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Molecules
2022 Mar 11;276:. doi: 10.3390/molecules27061846.
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Dihydropyridines Potentiate ATP-Induced Currents Mediated by the Full-Length Human P2X5 Receptor.
Schiller IC
,
Jacobson KA
,
Wen Z
,
Malisetty A
,
Schmalzing G
,
Markwardt F
.
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The P2X5 receptor, an ATP-gated cation channel, is believed to be involved in tumor development, inflammatory bone loss and inflammasome activation after bacterial infection. Therefore, it is a worthwhile pharmacological target to treat the corresponding diseases, especially in minority populations that have a gene variant coding for functional homotrimeric P2X5 channels. Here, we investigated the effects of dihydropyridines on the human full-length P2X5 receptor (hP2X5FL) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage clamp method. Agonist dependency, kinetics and permeation behavior, including Cl- permeability, were similar to hP2X5FL expressed in HEK293 or 1321N1 cells. Additionally, 1,4-dihydropyridines have been shown to interact with various other purinergic receptors, and we have examined them as potential hP2X5 modulators. Of seven commercially available and four newly synthesized dihydropyridines tested at hP2X5FL, only amlodipine exerted an inhibitory effect, but only at a high concentration of 300 µM. Isradipine and-even more-nimodipine stimulated ATP-induced currents in the low micromolar range. We conclude that common dihydropyridines or four new derivatives of amlodipine are not suitable as hP2X5 antagonists, but amlodipine might serve as a lead for future synthesis to increase its affinity. Furthermore, a side effect of nimodipine therapy could be a stimulatory effect on inflammatory processes.
Figure 1. Examples of hP2X5FL-mediated current traces in X. laevis oocytes elicited by ATP. ATP was applied for 6 s (A) or 3 s (B) at the indicated ATP concentration and for the duration indicated by the horizontal line. In (B), a 2 min washout period was inserted between the first and second ATP applications.
Figure 2. Agonist dependence of hP2X5FL-mediated ion currents in X. leavis oocytes. (A) Examples of current traces elicited consecutively by ATP or BzATP as indicated in the same hP2X5FL-expressing oocyte. (B) Concentration dependence of currents evoked by the indicated agonist. Iact,rel,0.01ATP was calculated according to Equation (1) and the approximation was performed using Equation (2). Data are means ± SEM from of 4–12 oocytes.
Figure 3. Permeation behavior of oocyte-expressed hP2X5FL ion channels. Time course of ramp currents during voltage ramps applied every second before and during application of 0.01 mM ATP for the time indicated by the horizontal bar before (A) and after (B) subtraction of the resting ion currents. The bathing solution was a NaCl-based Ringer’s solution. (C) Current ramp from a section of B as indicated. The red straight line is the linear fit of the current around the reversal potential. (D) Voltage ramp protocol. (E–H) Examples of ramp currents induced by 0.01 mM ATP in bathing solutions with the indicated main extracellular ions. The ramp current recorded before the ATP application was subtracted. The red lines indicate the linear approximation of the current–voltage dependence near I = 0, from which the slope conductance G and the reversal potential Vrev are obtained as indicated. Dependence of G (I) and Vrev (J,K) on the extracellular ions of hP2X5FL (I,J) and hP2X7 (K) expressing oocytes. All mean values are significantly different except the reversal potentials in NaCl and Na glutamate solution for hP2X7 expressing oocytes. Means from 13–37 oocytes.
Figure 4. Voltage dependence of oocyte-expressed hP2X5FL ion channels. Characteristic example of hP2X5FL-dependent current evoked by 0.01 mM ATP for the time indicated by the horizontal bar at a membrane potential of −80 mV (A) or +40 mV (B), respectively. The exponential fits of the deactivation time courses according to eq. 3 are plotted as red lines, with the determined rate constants as indicated. (C) Statistics of the effect of the membrane potential on the deactivation rate constant. All mean values are significantly different from each other. Means are from 9–19 oocytes.
Figure 5. Effect of different dihydropyridines and the P2X7-specific blocker A438079 on hP2X5FL-mediated currents. Examples of the inhibitory effect of amlodipine (A) the effect of nifedipine (B) and the stimulatory effect of isradipine (C) on ATP-induced currents. Currents were evoked by ATP in bathing solution and 2 min later in bathing solution supplemented with the indicated dihydropyridine derivative. (D) Summary of the effects of A438079 and various dihydropyridines on ATP-induced currents. Means are normalized to the mean value of the currents measured during the second ATP application without additional substances (control). Means of 9–50 oocytes. Values that differ significantly from the ATP-only control are marked by asterisks.
Figure 6. Concentration-dependent effect of nimodipine on hP2X5FL-mediated currents. (A) Example current traces consecutively evoked by ATP and ATP + nimodipine. (B) Concentration dependence of the effect of nimodipine on ATP-elicited hP2X5FL-mediated currents. Means are from of 6–40 oocytes. The structure of nimodipine is shown in the inset.
Figure 7. Effects of amlodipine-derived compounds on hP2X5FL-mediated currents. (A) Structures of amlodipine and four newly synthesized DHPs derived from amlodipine. (B) Statistics of the effect of the MRS compounds shown in A. Means are from of 7–9 oocytes.
Figure 8. Synthesis of new dihydropyridine derivatives. (A) Preparation of amide derivatives 1 and 2 of amlodipine. Reagents and conditions: (a) acetyl chloride, pyridine, 0 °C—rt., overnight, 44%; (b) phenylacetyl chloride, TEA, CH2Cl2, 0 °C—rt, overnight, 93%. (B) Preparation of N-benzylated derivatives of amlodipine. Reagents and conditions: (a) benzyl bromide, K2CO3, reflux, 3.5 h, 37% 3 and 39% 4.
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