Secretory granules in inositol 1,4,5-trisphosphate-dependent Ca²⁺ signaling in the cytoplasm of neuroendocrine cells

Abstract

Of all the intracellular organelles, secretory granules contain by far the highest calcium concentration; secretory granules of typical neuroendocrine chromaffin cells contain ∼40 mM Ca²⁺ and occupy ∼20% cell volume, accounting for >60% of total cellular calcium. They also contain the majority of cellular inositol 1,4,5-trisphosphate receptors (IP₃Rs) in addition to the presence of >2 mM of chromogranins A and B that function as high-capacity, low-affinity Ca²⁺ storage proteins. Chromogranins A and B also interact with the IP₃Rs and activate the IP₃R/Ca²⁺ channels. In experiments with both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells, in which the number of secretory granules present was changed by either suppression or induction of secretory granule formation, secretory granules were demonstrated to account for >70% of the IP₃-induced Ca²⁺ releases in the cytoplasm. Moreover, the IP₃ sensitivity of secretory granule IP₃R/Ca²⁺ channels is at least ∼6- to 7-fold more sensitive than those of the endoplasmic reticulum, thus enabling secretory granules to release Ca²⁺ ahead of the endoplasmic reticulum. Further, there is a direct correlation between the number of secretory granules and the IP₃ sensitivity of cytoplasmic IP₃R/Ca²⁺ channels and the increased ratio of IP₃-induced cytoplasmic Ca²⁺ release, highlighting the importance of secretory granules in the IP₃-dependent Ca²⁺ signaling. Given that secretory granules are present in all secretory cells, these results presage critical roles of secretory granules in the control of cytoplasmic Ca²⁺ concentrations in other secretory cells.—Yoo, S. H. Secretory granules in inositol 1,4,5-trisphosphate-dependent Ca²⁺ signaling in the cytoplasm of neuroendocrine cells.

Several cytoplasmic organelles, including the endoplasmic reticulum (ER), secretory granules, mitochondria (1,2,3,4), lysosomes, and endosomes (5,6,7) have been shown to function as intracellular Ca²⁺ stores. Yet studies on the intracellular Ca²⁺ stores in the past have often centered around the ER (8,9,10), presumably because of the first identification of the inositol 1,4,5-trisphosphate receptor (IP₃R) in the ER (11) and the importance of the ER in the cell. The possibility of secretory granules serving as an intracellular Ca²⁺ store was first raised in chromaffin cells when chromaffin granules rapidly released Ca²⁺ in response to IP₃ (12), and then in pancreatic acinar cells (13). IP₃ releases substantial amounts of Ca²⁺ rapidly from secretory granules, which is followed by immediate sequestration of the released Ca²⁺ into secretory granules, both in the absence of exogenous energy supply (12,13,14). The IP₃-induced release and sequestration of granular Ca²⁺ can be repeated several times without diminution of the magnitude and speed of Ca²⁺ release and sequestration (Fig. 1). The IP₃-mediated Ca²⁺ release from secretory granules has now been demonstrated in many types of secretory cells (15,16,17,18,19,20), and the acidic intragranular milieu (pH 5.5) is considered to be responsible for the energy-independent Ca²⁺ sequestration (20). In a recent review (7), participation of secretory granules, along with the ER and other intracellular organelles, in the control of intracellular Ca²⁺ concentrations in exocrine cells is discussed. Hence, in light of increasing evidence demonstrating the major role of secretory granules in the control of IP₃-dependent Ca²⁺ mobilization in the cytoplasm of neuroendocrine cells, the molecular basis that gives rise to the primary role of secretory granules in IP₃-dependent Ca²⁺ signaling in the cytoplasm is reviewed here.

Figure 1.

IP₃-induced Ca²⁺ release from secretory granules of chromaffin cells. IP₃-induced Ca²⁺ release from isolated secretory granules of bovine chromaffin cells was measured using fluorescence microscopy after adding 1 μl IP₃ (1 μM final) aliquots to 200 μl of secretory granule solution (1 mg protein/ml) in the presence of 15 μM Calcium Green-1 (111). To perform IP₃-induced Ca²⁺ release experiments, secretory granules that had been prepared as described previously (12) were resuspended in buffer I (10 mM HEPES/KOH, pH 7.2; 140 mM KCl; 0.25 M sucrose). Final Ca²⁺ concentration of the secretory granule solution was adjusted to 0.3 μM by adding appropriate amounts of EGTA. Sample chamber containing Calcium Green-1 was placed on the stage of a Zeiss Axiovert S 100 microscope (Carl Zeiss, Jena, Germany), and 1 μl IP₃ (1 μM final) aliquots were added. IP₃-induced Ca²⁺ release was analyzed by excitation of Calcium Green-1 at 495 nm with a Lambda LS xenon arc lamp and Lambda 10-2 optical filter changer (Sutter Instrument, Novato, CA, USA). The emission fluorescence signals at 530 nm were collected using a bandpass filter of D510/40 nm (Chroma Technology Corp., Rockingham, VT, USA) and Hamamatsu C4742-95 digital CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). Approximately 4 images/s were acquired; imaging continued with successive addition of IP₃. Changes of fluorescence were calculated by MetaFluor software (Meta Imaging 6.1; Universal Imaging Corporation, West Chester, PA, USA).

