The calcium loading of secretory granules. A possible key event in stimulus-secretion coupling
G h i s l a i n N i c a i s e ~, K a t i a M a g g i o ~, Sy lv i e T h i r i o n i, M a r i a n n e H o r o y a n 2, E r i c k K e i c h e r
I Laboratoire de Physiologie Cellulaire et Comparde, Universit~ de Nice-Sophia Antipolis, 06108 Nice Cedex 2; 2Laboratoire d'Immunologie, H6pital Sainte Marguerite, 13274 Marseille Cedex 9, France
(Received 25 February 1992; accepted 15 June 1992)
Summary - The review focuses on calcium accumulation by secretory organelles. The observation that secretory granules contain variable and often important quantities of calcium (1-200 mM of total calcium) can be interpreted as a maturation index. A progres- sive loading with calcium would be permitted by a Ca2+-transport mechanism on the granular membrane and calcium-binding molecules in the granular core. The saturation of this store by the stimulus-induced calcium transient would permit in mature (calcium- loaded) granules the ionic crisis leading to exocytosis. The inside of secretory organelles being acidic, calcium influx into the granule can be driven by calcium-proton exchange. The calcium-proton exchanger could be a Ca2÷-ATPase.
secretory granules / calcium / protons / Ca 2÷ ATPase
Introduction
It has been well established since the early work of Doug- las and co-workers that calcium ions (Ca 2+) play a criti- cal role in regulated exocytotic secretion. This concept was developed from the study of the adrenal medulla and the neurohypohysis and defined as stimulus-secretion coupling [37, 38]. Thirty years later, the mechanism by which calcium ions trigger exocytosis is still unknown.
The present article reviews data suggesting that the to- tal calcium content of secretory granules increases prior to secretion, as a consequence of the well-documented in- crease in cytosolic Ca 2 +. This increase in granular calci- um, possibly critical for secretion, would be due to a proton gradient and to a calcium-proton exchange.
Secretory granules contain high, often variable, amounts of total calcium
Most secretory granules tested so far contain millimolar or higher concentrations of total calcium, ie several orders of magnitude larger than the concentrations of free calcium ions found in the cytosol (table I). This has been shown either with biochemical techniques (table II), or with X-ray mic.roprobe analysis (table III). The biochemical methods (grinding and centrifuging) have been proven to induce some redistribution of calcium [34, 112] and present the inconvenience of averaging the contents of numerous organelles, regardless of their situation in the cell. If the microprobe techniques are performed after quick-freezing to immobilize calcium in situ, there are good reasons to believe that the risk of redistribution is minimal. Thus it is possible to quantify the content of individual granules or small groups of vesicles. In the studies published so far on various secretory organelles, the quick-freezing step has been followed either by resin embedding in presence of oxalic acid (to prevent translocation of calcium), which
gives a result in mmol of Ca per kg of resin-embedded tis- sue (see [104]) or more often by cryosection and freeze- drying, which gives a result in mmol/kg of dry weight (see [51], and table III). Quantitative microprobe analysis of individual granules most often reveals that the total calcium concentration is highly variable, even in a given experimental situation. The frequency histogram of figure 1 illustrates the variability of calcium content in Aplysia gliagrana. Mytilus interstitial granules present an even wider range of concentrations, from non-detectable (sub mM) to 200 mM [16, 103]. Most authors do not present the results in such detail but a similarly high dis- persion of the values can be inferred from the standard errors accompanying the data [65, 136].
Secretory granules contain calcium-binding molecules, with a finite capacity
Once transported into the secretory granules calcium ions are likely to be trapped by calcium-sequestering organic molecules; this until the capacity of these molecules is over- loaded. Direct evidence that calcium trapping occurs is provided by the comparison between total and free calcium inside the secretory granules. In platelet u-granules, the free calcium is about 12 ~M, for a total calcium of 32 mM [49], in chromaffin granules the respective values are simi- lar [22, 52].
The organic molecules responsible for calcium trapping can be the acidic glycoproteins chromogranins, first dis- covered in the granules of adrenal chromaffin cells, then found in a variety of other secretory cells; they show a good affinity and a high binding capacity for calcium, which makes them the most likely ligand for the chro- maffin granule calcium load according to Reiffen and Gratzl [120]. The storage molecule could also be glycosaminoglycans or proteoglycans; sulfated proteogly- cans, with a high negative charge, are found in a wide
90 G Nicaise et al
Table I. High ( > mM) in t ragranu la r Ca concent ra t ion , Ca 2+ ATPase activity and low ( < 7) pH~ in various secretory organelles. The numbers in brackets cor respond to selected l i terature references (for reasons discussed in the text, X-ray mic roprobe is preferred to biochemical analysis). The quest ion marks refer to dubious or cont radic tory data .
