Effects of extracellular calcium and magnesium on cytosolic calcium concentration in fura-2-loaded bovine parathyroid cells

Abstract

A newly developed calcium-sensitive dye, Fura-2, was employed in dispersed bovine parathyroid cells to study the effects of extracellular calcium and magnesium on cytosolic calcium concentration and parathyroid hormone (PTH) release. In comparison with control cells, Fura-2-loaded parathyroid cells showed the same maximal rate of PTH release, set-point for extracellular Ca⁺⁺ (the calcium concentration producing half of the maximal inhibition of PTH release), and maximal inhibition of PTH release (71.6%) by high extracellular Ca⁺⁺. At an extracellular Mg⁺⁺ concentration of 0.5 mM, raising extracellular Ca⁺⁺ in a stepwise fashion from 0.5 mM to 2.0 mM produced a dose-dependent, statistically significant (p < 0.01) increase in cytosolic Ca⁺⁺ from 198 ± 24 nM (0.5 mM Ca⁺⁺) to 411 ± 21 nM (2.0 mM Ca⁺⁺) which closely paralleled the concomitant decrease in PTH release. An elevation of extracellular Mg⁺⁺ from 0.5 mM to 5 mM, at an extracellular Ca⁺⁺ of 0.5 mM, resulted in a transient spike of cytosolic Ca⁺⁺ which lasted for approximately 30 seconds, followed by a small but stable increase in the cytosolic Ca⁺⁺ concentration (174 ± 7 nM vs. 237 ± 10 nM, n = 4, p < 0.01). Prior removal of extracellular calcium by addition of an excess of EGTA did not abolish the transient spike induced by high extracellular magnesium concentrations in Fura-2-loaded cells, suggesting that this rapid increase in cytosolic Ca⁺⁺ arises, at least in part, from intracellular stores of Ca⁺⁺. This is supported by the observation that pretreating cells with ionomycin resulted in disappearance of the magnesium-induced spike. In parallel experiments, the values for cytosolic calcium concentration at high and low extracellular calcium and magnesium concentrations in cells loaded with Fura-2 were comparable to those in cells loaded with Quin-2. These results show that Fura-2 may be employed to measure the cytosolic Ca⁺⁺ concentration in dispersed bovine parathyroid cells and that extracellular Ca⁺⁺ and Mg⁺⁺ produced sustained increases in cytosolic Ca⁺⁺ in cells loaded with this dye which are comparable to those seen with Quin-2-loaded cells. In addition, however, probably because of the lower loading concentrations possible with Fura-2, cells loaded with this dye show Mg⁺⁺-induced spikes in intracellular Ca⁺⁺, resulting, in part, from release of intracellular Ca⁺⁺. These Ca⁺⁺ transients support other data suggesting the possibility of an extracellular divalent cation receptor on parathyroid cells, which may produce some of its effects on parathyroid function by mobilizing intracellular Ca⁺⁺.