Ca²⁺ CONCENTRATIONS IN SECRETORY GRANULES

Secretory granules of secretory cells are known to contain the most intracellular calcium in the cytoplasm, containing 15–40 mM (7, 21,22,23). However, most of the intragranular calcium stays bound to chromogranins (24), and secretory granules maintain free Ca²⁺ concentrations of only 28–100 μM (13, 14, 19, 20, 24, 25) although 1.4 μM (26) and 300 μM (18) are also reported. Despite the seemingly low free-Ca²⁺ concentrations inside secretory granules, IP₃ triggers a rapid Ca²⁺ release from secretory granules (12,13,14, 17,18,19,20, 27). In addition to 40 mM calcium in secretory granules (21, 22), there are on average ∼15,000–30,000 secretory granules in each adrenal chromaffin cell (28,29,30,31,32), occupying ∼20% of the total cell volume (28,29,30) and accounting for >60% of total cellular calcium of these cells (21) (Table 1). On the other hand, the Ca²⁺ concentrations of the ER are known to be ∼3 mM (10, 33), and the ER of bovine adrenal chromaffin cells occupies ∼14% of the cell volume (28), which indicates that ∼5% of total cellular calcium of chromaffin cells is stored in the ER (Table 1). The unusually high calcium-storing capability of secretory granules is attributed to the presence of high concentrations of Ca²⁺-storage proteins chromogranins A and B (CGA and CGB), and secretogranin II (SgII) in secretory granules (Table 2) (24, 34,35,36).

TABLE 1.

Calcium and IP₃R concentrations in the ER and secretory granules of bovine chromaffin cells

Location	Cell volume (%)	Calcium concentration (mM)	Total calcium (% of cell)	IP3R1		IP3R2		IP3R3
				Relative concentration	Total IP3R1 (% of cell)	Relative concentration	Total IP3R2 (% of cell)	Relative concentration	Total IP3R3 (% of cell)
Endoplasmic reticulum	14.2	3	5	1.20	15.2	1.55	19.8	0.74	15.9
Secretory granule	20.5	40	60	3.77	69.3	3.48	64.3	1.88	58.1

Percentages of cell volume are from ref. 28. Calcium concentrations for the ER and secretory granules are from refs. 10 and 21, 22, respectively. Percentage of ER calcium is calculated assuming that 60% of total cell calcium is stored in secretory granules (21). Relative concentration values are obtained assuming the relative concentration of each IP₃R isoform of the nucleus as 1 (41). 

TABLE 2.

Ca²⁺-binding properties of chromogranins/secretogranin II and their respective concentrations in the ER and secretory granules of bovine chromaffin cells

Protein	Ca2+ binding (mol/mol protein)	Kd (mM)	Concentration (μM)
			Endoplasmic reticulum	Secretory granule
Chromogranin A	32–55	2.7–4.0	>140	>1800
Chromogranin B	50–93	1.5–3.1	>120	>200
Secretogranin II	30–61	2.2–3.0	>10	>30

Ca²⁺-binding and K_d values from refs. 35 (chromogranin A), 34, 36 (chromogranin B), and 36 (secretogranin II). Concentration values from refs. 28, 36. 

IP₃R/Ca²⁺ CHANNELS IN SECRETORY GRANULES

As the first IP₃-induced Ca²⁺ release from secretory granules was demonstrated with chromaffin granules (12), so was the first direct evidence for the existence of the IP₃Rs in the membranes of secretory granules obtained from chromaffin granules (37). In the same year, the insulin-containing secretory granules of pancreatic β cells were reported to contain type 3 IP₃R (IP₃R3) (38), but this report was questioned by another group (39) who raised the possibility of cross-reactivity of the IP₃R3 antibody, which had been used in previous studies with insulin molecules that are present inside the insulin-containing granules of pancreatic β cells. Yet, subsequent studies confirmed not only the presence of IP₃Rs (17) but also of all three types (types 1, 2, and 3) of IP₃Rs in the secretory granules of pancreatic β cells (16). It appears that depending on the epitope specificity of the antibodies used, the presence of the IP₃Rs in secretory granules has occasionally not been detected. On the other hand, in a study using vesicle membrane protein synaptobrevin-aequorin chimera that is expressed in a β-cell line (MIN6 cells) as a reporter, IP₃ was shown not to lower the inside Ca²⁺ concentrations of secretory granules ([Ca²⁺]_SG) in permeabilized MIN6 cells, whereas the ryanodine receptor activator caffeine caused a dramatic fall in [Ca²⁺]_SG of intact MIN6 cells (25), leading the researchers to conclude that only the ryanodine receptors, not the IP₃Rs, localize in β-cell granules. Given that the IP₃ effect was tested with permeabilized cells while the caffeine effect was with intact cells, it is likely that either permeabilization destroyed the IP₃Rs, and the signal receiving machinery on secretory granules or synaptobrevin-aequorin chimera was not positioned in the right structure along with other membrane components in the granules to respond to IP₃.

In addition to the IP₃Rs in secretory granules of chromaffin cells, pancreatic β and acinar cells, secretory granules of airway goblet cells (14) and mast cells (15, 20) are also known to contain the IP₃Rs. Utilizing the thin optical sectioning method and measuring the changes in Ca²⁺ concentrations in both secretory granules and the surrounding cytoplasm simultaneously, Verdugo and colleagues (14) showed that the IP₃-induced Ca²⁺ releases from secretory granules dictate the Ca²⁺ concentrations in the neighboring cytoplasm of goblet cells. Again taking advantage of the mast cell granules of mutant beige mice, which can reach up to 2–3 μm in diameter, both the IP₃-induced Ca²⁺ release from secretory granules and the IP₃-dependent intracellular Ca²⁺ oscillator role of secretory granules were unequivocally demonstrated (15, 20), thus establishing the presence of the IP₃Rs in secretory granules of airway goblet and mast cells.

