S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: induction by oxidant stress and reversal by reducing agents.
Marked increase in cell permeability ascribed to open connexin (Cx)43 hemichannels is induced by metabolic inhibition (MI) of cortical astrocytes in culture, but the molecular mechanisms are not established. Dephosphorylation and/or oxidation of Cx43 hemichannels was proposed as a potential mechanism to increase their open probability. We now demonstrate that MI increases the number of hemichannels on the cell surface assayed by biotinylation and Western blot, and that this change is followed by increased dephosphorylation and S-nitrosylation. The increase in rate of dye uptake caused by MI is comparable to the increase in surface expression; thus, open probability and permeation per hemichannel may be unchanged. Reducing agents did not affect dephosphorylation of Cx43 hemichannels but reduced dye uptake and S-nitrosylation. Uptake was also reduced by elevated intracellular but not extracellular levels of reduced glutathione. Moreover, nitric oxide donors induced dye uptake and nitrosylation of surface Cx43 but did not affect its abundance or phosphorylation. Thus, permeability per channel is increased, presumably because of increase in open probability. We propose that increased dye uptake induced by MI is mediated by an increased number of Cx43 hemichannels in the surface and is associated with multiple molecular changes, among which nitrosylation of intracellular Cx43 cysteine residues may be a critical factor.
Departamento de Ciencias Fisiológicas, Pontificia Universidad Católica de Chile, Santiago 6513492, Chile.
Mauricio A
MA
Retamal
Constanza J
CJ
Cortés
Luis
L
Reuss
Michael V L
MV
Bennett
Juan C
JC
Sáez
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport; D019921:Biotinylation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D018031:Connexin 43 / Q000032:analysis / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D018384:Oxidative Stress; D010766:Phosphorylation; D051381:Rats; D019163:Reducing Agents / Q000494:pharmacology
D018031:Connexin 43; D020030:Nitric Oxide Donors; D019163:Reducing Agents; D005978:Glutathione; D003545:Cysteine