Nicotine protects against arachidonic-acid-induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons

Hydrolysis of membrane phospholipids of spinal cord neurons is one of the first events initiated in spinal cord trauma. In this process, free fatty acids, and in particular arachidonic acid, are released. Exposure of spinal cord neurons to free arachidonic acid can compromise cell survival and initiate apoptotic cell death. In order to determine potential mechanisms of apoptosis induced by arachidonic acid, activation of caspases -3, -8, and -9, as well as the release of cytochrome c into the cytoplasm were measured in cultured spinal cord neurons exposed to 10 microM of this fatty acid. In addition, because nicotine can exert a variety of neuroprotective effects, we hypothesized that it can prevent arachidonic acid induced apoptosis of spinal cord neurons. To study this hypothesis, spinal cord neurons were pretreated with nicotine (10 microM for 2 h) before arachidonic acid exposure and caspase activation as well as markers of apoptotic cell death were studied. Treatment of spinal cord neurons with arachidonic acid for up to 24 h significantly increased cytoplasmic levels of cytochrome c, induced caspase activation and induced DNA laddering, a hallmark of apoptotic cell death. Nicotine pretreatment markedly attenuated all these effects. In addition, antagonist studies suggest that the alpha7 nicotinic receptor is primarily responsible for these anti-apoptotic effects of nicotine. These results indicate that nicotine can exert potent neuroprotective effects by inhibiting arachidonic acid induced apoptotic cascades of spinal cord neurons.     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

大鼠内侧前额叶皮质内ERK1/2 活化参与脊髓损伤后抑郁情绪

目的:观察细胞外信号调节激酶1/2(ERK1/2)的活化在脊髓损伤引起抑郁中的作用。方法:应用Western blot和行为药理学方法,观察脊髓损伤后(SCI)大鼠内侧前额叶皮质内(mPFC)ERK1/2及磷酸化-ERK1/2(p-ERK1/2)的表达情况及ERK1/2磷酸化抑制剂U0126对抑郁样行为的影响。结果:脊髓损伤后的第2天到第8周,SCI模型大鼠的BBB评分均显著低于假手术组,差异具有统计学意义(p0.05)。脊髓损伤后8周-12周,SCI模型大鼠强迫游泳不动时间与假手术组相比明显缩短,mPFC内pERK1/2蛋白表达水平明显升高,总ERK 1/2的蛋白水平则未见组间差异,而给予U0126的大鼠的不动时间与给药之前相比明显延长增加,mPFC内pERK1/2蛋白表达水平较SCI模型大鼠明显降低,差异均具有统计学意义(P0.05)。结论:内侧前额叶皮质内ERK1/2的激活参与了脊髓损伤后引起的突触可塑性,在相关的抑郁样行为的产生中发挥了重要的作用。     

Protein kinase C regulation of dopamine transporter initiated by nicotinic receptor activation in slices of rat prefrontal cortex

We previously reported that activation of nicotinic receptors causes an enhancement in amphetamine-stimulated release of dopamine via its transporter from slices of prefrontal cortex, but no such enhancement of release from slices of nucleus accumbens or striatum. The nicotinic receptors mediating the enhancement most likely contain alpha4 and beta2 subunits based upon pharmacological characterization. In this study, we sought to characterize the second messenger systems associated with the nicotine-mediated response. Sodium channel involvement was confirmed by the observation that tetrodotoxin blocked nicotine-mediated enhancement, whereas veratridine or elevated K(+) mimicked the enhancement seen with nicotine. Inclusion of EGTA blocked nicotine-mediated enhancement, suggesting that, even though no exogenous Ca(2+) was added, endogenous stores were required for the enhancement. The enhancement by nicotine was also abolished by the L-type voltage-dependent calcium channel (VDCC) antagonist nitrendipine, but not by the N-type VDCC antagonist omega-conotoxin GVIA. Finally, inhibition of protein kinase C also abolished the nicotine-mediated enhancement of amphetamine-stimulated dopamine release, whereas inhibitors of Ca(2+)/calmodulin kinase II did not. These findings establish that nicotine can exert selective effects on dopamine transporter activity in prefrontal cortex, an area involved in cognition and learning.     

Involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in amylin's eating inhibitory effect

Peripheral amylin inhibits eating via the area postrema (AP). Because amylin activates the extracellular-signal regulated kinase 1/2 (ERK) pathway in some tissues, and because ERK1/2 phosphorylation (pERK) leads to acute neuronal responses, we postulated that it may be involved in amylin's eating inhibitory effect. Amylin-induced ERK phosphorylation (pERK) was investigated by immunohistochemistry in brain sections containing the AP. pERK-positive AP neurons were double-stained for the calcitonin 1a/b receptor, which is part of the functional amylin-receptor. AP sections were also phenotyped using dopamine-β-hydroxylase (DBH) as a marker of noradrenergic neurons. The effect of fourth ventricular administration of the ERK cascade blocker U0126 on amylin's eating inhibitory action was tested in feeding trials. The number of pERK-positive neurons in the AP was highest ～10-15 min after amylin treatment; the effect appeared to be dose-dependent (5-20 μg/kg amylin). A portion of pERK-positive neurons in the AP carried the amylin-receptor and 22% of the pERK-positive neurons were noradrenergic. Pretreatment of rats with U0126 decreased the number of pERK-positive neurons in the AP after amylin injection. U0126 also attenuated the ability of amylin to reduce eating, at least when the animals had been fasted 24 h prior to the feeding trial. Overall, our results suggest that amylin directly stimulates pERK in AP neurons in a time- and dose-dependent manner. Part of the AP neurons displaying pERK were noradrenergic. At least under fasting conditions, pERK was shown to be a necessary part in the signaling cascade mediating amylin's anorectic effect.     

Nicotine stimulates adhesion molecular expression via calcium influx and mitogen-activated protein kinases in human endothelial cells

To evaluate the effect of nicotine on endothelium dysfunction and development of vascular diseases, we investigated the influence on adhesion molecular expression mediated by nicotine and the mechanism of this effect in human umbilical vein endothelial cells (HUVECs). The result showed that nicotine could induce surface/soluble vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin) expression in a time-response decline manner and the peak appeared at 15 min. This action could be mediated by mitogen-activated protein kinase/extracellular signal regulated kinase 1/2 (MAPK/ERK1/2) and MAPK/p38 because their activation could be distinctly blocked by MAPK inhibitors, PD098059 or SB203580. Mecamylamine (non-selective nicotinic receptor inhibitor), alpha-bungarotoxin (alpha7 nicotinic receptor inhibitor) could block Ca2+ accumulation, and then, prevented the phosphorylation on ERK1/2 and p38. They also inhibited the surface/soluble VCAM-1, E-selectin production of HUVECs modulated by nicotine. Therefore, we concluded that: (i) nicotine obviously up-regulates VCAM-1 and E-selectin expression at 15 min in HUVECs, (ii) nicotine activates HUVECs triggered by the ERK1/2 and p38 phosphorylation with an involvement of intracellular calcium mobilization chiefly mediated by alpha7 nicotinic receptor, (iii) intracellular Ca2+ activates a sequential pathway from alpha7 nicotinic receptor to the phosphorylation of ERK1/2, p38. These elucidate that nicotine activates HUVECs through fast signal transduction pathway and arguments their capacity of adhesion molecular production. Further more nicotine may contribute its influence to the progression of vascular disease such as atherosclerotic lesion.     

Phosphorylation of Extracellular Signal-Regulated Kinases 1/2 Predominantly Enhanced in the Microglia of the Rat Spinal Cord Following Lipopolysaccharide Injection

The present study was initiated to investigate the role of extracellular signal-regulated kinases (ERK) 1/2 signaling pathway in the early response of spinal cord to systemic inflammation by using Western blotting and immunohistochemical techniques in a rat model intraperitoneally injected with 10 mg/kg of lipopolysaccharide (LPS). The results showed that there was a considerable amount of phosphorylated ERK 1/2 protein in the spinal cord of inflamed animals killed under pentobarbital anesthesia. The result of Western blotting showed that the phosphorylation level of ERK 1/2 in the spinal cord was increased at one hour; then 12 and 24 h after LPS injection the level decreased, while the total ERK 1/2 level seemed unchanged. The phosphorylated ERK 1/2 dominantly existed in the microglia cells of the gray matter of spinal cord, as demonstrated with double immunofluorescent staining 1 h after LPS injection. Collectively, the present results suggest that ERK signal pathway involve the cellular activation in the spinal cord following systemic inflammation, with ERK mainly in microglia. The increase of phosphorylation of ERK 1/2 in microglia of spinal cord after LPS injection implicates that ERK signaling pathway involves intracellular activity of microglia responding to the inflammation. Dan Zhou and Min Fei contributed equally to this work.     

