Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity.

The 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3' UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3' UTR and stable 3' UTR-derived fragments of unknown function. Cleavage of the long 3' UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3' UTR-derived RNAs.

Colocalization of nitric oxide synthase, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities in postganglionic neurons of the quail superior cervical ganglion.

The colocalization of immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH) in the superior cervical ganglion (SCG) was investigated in the quail. In this bird, a substantial amount of NOS-immunoreactive (IR) cells were consistently found in the SCG without colchicine treatment or nerve ligation. The finding worthy of pointing out was that three-fourths of these NOS-IR cells were positive for TH. VIP-IR cells appeared with markedly low frequency than NOS-IR cells. They were generally small in size and often located in the ganglion peripheral. There were no VIP-IR cells positive for TH or negative for NOS: VIP immunoreactivity always appears in NOS-IR cells negative for TH. Thus, the results of the present study clearly showed the existence of two distinct subpopulations of postganglionic NOS-IR neurons (one is catecholaminergic and negative for VIP, and the other is non-catecholaminergic and positive for VIP). This suggests that nitric oxide (NO) and possibly VIP act as postganglionic neurotransmitters or neuromodulators in the quail SCG. The predominant appearance of the former category of NOS-IR cells must be considered in relation to some specific NO-induced controlling mechanisms of SCG neurons.

Effect of oxygen on phosphodiesterases (PDE) 3 and 4 isoforms and PKA activity in the superior cervical ganglia.

UNLABELLED: The cAMP-protein kinase A (PKA) signaling pathway is involved in regulating the release of transmitters from neurons and other cells. Multiple phosphodiesterase (PDE) isoforms regulate this pathway, however, the pattern of isoform expression and stimulus response across tissues has not been fully characterized.Using fluorescent resonance energy transfer (FRET)-based imaging in primary superior cervical ganglia (SCG) neurons and real-time qPCR, we explored the role of PDE3 and PDE4 isoforms and oxygen tension in the activation of PKA and changes in gene expression. These primary neurons were infected with an adenovirus containing A-Kinase activity reporter (AKAR3) and assayed for responses to PDE inhibitors: rolipram (ROL, 1 µM), milrinone (MIL, 10 µM) and IBMX (100 µM), and adenylyl cyclase activator forskolin (FSK, 50 µM). Different PDE activity patterns were observed in different cells: high PDE4 activity (n = 3), high PDE3 activity (n = 3) and presence of activity of other PDEs (n = 3). Addition of PKA inhibitor H89 (10 µM) completely reversed the response. We further studied the effect of oxygen in the PKA activity induced by PDE inhibition. Both normoxia (20%O(2)/5%CO(2)) and hypoxia (0%O(2)/5%CO(2)) induced a similar increase in the FRET emission ratio (14.5 ± 0.8 and 14.7 ± 0.8, respectively).PDE3a, PDE4b and PDE4d isoforms mRNAs were highly expressed in the whole SCG with no modulation by hypoxia. CONCLUSION: Using a FRET-based PKA activity sensor, we show that primary SCG neurons can be used as a model system to dissect the contribution of different PDE isoforms in regulating cAMP/PKA signaling. The differential patterns of PDE regulation potentially represent subpopulations of ganglion cells with different physiological functions.

PKR and RNase L contribute to protection against lethal West Nile Virus infection by controlling early viral spread in the periphery and replication in neurons.

West Nile virus (WNV) is a neurotropic, mosquito-borne flavivirus that can cause lethal meningoencephalitis. Type I interferon (IFN) plays a critical role in controlling WNV replication, spread, and tropism. In this study, we begin to examine the effector mechanisms by which type I IFN inhibits WNV infection. Mice lacking both the interferon-induced, double-stranded-RNA-activated protein kinase (PKR) and the endoribonuclease of the 2',5'-oligoadenylate synthetase-RNase L system (PKR(-/-) x RL(-/-)) were highly susceptible to subcutaneous WNV infection, with a 90% mortality rate compared to the 30% mortality rate observed in congenic wild-type mice. PKR(-/-) x RL(-/-) mice had increased viral loads in their draining lymph nodes, sera, and spleens, which led to early viral entry into the central nervous system (CNS) and higher viral burden in neuronal tissues. Although mice lacking RNase L showed a higher CNS viral burden and an increased mortality, they were less susceptible than the PKR(-/-) x RL(-/-) mice; thus, we also infer an antiviral role for PKR in the control of WNV infection. Notably, a deficiency in both PKR and RNase L resulted in a decreased ability of type I IFN to inhibit WNV in primary macrophages and cortical neurons. In contrast, the peripheral neurons of the superior cervical ganglia of PKR(-/-) x RL(-/-) mice showed no deficiency in the IFN-mediated inhibition of WNV. Our data suggest that PKR and RNase L contribute to IFN-mediated protection in a cell-restricted manner and control WNV infection in peripheral tissues and some neuronal subtypes.

