Brain stem cardiovascular regulatory dysfunction during brain stem 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 stem death ensues. At the same time, the ubiquitin-proteasome system (UPS) is involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain stem death by engaging in both synthesis and degradation of NOS II in RVLM. In a clinically relevant experimental model of brain stem 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 or 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-£eB or binding between NF-£eB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated I£eB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-£eB 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. We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain stem 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.

Mevinphos (Mev) is an organophosphate insecticide that acts on the rostral ventrolateral medulla (RVLM), the origin of sympathetic vasomotor tone, to induce cardiovascular responses. This study investigated the role of Trk (tropomyosin-related kinase) (receptor form) or Src (non-receptor form) tyrosine kinase at the RVLM in Mev-induced cardiovascular responses. Bilateral microinjection of Mev (10 nmol) into the RVLM elicited two distinct phases of cardiovascular responses, designated Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. Western blot assay showed that whereas p-Trk490 was increased during Phase I, p-Src416 was increased only during Phase II Mev intoxication. Interestingly, application of a Trk specific inhibitor (K252a; 1 pmol) or Src specific inhibitor (SU6656; 100 pmol) into the bilateral RVLM blunted the Mev-elicited sympathoexcitatory or sympathoinhibitory effect, respectively. Besides, K252a was limited to block NOS I protein expression in the RVLM during Mev intoxication, SU6656 only inhibited NOS II protein expression in the RVLM during Mev intoxication. We conclude that Trk tyrosine kinase (p-Trk490) in the RVLM participates in the Phase I cardiovascular responses during Mev intoxication, Src tyrosine kinase (p-Src416) in the RVLM participates in the Phase II cardiovascular responses associated with Mev intoxication.