Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

Figures

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  Fig 1. TRPV4 mRNA transcript and protein expression in FHH rat cerebral arterial myocytes. (A) (i) Use of TRPV4 channel gene specific primer amplified a PCR product of 351-bp as determined by semiquantitative RT-PCR. Shown in (ii) is the internal control GAPDH. (B) Western blot analysis using homogenates of freshly isolated FHH rat cerebral arterial myocytes lysates and the polyclonal anti-TRPV4 antibody (Alomone ACC-034 at 1:200) detected expression of the right molecular size for TRPV4 channel protein (n = 2–3 separate trials for each group).

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  Fig 2. Differentiation by immunofluorescent staining of a population of freshly isolated FHH rat cerebral arterial myocytes and immunolocalization of TRPV4 channel protein expression. (A) Characterization and differentiation by immunofluorescent staining of a population of freshly isolated cerebral arterial myocytes using the nuclei stain DAPI, the endothelial cell marker anti-CD31 (PECAM-1) antibody and anti-smooth muscle α-actin (Anti-SM-α-actin) antibody visualized with donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody. These findings revealed that population of freshly isolated cerebral arterial myocytes obtained using the method described in this study are smooth muscle α-actin positive cells that are free of endothelial cell contamination. (B) Depiction of single freshly isolated rat cerebral arterial myocyte stained with DAPI alone (nuclei stained blue) or co-stained with either anti-SM-α-actin antibody (red) or with anti-TRPV4 antibody [36] and visualized with donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody. The image of the arterial myocyte stained with anti-TRPV4 antibody demonstrates expression of TRPV4 channel protein in the cerebral arterial…

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  Fig 3. TRPV4 channel protein expression in cerebral arterial sections. Immunohistochemically detection of expression of TRPV4 channel protein using 4 μm sections of FHH rat cerebral arterial segment by treatment with either a secondary antibody alone (secondary antibody Donkey anti-Rabbit Alexa Fluor 488, 1:750, Thermo Fisher Scientific, upper panel) or with a primary polyclonal anti-TRPV4 antibody (Alomone ACC-034, 1:200, lower panel) revealing expression of TRPV4 protein in FHH rat cerebral arterial muscle cells as shown by punctate staining pattern both at the intima and media layers of the arterial wall. Blue: indicates nuclear staining with DAPI of the fixed arterial sections.

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  Fig 4. TRPV4-like single-channel cationic current conductance. Single-channel cationic currents recorded from FHH rat cerebral arterial myocytes exhibited two slope conductances: 85 ± 2 pS at more hyperpolarizing potentials and 96 ± 3 pS at more depolarizing potentials. (A) The single-channel cationic currents recorded at patch holding potential (PP) of 60 mV were sensitive to inhibition by the TRPV4 channel antagonist RN 1734 (5 μM) (B) or by HC 067047 (300 nM) (C) (n = 5–6 cells).

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  Fig 5. Characterization of the TRPV4-like single-channel cationic currents. The TRPV4-like cationic single-channel current recorded in cell-attached patches of FHH rat cerebral arterial myocytes was activated by the specific TRPV4 channel agonist GSK1016790A (100 nM) that was reduced or suppressed by the TRPV4 channel inhibitor HC 067047 (300 nM) (A and B). * denotes statistically significant difference from control at P < 0.05, n = 5–6 cells).

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  Fig 6. Sensitivity of TRPV4-like single-channel cationic current to membrane stretch. (A) Representative single TRPV4-like single-channel cationic currents recorded at a holding potential of 60 mV from cell attached patches of cerebral arterial myocytes shown in slower (a) and faster time spans (b) under control condition and following membrane stretch by application of negative pressure of 10 mm H2O or 30 mm H2O and (c) attenuation of the 30 mm H2O negative pressure induced activation of the single-channel cationic current by pretreatment with the TRPV4 channel inhibitor HC 067047. (B) Summary of application of a negative pressure of 10 cm H2O or 30 cm H2O induced marked increase in the open state probability of (NPo) of the TRPV4-like single-channel cationic current and the attenuation of the negative pressure of 30 mm H20 induced increase in NPo following treatment with the TRPV4 channel inhibitor HC 067047 (300 nM) (*, †, P < 0.05, n = 5–6 cells for each group).

