Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: Analyses of ASIC1 and ASIC3 expression in primary cultured human NP-MSCs. ( A ) Western blotting analyses of ASIC1 and ASIC3 expression when exposed to extracellular acid (pH6.6) with or without treatment of inhibitor Amiloride, PcTx1 or APETx2. ( B ) The relative expression of ASIC1 and ASIC3 using densitometric analysis of specific bands revealed by Western blotting as per ( A ). The samples were from case 4. Data are mean ± SD. With * indicating P≤0.05.

Article Snippet: As indicated, 100 μM Amiloride (Sigma), 20 nM PcTx1 (MCE) or 100 nM APETx2 (Abcam) was added, respectively.

Techniques: Expressing, Cell Culture, Western Blot

Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: The effect of acidic culture conditions on the proliferation of human NP-MSCs. The proliferation ability of human NP-MSCs derived from patients with Pfirrmann grade II ( A ), III ( B ) and IV ( C ) degeneration was determined using CCK-8 assays under control conditions or after exposure to extracellular acid (pH6.6) alone and in combination with addition of the inhibitors Amiloride, PcTx1 or APETx2. n=3 for each group; data are mean ± SD.

Article Snippet: As indicated, 100 μM Amiloride (Sigma), 20 nM PcTx1 (MCE) or 100 nM APETx2 (Abcam) was added, respectively.

Techniques: Derivative Assay, CCK-8 Assay, Control

Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: Acidic culture conditions inhibit cell cycle progression in human NP-MSCs which can be reversed using ASICs inhibitors. Flow cytometric DNA content profiles comparing cultured NP-MSCs derived from Pfirrmann grade II ( A ) and IV ( B ) cases cultured under control or acidic pH conditions in combination with or without treatment using Amiloride, PcTx1, APETx2, respectively. G0/G1, S and G2/M cell cycle phases were estimated as % totals using the ModFit software. The samples in ( A ) were from case 6 and that in ( B ) from case 11.

Article Snippet: As indicated, 100 μM Amiloride (Sigma), 20 nM PcTx1 (MCE) or 100 nM APETx2 (Abcam) was added, respectively.

Techniques: Cell Culture, Derivative Assay, Control, Software

Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: The induction of cellular senescence in human NP-MSCs exposed to acidic conditions can be reversed by ASICs inhibition. ( A ) Representative images (left) and the calculated percentage (right) of cultured Pfirrmann grade IV NP-MSCs undergoing senescence determined using SA-β-gal staining after exposure to acidic pH 6.6 conditions for 0, 24, 72 and 120 hours. n=3; data are mean ± SD, * P≤0.05 compared to 0 h group. ( B ) Comparative rates of senescence determined as per ( A ) in NP-MSCs derived from patients with Pfirrmann III and IV grades IVDD cultured under control or acidic pH conditions (pH 6.6) for 72 h. n=3; data are mean ± SD, * P≤0.05 compared to corresponding control group. ( C ) Effects of ASIC inhibitors on senescence induction in Pfirrmann grade II NP-MSCs cultured in control versus pH 6.6 for 120 h with or without treatment using Amiloride, PcTx1, APETx2 as indicated. The samples in ( A ) were from case 10, that in ( B ) of Pfirrmann III and IV group from case 7 and 11, respectively, and that in ( C ) from case 6. n=3; data are mean ±SD, * indicates P≤0.05 compared to control group; # indicates P≤0.05 compared to pH 6.6 group.

Article Snippet: As indicated, 100 μM Amiloride (Sigma), 20 nM PcTx1 (MCE) or 100 nM APETx2 (Abcam) was added, respectively.

Techniques: Inhibition, Cell Culture, Staining, Derivative Assay, Control

Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: The production of secreted SASP cytokines by human NP-MSCs is increased by acidic culture and partially reversed by ASICs inhibition. ELISA assays measuring secreted IL-6 ( A ) and IL-8 ( B ) by human NP-MSCs after culture in control or acidic medium (pH 6.6) with or without treatment using the inhibitors Amiloride, PcTx1 or APETx2 as indicated. n=3; data are mean ±SD, * indicating P≤0.01 compared to control group at the same time points; # indicating P≤0.01 compared to pH 6.6 group at the same time points. The samples were from case 8.

