Abstract
The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium–dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes. Patch clamp recording of single-channel current activity identified the presence of a single-channel cationic current with unitary conductance of ~85 pS and ~96 pS at hyperpolarizing and depolarizing potentials, respectively, that was inhibited by the TRPV4 channel antagonist RN 1734 or HC 067074 and activated by the potent TRPV4 channel agonist GSK1016790A. Application of negative pressure via the interior of the patch pipette increased the NPo of the TRPV4-like single-channel cationic current recorded in cell-attached patches at a patch potential of 60 mV that was inhibited by prior application of the TRPV4 channel antagonist RN 1734 or HC 067047. Treatment with the TRPV4 channel agonist GSK1016790A caused concentration-dependent increase in the NPo of KCa single-channel current recorded in cell-attached patches of cerebral arterial myocytes at a patch potential of 40 mV, which was not influenced by pretreatment with the voltage-gated L-type Ca2+ channel blocker nifedipine or the T-type Ca2+ channel blocker Ni2+. These findings demonstrate that FHH rat cerebral arterial myocytes express mRNA transcript and translated protein for TRPV4 channel and display TRPV4-like single-channel cationic current activity that was stretch-sensitive and activation of which increased the open state probability of KCa single-channel current in these arterial myocytes.
Highlights
Transient receptor potential vallinoid type 4 (TRPV4) channel, a member of the transient receptor potential (TRP) channel superfamily, is a non-selective cationic channel permeable to Na+, Ca2+, Mg2+ and is widely distributed in different cell types, including those of the brain [1, 2]
Immunofluorescent staining studies of freshly dissociated Fawn Hooded hypertensive (FHH) rat cerebral arterial myocytes (Fig 2A) revealed that a population of freshly isolated cerebral arterial myocytes obtained using the method described in the present study are smooth muscle α-actin positive cells that are stained blue with DAPI, and are free of endothelial cell contamination as confirmed by negative staining of the cells with the endothelial cell marker anti-CD31-antibody
The merged images of FHH rat cerebral arterial myocyte stained with anti-SM-α-actin antibody and anti-TRPV4 antibody [36] appeared in mixed colors of yellow and green demonstrating expression and co-localization of smooth muscle α-actin and TRPV4 channel protein exhibiting a punctate like pattern both in the cytosol and the cell membrane
Summary
Transient receptor potential vallinoid type 4 (TRPV4) channel, a member of the transient receptor potential (TRP) channel superfamily, is a non-selective cationic channel permeable to Na+, Ca2+, Mg2+ and is widely distributed in different cell types, including those of the brain [1, 2]. TRPV4 channel is extensively expressed in brain astrocytes and was identified to function as a key molecular sensor of hemodynamic stimuli and regulator of parenchymal arteriole tone [13]. Previous reports indicate that hypoxia/ischemia increases expression and activity of TRPV4 channel in brain astrocytes contributing to Ca2+ overload in the astroglial syncytium leading to cellular damage [15]. TRPV4 was proposed to form a novel Ca2+ signaling complex with the ryanodine receptors and BKCa / KCa channel. Simulation of this complex with the epoxide 11,12-epoxyeicosatrienoic acid (11,12-EET) initiates smooth muscle hyperpolarization and vasodilation [17]. TRPV4 channel has been reported to interact with PKC and AKAP150 (a kinase anchor protein 5) to form a dynamic signaling domains that control Ca2+ influx in arterial myocytes to oppose vasoconstriction in this rat strain [18]
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