The amounts of IP₃R isoforms that exist in each cellular organelle vary depending on the type of cells (40), yet the majority of cellular IP₃Rs in chromaffin cells is localized in secretory granules (41). Assuming the relative concentration of each IP₃R isoform in the nucleus as 1 and taking the cell volume of each organelle of bovine adrenal chromaffin cells (28) into consideration, the relative concentration and the total amount of each IP₃R isoform in each organelle can be estimated (Table 1). As summarized in Table 1, secretory granules contain ∼58–69% of total cellular IP₃Rs, with relative concentrations of 1.8–3.7, while the ER contains ∼15–20% of total cellular IP₃Rs, with relative concentrations of 0.7–1.5 (41). This indicates that the total amounts and relative concentrations of the IP₃Rs in secretory granules are higher than those of the ER by 3- to 4-fold and 2- to 3-fold, respectively (41, 42).

CHROMOGRANINS IN SECRETORY GRANULES

The major proteins of secretory granules are chromogranins A and B (43,44,45,46); thus, they are called marker proteins of secretory granules (47). Bovine secretory granules contain >1.8 mM CGA and >0.2 mM CGB (43, 44), while the ER contains >0.14 mM CGA and >0.12 mM CGB (Table 2). A third major member of the granin family proteins, secretogranin II, is also found in secretory granules, although its concentration is significantly lower than that of chromogranins (43, 44) (Table 2). Chromogranins A and B are multifunctional proteins (see below) that are present primarily in secretory cells though they are also found in cells such as cardiomyocytes (48) that are not traditionally considered secretory cells.

Granulogenesis

Chromogranins A and B participate directly in secretory granule biogenesis of secretory cells (49,50,51). Studies that had been done with both neuroendocrine and nonneuroendocrine cells have demonstrated the granulogenic roles of chromogranins. Expression of the granins in nonneuroendocrine cells, such as NIH3T3 or COS cells that do not normally contain secretory granules, induce formation of secretory granules in these cells, whereas suppression of granin expression in neuroendocrine cells substantially reduce the number of secretory granules formed in these cells (49,50,51). Nonetheless, it was initially reported that only CGA, not CGB, is capable of functioning as the granulogenic factor (51), which proved not to be the case in later studies (49, 50). The granulogenic ability of CGB is, in fact, shown to be ∼60% greater than that of CGA in certain conditions (50), and this appeared to reflect the markedly stronger ability of CGB than CGA to interact with other proteins in secretory granules (52, 53). Analogous to the granulogenic roles of chromogranins A and B, secretogranins have also been shown to participate in secretory granule formation (49, 54).

Ca²⁺ storage, and catecholamine and ATP binding

The high Ca²⁺ concentrations in secretory granules are due to the presence of large amounts of acidic protein chromogranins that have a high-capacity, low-affinity Ca²⁺-binding property (Table 2). Chromogranin A binds 32–55 mol of Ca²⁺/mol, with dissociation constants (K_d) of 2.7–4 mM (35), whereas chromogranin B binds 50–93 mol of Ca²⁺/mol, with K_d of 1.5–3.1 mM (34, 36). As a result, most (>99.9%) of the intragranular calcium stays bound to chromogranins, and only a very small amount of Ca²⁺ stays in free state, ranging from ∼28 to 100 μM (13, 14, 19, 20, 24, 25).

In light of the presence of >2 mM chromogranins in secretory granules, the high-capacity Ca²⁺ binding property of chromogranins would be sufficient to store ∼40 mM Ca²⁺ inside secretory granules. Moreover, the low affinity of chromogranins for Ca²⁺ would facilitate ready exchange of bound and free Ca²⁺ within secretory granules, so that intragranular calcium could actively participate in the Ca²⁺ flow through the IP₃R/Ca²⁺ channels of secretory granules. Analogous to the high-capacity, low-affinity Ca²⁺-binding property of chromogranins A and B, secretogranin II also binds 30–61 mol of Ca²⁺/mol with K_d of 2.2–3.0 mM (Table 2), implying active roles of SgII in the storage of Ca²⁺ in secretory granules.

Chromogranins are also known to bind catecholamines (55) and ATP (56) that exist in secretory granules at ∼600 mM and ∼150 mM, respectively (22, 57). Chromogranin A is reported to bind 32 mol of norepinephrine/mol, with a K_d of 2.1 mM (55). However, considering the intragranular concentrations of chromogranins and catecholamines, the binding of 32 mol of norepinephrine/mol of CGA is estimated to account for only ∼5.3% of norepinephrine present inside secretory granules (55). This result suggests that chromogranins are partly responsible for the accumulation of catecholamines in secretory granules. In line with this finding, the intragranular content, as well as the amount of catecholamines released in chromaffin cells from CGA-knockout mice, were shown to be reduced ∼30% (58), suggesting the role of chromogranin A in the storage of catecholamines. Chromogranin A also interacts with ATP through the adenine base of the nucleotide (56), yet its binding stoichiometry or affinity is not known. In view of the coexistence of catecholamines, ATP, the granins and divalent cations in high concentrations and the tendency of these molecules to interact with each other, it is likely that the intragranular residents form complexes, as has been proposed previously (59).

