Model Of Sympathetic Neurons Research Articles

156. Immune-cell products from mice with an autoimmune disease affect apoptosis, proliferation and metabolism of PC12 cells, a model of sympathetic neurons

Splenic sympathetic innervation decreases with the progression of the autoimmune, lymphoproliferative disease that Fas-deficientlpr/lpr mice develop, an alteration that contributes to its pathogenesis. We studied whether this derangement could be related to the hyperactive lpr/lpr immune system, and whether immune-derived products can influence the viability of sympathetic neurons. For this purpose, non-differentiated PC12 cells, an in vitro model of sympathetic neurons, were treated with supernatants from cultures of splenocytes from 11- and 40-week-old male and female C57BL/6 J (wild-type) and C57BL/6 J lpr/lpr mice. Immune-derived products from both lpr/lpr and wild-type mice induced apoptosis, but also supported proliferation, altered dopamine production and decreased the metabolic activity of PC12 cells. However, supernatants derived fromlpr/lpr immune cells induced more marked changes in all these parameters than those obtained from wild-type mice. Furthermore, supernatants obtained from lpr/lpr mice at more advanced stages of the disease appear to be more powerful in influencing the mentioned parameters than those derived from younger animals in which the disease was still not fully manifested. In contrast to the lpr/lpr-derived supernatants, the capacity of supernatants from lymphoid cells of wild-type mice to support PC12 proliferation seemed to predominate over its pro-apoptotic effect. These results strengthen the possibility that immune-derived products contribute to the alterations in splenic sympathetic innervation observed during the development of the autoimmune disease in lpr/lpr mice.

Stimulation of norepinephrine transporter function by fasudil, a Rho kinase inhibitor, in cultured bovine adrenal medullary cells

Norepinephrine transporter (NET) regulates noradrenergic synaptic transmission by controlling extracellular levels of norepinephrine (NE). The small GTPase, RhoA, and its downstream effector Rho kinase (ROCK) are involved in the regulation of actin cytoskeleton and focal adhesion/stress fiber formation, which may play an important role in various functions of the sympathetic nervous system. We report here the effect of fasudil, a ROCK inhibitor, on the functions of NET in cultured bovine adrenal medullary cells as a model of sympathetic neurons. Treatment of bovine adrenal medullary cells with fasudil caused an increase in [(3)H]NE uptake in time (8-120h) and concentration (10-100μM)-dependent manner. Another ROCK inhibitor, Y-27632 (10-100μM, 1day), also increased [(3)H]NE uptake by the cells. Kinetics analysis of the effect of fasudil on NE transport showed a significant increase in the V (max) of NE transport with little change in K (m). When both extracellular and intracellular Ca(2+) were removed by the deprivation of extracellular Ca(2+) and BAPTA-AM, a cell-permeable Ca(2+) chelator, [(3)H]NE uptake induced by fasudil was completely abolished. Nocodazole, an inhibitor of microtubule polymerization, but not cytochalasin D, an inhibitor of actin polymerization, suppressed the stimulatory effect of fasudil on [(3)H]NE uptake. The present findings suggest that the ROCK inhibitor fasudil up-regulates NET function in a Ca(2+)-dependent and/or nocodazole-sensitive pathway in adrenal medullary cells.

Neurites from PC12 cells are connected to each other by synapse‐like structures

PC12 cells have been used as a model of sympathetic neurons. Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and cAMP induce neurite outgrowth from PC12 cells. cAMP induced a greater number of neurites than did NGF. In particular, we attempted to elucidate whether PC12 cell neurites, induced by several factors including NGF, bFGF, and cAMP, form synapses, and whether each neurite has presynaptic and postsynaptic properties. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we observed that neurites are connected to each other. The connected regions presented dense core vesicles and a clathrin-coated membrane invagination. In addition, typical maker proteins for axon and dendrite were identified by an immuno-staining method. Tau-1, an axonal marker in neurons, was localized at a high concentration in the terminal tips of neurites from PC12 cells, which were connected to neurite processes containing MAP-2, a dendritic marker in neurons. Furthermore, neurites containing SV2 and synaptotagmin, markers of synaptic vesicles, were in contact with neurites harboring drebrin, a marker of the postsynaptic membrane, suggesting that neurites from PC12 cells induced by NGF, bFGF, and cAMP may form synapse-like structures. Tat-C3 toxin, a Rho inhibitor, augmented neurite outgrowth induced by NGF, bFGF, and cAMP. Tat-C3 toxin together with neurotrophins also exhibited synapse-like structures between neurites. However, it remains to be studied whether RhoA inhibition plays a role in the formation of synapse-like structures in PC12 cells.

