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Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild-type but not connexin40 null mice in oxidant- and PKA-dependent manner

Michael L. Bolon^*,, Yves Ouellette, Fuyan Li^*, and Karel Tyml^*,,,1

^* Lawson Health Research Institute, London, Ontario, Canada;
Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada;
Department of Pediatrics, Mayo Clinic College of Medicine, Rochester, Minnesota, USA; and
Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada

¹ Correspondence: Lawson Health Research Institute, The Centre for Critical Illness Research, Victoria Research Laboratory, 6th Floor, 800 Commissioners Rd., East London, Ontario N6C 2V5, Canada. E-mail: ktyml{at}lhsc.on.ca

SPECIFIC AIMS

Electrical coupling between cells of the vascular wall plays a key role in control of vascular resistance. We aimed to determine the pathophysiological effect of hypoxia and reoxygenation (H/R) on this coupling, the signaling pathway, and its target protein in a model of cultured microvascular endothelial cells (EC) monolayers derived from mouse skeletal muscle. We focused on the role of the gap junction protein connexin 40 (Cx40), as well as H/R-induced reactive oxygen species (ROS) formation and protein kinase A (PKA) signaling.

PRINICPAL FINDINGS

1. Hypoxia followed by abrupt but not slow reoxygenation reduces electrical coupling between wild-type (WT) mouse microvasular endothelial cells
To assess electrical coupling, we used a current injection technique and Bessel function model to compute the monolayer intercellular resistance (r_i, an inverse measure of cell coupling). Hypoxia (0.1% O₂, 1 h) followed by an abrupt reoxygenation (hypoxic culture medium replaced with normoxic medium) (H/R_a) resulted in a significant increase in r_i (Fig. 1 A, bars 1, 2), without any changes in expression of Cx37, Cx40, and Cx43 (i.e., connexins found in microvasculature ECs), or morphological evidence of cell injury. During 3.5 h of reoxygenation, r_i remained elevated for the first 90 min, but it then gradually returned to control. Significantly, hypoxia (0.1% O₂, 1 h) followed by slow reoxygenation (a procedure intended to generate reduced levels of ROS) did not increase r_i (Fig. 1A , bar 4).

View larger version (17K): [in this window] [in a new window]   Figure 1. Effect of hypoxia followed by abrupt (H/Ra) or slow reoxygenation (H/Rs), deletion of Cx40, and activation/inhibition of PKA activity on intercellular resistance in mouse endothelial cell (EC) monolayers. A) Bars 1 and 2 demonstrate the effect of H (1 h)/Ra (5–60 min) (cells abruptly reoxygenated with fresh normoxic culture medium) on intercellular resistance. Bar 3 shows the same effect of abrupt reoxygenation when the cells were reoxygenated with normoxic medium conditioned by separate cells previously subjected to 1 h hypoxia (H/Rac). Bar 4 shows that hypoxia (1 h) followed by slow reoxygenation (5–60 min) resulted in no change in resistance (slow reoxygenation consisted of introduction of room air oxygen to cell monolayer via diffusion through 2 mm thick unstirred hypoxic cell culture medium). Bars 5 and 6 demonstrate the lack of effect of H (1 h)/Ra (5–60 min) on intercellular resistance in Cx40–/– monolayers. *significant difference from the appropriate control group. P < 0.05, n = 28, 28, 4, 4, 8, and 8 for bars 1–6, respectively. B) Wild-type mouse EC monolayers pretreated with cell permeable cAMP analoge, 8-bromo cAMP (1 mM, 5 min), and subsequently exposed to H (1 h)/Ra (5–60 min) yielded no increase in resistance (bars 7 and 8). Control WT monolayer pretreated with PKA inhibitor 6–22 amide (10 nM, 1 h) yielded an increase in resistance similar to that caused by H/Ra alone (bar 9 vs. 1). #Significant difference from control in bar 1. P < 0.05, n = 4 for each of bars 7–10.

2. Knock out of Cx40 prevents the H/R_a-induced decrease in coupling
Among the 3 connexins, Cx40 plays the central role in vascular cell coupling along the length of the arteriole in vivo. To elucidate the role of Cx40 in the H/R_a-induced decrease in coupling, r_i was determined in EC monolayers derived from Cx40 knockout (Cx40^–/–) mice (littermates to WT mice). Significantly, H/R_a did not alter r_i (Fig. 1A , bars 5, 6), indicating that Cx40 plays a key role in H/R_a-induced reduction in electrical coupling in the monolayer.

