| Autor: |
Fjaervoll HK; Division of Head, Neck and Reconstructive Surgery, Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.; Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.; Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Oslo, Norway., Fjaervoll KA; Division of Head, Neck and Reconstructive Surgery, Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.; Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.; Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Oslo, Norway., Yang M; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States., Reiten OK; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States., Bair J; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States., Lee C; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States., Utheim TP; Division of Head, Neck and Reconstructive Surgery, Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.; Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.; Department of Plastic and Reconstructive Surgery, Oslo University Hospital, Oslo, Norway., Dartt D; Department of Ophthalmology, Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States. |
| Abstrakt: |
ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca 2+ i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca 2+ ] i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1 ) preincubation with the Ca 2+ chelator EGTA, 2 ) preincubation with the SERCA inhibitor thapsigargin (10 -6 M), which depletes ER Ca 2+ stores, 3 ) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4 ) preincubation with the voltage-gated calcium channel antagonist nifedipine (10 -5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10 -5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca 2+ ] i was significantly lower after chelating Ca 2+ i with 2 mM EGTA in Ca 2+ -free buffer. The peak [Ca 2+ ] i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10 -5 M and BzATP at 10 -4 M. Incubating CGCs with dantrolene alone decreased [Ca 2+ ] i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca 2+ i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca 2+ ] i . This study contributes to the understanding of diseases arising from defective Ca 2+ signaling in nonexcitable cells. NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca 2+ ] i ) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca 2+ ] i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca 2+ ] i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca 2+ ] i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca 2+ i homeostasis. |
