BBA - Molecular Cell Research (v.1539, #3)

Palmitoylation of the canine histamine H2 receptor occurs at Cys305 and is important for cell surface targeting by Yasushi Fukushima; Toshihito Saitoh; Motonobu Anai; Takehide Ogihara; Kouichi Inukai; Makoto Funaki; Hideyuki Sakoda; Yukiko Onishi; Hiraku Ono; Midori Fujishiro; Takashi Ishikawa; Kuniaki Takata; Ryozo Nagai; Masao Omata; Tomoichiro Asano (181-191).
To determine the presence and functional role of the histamine H2 receptor (H2R) palmitoylation, a receptor with a Cys305 to Ala (A305 receptor) mutation was generated. Wild-type (WT) and A305 receptors were tagged at their N-termini with a hemagglutinin (HA) epitope. WT, but not A305, receptors incorporated [3H]palmitate by metabolic labeling, indicating that the H2R is palmitoylated at Cys305. Immunocytochemistry of WT and A305 receptors expressed in COS7 cells revealed WT receptors to be distributed at the plasma membrane, while the majority of A305 receptors were localized intracellularly with only a small portion being at the plasma membrane. However, the affinity of the A305 receptor for tiotidine was comparable to that of the WT receptor. In addition, when the amounts of cell surface receptors as determined by anti-HA antibody binding were equivalent, A305 receptors mediated production of more cAMP than WT receptors. Preincubation of COS7 cells expressing each receptor with 10−5 M histamine for 30 min reduced subsequent cAMP production in response to histamine via the receptors to similar extents, indicating that palmitoylation is not necessary for desensitization. In addition, cell surface A305 receptors were capable of being internalized from the cell surface at a rate and extent similar to those of WT receptors. Finally, CHO cell lines stably expressing either WT or A305 receptors were incubated with 10−5 M histamine for 1, 6, 12 and 24 h. Total amounts of WT and A305 receptors, as determined by tiotidine binding, were reduced by incubation, indicating downregulation. Downregulation of the A305 receptor was more extensive than that of the WT receptor. Thus, palmitoylation of the H2R might be important for targeting to the cell surface and stability.
Keywords: Histamine H2 receptor; Palmitoylation; Subcellular localization; Site-directed mutagenesis;

NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide–thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37°C) whereas the rate of NADH oxidation approximately doubled with each 10°C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.
Keywords: Hydroquinone (NADH) oxidase; Tumor associated hydroquinone (NADH) oxidase; Oxidation of NADH; Cell enlargement; Growth; Temperature compensation; Ubiquinone; HeLa cell; Coenzyme Q;

Block copolymers modify the internalization of micelle-incorporated probes into neural cells by Dusica Maysinger; Oksana Berezovska; Radoslav Savic; Patrick Lim Soo; Adi Eisenberg (205-217).
An important therapeutic concern is rate and extent of internalization of drugs into cells. Hydrophilic agents often internalize poorly and slowly, and highly lipophilic ones too rapidly. The incorporation of drugs into micelles allows regulation of their internalization parameters, and newly-described block copolymers can be selectively tailored to suit specific drugs. This report compares internalization of Cell Tracker CM-DiI (DiI), a highly lipophilic non-cytotoxic fluorescent probe in common use in biology, from the freely-presented (non-micelle-incorporated) and micelle-incorporated states. DiI was effectively incorporated (>60%) into 25–50 nm diameter spherical micelles made from polycaprolactone-b-polyethylene oxide block copolymer. Confocal microscopy was used to evaluate the internalization of DiI into mixed neuron–glia cultures (2–14 days in vitro, 2DIV–14DIV). Incorporation of DiI into micelles strikingly reduced the rate and extent of its internalization in both 2DIV and 14DIV cultures. Both the age of the cultures and the block copolymer employed to construct the micelles significantly influence the internalization of micelle-incorporated probe.
Keywords: Block copolymer micelle; DiI; Neuron; Glia; Confocal microscopy;

Colchicine inhibits taurodeoxycholate transport in pericentral but not in periportal hepatocytes by Ulrich Baumgartner; Peter Baier; Ulrich Schöffel; Eduard H Farthmann (218-224).
Indirect evidence for a microtubule-dependent vesicular hepatocellular transport of bile acids has accumulated. Since inhibition of this transport by colchicine can be achieved only at high but not at low bile acid infusion rates we were wondering whether this transport pathway shows a hepatic zonation or not. To answer this question we perfused isolated rat livers antegradely or retrogradely, respectively, with unlabeled and labeled taurocholate or taurodeoxycholate. Inhibition of microtubule-dependent bile acid transport was aimed at co-infusion of colchicine. Periportal cells eliminated the likewise hydrophobic taurodeoxycholate as fast as the more hydrophilic taurocholate. In contrast, pericentral cells excreted taurodeoxycholate much slower than taurocholate. Colchicine did not change the biliary taurocholate excretion profile in periportal and pericentral cells. However, colchicine reduced significantly taurodeoxycholate excretion in pericentral but not in periportal cells. It is concluded that a microtubule-dependent vesicular, colchicine-sensitive transport pathway seems to be involved in the translocation of taurodeoxycholate in pericentral but not in periportal cells. Since such a vesicular bile acid transport is regarded to be much slower than transcellular transport by diffusion, this observation may explain the much slower excretion of hydrophobic bile acids like taurodeoxycholate in pericentral than in periportal cells under physiological conditions.
Keywords: Bile acid transport; Liver heterogeneity; Liver perfusion; Microtubule-dependent bile acid transport; Colchicine-sensitive bile acid transport;

