Many hereditary and sporadic neurodegenerative disorders are characterized by the accumulation of aberrant proteins. In sporadic Parkinson's disease, representing the most prevalent movement disorder, oxidative and nitrosative stress are believed to contribute to disease pathogenesis, but the exact molecular basis for protein aggregation remains unclear. In the case of autosomal recessive-juvenile Parkinsonism, mutation in the E3 ubiquitin ligase protein parkin is linked to death of dopaminergic neurons. Here we show both in vitro and in vivo that nitrosative stress leads to S-nitrosylation of wild-type parkin and, initially, to a dramatic increase followed by a decrease in the E3 ligase-ubiquitin-proteasome degradative pathway. The initial increase in parkin's E3 ubiquitin ligase activity leads to autoubiquitination of parkin and subsequent inhibition of its activity, which would impair ubiquitination and clearance of parkin substrates. These findings may thus provide a molecular link between free radical toxicity and protein accumulation in sporadic Parkinson's disease

The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro. In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression. Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia. Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia. Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia. Transient transfection of cells stimulated with interleukin-1beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.

Objective-To determine whether S-nitrosylation of connexins (Cxs) modulates gap junction communication between endothelium and smooth muscle. Methods and Results-Heterocellular communication is essential for endothelium control of smooth muscle constriction; however, the exact mechanism governing this action remains unknown. Cxs and NO have been implicated in regulating heterocellular communication in the vessel wall. The myoendothelial junction serves as a conduit to facilitate gap junction communication between endothelial cells and vascular smooth muscle cells within the resistance vasculature. By using isolated vessels and a vascular cell coculture, we found that Cx43 is constitutively S-nitrosylated on cysteine 271 because of active endothelial NO synthase compartmentalized at the myoendothelial junction. Conversely, we found that stimulation of smooth muscle cells with the constrictor phenylephrine caused Cx43 to become denitrosylated because of compartmentalized S-nitrosoglutathione reductase, which attenuated channel permeability. We measured S-nitrosoglutathione breakdown and NO x concentrations at the myoendothelial junction and found S-nitrosoglutathione reductase activity to precede NO release. Key Words: NO Ⅲ GSNO-R Ⅲ connexin Ⅲ myoendothelial junction Ⅲ nitrosylation W ithin the vessel wall of resistance arteries, coordinated vascular smooth muscle cell (SMC) and endothelial cell (EC) function is integrated by complex intercellular signaling to regulate the constriction and dilation of the artery. The anatomic structures that facilitate direct SMC and EC communication within the resistance artery are myoendothelial junctions (MEJs), which are cellular extensions from ECs or SMCs that project through the internal elastic lamina [1][2][3] and link the plasma membranes of the 2 different cell types together. The gap junctions (GJs) at the MEJ provide a conduit for second messenger and electric signaling between the 2 cell types. 2,4,5 For example, phenylephrine (PE) stimulation of SMCs induces inositol 1,4,5-triphosphate (IP 3 ) generation and an increase in [Ca 2ϩ ] i concentrations, constricting the artery. It is thought that the IP 3 progresses to the adjacent EC through GJs at the MEJ, initiating an increase in [Ca 2ϩ ] i and the release of NO to modulate the magnitude of vasoconstriction, thereby regulating the tone of the artery. 6 -8 Elucidation of the mechanisms regulating this process could provide novel insight into blood pressure regulation; however, the process remains uncharacterized. Conclusion-This study provides evidence for compartmentalized S-nitrosylation/denitrosylationGJs are intracellular signaling channels formed by 2 hexameric hemichannels, with each adjacent cell contributing 1 hemichannel. Connexin (Cx) proteins compose the channels, of which 4 different Cxs have been identified in the vasculature, with multiple studies demonstrating a potentially important role for Cx43 at the MEJ. 9 Recent studies have demonstrated that GJ communication and trafficking of Cx43 are mod...