IP₃R/Ca²⁺ channel modulation

The IP₃Rs bind chromogranins A and B directly at the intragranular pH 5.5 (60), and the bound chromogranins activate the IP₃R/Ca²⁺ channels to an open-ready state (61), increasing the open probability of the channels 8- to 16-fold and the mean open time 9- to 42-fold (62, 63). In contrast to CGA that fails to interact with the IP₃Rs at a near physiological pH 7.5, CGB still binds the IP₃Rs even at pH 7.5 (60) and increases the open probability of the Ca²⁺ channels 8-fold and the mean open time 24-fold (62, 63), suggesting critical roles chromogranins play in both storage and flux of the intragranular calcium through the IP₃R/Ca²⁺ channels (64). Further, the IP₃Rs undergo conformational changes on IP₃ binding (65), which will, in turn, lead to conformational changes of coupled chromogranins (60, 64). Given that the Ca²⁺-binding affinity and capacity of chromogranins differ depending on the conformation (34,35,36), the IP₃-induced conformational changes of chromogranins will lead to changes in the Ca²⁺-binding affinity and capacity of the coupled chromogranins, thereby enabling rapid exchanges of bound and free Ca²⁺ inside the granules. This rapid exchange of bound and free Ca²⁺ may serve as a basis for secretory granules to be able to repeatedly release and sequester Ca²⁺ on multiple IP₃ challenges.

Moreover, at pH 5.5, chromogranin A not only self-associates to form a CGA homotetramer (66, 67) but also interacts with chromogranin B to form a CGA₂CGB₂ heterotetramer (68). Given that the IP₃Rs form a homotetrameric or heterotetrameric complex to function as a Ca²⁺ channel (40), formation of a homotetrameric or heterotetrameric chromogranin complex in secretory granules implies coupling of tetrameric chromogranins with and activation to an open-ready state of the tetrameric IP₃R/Ca²⁺ channels (64). Nonetheless, this will not be the case in the ER, where the pH is maintained at ∼7.4 (69,70,71); chromogranin A fails to bind the IP₃Rs at a near physiological pH (60), although it interacts with chromogranin B to form a CGA-CGB heterodimer at pH 7.5 (68). In contrast, chromogranin B binds the IP₃Rs and activates the IP₃R/Ca²⁺ channels to an open-ready state even at a near-physiological pH 7.5 (63), thereby suggesting additional Ca²⁺ channel modulatory roles of CGB in organelles other than secretory granules. Accordingly, chromogranin B has indeed been shown to play essential roles in the activity of IP₃R/Ca²⁺ channels of the ER in neuronally differentiated PC12 cells (72) and neurons (73), thus controlling the IP₃-dependent Ca²⁺ release from the ER and the intracellular Ca²⁺ concentrations. It is also shown in cardiomyocytes that CGB controls the IP₃-mediated Ca²⁺ release in these cells, which is closely linked to ventricular cardiac hypertrophy (48).

As a source of bioactive peptides

In addition, chromogranins further exert important physiological functions even after leaving secretory granules, i.e., after exocytosis. Although intragranular chromogranins A and B remain bound to the membranes of secretory granule at the intragranular pH 5.5 through their binding to the integral membrane protein IP₃Rs (60), this state will change during exocytosis when they are exposed to the pH of the extracellular fluid; chromogranin A dissociates completely from the IP₃Rs, while chromogranin B dissociates partially at that pH (60). Therefore, large amounts of chromogranins will dissociate from the granules during exocytosis and be released into the bloodstream. The secreted chromogranins serve as the source of many bioactive peptides, including catestatin, vasostatin, pancreastatin, and secretoneurin, that circulate in the bloodstream (74,75,76,77,78,79,80,81,82). Among a variety of functions that are attributed to these peptides, catestatin and vasostatin are known to control the release of catecholamines (74, 75) and blood vessel dilation (76, 77), respectively, whereas pancreastatin and secretoneurin are known to reduce glucose metabolism (80) and to potentiate angiogenesis (78, 79), respectively.

SECRETORY GRANULES ARE RESPONSIBLE FOR THE MAJORITY OF IP₃-INDUCED Ca²⁺ RELEASES IN THE CYTOPLASM

In view of the presence of the majority of cellular calcium, IP₃R/Ca²⁺ channels, and chromogranins in secretory granules, which is necessary for an organelle to function as the major IP₃-sensitive intracellular Ca²⁺ store, it would be natural for secretory granules to play the most significant role in the IP₃-dependent intracellular Ca²⁺ control mechanism. Whether secretory granules play the major role in the cytoplasmic IP₃-dependent Ca²⁺ control mechanism could be discerned by comparing the magnitudes of the IP₃-mediated Ca²⁺ releases between the cells that contain different numbers of secretory granules (27, 83).

Although both chromogranins A and B play granulogenic roles (49,50,51), CGB can induce ∼60% more secretory granule formation than CGA (50). Hence, by taking advantage of the different granulogenic effects of CGA and CGB, the number of secretory granules produced in nonneuroendocrine cells can be controlled. Moreover, the granulogenic role of chromogranins can also be used in controlling the number of secretory granules formed in neuroendocrine cells; the number of secretory granules formed in neuroendocrine cells decreases by suppressing the intrinsic chromogranin expression in these cells by treating the cells with siRNA-chromogranin. Yet the extent of reduction in the number of secretory granules formed differs markedly depending on whether CGA or CGB expression is suppressed (50). Reflecting the stronger granulogenic role of CGB, suppression of CGB expression is far more effective in reducing the secretory granule formation that the CGB suppression in PC12 cells decreases the number of secretory granules formed ∼90% more than can be achieved with the CGA suppression (50). Thus utilizing this property of chromogranins, the number of secretory granules formed in both neuroendocrine and nonneuroendocrine cells can be controlled, thereby making it possible to examine the specific role of secretory granules in the IP₃-dependent Ca²⁺ releases in the cell.

