Compartmentalized Connexin 43 S -Nitrosylation/Denitrosylation Regulates Heterocellular Communication in the Vessel Wall

Abstract

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...

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…This method allows for visualization of large areas of antigen distribution. In agreement with our immuno-TEM data, there were no pannexin isoforms detectable; however, Cx43, as previously found to be located at MEJs via quantified immuno-TEM [215]; as used above), was localized to these holes In all images, green is autofluorescence of matrix proteins and red blood cells (E-H), red is the pannexin isoform of interest, and blue represents DAPI stained nuclei. Scale bars in A and E are both 20 μm.…”

“…The biotin switch assay was performed as previously described [215,216]. Briefly, cell monolayers were treated with 100 µM GSNO, 50 µM DEANONOate or vehicle for 10 minutes at 37°C.…”

“…The biotin switch assay was performed as previously described [215,216]. Briefly, cell monolayers were treated with 100 µM GSNO, 50 µM DEA- Immunofluorescence microscopy: Transfected HEK cells were fixed in 4% PFA for 15 minutes and subjected to standard immunocytochemistry as previously described [11].…”

“…even a single cysteine residue dramatically altering protein activity [215,251]. This modification is known to regulate the activity of several membrane channels including, among others, connexin43 (Cx43) gap junctions and hemichannels [215,252], the cardiac slowly activating delayed rectifier potassium channel KCNQ1 [253], the transient receptor potential channel TRPC5 [254] and the ryanodine receptor type 1 [251].…”

The cellular life of pannexins

“…However, it remains to be determined if cysteine residues of Panx1 are bona fide targets of S-nitrosylation as it appears to be the case for Cx43. 34 …”