As illustrated in the previous section, astrocytes along with many other functions participate in CNS inflammatory responses, both as instigator and effector. The major point to be illustrated in this section is that inflammatory stimuli of different kinds can summate to cause connexin and pannexin hemichannels to open. The data are from published studies (Orellana et al., 2010
Hypoxia glucose permeabilizes cortical astrocytes in culture by opening connexin hemichannels, and this effect is greater in abnormal glucose. In these experiments the cells are subjected to different periods of hypoxia in glucose concentrations ranging from zero to normal (5 mM) to high (27 – 37 mM) followed by reoxygenation, that is restoration of oxygenated normal saline solution with normal glucose (corresponding to “reperfusion” in situ
). Here there are two stressors, hypoxia and abnormal glucose during the hypoxia. Injury is measured as uptake of ethidium ions (Etd+
) through opening of connexin hemichannels, or after greater stress, uptake of labeled dextran, ascribed to membrane breakdown in dying or dead cells. The medium during hypoxia is an “ischemic saline”, which mimics the ionic milieu during cessation of blood flow in the CNS and is lower in Na+
and higher in K+
than normal medium (Bondarenko and Chesler, 2001
) [and normal medium during the hypoxia prevents most or all of the effects to be described]. Astrocytes subject to 3 h hypoxia in normal (5mM) glucose show no signs of damage after reoxygenation (, ). After 3 h hypoxia in high glucose, astrocytes are transiently permeable to Etd+
after 1 h reoxygenation and are fully recovered after 3 h (, ). The uptake is through Cx43 hemichannels, because the permeability increase is absent in astrocytes from (germ line) Cx43 KO mice and is reduced by connexin but not pannexin hemichannel blockers (Gap26, , Gap27 or La3+
, but not 10
panx1 or the purinergic blocker, oATP). (The point of the purinergic blocker is that activation of P2X7 receptors can lead to opening of pannexin hemichannels.) If the hypoxic exposure is for 6 h in normal glucose, there is still little change during the hypoxic period, but uptake increases to a plateau at 1 to 2 h reoxygenation (). 6 h hypoxia in high glucose produces some increase during the hypoxic period and then rise to a higher plateau during reoxygenation. In each case hypoxia in low glucose causes permeabilization that is greater than in normal glucose and not so great as in high glucose (). The relation between glucose concentration during hypoxia and dye uptake rate after 1 h reoxygenation is U-shaped (). With a 3 h hypoxic period, elevated glucose for 24 h before or 1h after has no effect on the Etd+
uptake (not illustrated); thus, the effect of abnormal glucose is initiated during the hypoxic period and does not require it before or after.
Permeabilization of astrocytes after 3 h hypoxia in high or normal glucose in “ischemic saline” followed by reoxygenation in normal glucose in normal saline.
Fig. 6 Influence of microglia on hypoxia/reoxygenation-induced permeabilization of astrocytes. A. 3h hypoxia in high (but not normal) glucose transiently increases Etd+ uptake during reoxygenation. B. Co-culture with microglia prevents the increase in Etd+ uptake (more ...)
As would be expected, prolonging the hypoxia further stresses cortical astrocytes. Although they survive for at least 6 h reoxygenation after 3 h hypoxia in high glucose, 6 h hypoxia and 6 h reoxygenation kills a fraction of the cells in normal glucose and many more of the cells in high glucose (). Cx43 hemichannels are implicated in cell death, because the cells are protected by connexin hemichannel blockers and not by pannexin hemichannel blockers (). An inhibitor of p38 MAPK, SB202190, applied before the hypoxia in high glucose, reduces dye uptake after 6 h reoxygenation and prevents cell death () and reduction in dye coupling (not shown); this fragmentary observation points to molecular mechanisms ultimately leading to cell death.
Fig. 5 Prolonged hypoxia causes death of astrocytes with greater mortality after hypoxia in high glucose. A – F. Micrographs of astrocyte cultures subjected to the indicated periods of hypoxia and concentrations of glucose followed by 6 h reoxygenation (more ...)
