摘要
Background The myocardial ATP sensitive potassium channel （KATP channel） has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using KATP agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that KATP channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial KATP channel by nicorandil.Methods Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4℃. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IKATP occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 μmol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4℃, 10℃, 20℃, 25℃ and 35℃ respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 （Graphpad, San Diego, CA, USA）. Nonlinear curve fitting was done in Clampfit （Axon） or Sigmaplot （SPSS）.Results At 4℃, 10℃, 20℃, 25℃ and 35℃, the time needed to open the myocardial KATP channel was （81.0±0） minutes, （50.54±11.7） minutes, （28.84±2.3） minutes, （9.4± 10.2） minutes and （2.3± 1.0） minutes respectively （P=0.003）. The linear relationship between temperature and time needed to open the channel was y （min） = （4348.790±124.277x）/60, where y （min） is time needed to open KATP channel, x is temperature, correlation coefficient r =-0.942 （P=0.00）, regression coefficient b =-124.277 （P=0.00）. The current densities among different temperatures were statistically different （P=0.022）, the current density was greater after the activation of KATP channel at higher temperatures. The lower the temperature, the fewer cells in which KATP channels could be opened. At 4℃, only one cell in which the KATP channel could be opened, took a quite long time （81 minutes）and the Ⅰ-Ⅴ curve was quite untypical.Conclusions KATP channel activated by nicorandil is temperature dependent and the temperature linearly related to time needed to open KATP channel; the lower the temperature, the longer the time needed to open channel and the smaller the current density. At profound hypothermia, it is difficult to activate KATP channels.
Background The myocardial ATP sensitive potassium channel （KATP channel） has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using KATP agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that KATP channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial KATP channel by nicorandil.Methods Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4℃. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IKATP occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 μmol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4℃, 10℃, 20℃, 25℃ and 35℃ respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 （Graphpad, San Diego, CA, USA）. Nonlinear curve fitting was done in Clampfit （Axon） or Sigmaplot （SPSS）.Results At 4℃, 10℃, 20℃, 25℃ and 35℃, the time needed to open the myocardial KATP channel was （81.0±0） minutes, （50.54±11.7） minutes, （28.84±2.3） minutes, （9.4± 10.2） minutes and （2.3± 1.0） minutes respectively （P=0.003）. The linear relationship between temperature and time needed to open the channel was y （min） = （4348.790±124.277x）/60, where y （min） is time needed to open KATP channel, x is temperature, correlation coefficient r =-0.942 （P=0.00）, regression coefficient b =-124.277 （P=0.00）. The current densities among different temperatures were statistically different （P=0.022）, the current density was greater after the activation of KATP channel at higher temperatures. The lower the temperature, the fewer cells in which KATP channels could be opened. At 4℃, only one cell in which the KATP channel could be opened, took a quite long time （81 minutes）and the Ⅰ-Ⅴ curve was quite untypical.Conclusions KATP channel activated by nicorandil is temperature dependent and the temperature linearly related to time needed to open KATP channel; the lower the temperature, the longer the time needed to open channel and the smaller the current density. At profound hypothermia, it is difficult to activate KATP channels.
基金
This work was supported by a grant from the Natural Science Foundation of China (No. 30371374).
