Abstract
The Ca2+-ATPase inhibitor, thapsigargin, activated Ca2+ entry into pancreatic acinar cells, a process known as capacitative calcium entry. In cells loaded with the calcium chelator BAPTA, the transient Ca2+ release was blunted and the rise of [Ca2+]i on readdition of Ca2+ was slowed. However, the steady-state [Ca2+]i due to Ca2+ entry was substantially augmented compared with control cells. This indicates that [Ca2+]i exerts a negative feedback on Ca2+ entry from a compartment buffered by BAPTA and separated from the bulk of cytoplasmic Ca2+. This interaction probably occurs close to the calcium channel where [Ca2+] is higher than in the bulk of the cytoplasm. In support of this interpretation, the slower Ca2+ chelator, EGTA, also blunted the release of Ca2+ and slowed the rise of the sustained [Ca2+]i phase but failed to augment steady-state [Ca2+]i. In contrast, Ca2+ entry in NIH 3T3 cells was characterized by a transient rise of [Ca2+]i that decays to near prestimulus levels. This decay in Ca2+ entry also results from negative feedback by Ca2+ because the decrease in Ca2+ entry was reversed by incubation in a Ca2+-deficient medium. However, unlike its effects in acinar cells, BAPTA neither augmented steady-state [Ca2+]i nor prevented the inactivation of entry. Rather, in BAPTA-loaded cells, [Ca2+]i failed to increase substantially suggesting that negative regulation by Ca2+ may occur at a site distinct from the cytoplasmic compartment and inaccessible to cytoplasmic BAPTA. These two distinct types of feedback behavior may indicate subtypes of store-operated calcium channels expressed in different cells or a single type of channel which is differentially regulated in a cell type-specific manner.
Highlights
Many hormones and neurotransmitters initiate cellular activation through the hydrolysis of phosphatidylinositol [4,5]bisphosphate generating the intracellular messenger inositol 1,4,5-triphosphate ((1,4,5)IP3),1 which mobilizes Ca2ϩ by binding to specific intracellular receptors
Addition of thapsigargin to fura-2-loaded pancreatic acinar cells incubated in a Ca2ϩ-free medium caused a transient increase of [Ca2ϩ]i which was followed on restoration of external Ca2ϩ by an elevated and sustained [Ca2ϩ]i signal (Fig. 1A)
We investigated the effects of intracellular BAPTA and EGTA on the thapsigargin-induced Ca2ϩ entry in pancreatic cells
Summary
Many hormones and neurotransmitters initiate cellular activation through the hydrolysis of phosphatidylinositol [4,5]bisphosphate generating the intracellular messenger inositol 1,4,5-triphosphate ((1,4,5)IP3), which mobilizes Ca2ϩ by binding to specific intracellular receptors. Gargin can activate Ca2ϩ influx without changes in inositol phosphate levels [3, 4] This entry of Ca2ϩ is presumed to occur through Ca2ϩ channels in the plasma membrane, termed “store-operated channels” [5, 6], the existence of such channels has not been proven unequivocally. Missiaen et al [12] demonstrated that, in HeLa cells, Ca2ϩ entry inactivates with a time course of tens of seconds giving rise in some cells to slow [Ca2ϩ]i oscillations. The relationship of this slow inactivation of Ca2ϩ entry to the [Ca2ϩ]i-dependent inactivation of Ca2ϩ current is not known It is not known whether these mechanisms are intrinsic to capacitative calcium entry in all instances or represent cell-specific modes of regulation. Our findings indicate that these mechanisms do not represent an intrinsic property of capacitative calcium entry in all cell types but rather appear to be differentially expressed in a cell type-specific manner
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