Modulation of temperature-sensitive TRP channels

Animals sense temperature--either cold or hot--by the direct activation of temperature-sensitive members of the TRP family of ion channels, the thermo-TRPs. To date, six TRP channels--TRPV1-4, TRPM8 and TRPA1--have been reported to be directly activated by heat and to be involved in thermosensation. Temperature sensing can be modulated by phosphorylation of intracellular residues by protein kinases or by insertion of new channels into the cell membrane. In this review we provide a brief overview of the properties of thermo-TRPs, and we summarise signalling pathways involved in their regulation.     

Gating and calcium-sensing mechanisms of TRPA1 channels revealed

Novel structures of the human TRPA1 channel were determined in the presence of the agonist iodoacetamide and the antagonist A-967079, to reveal the open and closed states of the channel, respectively. The structures further revealed the location of Ca²⁺ modulatory site that is likely conserved among several TRP subgroups.     

辣椒素及其受体

可以感受痛觉刺激的初级感觉神经元的周围末梢被称为伤害性感受器。这些小直径神经元的末梢可将化学、机械和热刺激信号转化为动作电位,并将这些信息上传到中枢,最后使机体产生痛觉或不舒服的感受。但到目前为止,人们对这些可探测到伤害性刺激的分子所知甚少。1997年成功克隆的辣椒素受体亚型1（vanilloid receptor subtype1,VR1)是近年来科学家们研究的“热点分子”,它是表达于伤害性感受器上的非选择性阳离子通道,已有诸多证据表明其可探测和整合诱发痛觉的化学和热刺激信号,基因敲除小鼠的研究分析也有力证明了该离子通道参与了疼痛及组织损伤后痛觉过敏的产生,而且是热诱发疼痛发生过程的关键分子。     

The effects of huwentoxin-I on the voltage-gated sodium channels of rat hippocampal and cockroach dorsal unpaired median neurons

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC₅₀ values of 66.1 ± 5.2 and 4.80 ± 0.58 nM, respectively. Taken together with our previous work on DRG neurons (IC₅₀ ≈ 55 nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM ? mammalian peripheral > mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.     

NT-3 and CNTF exert dose-dependent, pleiotropic effects on cells in the immature dorsal root ganglion: neuregulin-mediated proliferation of progenitor cells and neuronal differentiation

Neurons in the nascent dorsal root ganglia are born and differentiate in a complex cellular milieu composed of postmitotic neurons, and mitotically active glial and neural progenitor cells. Neurotrophic factors such as NT-3 are critically important for promoting the survival of postmitotic neurons in the DRG. However, the factors that regulate earlier events in the development of the DRG such as the mitogenesis of DRG progenitor cells and the differentiation of neurons are less defined. Here we demonstrate that both NT-3 and CNTF induce distinct dose-dependent responses on cells in the immature DRG: at low concentrations, they induce the proliferation of progenitor cells while at higher concentrations they promote neuronal differentiation. Furthermore, the mitogenic response is indirect; that is, NT-3 and CNTF first bind to nascent neurons in the DRG--which then stimulates those neurons to release mitogenic factors including neuregulin. Blockade of this endogenous neuregulin activity completely blocks the CNTF-induced proliferation and reduces about half of the NT-3-mediated proliferation. Thus, the genesis and differentiation of neurons and glia in the DRG are dependent upon reciprocal interactions among nascent neurons, glia, and mitotically active progenitor cells.     

Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels

A T-1-conotoxin, lt5d, was purified and characterized from the venom of vermivorous hunting cone snails Conus litteratus. The complete amino acid sequence of lt5d (DCCPAKLLCCNP) has been determined by Edman degradation. With two disulfide bonds, the calculated average mass is 1274.57 Da, which is confirmed by MALDI-TOF mass spectrometry (average mass 1274.8778). Under whole cell patch-clamp mode, lt5d inhibits tetrodotoxin-sensitive sodium currents on adult rat dorsal root ganglion neurons, but has no effects on tetrodotoxin-resistant sodium currents. The inhibition of TTX-sensitive sodium currents by lt5d was found to be concentration-dependent with the IC₅₀ value of 156.16 nM. Thus, this is the first T-superfamily conotoxin identified to block TTX-sensitive sodium channels.     

Ion channels and transporters involved in cell volume regulation and sensor mechanisms

All animal cell types have an appropriate volume. Even under physiological conditions of constant extracellular osmolarity, cells must regulate their volume. Cell volume is subjected to alterations because of persistent physicochemical osmotic load resulting from Donnan-type colloid osmotic pressure and of cell activity-associated changes in intracellular osmolarity resulting from osmolyte transport and metabolism. The strategy adopted by animal cells for coping with volume regulation on osmotic perturbation is to activate transport pathways, including channels and transporters, mainly for inorganic osmolytes to drive water flow. Under normotonic conditions, cells undergo volume regulation by pump-mediated mechanisms. Under anisotonic conditions, volume regulation occurs by additional channel/transporter-mediated mechanisms. Cell volume regulation is also attained through adjustment of intracellular levels not only of inorganic but also of organic osmolytes with changing the expression of their transporters or regulation of metabolism. In cell volume regulation mechanism, several "volume sensors" are thought to be involved. A volume-sensitive Cl- channel has lately attracted considerable attention in this regard.     

