Combination Of Nerve Growth Factor Research Articles

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

Objectives Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MBcells under NGF exposition. Methods The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MBcell cultures was studied by fluorescence in situ hybridization. Results We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GBcells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MBcells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MBcells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MBcells. Conclusions The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GBcells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MBcell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MBcells under NGF exposition. The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MBcell cultures was studied by fluorescence in situ hybridization. We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GBcells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MBcells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MBcells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MBcells. The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GBcells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MBcell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

Qualitative Assay to Detect Dopamine Release by Ligand Action on Nicotinic Acetylcholine Receptors.

A pheochromocytoma of the rat adrenal medulla derived (a.k.a. PC12) cell-based assay for dopamine measurement by luminescence detection was customized for the qualitative evaluation of agonists and antagonists of nicotinic acetylcholine receptors (nAChRs). The assay mechanism begins with ligand binding to transmembrane nAChRs, altering ion flow into the cell and inducing dopamine release from the cell. Following release, dopamine is oxidized by monoamine oxidase generating hydrogen peroxide that catalyzes a chemiluminescence reaction involving luminol and horseradish peroxidase, thus producing a detectable response. Results are presented for the action of nAChR agonists (acetylcholine, nicotine, and cytisine), and antagonists (α-conotoxins (α-CTxs) MII, ImI, LvIA, and PeIA) that demonstrate a luminescence response correlating to the increase or decrease of dopamine release. A survey of cell growth and treatment conditions, including nerve growth factor, nicotine, ethanol, and temperature, led to optimal assay requirements to achieve maximal signal intensity and consistent response to ligand treatment. It was determined that PC12 cells treated with a combination of nerve growth factor and nicotine, and incubated at 37 °C, provided favorable results for a reduction in luminescence signal upon treatment of cells with α-CTxs. The PC12 assay is intended for use as a fast, efficient, and economic qualitative method to assess the bioactivity of molecules that act on nAChRs, in which testing of ligand–nAChR binding hypotheses and computational predictions can be validated. As a screening method for nAChR bioactivity, lead compounds can be assessed for their likelihood of exhibiting desired bioactivity prior to being subjected to more complex quantitative methods, such as electrophysiology or live animal studies.

Nerve growth factor in combination with Oxiracetam in the treatment of Hypertensive Cerebral Hemorrhage.

Objective:To compare the clinical efficacy of nerve growth factor (NGF) in combination with oxiracetam and single use of oxiracetam in the treatment of hypertensive cerebral hemorrhage.Methods:One hundred and forty patients with hypertensive cerebral hemorrhage who were admitted to the hospital from July 2015 to September 2016 were selected as research subjects and randomly divided into a treatment group which was treated by NGF in combination with oxiracetam and a control group which was treated by oxiracetam only. The clinical efficacy was observed, and the death of both groups was recorded.Results:The National Institutes of Health Stroke Scale (NIHSS) score, Glasgow Coma Scale (GCS) score and limbs muscle force of both groups improved after treatment, and the improvement of the treatment was superior to that of the control group, suggesting a significant difference (P<0.05). The reduction of serum inflammatory factor level of the treatment group was much larger than that of the control group after treatment, and the difference had statistical significance (P<0.05). The survival analysis suggested that the survival rates of the two groups had a statistically significant difference (P<0.05).Conclusion:NGF in combination with oxiracetam is significantly effective in treating hypertensive cerebral hemorrhage as it can apparently recover neurologic impairment and limbs muscle force. The therapy has important clinical application values.

Effect of Macrophage Migration Inhibitory Factor on Corneal Sensitivity after Laser In Situ Keratomileusis in Rabbit