HGF promotes survival and growth of maturing sympathetic neurons by PI-3 kinase- and MAP kinase-dependent mechanisms.

Hepatocyte growth factor (HGF) is a pleiotrophic factor whose many functions include promoting neuronal survival and growth. Hitherto, these effects have been observed in the presence of other neurotrophic factors like NGF and CNTF, and this requirement for an accessory factor has made it difficult to elucidate the signaling pathways that mediate its survival and growth-enhancing effects. Here, we show that HGF promotes the survival of mature sympathetic neurons of the superior cervical ganglion (SCG) grown at low density in defined medium lacking other neurotrophic factors. This effect was first clearly observed in cultures established from postnatal day 20 (P20) mice and became maximal by P40. HGF also enhanced the growth of neurite arbors from neurons throughout postnatal development and in the adult. HGF treatment resulted in phosphorylation of Akt and ERK1/ERK2. Preventing Akt activation with the phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 blocked the HGF survival response, and inhibition of ERK activation with the MEK inhibitors PD98059 or U0126 reduced the HGF survival response and the neurite growth-promoting effects of HGF. These results indicate that HGF promotes the survival and growth of maturing sympathetic neurons by both PI-3 kinase- and MAP kinase-dependent mechanisms.

BDNF activated TrkB/IRR receptor chimera promotes survival of sympathetic neurons through Ras and PI-3 kinase signaling.

Insulin receptor-related receptor (IRR) expression is tightly coupled to the nerve growth factor (NGF) receptor, TrkA, throughout development. Expression of both receptors is primarily localized to neural crest derived sensory and sympathetic neurons. In contrast to TrkA, however, the physiological ligand for IRR is unknown. To analyze the intracellular signaling and potential function of the orphan IRR in neurons, an adenovirus expressing a TrkB/IRR chimeric receptor was used to infect cultured mouse superior cervical ganglion neurons that normally require NGF for survival. Brain derived neurotrophic factor (BDNF)-activated TrkB/IRR induced neuronal survival. We utilized numerous receptor mutants in order to identify the intracellular domains of IRR necessary for signaling and neuron survival. Finally, we employed adenovirus encoding dominant negative forms of the extracellular signal-regulated kinase (ERK) signaling cascade to demonstrate that IRR, like TrkA, requires ras activation to promote neuron survival. Therefore, by use of the chimeric TrkB/IRR receptor, we have demonstrated the ability of IRR to elicit activation of signaling cascades resulting in a biological response in superior cervical ganglion (SCG) neurons.

Involvement of TLCK-sensitive serine protease in colchicine-induced cell death of sympathetic neurons in culture.

Superior cervical ganglion (SCG) cells from neonatal rats underwent apoptosis upon treatment with colchicine, a microtubule-disrupting agent. Western blotting and activity measurements showed that caspase-3 was indeed activated, but its peptide inhibitor (Ac-DEVD-CHO) neither suppressed nuclear fragmentation nor rescued the neurons from cell death. z-VAD-fmk, the general inhibitor of caspases, prevented nuclear fragmentation and delayed the cell death. Moreover, N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), but not N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK), prevented nuclear fragmentation and provided neuronal protection as well. The combination of both z-VAD-fmk and TLCK provided a long-term neuronal protection (>4 days), whereas neither one alone could do so, suggesting that there are both caspase-dependent and -independent pathways. TLCK-sensitive serine protease is also likely to act upstream of caspase-3 in a caspase-dependent pathway. Electron microscopic observations demonstrated that z-VAD-fmk suppressed nuclear fragmentation and improved mitochondrial swelling, but failed to prevent vesicular formation, which resulted in a slowly-occurring necrosis. More importantly, TLCK effectively blocked this abundant vesicular formation along with suppressing chromatin condensation. Thus, the combination of both conferred a nearly normal morphology, which is consistent with the results of cell survival experiments. These findings clearly indicate that TLCK-sensitive serine protease plays multiple roles in caspase-dependent and -independent pathways of colchicine-induced cell death, and suggest a novel mechanism underlying a necrotic pathway involving ER swelling and vesicular formation.