Animal group Granule High total Ca Ca :+ A TPase Acidic ApH
Mammals C h r o m a f f i n + [112] ? [29] + see [29] Syn ves + [9] + [64, 95] + [94] His tamine _+ [74] + [33] + [66] Atrial + [136] + [138] + [136] Ant pi tui tary + [85] + [135] + [28] Post pi tui tary + [1441 9 [126] + [129] Insulin + [60] + see [61] + [1] Platelet ~t + [491 ? [35] + [48] Paro t id + see [1481 ? + [11] Zymogen + [121, 122] + [53]
Molluscs Centra l glia + [69] + [89] + [87] Interst i t ial + [103] + [891 + [88]
Sea-urchins Cortical + [43] + [43] ? see text Earthworms Chloragosome + [97] + [40]
Table II. Quant i ta t ive biochemical measurements of total calcium concen t ra t ion in isolated secretory vesicles, given in n m o l / m g of prote in ( n m o l / m g ) and conver ted in m m o l / k g of wet tissue (wet), using the da ta of the au thors or assuming 20 °70 prote in .
Species Organ Cell Organelle nmol/mg wet Re f
Bovine Pi tu i tary Anter ior Granules 14 3 [85] Bovine Post pi tui tary Neurosecretory Granules 35 7 [144] Guinea-pig Pancreas Exocrine Granules 37 7 [34] Pig Blood Platelet a -Granules 31 8 [49] Mouse Pancreas Endocr ine Granules 46 9 [6] Rat Paro t id Epithelial Serous gr 60 12 [148] Torpedo Electric Nerve end Syn ves 14 [64] Torpedo Electric Nerve end Syn ves 70/100 [72] Bovine Adrena l C h r o m a f f i n Granules 100 17 [115] Bovine Cont ro l adr C h r o m a f f i n Granules 125 21 [130]
stimul adr 242 40
Table III. Quant i ta t ive X-ray microprobe measurements of Ca concent ra t ions in secretory vesicles, given in m m o l / k g of dry weight (dry) or m m o l / k g of res in-embedded tissue (res) and conver ted in m m o l / k g of wet tissue (wet), using the water conten t (H,O in 070) or assuming 60 070 of water when the water content of the granular core was unknown .
Species Organ Cell Organelle dry res wet 1-t20 Re f
Mouse Cerebel lum Neurones Syn ves 0.7 - 0.3 - [9] Rat • Thymus Mast cell Low K gr 4 - 2 [74] Ox Adrena l C h r o m a f f i n Granules 13 - 4 66 [112] Rat Pancreas Exocrine Zymogen gr 16 - 6 [121] Dog Pancreas Exocrine Zymogen gr 19 - 7 63 [101] ? Pancreas Exocrine Zymogen gr 15 - 6 - [I 3] A punctata Centra l glia Gl iagrana - 8 9 - [69] Rat Thymus Mast cell Lucent gr 40 - 16 - [73] Rat Paro t id Epithelial Serous gr 42 - 17 - [99] Rabbi t Ileum Goble t Mucous gr 48 - 19 - [51] Rat M a m m a r y Epi thel ium Secret vac 47 - 19 - [25] Rat Pancreas Exocrine Zymogen gr 54 - 22 - [122] Rat Paro t id Epithelial Serous gr 69 - 28 [65] Rat Auricle Muscle Atr ial gr 75 - 30 - [136] Heterocentrotus Juxta l igamenta l Granule - 10/45 30 - [104] A californica Centra l glia Gl iagrana - 30 33 - [69] Rhesus Sweat g land Dark cell Granule 85 - 34 - [128] Mytilus A B R M Intersti t ial Granules 214 44 49 - [16] Rat Blood Platelet Dense gr 169 a - 68 - [17] E a r t h w o r m Chloragogenous Ch lo ragosome 412 - 165 - [97]
cell
aAssuming that the dense granules have a d iameter o f 0.3 t~m.