Ratios between number of neuroglial cells and number and volume of nerve cells in the spinal ganglia of two species of reptiles and three species of mammals

We studied the ratios between number of neuroglial (=satellite) cells and number and volume of neurons with which they are associated in the spinal ganglia of two species of reptiles (lizard and gecko) and three species of mammals (mouse, rat, and rabbit). In all five species, we found that the number of satellite cells associated with a nerve cell body increased with increasing volume of the latter. This result shows that there is a quantitative balance between neuroglia and nerve tissue in spinal ganglia. This balance seems to be maintained by a tight regulation of the number of satellite cells. We also found that the mean volume of nerve cell body corresponding to a satellite cell was lower for small neurons than for large ones. Since satellite cells metabolically support spinal ganglion neurons, the metabolic needs of small neurons are better satisfied than those of large ones. For a nerve cell body of a given size, the number of associated satellite cells did not differ between the lizard and gecko, nor between the mouse, rat, and rabbit. However, this number was significantly smaller in the reptiles than in the mammals. This result could be explained by the lower metabolic rate in the nervous system of poikilotherms than mammals, or could have a phylogenetic significance. These two interpretations are not mutually exclusive.     

Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia.     

TRP channels in thermosensation

The ability to sense external temperature is assumed by somatosensory neurons, in which temperature information is converted to neural activity by afferent input to the central nervous system. Somatosensory neurons consist of various populations with specialized gene expression, including thermosensitive transient receptor potential ion channels (thermo-TRPs). Thermo-TRPs are responsible for thermal transduction at the peripheral ends of somatosensory neurons and over a wide range of temperatures. In this review, we focus on several thermo-TRPs expressed in sensory neurons: TRPV1, TRPV4, TRPM2, TRPM3, TRPM8, TRPC5, and TRPA1. TRPV3, TRPV4, and TRPC5 expressed in non-neuronal cells that are also involved in somatosensation are also discussed, whereas TRPM2 and TRPM8 are involved in thermosensation in the brain.     

Gas7在成年大鼠脊髓和脊神经节的表达

目的 研究生长休止蛋白7（Gas7）在成年大鼠脊髓和脊神经节的表达.方法 成年SD大鼠12只,采用逆转录聚合酶链反应（RT-PCR）方法、焦油紫染色以及免疫组织化学方法来观察Gas7基因核酸和蛋白在成年SD大鼠脊髓和脊神经节的表达.结果 RT-PCR结果显示,脊髓和脊神经节有较丰富的Gas7 mRNA表达.免疫组化结果显示：与焦油紫染色相对照,脊髓灰质各板层神经元均表达Gas7蛋白,与其它版层相比较,后角Ⅱ版层胶状质的小细胞和前角Ⅸ版层的运动神经元显色较深且数量较多.脊髓白质Gas7免疫阳性反应较弱且分布均匀.脊神经节内大型感觉神经元呈Gas7免疫强阳性反应,中、小型感觉神经元为弱阳性反应.结论 本文首次描述了Gas7在成年大鼠脊髓和脊神经节的表达,为进一步研究Gas7在成年神经系统再生和修复过程中的功能提供形态学基础.     

Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRP channels as molecular targets

Tumor vascularization is a critical process that determines tumor growth and metastasis. In the last decade new experimental evidence obtained from in vitro and in vivo studies have challenged the classical angiogenesis model forcing us to consider new scenarios for tumor neovascularization. In particular, the genetic stability of tumor-derived endothelial cells (TECs) has been recently questioned in several studies, which show that TECs, as well as pericytes, differ significantly from their normal counterparts at genetic and functional levels. In addition to such an epigenetic action of tumor microenvironment on endothelial cells (ECs) commitment, the distinct characteristics of TECs could be due to differences in their origin compared with preexisting differentiated ECs. Intracellular Ca(2+) signals are involved at different critical phases in the regulation of the complex process of angiogenesis and tumor progression. These signals are generated by a wide variety of intrinsic and extrinsic factors. Several key components of Ca(2+) signaling including Ca(2+) channels in the plasma membrane, endoplasmic reticulum, calcium pumps, and mitochondria contribute to the generation, amplitude, and frequency of these Ca(2+) change. In particular, several members of the transient receptor potential (TRP) family of calcium-permeable channels have profound effects on the function of ECs. Because of its multifaceted role in the control of cell function, proliferation, and motility, TRP channels have been suggested as a potential molecular target for control of tumor neovascularization. Since plasma membrane Ca(2+) channels are easily and directly accessible via the bloodstream, they are potential targets for a number of pharmacological and antibody-targeted therapeutic strategies, with specificity being the main limitation. In this review we discuss recent advances in understanding the role of Ca(2+) channels, with specific reference to TRP channels, in tumor vascularization process.     

