CALBINDIN-D-28K APPEARS TO BUFFER INTRACELLULAR CA2-TREATED RAT INSULINOMA CELL-LINE( IN BUTYRATE)

Intracellular Ca2+ is a key second messenger within a dynamic signalin g pathway that regulates many cellular processes including insulin sec retion. Vitamin D-dependent calcium-binding protein, calbindin-D-28k, may efficiently buffer intracellular free calcium ([Ca2+](i)) because of its apparent high affinity for Ca2+ and fast kinetics. We employed fluorescent ratio imaging of fura-2-loaded rat insulinoma cells (RINr1 046-38) with normal and elevated (with butyrate) levels of calbindin-D -28k to study changes in [Ca2+](i) in response to insulin secretogogue s and calcium-modulating agents. The basal level of [Ca2+](i) in RINr1 046-38 cells was 110 +/- 2 nM. Challenge of the cells with glucose (30 mM) or their depolarization with high K+ (70 mM) resulted in a single Ca2+ transient with a peak height of 294 +/- 19 nM and 506 +/- 24 nM, respectively, and a peak width of 1-2 min. The Ca2+ channel agonist B ay K 8644 (2 mu M) induced similar Ca2+ transients which averaged 224 +/- 8 nM. Thapsigargin (5 mu M), a mobilizer of Ca2+ stores, elicited elevations in [Ca2+](i) to 306 +/- 57 nM. The calcium ionophore ionomy cin (0.5-2 mu M) produced rapid dramatic increases in [Ca2+](i) to 500 -2000 nM. In the absence of extracellular Ca-i(2+) the addition of ion omycin led to a transient elevation of [Ca2+](i) to 316 +/- 32 nM. Gen erally, changes in [Ca2+](i) within the cell population were heterogen eous in respect to magnitude but highly synchronized. Treatment of the cells with butyrate (2 mM for 24-72 h), which increased levels of cyt osolic calbindin-D-28k 2.6-3.4-fold, attenuated cellular responses to glucose, K+-depolarization, Bay K 8644, thapsigargin, and low concentr ations (0.5-2 mu M) of ionomycin. Cells loaded with the nonfluoresent, membrane-permeable Ca2+-chelator BAPTA/AM (50 mu M) in Ca2+ buffer (1 mM) demonstrated the [Ca2+](i) responses similar to those of butyrate -treated cells. The lack of [Ca2+](i) responses in butyrate-treated ce lls suggests a large increase in cytoplasmic Ca2+ buffering capacity c onsistent with elevated levels of calbindin-D-28k.