To determine the effect of TRPV4 activation on the expression of chondrogenic markers, ATDC5 cells were stimulated with 10–10000 nM of the selective TRPV4 agonist, GSK1016790A (GSK) (Sigma, MO, USA), or with a vehicle control, dimethyl sulfoxide (DMSO), for 6–72 h in serum-free medium followed by incubation with 1% ITS for 48 h. The mRNA expression levels of SOX9, Aggrecan, type 2 collagen, and TRPV4 were quantitatively measured by real-time PCR.
Gsk1016790a
Manufactured by GlaxoSmithKline
Sourced in United States
GSK1016790A is a laboratory compound developed by GlaxoSmithKline. It is a synthetic chemical substance intended for research and experimental use. The core function of GSK1016790A is to serve as a tool for scientific investigation, without extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 acetoxymethyl ester, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (1 mentions)
U46619, by Cayman Chemical (1 mentions)
Pregnenolone sulfate, by Merck Group (1 mentions)
Ionomycin calcium salt, by Merck Group (1 mentions)
Camphor, by Fujifilm (1 mentions)
Lysophosphatidylcholine, by Merck Group (1 mentions)
Xtt kit, by Biotium (1 mentions)
7 protocols using gsk1016790a
1
TRPV4 Activation Modulates Chondrogenic Markers
2
Fura-2 Calcium Imaging of Primary Mouse Microglia
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Primary mouse microglia from WT, M2KO, or V4KO mice on coverslips were incubated at 37 °C for 30 min in culture medium containing 5 μM fura-2-acetoxymethyl ester (Molecular Probes). The coverslips were washed with a standard bath solution that was identical to the standard solution used in the patch-clamp recordings as described below. Fura-2 fluorescence was measured in a standard bath solution with Fura-2 excitation wavelengths of 340 and 380 nm and monitoring of fluorescence emission at 510 nm with a CoolSnap ES charge coupled device (CCD) camera (Roper Scientific/Photometrics) at room temperature. GSK-1016790A (GSK) was applied as described below for the patch-clamp recordings. Data were acquired using IPlab software (Scanalytics) and analyzed with ImageJ (NIH) and Excel software (Microsoft). To prepare the Ca2+ (−) extracellular solution, 5 mM EGTA was added to the standard bath solution without Ca2+. Ionomycin (5 μM, Sigma-Aldrich) was applied to confirm cell viability.
3
Intravesical GSK1016790A and LPS Protocol
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GSK1016790A (GSK) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, JAPAN). Stock solutions of GSK were dissolved in 100% DMSO and adjusted with saline to a final concentration of 0.1%. Intravesical GSK was instilled in a mixture with LPS using the same protocol, as shown in Figure 1A
4
Vasomotor Regulation Protocol
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U46619 was obtained from Cayman Chemical Company (Ann Arbor, MI). GSK1016790A was kindly provided by GlaxoSmithKline Pharmaceuticals. All other chemicals were purchased from Sigma‐Aldrich. Stock solutions were made in distilled water, except U46619 (ethanol), GSK1016790A (dimethyl sulfoxide [DMSO]), 4α‐PDD (DMSO), and indomethacin (0.2 mol/L Na2CO3).
5
Calcium Signaling Pathway Activators
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GSK1016790A (GSK), capsaicin, pregnenolone sulfate (PS), lysophosphatidylcholine (LPC) and ionomycin calcium salt were purchased from Sigma-Aldrich; Allyl isothiocyanate (AITC) was purchased from KANTO; Camphor was purchased from Wako; Fura-2-acetoxymethyl ester was purchased from Invitrogen.
6
Evaluating Cell Proliferation and Viability
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Cell proliferation was determined using XTT kit (Biotium) and cell viability kit (Enzo biosciences). Cultured EC were trypsinized, counted, and plated in a 96-well plate (1000–2000 cells/well). 24–48 h post plating, media was removed and cells were treated with GSK1016790A (GSK) (10–500 nM) in serum free media for overnight. Cells were washed once with PBS; and XTT or Calcein dye was added to the cells and incubated for 30 min-24 h. Wells containing no cells with the dye served as controls to determine the level of the background signal. Absorbance was read at 485–535 nm or 450–500 nm, respectively for Calcein and XTT.
7
Vascular Function Assessment of Posterior Cerebral Artery
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Segments (~2 mm long) of the posterior cerebral artery (PCA, 3−5 mice/group) were cannulated, pressurized (60 mmHg) and superfused with a Krebs’ solution in a chamber for on-line videomicroscopy, as described before15 (link),22 (link). Dilations to ACh (10−11−10−4 M) and CGRP (10−11−10−4 M) were tested on vessels pre-constricted sub-maximally with phenylephrine (PE, 2 × 10−7M). Contractions to ET-1 (10−11–10−5 M) and the tonic production of the vasodilator NO were measured in vessels at basal tone, the latter after inhibition of NOS with L-NNA (10−5 M, 40 min). KATP and TRPV4 channels was assessed in pre-constricted vessels with the selective KATP (levcromakalim, LEV, 10−11–10−4 M)22 (link) and TRPV4 (GSK1016790A, GSK, 10−11–10−4 M) channel openers25 (link).
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