Journal: American Journal of Physiology - Renal Physiology

Article Title: Dynamic coupling between TRPV4 and Ca 2+ -activated SK1/3 and IK1 K + channels plays a critical role in regulating the K + -secretory BK channel in kidney collecting duct cells

doi: 10.1152/ajprenal.00037.2017

Figure Lengend Snippet: SK1/3 and IK1 KCa play a dominant role in controlling [Ca2+]i rise and BK channel activation in response to TRPV4 activation. All studies were done on a paired basis where a coverslip of mCCDcl1 cells grown to confluency was split in half with 1 part used for control (untreated) and the other part treated with apamin + TRAM-34. A: example showing GSK101-evoked [Ca2+]i increase through activation of TRPV4 in control cells, with the expected IbTX-sensitive component indicating BK activation. B: example showing that prior blocking of SK1/3 (apamin) and IK1 (TRAM-34) channels markedly reduced the GSK101-induced [Ca2+]i increase and the IbTX-sensitive component reflecting BK activity was nearly abolished. C: summary analysis showing that in the presence of apamin and TRAM-34, the GSK101-induced peak [Ca2+]i, or the observed maximum [Ca2+]i, was reduced from control values of 766 ± 25 nM (n = 30) to 348 ± 40 nM (n = 30) in the presence of apamin and TRAM-34. D: the initial rate of GSK101-induced [Ca2+]i rise was also markedly reduced in the presence of apamin and TRAM-34, as shown by the dashed line in A and B, being reduced from 215 ± 14 nmol·l−1·min−1 (n = 30) in the control to only 31 ± 6 nmol·l−1·min−1 with apamin + TRAM-34 treatment (n = 30). E: summary data showing that the IbTX-induced reduction in [Ca2+]i in GSK101-treated cells (−Δ[Ca2+]i)was significantly reduced from 384 ± 29 to 90 ± 27 nM (n = 30) in the presence of apamin and TRAM-34 (n = 30). ***P < 0.001.

Article Snippet: The chemicals and drugs used in the study were GSK101 (GSK1016790A; Santa Cruz Biotechnology) from a 1 mM stock solution in DMSO, apamin (Alomone) from a 1 mM stock solution in PBS, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; Tocris) from a 10 mM stock solution in DMSO, and iberiotoxin (IbTX; Alomone) from a 0.1 mM stock solution in PBS.

Techniques: Activation Assay, Control, Blocking Assay, Activity Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Dynamic coupling between TRPV4 and Ca 2+ -activated SK1/3 and IK1 K + channels plays a critical role in regulating the K + -secretory BK channel in kidney collecting duct cells

doi: 10.1152/ajprenal.00037.2017

Figure Lengend Snippet: The initial [Ca2+]i attained by TRPV4 activation is directly correlated with BK activation. A: pooled data from individual cells showing that the IbTX-sensitive [Ca2+]i, −Δ[Ca2+]i due to addition of IbTX, was directly proportional to the Initial [Ca2+]i (pseudoplateau) following TRPV4 activation (6 nM GSK101) in the absence of IbTX. Linear regression (LR) analysis showed a strong correlation (r = 0.8494, n = 72; P < 0.001), which, on extrapolation of the linear regression line (see figure), yielded an x-intercept value of 197 nM [Ca2+]i, the Initial [Ca2+]i level where the IbTX response would largely be abolished. B: blocking SK1/3 and IK1 channels by Apamin + TRAM-34 suppressed both the TRPV4-induced Initial [Ca2+]i levels and −Δ[Ca2+]i induced by IbTX. Limiting the regression analysis to Initial [Ca2+]i data points greater than the 197 nM x-intercept point defined in A, i.e., to non-0 Δ[Ca2+]i points, again displays a strong correlation (r = 0.9150, n = 26; P < 0.001) with the LR line defined as: y = 0.6726x − 113.4 (dashed line). Numerous data points also now appear to cluster at the lower Initial [Ca2+]i levels where the IbTX-sensitive −Δ[Ca2+]i for data points with an Initial [Ca2+]i of below ~200 nM were largely abolished. The solid line is the LR line (Control) from the control data defined in A. C: pooled control data comparing the Initial [Ca2+]i levels and IbTX-sensitive components from control studies of A (Control; GSK101 = 6 nM, extracellular Ca2+ = 1.0 mM) with results obtained where the Initial [Ca2+]i level was reduced (to mimic the case with Apamin + TRAM-34 in B) by employing low TRPV4 agonist levels (GSK101 = 3 nM) or exposing cells to low extracellular Ca2+ levels (0.1 mM; Control-Ex). The combined data sets again show a strong correlation between the Initial [Ca2+]i level and the IbTX-sensitive [Ca2+]i (r = 0.9139, n = 120) but where the results with the low Initial [Ca2+]i values (△) cluster near the low end of the scale, where the IbTX-sensitive component is largely abolished, just like for the case with Apamin + TRAM-34. The solid line is the linear regression line from the control data of A. D: superimposing the apamin + TRAM-34 data of B onto the control data of C shows that the same correlation holds true in the absence and presence of apamin and TRAM-34.