Article Snippet: As indicated, 100 μM Amiloride (Sigma), 20 nM PcTx1 (MCE) or 100 nM APETx2 (Abcam) was added, respectively.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Control

Journal: Aging (Albany NY)

Article Title: ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration

doi: 10.18632/aging.202850

Figure Lengend Snippet: The effect of acidic culture conditions on senescence-related protein expression in primary cultured human NP-MSCs. Western blotting analyses were performed against Rb1, p53, p27, p21, p16, Aggrecan and Collagen II, MMP9 and MMP3 expression in Pfirrmann grade III NP-MSCs exposed to pH 6.6 culture conditions with or without treatment using the inhibitors Amiloride, PcTx1 or APETx2, respectively. The samples were from case 5, 9 and 12, respectively.

Article Snippet: As indicated, 100 μM Amiloride (Sigma), 20 nM PcTx1 (MCE) or 100 nM APETx2 (Abcam) was added, respectively.

Techniques: Expressing, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3

doi: 10.1101/704445

Figure Lengend Snippet: (A) Example traces of the effect for the 5 different selected drugs (500 µM) on pH 7-induced rASIC3 activation together with example trace showing the effect of the prototypic rASIC3 modulator GMQ (500 µM). In all cases drugs were applied at pH 7. (B-C) Bar plots showing the effect of the selected drugs and GMQ on transient (peak) (B) and the sustained current (expressed as the ratio I 5s /I peak ) (C). (D) Example traces of the effect for the 5 different selected drugs (500 µM) on pH 6-induced rASIC3 activation together with example traces showing the effect of known rASIC3 modulators, APETx2 (1 µM) and GMQ (500 µM). In all cases drugs were applied for 30 s prior to pH 6-induced rASIC3 activation. (B-D) Bar plots showing the effect of the selected drugs, APETx2 and GMQ on transient (peak) (B) and sustained current (C), and inactivation time constant (D) of rASIC3 activation. All values were normalised to baseline pH 7 or pH 6 rASIC3 activation and expressed as means ± SEM (n = 6-11, paired t-test, *p <0.05 and ***p ≤ 0.005 vs baseline pH 7 or pH 6 activation).

Article Snippet: All the small molecules used in this study were purchased from Sigma (Gilliangham, UK) except for tizanidine (Tocris, Bristol, UK) and APETx2 (Smartox, Saint egrève, France).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Fluid-pulse stimulation (20 p.s.i., 80 ms) elicited action potential responses in control conditions (black) in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). Application of the ASIC3 blocker APETx2 (2 μM) abolished responses (blue), which partially recovered upon washout (green). ( b ) Raster plot showing reliable responses to pressure stimulations (20 p.s.i., 80 ms) in control, and which were completely abolished by application of APETx2. Responses reappeared upon washout. ( c ) The receptor potential elicited by fluid pulses (20 p.s.i., 60 ms) was also blocked by APETx2 in the presence of TTX (1.5 μM). ( d ) Complete blockade of responses after application of APETx2 ( n =6). Means±s.e.m.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques: Control

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) CSF-c neuron showing mechanosensitivity to a fluid pressure pulse (80 ms, 20 p.s.i.). ( b , c ) In the same neuron, the action potential frequency increased at pH 6.9 in the presence of gabazine (20 μM), AP5 (100 μM) and NBQX (40 μM). ( d ) Application of APETx2 (2 μM) abolished the response to fluid-pulse stimulation. ( e , f ) APETx2 also abolished the response to pH lowering. ( g ) Firing of CSF-c neurons before and after application of APETx2. The values are means±s.e.m. during 1 min of recording, normalized to basal activity at pH 7.4 ( n =5). Student's t -test: *** P <0.001, significant difference at pH 6.9 compared with 7.4 only in control conditions in the absence of APETx2. ( h ) Decreases in pH resulted in depolarizing potentials also in the presence of TTX (1.5 μM), that reversed at positive holding potentials. ( i ) Addition of APETx2 (1 μM) completely blocked the response to lowered pH in the presence of TTX (1.5 μM).