Phosphatidylinositol 3-kinase is required for the trophic, but not the survival-promoting, actions of NGF on sympathetic neurons.

Nerve growth factor (NGF) supports target-dependent survival of sympathetic and other neurons during development; however, the NGF-regulated signaling pathways required for survival are not fully understood. Sympathetic neurons are able to abort acutely the cell death pathway initiated by NGF deprivation at early, as well as late, time points after readdition of NGF. We found that NGF-dependent phosphatidylinositol 3-kinase (PI-3-K) activity inhibited an early cell death event proximal to c-Jun phosphorylation. However, PI-3-K activity was not required for NGF to inhibit the translocation of Bax from the cytoplasm to the mitochondria, nor was it required for NGF to inhibit the subsequent release of mitochondrial cytochrome c, two events required for NGF deprivation-induced apoptosis. MEK/MAPK activity did not account for any of these NGF-dependent events. When subjected to long-term PI-3-K inhibition in the presence of NGF, the majority of sympathetic neurons did not die. Those that did die exhibited significant differences in the characteristics of death caused by PI-3-K inhibition as compared with NGF deprivation. Additionally, PI-3-K inhibition in the presence of NGF did not induce release of mitochondrial cytochrome c, indicating that these neurons were unable to complete the apoptotic program. In contrast to its modest effects on survival, inhibition of PI-3-K induced marked decreases in somal diameter and metabolic function, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, suggesting that PI-3-K is required for the trophic effects of NGF. Therefore, although PI-3-K is important for the trophic effects of NGF, it is not required for survival. Other, or at least additional, signaling pathways contribute to NGF-mediated survival of sympathetic neurons.

Intrinsic choroidal neurons in the duck eye express galanin.

Recently, it has been shown that the choroid of the duck eye harbours approximately 1,000 intrinsic choroidal neurons positive for vasoactive intestinal polypeptide and neuronal nitric oxide synthase. Their connections and functional significance are largely unknown. This study was performed to establish a typical chemical code for these neurons and to define their targets by using immunocytochemistry and confocal laser scanning microscopy. Almost all intrinsic choroidal neurons coexpressed galanin (GAL), vasoactive intestinal polypeptide (VIP), and neuronal nitric oxide synthase (nNOS)/NADPH-diaphorase. A few stained for GAL and/or nNOS only. Among extrinsic ganglia, GAL/VIP/nNOS coexpressing neurons were only found in the pterygopalatine ganglion where they accounted for approximately 30% of the neuronal population. Thus, GAL/VIP/nNOS-positive nerve fibres around branches of the ciliary artery and within the nonvascular smooth muscle stroma of the choroid may originate mainly from intrinsic neurons and to some extent in a subpopulation of pterygopalatine ganglionic neurons exhibiting the same chemical coding. Close contacts of GAL-positive fibres upon intrinsic choroidal neurons may indicate reciprocal connections between them. Thus, intrinsic choroidal neurons may represent peripherally displaced pterygopalatine ganglion neurons forming a local network for regulation of vascular and nonvascular smooth muscle tone in the duck choroid. They may be integrated in the neuronal circuitry controlling intraocular pressure, choroidal thickness, accommodation, and axial bulbus length.

Ecto-nucleotidase of cultured rat superior cervical ganglia: dipyridamole is a novel inhibitor.

