No abstract

While large, myelinated dorsal root ganglion (DRG) neurons are capable of firing at high frequencies, small unmyelinated DRG neurons typically display much lower maximum firing frequencies. However, the molecular basis for this difference has not been delineated. Because the sodium currents in large DRG neurons exhibit rapid repriming (recovery from inactivation) kinetics and the sodium currents in small DRG neurons exhibit predominantly slow repriming kinetics, it has been proposed that differences in sodium channels might contribute to the determination of repetitive firing properties in DRG neurons. A recent study demonstrated that Nav1.7 expression is negatively correlated with conduction velocity and DRG cell size, while the Nav1.6 voltage-gated sodium channel has been implicated as the predominant isoform present at nodes of Ranvier of myelinated fibres. Therefore we characterized and compared the functional properties, including repriming, of recombinant Nav1.6 and Nav1.7 channels expressed in mouse DRG neurons. Both Nav1.6 and Nav1.7 channels generated fast-activating and fast-inactivating currents. However recovery from inactivation was significantly faster (approximately 5-fold at -70 mV) for Nav1.6 currents than for Nav1.7 currents. The recovery from inactivation of Nav1.6 channels was also much faster than that of native tetrodotoxin-sensitive sodium currents recorded from small spinal sensory neurons, but similar to that of tetrodotoxin-sensitive sodium currents recorded from large spinal sensory neurons. Development of closed-state inactivation was also much faster for Nav1.6 currents than for Nav1.7 currents. Our results indicate that the firing properties of DRG neurons can be tuned by regulating expression of different sodium channel isoforms that have distinct repriming and closed-state inactivation kinetics.

The detrimental effect of severe hypoxia (SH) on neurons can be mitigated by hypoxic preconditioning (HPC), but the molecular mechanisms involved remain unclear, and an understanding of these may provide novel solutions for hypoxic/ischemic disorders (e.g. stroke). Here, we show that the ␦-opioid receptor (DOR), an oxygen-sensitive membrane protein, mediates the HPC protection through specific signaling pathways. Although SH caused a decrease in DOR expression and neuronal injury, HPC induced an increase in DOR mRNA and protein levels and reversed the reduction in levels of the endogenous DOR peptide, leucine enkephalin, normally seen during SH, thus protecting the neurons from SH insult. The HPC-induced protection could be blocked by DOR antagonists. The DOR-mediated HPC protection depended on an increase in ERK and Bcl 2 activity, which counteracted the SH-induced increase in p38 MAPK activities and cytochrome c release. The cross-talk between ERK and p38 MAPKs displays a "yinyang" antagonism under the control of the DOR-G protein-protein kinase C pathway. Our findings demonstrate a novel mechanism of HPC neuroprotection (i.e. the intracellular up-regulation of DOR-regulated survival signals).Neuronal death as a result of neuronal injury following hypoxic/ischemic insults, such as stroke, is an irreversible process that leads to long term neurological deficit. The prevention of neuronal injury is therefore critical in rescuing the brain from neurological disaster. However, clinical strategies that may help mitigate the effects of hypoxic/ischemic injury are still very limited.One strategy that has been shown to provide effective protection from harmful stress is known as preconditioning. This involves transient, but sublethal, exposure to a stress, resulting in enhanced cellular resistance to subsequent severe stress. It was initially demonstrated in the heart (1) and subsequently found to work in other organ beds (2). The effects of preconditioning have been widely studied in the whole brain (3, 4), as well as in vitro in brain slices (5, 6) and neuronal cultures (7-9). Most studies to date have shown the beneficial effects of preconditioning on rescuing neurons from cell injury in response to subsequent severe insults. Hypoxic preconditioning (HPC), 1 for example, caused by lowering the oxygen content or by combined oxygen and glucose deprivation, protects against subsequent hypoxic injury (4,7,8,10). However, a recent study failed to show neuronal protection with HPC treatment (11), suggesting that the neuronal response to preconditioning may vary depending on neuronal situation and involve complex mechanisms. These molecular mechanisms remain unclear, especially with regard to intracellular signal transduction. In this study, we demonstrated that, in neurons in culture, HPCinduced neuroprotection is dependent on specific factors and that the effect is mediated by intracellular up-regulation of ␦-opioid receptor (DOR)-regulated survival signals, which suppress the increased activity of intracellular death s...

The skeletal muscle ryanodine receptor plays a crucial role in excitation–contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca2+ release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.

Modulation of sarcoplasmic reticulum Ca 2ϩ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1. Am J Physiol Cell Physiol 300: C998 -C1012, 2011. First published February 2, 2011 doi:10.1152/ajpcell.00370.2010 and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca 2ϩ concentrations, whereas at micromolar Ca 2ϩ concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1 The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca 2ϩ release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