Fig 1. Frequency distribution of the calcium concentrations meas- ured by X-ray microanalysis on glial granules of A californica and A punctata. The calcium concentrations (Ca) are expressed in mmol kg -~ of epoxy-embedded tissue (which can be multi- plied by !.11 to obtain mmol kg -t of wet tissue). The limit at the origin of the abscissae does not represent an absence of cal- cium but the sensitivity limit of the method (3-5 mM Ca). From Keicher et al [69].
range of secretory vesicles (see refs in [71]). The variety of secretion products, secretory cell types and physiologi- cal conditions of secretion may be reflected in part by a certain variety in calcium storage properties. Synaptic microvesicles, for example, are likely to be more readily saturated than large dense-core secretory granules.
The calcium overload of secretory organelles is likely to occur in the vicinity of the plasma membrane, where stimulus-secretion coupling takes place (see [23]). A direct osmotic effect of the intragranular calcium overload seems to be ruled out (or secondary), at least in the systems where it is admitted that the formation of the exocytotic pore precedes granule swelling [19, 116, 153] but a consequence of intragranular calcium loading is to build up a ApCa which may be needed for the formation of the exocytotic pore. Recent data have been presented, which favor the formation of this pore by fusogenic proteins like synap- tophysin [3, 23, 84, 116, 143]. It is particularly interesting for otrr intragranular calcium loading model that the primary structure of synaptophysin "suggests that the Ca2+-binding site is intravesicular" [139]. Even if these putative fusion proteins are not related to connexin [140], they may share functions with gap-junctions, the opening of which is modulated by transmembrane ApCa and/or ApH [137].
In a study of pancreatic fl-cell secretion, Hellman et a! [55] proposed " tha t Ca 2+ must reach a certain concen- tration in the granule sac before the granule content can
be extruded by exocytosis". A direct action of intragranu- lar calcium in the exocytotic process has also been briefly evoked by Zimmerberg and Liu [154] as a possible expla- nation for the secretion of sea urchin cortical granules.
The granule calc ium content may change upon secretory s t imulat ion
It is possible to measure the total calcium concentration of secretory granule populations after stimulation of the secretory process, as compared to controls: this has been done by isolation of organelles and also more accurately by X-ray microanalysis of rapidly frozen secretory tissues, Supposing that an increase in granular total calcium concentration always precedes secretion, two types of changes can be predicted: if the sampling is made before completion of the secretory process, the calcium content of the granule population should be higher than the control, but later this content (in the granules which have not been secreted) should be equal or even inferior to the control.
Isolated chromaffin granules of stimulated adrenal medulla are richer in 4SCa but not richer in total calcium than the controls [18], however, repeated stimulation in- creases their calcium content [130]. It was demonstrated that 45Ca incorporated by chromaffin granules is released directly with catecholamines by exocytosis [47]. 45Ca in- corporation into the granular pool is slow and the 45Ca presumed to enter the cell during short-term stimulation is not found in the releasable pool during a second period of triggered secretion [47]. The experiment therefore failed to demonstrate that exocytosis was actually terminating the stimulating increase in cytoplasmic calcium. However, these authors could not rule out the possibility that the calcium which enters the cell during stimulation is se- questered by the same granules which are released as a direct effect of this stimulation.
Glucose stimulation of insulin-secreting/3-cells induces a small (not statistically significant) increase in total cal- cium content of the secretory granule population but a large significant increase in 45Ca turnover inside the granules [6].
Comparative X-ray microanalysis of resting and salivat- ing rat parotid glands shows a slight (not statistically significant) increase in granular calcium [65], similarly non- significant decreases in granular calcium were reported in dog submandibular gland (Sasaki et al, 1983 in [65]) and dog pancreas [101]. In a preliminary report, Barnard [13] mentions an increase in the calcium content of zymogen pancreatic granules, which was fully confirmed by Roo- mans and Wei [121] who found a significant increase from 16 to 22 mmol kg -~ dry, under carbachol stimulation.
However, the calcium content of the granule popula- tion was found to decrease significantly in interstitial cells of Myti lus retractor muscle after potassium stimulation [16], a treatment which was found to induce neutraliza- tion of the granular acidity, and decrease in granule abun- dance (unpublished); it is therefore likely that calcium-rich granules did undergo preferential exocytosis. A milder treatment, by superfusing this same tissue with calcium- rich saline (artificial sea water, Ca 2÷ = 12.2 mM, instead of 8.36 mM), also neutralized granular acidity but did not appear to induce exocytosis (fig 2); in this case the calcium concentration was found to increase significantly in the granule population (fig 3).
