of membranes with pertussis toxin before formation of the covalent galanin-receptor complex, dramatically reduced the labeling of the Mr 57,000 species. Covalent Mr 57,000 galanin-receptor complexes solubilized by Triton X-100 bound specifically to wheat germ agglutinin-concanavalin A-, and soybean-coupled Sepharose, supporting the glycoproteic nature of the galanin receptor. Assuming one molecule of 125I-galanin (Mr 3,000) was bound per molecule of protein, these results suggest that the pancreatic galanin receptor is a glycoprotein with a Mr of 54,000 bearing the recognition site for the ligand and which is coupled with a pertussis toxin-sensitive G protein in the plasma membrane.     

Galanin stimulates rat pituitary growth hormone secretion in vitro

The effect of galanin on growth hormone (GH) secretion was investigated in monolayer cultures of rat anterior pituitary cells. Galanin caused a gradual increase in GH concentrations into the culture medium that was maximal at 90 minutes and sustained after 180 minutes. The ED50 for galanin-stimulated GH secretion was approximately 200 nM compared to an ED50 for rat GH-releasing factor (rGRF)-stimulated GH secretion of 10pM. Galanin and rGRF were additive in increasing GH release into the incubation medium. These data indicate that porcine-derived galanin has a direct effect on pituitary GH secretion in vitro.     

Desmethylimipramine pretreatment prevents 6-hydroxydopamine induced somatostatin receptor reduction in the rat hippocampus.

Several studies have shown anatomical and functional interconnections between catecholaminergic and somatostatinergic systems. To assess whether somatostatin (SS) may act presynaptically on catecholamine neurons, SS receptors were measured using radioligand test-tube binding assays on synaptosomes from hippocampus and frontoparietal cortex--areas that are innervated by catecholaminergic neurons with different densities and that have a high number of SS receptors--from control and 6-hydroxydopamine (6-OHDA)-treated rats. Intracerebroventricular (i.c.v.) injection of the catecholamine neurotoxin 6-OHDA (0.78 mg free base/kg of body weight in saline with 0.1% ascorbic acid) lowered hippocampal and frontoparietal cortical noradrenaline (NA) and dopamine (DA) levels at 1 week following the injection. Pretreatment of rats with desmethylimipramine (DMI) (40 mg/kg, intraperitoneal) prevented the drop in NA levels, but was not effective in attenuating DA depletion in the two brain areas studied. Treatment with 6-OHDA lowered the number of 125I-Tyr11-SS receptors in the hippocampus (130 +/- 19 vs. 266 +/- 16 fmol/mg protein, P < 0.001), whereas in the frontoparietal cortex a non significant 20% reduction in receptor number was found. The dissociation constants of 125I-Tyr11-SS binding to synaptosomes from frontoparietal cortex (0.65 +/- 0.06 vs. 0.60 +/- 0.04, P not significant) and hippocampus (0.44 +/- 0.04 vs. 0.63 +/- 0.14, P not significant) were similar in control and treated groups. Pretreatment with DMI reversed up to 80% of the effect of 6-OHDA on hippocampus SS receptors. DMI alone had no observable effect on the number and affinity of SS receptors. The 6-OHDA and the DMI treatment did not affect SLI levels in the brain areas studied. These results suggest that a portion of the hippocampal SS receptors may be localized presynaptically on the noradrenergic and dopaminergic nerve terminals.     

Galanin potentiates electrical stimulation and exogenous norepinephrine-induced contractions in the rat vas deferens

In order to evaluate the mode of action of galanin (GAL) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of this peptide were tested on the electrical stimulated and the unstimulated preparations of the isolated rat vas deferens in the presence of 10(-7) M atropine. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers were dose-dependently potentiated by GAL in concentrations ranging from 1 to 50 nM. The facilitatory action induced by GAL in high concentrations (greater than 10 nM) usually returned to the control level at 2-3 min and were tachyphylactic. The potentiating action of GAL was not modified by pretreatment with 10(-7) M propranolol. Contractions produced by exogenous norepinephrine (NE) in the unstimulated preparations were not affected by pretreatment with low concentrations (less than 5 nM) of GAL. On the other hand, the contractions were dose-dependently potentiated 1 min after pretreatment with higher concentrations (greater than 10 nM) of GAL, which recovered 15 min after constant flow washout. Contractions developed by exogenous 5-hydroxytryptamine were not affected, or slightly inhibited, by GAL (1-50 nM). In some preparations without electrical stimulation, high concentrations of GAL caused a slight contraction, which was not blocked by pretreatment with 10(-6) M phentolamine and 10(-6) M tetrodotoxin. These results suggest that GAL receptors exist presynaptically in the rat vas deferens and that stimulation of the receptors by GAL potentiates the release of NE from the nerve terminals during postganglionic sympathetic nerve stimulation. Other mechanisms for GAL action, such as influence on neuronal uptake and catecholamine metabolism, cannot be ruled out.