In this master thesis we checked the response of the callus-derived maize cells and protoplasts to acetylcholine and acetylcholine receptor agonists. Callus formation was induced by inoculation of young shoots of maize on Murashige-Skoog medium with addition of synthetic auxin 2,4-D [3 mg/L]. We followed the responses of cells and protoplasts to stimulation by 60 µM acetylcholine-iodide, nicotine or pilocarpine-hydrochloride, by alterations in fluorescence intensity. Fluorescent calcium indicator, Fluo-4-AM was used to follow the alterations in [Ca2+]i. We did not observe statistically significant differences between responses of cells and protoplasts to stimulants and negative control (plant cells and protoplasts stimulated by nutrient solution). Increased [Ca2+]i was observed at the time of stimulation, but probably as a consequence of mechanical stimulation when solutions were added to cells and protoplasts. Results show absence of achetilcholine receptors on the dediferentiated maize callus cells or, if expressed, their unresponsiveness to used concentration – 60 µM of acetylcholine-iodide, nicotine or pilocarpine-hydrochloride. The response to ionomycin was uncommon. HUVEC cells were used as positive control. When cells were exposed to acetylcholine-iodide, nicotine and pilocarpine-hydrochloride, statistically significant differenences from negative control (HUVEC stimulated by extracellular solution with 10 mM glucose) were observed in responses, thus increased [Ca2+]i. Results confirm sensing of acetylcholine-iodide, nicotine and pilocarpine-hydrochloride, and responsiveness of HUVEC cells. Alteration in [Ca2+]i after addition of ionomycine to HUVEC was statistically significant.
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