Purpose: To study spontaneous and evoked calcium oscillations in the ciliary epithelial bilayer by muscarine, examine whether these exist in one or both layers of the bilayer, and examine their patterns. Methods: The ciliary epithelial bilayer was isolated, loaded with 5-10 μM Fluo-3 acetoxymethyl ester (Fluo-3 AM; Molecular Probes, Inc., OR, USA), and mounted in a specially designed laminar perfusion chamber. Observations and measurements were made with a real time confocal laser scanning device (Odyssey, Noran Instruments, CA, USA) interfaced with a Zeiss Axioscope provided with Image-1 software and equipped with an Argon laser (Omnichrome, CA, USA). Optical slices were about 5 μM thick and acquisition was performed at 25 frames/second. Images were stored on a video recorder and analyzed off line. All calculations and plots were done with Sigmaplot software. Results: Calcium imaging under confocal microscopy of the isolated secretory epithelium revealed spontaneous calcium oscillations, especially of the inner layer. These were observed in multiple patterns. Oscillations could also be induced by stimulation of the muscarinic receptor. The onset of these induced oscillations coincide with the synthesis and release of IP3. Caffeine 5 μM induced a calcium transient and blocked spontaneous and muscarinic induced (0.1 μM) oscillations. Thapsigargin (1 μM) prevented a muscarinic response and inhibited calcium oscillations. Muscarine and caffeine induced transients have significantly greater amplitude in NPE than PE, ΔF/F=2.5±0.42(64) and 1.99±0.27(27) versus 0.52±0.06(41) and 0.91±0.41(11) respectively. Similarly, calcium transients and frequencies of spontaneous oscillations are greater in NPE than PE. Muscarine induced oscillations are observed in NPE only and with higher frequencies than the spontaneous ones. γ=0.24±0.05Hz(8) versus 0.08±0.01 Hz(18). Conclusions: These repetitive responses, spontaneous and receptor coupled, probably indicate intracellular coding of information. If modulation of aqueous secretion occurs via a muscarine induced calcium signal, it probably is located in the NPE.
|Journal||Investigative Ophthalmology and Visual Science|
|Publication status||Published - Feb 15 1996|
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