Neurochemical Characteristics of a Novel Dorsal Root Ganglion X Neuroblastoma Hybrid Cell Line, F-11

We investigated the properties of the novel dorsal root ganglion (DRG) hybrid cell line F-11 to see how closely these cells resembled normal DRG cells. Under normal growth conditions, F-11 cells appeared to contain several short neurite-like processes. However, these cells could also be grown under conditions in which they showed a much more extensive neuronal morphology, exhibiting many long neurites. Several differentiated features of DRG cells were present on F-11 cells. These included the presence of delta-opioid receptors, receptors for prostaglandins and bradykinin, and dihydropyridine-sensitive calcium channels. F-11 cells also synthesized and released a substance P-like compound, as determined by immunoreactivity. Both the number of bradykinin receptors and the voltage-sensitive calcium influx increased on cell differentiation. Opioid agonists (delta-specificity) were found to decrease cyclic AMP levels in F-11 cells in a naloxone- and pertussis toxin-reversible fashion. Bradykinin stimulated the synthesis of inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate. Ca2+ channel agonists stimulated voltage-sensitive Ca2+ influx in a dose-dependent, stereospecific manner, whereas Ca2+ channel antagonists inhibited Ca2+ influx. F-11 cells should, therefore, prove useful as models for authentic DRG neurons.     

Formation of Neuromuscular Junctions and Synthesis of Sensory Neuropeptides in the Co-cultures of Dorsal Root Ganglion and Cardiac Myocytes

Aim The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. Methods The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). Results In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. Conclusions The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes. Zhen Liu and Huaxiang Liu contributed equally to this article.     

Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons

Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo-[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 microM bradykinin for time intervals of 5-300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion-exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10-fold increased accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl-monoacylglycerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyceride suggested the involvement of di- and monoglyceride lipases in arachidonic acid mobilization. A role for phospholipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin-stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 microM), or EDTA (2.5 mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 microM).     

Effects of Electro-acupuncture on PDGF Expression in Spared Dorsal Root Ganglion and Associated Dorsal Horn Subjected to Partial Dorsal Root Ganglionectomy in Cats

The effects of electro-acupuncture (EA) on the expression of platelet derived growth factor (PDGF) in spared dorsal root ganglion (DRG) and associated dorsal horns were evaluated in cats subjected to bilateral removal of L₁–L₅ and L₇–S₂ DRG, while sparing L₆ DRG and were demonstrated using Immunohistochemistry, Western blot and RT-PCR techniques. On the acupunctured side, there was a significant increase in the total number of PDGF positive neurons. Large neurons of the L₆ DRG at 7 days post operation (dpo), and small to medium-sized neurons at 14 dpo, as well as in the lamina II of the L₆ spinal cord at 14 dpo was observed. The expression of PDGF protein increased significantly in the L₆ DRG at 7 and 14 dpo and in the dorsal horn of the L₆ spinal cord at 14 dpo while the upregulation of PDGF mRNA was seen at 3 dpo in the L₆ DRG and the dorsal horn of the L₃ and L₆ spinal cord. These findings demonstrate that intrinsic PDGF has been upregulated in cats subjected to partial dorsal root ganglionectomy following EA, indicating endogenous PDGF is involved in promoting spinal plasticity following EA.     

μ-Opioid Receptor Stimulation of Inositol (1,4,5)Trisphosphate Formation via a Pertussis Toxin-Sensitive G Protein

Abstract: The cellular mechanisms underlying opioid action remain to be fully determined, although there is now growing indirect evidence that some opioid receptors may be coupled to phospholipase C. Using SH-SY5Y human neuroblastoma cells (expressing both μ-and δ-opioid receptors), we demonstrated that fentanyl, a μ-preferring opioid, caused a dose-dependent (EC₅₀= 16 n M ) monophasic increase in inositol (1,4,5)trisphosphate mass formation that peaked at 15 s and returned to basal within 1–2 min. This response was of similar magnitude (25.4 ± 0.8 pmol/mg of protein for 0.1 μ M fentanyl) to that found in the plateau phase (5 min) following stimulation with 1 m M carbachol (18.3 ± 1.4 pmol/mg of protein), and was naloxone-, but not naltrindole-(a δ antagonist), reversible. Further studies using [ d -Ala², MePhe⁴, Gly(ol)⁵]enkephalin and [ d -Pen^2,5]enkephalin confirmed that the response was specific for the μ receptor. Incubation with Ni²⁺ (2.5 m M ) or in Ca²⁺-free buffer abolished the response, as did pretreatment (100 ng/ml for 24 h) with pertussis toxin (control plus 0.1 μ M fentanyl, 26.9 ± 1.5 pmol/mg of protein; pertussis-treated plus 0.1 μ M fentanyl, 5.1 ± 1.3 pmol/mg of protein). In summary, we have demonstrated a μ-opioid receptor-mediated activation of phospholipase C, via a pertussis toxin-sensitive G protein, that is Ca²⁺-dependent. This stimulatory effect of opioids on phospholipase C, and the potential inositol (1,4,5)trisphosphate-mediated rises in intracellular Ca²⁺, could play a part in the cellular mechanisms of opioid action.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.     

