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    • Fig summarizes the results for M concentration of

    2022-01-17

    Fig. 1 summarizes the results for 500 μM concentration of the drugs. Action of histamine is presented for comparison. Both 1-methylhistamine and Nα-methylhistamine strongly potentiated ASIC1a responses. The concentration-dependence of action of Nα-methylhistamine was measured (Fig. 1B). Fitting by Hill equation suggests that Nα-methylhistamine has larger efficacy than histamine has maximal potentiation reaches 340% the EC50 values were estimated as 350 ± 90 μM. At 1 mM concentration both 1-methylhistamine and Nα-methylhistamine were significantly more potent than histamine (paired t-test, n = 7, P < 0.05). Dimaprit also potentiated ASIC1a but with lesser efficiency when compared with histamine: 500 μM of the drug caused 54 ± 9% (n = 6) potentiation. Concentration-dependence of dimaprit action revealed maximal effect of 92 ± 50% (n = 6) at 1 mM, yet the effect did not reach the saturation. Imetit demonstrated the same tendency, but the potentiating effect was not statistically significant (36 ± 32%, n = 7, P > 0.1). Thus, it appears that both the amino group and imidazole ring of histamine are essential for potentiating action and replacement of these groups by the isothiourea moiety reduces the effect. Diphenhydramine and tripelenamine were found to be weak potentiators of ASIC1a - they caused less than 50% potentiation in the concentration 500 μM. Thioperamide, while applied in the same conditions resulted in 78 ± 72% (n = 12) potentiation, however, it demonstrated large cell-to-cells variations making more detailed analysis impractical. In PF-573228 sale to the other drugs tested, A943931 caused inhibition of ASIC1a-mediated currents by 54 ± 14%. Analysis of the concentration dependence of inhibitory action suggested IC50 of 665 ± 236 μM (n = 7). In the previous structure-activity study [10] we proposed that action of amines on ASICs can be mediated by at least two independent mechanisms. To analyze mechanisms of action of ASIC1a potentiators and inhibitors we compared voltage- and pH-dependence of effects of A943931 and dimaprit (Fig. 2). Potentiating effect of dimaprit was found voltage-independent, potentiation at −40 mV or −140 mV did not differ significantly from the action at −80 mV (n = 6, one-way ANOVA, P = 0.195). In contrast, inhibition by A943931 demonstrated significant voltage-dependence, depolarization to −40 mV caused decrease of the effect to 41 ± 15% (n = 5) and hyperpolarization to −140 mV enhanced the inhibition to 74 ± 9% (n = 5). The values at −40 mV and at −140 mV were significantly different (n = 5, one-way ANOVA, P < 0.05). Dimaprit and A943931 also demonstrated drastically different dependence on the value of activating pH. Activation of ASIC1a by saturating pH 5.0 nearly abolished potentiating effect of dimaprit 9 ± 6% (n = 6) but did not affect inhibitory action of A943931 53 ± 29% (n = 6). Thus, mechanism of ASIC1a potentiation by dimaprit is likely the same as demonstrated by histamine [11] and by other amines potentiating ASICs [9]. It can affect activation of the channel by direct donation of protons to the proton-sensitive site or by an allosteric mechanism. Mechanism of inhibition by A943931 is obviously different. Lack of pH-dependence and pronounced voltage-dependence suggest that it blocks the ion conducting pore. Action of histamine receptor ligands on ASIC2a. Due to low sensitivity of ASIC2a to protons, these receptors were activated by pH drops from 7.4 to 5.0. Also, ASIC2a desensitize slower than ASIC1a, hence the activating applications of acidic solutions were 40 s in duration. Some monoamines potentiate ASIC2a under these conditions [9] but histamine did not show significant effect [11]. In the present work application of 500 μM of the histamine receptor ligands caused only minor effects from 23 ± 16% (n = 5) potentiation by thioperamide to 19 ± 10% (n = 4) inhibition by 1-methylhistamine (Fig. 3). Surprisingly, A943931 was also inactive against ASIC2a. Taking into account the voltage-dependence of its action on ASIC1a, we repeated the experiments at −140 and −40 mV holding voltage. However, even at hyperpolarized voltages the effect was not statistically significant (n = 5, P > 0.1). Increase of imetit and dimaprit concentration to 1 mM also did not reveal significant effect while potentiation by thioperamide increased to 38 ± 10% (n = 5). All these drugs were inactive at lower concentrations (Fig. 3B).