Ion channels in asthma

Ion channels are specialized transmembrane proteins that permit the passive flow of ions following their electrochemical gradients. In the airways, ion channels participate in the production of epithelium-based hydroelectrolytic secretions and in the control of intracellular Ca(2+) levels that will ultimately activate almost all lung cells, either resident or circulating. Thus, ion channels have been the center of many studies aiming to understand asthma pathophysiological mechanisms or to identify therapeutic targets for better control of the disease. In this minireview, we focus on molecular, genetic, and animal model studies associating ion channels with asthma.     

Altered Neurotrophin mRNA Levels in Peripheral Nerve and Skeletal Muscle of Experimentally Diabetic Rats

Abstract: The levels of neurotrophin mRNA in sensory ganglia, sciatic nerve, and skeletal muscle were measured in the streptozotocin-diabetic rat using northern blotting. Periods of diabetes of 4, 6, and 12 weeks significantly elevated brain-derived neurotrophic factor (BDNF) mRNA levels in soleus muscle compared with age-matched controls, the increase being highest at 6 weeks. At all time periods studied, the levels of nerve growth factor (NGF) mRNA in soleus muscle were decreased by 21–47%. Following 12 weeks of diabetes, BDNF mRNA levels were increased approximately two-to threefold in L4 and L5 dorsal root ganglia (DRG), and in sciatic nerve, NGF mRNA levels were raised 1.65-fold. Intensive insulin treatment of diabetic rats for the final 4 weeks of the 12-week period of diabetes reversed the up-regulation of BDNF mRNA in DRG and muscle and NGF mRNA in sciatic nerve. All diabetes-induced changes in neurotrophin mRNA were not paralleled by similar alterations in the levels of β-actin mRNA in muscle and nerve, or of GAP-43 mRNA in DRG and nerve. It is proposed that the up-regulation of neurotrophin mRNA is an endogenous protective and/or repair mechanism induced by insult and, as such, appears as an early marker of peripheral nerve and muscle damage in experimental diabetes.     

Diamine Oxidase Induces Neurite Outgrowth in Chick Dorsal Root Ganglia by a Nonenzymatic Mechanism

Abstract: The enzyme diamine oxidase (DAO) catalyzes the oxidative deamination of histamine, diamines, and polyamines. DAO has been localized to several tissues, including thymus, kidney, intestine, seminal vesicles, placenta, and pregnancy plasma. DAO is not constitutively expressed in the mammalian brain, but it becomes detectable following focal injury. Although the physiologic role of DAO remains unknown, the observation that it is present at the interface between rapidly dividing and quiescent cells in several tissues suggests that it might be involved in regulating cell division or differentiation at tissue boundaries. In addition, the observation that DAO is expressed in the brain following injury suggests that the protein might play a role in the CNS response to focal neuronal damage. To test that hypothesis, we assessed the ability of purified DAO to alter the pattern of neuronal differentiation and nerve growth in vitro. In chick dorsal root ganglion explant cultures, purified porcine DAO induced neurite outgrowth in the low nanomolar range. Addition of aminoguanidine, which inhibits DAO enzyme activity, did not inhibit the protein's neurotrophic activity. These findings suggest that DAO can function as a neurotrophic ligand independent of its enzymatic activity.     

Outer segment growth and periciliary vesicle turnover in developing photoreceptors of Xenopus laevis

Summary Cytoskeletal alterations in the cytoplasm of chromatolytic neurons of the dorsal root ganglia were studied in chickens after transection of the sciatic nerves. These studies were carried out using cryofixation with a nitrogencooled propane jet. By this method, the morphological complexity of the cytoskeleton in normal perikarya and cell processes can be visualized. The cytoskeleton of the dorsal root ganglion cells (DRG) is composed of an intricate network of microtubules, neurofilaments and microfilaments. The membrane-bounded cell organelles, as well as the cell nucleus and the plasmalemma, are linked to the microtubules and neurofilaments by microfilaments (or crosslinkers). As a result of the transection of the axon, chromatolysis takes place, characterized by dislocation of cell organelles, eccentric position of the nucleus and dispersion of the parallel cisternae of the rough endoplasmic reticulum throughout the cytoplasm. This characteristic phenomenon coincides with a regression of the neurocytoskeletal network. The neurofilaments and microtubules become shorter, and the microfilaments are replaced by strands of globular or granular material. The temporary regression of the microfilaments leads to a dispersion of the cell organelles. During the remodelling of the cytoskeletal structures, proliferation of the neurofilaments in the regenerating neurons may occasionally be observed. These results show that the cytoskeletal structures are responsible not only for the preservation of cell shape, but also for the maintenance of the normal distributional pattern (location and mobility) of the intracellular components.