Bradykinin induces a rapid release of inositol trisphosphate from a neuroblastoma hybrid cell line NCB-20 that is not antagonized by enkephalin

In mouse neuroblastoma x Chinese hamster brain clonal cell line NCB-20, bradykinin (BK) receptor stimulation causes phosphoinositide hydrolysis and release of inositol phosphates. Maximum stimulation (4-fold) of [2-3H]inositol trisphosphate (IP3) release in the absence of Li+ from NCB-20's prelabelled for 20-24 hours with [2-3H]myo-inositol (15 microCi/confluent 60mm dish) occurred after 5-10 seconds of bradykinin exposure, with an EC50 of approximately 100nM. Inositol bisphosphate (IP2) and inositol monophosphate (IP1) also showed increases (2.9-fold and 1.5 fold, respectively), with peaks at 15-20 seconds and 50 seconds, respectively. Under these same conditions, D-Ala2-D-Leu5 enkephalin (DADLE) (10 microM), an opiate agonist with 2nM affinity, gave no stimulation of IP3 release. Furthermore, it did not block BK-initiated release, both when applied simultaneously with BK and when cells were preincubated with DADLE for 100 minutes to lower cyclic AMP levels. These results show that pain-inducing BK has a major acute stimulatory effect on receptor-phospholipase C-coupled IP3 release, the opioid peptide DADLE has no such effect and, DADLE does not block the IP3 release induced by BK.     

Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.     

Expression of human neuronal antigens in a mouse neuroblastoma x human dorsal root ganglion cell hybrid

Clonal mouse neuroblastoma cells were fused with cells from human foetal dorsal root ganglia and several continuously-growing hybrid clones isolated. One hybrid cell line (F2.1D1) containing a number of human chromosomes, was shown to retain the ability to extend neurites in response to dibutyryl cyclic AMP and to express various antigens characteristic of human foetal dorsal root ganglion neurons. The X-chromosome-controlled 12E7 antigen, human Thy-1 and the neuron-specific F12.A2B5 antigen were identified as surface components of the hybrid cells. None of these antigens were detected in the parental neuroblastoma cell line. In addition, using a species-specific monoclonal antibody, the hybrid cells were shown to synthesize human neurofilament protein. This is the first demonstration of the continued expression of a human species- and neuron-specific gene product in a human-mouse somatic cell hybrid.     

Epigenetic regulation of sensory neurogenesis in the dorsal root ganglion cell line ND7 by folic acid

The epigenetic mechanism of folic acid (FA) action on dorsal root ganglion (DRG) cell proliferation and sensory neuron differentiation is not well understood. In this study, the ND7 cell line, derived from DRG cells, was used to elucidate this mechanism. In ND7 cells differentiated with dbcAMP and NGF, Hes1 and Pax3 levels decreased, whereas Neurog2 levels showed a modest increase. Chromatin immunoprecipitation (ChIP) assays examining epigenetic marks at the Hes1 promoter showed that FA favored increased H3K9 and H3K19 acetylation and decreased H3K27 methylation. Hence, FA plays a positive role in cell proliferation. In differentiated ND7 cells, H3K27 methylation decreased, whereas H3K9 and H3K18 acetylation increased at the Neurog2 promoter. FA did not favor this phenotypic outcome. Additionally, in differentiated ND7 Neurog2 associated with the NeuroD1 promoter, FA decreased this association. The results suggest that the switch from proliferation to sensory neuron differentiation in DRG cells is regulated by alterations in epigenetic marks, H3K9/18 acetylation and H3K27 methylation, at Hes1 and Neurog2 promoters, as well as by Neurog2 association with NeuroD1 promoter. FA although positive for proliferation, does not appear to play a role in differentiation.     

Epigenetic regulation of sensory neurogenesis in the dorsal root ganglion cell line ND7 by folic acid