Based on studies of agonist potencies on intact rat superior cervical ganglia, it has been suggested that this ganglion possesses distinct receptors for purine and pyrimidine nucleotides. However, the potency of an agonist is dependent upon whether it is susceptible to extracellular metabolism by the tissue. The aim of this investigation was to study the metabolism of uridine or adenosine nucleotides and nucleosides and the effects of dipyridamole and an ecto-ATPase inhibitor ARL 67156 (6-N, N-diethyl-D-beta-gamma-dibromomethylene-ATP) on their metabolism. Adenosine- and uridine-5'-triphosphates (ATP and UTP) were catabolised by cultured rat superior cervical ganglia, to their di- and monophosphates. Both ATP and UTP breakdown was significantly inhibited by dipyridamole (10 mcM), whereas ARL 67156 (100 mcM), was a weaker inhibitor of ATP degradation and inhibited UTP breakdown by approximately 40%. Metabolism of ATP and UTP by cultured rat superior cervical ganglia was reduced after treatment with cytosine-beta-arabinoside, suggesting that non-neuronal cells along with neuronal cells contribute to their breakdown. In conclusion, these results indicate that rat superior cervical ganglia possess ecto-nucleotidases capable of catabolising purine and pyrimidine nucleotides to their nucleosides, and that dipyridamole is a potent inhibitor of ecto-nucleotidase activity.

Reversible physiological alterations in sympathetic neurons deprived of NGF but protected from apoptosis by caspase inhibition or Bax deletion.

Cell death in nervous system development and in many neurodegenerative diseases appears to be apoptotic or programmed. Withdrawal of nerve growth factor (NGF) from cultures of superior cervical ganglia neurons (SCG) is an excellent model of programmed cell death (PCD), producing apoptosis within 24-48 h. This death can be prevented by treatment with caspase inhibitors or deletion of the proapoptotic Bax gene. Since inhibition of apoptosis is an attractive strategy for the therapy of many neurological diseases and little is known about the function of neurons when apoptosis has been aborted, we examined the electrophysiological properties of NGF-deprived SCG neurons from rats and mice, saved by the caspase inhibitor boc-aspartyl(OMe)fluoromethyl ketone (BAF) or by Bax deletion. Compared to NGF-maintained controls, the resting membrane potentials of BAF-saved neurons were depolarized by 9 mV and the action potentials were prolonged by over 50%. Nicotinic cholinergic current density was depressed by about 50%. Electrophysiological parameters returned to normal within 4 days after NGF restoration. Neurons from Bax-deficient mice were altered differently by NGF withdrawal. There were no detectable changes in resting or action potentials. However, nicotinic current density was reduced just as in BAF-saved rat neurons. There were no observable changes in the processes of individual neurons after 6 days of NGF deprivation in the presence of BAF. Our results indicate that neurons are physiologically altered during pharmacological inhibition of PCD, but fully recover after trophic support is returned.

Synergistic effects of muscarinic agonists and secretin or vasoactive intestinal peptide on the regulation of tyrosine hydroxylase activity in sympathetic neurons.

Cholinergic agonists and certain peptides of the glucagon-secretin family acutely increase tyrosine hydroxylase activity in the superior cervical ganglion in vitro. The present study was designed to investigate possible interactions between these two classes of agonists in regulating catecholamine biosynthesis. Synergistic effects were found between carbachol and either secretin or vasoactive intestinal peptide in the regulation of DOPA (dihydroxyphenylalanine) synthesis. In addition, synergism was found at the level of the accumulation of cyclic adenosine monophosphate, the likely second messenger in the peptidergic regulation of tyrosine hydroxylase activity. The synergism seen with carbachol was blocked by a muscarinic, but not by a nicotinic, antagonist. Synergism was also found between bethanechol, a muscarinic agonist, and secretin, but not between secretin and dimethylphenylpiperazinium, a nicotinic agonist. Since previous immunohistochemical results suggest that vasoactive intestinal peptide and acetylcholine are colocalized in some preganglionic sympathetic neurons, the present data raise the possibility that the two might act synergistically in vivo in regulating catecholamine biosynthesis. Synergistic postsynaptic actions may be a common feature at synapses where peptides of the secretin-glucagon and acetylcholine are colocalized.

SERCA function declines with age in adrenergic nerves from the superior cervical ganglion.