Nicotine Promotes Proliferation of Human Nasopharyngeal Carcinoma Cells by Regulating α7AChR, ERK, HIF-1α and VEGF/PEDF Signaling

Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis, but the precise mechanisms involved remain largely unknown. Here, we investigated the mechanism of action of nicotine in human nasopharyngeal carcinoma (NPC) cells. Nicotine significantly promoted cell proliferation in a dose and time-dependent manner in human NPC cells. The mechanism studies showed that the observed stimulation of proliferation was accompanied by the nicotine-mediated simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling. Treatment of NPC cells with nicotine markedly upregulated the expression of α7AChR and HIF-1α proteins. Transfection with a α7AChR or HIF-1α-specific siRNA or a α7AChR-selective inhibitor significantly attenuated the nicotine-mediated promotion of NPC cell proliferation. Nicotine also promoted the phosphorylation of ERK1/2 but not JNK and p38 proteins, thereby induced the activation of ERK/MAPK signaling pathway. Pretreatment with an ERK-selective inhibitor effectively reduced the nicotine-induced proliferation of NPC cells. Moreover, nicotine upregulated the expression of VEGF but suppressed the expression of PEDF at mRNA and protein levels, leading to a significant increase of the ratio of VEGF/PEDF in NPC cells. Pretreatment with a α7AChR or ERK-selective inhibitor or transfection with a HIF-1α-specific siRNA in NPC cells significantly inhibited the nicotine-induced HIF-1α expression and VEGF/PEDF ratio. These results therefore indicate that nicotine promotes proliferation of human NPC cells in vitro through simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling and suggest that the related molecules such as HIF-1α might be the potential therapeutic targets for tobacco-associated diseases such as nasopharyngeal carcinomas.     

Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity

The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.     

Nicotine stimulates prolactin-releasing peptide (PrRP) cells and non-PrRP cells in the solitary nucleus

Nicotine has been reported to regulate food intake and body weight. But the mechanisms underlying these roles have not been fully elucidated. In the present study, we showed that acute administration of nicotine (0.5 mg/kg s.c.) could activate prolactin-releasing peptide (PrRP)-bearing neurons in the A2 area of the NTS of rats, suggesting that PrRP may be associated with nicotine-induced effects in the central nervous system (CNS). We next treated rats with nicotine chronically (4 mg/kg/day for 7 days i.p.), and the results showed that the body weight was strongly reduced and food intake was greatly suppressed compared to the vehicle control group (p<0.01). Immunocytochemical studies revealed that PrRP-bearing neurons in the NTS were evidently activated after chronic administration of nicotine, suggesting that PrRP was involved in the regulation of nicotine-mediated body weight loss and food intake suppression in rats. We also found that acute/chronic administration of nicotine activated PrRP-negative neurons in the NTS, and the majority of these neurons were shown to be TH-negative, suggesting that noncatecholaminergic, PrRP-negative neurons in the NTS are associated with the roles of nicotine. Nicotine has also been shown to stimulate the secretion of ACTH, a stress responsive hormone. In the present study, rats received nicotine (0.5 mg/kg s.c.) or saline followed by restraint stress for 30 min. The immunocytochemical results showed that nicotine/stress and saline/stress both activated the majority of the PrRP neurons in the NTS, there being no significant difference between the two treatments (p>0.05). Nicotine/stress also greatly activated PrRP/TH-negative neurons in the NTS. Saline/stress, however, caused much lower effect on the activation of PrRP/TH-negative neurons. In addition, the activation effect of nicotine/stress on PrRP/TH-negative neurons was much stronger than that of nicotine alone (p<0.01). These results indicated that PrRP was associated with stress responses, but it had little effect on nicotine-mediated stress responses. On the other hand, nicotine and restraint stress may synergistically activate PrRP/TH-negative neurons in the NTS. Taken together, our data show that PrRP is involved in the nicotine-induced regulation of body weight and food intake, but may not be involved in the mediation of nicotine on stress responses. PrRP/TH-negative neurons in the NTS are also associated with the roles of nicotine in the CNS.     

