Letter to the editor

IP₃ receptors and secretory granules

Cellular Ca²⁺signaling results from a complex interplay among a variety of Ca²⁺ fluxes going across the plasma membrane and across the membranes of several organelles, together with the buffering effect of large numbers of Ca²⁺-binding sites distributed along the cell architecture. Endoplasmic and sarcoplasmic reticulum, mitochondria and even nucleus have all been involved in cellular Ca²⁺ signaling, and the mechanisms for Ca²⁺ uptake and release from these organelles are well known. In neuroendocrine cells, the secretory granules also constitute a very important Ca²⁺-storing organelle, and the possible role of the stored Ca²⁺ as a trigger for secretion has attracted considerable attention. However, this possibility is frequently overlooked, and the main reason for that is that there is still considerable uncertainty on the main questions related with granular Ca²⁺ dynamics, e.g., the free granular [Ca²⁺], the physical state of the stored Ca²⁺ or the mechanisms for Ca²⁺ accumulation and release from the granules. This review will give a critical overview of the present state of knowledge and the main conflicting points on secretory granule Ca²⁺ homeostasis in neuroendocrine cells.

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray microanalysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components.

In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines:

• It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue.

• Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in theDrosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca²⁺]_i) and intracellular pH (pH_i) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH₄Cl, 15 mM) and a Na⁺/H⁺ionophore (monensin, 10 μM) evoked cytosolic alkalinization followed by Ca²⁺release from acidic intracellular Ca²⁺stores. Pretreatment of S2 cells with either thapsigargin (1 μM), an inhibitor of endoplasmic reticulum Ca²⁺-ATPases, or with the Ca²⁺ionophore ionomycin (10 μM) was without effect on the amplitude of Ca²⁺release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 μM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP₃-sensitive Ca²⁺store but failed to affect the amplitude of alkalinization-evoked Ca²⁺release. Glycyl-L-phenylalanine-β-naphthylamide (200 μM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca²⁺release from internal stores, while application of brefeldin A (10 μM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca²⁺]_i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH₄Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca²⁺stores did not elicit depletion-operated Ca²⁺entry. They were refilled upon re-exposure of cells to normal saline ([Ca²⁺]_o= 2 mM), but not by thapsigargin-induced [Ca²⁺]_ielevation in Ca²⁺-free saline.

Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca²⁺ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 μM Ca²⁺ induced an intra-organelle [Ca²⁺] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca²⁺] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca²⁺] increase. These results taken together suggest the participation of $\text{Na}\text{Ca}$ exchange, known to exist in the membrane of these secretory vesicles. In addition to $\text{Na}\text{Ca}$ exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca²⁺] increase obtained under acidic compartments: neutralization with NH₄Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca²⁺] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca²⁺ changes would be involved in stimulus-secretion coupling.