Günter Glassmeier

Activation of T Cell Calcium Influx by the Second Messenger ADP-ribose

Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (ICRAC). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.

Aromatase Inhibition Abolishes LTP Generation in Female But Not in Male Mice

Inhibitors of aromatase, the final enzyme of estradiol synthesis, are suspected of inducing memory deficits in women. In previous experiments, we found hippocampal spine synapse loss in female mice that had been treated with letrozole, a potent aromatase inhibitor. In this study, we therefore focused on the effects of letrozole on long-term potentiation (LTP), which is an electrophysiological parameter of memory and is known to induce spines, and on phosphorylation of cofilin, which stabilizes the spine cytoskeleton and is required for LTP in mice. In acute slices of letrozole-treated female mice with reduced estradiol serum concentrations, impairment of LTP started as early as after 6 h of treatment and progressed further, together with dephosphorylation of cofilin in the same slices. Theta-burst stimulation failed to induce LTP after 1 week of treatment. Impairment of LTP was followed by spine and spine synapse loss. The effects were confirmed in vitro by using hippocampal slice cultures of female mice. The sequence of effects in response to letrozole were similar in ovariectomized female and male mice, with, however, differences as to the degree of downregulation. Our data strongly suggest that impairment of LTP, followed by loss of mushroom spines and spine synapses in females, may have implications for memory deficits in women treated with letrozole.

HERG K(+) currents in human prolactin-secreting adenoma cells.

Abstract. To investigate the presence and possible function of ether-à-go-go-related gene (erg) K+ channels in human lactotroph cells (HERG channels), primary cultures were prepared from human prolactinoma tissue. In almost all primary cultures, HERG currents could be recorded in identified prolactin cells using an external high-K+ solution. The antiarrhythmic agent E-4031, a specific blocker of erg channels, served to isolate HERG currents as the drug-sensitive currents. In cells of two tumours tested, thyrotropin-releasing hormone significantly reduced the amplitude of the HERG currents. The potential dependence of HERG current availability and the deactivation kinetics differed significantly even between prolactin cells derived from one adenoma. For comparison, corresponding values were obtained for heterologously expressed rat erg1, erg2 and erg3 channels. The expression of the three HERG channel subunits was investigated in nine human adenomas using RT-PCR. Transcripts for HERG1 were present in all adenomas and although transcripts for HERG2 and HERG3 were also detected, their expression level was more variable. The results demonstrate the functional expression of HERG channels in human prolactin-secreting tumours and are compatible with a physiological role for these channels in the control of prolactin secretion, as has been shown in normal rat lactotroph cells.

Interaction of the human somatostatin receptor 3 with the multiple PDZ domain protein MUPP1 enables somatostatin to control permeability of epithelial tight junctions

MINT‐6800607, MINT‐6801122:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by anti bait coimmunoprecipitation (MI:0006)

Erg K+ channels modulate contractile activity in the bovine epididymal duct

The expression and functional role of ether-à-go-go-related gene (erg) K+ channels were examined in the bovine epididymal duct. Sperm transit through the epididymal duct relies on spontaneous phasic contractions (SC) of the peritubular smooth muscle wall. Isometric tension studies revealed SC-enhancing effects of the erg channel blockers E-4031, dofetilide, cisapride, and haloperidol and SC-suppressing effects of the activator NS-1643. In the corpus epididymidis, EC50 values of 32 nM and 8.3 microM were determined for E-4031 and NS-1643, respectively. E-4031 was also able to elicit contraction in epithelium-denuded corpus segments, which lacked SC. In the cauda region, E-4031 and NS-1643 exerted effects on agonist-induced contraction similar to those observed in the proximal duct. Experiments with nifedipine and thapsigargin suggested that the excitatory effects of E-4031 depended mainly on external calcium influx and not on intracellular calcium release. Western blot and RT-PCR assays revealed the expression of both, erg1a and erg1b, in all duct regions. Because erg1b appears to predominate in the epididymal duct, patch-clamp experiments were performed on heterologously expressed erg1b channels to investigate the sensitivity of this splice variant to NS-1643. In contrast to its effects on erg1a, NS-1643 induced a concentration-dependent current increase mainly due to a marked leftward shift in erg1b channel activation by approximately 30 mV at 10 microM, explaining the inhibitory effect of the drug on epididymal SC. In summary, these data provide strong evidence for a physiological role of erg1 channels in regulating epididymal motility patterns.

Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b

TRPM4b is a Ca2+‐activated, voltage‐dependent monovalent cation channel that has been shown to act as a negative regulator of Ca2+ entry and to be involved in the generation of oscillations of Ca2+ influx in Jurkat T‐lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca2+‐dependent inward and outward currents in whole cell patch clamp recordings. HEK‐293 cells stably overexpressing TRPM4b showed higher ionomycin‐activated Ca2+ influx than wild‐type cells. In addition, analysis of the membrane potential using the potentiometric dye bis‐(1,3‐dibutylbarbituric acid)‐trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca2+ entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca2+ influx. In conclusion, in cells with a small negative membrane potential, such as HEK‐293 cells, TRPM4b acts as a positive regulator of Ca2+ entry.

Strong Activation of ether-a`-go-go-Related Gene 1 K Channel Isoforms by NS1643 in Human Embryonic Kidney 293 and Chinese Hamster Ovary Cells

Two different mechanisms leading to increased current have been described for the small-molecule human ether-à-go-go-related gene (herg) activator NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea]. On herg1a channels expressed in Xenopus laevis oocytes, it mainly acts via attenuation of inactivation and for rat (r) erg1b channels expressed in human embryonic kidney (HEK)-293 cells, it strongly shifts the activation curve to the left. We now investigated the NS1643 effects on erg1b channels in more detail and performed comparative experiments with rat and human erg1a in different expression systems. Significant differences were observed between expression systems, but not between the rat and human isoform. In HEK-293 or Chinese hamster ovary (CHO) cells, activation of rat erg1b channels occurred in a dose-dependent manner with a maximum current increase of 300% obtained with 10 μM NS1643. In contrast, the NS1643-induced strong leftward shift in the voltage dependence of activation further increased with higher drug concentration, needed more time to develop, and exhibited use dependence. Coexpression of KCNE1 or KCNE2 did not attenuate this NS1643 effect on erg1 channel activation and did thus not mimic the lower drug potency on this parameter observed in oocytes. NS1643 (10 μM) slowed erg1b channel deactivation and recovery from inactivation without significant changes in activation and inactivation kinetics. With the exception of accelerated activation, NS1643 affected erg1a channels similarly, but the effect was less pronounced than in erg1b or erg1a/1b channels. It is noteworthy that rerg1b and herg1a inactivation estimated from fully activated current voltage relationships were unaltered in the continued presence of 10 μM NS1643 in the mammalian expression systems, indicating qualitative differences from NS1643 effects in X. laevis oocytes.

Neuronal differentiation by indomethacin and IBMX inhibits proliferation of small cell lung cancer cells in vitro

BACKGROUND Small cell lung cancer (SCLC) is one of the most aggressive malignancies implying a very poor prognosis for patients even under therapy. Since it is known that SCLC cells exhibit neurone-like characteristics, we investigated whether a neuronal induction medium (NID) consisting of indomethacin (200 μM), 3-isobutyl-1-methylxanthine (IBMX, 500 μM) and insulin (5 μg/ml) induces neuronal differentiation and by this reduces malignancy of SCLC in vitro. METHODS Anti-proliferative effects were tested by incubating five SCLC cell lines (OH1, OH3, SW2, H69 and H82) with NID for 72 h (XTT-assay). Afterwards, anti-proliferative as well as cytotoxic effects (lactate dehydrogenase [LDH] assay, electron microscopy) of a range of drug concentrations (indomethacin 6.25-800 μM, IBMX 15.625-2000 μM and combinations of both) regarding H82 and SW2 were analysed. We further investigated the presence of cyclooxygenase- (COX-) 1 and 2 (IHC, Western blot) as well as levels of COX-2 before and after treatment. Neuronal differentiation was evaluated by morphological analyses (electron microscopy), detection of CD 56 and CD 171 (FACS) and recording Na(+) and K(+) currents (patch clamp). RESULTS Proliferation of all cell lines was inhibited significantly in a dose dependent manner (linear regression), whereas SW2 and H82 were most sensitive. Treatment with insulin alone had no effect at all. Cytotoxic effects were only observed after incubation with high concentrations of indomethacin (H82) and combined treatment (SW2). COX-1 and 2 were detectable in H82 and SW2, whereas the level of COX-2 remained unaffected under treatment. By electron microscopy, we could not observe distinct neurone-like morphological changes after 72 h of treatment. However, the majority of H82 and SW2 cells expressed both CD 56 (NCAM) and CD 171 (L1), showing an increase of NCAM and L1 intensity at the cell surface after 7 and 14 days of treatment. We further demonstrated an up-regulation of neurone-specific Na(+) currents as well as a significant down-regulation of herg K(+) currents after NID treatment. CONCLUSION Our findings demonstrate significant anti-proliferative, non-toxic effects of indomethacin and IBMX on SCLC cells in vitro. Treated SCLC cells further possess increased neuronal characteristics in vitro, possibly leading to a reduced malignant potential.