The epigenetic mechanism of folic acid (FA) action on dorsal root ganglion (DRG) cell proliferation and sensory neuron differentiation is not well understood. In this study, the ND7 cell line, derived from DRG cells, was used to elucidate this mechanism. In ND7 cells differentiated with dbcAMP and NGF, Hes1 and Pax3 levels decreased, whereas Neurog2 levels showed a modest increase. Chromatin immunoprecipitation (ChIP) assays examining epigenetic marks at the Hes1 promoter showed that FA favored increased H3K9 and H3K19 acetylation and decreased H3K27 methylation. Hence, FA plays a positive role in cell proliferation. In differentiated ND7 cells, H3K27 methylation decreased, whereas H3K9 and H3K18 acetylation increased at the Neurog2 promoter. FA did not favor this phenotypic outcome. Additionally, in differentiated ND7 Neurog2 associated with the NeuroD1 promoter, FA decreased this association. The results suggest that the switch from proliferation to sensory neuron differentiation in DRG cells is regulated by alterations in epigenetic marks, H3K9/18 acetylation and H3K27 methylation, at Hes1 and Neurog2 promoters, as well as by Neurog2 association with NeuroD1 promoter. FA although positive for proliferation, does not appear to play a role in differentiation.     

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X_{1 –7}). P2X₃ homomultimer and P2X_2/3 heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X₅ receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X₅ receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X₅ receptors in cat. In mouse DRG, the P2X₅ receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X₅ receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X₅ receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X₅-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X₅-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X₅ receptors were coexpressed with NF-200 or with NOS. Only a few P2X₅-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X₅ receptors.     

Prenatal expression of purinergic receptor P2X3 in human dorsal root ganglion

The dorsal root ganglion (DRG) is consisted of neurons that relay multiple types of spinal sensory stimuli to the central nervous system. Several neuroactive molecules may be involved in sensory modulation especially pain processing at the DRG, including the purinergic receptor P2X3 and calcitonin-gene-related peptide (CGRP). P2X3 receptor has been considered a promising pharmaceutical target for the development of new pain medicine. Currently, litter is known about the expression of P2X3 in the human DRG. The present study characterized the localization of P2X3 in prenatal human DRG obtained from fetuses at 4-8 gestational months, by comparing to CGRP expression as well as binding pattern of isolectin-B4 (IB4), a marker of small DRG neurons presumably relevant to nociception. P2X3 immunoreactivity (IR) appeared in most neuron-like perikarya, with their numerical density reduced during the gestational period studied. P2X3 IR was co-labeled very commonly with IB4 binding and infrequently with CGRP IR and was not colocalized with IR for the gliocyte marker glutamine synthetase. Together, the data show an early and broad expression of P2X3 in prenatal human DRG neurons, pointing to a biological role of purinergic signaling during the development of spinal sensory system.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion

After injection of the L7 dorsal root ganglion with ³H-leucine, fast axoplasmic transport carries some 3–5 × more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Multiple and alternative adhesive responses on defined substrata of an immortalized dorsal root neuron hybrid cell line

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term acrylamide intoxication induces atrophy of dorsal root ganglion A-cells and of myelinated sensory axons

We have examined the effects of acrylamide on primary sensory nerve cell bodies and their myelinated axons in chronic acrylamide intoxication. The numbers and sizes of dorsal root ganglion cell bodies (L5) and myelinated nerve fibers were estimated with sterelogical techniques in severely disabled rats which had been treated with 33.3 mg/kg acrylamide twice a week for 7.5 weeks. There was no loss of dorsal root ganglion cells or myelinated nerve fibers in the roots, the sciatic nerve, sural nerve, and a tibial nerve branch. The mean perikaryal volume of A-cells was reduced by 20% (2P < 0.001) from 50000 μm³ in controls (CV = 0.13) to 40000 μm³ (0.12), whereas B-cell volume was unchanged. All size-frequency distribution curves of myelinated axon area of peripheral nerves and sensory roots were shifted to the left towards smaller values in rats exposed to acrylamide. In the L5 sensory root 3 mm from the ganglion, there was a significant reduction of mean cross sectional area of myelinated axons by 14% (2P < 0.05) from 7.6 μm² (0.11) in controls to 6.5 μm² (0.13) in intoxicated rats. The mean cross sectional area of myelinated sural nerve axons was reduced by 22% (2P < 0.001) from 8.6 μm² (0.08) in controls to 6.7 μm² (0.17) in intoxicated rats. We conclude that chronic intoxication with acrylamide leads to selective atrophy of type A dorsal root ganglion cell bodies and simultaneous atrophy along their peripheral axons, whereas neuronal B-cell bodies and motor axons are spared. It is suggested that the neuronal atrophy might well represent a defect of neurofilament synthesis and transport.     

Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion.

