Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Role of TRPV4 and K + channels in AA‐induced dilation of HCAs. A, AA dilated HCAs in a concentration‐dependent manner. The dilation was markedly inhibited by the TRPV4 antagonist RN‐1734 (20 μmol/L). B, AA‐induced dilation was inhibited by the combination of TRAM‐34 (1 μmol/L) and apamin (1 μmol/L), which selectively inhibit intermediate‐conductance K Ca (IK Ca ) and small‐conductance K Ca (SK Ca ) channels, respectively. C, AA‐induced dilation was eliminated by high extracellular K + (80 mmol/L); n=5 to 7 patients/each group. * P <0.05 vs control. TRPV4 indicates transient receptor potential vanilloid 4; AA, arachidonic acid; HCA, human coronary artery.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Concentration Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: AA‐induced increase of [Ca 2+ ] i in native HCAECs. A, AA (3 μmol/L) increased [Ca 2+ ] i , which was nearly eliminated in the absence of extracellular Ca 2+ (Ca 2+ free). B, AA‐elicited [Ca 2+ ] i increase was inhibited by 3 CYP pathway inhibitors: ETYA (30 μmol/L), 17‐ODYA (10 μmol/L), and MS‐PPOH (30 μmol/L). C, Treatment of cells with TRPV4 antagonists RN‐1734 (20 μmol/L), HC‐067047 (1 μmol/L), and ruthenium red (RuR; 1 μmol/L) also markedly inhibited AA‐induced [Ca 2+ ] i elevation. D, The TRPV4 agonist GSK1016790A (GSK; 10 nmol/L) increased [Ca 2+ ] i , which was inhibited by RN‐1734. All data represent mean±SEM of ≥60 cells analyzed in 3 to 5 independent experiments. * P <0.05, ** P <0.01 versus control. AA indicates arachidonic acid; ETYA, eicosatetraynoic acid; 17‐ODYA, 17‐octadecynoic acid; MS‐PPOH, N‐(methylsulfonyl)‐2‐(2‐propynyloxy)‐benzenehexanamide; HCAEC, human coronary artery endothelial cell; CYP, cytochrome P450; TRPV4, transient receptor potential vanilloid 4.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Ca 2+ responses in hTRPV4‐overexpressing HCAECs. A, Representative images of endothelial cells expressing hTRPV4‐GFP (top) and corresponding cells loaded with fura‐2 before (middle) and after (bottom) AA treatment. B, Compared with nontransduced (NT) cells, AA (3 μmol/L) elicited augmented [Ca 2+ ] i elevation in hTRPV4‐expressing cells. C, The AA‐induced Ca 2+ response was inhibited by CYP pathway inhibitors ETYA (30 μmol/L) and 17‐ODYA (10 μmol/L). D, None of 4 isomeric EETs (3 or 10 μmol/L in some experiments) showed substantial activation of TRPV4 channels compared with AA. All data represent mean±SEM of ≥60 cells analyzed in 3 to 5 independent experiments. * P <0.05 versus control. hTRPV4 indicates human transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell; GFP, green fluorescent protein; AA, arachidonic acid; CYP, cytochrome P450; EET, epoxyeicosatrienoic acid.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Expressing, Activation Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Modulation of TRPV4‐mediated Ca 2+ influx by membrane potential. A, AA (3 μmol/L)‐elicited [Ca 2+ ] i increase was blunted in the presence of 60 mmol/L extracellular K + . B, AA induced membrane hyperpolarization in HCAECs, as indicated by a decrease in PMPI fluorescence intensity (F480). In contrast, the TRPV4 agonist 4α‐PDD (3 μmol/L) depolarized the membrane potential. Valinomycin (K + ‐selective ionophore, 2 μmol/L) was used as a positive control to indicate membrane hyperpolarization. C, The TRPV4 specific agonist 4α‐PDD (5 μmol/L) increased [Ca 2+ ] i in HCAECs, and the increase was not affected by 60 mmol/L K + . D, Membrane hyperpolarization by valinomycin resulted in Ca 2+ influx in hTRPV4‐expressing HCAECs, which was inhibited by HC‐067047. The data represent mean±SEM of ≥60 cells analyzed in 3 to 5 independent experiments. * P <0.05 vs control, ** P <0.01 vs vehicle, † P <0.05 vs valinomycin (5 μmol/L). TRPV4 indicates transient receptor potential vanilloid 4; AA, arachidonic acid; 4α‐PDD, 4α‐phorbol‐12,13‐didecanoate; HCAEC, human coronary artery endothelial cell; PMPI, plasma membrane potential indicator.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Fluorescence, Positive Control, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Regulation of TRPV4 activation by protein phosphorylation. A, In hTRPV4‐expressing HCAECs, the AA‐induced [Ca 2+ ] i increase was almost abolished by the PKA inhibitor PKI (1 μmol/L), whereas this AA response was only partially but not significantly attenuated by the PKC inhibitor GF 109203X (1 μmol/L). B, TRPV4 is phosphorylated at serine‐824 in unstimulated HCAECs expressing the hTRPV4‐GFP fusion protein. The level of serine‐824 phosphorylation was not further increased by AA (3 μmol/L). PKI (1 μmol/L) significantly inhibited serine‐824 phosphorylation. Endothelial cells were treated with the indicated reagents, and TRPV4‐GFP was immunoprecipitated with GFP antibodies and detected with pS824 antibodies (upper panel). The same membrane was reprobed with GFP antibodies to detect total TRPV4 protein (middle panel). All data represent mean±SEM from 3 to 5 independent experiments. At least 60 cells analyzed in (A). * P <0.05, ** P <0.01 vs control. TRPV4 indicates transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell; AA, arachidonic acid; PKA, protein kinase A; PKI, protein kinase A inhibitor; IP, immunoprecipitation; IB, immunoblotting; GFP, green fluorescent protein.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Activation Assay, Expressing, Immunoprecipitation, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Single‐channel properties of hTRPV4 overexpressed in HCAECs. A, TRPV4 single‐channel currents (left) were recorded from a cell‐attached patch at the indicated membrane potentials (Vm). Right panel depicts average current–voltage relationship for single‐channel currents, with a calculated slope conductance determined over voltage ranges of 0 to 60 and −40 to 0 mV, respectively. Data are mean±SE from 5 membrane patches. B, Single‐channel activity of TRPV4 (left) was low under basal conditions but markedly increased following bath perfusion of the TRPV4 agonist 4α‐PDD (1 μmol/L). Shown on the right are the corresponding amplitude histograms in relation to open‐state probability (NPo). Data are representative of >5 membrane patches. Dashed lines indicate current level when the channel is closed. Cell‐attached recordings were obtained with a normal extracellular solution (140 Na + , 5 Cs + ) in the pipette and a high‐K + (140 K + ) bath solution to zero the cell membrane potential. TRPV4 indicates transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Activity Assay, Transferring

