Xu Hao-Liang, Gavrilyuk Vitaliy, Wolde Hailemariam M, Baughman Verna L, Pelligrino Dale A
Neuroanesthesia Research Laboratory, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Am J Physiol Heart Circ Physiol. 2004 May;286(5):H2020-7. doi: 10.1152/ajpheart.01105.2003. Epub 2004 Jan 8.
Multidrug resistance protein 5 (MRP5) has been linked to cGMP cellular export in peripheral vascular smooth muscle cells (VSMCs) and is widely expressed in brain vascular tissue. In the present study, we examined whether knockdown of MRP5 in pial arterioles [via antisense oligodeoxynucleotide (ODN) applications] affected nitric oxide (NO)/cGMP-induced dilations. The antisense or (as a control) missense ODN was applied to the cortical surface approximately 24 h before study via closed cranial windows. The efficacy of the antisense vs. missense ODN in eliciting selective reductions in MRP5 expression was confirmed by analysis of MRP5 mRNA in pial tissue. Unexpectedly, in initial studies, a significantly lower maximal pial arteriolar diameter increase in the presence of the NO donor S-nitrosoacetylpenicillamine (SNAP) was seen in the antisense vs. missense ODN-treated rats (35 vs. 48% diameter increase, respectively). It was suspected that this related to a reduced vascular smooth muscle cell sensitivity to cGMP due to prolonged exposure to increased intracellular cGMP levels elevated by overnight restriction of cGMP efflux. That postulate was supported by a finding of a diminished vasodilating response to the cGMP-dependent protein kinase-activating cGMP analog 8-p-chlorophenylthio-cGMP in antisense vs. missense ODN-treated rats. To prevent desensitization, additional rats were studied in the presence of chronic NOS inhibition via Nomega-nitro-L-arginine. In the NO synthase (NOS)-inhibited rats, the maximal SNAP response was much higher in the antisense (62% increase) vs. the missense ODN (40% increase) group. A similar result was obtained when monitoring responses to the soluble guanylyl cyclase-activating drugs YC-1 and BAY 41-2272. Moreover, in the presence of NOS inhibition, the normal SNAP-induced rise in periarachnoid cerebrospinal fluid cGMP levels, which reflects cGMP efflux, was absent in the antisense ODN-treated rats, a finding consistent with loss of MRP5 function. In conclusion, if one minimizes the confounding effects of basal cGMP production, a clearer picture emerges, one that indicates an important role for MRP5-mediated cGMP efflux in the regulation of NO-induced cerebral arteriolar relaxation.
多药耐药蛋白5（MRP5）与外周血管平滑肌细胞（VSMC）中的环磷酸鸟苷（cGMP）细胞输出有关，并且在脑血管组织中广泛表达。在本研究中，我们研究了通过反义寡脱氧核苷酸（ODN）应用敲低软脑膜小动脉中的MRP5是否会影响一氧化氮（NO）/cGMP诱导的血管舒张。在研究前约24小时，通过闭合的颅骨视窗将反义或（作为对照）错义ODN应用于皮质表面。通过分析软脑膜组织中的MRP5 mRNA证实了反义ODN与错义ODN在引起MRP5表达选择性降低方面的效果。出乎意料的是，在初步研究中，在反义ODN处理的大鼠与错义ODN处理的大鼠中，在存在NO供体S-亚硝基乙酰青霉胺（SNAP）的情况下，软脑膜小动脉最大直径增加明显更低（分别为直径增加35%和48%）。怀疑这与由于通过过夜限制cGMP流出而使细胞内cGMP水平升高导致血管平滑肌细胞对cGMP的敏感性降低有关。这一假设得到了以下发现的支持：在反义ODN处理的大鼠与错义ODN处理的大鼠中，对cGMP依赖性蛋白激酶激活的cGMP类似物8-对氯苯硫基-cGMP的血管舒张反应减弱。为了防止脱敏，在存在通过Nω-硝基-L-精氨酸进行慢性一氧化氮合酶（NOS）抑制的情况下，对另外的大鼠进行了研究。在NOS抑制的大鼠中，反义组（增加62%）的最大SNAP反应比对错义ODN组（增加40%）高得多。在监测对可溶性鸟苷酸环化酶激活药物YC-1和BAY 41-2272的反应时也获得了类似的结果。此外，在存在NOS抑制的情况下，反义ODN处理的大鼠中不存在正常的SNAP诱导的蛛网膜下腔脑脊液cGMP水平升高，这反映了cGMP流出，这一发现与MRP5功能丧失一致。总之，如果将基础cGMP产生的混杂效应降至最低，就会出现更清晰的情况，即表明MRP5介导的cGMP流出在调节NO诱导的脑动脉舒张中起重要作用。