PurposeTo investigate the effect of macrophage migration inhibitory factor (MIF) on corneal sensitivity after laser in situ keratomileusis (LASIK) surgery.MethodsNew Zealand white rabbits were used in this study. A hinged corneal flap (160-µm thick) was created with a microkeratome, and -3.0 diopter excimer laser ablation was performed. Expressions of MIF mRNA in the corneal epithelial cells and surrounding inflammatory cells were analyzed using reverse transcription polymerase chain reaction at 48 hours after LASIK. After LASIK surgery, the rabbits were topically given either 1) a balanced salt solution (BSS), 2) MIF (100 ng/mL) alone, or 3) a combination of nerve growth factor (NGF, 100 ug/mL), neurotrophine-3 (NT-3, 100 ng/mL), interleukin-6 (IL-6, 5 ng/mL), and leukemia inhibitory factor (LIF, 5 ng/mL) four times a day for three days. Preoperative and postoperative corneal sensitivity at two weeks and at 10 weeks were assessed using the Cochet-Bonnet esthesiometer.ResultsExpression of MIF mRNA was 2.5-fold upregulated in the corneal epithelium and 1.5-fold upregulated in the surrounding inflammatory cells as compared with the control eyes. Preoperative baseline corneal sensitivity was 40.56 ± 2.36 mm. At two weeks after LASIK, corneal sensitivity was 9.17 ± 5.57 mm in the BSS treated group, 21.92 ± 2.44 mm in the MIF treated group, and 22.42 ± 1.59 mm in the neuronal growth factors-treated group (MIF vs. BSS, p < 0.0001; neuronal growth factors vs. BSS, p < 0.0001; MIF vs. neuronal growth factors, p = 0.815). At 10 weeks after LASIK, corneal sensitivity was 15.00 ± 9.65, 35.00 ± 5.48, and 29.58 ± 4.31 mm respectively (MIF vs. BSS, p = 0.0001; neuronal growth factors vs. BSS, p = 0.002; MIF vs. neuronal growth factors, p = 0.192). Treatment with MIF alone could achieve as much of an effect on recovery of corneal sensation as treatment with combination of NGF, NT-3, IL-6, and LIF.ConclusionsTopically administered MIF plays a significant role in the early recovery of corneal sensitivity after LASIK in the experimental animal model.

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

The effect of neurotrophic factors on morphology, TRPV1 expression and capsaicin responses of cultured human DRG sensory neurons

We have studied the effect of key neurotrophic factors (NTFs) on morphology, levels of the vanilloid receptor-1 (TRPV1) and responses to capsaicin in adult human sensory neurons in vitro. Avulsed dorsal root ganglia (DRG, n = 5) were cultured with or without a combination of nerve growth factor (NGF), glial cell (line)-derived growth factor (GDNF) and neurotrophin3 (NT3) for 5 days. In the absence of NTFs, the diameter of neurons ranged from 20 to 100 μm (mean 42 ± 4 μm). Adding NTFs caused a significant increase in neuronal sizes, up to 120 μm (mean diameter 62 ± 5 μm, P < 0.01, t-test), an overall 35% increase of TRPV1-positive neurons ( P < 0.003), and notably of large TRPV1-positive neurons >80 μm ( P < 0.05). Responses to capsaicin were significantly enhanced with calcium ratiometry ( P < 0.0001). Short duration (1 h) exposure of dissociated sensory neurons to NTFs increased numbers of TRPV1-positive neurons, but not of TRPV3, Nav 1.8 and IK1 and the morphological size-distribution remained similar to intact post-mortem DRG neurons. NTFs thus increase size, elevate TRPV1 levels and enhance capsaicin responses in cultured human DRG neurons; these changes may relate to pathophysiology in disease states, and provide an in vitro model to study novel analgesics.

Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases.

In the present work, nerve growth factor (NGF) was used in combination with the calcium ionophore, ionomycin or dibutyryl cyclic AMP (dbcAMP), to study the connection between neuronal differentiation and extracellular signal-regulated kinase (ERK) activation of PC12 rat pheochromocytoma cells expressing a dominant negative, Ha-Ras Asn17 protein. Due to the block of endogenous Ras activity, neurite outgrowth in response to NGF is completely inhibited in these cells. However, this blockade can be bypassed by combined treatment with NGF plus ionomycin or NGF plus dbcAMP. The mitogen-activated protein kinase (MAPK) /ERK kinase inhibitor, PD98059, proved to be insufficient in inhibiting the neurite outgrowth under these conditions. Moreover, although both long-term ERK activation and nuclear translocation of ERKs are believed to be key events in neuronal differentiation, neither detectable ERK phosphorylation, nor nuclear translocation of these enzymes, occurred upon combination treatments in our experimental system. However, the neuritogenesis induced by either the combination of NGF/ionomycin or NGF/dbcAMP was inhibited by the Trk inhibitor, K252a. Ras-independent pathways, originating from the NGF receptor, can thus synergize with second messenger analogues bypassing the ERK cascade but leading to the same biological result--neurite formation.