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques: Activity Assay, Control

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) The amplitude of the receptor current increases at hyperpolarised holding potentials and decreases upon depolarization in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM). ( b ) The receptor current reversed at ∼+25 mV ( n =5). ( c ) The receptor current evoked by fluid pulse was eliminated in the presence of the ASIC3 blocker APETx2 (1 μM) and returned after washout. The receptor currents in a – c were elicited by a fluid pulse of 60 ms, 20 p.s.i. ( n =6). ( d ) No current events were seen at pH 7.4 in the presence of gabazine (20 μM), AP5 (100 μM), NBQX (40 μM) and TTX (1.5 μM). After a decrease in extracellular pH to 6.5, inward current deflections appeared that decreased in amplitude and frequency at more depolarized holding potentials and were reversed in sign at +35 mV ( n =5). ( e ) At pH 6.5 discrete current deflections were recorded, which may correspond to single-channel openings. ( f ) The inward currents recorded at pH 6.5 were completely blocked in the presence of APETx2 (1 μM; n =3). The data in b are represented as means±s.e.m.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques:

Journal: Nature Communications

Article Title: Ciliated neurons lining the central canal sense both fluid movement and pH through ASIC3

doi: 10.1038/ncomms10002

Figure Lengend Snippet: ( a ) Illustration of arrangement for ventral root recordings with suction electrodes in the intact, isolated spinal cord preparation (VR-L, VR-R, left and right side ventral root, respectively). ( b ) Bilateral ventral root recording during NMDA (100 μM)-induced fictive locomotion in the isolated lamprey spinal cord, during control conditions (pH 7.4; black traces) and during lowered pH of 6.9 (red traces) and 6.5 (orange traces). ( c ) Decreases in extracellular pH prolonged the cycle period. The mean period was determined during 20 cycles for each of the conditions and normalized to the value during control conditions (% of control; n =7). ( d ) Bilateral ventral root recording during control conditions (pH 7.4; black traces), during pH 6.9 (red traces) and in the presence of the ASIC3 blocker APETx2 (1 μM; blue traces). ( e ) Application of APETx2 blocked the effect of lowered extracellular pH (6.9) on the cycle period, which recovered upon washout (mean values calculated for 20 cycles during each condition; n =2 preparations). ( f ) Effect of somatostatin (10 nM, 100 nM and 1μΜ) on the cycle period of the locomotor activity. Somatostatin significantly increased the period at all tested concentrations ( n =19). ( g ) Bilateral ventral root recording in the isolated spinal cord, during control conditions (pH 7.4; black traces), during pH 6.5 (orange traces) and following application of the somatostatin receptor sst 2 antagonist CYN-154806 (2 μM; green traces). ( h ) Application of CYN-154806 lead to a shortening of the period length at control pH 7.4 (dark green; n =4). In the presence of the antagonist, a decrease of pH (here to 6.5; light green) had no effect on the cycle period. The data are represented as means±s.e.m. ( c , f,h ) and ±s.d. ( e ); Student's t -test: *** P <0.001; ** P <0.01, significant difference compared with control; NS: non-significant, NS P >0.5.

Article Snippet: The following drugs were added to the extracellular solution and applied by bath perfusion: the specific ASCI3 blocker APETx2 (1–2 μM, Merck Chemicals Ltd., Nottingham, UK), the GABA A receptor antagonist gabazine (20 μM, Tocris, Ellisville, MO, USA), the NMDA receptor antagonist AP5 (100 μM, Tocris), the AMPA receptor antagonist NBQX (40 μM, Tocris) and TTX (1.5 μM; Sigma).

Techniques: Isolation, Control, Activity Assay