3. H/R_a-induced reduction in coupling is oxidant- and PKA-dependent
Since it has been shown that Cx40 protein contains a consensus sequence for PKA, and that PKA activation increases the Cx40 macroscopic gap junctional conductance, we tested for the involvement of PKA pathway in the H/R_a-induced effect on r_i in WT monolayers. Figure 1B (bars 7, 8) shows that activation of PKA by 8-bromo-cAMP (cell permeable cAMP analog) resulted in elimination of H/R_a-induced increase in r_i. Conversely, inhibition of PKA by the peptide inhibitor 6-22 amide mimicked the effect of H/R_a in control normoxic cells (Fig. 1B , bar 9). Data in Fig. 2A (bars 1–3) support the role for PKA signaling in the H/R_a-reduced coupling, since PKA reduction was observed after H/R_a but not after H/R_s treatment (i.e., H/R_s did not alter coupling).

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of hypoxia followed by abrupt or slow reoxygenation, deletion of Cx40, and ascorbate on PKA activity and reactive oxygen species (ROS) formation. A) In both WT and Cx40–/–cells, hypoxia followed by an abrupt reoxygenation significantly decreased PKA activity (bar 2 vs. 1, and bar 5 vs. 4). In contrast, WT cells exposed to hypoxia followed by slow reoxygenation displayed no change in PKA activity (bar 3 vs. 1). Preloading of WT cells with the antioxidant ascorbate (200 µM for 4 h) also showed no change in PKA activity after H/Ra (bar 7 vs. bar 6 or 1). *Significant difference from the appropriate control group. P < 0.05, n = 4–6 monolayers per group in bars 1–7. B) In both WT and Cx40–/–cells, hypoxia followed by abrupt reoxygenation resulted in increased intracellular ROS production. In WT cells, hypoxia followed by slow reoxygenation resulted in no increase in ROS, as compared with control (bar 3 vs. 1). *Significant difference from the appropriate control group. P < 0.05, n = 5 monolayers per group in bars 1–5.

Figure 2B confirms that our H/R_a and H/R_s treatments indeed yielded increased and unchanged levels of ROS, respectively, and underscores the possibility that ROS is the upstream inhibitor of PKA activity, as others have suggested. In the present study, we substantiate this possibility by showing that the antioxidant ascorbate (200 µM, 4 h pretreatment) prevents the H/R_a-mediated decrease in PKA activity (Fig. 2A , bars 6, 7). We have further demonstrated that the H/R_a-reduced coupling is oxidant-dependent, since pretreatment of WT monolayer with ascorbate prevented the effect of H/R_a on intercellular coupling.

H/R_a-induced changes in PKA activity and ROS production were not altered by Cx40 deletion (Fig. 2A, B , bars 5, 6) suggesting that prevention of H/R_a-induced decrease in coupling was due to the absence of Cx40 rather than due to nonspecific alteration in ROS generation or PKA activity caused by the knock out procedure.

4. Ischemia followed by abrupt reperfusion reduces arteriolar conducted response in the mouse cremaster muscle in vivo in PKA-dependent manner
We tested the relevance of our findings in monolayer in vitro, in our model of vascular cell coupling along the arteriole in vivo (i.e., the arteriolar conducted response). KCl puff introduced to the arteriole by micropipette initiated local constriction that conducted along the arteriole. We assessed conduction by measuring diameter at the local site and at 500 µm upstream site. One hour complete ischemia followed by abrupt reperfusion (I/R) significantly reduced conducted response during the first 30 min of reperfusion (response recovered at 60 min). Significantly, PKA activation by 8-bromo cAMP prevented this effect of I/R, while inhibition of PKA mimicked I/R in the control pre-ischemic arteriole and prevented the recovery at 60 min of reperfusion. Thus, the two features tested our in vivo model (i.e., temporary reduction in conduction and PKA dependency) were qualitatively identical to those present in the in vitro model.

CONCLUSIONS AND SIGNIFICANCE

The present study demonstrates for the first time that 1 h hypoxia followed by abrupt, but not slow, reoxygenation reduces electrical coupling between WT mouse microvascular endothelial cells in vitro. This reduction is prevented by the antioxidant ascorbate and by PKA activation with 8-bromo cAMP. Notably, abrupt reoxygenation does not reduce coupling in cells from Cx40^–/– mice. We propose that H/R_a reduces electrical coupling via oxidant- and PKA-dependent signaling that targets Cx40 (Fig. 3 ). Based on two features tested in vivo (time course and PKA dependency of I/R-induced deficit in arteriolar conducted response), our results underscore the physiological significance of these findings and suggest that this mechanism contributes to the compromised arteriolar function after I/R.

View larger version (18K): [in this window] [in a new window]   Figure 3. Proposed model of reduced interendothelial cell electrical coupling following hypoxia and abrupt reoxygenation. Baseline protein kinase A (PKA) activity keeps connexin (Cx) 40-containing gap junctions open to maintain coupling. Quickly after the onset of reoxygenation (within 5 min), elevated production of reactive oxygen species (ROS) inhibits PKA activity and reduces Cx40-dependent coupling. Deletion of Cx40, ROS inhibition, or PKA activation after H/Ra prevents this reduction. PKA inhibition in normoxic WT cells mimics this reduction in coupling.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3446fje; doi: 10.1096/fj.04-3446fje

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