In rat neurohypophysis isolated nerve endings,
92 G Nicaise et al
N
a
0 20 40 60 80 100 120 140 160 180
[Ca]
Fig 2. Acridine orange stained glio-intersfitial granules in Myti- lus byssal retractor muscle, after 3 -4 days of adaptation in Ca-'* poor (containing 8.36 mM Ca '-+) artificial sea water (ASW). The interstitial cell process contains a row of in- tracytoplasmic red fluorescent granules (a). When submitted to a calcium-enriched ASW (12.2 mM Ca -'÷), the granule fluores- cence disappears or is significantly decreased (b). This is not due to granule exocytosis: the granules can still be seen after calcium- enriched ASW treatment under Nomarski phase contrast (e). × 1200. From Maggio et al [88].
potassium stimulation induces a non-un i fo rm increase in the total calcium content o f secretory granules; after rinsing in normal saline, the calcium concentrat ions are not significantly different f rom controls, but it is not known if this is due to calcium loss or to preferential ex- ocytosis o f the calcium-rich granules [142].
Further experiments are needed to resolve the time course o f granular calcium loading under secretory stimu- lation.
Secretion can be independent f rom or even inhibited by calcium increase
Obviously the examples o f Ca 2+ independent secretion [39, 45, 63, 84, 107] suggest that increasing cytosolic free
20 40 60 go IO0 120 140 160 180
[Ca]
b
Fig 3. Frequency distribution of the calcium concentrations of gtio-interstitial granules (in mmol/kg of epoxy-embedded tissue) averaged for each block level, in Mytilus retractor muscle incubat- ed in control artificial sea-water containing 8.36 mmol/l of Ca 2÷ (a), or incubated in calcium-enriched artificial sea-water (b; Ca 2. 12.2 mM), a treatment which normally quenches the granular acidity (fig 2). Using a Mann-Whitney test, it can be shown that the calcium concentration of the granules in tissues exposed to calcium-enriched sea water is significantly higher than in the control (P<0.001). It is assumed that the increase in ex- ternal calcium induced an increase in free internal Ca 2+ concen- tration. From Maggio et al [88].
Ca 2÷ is not the only way to exocytosis, a n d / o r is not the final step. However , when a decrease in cytosolic calcium is needed for the secretory process as in intact parathyroid cells [21, l 17, 131 ], or renin-secreting juxtaglomerular cells [41,123], the consequence is probably an increase in granu- lar ApCa, ending in a situation similar to that predicted in our intragranular calcium loading model. The obser- vation that in permeabilized para thyroid cells, increasing free internal Ca 2 . above 2 × 10 -6 M leads to secretion [109] instead o f inhibiting it could also be explained by the need of a granular calcium load: the calcium-free ' in- tracellular solut ion ' used a round permeabilized cells is likely to deplete the calcium store o f the granules, as it does in mast cells (see refs in [123]). Similarly, the rein- t roduct ion o f calcium after 3 h o f E G T A has been found to paradoxical ly stimulate renin release [82]: we propose that the renin granules could have reversibly lost their cal- cium load and ipso f a c t o their 'secretabil i ty ' during the period o f calcium deprivation. Unfor tunate ly , we cannot comment further on renin or para thyro id granules: they were not included in table I because we could not find liter- ature data on their calcium content, pH, or Ca2+-ATPase activity.
The calcium loading of secretory granules 93
Excretion of calcium can be achieved by granule exocytosis
The secretory products are often rich in calcium: calcium ions can be extruded by the calcium pump or Na ÷/Ca 2 ÷ exchanger of the plasma membrane to restore a normal cytosolic level or, if the granular core contains calcium, it is only logical that calcium is released as a consequence of exocytosis; calcium excretion via secretory granule exo- cytosis was observed in various secretory cell types, such as blood platelets [100], parotid gland cells [149], exocrine pancreas acinar cells [31], adrenal chromaffin cells [47], or sea-urchin eggs [43].
The hypothesis that the calcium-induced activation of secretion could be self-terminating by calcium sequestra- tion into the secreted vesicles has been suggested before, particularly in the case of synaptic vesicles [64, 141], and chromaffin granules [46] but calcium secretion may also have other functions. Calcium secretion of the parotid gland is well documented and classically considered as a particular requirement of buccal biology [148]. The calcium content of mammary epithelium secretion, milk, is generally believed to be of dietary value for the suck- ling. The calcium released by platelets dense granules is considered to play a significant role in haemostasis [100, 133]. Echinoderm juxtaligamental cells may increase the stiffness of collagen tendons [57, 150] by releasing their granular calcium stores. It has also been proposed that glial granules could function as a store to regulate the perineuronal calcium concentration in molluscs [69, 70, 102] as well as vertebrates [42]. But many other secretory granules are likely to release significant amounts of calcium (tables II, III) when they undergo partial or total exocytosis.