SummaryThe role of NO and cGMP signaling in tumor biology has been extensively studied during the past three decades. However, whether the pathway is beneficial or detrimental in cancer is still open to question. We suggest several reasons for this ambiguity: first, although NO participates in normal signaling (e.g., vasodilation and neurotransmission), NO is also a cytotoxic or apoptotic molecule when produced at high concentrations by inducible nitric-oxide synthase (iNOS or NOS-2). In addition, the cGMP-dependent (NO/sGC/cGMP pathway) and cGMP-independent (NO oxidative pathway) components may vary among different tissues and cell types. Furthermore, solid tumors contain two compartments: the parenchyma (neoplastic cells) and the stroma (nonmalignant supporting tissues including connective tissue, blood vessels, and inflammatory cells) with different NO biology. Thus, the NO/sGC/cGMP signaling molecules in tumors as well as the surrounding tissue must be further characterized before targeting this signaling pathway for tumor therapy. In this review, we focus on the NOS-2 expression in tumor and surrounding cells and summarized research outcome in terms of cancer therapy. We propose that a normal function of the sGC-cGMP signaling axis may be important for the prevention and/or treatment of malignant tumors. Inhibiting NOS-2 overexpression and the tumor inflammatory microenvironment, combined with normalization of the sGC/cGMP signaling may be a favorable alternative to chemotherapy and radiotherapy for malignant tumors.2012 IUBMB IUBMB Life, 64(8): 676-683, 2012

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479 -1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained ϳ32% of wild type transport activity and ϳ56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na ؉ and at 4°C, their ability to protect Cys-357 required Na ؉ and was prevented at 4°C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K ؉ ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity. Serotonin transporter (SERT)1 is a member of a large family of homologous integral membrane proteins (1-4). These transporters take up extracellular substrate in a process that is coupled to the transmembrane movement of Na ϩ , Cl Ϫ , and, in some cases, K ϩ (5). In SERT, serotonin (5-HT) reuptake into neurons and peripheral cells such as platelets is believed to occur through cotransport with Na ϩ and Cl Ϫ and countertransport with K ϩ (5). A widely studied aspect of these proteins is their role in the removal of neurotransmitter after its release into the synaptic cleft of neurons, by which they regulate synaptic activity. The role of SERT in behavior is demonstrated by the action of SERT inhibitors, which are clinically effective as antidepressants (6). SERT also interacts with psychostimulants, some of which, such as cocaine, are inhibitors (7), while others, such as amphetamine derivatives, are alternative substrates (8). Members of this family include transporters for dopamine, norepinephrine, glycine, ␥-aminobutyric acid, proline, creatine, and betaine (9 -21). SERT is most closely related to transporters for the catecholamines dopamine and norepinephrine (DAT and NET, respectively) (1, 22). These biogenic amine transporters stand out as a distinct subfamily. They are all inhibited by cocaine and share many structural and functional properties.Hydropathy analysis of the cDNA sequence coding for SERT (23-25) predicted 12 ␣-helical transmembrane...

Ambient oxygen concentration and vascular endothelial growth factor (VEGF)-A are vital in lung development. Since hypoxia stimulates VEGF-A production and hyperoxia reduces it, we hypothesized that VEGF-A down-regulation by exposure of airways to hyperoxia may result in abnormal lung development. An established model of in vitro rat lung development was used to examine the effects of hyperoxia on embryonic lung morphogenesis and VEGF-A expression. Under physiologic conditions, lung explant growth and branching is similar to that seen in vivo. However, in hyperoxia (50% O 2 ) the number of terminal buds and branch length was significantly reduced after 4 d of culture. This effect correlated with a significant increase in cellular apoptosis and decrease in proliferation compared with culture under physiologic conditions. mRNA for Vegf164 and Vegf188 was reduced during hyperoxia and addition of VEGF165, but not VEGF121, to explants grown in 50% O 2 resulted in partial reversal of the decrease in lung branching, correlating with a decrease in cell apoptosis. Thus, hyperoxia suppresses VEGF-A expression and inhibits airway growth and branching. The ability of exogenous VEGF165 to partially reverse apoptotic effects suggests this may be a potential approach for the prevention of hyperoxic injury. T he lung develops by outgrowth, elongation, and reiterated subdivision of the distal embryonic lung bud (1). Rodent embryonic lung explants appear to retain this stereotypic pattern of growth, thus allowing investigation of the pathways that can regulate lung development and how lung maturation may be disrupted under conditions of cellular stress.In the mouse, lung branching morphogenesis begins at embryonic day 9.5 (E9.5) (2) and is regulated by growth factors, cytokines, and the ambient oxygen concentration (3). Low oxygen tensions are a persistent feature of embryonic life (4) and experiments in explanted lungs reveal that oxygen concentrations between 3% and 5% stimulate the process of bud branching and cell proliferation compared with ambient oxygen tension (5). Under similar conditions, cultured developing kidneys exhibit enhanced growth and greater numbers of tubules and blood vessels (6).The ability of hypoxia to stimulate organ development has been attributed to the transcriptional up-regulation of hypoxiainducible factor (HIF)-dependent pathways such as Vegf-A (7) and its receptors 1 (Vegf-r1 or Flt-1) and 2 (Vegf-r2 or Flk-1) (8). VEGF-A is secreted by multiple cell types, including the airway epithelium, and activates VEGF receptors on nearby endothelial cells, stimulating vascular growth. Both Vegf-r1 and Vegf-r2 have been localized to endothelial cells and are expressed throughout development. Recent studies have shown that Vegf-A can also play a role in epithelial cell morphogenesis in both the lung and the kidney (9,10). This may be due to an indirect effect of VEGF-stimulated vascular development on adjacent epithelial structures, or a direct effect of VEGF-A on the epithelial cells themselves.Alte...