The increases in dye uptake are associated with increase in surface expression of Cx43 as determined by biotinylation and western analysis, while total Cx43 is unchanged (Orellana et al., 2010
). [The recovery of permeability following the transient increase in uptake caused by 3 h hypoxia in high glucose is associated with return to basal surface expression.] The increase in surface expression (to ~3 times control) is not quite sufficient to account for the increased uptake (to ~6 times control), and an increase in open probability and/or increase in permeability per open channel may occur. In astrocytes subjected to metabolic inhibition, insertion of hemichannels measured by biotinylation without change in open probability can account for the entire increase in dye uptake rate (Retamal et al., 2006
). The loss of coupling in the insulted astrocytes is associated with reduction in the number of large Cx43 immunoreactive plaques, presumptive gap junctions, and increase in the number of small immunoreactive puncta likely to represent trafficking vesicles associated with internalization, as well as supplying new hemichannels for cell-cell channel formation (Orellana et al., 2010
). The loss of large Cx43 plaques after hypoxia/reoxygenation suggests that the decrease in coupling is contributed to by internalization of junctions, but there could also be a contribution from closing of the cell-cell channels. With the Cx43 antibody used, we did not visualize the increased surface expression indicated by biotinylation; the distribution in the membrane could well have been too diffuse to detect with our methods. Although the increase of surface permeability is paralleled by decrease in coupling, the change in permeability is unlikely to be due to separation of cell-cell channels to form hemichannels connected to the external milieu. In general connexin cell-cell channels do not split into two hemichannels facing the extracellular space (although innexin/pannexin channels may do so, Lane and Swales, 1978
), but small clusters of channels are internalized into one cell as double walled vesicles that are transported to lysosomes for degradation; the vesicles include both cell membranes and a small amount of cytoplasm from the apposed cell (“annular gap junctions”, Gaietta et al., 2002
Connexin hemichannel blockers have no discernable effect on the ischemia-induced decrease in coupling indicating that increased leak of dye through these hemichannels does not cause the decrease in dye coupling (see ). (The > 50% decrease in dye coupling associated with 6 h Gap26 treatment without hypoxia likely results from block of cell-cell channel formation with continuing internalization of existing channels. The decrease caused by Gap26 treatment does not add to the decrease caused by 6 h hypoxia in high glucose followed by 6 h reoxygenation; this observation raises the possibility of occlusion in the uncoupling processes.)
Dye uptake after 6 h hypoxia in high glucose and 1 h reoxygenation is reduced within minutes by DTT applied during dye application (Orellana et al., 2010
), consistent with previous results with metabolic inhibition (Retamal et al., 2007a
) and suggesting oxidation of the hemichannels or an associated molecule as a result of hypoxia/reoxygenation. [DTT increases dye uptake in control cells and little effect after 3 h hypoxia in high glucose and 1 h reoxygenation.] DTT does not reverse the reduction in dye coupling over the same time course. The differential effect of DTT on permeability and dye coupling is consistent with internalization of the gap junctions as the cause of uncoupling. The inhibitor of p38 MAPK, SB202190, applied before hypoxia in high glucose, reduces dye uptake after 1 h reoxygenation and but does not prevent reduction in dye coupling between cells, again indicating different mechanisms.
To address the question of whether the action of hypoxia/reoxygenation on Cx43 in astrocytes was cell type specific, we examined Etd+
uptake in HeLa cells transfected with Cx43-EGFP (Orellana et al., 2010
). As observed previously (Contreras et al., 2002
), rate of uptake in control conditions is proportional to amount of Cx43-EGFP expressed. Rate of uptake is increased by 6 h hypoxia, and the degree of permeabilization is greater in 27 mM than in 5 mM glucose. The magnitude is ~5 fold and comparable to the effects in astrocytes. However, the permeability increase is maximal immediately after reoxygenation and presumably develops during the hypoxia. Moreover, the permeability returns to near normal in ~2 h reoxygenation, in contrast to the delayed and longer lasting permeability increases in astrocytes. The resilience of HeLa cells has been well documented and may reflect their tumor origin and years of selection in laboratories (Anderson et al., 2006
; Skloot, 2010
Now we introduce microglia. Co-culture of cortical astrocytes with microglia for 24 h prior to 3h hypoxia in high glucose prevents the transient rise in astrocyte permeability that occurs in the absence of microglia; the microglia appear to protect the astrocytes (). Contrasting results are obtained when astrocytes are co-cultured for 24 h with microglia and a toxic peptide fragment of amyloid precursor protein, Aβ25–35
, prior to 3h hypoxia in high glucose; Etd+
uptake after reoxygenation is greatly increased and prolonged (). If the hypoxia is in normal glucose, uptake is also increased, but to a lesser extent. Aβ25–35
is another potential stressor (Pike et al., 1995
), but it is acting through the microglia and has little direct effect on the astrocytes (compare to ). Medium conditioned by 24 h culture of microglia in the presence of Aβ25–35
(CM-Aβ) has the same effect as co-culture with microglia and the peptide (). The conclusion is that Aβ causes microglia to release a toxic substance or substances that sensitize astrocytes to the hypoxia in hyperglycemic conditions. Activated microglia are known to release TNF-α and IL-1β (Retamal, 2007b
), and 24 h treatment with these agents mimics the effect of microglia plus Aβ or of CM-Aβ (). Moreover, blocking agents indicate that the entire effect of the activated microglia is due to the summated actions of TNF-α and of IL-1β alone. Not one of the 24 h pretreatments by itself permeabilizes the cells (not shown).
Again, the permeabilization is due to opening of Cx43 hemichannels. None of the protocols increase permeability in astrocytes from Cx43 KO mice. For CM-Aβ and TNF-α + IL-1β treatments, connexin hemichannel blockers prevent the increases in permeability, as well as reduce the basal permeability in control cells. None of the Px1 hemichannel blockers are effective.
As above, the increases in astrocyte dye uptake are associated with increased surface expression of Cx43.
As above, in all of the conditions in which astrocytes are permeabilized, dye coupling is decreased with a similar time course and magnitude. Moreover, block of hemichannels does not increase the dye coupling, indicating that the reduced coupling is not due to leak from hemichannels.