The exocytotic secretion of calcium could have been a regulatory mechanism in early cells
We would like to evoke the possibility that the mechan- ism of exocytosis has appeared during the evolution of cells as a way to excrete calcium, and has retained certain properties of this early function. From the viewpoint of a single cell, secretion can be considered as excretion, and it is reasonable to assume that calcium excretion after prior storage into a membrane-bound organelle is beneficial for the cell. Above pCa 5.2, phosphate would precipitate and could not be used for phosphorylation: therefore a low cytosolic Ca 2÷ concentration must have been invented very early in evolution [78]. All present day cells main- tain this low cytosolic Ca 2÷ concentration by calcium pumps [26] and they all have calcium pumps on their plas- ma membranes [27]; some possess in addition Ca2÷/Na ÷ exchangers [68, 80]. The surface regulation of these plas- ma membranes supposes endocytosis as a necessary sym- metrical eyent, following (or preceding) exocytosis, and this symmetry has been verified in certain systems [75, 96, 98, 119] although there are exceptions [116]. When endocy- tosis takes place, it is bound to produce vesicles with an innate capacity to extract calcium from the cytosol, ac- cumulating this ion into the vesicles instead of pumping it outside.
The sequestration of calcium in membrane-bound sub- cellular compartments has obvious advantages adding to simple active pumping via a calcium ATPase at the level of the plasma membrane, such as: 1) increased surface of membrane where exchange occurs; 2) the possibility of a faster relaxation after calcium transients; and 3) downhill
(exergonic) clearance of calcium at a time when ATP can be rare (this would occur if a proton gradient has been built and used for calcium uptake by the vesicle). The sug- gestion that secretory granules could actually regulate the cytosolic Ca 2÷ concentration by calcium uptake has been made by several investigators [7, 46, 55, 122, 123, 130, 132] although other authors came to opposite conclusions on similar secretory models [60].
Secretory granules are acidic
Practically all acidic intracellular organelles belong either to the endocytotic or to the exocytotic pathway [5, 93]. Intracellular organelles of the secretory pathway are gener- ally acidic ([93, 124] and table I): this statement has been verified for many secretory granules or vesicles, such as amine-containing granules of the adrenal medulla, plate- lets, and mast cells [29], peptide-secreting granules of adenohypophysis [28], neurohypophysis [129], and endo- crine pancreas (insulin secreting)/3 cells [1, 110], as well as cholinergic synaptic vesicles [4, 94, 146]. Adrenal chro- maffin granules, the best studied so far of all secretory granules, have an average internal pH of 5.5-5.7 (see refs in [29]) or perhaps slightly higher (6.2 according to Bulenda and Gratzl [22]). Similarly, the intragranular pH in other tissues has also been shown to be acidic: mast cell gran- ules 6.1 [66], platelet dense granules approximately 5.7 [67], platelet ~-granules 6 [48], insulin-containing pancreat- ic granules 5 - 6 [62], anterior pituitary granules 6.0 [28]; the posterior pituitary (neurosecretory) granules would have a pH of 5.8 following Russel and Holz [125] but aged neurosecretory granules would have a pH of 6.4 and newly formed neurosecretory granules a pH of 6.6 according to Scherman and Nordmann [129]. Amylase-containing gran- ules of parotid acinar cells are the least acidic secretory granules to date with a pH of 6.8 [11] that may become alkaline in rats which have been chronically stimulated with isoproterenol [10]; they do not accumulate the fluorescent dye acridine orange [11] and the authors suggest that they could be more alkaline than the cytosol [10]. Cortical gran- ules of the sea-urchin egg do not seem to accumulate fluorescent amines either and may also have a weak ApH although this has not yet been measured (R Christen, per- sonal communication).
The zapH of secretory granules is maintained by a low membrane permeability to H ÷ ions and a H ÷-ATPase [2, 124] and in some cases by Donnan equilibrium [129].
Three main functions have been attributed to this ApH: uptake and maturation of secretory products; intracellu- lar movement; and energy storage for the exocytotic climax.
Uptake and maturation o f secretory products
It has been carefully demonstrated that this ATP- dependent proton gradient is determinant in the accumu- lation of biogenic amines by chromaffin granules (see [29]) and of various neurotransmitter by synaptic vesicles [139]. The maturation of insulin in endocrine pancreas [110] and of neuropeptides (see refs in [59]) also depend on an acid- ic intragranular pH.