1. Intracellular calcium is a universal second messenger integrating numerous cellular pathways. An age-related breakdown in the mechanisms controlling [Ca2+]i homeostasis could contribute to neuronal degeneration. One component of neuronal calcium regulation believed to decline with age is the function of sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. 2. Therefore we investigated the impact of age on the capacity of SERCA pumps to control high (68 mM) [K+]-evoked [Ca2+]i-transients in acutely dissociated superior cervical ganglion (SCG) cells from 6- and 20-month-old Fisher-344 rats. Calcium transients were measured by fura-2 microfluorometry in the presence of vanadate (0.1 microM) to selectively block plasma membrane calcium ATPase (PMCA) pumps, dinitrophenol (100 microM) to block mitochondrial calcium uptake and extracellular sodium replaced with tetraethylammonium to block Na+/Ca2+-exchanger, thus forcing the neuronal cells to rely on SERCA uptake to control [Ca2+]i homeostasis. 3. In the presence of these calcium buffering blockers, the rate of recovery of [Ca2+]i was significantly slower and time to recover to approximately 90% of resting [Ca2+]i was significantly greater in SCG cells from old (20 months) compared with young (6 months) animals. 4. This age-related change in the recovery phase of [K+]-evoked [Ca2+]i-transients could not be explained by differences in the sensitivity of SCG cells to the calcium buffering blockers, as no age-related difference in basal [Ca2+]i was observed. 5. These studies illustrate that when rat SCG cells are forced to rely on SERCAs to buffer [K+]-evoked [Ca2+]i-transients, an age-related decline in SERCA function is revealed. Such age-related declines in calcium regulation coupled with neuronal sensitivity to calcium overload underscore the importance of understanding the components of [Ca2+]i homeostasis and the functional compensation that may occur with advancing age.

Protein phosphorylation signaling mechanisms in carotid body chemoreception.

Chemotransduction in the carotid body occurs in specialized type I cells and likely involves a complex series of regulated events which culminates in the release of neurotransmitter agents and the excitation of afferent nerve fibers. Previous studies have shown that multiple factors, including the levels of calcium and cyclic nucleotide second messengers, are important regulators of the chemoreceptor transduction cascade in type I cells. In addition, increases in electrical excitability induced in type I cells by chronic exposure to hypoxia are mimicked by agents which elevate intracellular cyclic AMP levels [Stea et al., J Neurosci 1995;15:2192-2202]. These and other findings suggest that protein kinases, and the phosphorylation of specific protein targets are important components of the hypoxic transduction machinery. Moreover, protein kinase-mediated cascades may participate in the well-known physiological adjustments which occur in the carotid body during prolonged stimulation. In the current study, our data demonstrate (1) the presence of specific protein kinases and target phosphoproteins in the carotid body, and also in the morphologically similar small intensely fluorescent cells of the superior cervical sympathetic ganglia. (2) Nitric oxide production and efferent inhibition in the chemosensory tissue is reduced in the presence of the specific tyrosine kinase inhibitor, lavendustin A. (3) Hypoxia-induced catecholamine release from type I cells is inhibited by the protein kinase A antagonist, Rp-cAMPs. And finally (4), exposure to chronic hypoxia up-regulates the expression of the tyrosine kinase, fyn, and an important growth regulatory phosphoprotein, growth associated protein-43 (GAP-43). These findings suggest that second messenger-mediated phosphorylation and dephosphorylation of specific protein targets is a mechanism capable of regulating diverse cellular functions in the carotid body during acute and chronic stimulation.

Plasticity in the phenotypic expression of catecholamines and vasoactive intestinal peptide in adult rat superior cervical and stellate ganglia after long-term hypoxia in vivo.