When the formation of secretory granules in PC12 cells that normally contain chromogranins and intrinsic secretory granules was suppressed by suppressing the intrinsic CGA expression, the magnitude of IP₃-induced Ca²⁺ release in the cytoplasm was reduced to less than a half that of control cells (Fig. 2A), but when the CGB expression was suppressed, the magnitude of IP₃-induced Ca²⁺ release was reduced to approximately one-quarter that of control cells (Fig. 2A). Given that only the number of secretory granules formed is reduced in the cytoplasm, these results indicate that secretory granules are responsible for the majority of IP₃-mediated cellular Ca²⁺ release in the cytoplasm of neuroendocrine PC12 cells (27). On the other hand, induction of secretory granule formation by expressing chromogranins in nonneuroendocrine NIH3T3 cells that normally do not contain any secretory granules markedly increased the magnitude of IP₃-induced Ca²⁺ release of these cells (27, 83). When secretory granule formation was induced by expressing CGA in NIH3T3 cells, the magnitude of IP₃-induced Ca²⁺ release in the cytoplasm of NIH3T3 cells increased ∼2.7-fold over the control (Fig. 2B). But when the number of newly formed secretory granules was further increased by expressing CGB in NIH3T3 cells, the magnitude of IP₃-induced Ca²⁺ release in the cytoplasm of NIH3T3 cells increased ∼3.7-fold over the control (Fig. 2B), further underscoring the major role of secretory granules in IP₃-dependent Ca²⁺ mobilization. Consistent with the results shown for the PC12 cells, these results demonstrate that secretory granules are the major IP₃-sensitive intracellular Ca²⁺ store, accounting for >70% of the cytoplasmic Ca²⁺ released by IP₃.

Figure 2.

Microinjection of IP₃ and Ca²⁺ mobilization in the cytoplasm of PC12 and NIH3T3 cells. A) IP₃-induced Ca²⁺ releases in the cytoplasm of control (normal), siCGA-treated (siCGA), and siCGB-treated (siCGB) PC12 cells were measured after microinjection of 10 nM IP₃ into the cytoplasm of the cell. B) IP₃-induced Ca²⁺ releases in the cytoplasm of control (normal), CGA-transfected (CGA/ECFP), and CGB-transfected (CGB/ECFP) NIH3T3 cells were measured after microinjection of 10 nM IP₃ into the cytoplasm of the cell. Figure is modified from Figs. 2 and 7 of ref. 83.

Although it is possible that the effect shown by CGB expression is a combined effect of secretory granules and the CGB-containing ER, the effect exerted by the CGB-containing ER will be very minimal compared to that shown by secretory granules because of the large difference in the total calcium stored and the IP₃R concentrations that exist in each organelle, and the significantly lower IP₃ sensitivity of the IP₃R/Ca²⁺ channels of the ER. In addition, the weaker binding of CGB to the IP₃Rs (84,85,86,87) and the lower Ca²⁺ binding capacity of CGB (36) at the pH of the ER compared to those shown at the intragranular pH could also contribute to the minimal effect. That the effect shown by chromogranin expression or suppression is very closely tied to the number of secretory granules formed or reduced further strengthens this assessment.

SECRETORY GRANULE IP₃R/Ca²⁺ CHANNELS ARE MORE SENSITIVE TO IP₃ THAN THE ER IP₃R/Ca²⁺ CHANNELS

By examining the IP₃ sensitivity of the IP₃R/Ca²⁺ channels in the cytoplasm of cells in which the formation of secretory granules is either induced or suppressed without disturbing the ER, it is possible to estimate the respective IP₃ sensitivity of the IP₃R/Ca²⁺ channels of the ER and secretory granules. The IP₃ sensitivity of the IP₃R/Ca²⁺ channels, which is normally expressed in EC₅₀ values of IP₃ concentration, will predict which IP₃R/Ca²⁺ channels open first in response to IP₃. In this regard, the EC₅₀ value of IP₃ concentration of the cytoplasmic IP₃R/Ca²⁺ channels in nonneuroendocrine NIH3T3 cells that do not have secretory granules is shown to be 110 nM (88), but in the NIH3T3 cells that contain newly formed secretory granules as a result of chromogranin A expression, the EC₅₀ value changes to 27 nM (Fig. 3A), increasing the IP₃ sensitivity of the cytoplasmic IP₃R/Ca²⁺ channels ∼4-fold (88). Further, expression of CGB in the same cells changes the EC₅₀ of cytoplasmic IP₃R/Ca²⁺ channels to 20 nM (Fig. 3A), increasing the IP₃ sensitivity of the cytoplasmic IP₃R/Ca²⁺ channels of the NIH3T3 cells ∼6-fold (88). Here, the effect of CGB expression is significantly greater than CGA. In view of the fact that induction of secretory granule formation by CGB expression increased the IP₃ sensitivity of the NIH3T3 cells ∼6-fold compared to the same NIH3T3 cells without secretory granules, it is apparent that the IP₃R/Ca²⁺ channels of secretory granules are at least 6-fold more sensitive to IP₃ than those of the ER.

Figure 3.

Interrelation between number of secretory granules and EC₅₀ of cytoplasmic IP₃Rs and magnitude of IP₃-induced Ca²⁺ release. Interrelation between number of secretory granules per square micrometer of cell area on one hand and EC₅₀ value of IP₃ concentrations for cytoplasmic IP₃Rs and increased ratio of IP₃-induced Ca²⁺ release in cytoplasm (in fold increase over resting state) after stimulation with IP₃-producing ATP on the other is shown for both nonneuroendocrine NIH3T3 and neuroendocrine PC12 cells. A) Control (normal), CGA-expressed, and CGB-expressed NIH3T3 cells. B) Control (normal), CGA-suppressed, and CGB-suppressed PC12 cells. Numbers of secretory granules per square micrometer of cell area are from Table 1 of ref. 50, EC₅₀ values are from Table 1 of ref. 88, and the IP₃-induced [Ca²⁺]_c increased ratio (fold increases) are from Figs. 1 and 6 of ref. 83.