Axonal regeneration stimulated by the combination of nerve growth factor and ciliary neurotrophic factor in an end-to-side model.

The aim of this study was to investigate the potential for stimulating axonal regeneration in the context of end-to-side coaptation using a combination of nerve growth factor and ciliary neurotrophic factor in the rat sciatic nerve model. Four experimental groups (n = 8) were used: end-to-side coaptation only, end-to-side coaptation plus growth factor injection, primary repair, and nontransferred gap control. Twenty weeks after surgery histologic analysis showed that the ratio of axon density was significantly increased for the growth factor injection group. Histologic evidence suggested contamination from the proximal peroneal stump. Electrical stimulation and muscle weights showed that the target muscles had been reinnervated in all groups except the nontransferred gap control group. These data support the conclusion that the use of nerve growth factor and ciliary neurotrophic factor in combination may enhance regeneration in the peripheral nervous system. This is consistent with previous reports on the central nervous system and suggests a potential application in future studies aimed at improving peripheral nerve regeneration. Another conclusion is that contamination from the proximal peroneal stump may explain the regeneration observed in the end-to-side model. Further study using retrograde labeling is needed to establish the origin of the regenerating axons. Finally, evidence suggests that regenerating axons can use the epineurium of an intact nerve to bridge a gap in continuity.

Triiodothyronine and nerve growth factor are required to induce cytoplasmic dynein expression in rat dorsal root ganglion cultures

Beside the several growth factors which play a crucial role in the development and regeneration of the nervous system, thyroid hormones also contribute to the normal development of the central and peripheral nervous system. In our previous work, we demonstrated that triiodothyronin (T 3 ) in physiological concentration enhances neurite outgrowth of primary sensory neurons in cultures. Neurite outgrowth requires microtubules and microtubule associated proteins (MAPs). Therefore the effects of exogenous T 3 or/and nerve growth factors (NGF) were tested on the expression of cytoskeletal proteins in primary sensory neurons. Dorsal root ganglia (DRG) from 19 day old rat embryos were cultured under four conditions: (1) control cultures in which explants were grown in the absence of T 3 and NGF, (2) cultures grown in the presence of NGF alone, (3) in the presence of T 3 alone or (4) in the presence of NGF and T 3 together. Analysis of proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of several proteins in the molecular weight region around 240 kDa. NGF and T 3 together induced the expression of one protein, in particular, with a molecular weight above 240 kDa, which was identified by an antibody against MAP1c, a protein also known as cytoplasmic dynein. The immunocytochemical detection nor explants treated with either NGF or T 3 alone expressed dynein. In conclusion, a combination of nerve growth factor and thyroid hormone is necessary to regulate the expression of cytoplasmic dynein, a protein that is involved in retrograde axonal transport.

Triiodothyronine and nerve growth factor are required to induce cytoplasmic dynein expression in rat dorsal root ganglion cultures

Beside the several growth factors which play a crucial role in the development and regeneration of the nervous system, thyroid hormones also contribute to the normal development of the central and peripheral nervous system. In our previous work, we demonstrated that triiodothyronin (T 3) in physiological concentration enhances neurite outgrowth of primary sensory neurons in cultures. Neurite outgrowth requires microtubules and microtubule associated proteins (MAPs). Therefore the effects of exogenous T 3 or/and nerve growth factors (NGF) were tested on the expression of cytoskeletal proteins in primary sensory neurons. Dorsal root ganglia (DRG) from 19 day old rat embryos were cultured under four conditions: (1) control cultures in which explants were grown in the absence of T 3 and NGF, (2) cultures grown in the presence of NGF alone, (3) in the presence of T 3 alone or (4) in the presence of NGF and T 3 together. Analysis of proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of several proteins in the molecular weight region around 240 kDa. NGF and T 3 together induced the expression of one protein, in particular, with a molecular weight above 240 kDa, which was identified by an antibody against MAP1c, a protein also known as cytoplasmic dynein. The immunocytochemical detection nor explants treated with either NGF or T 3 alone expressed dynein. In conclusion, a combination of nerve growth factor and thyroid hormone is necessary to regulate the expression of cytoplasmic dynein, a protein that is involved in retrograde axonal transport.