Different signaling molecules responsible for IL-1beta-induced oxytocinergic and vasopressinergic neuron activation in the hypothalamic paraventricular nucleus of the rat

It has been well known that oxytocin (OT)-ergic and arginine vasopressin (AVP)-ergic neurons located in the hypothalamic paraventricular nucleus (PVN) and super optic nucleus (SON) are two kinds of neuroendocrine cells with diverse functions. It has also been demonstrated that immune stimuli can activate these neurons to secret OT and AVP. However, the intracellular signal transduction molecules responsible for the activation of these OT-ergic and AVP-ergic neurons in PVN by immune stimuli are still unclear. In this experiment, the roles of Fos, a protein product of immediate early gene c-fos, and extracellular signal-regulated protein kinase (ERK) 1/2, a signal transduction molecule of mitogen-activated protein kinase (MAPK) family, in these processes were studied in the PVN of the rat following IL-1beta stimulation. The Sprague-Dawley rats were received either 750 ng/kg IL-1beta or equal volume normal saline (NS) injection intravenously (i.v.), and perfused transcardially by 4% paraformaldehyde 3h later. Fos and phosphorylated ERK1/2 (pERK1/2)-immunoreactivity (-ir) was observed in PVN by ABC immunohistochemical staining. Meanwhile, the double staining for OT/Fos, AVP/Fos, OT/pERK1/2 and AVP/pERK1/2 were also processed. The ABC immunohistochemical staining results showed that after an i.v. injection of IL-1beta, the expressions of Fos and pERK1/2 increased evidently in the PVN. Double-staining results showed that a large number of OT-ir cells contained strong Fos-ir products in their nuclei, while only a few of OT cells were double labeled with pERK1/2. As to AVP neurons, great quantities of AVP cells were strongly double labeled with pERK1/2 while there were nearly no Fos-ir nuclei in AVP-ir cells. We conclude from these results that the intracellular IL-1beta-induced events in OT and AVP neurons in PVN are quite different. The OT neurons are mainly activated via Fos without involvement of ERK1/2 pathway, while the latter, but not Fos, involves the intracellular event in AVP neurons activated by IL-1beta.     

Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria

Familial amyotrophic lateral sclerosis (ALS)-linked mutations in the copper-zinc superoxide dismutase (SOD1) gene cause motor neuron death in about 3% of ALS cases. While the wild-type (wt) protein is anti-apoptotic, mutant SOD1 promotes apoptosis. We now demonstrate that both wt and mutant SOD1 bind the anti-apoptotic protein Bcl-2, providing evidence of a direct link between SOD1 and an apoptotic pathway. This interaction is evident in vitro and in vivo in mouse and human spinal cord. We also demonstrate that in mice and humans, Bcl-2 binds to high molecular weight SDS-resistant mutant SOD1 containing aggregates that are present in mitochondria from spinal cord but not liver. These findings provide new insights into the anti-apoptotic function of SOD1 and suggest that entrapment of Bcl-2 by large SOD1 aggregates may deplete motor neurons of this anti-apoptotic protein.     

Capsaicin-Induced Activation of ERK1/2 and Its Involvement in GAP-43 Expression and CGRP Depletion in Organotypically Cultured DRG Neurons

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.     

Nicotine-induced phosphorylation of ERK in mouse primary cortical neurons: evidence for involvement of glutamatergic signaling and CaMKII

