| Patch clamp and laser confocal scanning microscopy techniques were used in the experiments to investigate the change of BKca in vascular hyporeactivity in hemorrhagic shock, and the results were as follows:1. Properties of BKca in ASMC. The current recorded was voltage dependent, high potassium selective and intracellular calcium sensitive. The reversal potential of BKca was 0.9 mV, slope conductance was 221? pS. [Ca2+]i was 3 X10-7 M when the channel was half maximum activated with 140 mM K- bathing and pipette solutions in inside-out configuration. NPo increased e-fold with 17mV positive change in membrane potential at 0.1 uM [Ca2+]j. Two exponentials were required to describe the observed open and closed time distribution of BKca, with open time constant of 0.7674?.1359, 6.0946?.8998 and closed time constant of 0.5964?.0929, 10.8426?.9037 respectively at 40 mV. At 1#M [Ca2+ ];, long term activation of BKCa was followed by long time inactivation, which was known as calcium dependent inactivation. BKca could be inhibited by extracellular amplication of TEA (200 uM) and ChTX (100 nM), indicating that BKca in ASMC was a TEA and ChTX sensitive potassium channel.2. Change of BKca in ASMC in hemorrhagic shock. The resting membrane potential of ASMC changed from -41.90?.25 mV in sham (n=10) to -66.21?.88 mV in hemorrhagic shock (n=8, p<0.01). The current amplitude of BKca increased significantly at holding potential of 20, 40, 60 mV respectively with cell attached patch clamp recordings, whereas the single channel conductance (145?1 and 145?5 pS respectively) had no alteration in shock group. The reversal potential of BKCa shifted negatively from 0.9?.9 mV in sham group to -13.0?.2 mV in shock group (p<0.01), resulting in the shift of equilibrium potential of potassium, which was consistent with theASMC hyperpolarization in hemorrhagic shock.An excised patch clamp configuration with symmetric high potassium (140 mM) solutions was used to further investigate if the property of BKca changed in hemorrhagic shock and the result showed that neither slope conductance (221 ? and 216? pS respectively) of the channel nor reversal potential was altered in shock group. The voltage dependence of BKca didn't change either, whereas the calcium dependence property of the channel greatly altered. [Ca2+]i causing inactivation of BKca shifted from 106 M in sham group to 10-5 M in shock group. Cytosolic calcium concentration ([Ca+]i) was further detected and the result showed that [Ca2+]i increased apparently from 130.5?5.4 nM in sham group to 202.6?3.6 nM in shock group, indicating that the calcium overload might be involved in the activation of BKCa in hemorrhagic shock.3. The properties of calcium spark and spontaneous outward current (STOC) and their change in hemorrhagic shock. Laser confocal scanning microscopy was used to detect the calcium spark in ASMC and the results showed that calcium spark localized mainly to the submembrane. 83.12?.83% of the cells tested had spontaneous calcium spark in resting conditions. When nifedipine (10 nM), a blocker of L-type voltage dependent calcium channel (L-VOC) and ryanodine(10 #M), an inhibitor of ryanodine receptor (RyR) were applied, only 22.22?.88% and 27.27?.87% of the cells detected had calcium spark respectively, suggesting that the calcium spark was closely related with L-VOC and RyR. The amplitude (F/F0), Dmax, Dave, FDHM, Wmax, Wave and FWHM of calcium spark all increased significantly in hemorrhagic shock (p<0.05, compared with sham group), indicating the elevation of local calcium release from RyR.With perforated whole cell patch clamp techniques, STOC in ASMC was recorded at holding potential of 0 mV, and the results demonstrated that the amplitude, rise time, decay time, duration, and FDHM of STOC increased apparently in hemorrhagic shock (p<0.01, compared with sham group),suggesting the activation of STOC during shock.4. Experimental treatment. To further confi...
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