Background: We have previously reported that high extracellular calcium ([Ca2+]o) levels elicited rapid increases in the cytosolic free calcium ([Ca2+]i) and insulin release from human insulinoma cells. In this study we further investigated the mechanism for stimulus-secretion coupling of insulinoma cells exposed to high levels of [Ca2+]o.
Methods: Insulinoma tissues were surgically obtained for primary culture. The changes of [Ca2+]i level in response to various agents were monitored by fluorometry. Total RNA was extracted from tissues and subjected to reverse transcription-polymerase chain reaction (RT-PCR) with calcium-sensing receptor (CaR)-specific primers. PCR products were subcloned and sequenced.
Results: When [Ca2+]o level was elevated, [Ca2+]i in insulinoma cells was immediately increased. Application of neomycin abolished the increase in [Ca2+]i level, although extracellular nifedipine and lanthanum chloride did not affect it. The depletion of intracellular calcium stores with thapsigargin or carbachol eliminated the increase in [Ca2+]i level. RT-PCR analysis identified the 682 bp product, of which the sequence was identical to the corresponding regions of human parathyroid CaR.
Conclusions: Intracellular Ca2+ release might be important in insulin release from insulinoma cells after exposing to high level of [Ca2+]o. CaR could be involved in this mechanism.