Biosynthesis of Prostaglandins D2 and E2 in Chick Dorsal Root Ganglion During Development

Newly formed prostaglandins (PGs), which are assumed to act as modulators of afferent sensory messages, were studied in chick dorsal root ganglia (DRG) during development. [1-14C]Arachidonic acid was converted by DRG homogenates from 1-week-old chickens into two major 14C-PGs: PGE2 and PGD2. The enzymatic conversion of arachidonic acid was characterized as follows: (a) Boiled preparations were inactivated; (b) synthesis of PGs was inhibited by pretreatment with aspirin or indomethacin and enhanced by esculetin, a protector of cyclooxygenase; and (c) [14C]PGE2 and [14C]PGD2 accumulation was a protein dose-dependent process. Further fractionation of crude homogenates indicated that PG endoperoxide synthetase (EC 1.14.99.1) and PGE2 synthetase (EC 5.3.99.3) were membrane-bound enzymes, whereas PGD2 synthetase (EC 5.3.99.2) was recovered in the cytosol. During development, from embryonic day 10 to day 14 after hatching, PGD2 synthetase activity remained constant; in contrast, a sharp rise in [14C]PGE2 synthesis was observed from embryonic day 14 to 18. The time curves of PGD2 and PGE2 synthetase specific activity may be related to changes taking place in the cell population of developing DRG. It is therefore suggested that arachidonic acid would be enzymatically converted early into PGD2 by maturing ganglion cells and then later into PGE2 by proliferating fibroblasts.     

海南捕鸟蛛毒素-IV对大鼠背根神经节细胞钠通道的抑制影响(英文)

海南捕鸟蛛毒素 IV(HNTX IV)是从中国捕鸟蛛Seleconosmiahainana粗毒中分离得到的一种肽类神经毒素 ,在成年大鼠背根神经节 (DRG)细胞上观察了该毒素对电压门控钠通道的影响。在全细胞膜片钳条件下 ,HNTX IV能明显抑制哺乳动物神经性河豚毒敏感型 (TTX S)钠电流 ,但不影响河豚毒不敏感型 (TTX R)钠电流。HNTX IV对DRG细胞TTX S钠电流的抑制作用具有浓度依从性 ,其有效半抑制浓度 (IC50 )为 44 .6nmol/L。该毒素不影响DRG钠电流的激活与失活时间特征 ,但能导致钠通道的半数稳态失活电压向超极化方向漂移约 10 .1mV。结果表明HNTX IV是一种新型的蜘蛛毒素 ,其影响电压门控钠通道的机制可能有别于那些结合于通道位点 3来延缓钠电流失活时间特征的蜘蛛毒素如δ 澳洲漏斗网蛛毒素、μ 美洲漏斗网蛛毒素I VI等。     

Voltage and Use Dependence of Sodium Channel Blockade by a New Analgesic,D57, in Rat Dorsal Root Ganglion Neurons

We studied the voltage- and use-dependent action of pyrrolo-imidazole derivative, D57, on sodium currents in different dorsal root ganglion neurons of rats. At the level of 50% of maximum tonic block, which corresponded to a concentration of 0.44 mM, the use-dependent block of tetrodotoxin resistant (TTXr) sodium currents reached 59 ± 12% of the remaining currents when neurons were stimulated by 6-msec-long impulses up to -10 mV with a 20 sec^-1 frequency, whereas for TTX sensitive (TTXs) currents this value was equal to 38 ± 9%. This block was dependent on the holding potential, and for cells with only TTXr currents the dependence was shifted to more positive potentials compared with that for neurons with only TTXs currents or with both of them.     

Regional Distribution of Calcitonin Gene-Related Peptide-, Substance P-, Cholecystokinin-, Met5-Enkephalin-, and Dynorphin A (1–8)-Like Materials in the Spinal Cord and Dorsal Root Ganglia of Adult Rats: Effects of Dorsal Rhizotomy and Neonatal Capsaicin

Biochemical mapping of five different peptide-like materials--calcitonin gene-related peptide (CGRP), substance P (SP), Met5-enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1-8) (DYN)--was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3-month-old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4-T2) or after neonatal administration of capsaicin (50 mg/kg s.c.). In control rats, all peptide-like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy-induced CGRP depletion (-85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (-60%), a finding suggesting that this peptide is contained in both capsaicin-sensitive (mostly unmyelinated) and -insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (-50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin-sensitive small-diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin-insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.     

Effects of Cell Division, Cell Density, and Cyclic Nucleotides on Choline Acetyltransferase Activity in a Cholinergic Neuroblastoma Cell Line (S-20Y)

We investigated the effects of a number of experimental perturbations on choline acetyltransferase (ChAT) in a cholinergic mouse neuroblastoma cell line (S-20Y). ChAT specific activity increased by 4.5-fold during growth, suggesting that enzyme activity is dependent on increased cell density. This was confirmed by assessing enzyme activity at differential initial seeding densities. ChAT activity was also markedly enhanced by 1 mM dibutyryl cyclic-3',5'-AMP (dBcAMP), an effect that was blocked by cycloheximide. Confirmation of the dBcAMP effect was achieved with forskolin, a compound known to enhance intracellular cyclic AMP; forskolin (100 microM) caused a significant increase in ChAT activity. After a 20-h latent interval ChAT activity was also enhanced significantly by cytosine arabinoside. The common element in these diverse effects on ChAT activity may be cessation of cell division, although cell-cell interactions at the level of the cell membrane may also be important in the control of ChAT in S-20Y.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.