Heme oxygenase-1 plays a pro-life role in experimental brain stem death via nitric oxide synthase I/protein kinase G signaling at rostral ventrolateral medulla

Abstract

Abstract Background Despite its clinical importance, a dearth of information exists on the cellular and molecular mechanisms that underpin brain stem death. A suitable neural substrate for mechanistic delineation on brain stem death resides in the rostral ventrolateral medulla (RVLM) because it is the origin of a life-and-death signal that sequentially increases (pro-life) and decreases (pro-death) to reflect the advancing central cardiovascular regulatory dysfunction during the progression towards brain stem death in critically ill patients. The present study evaluated the hypothesis that heme oxygnase-1 (HO-1) may play a pro-life role as an interposing signal between hypoxia-inducible factor-1 (HIF-1) and nitric oxide synthase I (NOS I)/protein kinase G (PKG) cascade in RVLM, which sustains central cardiovascular regulatory functions during brain stem death. Methods We performed cardiovascular, pharmacological, biochemical and confocal microscopy experiments in conjunction with an experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of adult male Sprague-Dawley rats. Results Western blot analysis coupled with laser scanning confocal microscopy revealed that augmented HO-1 expression that was confined to the cytoplasm of RVLM neurons occurred preferentially during the pro-life phase of experimental brain stem death and was antagonized by immunoneutralization of HIF-1α or HIF-1β in RVLM. On the other hand, the cytoplasmic presence of HO-2 in RVLM neurons manifested insignificant changes during both phases. Furthermore, immunoneutralization of HO-1 or knockdown of ho-1 gene in RVLM blunted the augmented life-and-death signals exhibited during the pro-life phase. Those pretreatments also blocked the upregulated pro-life NOS I/PKG signaling without affecting the pro-death NOS II/peroxynitrite cascade in RVLM. Conclusions We conclude that transcriptional upregulation of HO-1 on activation by HIF-1 in RVLM plays a preferential pro-life role by sustaining central cardiovascular regulatory functions during brain stem death via upregulation of NOS I/PKG signaling pathway. Our results further showed that the pro-dead NOS II/peroxynitrite cascade in RVLM is not included in this repertoire of cellular events.