Cytoplasmic Ca2+ concentrations in intact Merkel cells of an isolated, functioning rat sinus hair preparation

Abstract

An isolated, functioning sinus hair preparation was developed to investigate cytoplasmic Ca2+ concentrations in intact Merkel cells using microfluorimetric techniques. Intracellular Ca2+ levels were monitored by means of photon counters in small groups of Merkel cells loaded with the calcium fluorescent indicators fura-2 or fluo-3. Mechanical stimulation of Merkel cells with fine glass rods resulted in small transient increases in intracellular Ca2+ levels (by about 20%) in the group of Merkel cells around the stimulating probe. A rise in Ca2+ is presumed to be essential for the postulated synaptic transmission to the afferent nerve terminal. Depolarization with a high concentration of potassium chloride (100 mM) caused increases in intracellular Ca2+ concentrations in Merkel cells (by about 70%) only in the presence of extracellular Ca2+, indicating an influx of Ca2+ through voltage-gated channels. The Ca2+ response was abolished neither by (+)-BayK8644 nor omega-conotoxin, suggesting that the Ca2+ channels are different from the classical L- or N-type channels. Extracellular application of ATP (10 microM to 5 mM) caused dose-dependent increases in intracellular Ca2+ levels in Merkel cells of up to sevenfold from the basal level of about 100 nM. Similar responses to ATP were also measured during superfusion with Ca(2+)-free medium, suggesting intracellular stores as the main Ca2+ source. Pre-incubation of Merkel cells with the purinoceptor antagonist suramin (100 microM) for 30 min reduced the Ca2+ responses to ATP by about 50% compared with control conditions. In conclusion, the results have demonstrated that a rise in intracellular Ca2+ in Merkel cells can be evoked by mechanical stimulation, membrane depolarization and chemical stimulation by ATP. These observations strongly suggest a possible contribution of Ca2+ to the normal responsiveness of Merkel cell mechanoreceptors, in turn supporting the hypothesis that Merkel cells are involved in the mechano-electric transduction process in sinus hair type I mechanoreceptors.