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  Fig 7. Effect of the TRPV4 channel agonist GSK1016790A on activities of a KCa single-channel current in FHH rat cerebral arterial myocytes. (A) Examples of single-channel current traces recorded at a patch holding potential of 40 mV using symmetrical (145 mM KCl) recording solution under control condition and following treatment with 10 nM or 100 nM GSK1016790A. Application of GSK1016790A induced concentration-dependent increase in the opening frequency of the single-channel KCa currents that was attenuated by treatment with the KCa channel blocker paxilline. (B) Summary of the effects of treatment with 10 nM or 100 nM GSK1016790A on the open state probability (NPo) of the KCa single-channel currents recorded at a patch holding potential of 40 mV. Treatment with 10 nM or 100 nM GSK1016790A significantly increased the NPo of the KCa single-channel currents compared to the control NPo value that was attenuated by treatment with the KCa channel inhibitor paxilline. (*,† denote P < 0.05, n = 5–6 cells for each group; c and o represent closed and open states of the channel, and 1,2,3 indicate stocked channel opening levels).

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  Fig 8. Effect of the L-type Ca2+ channel inhibitor nifedipine on the TRPV4 channel agonist GSK1016790A induced activation of KCa single-channel current in FHH rat cerebral arterial myocytes. (A) Representative KCa single-channel current traces recorded at a patch holding potential of 40 mV from cell-attached patches under control condition and following treatment with 100 nM GSK1016790A before and after addition of 1 μM nifedipine or the KCa channel inhibitor 1 μM paxilline to the bath. Treatment with 100 nM GSK1016790A increased the opening frequency of the KCa single-channel currents that was not influenced by treatment with the L-type Ca2+ channel inhibitor 1 μM nifedipine, but attenuated by the KCa channel inhibitor 1 μM paxilline. (B) Summary of the effects of treatment with 100 nM GSK1016790A on the NPo of the KCa single-channel currents in the absence and presence of the L-type Ca2+ channel inhibitor 1 μM nifedipine or the KCa channel inhibitor paxilline (1 μM). Treatment with 100 nM GSK1016790A induced a significant increase in NPo of the KCa single-channel current that was not affected by pretreatment with the L-type Ca2+ channel inhibitor 1 μM nifedipine, but attenuated in the…

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  Fig 9. Effect of the T-type Ca2+ channel inhibitor nickel on the TRPV4 channel agonist GSK1016790A induced activation of KCa single-channel current in FHH rat cerebral arterial myocytes. (A) Representative single-channel current traces recorded at a patch holding potential of 40 mV from cell attached patches under control condition and following treatment with 100 nM GSK1016790A before and after addition of the T-type Ca2+ channel inhibitor 50 μM nickel (Ni2+) or the KCa channel inhibitor 1 μM paxilline to the bath. Treatment with 100 nM GSK1016790A increased the opening frequency of the KCa single-channel currents that was not influenced by pretreatment with the T-type Ca2+ channel inhibitor 50 μM Ni2+ but attenuated by the KCa channel inhibitor 1 μM paxilline. (B) Summary of the effects of treatment with 100 nM GSK1016790A on the NPo of single-channel KCa currents in the absence and presence of the T-type Ca2+ channel inhibitor 50 μM Ni2+ or the KCa channel inhibitor paxilline (1 μM). Treatment with 100 nM GSK1016790A induced significant increase in NPo of the KCa single-channel current that was not affected by pretreatment with the T-type Ca2+ channel inhibitor 50 μM Ni2+, but…

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  Fig 10. Effects of TRPV4 channel inhibition on intraluminal pressure-dependent reductions in diameter of endothelium-denuded and cannulated cerebral arterial segments of Sprague Dawley (A, SD) and FHH (B, FHH) rats. Pretreatment with the specific TRPV4 channel inhibitor HC067047 (1 μM) had no significant effect on increases in intraluminal pressure induced reductions in diameter of the cerebral arterial segments of SD rat (A) and the FHH rat (B) (n = 5). In Ca2+-free PSS bath solution the cannulated cerebral arterial segments of FHH rat exhibited an average passive dilation of 11.2 ± 2.2% whereas the Sprague Dawley rats elicited a mean passive dilation of 31.8 ± 5% (n = 5, < 0.05). Presented in (C) is Western blot showing the level of expression of TRPV4 channel protein with bar graphs depicting summary of the normalized TRPV4 channel protein with the internal control β-actin determined using brain homogenates of SD and FHH rats which are not significantly different (n = 3 independent trials).