Effect of acyclic monoterpene alcohols and their derivatives on TRP channels

A series of thirty-six geraniol, nerol, citronellol, geranylamine, and nerylamine derivatives was synthesized and tested on TRPA1, TRPM8, and TRPV1 channels. Most of them acted as strong modulators of TRPA1 channels with EC₅₀ and/or IC₅₀ values <1 μM. None was able to significantly activate TRPM8 channels, while thirteen of them behaved as ‘true’ TRPM8 antagonists. Little or no effect was generally observed on TRPV1 channels. Some of the compounds examined, that is, compounds 1d,g,n, 2c,d,h,i,o, 3b,e exhibited an appreciable selectivity for TRPA1 subtype.     

Effects of activation of skin ion channels TRPM8, TRPV1, and TRPA1 on the immune response. Comparison with effects of cold and heat exposure

The effects of pharmacological stimulation of skin ion channels TRPA1, TRPM8, TRPV1 on the immune response are presented. These effects are compared with the effects of different types of temperature exposures - skin cooling, deep cooling, and deep heating. This analysis allows us to clear the differences in the influence on the immune response of thermosensitive ion channels localized in the skin; (2) whether the changes in the immune response under temperature exposures are due to these thermosensitive ion channels. Experiments were performed on Wistar rats. For stimulation of TRPM8 ion channel, an application to the skin of 1% menthol was used, for TRPA1 - 0.04% allylisotiocianate, and for TRPV1 - capsaicin in a concentration of 0.001.The antigen binding in the spleen was two-times stimulated by activation of the cold-sensitive ion channel TRPM8 and much weaker by activation of warm-sensitive TRPV1 (by 15%), and another cold-sensitive ion channel TRPA1 (by 40%). Only the stimulation of TRPA1 significantly (by 140%) increased antibody formation in the spleen, while TRPM8 had practically no effect on this process, and activation of TRPV1 significantly (by 60%) inhibited antibody formation. Stimulation of the TRPM8 ion channel significantly (by 60%) reduced the level of IgG in the blood, which is believed to control of infectious diseases.The obtained results show that pharmacological activation of the skin TRPA1, TRPM8, TRPV1 ion channels can differently affect the immune system. At the epicenter of changes there were the antigen binding and antibody formation in the spleen, as well as the level of IgG in the blood. Exactly stimulation of the TRPM8 ion channel determines the changes in the immune response when only the skin is cooling, while at deep body heating, the changes in the immune response are mostly determined by the activation of the skin TRPV1 ion channel.     

Chronic fluoxetine administration increases expression of the L-channel gene Cav1.2 in astrocytes from the brain of treated mice and in culture and augments K-induced increase in [Ca]i

We have recently shown that freshly isolated astrocytes from the mouse brain express mRNA for the L-channel gene Ca_v1.3 to at least the same degree (per mg mRNA) as corresponding neurons. The amount of extracellular Ca²⁺ actually entering cultured astrocytes by its opening is modest, but due to secondary Ca²⁺-mediated stimulation of the ryanodine receptor (RyR) the increase in free cytosolic Ca²⁺ [Ca²⁺]_i is substantial. The other Ca_v1 subtype expressed in brain is Ca_v1.2, which is even expressed in higher density. Although the different primers used for the two genes preclude exact quantitative comparison, the present study suggests that this is also the case in the freshly isolated astrocytes and neurons, which express equal Ca_v1.2 densities. Again, most of the increase in [Ca²⁺]_i occurred by RyR activity. In contrast to Ca_v1.3 the expression of Ca_v1.2 was greatly increased (doubled) after two weeks of treatment with fluoxetine hydrochloride (10 mg/kg). Accordingly [Ca²⁺]_i in cultured astrocytes exposed to the addition of 10–60 mM KCl increased substantially in cultured astrocytes treated chronically with fluoxetine with the lag time until the effect was observed depending upon the fluoxetine concentration. This effect was inhibited by nifedipine or siRNA against Ca_v1.2. The increase in K⁺-induced rise in [Ca²⁺]_i after fluoxetine treatment is directly opposite to a decrease in [Ca²⁺]_i after treatment with any of the anti-bipolar drugs lithium, carbamazepine or valproic acid, due to reduced capacitative Ca²⁺ influx. We have previously shown a similar effect after fluoxetine treatment, but it becomes overridden by the Ca_v1.2 up-regulation.     