Extracellular signal-regulated kinase (ERK) is activated in vivo in a number of brain areas by nicotine and other drugs of abuse. Here we show that nicotine stimulation of cultured mouse cortical neurons leads to a robust induction of ERK phosphorylation that is dependent on nicotine concentration and duration of exposure. Calcium/calmodulin-dependent protein kinase II activity is necessary for nicotine-induced ERK phosphorylation and neither cAMP-dependent protein kinase or protein kinase C appear to be involved. Activity of glutamate receptors, L-type voltage-gated calcium channels, and voltage-gated sodium channels are also required for nicotine-induced ERK phosphorylation. Nicotine-induced ERK phosphorylation was inhibited by high concentrations of mecamylamine, however it was not blocked by other broad nicotinic acetylcholine receptor (nAChR) inhibitors (including hexamethonium and chlorisondamine) or nAChR subtype selective inhibitors (such as methyllycaconitine, alpha-bungarotoxin, dihydro-beta-erythroidine, and alpha-conotoxin Au1B). In accord with these pharmacological results, nicotine-induced ERK phosphorylation was normal in primary cultures made from beta2 or alpha7 nAChR subunit knockout mice. The alpha3/beta4 nAChR agonist cytisine did not induce ERK phosphorylation suggesting that alpha3/beta4 nAChRs were not involved in this process. Taken together, these data define a necessary role for glutamatergic signaling and calcium/calmodulin-dependent protein kinase II in nicotine-induced ERK phosphorylation in cortical neurons and do not provide evidence for the involvement of classical nAChRs.     

Effects of low concentration of endosulfan on proliferation, ERK1/2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes

Endosulfan is a potent organochlorinated pesticide that is known to induce side effects in aquatic organisms, including Oreochromis niloticus (Nile tilapia). It has been previously shown that endosulfan induces oxidative stress and non-specific activation of splenic macrophages and exacerbated serum interleukin-2 synthesis in Nile tilapia. Endosulfan may promote proliferation of T cells through MAP kinase (MAPK) activated signal transductions. The ERK family of MAPKs includes ERK1 and ERK2. Phosphorylated ERK1/2 (pERK1/2) molecules are involved in many aspects of cellular survival, and are important for apoptosis or oxidative stress-induced senescence. In order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide on proliferation, the ERK1/2 pathway, apoptosis and cell senescence in splenocytes from Nile tilapia. Lymphoproliferation was evaluated by colorimetric method using the WST-1 assay. Flow cytometry was used to assess pERK1/2, apoptosis and senescence, using Annexin V-FITC and β-galactosidase respectively. Experimental data showed that exposure to 7 μg mL(-1) of endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA + ionomycin. Splenocytes exposed to endosulfan for 15-180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence. In conclusion, exposure of immune cells to a low concentration of endosulfan deregulates their function and may facilitate the development of multiple diseases.     

脊髓细胞外调节激酶1/2在大鼠后足切割痛中的表达和作用

目的：探讨大鼠后足切割后脊髓ERK的表达情况。方法：以大鼠右后足切割作为急性疼痛模型;用免疫组织化学法测试脊髓磷酸化ERK（pERK）表达情况。ERK抑制剂U0126（1μg）在切割前20min或切割后20min鞘内注射。用von Frey纤维测试大鼠机械性痛敏。结果：大鼠后足切割后1min,在切割侧L4-L5脊髓浅层背侧角（板层Ⅰ和板层Ⅱ）ERK被迅速地激活,并在5min达到峰值,随后恢复到基础值。切割前鞘内给予U0126能显著减轻机械性痛敏,然而,切割后鞘内给予U0126对机械性痛敏的作用并不明显。结论：脊髓ERK在大鼠后足切割痛中产生机械性痛敏发挥了重要的作用。     

Activation of Tyrosine Hydroxylase by Nicotine in Rat Adrenal Gland

The administration of nicotine activates tyrosine hydroxylase in the rat adrenal gland. This activation is apparently maximal 25 min after a single subcutaneous injection of nicotine at 2.3 mg/kg. Repeated injections of nicotine (seven injections once every 30 min) are associated with a persistent activation of adrenal tyrosine hydroxylase for at least 3 h. The nicotinic receptor antagonist hexamethonium does not significantly inhibit the nicotine-mediated activation of tyrosine hydroxylase in innervated adrenal glands. However, hexamethonium completely blocks the activation of adrenal tyrosine hydroxylase by nicotine in denervated adrenal glands. Furthermore, even though a single injection of nicotine activates tyrosine hydroxylase in both innervated and denervated adrenal glands, repeated injections of nicotine do not activate tyrosine hydroxylase in denervated adrenal glands. Our results suggest that the systemic administration of nicotine activates adrenal tyrosine hydroxylase by two mechanisms: (1) via direct interaction with adrenal chromaffin cell nicotinic receptors; and (2) via stimulation of the CNS leading to the release from the splanchnic nerve of substances that interact with adrenal chromaffin cell receptors other than the nicotinic receptor.     

Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10^-8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.