Exposure of endothelial cells to cyclic strain induces elevations of cytosolic Ca2+ concentration through mobilization of intracellular and extracellular pools.
We have previously reported that exposure of endothelial cells to cyclic strain elicited a rapid but transient generation of inositol 1,4,5-trisphosphate (IP3), which reached a peak 10 s after the initiation of cyclic deformation. To address the effect of cyclic strain on intracellular Ca2+ concentration ([Ca2+]i) and its temporal relationship to IP3 generation, confluent bovine aortic endothelial cells were grown on flexible membranes, loaded with aequorin and the membranes placed in a custom-designed flow-through chamber. The chamber was housed inside a photomultiplier tube, and vacuum was utilized to deform the membranes. Our results indicate that the initiation of 10% average strain induced a rapid increase in [Ca2+]i which contained two distinct components: a large initial peak 12 s after the initiation of stretch which closely followed the IP3 peak, and a subsequent lower but sustained phase. Pretreatment with 5 microM GdCl3 for 10 min or nominally Ca2+-free medium (CFM) for 3 min reduced the magnitude of the initial rise and abolished the sustained phase. Repetitive 10% average strain at a frequency of 60 cycles/min also elicited a single IP3 peak at 10 s. However, there was also a large initial [Ca2+]i peak followed by multiple smaller transient [Ca2+]i elevations. Preincubation with 5 microM GdCl3 or CFM diminished the initial [Ca2+]i transient and markedly inhibited the late-phase component. Preincubation with 25 microM 2,5-di-(t-butyl)-1,4-benzohydroquinone (BHQ) attenuated the initial [Ca2+]i transient. Cyclic-strain-mediated IP3 formation in confluent endothelial cells at 10 s, however, was not modified by pretreatment with 25 microM BHQ, 500 microM NiCl2, 10 nM charybdotoxin, 5 microM GdCl3 or CFM. We conclude that in endothelial cells exposed to cyclic strain, Ca2+ enters the cytosol from intracellular and extracellular pools but IP3 formation is not dependent on Ca2+ entry via the plasma membrane.
Rosales
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Department of Medicine, Yale University School of Medicine, New Haven, CT 06510, USA.
Isales
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Barrett
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Brophy
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Sumpio
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D000818:Animals; D001013:Aorta, Thoracic; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D005682:Gadolinium / Q000494:pharmacology; D006873:Hydroquinones / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D013314:Stress, Mechanical
D003470:Culture Media; D006873:Hydroquinones; C019359:2,5-di-tert-butylhydroquinone; D015544:Inositol 1,4,5-Trisphosphate; D005682:Gadolinium; C038958:gadolinium chloride; D002118:Calcium