Src, ras, and rac mediate the migratory response elicited by NGF and PMA in PC12 cells.

The combination of nerve growth factor (NGF) and phorbol 12-myristate 13-acetate (PMA) rapidly induced the locomotion of PC12 cells by sequentially stimulating lamellar spreading, ruffling with pinocytosis, and polarization by retraction from the substratum. During migration, cells acquired long processes as a result of several undisrupted cell-substratum attachment points. The effect of NGF on PC12 migration was blocked by K-252a, a selective inhibitor of the trk family of receptor tyrosine kinases. When PMA was added to cells expressing pp60v-src, the cells displayed the same morphological behavior as they did with NGF and PMA addition. Activated ras only partially substituted for the effects of NGF; but, when ras was inhibited, the number of migrating cells decreased significantly due to a defect in spreading and retraction. Expression of an activated form of rac stimulated spontaneous growth of lamellipodia and enhanced cell migration in response to PMA. Expression of a dominant negative form of rac inhibited cell spreading and motility. Also, as a later effect, rac-inhibited cells extended much shorter neurites than wild type cells in response to NGF alone. These results indicate that the cytoarchitectural changes induced by NGF and PMA in PC12 cells are mediated by src, ras, and rac. Whereas ras and rac activation affect lamellipodia extension and retraction but not pinocytotic ruffling, src activation is involved in all three events.

Synergistic induction of neurotensin gene transcription in PC12 cells parallels changes in AP-1 activity

A consensus AP-1 site in the promoter of the rat neurotensin/neuromedin N (NT/N) gene is a critical regulatory element required for synergistic regulation by combinations of nerve growth factor (NGF), lithium, glucocorticoids, and adenylate cyclase activators. A rapid RNase protection assay was developed to examine the kinetics of NT/N gene activation and to determine whether activation requires newly synthesized proteins. Either NGF or lithium in combination with dexamethasone and forskolin transiently activated NT/N gene expression, but with distinct kinetics. Protein synthesis was not required for activation when NGF was used as the permissive inducer, but was required for the rapid down-regulation of the response. In contrast, lithium responses were attenuated in the absence of protein synthesis, consistent with a requirement for newly synthesized AP-1 complexes in activation. In all cases, increases in NT/N gene expression closely paralleled increases in AP-1 binding activity. Lithium in combination with other inducers caused delayed increases in both AP-1 binding activity and c-jun, c-fos and fra-1 gene expression. These results indicate that NGF and lithium exert their effects on NT/N gene expression through distinct pathways. The lithium pathway is active in neuronally-differentiated PC12 cells and could potentially be involved in the regulation of NT/N gene expression in the nervous system.

The stimulatory effect of brain-derived neurotrophic factor on dopaminergic phenotype expression of embryonic rat cortical neurons in vitro

Cells of embryonic (E12–16) rat cerebral cortex were cultured for 7 days in vitro (7DIV) in the presence of either brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), with or without dopamine (DA). Chronic treatment of cells with DA or BDNF alone increased (300% and 600%, respectively) the number of the cells that expressed tyrosine hydroxylase (TH). However, the combination of BDNF and DA treatment greatly increased the expression of TH in E14 cortical cells in a dose-dependent manner, to a much greater extent than DA or BDNF alone. This marked response due to treatment with both BDNF and DA was greater in cortical tissue from E12 embryos than that from E14 embryos. The combination of CNTF and DA also increased expression of the dopaminergic phenotype whilst CNTF alone was ineffective, but this effect was largely due to DA. No effect of DA, or of neurotrophic factors, was observed on cortical cells from E16 embryos under any of the treatment conditions. The present study reveals how chemical environment plays an important role in determining the final phenotype of cortical neurons during early periods in brain development. BDNF, but not CNTF, may influence the differentiation of fetal cortical cells towards a dopaminergic phenotype via a unique mechanism, different from that due to DA. This combination of nerve growth factor and neurotransmitter may be of general importance in phenotype determination in the early developmental stages of the nervous system.

