A Double-Edged Sword Role for Ubiquitin-Proteasome System in Brain Stem Cardiovascular Regulation During Experimental Brain Death

Abstract

BackgroundBrain stem cardiovascular regulatory dysfunction during brain death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain death ensues. Furthermore, the ubiquitin-proteasome system (UPS) may be involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain death by engaging in both synthesis and degradation of NOS II in RVLM.Methodology/Principal FindingsIn a clinically relevant experimental model of brain death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1) potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-κB or binding between NF-κB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated IκB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-κB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated.Conclusions/SignificanceWe conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.

Sumoylation of IkB attenuates NF-kB-induced nitrosative stress at rostral ventrolateral medulla and cardiovascular depression in experimental brain death

“…Figure 1 showed that, similar to our previous studies [ 29 , 30 , 36 ], intravenous administration of LPS (10 mg kg −1 ) in our control animals pretreated with microinjection bilaterally into RVLM of normal serum (1:20) elicited a reduction (Phase I), augmentation (Phase II; pro-life phase) and a secondary decrease (Phase III; pro-death phase) in the power density of the BLF component of AP signals. MAP underwent typically a significant decrease during Phase I, followed by a rebound (Phase II) and progressive hypotension during Phase III.…”

“…An experimental endotoxemia model of brain death [ 21 ], which mimics clinically the progression towards brain death in patients died of systemic inflammatory response syndrome [ 23 ] was used. Escherichia coli lipopolysaccharide (LPS; 0111:B4 strain; InvivoGen, San Diego, CA) was administrated intravenously at 10 mg kg −1 [ 30 ], with saline serving as the vehicle control. Temporal changes in pulsatile AP, mean AP (MAP), HR and power density of the BLF component were routinely followed for 300 min, or until the animal succumbed to endotoxemia.…”

“…It follows that this brain stem site is a suitable neural substrate for mechanistic delineation of brain death [ 21 ]. In an endotoxemia model of brain death, our laboratory showed previously that the progression towards brain death is causally related to transcriptional upregulation of nitric oxide synthase II (NOS II) induced by NF-kB in the RVLM [ 28 – 30 ]. The eventual fatal culprit is nitrosative stress elicited by the formation of peroxynitrite via a reaction between NOS II-produced nitric oxide (NO) and superoxide anion in the RVLM, of which underpins the loss of BLF power that precedes hypotension and asystole during the progression towards brain death [ 30 , 31 ].…”

Nitrosative Stress-Induced Disruption of Baroreflex Neural Circuits in a Rat Model of Hepatic Encephalopathy: A DTI Study

“…In addition, the high level of glutamine-derived ammonia within mitochondria reportedly brings about generation of ROS/RNS or cerebral RNA oxidation743. It should be pointed out that previous work from our laboratory16171819202122232425 emphasizes that it is nitrosative stress induced by peroxynitrite in key nuclei of the baroreflex circuit, rather than oxidative stress, that underpins defunct baroreflex-medicated sympathetic vasomotor tone, leading to brain death. This notion is again substantiated in the present study.…”

“…Nonetheless, under pathological conditions, our laboratory has demonstrated in comatose patients that irreversible loss of baroreflex-mediated sympathetic vasomotor tone is a hallmark for brain death12131415. We further identified that nitrosative stress in key nuclei of the neural circuit is the culprit for the defunct baroreflex16 in animal models of brain death16171819202122232425. Intriguingly, patients with alcoholic cirrhosis exhibit reduced baroreflex sensitivity26, and the degree of decreased HR variability is related to the severity of HE in patients with cirrhosis27.…”