Mechanisms for the coupling of cannabinoid receptors to intracellular calcium mobilization in rat insulinoma beta-cells

In RIN m5F rat insulinoma beta-cells, agonists at cannabinoid CB(1) receptors modulate insulin release. Here we investigated in these cells the effect of the activation of cannabinoid CB(1) and CB(2) receptors on intracellular Ca(2+) ([Ca(2+)](i)). The CB(1) agonist arachidonoyl-chloro-ethanolamide (ACEA), and the CB(2) agonist JWH133, elevated [Ca(2+)](i) in a way sensitive to the inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), U73122 (but not to pertussis toxin and forskolin), and independently from extracellular Ca(2+). PI-PLC-dependent Ca(2+) mobilization by ACEA was entirely accounted for by activation of inositol-1,3,4-phosphate (IP(3)) receptors on the endoplasmic reticulum (ER), whereas the effect of JWH133 was not sensitive to all tested inhibitors of IP(3) and ryanodine receptors. ACEA, but not JWH133, significantly inhibited the effect on [Ca(2+)](i) of bombesin, which acts via G(q/11)- and PI-PLC-coupled receptors in insulinoma cells. The endogenous CB(1) agonists, anandamide and N-arachidonoyldopamine, which also activate transient receptor potential vanilloid type 1 (TRPV1) receptors expressed in RIN m5F cells, elevated [Ca(2+)](i) in the presence of extracellular Ca(2+) in a way sensitive to both CB(1) and TRPV1 antagonists. These results suggest that, in RIN m5F cells, CB(1) receptors are coupled to PI-PLC-mediated mobilization of [Ca(2+)](i) and might inhibit bombesin signaling.     

Insights and perspectives on calcium channel functions in the cockpit of cancerous space invaders

Development of metastasis causes the most serious clinical consequences of cancer and is responsible for over 90 % of cancer-related deaths. Hence, a better understanding of the mechanisms that drive metastasis formation appears critical for drug development designed to prevent the spread of cancer and related mortality. Metastasis dissemination is a multistep process supported by the increased motility and invasiveness capacities of tumor cells. To succeed in overcoming the mechanical constraints imposed by the basement membrane and surrounding tissues, cancer cells reorganize their focal adhesions or extend acto-adhesive cellular protrusions, called invadosomes, that can both contact the extracellular matrix and tune its degradation through metalloprotease activity. Over the last decade, accumulating evidence has demonstrated that altered Ca²⁺ channel activities and/or expression promote tumor cell-specific phenotypic changes, such as exacerbated migration and invasion capacities, leading to metastasis formation. While several studies have addressed the molecular basis of Ca²⁺ channel-dependent cancer cell migration, we are still far from having a comprehensive vision of the Ca²⁺ channel-regulated mechanisms of migration/invasion. This is especially true regarding the specific context of invadosome-driven invasion. This review aims to provide an overview of the current evidence supporting a central role for Ca²⁺ channel-dependent signaling in the regulation of these dynamic degradative structures. It will present available data on the few Ca²⁺ channels that have been studied in that specific context and discuss some potential interesting actors that have not been fully explored yet.     

Localization of plasma membrane and secretory calcium pumps in the mammary gland

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.     

Salmonella enterica serovar Typhimurium-induced internalization and IL-8 expression in HeLa cells does not have a direct relationship with intracellular Ca levels

The invasion-associated type III secretion system (T3SS-1) of S. Typhimurium is required to initiate and sustain an acute inflammatory response in the intestine. We investigated the relationship of S. Typhimurium T3SS-1-induced IL-8 expression and invasion with intracellular Ca²⁺ mobilization in HeLa cells. Compared to the sipAsopABDE2 mutant, strains carrying a mutation in sipA, or mutations in sopABDE2 induced higher levels of IL-8 and greater bacterial internalization despite the fact that these mutants elicited similarly low intracellular concentrations of Ca²⁺. Likewise, complemented sipAsopABDE2 mutant with sopE2 did not affect intracellular Ca²⁺ concentrations or IL-8 expression, but significantly increased bacterial internalization. Treating HeLa cells with the calcium chelator BAPTA-AM or with D-BAPTA-AM, a derivative with greatly reduced Ca²⁺ chelating activity, yielded strong evidence that BAPTA-AM does not affect invasion and inhibits IL-8 secretion by a calcium-dependent mechanism. These findings suggest that, although wild-type S. Typhimurium-induced IL-8 expression and bacterial internalization in HeLa cells coincides with increased cytosolic Ca²⁺, the differing levels of IL-8 and invasion induced by strains carrying different effector proteins are unrelated to levels of intracellular Ca²⁺.     