Sympathetic ganglia in the adult rat contain various populations of nerve cells which demonstrate plasticity with respect to their transmitter phenotype. The plasticity of the neuronal cell bodies and of the small intensely fluorescent cells in the superior cervical and stellate ganglia in response to hypoxia in vivo (10% O2 for seven days) was assessed by studying the expression of catecholamines and vasoactive intestinal peptide. The levels of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid and vasoactive intestinal peptide immunoreactivity were determined. In addition, the density of the immunohistochemical staining of cells for tyrosine hydroxylase and vasoactive intestinal peptide was evaluated. In the intact superior cervical ganglion, hypoxia increased the dopamine level as well as the density of small intensely fluorescent cells immunolabelled for tyrosine hydroxylase and vasoactive intestinal peptide. In the axotomized ganglion, hypoxia elicited a twofold rise in the level of the vasoactive intestinal peptide as well as enhancing the density of neuronal cell bodies immunostained for this peptide. Thus, the effect of hypoxia on the expression of vasoactive intestinal peptide expression in neurons was dependent on neural interactions. In the intact stellate ganglion, hypoxia alone induced a 1.5-fold increase in the density of neuronal cell bodies immunostained for vasoactive intestinal peptide. Thus, ganglia-specific factors appeared to play a role in determining changes in neuronal phenotype in response to hypoxia. The present study provides evidence for the involvement of dopamine and vasoactive intestinal peptide in ganglionic responses to long-term hypoxia as well as for differential responses by the two ganglionic cell populations, i.e. neuronal cell bodies and small intensely fluorescent cells. Changes in the expression of the vasoactive intestinal peptide during long-term hypoxia may be of energetic, trophic and/or synaptic significance. Hypoxia may be considered to be a vasoactive intestinal peptide-inducing factor in sympathetic ganglia.

Autocrine hepatocyte growth factor provides a local mechanism for promoting axonal growth.

In this report, we describe a novel local mechanism necessary for optimal axonal growth that involves hepatocyte growth factor (HGF). Sympathetic neurons of the superior cervical ganglion coexpress bioactive HGF and its receptor, the Met tyrosine kinase, both in vivo and in vitro. Exogenous HGF selectively promotes the growth but not survival of cultured sympathetic neurons; the magnitude of this growth effect is similar to that observed with exogenous NGF. Conversely, HGF antibodies that inhibit endogenous HGF decrease sympathetic neuron growth but have no effect on survival. This autocrine HGF is required locally by sympathetic axons for optimal growth, as demonstrated using compartmented cultures. Thus, autocrine HGF provides a local, intrinsic mechanism for promoting neuronal growth without affecting survival, a role that may be essential during developmental axogenesis or after neuronal injury.

Role of the Jun kinase pathway in the regulation of c-Jun expression and apoptosis in sympathetic neurons.

When deprived of nerve growth factor (NGF), developing sympathetic neurons die by apoptosis. This death is associated with an increase in the level of c-Jun protein and is blocked by expression of a c-Jun dominant negative mutant. Here we have investigated whether NGF withdrawal activates Jun kinases, a family of stress-activated protein kinases that can stimulate the transcriptional activity of c-Jun by phosphorylating serines 63 and 73 in the transactivation domain and which can activate c-jun gene expression. We found that sympathetic neurons contained high basal levels of Jun kinase activity that increased further after NGF deprivation. In contrast, p38 kinase, another stress-activated protein kinase that can also stimulate c-jun gene expression, was not activated after NGF withdrawal. Consistent with Jun kinase activation, we found using a phospho-c-Jun-specific antibody that c-Jun was phosphorylated on serine 63 after NGF withdrawal. Furthermore, expression of a constitutively active form of MEK kinase 1 (MEKK1), which strongly activates the Jun kinase pathway, increased c-Jun protein levels and c-Jun phosphorylation and induced apoptosis in the presence of NGF. This death could be prevented by co-expression of SEKAL, a dominant negative mutant of SAPK/ERK kinase 1 (SEK1), an activator of Jun kinase that is a target of MEKK1. In contrast, expression of SEKAL alone did not prevent c-Jun expression, increases in c-Jun phosphorylation, or cell death after NGF withdrawal. Thus, activation of Jun kinase and increases in c-Jun phosphorylation and c-Jun protein levels occur at the same time after NGF withdrawal, but c-Jun levels and phosphorylation are regulated by an SEK1-independent pathway.

[Intracellular regulation of neuronal nicotinic cholinergic receptors]. / Vnutrikletochnaia reguliatsiia neironal'nykh nikotinovykh kholinoretseptorov.

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.