Activation of the plasma membrane Ca²⁺ pump during agonist stimulation of pancreatic acini

The role of internal stores and plasma membrane Ca²⁺ pumps in controlling [Ca²⁺](i) during agonist stimulation and their regulation by agonists are not well understood. We report here measurements of intracellular ([Ca²⁺](i)) and extracellular ([Ca²⁺](o)) Ca²⁺ concentrations in agonist-stimulated pancreatic acini in an effort to directly address these questions. Stimulation of acini suspended in Ca²⁺- free or Ca²⁺-containing medium with Ca²⁺ mobilizing agonists resulted in a typical transient increase in [Ca²⁺](i). Thapsigargin, a specific inhibitor of internal Ca²⁺ pumps, inhibited the rate of [Ca²⁺](i) reduction after agonist stimulation by approximately 40%. Under the same conditions, thapsigargin had no effect on the rate of the unidirectional Ca²⁺ efflux across the plasma membrane as revealed by measurements of [Ca²⁺](o). These findings suggest that internal Ca²⁺ pumps actively remove Ca²⁺ from the cytosol during continued agonist stimulation. The correlation between the reduction in [Ca²⁺](i) and the increase in [Ca²⁺](o) showed that Ca²⁺ efflux from cells stimulated with agonist and thapsigargin represent Ca²⁺ efflux across the plasma membrane. Inhibition of cells exposed to agonist and thapsigargin with a specific antagonist sharply reduced the rates of the [Ca²⁺](i) decrease and the accompanied [Ca²⁺](o) increase. Hence, at comparable [Ca²⁺](i), Ca²⁺ efflux from stimulated cells was about 3-fold faster than that from resting cells, indicating that agonists directly activate the plasma membrane Ca²⁺ pump. To study the role of [Ca²⁺](i) increase in plasma membrane Ca²⁺ pump activation the acini were loaded with 1,2-bis-(2-aminophenoxyethane- N,N,N',N')-tetraacetic acid (BAPTA), and [Ca²⁺](o) was measured during agonist stimulation. Surprisingly, although BAPTA completely prevented the increase in [Ca²⁺](i), Ca²⁺ efflux rate was reduced by only 34%. These findings provide the first evidence for Ca²⁺-independent activation of the plasma membrane Ca²⁺ pump by Ca²⁺ mobilizing agonists.