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  • The finding of histamine action on


    The finding of histamine action on ASICs also raises the question about possible action of other histamine receptor ligands. In the present work we selected several compounds and performed electrophysiological testing of their action on recombinant homomeric ASIC1a and ASIC2a. Nα-methylhistamine is a H3R agonist [14]. This compound together with stable histamine metabolite 1-methylhistamine [15] represents minor modifications of the histamine structure. The H3R agonist imetit and H2R agonist dimaprit [16] were selected to test how replacement of the amino group and imidazole ring by the isothiourea moiety affects the action on ASICs. The inverse agonist of H1R diphenhydramine and antagonist of H1R tripelenamine were selected because of their structural similarity with recently found ASIC ligand IEM-2044. In contrast, H3R antagonist thioperamide [17] and H4R antagonist A943931 [18] were selected to test if these distinct structures can affect ASICs. Thus, the series of drugs tested in the present study includes structurally different compounds that exhibit different selectivity for histamine receptors.
    Methods ASIC1a and ASIC2a homomers were expressed in Chinese hamster ovary (CHO) cells. CHO cells were cultured at 37 °C in a humidified Fructose of 5% CO2. Cells were maintained with standard culture conditions (Dulbecco's modified Eagle's medium DMEM plus 10% fetal bovine serum and 5% gentamycin). Plasmids encoding ASIC subunits were transfected using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer's protocol. Expression vectors encoding rat ASIC1a and ASIC2a were a kind gift from Dr. A. Staruschenko. For the expression of the channels, cells were transfected with 0.5 μg rASIC1a or rASIC2a cDNA per 35 mm2 dish together with 0.5 μg GFP. Electrophysiological experiments were performed 48–72 h after transfection. Transfected cells were identified by green fluorescence using the Leica DMIL LED microscope. Whole cell currents were recorded using patch clamp technique at holding potential of −80 mV. For this purposes EPC-8 amplifier (HEKA Electronics, Lambrecht, Germany) was used. The signal was filtered in the frequency band of 0–5 kHz, digitized at the sampling rate of 1 kHz and recorded on a personal computer using the Patchmaster software. Series resistance of about 10MΩ was compensated by 70–80% and monitored during the experiments. Only recordings where access resistance and capacitance changed less than 10% were used. All experiments were performed at room temperature (23–25 °C). The pipette solution contained (in mM):100CsF, 40CsCl, 5NaCl, 0.5CaCl2, 5EGTA and 10HEPES adjusted to 7.2 by adding CsOH. Cells were continuously perfused with an extracellular solution contained the following (in mM):143NaCl, 5KCl, 2.5CaCl2, 2 MgCl2, 18D-glucose, 10HEPES and 10MES adjusted to 7.4 by adding NaOH. Test solutions were prepared from this extracellular solution by addition of the drugs studied and final adjusting of pH by to the required value. All solutions were filtered through micropore cellulose membranes using a vacuum glass filter (Sartorius AG, Germany). For fast drug application the ALA-VM8 micromanifold system (ALA Scientific Instruments, USA) was used. Compounds were purchased from Sigma, Tocris and Abcam. All data are presented as a “mean ± standard deviation” calculated from at least five experiments. The statistical significance of the effects was evaluated using the paired t-test with P < 0.05 (the value of the response amplitude in the presence of a compound relative to the control).
    Results Action of histamine receptor ligands on ASIC1a. Recombinant homomeric ASIC1a were activated by 20 s pH drops from 7.4 to 6.5. Interval between activations was 30 s to ensure full recovery from desensitization. After recording of at least three control responses both neutral and activating solutions were replaced by the ligand-containing solutions, so the ligand perfused the cell continuously. At least three responses were recorded in the presence of the ligand to prove the stability of the effect. Next, the ligand was washed out and another series of responses was recorded to demonstrate the effect reversibility. In the case of a response rundown during the experiment, the drug effect was measured relative to the averaged response amplitude of control series and the responses after wash out.