Examination of the role of TRPM8 in human mast cell activation and its relevance to the etiology of cold-induced urticaria

Mast cells are considered the primary initiators of allergic diseases as a consequence of the release of multiple inflammatory mediators on activation. Although predominately activated through antigen-mediated aggregation of IgE-occupied-Fc?RI, they can also be induced to release mediators by other receptors and environmental stimuli. Based on studies conducted in the RBL 2H3 rodent mast cell line, the transient receptor potential melastatin 8 (TRPM8) cation channel has been implicated in the activation of mast cells in response to cold and, by inference, the development of urticaria. Here we investigated the expression and role of TRPM8 receptor, in both human and mouse non-transformed cells, with the aim of exploring the potential link between TRPM8 and the pathology of cold urticaria in humans. Although expressed in mouse mast cells, we found no evidence of TRPM8 expression in human mast cells or functional mutations in TRPM8 in cold urticaria patients. Furthermore, neither mouse nor human primary cultured mast cells degranulated in response to cold challenge or TRPM8 agonists and mast cell reactivity was unaffected in Trpm8^−/− mice. From these data, we conclude that TRPM8 is unlikely to directly regulate mast cell activation in cold urticaria. Thus, alternative mechanisms likely exist for the pathogenesis of this disease.     

Spotting the right location— imaging approaches to resolve the intracellular localization of invasive pathogens

Background

A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.

Scope of Review

Imaging has been instrumental to unravel paradigms of pathogen entry, to identify their exact intracellular location, and to understand the underlying mechanisms for the formation of pathogen-containing niches. Here, we provide an overview of imaging techniques that have been applied to monitor the intracellular lifestyle of pathogens, focusing mainly on bacteria that either remain in vacuolar-bound compartments or rupture the endocytic vacuole to escape into the host's cellular cytoplasm.

Major Conclusions

We will depict common molecular and cellular paradigms that are preferentially exploited by pathogens. A combination of electron microscopy, fluorescence microscopy, and time-lapse microscopy has been the driving force to reveal underlying cell biological processes. Furthermore, the development of highly sensitive and specific fluorescent sensor molecules has allowed for the identification of functional aspects of niche formation by intracellular pathogens.

General Significance

Currently, we are beginning to understand the sophistication of the invasion strategies used by bacterial pathogens during the infection process- innovative imaging has been a key ingredient for this.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Icilin evokes a dose- and time-dependent increase in glutamate within the dorsal striatum of rats

Summary. Icilin, the peripheral cold channel agonist, activates TRPM8 and TRPA1, localized on dorsal root ganglia and trigeminal neurons in rats. Icilin precipitates immediate wet-dog shakes in this species, which are antagonized by centrally acting mu and kappa opioid agonists, implicating the central nervous system in the behavioral response. We studied the effect icilin has on glutamate levels in the dorsal striatum, a brain region involved in movement. Icilin (0.25, 0.5 and 0.75 mg/kg, i.p.) elicited a dose- and time-dependent increase in glutamate within the striatum, indicative of icilin’s neurochemical effect in rats.     

Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting

Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

IL-18BP mediates the balance between protective and pathological immune responses to Toxoplasma gondii

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强筋与弱筋小麦籽粒蛋白质组分与加工品质对灌浆期弱光的响应

选用强筋小麦品种济麦20和弱筋小麦品种山农1391,在大田试验条件下,分别于籽粒灌浆前期(花后6—9 d)、中期(花后16—19 d)和后期(花后26—29 d)对小麦进行弱光照处理,研究了籽粒产量、蛋白质组分及加工品质的变化。灌浆期弱光显著降低小麦籽粒产量,灌浆中期对济麦20和灌浆后期对山农1391的产量降幅最大。弱光处理后,籽粒氮素积累量及氮素收获指数减少。但弱光使籽粒蛋白质含量显著升高,其中灌浆中期弱光升幅最大,原因可能是由于其粒重降低造成的。弱光对可溶性谷蛋白无显著影响,但增加不溶性谷蛋白含量,使谷蛋白聚合指数显著升高,面团形成时间和稳定时间亦升高,籽粒灌浆中、后期弱光对上述指标的影响较前期大。灌浆期短暂的弱光照对改善强筋小麦粉质仪参数有利,但使弱筋小麦变劣;并均伴随籽粒产量的显著降低这一不利影响。     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.