In contrast, when the formation of intrinsic secretory granules in PC12 cells is suppressed by inhibiting the CGA expression, the EC₅₀ of IP₃ concentration of the cytoplasmic IP₃R/Ca²⁺ channels changes from a normal value of 17 to 47 nM (Fig. 3B), decreasing the IP₃ sensitivity of the PC12 cells that contain the reduced number of secretory granules ∼3-fold (88). However, inhibition of the CGB expression in the same cells changes the EC₅₀ from 17 to 102 nM (Fig. 3B), decreasing the IP₃ sensitivity of the cytoplasmic IP₃R/Ca²⁺ channels of the PC12 cells that contain the reduced number of secretory granules ∼6-fold (88). Again, the effect that results from CGB suppression is markedly greater than that of CGA suppression (50). These results indicate that secretory granules can release Ca²⁺ in response to less than one-sixth the IP₃ concentration that is required to release Ca²⁺ from the ER. Considering that a mere reduction of the number of secretory granules, not a complete absence, made the cytoplasmic IP₃R/Ca²⁺ channels of PC12 cells ∼6-fold less sensitive in the presence of the identical ER, it is clear that the IP₃R/Ca²⁺ channels of secretory granules are at least 6- to 7-fold more sensitive to IP₃ than those of the ER. This, in turn, predicts that secretory granules of secretory cells will release Ca²⁺ in response to IP₃, whose concentrations are too low for the ER of both secretory and nonsecretory cells to even sense the presence of IP₃, not to mention of releasing Ca²⁺.

DIRECT CORRELATION BETWEEN THE NUMBER OF SECRETORY GRANULES AND THE EC₅₀ OF CYTOPLASMIC IP₃R/Ca²⁺ CHANNELS AND THE INCREASED RATIO OF IP₃-INDUCED Ca²⁺ RELEASE

As discussed above, the IP₃-mediated Ca²⁺ release properties of the cells undergo profound changes depending on the number of secretory granules in the cells, underscoring the major role of secretory granules in the IP₃-dependent intracellular Ca²⁺ control. Not only is the IP₃ sensitivity of the cytoplasmic IP₃R/Ca²⁺ channels of secretory cells 6- to 7-fold higher than those of nonsecretory cells (Fig. 3A, B) but also the magnitude of IP₃-induced Ca²⁺ release of secretory cells is severalfold higher than that of nonsecretory cells (27, 83). This result indicates that the cells that contain secretory granules release far greater amounts of Ca²⁺ in response to IP₃ than the cells without granules or with reduced number of secretory granules (27, 50). Therefore, it appears that the IP₃ sensitivity of the IP₃R/Ca²⁺ channels and the magnitude of IP₃-induced Ca²⁺ release of secretory cells reflect the number of secretory granules present in the cells, as these parameters either increase or decrease, depending on the induction or suppression of secretory granule formation in the cells. In line with the critical roles of secretory granules in the control of IP₃-dependent cytoplasmic Ca²⁺ concentrations, the IP₃sensitivity of the cytoplasmic IP₃R/Ca²⁺ channels of bovine chromaffin cells that contain far more secretory granules than PC12 cells was ∼4-fold higher than that of PC12 cells, and the magnitude of IP₃-induced Ca²⁺ release in chromaffin cells was also significantly higher than that in PC12 cells (unpublished results).

The relevant relationship becomes apparent by expressing 3 parameters in the published literature, i.e., the number of secretory granules/unit area, the EC₅₀ values of IP₃ concentration of the cytoplasmic IP₃Rs, and the increased ratio of IP₃-induced cytoplasmic Ca²⁺ release ([Ca²⁺]_c) over the resting state, in one figure (Fig. 3). Figure 3A shows the relationship obtained from both the normal and chromogranin-transfected NIH3T3 cells: when the secretory granule formation was induced from 0 to 0.0287 and 0.0445 granules/μm² of cell area (50), the increased ratio in IP₃-induced Ca²⁺ release over the resting state increased from 2.39 to 3.49 and 3.78, respectively (83). Moreover, in accordance with the increase in the number of secretory granules formed, the IP₃ sensitivity also increased (Fig. 3A). Taken together, in the NIH3T3 cells that contain the induced secretory granules, the IP₃ sensitivity of the cytoplasmic IP₃Rs and the increased ratio of the IP₃-induced cytoplasmic Ca²⁺ release show a linear relationship with the increase in the number of secretory granules in the cell (Fig. 3A), although in normal NIH3T3 cells without secretory granules, the linear relationship appears to deviate slightly. The slight deviation is not unexpected, considering that the CGA-induced secretory granules contain only CGA, while the CGB-induced secretory granules contain only CGB in the newly formed granules, features that could easily influence both the Ca²⁺ storage and the IP₃-dependent Ca²⁺ release properties of secretory granules.