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein by Tomonori Fujiwara; Tetsuo Yamamori; Kimio Akagawa (225-232).
It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.
Keywords: HPC-1/syntaxin 1A; SNARE; HIV Tat;

Inhibition of the Na+/H+ antiporter suppresses IL-12 p40 production by mouse macrophages by Zoltán H. Németh; John G. Mabley; Edwin A. Deitch; Csaba Szabó; György Haskó (233-242).
The amiloride-inhibitable Na+/H+ antiporter plays an important role in macrophage activation. The intracellular pathways leading to interleukin (IL)-12 p40 production by activated macrophages are incompletely understood. In the present study, we examined the contribution of the Na+/H+ antiporter to the production of IL-12 p40. Amiloride or its analogs decreased the production of IL-12 p40 in macrophages stimulated with bacterial lipopolysaccharide and interferon-γ. The order of potency of amiloride analogs was consistent with the proposition that the effect of amiloride is mediated by the inhibition of the Na+/H+ antiporter. The effect of amiloride was post-transcriptional, as IL-12 p40 mRNA levels induced by lipopolysaccharide and interferon-γ were not affected by this inhibitor. Furthermore, the inhibitory effect of amiloride on IL-12 p40 production was not a result of interference with the activation of the p38 and p42/44 mitogen-activated protein kinases or c-Jun kinase. In summary, the production of IL-12 p40 requires a functional Na+/H+ antiporter.
Keywords: Cytokine; Monocyte; Autoimmune disease; Inflammation; Shock; T helper 1/T helper 2;

Expression of transient receptor potential mRNA isoforms and Ca2+ influx in differentiating human stem cells and platelets by Els den Dekker; Daniel G.M Molin; Githa Breikers; René van Oerle; Jan-Willem N Akkerman; Guillaume J.J.M van Eys; Johan W.M Heemskerk (243-255).
Store-regulated Ca2+ entry (SOCE) is an important mechanism of elevating cytosolic [Ca2+]i in platelets, though the Ca2+ influx channels involved are still unclear. We screened human platelets and their precursor cells (human stem cells and megakaryocytes) for the presence of candidate influx channels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34+) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61+/CD42blow) and mature (CD61+/CD42bhigh) megakaryocytes as well as platelets expressed in addition unspliced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet lineage, since immature (CD14+/CD61/CD42b) and mature monocytes expressed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A, 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell line, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem cells to mature megakaryocytes and platelets was accompanied by increased influx of extracellular Ca2+ after pretreatment of the cells with thapsigargin or thrombin. Expression of new Trp isoforms in the differentiated cells is thus accompanied by increased SOCE.
Keywords: Calcium channel; Megakaryocyte; Monocyte; Stem cell; Trp; Platelet;

Inhibition of phosphatidylserine synthesis in Jurkat T cells by hydrogen peroxide by Claudette Pelassy; Jean Philippe Breittmayer; Claude Aussel (256-264).
Incubation of Jurkat cells in the presence of H2O2 either directly added to the culture medium or generated with glucose oxidase, menadione or the couple xanthine/xanthine oxidase induced a marked decrease of phosphatidylserine synthesis in the absence of changes in the synthesis of phosphatidylcholine and phosphatidylethanolamine. Concentration dependent response curves indicated that H2O2 induced inhibition of phosphatidylserine synthesis with an IC50=5 μM while both induction of tyrosine phosphorylation of proteins and Ca2+ signals were obtained with an EC50=300 μM. The tyrosine kinase and Ca2+ independent mechanism was confirmed by comparing the H2O2-induced and the CD3-induced inhibition of phosphatidylserine synthesis using several Jurkat clones differing in the expression of cell surface receptors such as CD3/TCR and CD45 and protein tyrosine kinase such as p72syk, ZAP-70 and p56lck. While CD3-induced inhibition of phosphatidylserine synthesis necessitates protein tyrosine phosphorylation and Ca2+ signals, H2O2 provoked its effect in all the clones studied independently of the presence or absence of the proteins previously shown to be key elements in T cell signal transduction. Conversely, the antioxidant molecule, butylated hydroxanisole, generates an increased PtdSer synthesis, suggesting that the synthesis of this phospholipid is regulated by the redox status of the cells.
Keywords: T cell activation; Tyrosine protein kinase; Calcium; Phosphatidylserine;

Author Index (265-266).

Cumulative Contents (267-268).