Intracellular movement
The acidity of secretory granules would play a role in their transit between the Golgi and site of release [111]. This
94 G Nicaise et al
is also true for the organelles of the endocytotic pathway. The role of vesicular pH is better documented for the en- docytotic pathway but it is likely that intracellular trans- port of vesicles shares common properties in both pathways; inhibiting acidification of endocytotic vesicles blocks their transport function [44]; when the proton gra- dient of lysosomes is experimentally decreased, these or- ganelles rapidly move towards the cell periphery and conversely this movement is reversed if the proton gradient is increased [56]. However, in AtT20 cells, the movement of ACTH secretory vesicles does not seem to depend on their internal pH [77].
Energy storage for the exocytot& climax
At the site of exocytosis, the granular proton gradient can be tapped as a chemiosmotic energy source for exocytotic events and particularly granule swelling [50, 118]. However, this line of research has lost most of its appeal after it had been shown that exocytosis could be induced in the absence of a proton gradient [59, 76] and that gran- ule swelling could occur after the formation of an exocytot- ic pore [3, 12, 19, 116].
The proton gradient may lead to ATP-independent Ca 2 + uptake into the secretory granules
Ca 2+ uptake by chromaffin granules, as mediated in vitro by the ionophore A23187, is dependent upon an in- tragranular proton store [22]: however, because A23187 is a calcium-proton exchanger, these data give little infor- mation on the physiological mechanism coupling Ca 2+ uptake and exocytosis. In lysosomes, organelles of the en- docytotic pathway which are both acidic inside and calcium-rich, like secretory granules, the ApH is used for an ATP-independent calcium uptake [81].
In molluscan glio-interstitial granules, the extinction of acridine orange-induced fluorescence in conditions of in- creased cytosolic Ca 2+ concentration is suggestive of a Ca 2 + /H + exchange, and the increase in granular calcium content can be confirmed by quantitative X-ray microana- lysis (figs 2, 3).
In pancreatic endocrine fl-cells, the proton ionophore monensin not only quenches the acridine orange-induced red fluorescence of the granules but also inhibits glucose- induced insulin secretion [113]. In neurohypophysis per- meabiliz~ed nerve endings, the proton ionophore CCCP in-
2+ hibits Ca -dependent hormone secretion [30]. Lowering the cytosolic pH (ie decreasing the ApH of secretory gran- ules, as their membrane is relatively impermeable to pro- tons) inhibits parathyroid hormone as well as platelet-dense granule secretion [118]. The possibility that the granular ApH plays a role in exocytosis of chromaffin granules has been tested by two groups on cultured [59] and on dis- sociated an d permeabilized cells [76]. In the first case, the authors presented the observation that diverse reagents ac- tually collapsing the granular proton gradient failed to in- hibit secretion, provided that they were used at low concentrations and did not reduce cytosolic ATP concen- tration [59]. In the permeabilized cells the results also showed "a small and in a few cases up to 50% reduction in exocytosis in the presence of high concentrations of the various agents used to collapse or modify pH i and ~m-Suggesting that the secretory vesicle pH and poten- tial may play a small modulating role in exocytosis" [76]. In this second model in particular, exocytosis depends both
on calcium and Mg-ATP, although the "mode of action (of ATP) is obscure" [76]. Metabolic inhibitors impair most secretions [84] but ATP is not considered anymore to be necessary at the time of exocytosis in normal condi- tions [116]. If, as we propose, calcium accumulation in- side the granule is a critical event, it could be performed by a calcium ATPase using either a proton gradient, or ATP if the proton gradient has been artificially abolished. It can be expected that neutralizing the intragranular acidi- ty may not necessarily inhibit secretion, as it mimics one half of the intragranular calcium-proton exchange. This may also explain why the proton ionophore FCCP tends to induce spontaneous degranulation in mast cells [20]. In addition, the dissipation of a proton gradient (by a pro- ton ionophore) can rapidly enhance the fusion of artifi- cial phospholipid vesicles, and neutralization also decreases the Ca 2+ threshold concentration required for aggrega- tion of the vesicles [151].
