title: Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells.
abstract: The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca(2+)]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca(2+)]i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca(2+)]i. Step changes in shear stress resulted in a rapid increase in [Ca(2+)]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca(2+)]i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm(2) stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm(2) stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca(2+)]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.
authors.affiliation: Department of Bioengineering, Imperial College, London, England;
authors.forename: M
authors.initials: M
authors.lastname: Jafarnejad
authors.affiliation: Department of Medical Physiology, Texas A&M Health Science Center, Temple, Texas; and.
authors.forename: W E
authors.initials: WE
authors.lastname: Cromer
authors.affiliation: Department of Biomedical Engineering, Texas A&M University, College Station, Texas.
authors.forename: R R
authors.initials: RR
authors.lastname: Kaunas
authors.affiliation: Department of Medical Physiology, Texas A&M Health Science Center, Temple, Texas; and.
authors.forename: S L
authors.initials: SL
authors.lastname: Zhang
authors.affiliation: Department of Medical Physiology, Texas A&M Health Science Center, Temple, Texas; and.
authors.forename: D C
authors.initials: DC
authors.lastname: Zawieja
authors.affiliation: Department of Bioengineering, Imperial College, London, England; james.moore.jr@imperial.ac.uk.
authors.forename: J E
authors.initials: JE
authors.lastname: Moore
mesh_terms: D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004729:Endothelium, Lymphatic / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D013314:Stress, Mechanical; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D000252:Calcium-Transporting ATPases
keywords: intracellular calcium; lymphatic endothelial cell; shear stress