Regulation of neuronal survival by the extracellular signal-regulated protein kinase 5

The extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase (MAPK) that phosphorylates and regulates various transcription factors in response to growth factors and extra-cellular stresses. To address its biological function during the development of the peripheral nervous system (PNS), we have engineered a novel model of sympathetic neurons in which the erk5 gene can be deleted in vitro. Our data provide for the first time genetic evidence that ERK5 is required to mediate the survival response of neurons to nerve growth factor (NGF). Increased cell death associated with the loss of ERK5 is caused by elevated expression of the BH3-only members of the Bcl-2 family, Bad and Bim. Further investigation indicated that ERK5 suppresses the transcription of the bad and the bim genes via Ca++/cAMP response element binding protein (CREB) and Forkhead box 03a (Foxo3a), respectively. Consistently, we found that the phosphorylation of both p90 ribosomal S6 kinase (RSK) and protein kinase B (PKB) is impaired in neurons lacking ERK5. Together these findings reveal a novel signaling mechanism that promotes neuronal survival during the development of the PNS.

Simvastatin Inhibits Catecholamine Secretion and Synthesis Induced by Acetylcholine via Blocking Na+and Ca2+Influx in Bovine Adrenal Medullary Cells

Simvastatin, an inhibitor of HMG-CoA reductase, is a potent inhibitor of cholesterol biosynthesis and has beneficial effects in the primary and secondary prevention of cardiovascular diseases. In this study, we report the effects of simvastatin on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells used as a model of sympathetic neurons. Simvastatin inhibited catecholamine secretion induced by acetylcholine, an agonist of the nicotinic acetylcholine receptor; by veratridine, an activator of voltage-dependent Na(+) channels; and by high K(+), an activator of voltage-dependent Ca(2+) channels (IC(50) = 3.8, 7.8, and 6.1 microM, respectively). Simvastatin also suppressed acetylcholine-induced (22)Na(+) influx (IC(50) = 4.3 microM) and (45)Ca(2+) influx (IC(50) = 6.1 microM), veratridine-induced (22)Na(+) influx (IC(50) = 6.6 microM) and (45)Ca(2+) influx (IC(50) = 12 microM), and high K(+)-induced (45)Ca(2+) influx (IC(50) = 11 microM). The reduction of catecholamine secretion caused by simvastatin was not overcome by increasing the concentration of acetylcholine or by treatment with mevalonate, the first metabolite of HMG-CoA. The inhibitory effect of simvastatin on histamine-induced secretion of catecholamines was observed in the presence of extracellular Ca(2+), but not in a Ca(2+)-free medium, suggesting that simvastatin does not interfere with histamine receptors nonselectively. Simvastatin also suppressed acetylcholine-induced [(14)C]catecholamine synthesis from [(14)C]tyrosine as well as tyrosine hydroxylase activity. These findings suggest that simvastatin inhibits catecholamine secretion and synthesis induced by acetylcholine through suppression of Na(+) and Ca(2+) influx in the adrenal medulla and probably in the sympathetic neurons.

Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H(2)O(2) injury.

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3-aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H(2) O(2) injury. Exposure to 0.5 mm H(2) O(2) resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12-24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H(2) O(2) -induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase-3 like activity was absent. Moreover, pretreatment with AB restored caspase-3-like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.

A New Perspective on the Inhibitory Role of Nitric Oxide in Sympathetic Neurotransmission

By using, as a model of sympathetic neurons, immortalized rat pheochromocytoma (PC12) cells differentiated by nerve growth factor (NGF), the effect of nitric oxide on sympathetic neurotransmission was examined. The NO donor sodium nitroprusside (SNP; 10−4-3×10−4 M) caused an apparent inhibition of dopamine release from PC12 cells, as measured by HPLC. Studies, in the absence of cells, involving the incubation of dopamine (20 ng/ml) or norepinephrine (15 ng/ml) with SNP (10−6-3×10−4 M) or authentic NO (6×10−6-3×10−5 M) revealed a similar reduction in the detection of the catecholamines. In addition, absorption spectroscopy studies showed dopamine and norepinephrine to be oxidized by NO resulting in the formation of their respective quinone products. These observations, coupled with the finding that the ability of dopamine to raise cAMP levels within PC12 cells was reduced after incubation with SNP, reveal that NO inhibits the biological activity rather than the release of catecholamines.

Efflux of noradrenaline from platelets in normotensive members of hypertensive families.

1. Platelets have been used as a model of sympathetic neurons to study the storage of noradrenaline in normotensive individuals belonging to families with essential hypertension for at least two generations. 2. The initial efflux rate (k) of noradrenaline was determined in 44 young relatives (mean age 29.2 years), in 18 middle-aged relatives (mean age 46.7 years) and in 31 young controls with no known family history of essential hypertension (mean age 29.8 years). From the groups of relatives all those with definite hypertension had been excluded a priori. 3. k was significantly higher in the young relatives (22.7 +/- 7.9) than in the middle-aged relatives (17.7 +/- 6.4) and in the controls (15.6 +/- 5.1). Of the relatives 27.3% had higher k values than any of the controls. A significant correlation was found between k and diastolic blood pressure in controls but not in young relatives.