Article Snippet: The chemicals and drugs used in the study were GSK101 (GSK1016790A; Santa Cruz Biotechnology) from a 1 mM stock solution in DMSO, apamin (Alomone) from a 1 mM stock solution in PBS, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; Tocris) from a 10 mM stock solution in DMSO, and iberiotoxin (IbTX; Alomone) from a 0.1 mM stock solution in PBS.

Techniques: Activation Assay, Blocking Assay, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Dynamic coupling between TRPV4 and Ca 2+ -activated SK1/3 and IK1 K + channels plays a critical role in regulating the K + -secretory BK channel in kidney collecting duct cells

doi: 10.1152/ajprenal.00037.2017

Figure Lengend Snippet: Electrophysiological examination of SK1/3 and IK1 dependence of BK channel activity during TRPV4 stimulation. A: representative Vm trace showing that TRPV4 activation by GSK101 (arrow 1) led to Vm depolarization (occasionally, this is preceded by an initial transient hyperpolarization as in the example shown) in mCCDcl1 cells, demonstrating that the depolarizing effect (upward deflection) of TRPV4 dominates over the hyperpolarizing effect (downward deflection) associated with activation of the KCa. Subsequent inhibition of BK by IbTX (100 nM; arrow 2) caused a further Vm depolarization (maximal depolarization indicated by arrow 3), indicating the involvement of BK channels to counter the depolarizing effect of TRPV4. B: representative Vm trace showing that the pretreatment with SK1/3 and IK1 blockers (apamin + TRAM-34) resulted in a much stronger Vm depolarization on TRPV4 activation, consistent with TRPV4 mediating the activation of SK1/3 and IK1. In the continued presence of apamin and TRAM-34, the application of IbTX (arrow 2) did not lead to further depolarization in this example, indicating that BK was not very active when SK1/3 and IK1 were inhibited. Arrow 3 reflects the apparent maximum depolarization observed in the presence of IbTX. C: summary data for A and B showing that in the absence of apamin and TRAM-34, GSK101 evoked a depolarization of 5.5 ± 1.0 mV (100 ± 17.2%, n = 17) from a resting Vm of −30.4 ± 1.2 mV, whereas in the presence of apamin and TRAM-34, the GSK101-evoked Vm depolarization was much larger, averaging 12.4 ± 1.9 mV (223 ± 35.0%, n = 13; **P < 0.01) from a resting Vm of −33.1 ± 1.9 mV. D: summary data for A and B showing that in the presence of apamin and TRAM-34, the IbTX-induced Vm depolarization on top of that evoked by GSK101 was much smaller (38.9 ± 14.9%, 0.7 ± 0.26 mV, n = 13; *P < 0.05) than that observed in the Control conditions (100 ± 22.6%, 1.8 ± 0.4 mV, n = 17).

Article Snippet: The chemicals and drugs used in the study were GSK101 (GSK1016790A; Santa Cruz Biotechnology) from a 1 mM stock solution in DMSO, apamin (Alomone) from a 1 mM stock solution in PBS, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; Tocris) from a 10 mM stock solution in DMSO, and iberiotoxin (IbTX; Alomone) from a 0.1 mM stock solution in PBS.

