Author + information
- Wang Na,
- Jun Cheng,
- Wenjun Huang,
- Xinrong Fan and
- Xiaorong Zeng
The goal of this study was to test the interaction between HSYA and BKCa channels and revealed HSYA on the function and mechanism of BKCa channels by using vasomotor assay and patch-clamp recordings, thus further explain vasodilatation function of HSYA is related to BKCa channels. This applies for HSYA provide basis for clinical treatment of hypertension and other cardiovascular diseases.
Separation of rat mesenteric artery rings and vasomotor assay: The branches of the superior mesenteric arteries were rapidly isolated from the anesthetized male Sprague-Dawley rats. The arteries were sectioned into four rings for the tension measurements and vasomotor assay.
Expression of BKCa channels on HEK293 cells and culture: The BKCa α-subunit cloned from human VSMCs (hSloα) was constructed in the vector pcDNA3.1 and stably expressed in HEK293 cells.
Patch-clamp recordings: Single channel current recordings were conducted under cell-attached configuration using a patch clamp amplifier (HEKE). Single channel currents were recorded by a membrane patch formation after 10 mins. Data were low-pass filtered at 1 kHz.
The relaxation effect of HSYA on the tension of rat mesenteric artery rings precontracted with NE could be obviously observed and weakened by 5 mM TEA which was non-selective potassium channel blocker (n=4). Importantly, IbTX (BKCa channel blocker) also extremely decreased the effect of HSYA, which indicated that the relaxation effect of HSYA partly resulted from the activation of BKCa channel. The electrophysiological results revealed that 30 μM and greater concentration of HSYA could obviously activate currents and increase the current amplitude. The activation function of HSYA was enhanced with the concentration increased. Single channel currents were recorded under cell-attached configuration. Further analysis of the NPO, TC and TO from single channel recorded currents of BKCa channel, showed that HSYA increased NPO and TO in concentration-dependent manner (n=4). Yet, the effect of HSYA on TC didn't change obviously compared with control group from 10 to 120 μM. However, 240 μM HSYA significantly reduced the TC.
The vasomotor assay indicates that vasodilatation function of HSYA is related to BKCa channels. By using patch-clamp technique, it further confirms that HSYA could directly activate BKCa channels expressed on HEK293 cells in concentration-dependent manner under cell-attached configuration. These results suggest that the activation function of HSYA on BKCa channels probably is an important mechanism in dilating superior mesenteric arteries.