Further, the results obtained from PC12 cells that contain intrinsic secretory granules reveal a clearer relationship (Fig. 3B): when the number of secretory granules was reduced from 3.8 to 2.2 and 0.86 granules/μm² of cell area (50), the increased ratio in IP₃-induced Ca²⁺ release over the resting state decreased from 5.03 to 4.04 and 3.41, respectively (83). Again, in accordance with the number of secretory granules reduced, the IP₃ sensitivity also decreased (Fig. 3B) (88). Remarkably, both the IP₃ sensitivity of the cytoplasmic IP₃Rs and the increased ratio of the IP₃-induced cytoplasmic Ca²⁺ release of the PC12 cells demonstrate a direct correlation with the number of secretory granules present in the cell (Fig. 3B). From these results, it is apparent that both the IP₃ sensitivity of the cytoplasmic IP₃R/Ca²⁺ channels and the increased ratio of IP₃-induced Ca²⁺ release in the cell that contain secretory granules, whether intrinsic or induced, are directly related to the number of secretory granules in the cell; the more the secretory granules in the cell, the higher the sensitivity of the IP₃R/Ca²⁺ channels and the increased ratio of IP₃-induced Ca²⁺ release of the cell (27, 50).

PHYSIOLOGICAL SIGNIFICANCE

It is of crucial physiological importance that secretory cells that secrete the secretory granule contents in response to a sudden increase of intracellular Ca²⁺ concentration, which can be initiated by IP₃-mediated Ca²⁺ release, are equipped with molecular machinery that responds to IP₃ with a significantly higher sensitivity than nonsecretory cells (88). In this regard, the high sensitivity of IP₃R/Ca²⁺ channels and the high magnitude of IP₃-induced Ca²⁺ release in the cytoplasm of neuroendocrine cells are in line with the physiological needs of secretory cells to quickly produce the secretory cargos and release at the site and precisely at the moment they are needed.

The roles of secretory granules and their key components chromogranins play in the physiology of neuroendocrine cells are shown in a schematic drawing (Fig. 4). As shown in Fig. 4, both chromogranins A and B are present in the ER and secretory granules, but given that only chromogranin B binds to the IP₃Rs at the pH of the ER (60), it would be chromogranin B, not chromogranin A, that couple to the IP₃R/Ca²⁺ channels in the ER and modulate the Ca²⁺ channel activity. However, in secretory granules, both CGA homotetramers and CGA₂CGB₂ heterotetramers will be present (66,67,68), but CGA₂CGB₂ heterotetramers will preferentially couple to the tetrameric IP₃R/Ca²⁺ channels due to the markedly stronger affinity of CGB for the IP₃Rs (84,85,86,87). The affinity of CGB for the IP₃R at pH 5.5 is several orders of magnitude stronger than that of CGA at the same pH and substantially stronger than that of CGB for the IP₃R at pH 7.5 (84,85,86,87).

Figure 4.

Secretory granule formation and IP₃-induced Ca²⁺ mobilization in cytoplasm of neuroendocrine cells. Process of secretory granule biogenesis is shown from the ER via the Golgi. Tetrameric IP₃R/Ca²⁺ channels are shown as red and blue columns, and chromogranins A and B are shown as open and hatched circles, respectively. Note that only chromogranin B can couple to tetrameric IP₃Rs in the ER, whereas both chromogranins A and B, which form a CGA₂CGB₂ heterotetrameric complex, couple to tetrameric IP₃Rs in secretory granules. Other contents of secretory granules are shown as twisted red structures. Generation of IP₃ at the plasma membrane as a result of agonist stimulation is the first step in IP₃-dependent Ca²⁺ release from intracellular Ca²⁺ stores in cytoplasm. Depending on amount of IP₃ produced and sensitivity of IP₃R/Ca²⁺ channels to IP₃, each intracellular Ca²⁺ store will respond differently to IP₃. The significantly higher sensitivity of secretory granule IP₃R/Ca²⁺ channels would enable secretory granules to release Ca²⁺ in response to low IP₃ concentrations (1) that are not high enough for the ER IP₃R/Ca²⁺ channels to sense the presence of IP₃ (2), thereby initiating the exocytotic processes. Ca²⁺ sensor molecules on the membranes of secretory granules and the plasma membranes, which participate in the Ca²⁺-dependent fusion process, are shown in blue and yellow, respectively. Exocytosed secretory granule contents will enter the bloodstream as mixtures of both intact and cleaved molecules.

As a result of the coupling of CGB to the IP₃R/Ca²⁺ channels at pH 5.5, both the channel open probability and the mean open time of the IP₃R/Ca²⁺ channels are ∼2-fold higher than those that result from the same coupling at pH 7.5 (63). The interaction of CGA or CGB with the IP₃Rs changes the conformation of the IP₃Rs (61, 63), and the stronger interaction of CGB with the IP₃Rs at pH 5.5 than at pH 7.5 changes the conformation of the IP₃Rs to a greater extent (63). It appears that chromogranin coupling to the IP₃Rs changes the conformation of the IP₃R/Ca²⁺ channels to a more ordered structure that can respond better to IP₃ (61,62,63), apparently explaining the higher sensitivity of secretory granule IP₃R/Ca²⁺ channels than the ER Ca²⁺ channels. From a mechanistic point, it is highly likely that the tightly coupled CGB maintains the IP₃R/Ca²⁺ channels in a high-tension state that is open-ready in case IP₃ arrives in the cytoplasmic side of the channels. This high-tension, open-ready state of the IP₃R/Ca²⁺ channels may not exist in the ER and is likely to differentiate the IP₃R/Ca²⁺ channels of secretory granules from those of the ER. Hence, the initial Ca²⁺ released from secretory granules would be able to serve as the trigger signal for the ensuing additional Ca²⁺ releases, influx of extracellular Ca²⁺, and the secretory processes.

