| A wide variety of studies have indicated that overexpression of inducible nitric oxide synthase (iNOS) with subsequent overproduction of NO plays a pivotal role in the pathogenesis and development of many diseases. However, recent findings have strongly implied that the cytotoxic effects, which were previously attributed to NO, are mainly mediated by ONOO", a product via the rapid reaction between NO and superoxide anion (O2 ~) rather than NO per se. Concurrently, the experimental results from our lab demonstrated that NO contributed vitally to vascular hyporeactivity during severe hemorrhagic and septic shock. A large amout of NO, which was released by a NO donor (SNAP), resulted in the ASMCs hyperpolarization and decreased intracellular calcium with low vasoreactivity. However, the NO action mentioned above could be abolished by pretreatment of tiron, a scavenger of O2 , which indicated that the participant of NO in the pathogenesis of vascular hyporeactivity might be due to ONOO" generation . On the other hand, our and other's results have unanimously revealed that membrane potassium channels activation with subsequent membrane hyperpolarization is crucially involved in vascular hyporeactivity during the late stage of severe shock. If ONOO" itself can decrease vascular reactivity to some extent, one should then consider whether BKca channel activation with membrane hyperpolarziation represents a possible access for ONOO" to exert its deleterious actions. To these ends, we performed a series of experimentswith the aids of some outstanding biological techniques including patch-9-clamp, confocal microscopy and micro-monitoring system. The results are listed below:1. The underlying mechanisms for ONOO" modulation on rat cremaster arteriolar reactivity1) Superfusion of isolated cremaster muscle with authentic ONOO" (0~100M) for 40 minutes induced a conspicuous decrease of arteriolar reactivity (AR) in a dose- and time-dependent manner. The arteriolar reactivity, which was expressed by the negative logarithm value of norepinephrine concentration in producing constriction of creamster muscle arterioles, was monitored at 0, 10th, 20th and 40th minute respectively. Continuous superfusion with Kreb's solution had little effect on AR and the AR values at the above time points were 8.00?.53, 8.43?.43, 8.29?.47 and 8.29?.47 respectively. 20M ONOO could only slightly but not significantly decrease AR and the AR values were 7.56 0.58 6.71 0.89 6.57 1.02 5.56?.93 at the above time points. However, when ONOO" concentration in the perfusion solution was increased to 50M, it could be clearly seen that AR was significantly decreased in a time-dependent fashion with the AR values of 8.33?.29, 5.11?.79 (p<0.05 vs that of 0 min), 3.89?.96 (p<0.01 vs that of 0 min) and 3.56?.86 (p<0.01 vs that of 0 min) at the above time points Washout of ONOO" could partially restore the decreased AR to 6.67?.88. Further elevation of ONOO" to 100M exerted more deleterious effect on AR and the AR values were decreased progressively from 8.30?.21 to 3.25?.62 (p<0.01 vs that of 0 min) at 10th min, 2.62?.63 (p<0.01 vs that of 0 min) at 20th min and 2.00?.56 (p<0.01 vs that of 0 min) at 40th min, whichcould receive partial recovery to 5.50?.10 after ONOO" washout. Bycontrast, treatment with 50 decomposed ONOO" failed to produce any influences on AR. We also found that arteriolar diameter (AD) was not affected by ONOO" administration at any of the above concentrations. The AD values were 42.8 5.4 and 43.5?.4m before and after control supefusion with Kreb's solution, which were 35.3?.7 and 36.3?.4m respectively in 20M ONOO" perfusion group. In addition, even if ONOO" concentration in the prefusate was increased to 50 or 100M, the AR underwent little changes. Prior to 50 or 100M ONOO" treatment, AR values were 44.6?.3 and 39.7?.8um respectively, which were 44.0?.2 and 38.7?0.1 urn posterior to ONOO" application.2) Superfusion with 20M ONOO" for 40 min caused a slight and not significant hyperpolarization in ASMCs. |
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