Neuropeptide expression by newborn and adult rat sensory neurons in culture: Effects of nerve growth factor and other neurotrophic factors

Adult rat dorsal root ganglion sensory neurons in culture require nerve growth factor for synthesis of substance P and calcitonin gene-related peptide but express vasoactive intestinal peptide independently of nerve growth factor. In contrast, the same neurons from newborn rats do not express detectable vasoactive intestinal polypeptide when cultured with nerve growth factor. To further explore the mechanisms regulating neuropeptide expression in these cells, I compared the effects of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor and leukaemia inhibitory factor on substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and somatostatin expression in rat dorsal root ganglion cultures. As with neurons from adult animals, newborn rat sensory neurons required nerve growth factor for synthesis of substance P and calcitonin gene-related peptide. This effect was independent of neuronal survival since most neurons capable of expressing these peptides appeared to survive without added neurotrophic factors. Neurons surviving in the absence of nerve growth factor also expressed vasoactive intestinal polypeptide, suggesting that nerve growth factor suppresses vasoactive intestinal polypeptide expression in immature neurons. However, nerve growth factor withdrawal after eight days' culture failed to cause vasoactive intestinal polypeptide induction which therefore appears to depend on other factors also. Neither ciliary neurotrophic factor nor leukaemia inhibitory factor affected peptide levels when used alone, but both inhibited nerve growth factor-stimulated expression of substance P and calcitonin gene-related peptide in adult rat neurons. They also stimulated vasoactive intestinal polypeptide expression in newborn rat neurons in the presence of nerve growth factor but not to such high levels as those seen under conditions of nerve growth factor deprivation. Neither brain-derived neurotrophic factor nor neurotrophin-3 affected peptide expression significantly. Somatostatin was detected in adult rat neurons, but was unaffected by neurotrophic factors. No somatostatin was detected in newborn rat neurons. These results suggest that in immature animals at least, the increased expression of vasoactive intestinal polypeptide seen in sensory neurons following peripheral nerve injury in vivo, could result from deprivation of target-derived nerve growth factor in combination with increased availability of ciliary neurotrophic factor or leukaemia inhibitory factor from the injured nerve.

PC12 cells can be induced to produce, but do not process, the neurotensin/neuromedin N precursor

Neurotensin and neuromedin N are two biologically active, related peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C- terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences, which are thought to represent putative processing sites, occur in the precursor molecule. Studies by others have shown that rat pheochromocytoma PC12 cells produced neurotensin and dramatically increased their neurotensin/neuromedin N precursor mRNA content in response to a combination of nerve growth factor, dexamethasone, forskolin, and Li +. Here, we investigated the effects of this combination of inducers on the posttranslational processing of the neurotensin/neuromedin N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and arginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doublets. These studies revealed that PC12 cells synthesized huge amounts (> 100 pmol/mg of protein) of unprocessed neurotensin/neuromedin N precursor in response to inducers, but largely lacked the capability to process this precursor at any of the four Lys-Arg doublets. Thus, mature neurotensin and neuromedin N represented less than 1% of the total precursor content in PC12 cells. The PC12 cell line may represent an interesting model with which one could transfect the recently cloned prohormone convertases PC1 and PC2, thereby allowing the study of the role of these enzymes in the processing of the neurotensin/neuromedin N precursor.

Lithium dramatically potentiates neurotensin/neuromedin N gene expression.

Lithium perturbs intracellular signal transduction pathways used by neurotransmitters, suggesting that changes in receptor signalling may underlie its actions in the treatment of manic depressive illness. Little attention, however, has been directed toward possible additional actions at the level of specific gene expression, particularly of genes encoding neurotransmitters or neuromodulators. In PC12 pheochromocytoma cells, lithium dramatically potentiates increases in intracellular levels of the neuropeptide neurotensin and the mRNA encoding it, caused by combinations of nerve growth factor, dexamethasone, and the adenylate cyclase activator, forskolin. This result demonstrates that lithium can profoundly influence the expression of a specific neuropeptide gene in a previously unanticipated manner and suggests that changes in gene expression might be involved in its therapeutic activity.

Induction of tyrosine hydroxylase synthesis in rat superior cervical ganglia [formula omitted] by nerve growth factor and dexamethasone

The addition of dexamethasone and nerve growth factor to organ cultures of superior cervical ganglia from young rats induces the synthesis of tyrosine hydroxylase. The combination of nerve growth factor and dexamethasone in vitro produces a differential rate of tyrosine hydroxylase synthesis which approaches that obtained by the in vivo administration of nerve growth factor.