Distinct Effects of Ca²⁺Sparks on Cerebral Artery and Airway Smooth Muscle Cell Tone in Mice and Humans

Figures

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  Figure 1. Effects of ryanodine on Ca2+ sparks and cell length. (A) Simultaneous recordings of Ca2+ sparks and cell length were performed in mouse CASMCs using an LMS 700 confocal microscope. Ca2+ sparks were abolished by ryanodine, and the cell was shortened. (B) The same experiments were conducted in mouse ASMCs. The cell length did not change. (C) Summary of the results. (D) Single mouse ASMCs were incubated with ryanodine or vehicle for 15 min in the suspended state. Their lengths were increased compared with those of the controls. NS: P> 0.05; ***: P< 0.001. These results indicate that Ca2+ sparks decreased CASMC tone and increased ASMC tone.

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  Figure 2. Ca2+ sparks-activated currents in mouse cells. (A) Ca2+ sparks-activated currents at potentials from -60 mV to +10 mV were recorded in CASMC. Only STOCs were observed. (B) The same recordings were performed in an ASMC, showing that Ca2+ sparks simultaneously triggered STOCs and STICs; however, the former occurred prior to the latter shown in the inset box. (C) STOC and STIC frequency and amplitude. (D) Whole-cell ramp currents induced by 10 mM caffeine were recorded in CASMCs and ASMCs. The paxilline-sensitive currents were observed only in CASMCs and these currents with NA-sensitive currents were noted in ASMCs. The paxilline-sensitive currents were larger in CASMCs than in ASMCs. *: P< 0.05. These data indicate that CASMCs had no functional Clca channels; however, they had more STOCs and caffeine-induced inward currents than ASMCs.

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  Figure 3. BKs and Clca channels mediate Vm changes in mouse cells. (A) Vm in a CASMC displayed smaller oscillations and TEA abolished oscillations and elevated the baseline. NA did not induce changes. All-point amplitude histograms were constructed, and S40 (the area to the right of -40 mV) and ST (the total area) were marked. (B) The ratios of S40/ST are summarized. (C-D) The same experiments and analyses were performed in ASMCs. *: P< 0.05; ***: P < 0.001. These results indicate that CASMCs had no functional Clca channels; in the resting state, the Po of LVDCCs was lower in CASMCs than in ASMCs, Clca channels-induced depolarization and LVDCC activation were higher than BKs-caused hyperpolarization and LVDCC inactivation in ASMCs, and that BKs-caused hyperpolarization and LVDCC inactivation were higher in CASMCs compared to ASMCs.

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  Figure 4. Voltage and Ca2+ sensitivity of BKs in mouse CASMCs and ASMCs. (A) Single BK currents were recorded using the inside-out technique at 0, 20, 40, and 60 mV under 1, 3, and 10 μM free Ca2+ conditions in an excised patch from a CASMC. The Po values are shown above each trace. The corresponding all-point amplitude histograms were constructed and fitted, and the single-channel currents were obtained. (B) The same recordings and analyses from an excised patch from an ASMC. (C) I-V curves were constructed, and the conductance values were calculated for CASMCs and ASMCs. (D) Po-voltage curves show that the Po was larger in CASMCs than in ASMCs following the increases in voltage and free Ca2+, indicating that BKs in the former had a higher voltage and Ca2+ sensitivity than in the latter. *: P < 0.05.

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  Figure 5. Effects of anti-β1 antibodies on single BK currents in mouse cells. (A) Single BKs-mediated currents were similarly recorded at 0, 20, 40 and 60 mV under 1 and 3 μM free Ca2+ on excised patches from CASMCs and ASMCs using the inside-out technique. After incubation with anti-β1 antibodies, the currents were recorded again. Po values before and after the incubation with antibodies were calculated, and Po-voltage curves were constructed. The results show that the antibodies induced decreases in Po values in both CASMCs and ASMCs, however, the decreases were larger in the former than in the latter. (B) The net decreases in the Po (i.e., ΔPo) values were calculated and used to plot the ΔPo-voltage curves, showing that the ΔPo values were larger following increases in Ca2+ concentration and voltage in CASMCs than in ASMCs. *: P < 0.05. These data demonstrate that β1 subunits mediated the Ca2+ and voltage sensitivity of BKs in both cells and the higher Ca2+ and voltage sensitivity in CASMCs than in ASMCs.

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  Figure 6. Effects of Ca2+ sparks on human cell tone and the underlying mechanism. (A) Human CASMCs and human ASMCs were incubated with vehicle and ryanodine, and the cell lengths were measured. (B) Only STOCs were recorded in human CASMCs, however, STICs and STOCs were observed in human ASMCs. (C) Average frequency and amplitude of STICs and STOCs, showing that the amplitude of STOCs was lower in CASMCs than in ASMCs. (D) Caffeine failed to induce inward currents in three human CASMCs that occurred in three human ASMCs. **: P < 0.01; ***: P < 0.001. These results indicate that Ca2+ sparks decreased human ASMC tone and increased human CASMC tone and the former had only BKs and the latter had both Clca channels and BKs.

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  Figure 7. Mechanisms of Ca2+ sparks-induced decreases in CAMSC tone and increases in ASMC tone. CASMCs lack functional Clca channels. Ca2+ sparks only activate BKs, which mediate relaxation via the STOC-Vm-LVDCC-Ca2+ influx termination pathway. However, this relaxation is partially counteracted by the contraction induced by the spontaneous depolarization of Vm. In ASMCs, except for this BKs-mediated relaxant pathway, Ca2+ sparks also activate Clca channels, which then mediate contraction through the STIC-Vm-LVDCC-Ca2+ influx pathway. However, the integrated result is contraction. The cause may be that Ca2+ sparks activate BKs prior to Clca channels, allowing the contraction is not exactly antagonized by the relaxation. Moreover, Clca channels-induced membrane depolarization and LVDCC activation are larger compared to BKs-caused membrane hyperpolarization and LVDCC inactivation. In addition, the Ca2+ spark frequency and amplitude, BK density, BK currents and β1-determined Ca2+ and voltage sensitivity of BKs were higher in CASMCs than in ASMCS. These allow Ca2+sparks to cause more relaxation in CASMCs than in ASMCs. In addition, TMEM16A will be one type of Clca channels in ASMCs that plays a…