If Ca2+/H + exchange is important for exocytosis, it should occur just before the formation of the exocytotic pore. The intragranular acidity, as seen by quinacrine fluorescence, has been monitored by Breckenridge and A1- mers [19] in parallel with the membrane capacitance changes indicating exocytosis of giant granules in beige mouse mast cells. In an elegant experiment, the authors recorded decreases in fluorescence accompanying step in- creases in membrane capacitance (single granule exocy- toses) but they consider that these extinctions are due to dye loss in the extracellular milieu; they would thus take place after the formation of the exocytotic pore. In par- ticular, they report two cases where the decrease in fluores- cence started at the end of a period of flicker (intermittent opening of the exocytotic pore, see [39, 84, 153]). This ex- periment is clearly contradictory to a role of the proton gradient as we suggest it. It is, however, possible that the size of the giant granules and/or the use of quinacrine did slow down the fluorescence changes which could have been better monitored with normal sized granules and a more classical pH probe like BCECF. With the help of recent data obtained on rat peritoneal mast cells (Horoyan et al, in preparation) we suggest that the experiments of Breck- enridge and Almers could be partially reinterpreted to admit that the neutralization of the granular acidity may take place before exocytosis.
Subcellular CaE+/H + exchange seem to be very common (eg [24, 58, 91, 147]) and membranes may use different solutions to exchange protons against calcium; in the next section, we shall insist on the Ca2+-ATPase but other exchangers have been proposed [15, 108]. More research is obviously needed to collect direct evidence of a Ca 2+/H + exchange at the level of secretory granules, however, the presence of a Ca 2+/H + exchanger such as a calcium-transporting ATPase would be a good indica- tion that such an exchange can take place in physiological conditions.
The Ca2+/H + exchange can be performed via a Ca 2+- ATPase
All calcium-transporting transmembrane pumps appear to behave as CaE+/H+ exchangers [27, 36, 83, 86, 90, 105, 134], but the generality of this mechanism has not been recognized until recently, and was overlooked in most studies on secretory granules. Information available on the presence (or absence) of Ca2+-transporting ATPases at the level of secretory granule membranes is based either
The calcium loading of secretory granules 95
on cytochemical evidence of a calcium-dependent ATPase activity or on ATP-dependent calcium accumulation in- side the isolated organelles (table I).
A calcium-activated ATPase activity has been ultrastruc- turally demonstrated in the Golgi zone and young (newly formed) secretory granules of chromaffin cells [14] and on all chromaffin granules in another study (Nakai et al, 1976, in [152]), as well as on atrial specific granules [138], rat mast cell granules [33], rat anterior pituitary granules [135] and on Aplysia gliagrana [89]. However, the cytochemistry of calcium-ATPases has considerably im- proved with time and the data obtained prior to the work of Ando and co-workers [8] must be considered with caution.
An ATP-dependent calcium uptake has been demon- strated on insulin granules [7], microvesicles of the neu- rosecretory endings [106], synaptic vesicles [64, 95], sea-urchin cortical granules [43] and isolated chromaffin granules [46, 54] although several failures were also report- ed for these last organelles (see [29]). Isolated zymogen granule membranes [53] and insulin storage pancreatic granules [61] also present a Ca2+-ATPase activity.
In the secretory pathway, the coexistence of a proton pump, or at least an acidic interior, and a calcium pump on the granular membrane has therefore been demonstrat- ed in many cell types, but seldom related to a possible Ca 2+/H ÷ exchange. For the endocytotic pathway, this possibility has been presented in the case of cultured rab- bit reticulocytes [108]; the authors suggest that a Ca2+/H + exchanger could slow down the centripetal movement of endocytotic vesicles by dissipating the ApH in conditions of high intracellular Ca 2+ concentration. However, these authors suggest that the Ca2+/H + ex- changer is not necessarily the Ca2+-ATPase.
In the case of mast cells at least, it has been demonstrat- ed that various inhibitors of the ATPase activity also in- hibit secretion, presumably by blocking the exocytotic process, and the author suggest that the inhibition would primarily affect the Ca 2 + - M g 2+ ATPase [32].
lntragranular sodium uptake could be an important con- sequence of the Ca 2+ crisis
Other mechanisms than a Ca2+/H ÷ exchange or a calcium-ATPase pump have been proposed for the calci- um accumulation inside secretory granules, and particu- larly Ca2+/Na ÷ exchange [92]. The membranes of chromaffin granules [79, 114] and posterior pituitary granules [127] appear to possess a Ca2+/Na ÷ antiport which, according to the authors, would cause under physiological conditions an intragranular accumulation of calcium. The finding that no sodium could be detected by X-ray microanalysis in chromaffin granules frozen in situ [112] seriously challenged that hypothesis. However, other secretory granules analyzed in situ contain measura- ble sodium, and the sodium concentration may increase after stimulation: from 5 mM (at rest) to 55 mM (when salivating) in rat parotid [65], from 6 mM (at rest) to 10 mM (when stimulated) in dog pancreas [101] but no changes could be seen in rat pancreas [121]. No data were found on stimulated chromaffin cells or neurosecretory endings.