Techniques: Activity Assay, Activation Assay, Inhibition, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Dynamic coupling between TRPV4 and Ca 2+ -activated SK1/3 and IK1 K + channels plays a critical role in regulating the K + -secretory BK channel in kidney collecting duct cells

doi: 10.1152/ajprenal.00037.2017

Figure Lengend Snippet: Cell model of TRPV4-induced activation of the BK channel and K+ secretion showing the critical initial events where TRPV4-mediated Ca2+ influx 1st activates the SK1/3 and IK1 channels, which leads to enhanced Ca2+ entry and subsequent activation of BK. Initial Events: the initial stimulus (e.g., high tubular flow) leads to activation of TRPV4 (step 1), which, in turn, produces both a depolarization of Vm (step 2a) and the pathway for Ca2+ influx (step 2b). Ca2+ influx is greatly limited at this point since the depolarization of Vm and flux of monovalent cations, like Na+, through the TRPV4 channel greatly limits Ca2+ influx. Without any further stimulation, as shown for the initial events where SK1/3 and IK1 channels are blocked (apamin + TRAM-34), the increase in [Ca2+]i is modest and may be spatially restricted, perhaps near the membrane, and only sufficient to activate a limited number of BK channels, if any. The modest elevation in [Ca2+]i is, however, more than adequate to activate the “Ca2+ sensor” channels SK1/3 and IK1 (step 3) once the blocking peptides are removed. Follow-on Events: the follow-on events are defined as the series of sequential/parallel events that are induced following the early rise in [Ca2+]i due to the initial activation of TRPV4 during the initial events. The modest rise in [Ca2+]i during the initial events leads to activation of both SK1/3 and IK1 channels (in the absence of apamin and TRAM-34) that generates a noted shift of Vm in the hyperpolarizing direction (step 4a). This results in a marked increase in the driving force for Ca2+ entry through the TRPV4 channel and a strong enhancement of the Ca2+ signal, i.e., increase in [Ca2+]i (step 4b). The SK1/3/IK1-induced hyperpolarization of Vm leads to a stimulation of Ca2+ influx, which is more than sufficient to bring about a strong activation of the BK channel and, supposedly, K+ secretion (step 5). The role of the SK1/3 and IK1 channels is central in the sequence of events since, if these channels are blocked, the rise in [Ca2+]i is too small to activate BK fully, leading to limited or no activation of the BK channel and K+ secretion.

Article Snippet: The chemicals and drugs used in the study were GSK101 (GSK1016790A; Santa Cruz Biotechnology) from a 1 mM stock solution in DMSO, apamin (Alomone) from a 1 mM stock solution in PBS, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; Tocris) from a 10 mM stock solution in DMSO, and iberiotoxin (IbTX; Alomone) from a 0.1 mM stock solution in PBS.

Techniques: Activation Assay, Membrane, Blocking Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: The Therapeutic Effect of STAT3 Signaling-Suppressed MSC on Pain and Articular Cartilage Damage in a Rat Model of Monosodium Iodoacetate-Induced Osteoarthritis

doi: 10.3389/fimmu.2018.02881

Figure Lengend Snippet: Effects of STAT3 inhibition on anti-inflammatory activity and migration potential in MSCs from osteoarthritis (OA) patients. (A) OA-MSCs were incubated with STA21 for 3 days and, ELISA was performed to measure IL-6, IL-8, IL-1β, and VEGF concentrations. (B) OA-MSCs were incubated with STA21 for 1 h. The expression of phospho (p)-STAT3 705, 727, and total STAT3 protein was analyzed by Western blotting. (C) mRNA levels for anti-inflammatory cytokines IL-10 and TGF-β were measured by real time-PCR (D) Expression of chemokine receptors in OA-MSCs incubated with STA21 was measured by real time-PCR. OA-MSC were isolated of fat tissues obtain from each 3 person with OA. Data represent the mean ± SD of 3 independent experiments ( * P < 0.05; ** p < 0.01).

Article Snippet: The cells were cultured for 4 days until 90% confluent (passage 0) and were then expanded for 2–3 passages and used for experiments. iSTAT3 OA-MSCs were prepared by treatment of OA-MSCs with 10 μM STA21 (Santa Cruz, Texas, USA) for 72 h.

Techniques: Inhibition, Activity Assay, Migration, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Isolation

Journal: iScience

Article Title: Identifying signatures of EV secretion in metastatic breast cancer through functional single-cell profiling

doi: 10.1016/j.isci.2023.106482

Figure Lengend Snippet: Key resources table

Article Snippet: ganetespib , medchemexpress , Cat#HY-15205.

Techniques: Recombinant, Protease Inhibitor, Membrane, Single Cell, Multiplexing, Amplification, BIA-KA, Luminex, Software