Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries

Abstract

Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lung's vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 microm from the target site, indicating that Ca2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.

Figure 1. Photolytic uncaging-induced Ca²⁺ responses in rat lung capillaries.

(A) Fluorescence images in pseudocolor of a rat capillary network loaded with fluo-4 and NP-EGTA show endothelial Ca²⁺ level at baseline (left) and after Ca²⁺ uncaging (right). Dashed lines and circle in baseline image represent capillary margins and uncaging target, respectively. Arrowheads indicate measurement sites on venules. The distance between measurement and uncaging sites was determined along the vascular length (dotted line). (B) Tracings show temporal decay of endothelial Ca²⁺ level at the uncaging site (black) and in a responding venule (red). (C) Filled circles represent mean Ca²⁺ responses (± SEM) to uncaging at indicated distances from the uncaging site. Each mean was higher than the baseline endothelial Ca²⁺ value (79 ± 10 nM). Line calculated by exponential regression of means (r² = 0.99). Values predicted by a computational model of diffusion (open triangles) were not significantly different from experimental values. The dashed line indicates 50% of initial. *P < 0.05 compared with baseline. (D) Tracings show responses to repeated uncaging in a capillary. As indicated, NP-EGTA was loaded after the first response (no. 1). Response nos. 1–3 were obtained at the same capillary site. Response no. 4 was obtained at a different site. The experiment was replicated 3 times. (E) Plot of fluo-4 fluorescence intensity against Ca²⁺ concentration determined at identical endothelial locations. Solid line calculated by linear regression (r² = 0.42). Dashed lines indicate 95% confidence intervals. Data are from 5 capillaries coloaded with fluo-4 and fura-2.

Figure 2. Distribution and functional role of Cx43 in mouse lung (WT, wild-type mice;Cx43^–/– , endothelium-specific Cx43-knockout mice).

(A and B) Three-micrometer confocal slices of Cx43 immunofluorescence show punctuate distribution. Lines indicate capillary and alveolar margins. n = 3. (C) Pseudocolor images show endothelial fluo-4 fluorescence after Ca²⁺ uncaging. Pseudocolor was red-orange at baseline (data not shown) and changed to green after uncaging. Note that the green pseudocolor is spatially extensive in WT but localized in Cx43^–/–. Dashed lines and circle represent vessel margins and the uncaging target, respectively. (D) Group data show responses to uncaging. Baseline endothelial Ca²⁺ levels (84 ± 12 nM) were similar in the 2 groups. Mean ± SEM; n = 3. *P < 0.05 compared with baseline.

Figure 4. Role of intracellular organelles in uncaging-induced Ca²⁺ responses in rat capillaries.

(A) Traces show mitochondrial Ca²⁺ responses in microvessels loaded with rhod-2. n = 4. (B) Uncaging-induced endothelial Ca²⁺ responses in capillaries and venules treated with the inhibitors t-BHQ and XeC. n = 3. The baseline Ca²⁺ level was 76 ± 10 nM. The control response at the uncaging site was an increase of 45 ± 5 nM after uncaging. Mean ± SEM; n = 3. *P < 0.05 compared with baseline. ^†P < 0.05 compared with respective responses at 0 μm from the uncaging site.

Figure 5. P-selectin expression in rat lungs.

(A) Images show venular P-selectin immunofluorescence before (left) and after (right) uncaging. The circle and lines represent the uncaging target and vessel margins, respectively. (B) Group data show expression in alveolar capillaries at the uncaging site (AC) and in venules located 100 μm distant. 2y, secondary Ab only; IgG, isotype-matched IgG to P-selectin Ab; KO, P-selectin–knockout mice. Mean ± SEM. n = 4. *P < 0.05 compared with the far-left bar. (C) Uncaging-induced expression in the presence of gap and scrambled gap peptides. Mean ± SEM. n = 4. *P < 0.05 compared with baseline.

Figure 6. Thrombin-induced increase in lung microvascular permeability in mice.

Thrombin doses were 2.5 (t-2) and 5.0 U/ml (t-5). Data show thrombin-induced responses at different thrombin concentrations (A), in gap or scrambled gap peptide–treated lungs (B), and in lungs of P-selectin–knockout mice (C). n = 4 for each bar in A and B and n = 3 for each bar in C. Data represent mean ± SEM. *P < 0.05 compared with baseline. Bas, baseline.

Figure 3. Inhibition of Ca²⁺ conduction in lung capillaries.

Shown are responses to uncaging in alveolar capillaries, before, during (Gap), and after infusion of gap26/27. (A and B) Data show attenuation of Ca²⁺ levels with distance from uncaging site (baseline endothelial Ca²⁺ level was 76 ± 5 nM; n = 4 for each bar) and responses to scrambled gap26/27 (scgap) showing no inhibition (baseline endothelial Ca²⁺ level was 79 ± 10 nM; n = 3 for each bar). (C) Data show uncaging-induced responses during infusion of gap26/27 in capillaries (Cap) and venules (Ven). Capillary and venular sites were 100 μm apart. Note that the lack of venular response to capillary uncaging (second bar) was rescued by direct venular uncaging (third bar). The baseline endothelial Ca²⁺ level was 80 ± 4 nM; n = 4 for each bar. Mean ± SEM. *P < 0.05 compared with baseline.