Fitting with the presence of the majority of cellular calcium and IP₃R/Ca²⁺ channels in secretory granules, Ca²⁺ release from secretory granules was shown to be essential for insulin secretion (89, 90) and sufficient to initiate exocytosis in chromaffin, pancreatic acinar, and β cells (13, 16, 17, 91,92,93,94,95). For example, application of acetylcholine, which is known to produce intracellular IP₃, to the basal surface of pancreatic acinar cells in the absence of external Ca²⁺ invoked the initial IP₃-induced Ca²⁺ release in the granule-rich apical zone and led to exocytosis (94). Although the initial IP₃-induced Ca²⁺ release in the granular zone of pancreatic acinar cells (13, 94, 95) has not always been ascribed to the secretory granules that are exclusively clustered in the granular zone, it appears plain that the IP₃-induced Ca²⁺ release from secretory granules in the absence of external Ca²⁺ is sufficient to cause exocytosis. In a more recent study, application of IP₃-producing ATP to pancreatic β cells in the absence of external Ca²⁺ caused release of Ca²⁺ from secretory granules of these cells, and this IP₃-induced Ca²⁺ release was shown to initiate exocytosis in pancreatic β cells (17). Therefore, it seems natural to think that the IP₃-induced Ca²⁺ release from secretory granules in the absence of external Ca²⁺ will not only be sufficient but also be the first signal to initiate exocytosis of secretory cells.

Yet, in exocrine pancreatic acinar cells, the results have not been consistent. Despite the early report of IP₃-induced Ca²⁺ release from secretory granules of exocrine pancreatic acinar cells (13), secretory granules of the same cells that had been purified by Percoll gradient failed to release Ca²⁺ in response to IP₃ in another study (96), although it is possible that residual Percoll, which is detrimental to IP₃-mediated Ca²⁺ release, disenabled the release process. In the same year, in a study using immunofluorescence microscopy with very low magnification, the IP₃Rs were reported not to localize in zymogen granules of the apical (granular) zone of acinar cells (97). Nonetheless, in a more recent and thorough study, it was further shown that secretory granules of exocrine pancreatic acinar cells release Ca²⁺ in response not only to IP₃ but also to cyclic ADP-ribose or nicotinic acid adenine dinucleotide phosphate (18).

Because one of the primary functions of secretory cells is to store and secrete molecules essential for cellular communication, and the trigger signal for secretion is Ca²⁺ (7,8,9, 33), the presence of most of cellular Ca²⁺ and IP₃R/Ca²⁺ channels along with the highest concentrations of chromogranins in secretory granules highlights the extraordinary efficiency of secretory cells to deal with their complex physiological needs. Moreover, secretory granules also contain Ca²⁺ sensor proteins, such as synaptotagmin and syntaxin (98,99,100), which are known to participate in the Ca²⁺-initiated membrane fusion and exocytotic processes (101,102,103). These membrane proteins will initiate the fusion process and pave the way for secretion of intragranular contents to the extracellular space and on to the bloodstream (Fig. 4). Once released, both intact chromogranins and chromogranin-derived peptides circulate in the bloodstream and serve as the source of a number of bioactive peptides (74,75,76,77,78,79,80). The concentration of chromogranin-derived proteins in the blood is a very good indicator of secretory activity of secretory cells in the body; hence, precise measurements of these proteins in the blood samples have been widely used in assessing a number of clinical symptoms that include tumors of secretory cell origin and blood pressure (104,105,106,107,108).

PERSPECTIVES

Traditionally, secretory granules have simply been regarded as organelles that store and transport secretory contents, and the unique and extraordinary roles exerted by secretory granules in the physiology of neuroendocrine cells have mostly not been duly appreciated. Secretory granules and the associated molecules are not stationary entities in the cytoplasm; rather, they are constantly on the move and are in the center of dynamic key cellular activities of secretory cells. Secretory granules and the associated molecules actively participate in the IP₃-dependent Ca²⁺ signaling in neuroendocrine cells, and the roles described above point out the hitherto underappreciated roles of these. The functions uncovered thus far strongly hint at a number of additional roles of major importance by this fascinating organelle and its companions.

The fate of secretory granules and the associated molecules is intimately tied together from the beginning of their birth in the cell to their final destination at the secretion site as transport vehicles and secreted products. Only at the secretion site will the secretory cargos separate from the granules and venture into a new environment (Fig. 4). Even at this point, the amounts and types of secreted molecules will differ from one secretion site to another and from one granule to another, depending on the nature of the interaction between the granule membranes and the granule contents. Therefore, the importance of future studies on secretory granules and the associated molecules goes far beyond the neuroendocrine cells and could extend to other types of secretory cells, covering a variety of cellular activities, such as secretory granule biogenesis, protein trafficking, control of intracellular Ca²⁺ concentration, membrane fusion, exocytosis, production of biopeptides, and control of hormone secretion.

On a new front, recent studies indicate the presence and operation of an IP₃-dependent Ca²⁺ store inside the nucleus (109, 110), which is independent of Ca²⁺ stores of cytoplasmic or nuclear envelope origin, and chromogranin B is shown to be a key component in the system (27, 88). Chromogranin B is present in the IP₃-sensitive nucleoplasmic Ca²⁺ store vesicles (109, 110), and the nuclear chromogranin B is presumed to play key roles in the IP₃-dependent Ca²⁺ control mechanism through its specific interaction with the IP₃R/Ca²⁺ channels. Moreover, secretogranin II is also shown recently to be present in the nucleus, localizing in the IP₃-sensitive nucleoplasmic Ca²⁺ store vesicles along with CGB and the IP₃Rs (36). This implies that secretogranin II may also assume an important role in the control of nuclear calcium in conjunction with other Ca²⁺ storage proteins and the Ca²⁺ channels. Hence, further studies on the function of the granins will be pivotal in obtaining a complete picture of the IP₃-dependent Ca²⁺ signaling mechanisms of neuroendocrine cells encompassing both the cytoplasm and the nucleus.