After injection of the L7 dorsal root ganglion with 3H-leucine, fast axoplasmic transport carries some 3--5 x more labeled materials down the sensory fibers branches entering the sciatic nerve as compared to the dorsal root fiber branches of the neurons. Freeze-substitution preparations taken from the two sides of the lumbar seventh dorsal root ganglia of cats and monkeys showed little difference in the histograms of nerve fiber diameters of the sensory nerve fiber branch of these neurons as compared to the dorsal root fiber branches. A similar density of microtubules and of neurofilaments in the dorsal root and sensory nerve fiber branches over a wide range of fiber diameters was found in electron micrograph preparations. In the absence of an anatomical difference in the fibers to account for the asymmetrical outflow, a functional explanation based on the transport filament model was advanced.     

Okadaic acid induces the rapid and reversible disruption of the neurofilament network in rat dorsal root ganglion neurons.

Treatment of 15-17 day old dissociated cultures of rat dorsal root ganglia with 1 microM okadaic acid caused a reduction in the mobilities of neurofilament subunits on SDS-polyacrylamide gels, signifying an increase in their phosphorylation levels. When cultures were exposed to okadaic acid for 0.5 hrs and harvested in buffer containing Triton X-100, NF-H was nearly completely redistributed to the detergent- soluble fraction while NF-M and NF-L required a longer exposure to the drug before undergoing a similar shift. This redistribution of subunits corresponded with striking changes in the immunofluorescence staining pattern for neurofilaments. Upon removal of okadaic acid from the culture medium following a 0.5 hr treatment, NF-L and NF-M returned to the Triton X-100 insoluble fraction within 2 hrs while NF-H required 10 hrs for recovery.     

Biological characteristics of rat dorsal root ganglion cell and human vascular endothelial cell in mono- and co-culture

This study aimed to evaluate the biological activity of rat dorsal root ganglion cell (DRGC) and human vascular endothelial cell (HMVEC) in mono- and co-culture. Expression levels of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) mRNA were measured by quantitative real-time RT-PCR (qRT-PCR). Western blot analysis was used to identify VEGF and NGF protein expressions. Cell injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay. The results showed that VEGF and NGF mRNA levels in the HMVEC+DRGC group were significantly higher than those in the DRGC and HMVEC groups (all p < 0.05). There were also greater increases in both VEGF and NGF protein expressions in the HMVEC+DRGC group than those in the DRGC and HMVEC groups (all p < 0.05). The results of MTT analysis revealed significant differences in cell viability among the HMVEC+DRGC group and the DRGC and HMVEC groups (all p < 0.05). In summary, our findings provide evidence that DRGC and HMVEC in co-culture may exhibit greater biological activity than DRGC in mono-culture.     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.     

Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line

Phosphatidylinositol metabolism and ⁴⁵Ca²⁺ efflux were examined in a vascular smooth muscle cell line (A₇r₅). [Arg⁸]Vasopressin stimulated the rapid formation (measurable at 1 sec) of inositol phosphates in a concentration-dependent manner. The time course for formation of inositol phosphates was similar to that for ⁴⁵Ca²⁺ efflux from preloaded cells. The efflux of ⁴⁵Ca²⁺ in response to [Arg⁸]vasopressin could be inhibited by a vasopressin antagonist. This supports the hypothesis that inositol 1,4,5-trisphosphate plays a role in vasopressin stimulated calcium mobilisation from an intracellular source in cultured vascular smooth muscle cells.     

Cobalt inhibits motility of axonal mitochondria and induces axonal degeneration in cultured dorsal root ganglion cells of rat

Cobalt is a trace element that localizes in the human body as cobalamin, also known as vitamin B12. Excessive cobalt exposure induces a peripheral neuropathy, the mechanisms of which are yet to be elucidated. We investigated how cobalt may affect mitochondrial motility in primary cultures of rat dorsal root ganglion (DRG). We observed mitochondrial motility by time-lapse imaging after DsRed2 tagging via lentivirus, mitochondrial structure using transmission electron microscopy (TEM), and axonal swelling using immunocytochemical staining. The concentration of cobaltous ion (Co²⁺) required to significantly suppress mitochondrial motility is lower than that required to induce axonal swelling following a 24-h treatment. Exposure to relatively low concentrations of Co²⁺ for 48 h suppressed mitochondrial motility without leading to axonal swelling. TEM images indicated that Co²⁺ induces mitochondrial destruction. Our results show that destruction of the axonal mitochondria precedes the axonal degeneration induced by Co²⁺ exposure.