Future investigations may confirm the presence of a Ca 2÷/Na ÷ antiport on various secretory granule mem- branes: it would be a simple consequence of the exocytotic- endocytotic cycle (see above), but this antiport is not neces-
2+
Ca 2+ / /
BM- Ca / t /Ca
Fig 4. One of the possible schematic interpretations of calcium loading in secretory granules, representing a typical acidic cytoplasmic granule (top drawing) and a granule of the same cell, caught in the act of stimulus-secretion coupling, docking on the inner side of the plasma membrane and saturated with calcium. BM: calcium-binding molecules of the granular matrix; Ca: bound calcium; Ca2÷: calcium ions; H+: hydrogen ions. Larger font sizes are used for higher concentrations. The clear mushroom on the granular membrane represents a proton ATPase, the dotted one a calcium ATPase, the dotted circle on the plasma membrane a calcium channel. The shaded arrow points to fusogenic molecules.
sarily used to accumulate calcium. If our calcium loading model is valid, the predicted climatic Ca 2+ increase should induce a sodium influx in the granule. This is par- ticularly interesting with regard to the observation that in the absence of calcium, at least in some systems, sodium by itself can be sufficient to induce exocytosis [107, 145].
Conclusion
The present article reviews three kinds of data, which may be complementary, as shown in figure 4.
The high calcium concentrations of secretory granules indicate that there are mechanisms responsible for calcium storage into these organelles, which function before the time of exocytosis. The same mechanisms are likely to
96 G Nicaise et al
induce a calcium overload at the time and place of stimulus-secretion coupling. I f granular calcium loading is one of the critical events leading to membrane fusion, and all granules are fitted to capture calcium ions, there must be a difference between granules, at least in the most common situation where only a part of the granules is secreted upon a given stimulation. This difference can obviously be the distance between the granule and the plasma membrane, but the maturation ('secretability') can also result f rom the saturation of calcium storage molecules which are known to be present in the granular core. The variety of calcium concentrations measured by X-ray microanalysis would indicate differences in secre- tability.
The hypothesis that intragranular calcium accumulation plays a role in stimulus-secretion coupling has explanatory value: for example if Ca 2+ ions exert their action from the inside of the secretory organelle, it is possible to describe the reverse calcium dependence of exocytosis, shown by non-permeabilized parathyroid cells for exam- ple, in the same terms as the classical calcium-dependent secretion (using ApCa, see above).
This hypothesis can be tested: for example the rise in intragranular free Ca 2+ at the time of exocytosis, when the stores are supposedly overloaded, can be monitored by calcium imaging (at least when granular esterases per- mit the use of acetoxy-methylester probes). The calcium load of non-immediately secretable granules (eg old, as op- posed to newly formed, neurosecretory granules) can be measured by X-ray microanalysis, as well as the predict- ed restoring of their calcium load in conditions of restored secretability.
The present article also tries to revive the classical state- ment that most secretory organelles being acidic, the secre- tory crisis can use the potential energy of a proton gradient. We adopt the hypothesis of Hellman et al [55], which differs from other proposals [50, 118] in the sense that Ca 2+ ions would not activate an exchanger but exchange themselves against protons. The reality of a calcium-proton exchange can be tested, particularly with pH specific fluorescent probes, in association with quantitative X-ray microanalysis of the granular total calcium content or with fluorescent imaging of intragranular ionized Ca 2+. However, as for the previous hypothesis, it will be impor- tant to decide wether the observed granules are taken just before exocytosis or are already communicating with the outside, and the results will be more conclusive if the meas- ured inttagranular calcium concentrations and pH raise above extracellular levels.
Finally we propose that, in certain cases at least, the calcium-proton exchanger would be a Ca2+-ATPase. This additional assumption explains why in the absence of a proton gradient, calcium can still be transported into the granule, at the expense of ATP. Although specific inhibi- tion of calcium pumps may be difficult to perform, the third hypothesis predicts that inhibition of ATPases is bound to suppress the granular calcium-proton exchange and impair exocytosis.
These three hypotheses are presented together with the hope that they will help to collect more significant data on the time course of intragranular calcium loading during exocytosis, calcium-proton exchange and even calcium-sodium exchange in secretory granules. They probably give a grossly oversimplified view of the role of calcium in exocytotic regulated secretion but they seem to be compatible with most of the available literature.
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