Localization of Three Types of the Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channel in the Secretory Granules and Coupling with the Ca2+ Storage Proteins Chromogranins A and B

Abstract

Although the role of secretory granules as the inositol 1,4,5-trisphosphate (IP 3 )-sensitive intracellular Ca 2؉ store and the presence of the IP 3 receptor (IP 3 R)/Ca 2؉ channel on the secretory granule membrane have been established, the identity of the IP 3 R types present in the secretory granules is not known. We have therefore investigated the presence of different types of IP 3 R in the secretory granules of bovine adrenal medullary chromaffin cells using immunogold electron microscopy and found the existence of all three types of IP 3 R in the secretory granules. To determine whether these IP 3 Rs interact with CGA and CGB, each IP 3 R isoform was cotransfected with CGA or CGB into NIH3T3 or COS-7 cells, and the expressed IP 3 R isoform and CGA or CGB were co-immunoprecipitated. From these studies it was shown that all three types of IP 3 R form complexes with CGA and CGB in the cells. To further confirm whether the IP 3 R isoforms and CGA and CGB form a complex in the secretory granules the potential interaction between all three isoforms of IP 3 R and CGA and CGB was tested by co-immunoprecipitation experiements of the mixture of secretory granule lysates and the granule membrane proteins. The three isoforms of IP 3 R were shown to form complexes with CGA and CGB, indicating the complex formation between the three isoforms of IP 3 R and CGA and CGB in the secretory granules. Moreover, the pH-dependent Ca 2؉ binding property of CGB was also studied using purified recombinant CGB, and it was shown that CGB bound 93 mol of Ca 2؉ /mol with a dissociation constant (K d ) of 1.5 mM at pH 5.5 but virtually no Ca 2؉ at pH 7.5. The high capacity, low affinity Ca 2؉ -binding property of CGB at pH 5.5 is comparable with that of CGA and is in line with its role as a Ca 2؉ storage protein in the secretory granules.The secretory granules of endocrine cells, neurons, and neuroendocrine cells contain many hormones, ions, peptides and proteins, including 40 mM Ca 2ϩ and 1-2 mM chromogranins A and B in addition to high concentrations of hormones (1-6). The secretory granule contents are secreted to the extracellular space and then into the bloodstream during exocytosis, which is initiated by a sudden increase of intracellular Ca 2ϩ concentration (7). In bovine adrenal medullary chromaffin cells the secretory granules occupy ϳ10% of the total cell volume (8), thereby storing a majority of the intracellular Ca 2ϩ of the cell in the secretory granules. Hence it appears inevitable for the secretory granules to participate in the control of intracellular Ca 2ϩ concentrations. Consistent with this notion, the secretory granules from adrenal medullary chromaffin cells (9), pancreatic acinar cells (10), and the goblet cells (11) concentrations but also in exocytotic processes. Despite the importance of the IP 3 -sensitive intracellular Ca 2ϩ store role of secretory granules, the study of IP 3 R/Ca 2ϩ channels in the secretory granules did not begin until the secretory granule Ca 2ϩ storage protein chromogranin A was shown to...

Organelles Containing Inositol Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

“…The foregoing results raised the possibility that the ER and secretory granules were immunoreactive to putative anti-InsP 3 R2 Abs, as shown in bovine AM cells [9]. However, this complexity might be ascribed to heterogeneous immunoreactivities of the Abs used.…”

“…Furthermore, the nucleoplasm was also shown to have InsP 3 R-like products, as was noted with the Sigma anti-InsP 3 R2 Ab. Thus according to a series of papers by Yoo and his colleagues [9,37], InsP 3 Rs turn out to be present almost everywhere in bovine chromaffin cells. This apparent complexity of InsP 3 R distributions, in our opinion, may at least in part be due to nonspecific binding.…”

“…The physiological significance of these intragranular Ca 2+ ions ([Ca 2+ ] g ), although suggested to be involved in secretion [7], still remains obscure. Chromaffin and zymogen granules were shown to release Ca 2+ ions in response to InsP 3 [8,17], and InsP 3 Rs were localized in chromaffin granules by the use of immunogold electron microscopy [9]. Consistent with the notion that secretory granules are dynamic Ca 2+ stores, the measurement of [Ca 2+ ] in insulin-containing granules of MIN6 cells with a vesicle-associated membrane protein aequorin chimera revealed a dramatic decrease in [Ca 2+ ] g in response to caffeine and cyclic ADP ribose [11].…”

“…In goblet cells, however, secretory granules were reported to release Ca 2+ in response to inositol 1,4,5-trisphosphate (InsP 3 ) [5], whereas in submandibular acinar cells InsP 3 receptors (InsP 3 Rs) were shown to be present in vesicles, but not in the rough ER [6]. Moreover, Yoo and his colleagues reported that all three subtypes of InsP 3 Rs resided in secretory granules as well as the ER in bovine adrenal medullary (AM) cells [7][8][9]. From a functional point of view, the presence of InsP 3 Rs in secretory granules is readily explained.…”

Calcium dynamics in the secretory granules of neuroendocrine cells

“…The presence of InsP 3 R in secretory granules has been a highly conflicting point over the [49] reported that inositol 1,4,5-trisphosphate (InsP 3 ) released Ca 2+ from chromaffin cells secretory vesicles. Immunocytochemical localization of all three types of InsP 3 R in serially sectioned bovine chromaffin cells showed that the secretory granules contained about 60% of the InsP 3 R in the cell [50], and additional evidence for the presence of InsP 3 R in the granules was the finding that both chromogranin A and B interact with InsP 3 R and modulate its activity [26,[51][52][53][54]. More recently, using targeted aequorin to measure [Ca 2+ ] in the secretory granules of chromaffin cells and the neurosecretory cell lines PC12 and INS1 [23,24], we have shown that InsP 3 and InsP 3 -releasing agonists can release Ca 2+ from the granular compartment (see Fig.…”