Nitric oxide specifically reduces the permeability of Cx37‐containing gap junctions to small molecules

Abstract

Gap junction intercellular communication (GJIC) plays a significant role in the vascular system. Regulation of GJIC is a dynamic process, with alterations in connexin (Cx) protein expression and post-translational modification as contributing mechanisms. We hypothesized that the endothelial autacoid nitric oxide (NO) would reduce dye coupling in human umbilical vein endothelial cells (HUVECs). In our subsequent experiments, we sought to isolate the specific Cx isoform(s) targeted by NO or NO-activated signaling pathways. Since HUVEC cells variably express three Cx (Cx37, Cx40, and Cx43), this latter aim required the use of transfected HeLa cells (HeLaCx37, HeLaCx43), which do not express Cx proteins in their wild type form. Dye coupling was measured by injecting fluorescent dye (e.g., Alexa Fluor 488) into a single cell and determining the number of stained adjacent cells. Application of the NO donor SNAP (2 mM, 20 min) reduced dye coupling in HUVEC by 30%. In HeLa cells, SNAP did not reduce dye transfer of cells expressing Cx43, but decreased the dye transfer from Cx37-expressing cells to Cx43-expressing cells by 76%. The effect of SNAP on dye coupling was not mediated via cGMP. In contrast to its effect on dye coupling, SNAP had no effect on electrical coupling, measured by a double patch clamp in whole cell mode. Our results demonstrate that NO inhibits the intercellular transfer of small molecules by a specific influence on Cx37, suggesting a potential role of NO in controlling certain aspects of vascular GJIC.

Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function

“…A moderate (twofold) increase in NO prevented the effects of ADMA, whereas a fourfold increase in NO induced endothelial leakage. In agreement with our data, others have reported that small increases in NO promote intercellular junction formation in HUVECs (Kameritsch et al, 2005), whereas a substantial (three-to fourfold) increase causes endothelial leakage (Miyawaki-Shimizu et al, 2006), likely to result from a significant increase in nitration and nitrosylation of proteins (Predescu et al, 2005).…”

Connexin 40 Mediates the Tubuloglomerular Feedback Contribution to Renal Blood Flow Autoregulation

“…27,28 Importantly, this NO influence depends on functional TGF, 27,28 suggesting neuronal NOS in macula densa cells as the source. Given the probable role of Cx40 in TGF signal transduction and the possibility of NO modulation of connexin conductance, 25,39 we postulated that the absence of Cx40 impacts on NO effects on the renal MR. This, however, was not the case, because inhibition of NO production has similar augmenting effects on the MR in wt, Cx40-ko, and Cx40KI45 mice.…”

“…NO is thought to modulate connexin conductance acutely 24 and expression chronically. 25 NO attenuates the magnitude of acute vasoconstriction elicited by receptor agonists such as Ang II and NE. 26 In addition, NO blunts the strength of the pressure-induced MR in RBF autoregulation, an effect specific to the kidney and dependent on JGA function and TGF.…”

Vascular Gap Junctions in Hypertension

“…Gap junctional permeability can be regulated by small ions, such as Ca ϩϩ and H ϩ , 3 phosphorylation, 4,5 and possibly NO. 6,7 Connexin isoforms manifest molecular charge, size, and solute selectivity, 8 -10 and the translation of molecular selectivity into function can be inferred from the fact that deletion of one of the connexin isoforms can result in a unique phenotype that cannot be rescued by insertion or "knock in" of another connexin isoform (eg, see . Hemichannels forming the gap junctions can be composed of mixtures of proteins that vary according to location within the cell.…”