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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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Cultured IEC exhibited a cobblestone morphology (light microscope 200x). (b) More than 90% of the attached cells were positive for alkaline phosphatase (light microscope 200x). (c) Microvilli on the surface of the IEC (transmission electron microscope 5000x). (d) Apoptotic body (Arrowed) (Transmission electron microscope, 5000x). Scale bar = 2 μm.
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fig1: Cultured IEC exhibited a cobblestone morphology (light microscope 200x). (b) More than 90% of the attached cells were positive for alkaline phosphatase (light microscope 200x). (c) Microvilli on the surface of the IEC (transmission electron microscope 5000x). (d) Apoptotic body (Arrowed) (Transmission electron microscope, 5000x). Scale bar = 2 μm.

Mentions: The cultured cells showed a high degree of attachment and appeared slabstone shaped under light microscopy. Immunocytochemical characterization showed that more than 90% of the attached cells were positive for alkaline phosphatase (Figures 1(a) and 1(b)). Proliferating colonies of IECs coalesced to form large confluent areas of cells. In contrast, nonepithelial cells had a smooth-muscle-cell-like appearance, were present infrequently, and tended to be located at the edges of the IEC colonies or at the periphery of the well. Using the transmission electron microscope, numerous microvilli were observed on the surface of the IECs (Figure 1(c)). In addition, tight junctions, gap junctions, and desmosomes were evident between cells. In adherent cells the cytoskeleton was evident adjacent to the basement membrane with the appearance of focal adhesion forming. Apoptotic bodies were observed in some cells undergoing apoptosis (Figure 1(d)).


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)

Cultured IEC exhibited a cobblestone morphology (light microscope 200x). (b) More than 90% of the attached cells were positive for alkaline phosphatase (light microscope 200x). (c) Microvilli on the surface of the IEC (transmission electron microscope 5000x). (d) Apoptotic body (Arrowed) (Transmission electron microscope, 5000x). Scale bar = 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369571&req=5

fig1: Cultured IEC exhibited a cobblestone morphology (light microscope 200x). (b) More than 90% of the attached cells were positive for alkaline phosphatase (light microscope 200x). (c) Microvilli on the surface of the IEC (transmission electron microscope 5000x). (d) Apoptotic body (Arrowed) (Transmission electron microscope, 5000x). Scale bar = 2 μm.
Mentions: The cultured cells showed a high degree of attachment and appeared slabstone shaped under light microscopy. Immunocytochemical characterization showed that more than 90% of the attached cells were positive for alkaline phosphatase (Figures 1(a) and 1(b)). Proliferating colonies of IECs coalesced to form large confluent areas of cells. In contrast, nonepithelial cells had a smooth-muscle-cell-like appearance, were present infrequently, and tended to be located at the edges of the IEC colonies or at the periphery of the well. Using the transmission electron microscope, numerous microvilli were observed on the surface of the IECs (Figure 1(c)). In addition, tight junctions, gap junctions, and desmosomes were evident between cells. In adherent cells the cytoskeleton was evident adjacent to the basement membrane with the appearance of focal adhesion forming. Apoptotic bodies were observed in some cells undergoing apoptosis (Figure 1(d)).

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