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca 2+ Entry

doi: 10.1161/JAHA.113.000080

Figure Lengend Snippet: Effect of AA on hTRPV4 single‐channel currents in HCAECs. AA (1 μmol/L) increased TRPV4 single‐channel currents in both cell‐attached patches (A) and inside‐out patches, which lacked intracellular constituents (B). Left, representative recordings at the indicated membrane potential (Vm). Note that c is the current level when the channel is closed. Right, summary of channel open‐state probability (NPo). Cell‐attached and inside‐out patch recordings before and after treatment with AA were performed with a normal extracellular solution (140 Na + ,5 Cs + ) in the pipette and a high‐K + (140 K + ) solution in the bath. n=6 patches/each group. * P <0.05 vs control. C, Possible mechanism for AA‐induced Ca 2+ entry through TRPV4 channels in human coronary endothelial cells and subsequent dilation of coronary arterioles. AA indicates arachidonic acid; TRPV4, transient receptor potential vanilloid 4; HCAEC, human coronary artery endothelial cell; PKA, protein kinase A; EDH, endothelium‐dependent hyperpolarization.

Article Snippet: The human TRPV4 (NM_021625) full‐length cDNA clone was obtained from Origene and shuttled into a pCMV6 mammalian expression vector, resulting in a TRPV4 fusion protein with its COOH‐terminus tagged with turbo green fluorescent protein (GFP).

Techniques: Transferring

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Quantification of TRPV4 expression. (a) Sites where tissue samples were taken and the quantification method for stained areas. Tissue was collected from 57 patients who underwent gastric resection. The following areas were sectioned in all layers: 1. greater curvature of the fornix; 2. greater curvature of the gastric body; 3. greater curvature of the antrum; 4. anterior wall of the gastric body; 5, posterior wall of the gastric body. (b) TRPV4 immunostaining in gastric tissue. (c) The images were captured using a microscope with BZ-X710 (Keyence, Osaka, Japan), and staining was quantified with hybrid cell count BZ-H4C.

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Expressing, Staining, Immunostaining, Microscopy, Cell Counting

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Comparison of stained areas between nonobese and obese cases. (a) Renal tissues were used as a positive control for TRPV4-immunostaining. (b) For the negative control, tissues were subjected to immunostaining without the primary antibody. (c) Immunostaining of the nonobese and obese stomach. (d) Comparison of stained areas in tissue samples collected from patients with and without obesity. Lines within the boxes represent median values; upper and lower lines of the boxes represent 25th and 75th percentiles, respectively; upper and lower bars outside the boxes represent the maximum and minimum, respectively ( ∗∗ p < 0.01).

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Comparison, Staining, Positive Control, Immunostaining, Negative Control

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: TRPV4 immunostaining in the stomach of patients with and without obesity.

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Immunostaining

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Fluorescence immunostaining of TRPV4 in MGN3-1 cells. TRPV4 protein expression was observed in MGN3-1 cells. Magnification: ×600. TRPV4-mediated change in [Ca 2+ ] i in MGN3-1 cells.

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Fluorescence, Immunostaining, Expressing

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: TRPV4-mediated changes in cytosolic Ca 2+ in MGN3-1 cells. The effects of GSK1016790 A and HC 067047 on [Ca 2+ ] i in MGN3-1 cells. Compared to the control level at 180 s, [Ca2+] i increased in MGN3-1 cells treated with GSK1016790 A ( n = 20, p < 0.01) but decreased in cells treated with GSK1016790 A and HC067047 ( n = 20, p < 0.01) (GSK1016790 A, TRPV4 agonist; HC067047, TRPV4 antagonist).

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: Control

Journal: International Journal of Endocrinology

Article Title: Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity

doi: 10.1155/2022/7241275

Figure Lengend Snippet: Regulation of ghrelin secretion by the TRPV4 agonist and antagonist in MGN3-1 cells. (a) The amount of secreted acyl ghrelin after four-hour treatment of MGN3-1 cells with GSK1016790 A (3 μ M) or HC067047 (20 μ M) + GSK1016790 A (3 μ M). (b) The amount of secreted des-acyl ghrelin after four-hour treatment of MGN3-1 cells with GSK1016790 A (3 μ M) or HC067047 (20 μ M) + GSK1016790 A (3 μ M) ( ∗ p < 0.05; ∗∗ p < 0.01; n = 6; error bars: standard error of the mean).

Article Snippet: The cells were then probed with a rabbit polyclonal anti-TRPV4 antibody (OST00265 G, Osenses Pty Ltd, Keswick, Australia) overnight at 4°C.

Techniques: