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Ischemia-induced apoptosis of intestinal epithelial cells correlates with altered integrin distribution and disassembly of F-actin triggered by calcium overload.

Jia Z, Chen Q, Qin H - J. Biomed. Biotechnol. (2012)

Bottom Line: Results.Verapamil attenuated both changes of integrins and F-actin.Conclusions.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China.

ABSTRACT
The present study examined intestinal epithelial cell (IEC) integrin distribution and disassembly of actin cytoskeleton in response to ischemia-anoxia. Protective effects of calcium channel blocker(CCB) were further examined to explore underlying mechanisms of cellular injury. Materials and Methods. Primary cultures of rat IECs and an in vitro model of ischemia/anoxia were established. IECs were exposed to ischemia/anoxia in the presence and absence of verapamil. The extent of exfoliation was determined using light microscopy while apoptosis rate was measured using flow cytometry. Changes in intracellular calcium, the distribution of integrins and the morphology of F-actin were assessed by confocal microscopy. Results. Detachment and apoptosis of IECs increased following ischemia/anoxia-induced injury. Treatment with verapamil inhibited the detachment and apoptosis. Under control conditions, the strongest fluorescent staining for integrins appeared on the basal surface of IECs while this re-distributed to the apical membrane in response to ischemic injury. Depolymerization of F-actin was also observed in the injured cells. Verapamil attenuated both changes of integrins and F-actin. Conclusions. Redistribution of integrins and disruption of F-actin under ischemia/anoxia injury is associated with IEC detachment and increased apoptosis. These events appeared to be triggered by an increase in Ca(2+)(i) suggesting a potential use for CCB in prevention and treatment of intestinal injury.

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(a) Fluorescence images of integrin α3 distribution (A: control group; B: injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (b) Fluorescence images of integrin α5 distribution (A: control group; B: Injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (c) Fluorescence images of integrin β1 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (d) Fluorescence images of integrin β5 distribution (A: Control group B: Injury group C: Verapamil group Planes 1→5: basal surface→apical surface; scale bar = 10 μm). (e) Fluorescence images of integrin α2 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm).
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fig5: (a) Fluorescence images of integrin α3 distribution (A: control group; B: injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (b) Fluorescence images of integrin α5 distribution (A: control group; B: Injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (c) Fluorescence images of integrin β1 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (d) Fluorescence images of integrin β5 distribution (A: Control group B: Injury group C: Verapamil group Planes 1→5: basal surface→apical surface; scale bar = 10 μm). (e) Fluorescence images of integrin α2 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm).

Mentions: In the control group, α3, α5, β1, and β5 integrins in the first z-stack section showed the strongest fluorescence intensity, consistent with these subunits being predominantly located on the basal surface of IECs. In contrast, the apical plasma membrane showed only a slight fluorescence for these integrin subunits. After ischemia/anoxia injury, the fluorescence intensity on this first layer diminished while the intensity on the other layers increased, especially on the top-most layer which indicated these subunits altered their original distribution, moving to the apical surface of the cells. In verapamil-treated cells, the strongest fluorescence intensity was again located toward the basal surface demonstrating an inhibitory effect of the Ca2+ channel blocker on integrin redistribution (Figure 5, Table 3).


Ischemia-induced apoptosis of intestinal epithelial cells correlates with altered integrin distribution and disassembly of F-actin triggered by calcium overload.

Jia Z, Chen Q, Qin H - J. Biomed. Biotechnol. (2012)

(a) Fluorescence images of integrin α3 distribution (A: control group; B: injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (b) Fluorescence images of integrin α5 distribution (A: control group; B: Injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (c) Fluorescence images of integrin β1 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (d) Fluorescence images of integrin β5 distribution (A: Control group B: Injury group C: Verapamil group Planes 1→5: basal surface→apical surface; scale bar = 10 μm). (e) Fluorescence images of integrin α2 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm).
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fig5: (a) Fluorescence images of integrin α3 distribution (A: control group; B: injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (b) Fluorescence images of integrin α5 distribution (A: control group; B: Injury group; C: verapamil-treated group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (c) Fluorescence images of integrin β1 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm). (d) Fluorescence images of integrin β5 distribution (A: Control group B: Injury group C: Verapamil group Planes 1→5: basal surface→apical surface; scale bar = 10 μm). (e) Fluorescence images of integrin α2 distribution (A: control group; B: injury group; C: verapamil group; planes 1→5: basal surface→apical surface; scale bar = 10 μm).
Mentions: In the control group, α3, α5, β1, and β5 integrins in the first z-stack section showed the strongest fluorescence intensity, consistent with these subunits being predominantly located on the basal surface of IECs. In contrast, the apical plasma membrane showed only a slight fluorescence for these integrin subunits. After ischemia/anoxia injury, the fluorescence intensity on this first layer diminished while the intensity on the other layers increased, especially on the top-most layer which indicated these subunits altered their original distribution, moving to the apical surface of the cells. In verapamil-treated cells, the strongest fluorescence intensity was again located toward the basal surface demonstrating an inhibitory effect of the Ca2+ channel blocker on integrin redistribution (Figure 5, Table 3).

Bottom Line: Results.Verapamil attenuated both changes of integrins and F-actin.Conclusions.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, China.

ABSTRACT
The present study examined intestinal epithelial cell (IEC) integrin distribution and disassembly of actin cytoskeleton in response to ischemia-anoxia. Protective effects of calcium channel blocker(CCB) were further examined to explore underlying mechanisms of cellular injury. Materials and Methods. Primary cultures of rat IECs and an in vitro model of ischemia/anoxia were established. IECs were exposed to ischemia/anoxia in the presence and absence of verapamil. The extent of exfoliation was determined using light microscopy while apoptosis rate was measured using flow cytometry. Changes in intracellular calcium, the distribution of integrins and the morphology of F-actin were assessed by confocal microscopy. Results. Detachment and apoptosis of IECs increased following ischemia/anoxia-induced injury. Treatment with verapamil inhibited the detachment and apoptosis. Under control conditions, the strongest fluorescent staining for integrins appeared on the basal surface of IECs while this re-distributed to the apical membrane in response to ischemic injury. Depolymerization of F-actin was also observed in the injured cells. Verapamil attenuated both changes of integrins and F-actin. Conclusions. Redistribution of integrins and disruption of F-actin under ischemia/anoxia injury is associated with IEC detachment and increased apoptosis. These events appeared to be triggered by an increase in Ca(2+)(i) suggesting a potential use for CCB in prevention and treatment of intestinal injury.

Show MeSH
Related in: MedlinePlus