Application of NSC-156529 as T-type calcium channel antagonist
A calcium channel antagonist and calcium channel technology, applied in the field of NSC-156529 as a T-type calcium channel antagonist, can solve the problems of unrevealed and undisclosed analgesic effects of NSC-156529 compounds
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Embodiment 1
[0029] Example 1: NSC-156529 can dose-dependently inhibit T-type calcium channel current in TG neurons
[0030] In this embodiment, the selected experimental materials are as follows:
[0031] ICR mice (18-22g) were provided by the Animal Center of Soochow University, animal certificate number: SYXK (Su) 2007-0035. NSC-156529 and electrophysiological reagents were purchased from Sigma.
[0032] The specific experimental plan is as follows:
[0033] Bilateral trigeminal ganglion (TG) of mice was collected by microsurgery, TG neurons were isolated acutely by enzymatic digestion, and the T-type calcium channel currents of small-diameter TG neurons were recorded by whole-cell patch clamp, and each group recorded 7- 9 cells.
[0034] When recording the T-type calcium channel current, the cell was clamped at -110mV, stimulated with a square wave to -40mV, and the stimulation time course was 40ms. The inner liquid of the recording electrode is: 110mM CsCl, 4mM MgATP, 0.3mM Na 2 ...
Embodiment 2
[0039] Example 2: NSC-156529 inhibits T-type calcium channel current independent of G protein and does not pass through the PKB / Akt pathway In this example, the selected experimental materials are as follows:
[0040] The G protein blocker GDP-β-S and the Akt kinase inhibitor Akt inhibitor III used in the experiment were purchased from Sigma, USA.
[0041] The specific experimental plan is as follows:
[0042] The method of acute cell separation is the same as above, the method of whole-cell patch clamp recording of T-type calcium channels and the components of the internal and external fluid of the electrodes are as described above. GDP-β-S and Akt inhibitor III block the signaling pathways mediated by G-protein and PDK1 / Akt, respectively, to detect whether NSC-156529 inhibits T-type calcium channels through the above pathways. Such as figure 2 As shown in A, 2C and 2B, 2D, the G-protein inhibitor GDP-β-S (1mM, figure 2 A, 2C) and Akt inhibitor III (10μM, figure 2 B, 2...
Embodiment 3
[0043] Example 3: NSC-156529 inhibits T-type calcium channel Cav3.1 and Cav3.2 subtypes, but has no effect on Cav3.3
[0044] In this embodiment, the selected experimental materials are as follows:
[0045] HEK293 cells were purchased from ATCC, USA; NSC-156529, transfection reagents and electrophysiological reagents were all purchased from Sigma.
[0046] The specific experimental plan is as follows:
[0047] Take a bottle of cultured cells, discard the culture medium, add 3ml of phosphate buffer saline to wash lightly twice, add about 1ml of 0.25% trypsin to digest, add DMEM culture medium after about 3-5 minutes, blow and beat, collect cells and centrifuge (1000 Turn / min) for 5 minutes, discard the supernatant, add about 5-10ml DMEM culture medium to suspend the cells. After slow blowing, passage to a 35mm petri dish covered with glass slides, and transfect after 24 hours. The classic calcium phosphate transfection method was used for cell transfection (transfection of C...
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