Regulated secretion in neurosecretory cells is mediated by two types of organelles, large dense-core vesicles (LVs) and small synaptic-like vesicles (SVs), that differ in their biogenesis and contents (Clift et al. 1990; Kelly, 1993; Itakura et al. 1999). The exocytoses of both LVs and SVs exhibit distinct ion selectivities. Exocytosis of LVs from endocrine cells is supported when external Ca2+ is replaced with Ba2+ (Berggren, 1981; Douglas et al. 1983; Brown et al. 1990; von Ruden et al. 1993; Seward et al. 1996; Borges et al. 1997; Nucifora & Fox, 1998). In contrast, synchronous SV exocytosis from presynaptic terminals is not maintained by Ba2+ in the external solution (Dodge et al. 1969; Alvarez-Leefmans et al. 1978; Augustine & Eckert, 1984; Medina et al. 1994; but see Ohno-Shosaku et al. 1994), although Ba2+ does support asynchronous slow neurotransmitter release (Silinsky, 1978; McMahon & Nicholls, 1993; Sihra et al. 1993; Verhage et al. 1995). None of these studies, however, examined the effects of divalent cations introduced directly into the cytosol.
The LVs and SVs of rat phaeochromocytoma (PC12) cells contain monoamines and acetylcholine, respectively (Greene & Tischler, 1976; Baumert et al. 1990; Schmidt et al. 1997). We have previously shown that abrupt increases in the cytosolic Ca2+ concentration ([Ca2+]i) generated by photolysis of caged-Ca2+ compounds trigger two components of exocytosis in PC12 cells with markedly different time constants of 30-100 ms and 10 s (Kasai et al. 1996). The slow component appears to be mediated by LVs, given that it is accompanied by monoamine secretion; conversely, the fast component is probably mediated by SVs, given that it is associated with secretion of acetylcholine, but not with that of monoamines (Ninomiya et al. 1997). Dissociation of the fast increase in membrane capacitance (Cm) from the amperometric detection of monoamine secretion has also been demonstrated in pancreatic β cells (Takahashi et al. 1997) and adrenal chromaffin cells (Ninomiya et al. 1997; Haller et al. 1998; Kasai, 1999). This dissociation is less marked in chromaffin cells (Ninomiya et al. 1997) and most increases in Cm in these cells at [Ca2+]i values of < 100 μm have been attributed to the exocytosis of LVs (Haller et al. 1998).
To characterise the ion selectivities of exocytosis of LVs and SVs, we have chosen to study PC12 cells, because of the pronounced differences in the corresponding time courses of exocytosis. We triggered exocytosis by inducing the photolysis of caged-Ca2+ compounds loaded with various metal ions, which results in direct increases in the cytosolic concentrations of these ions, and we monitored exocytosis by measurement of Cm and amperometry. We detected marked differences in ion selectivity between exocytosis of LVs and that of SVs and these selectivities are similar to those of endocrine secretion and synchronous synaptic neurotransmitter release, respectively. The ion selectivities of exocytosis in PC12 cells support a role for synaptotagmin- phospholipid as the Ca2+ sensor (Brose et al. 1992; Bommert et al. 1993; Elferink et al. 1993; Südhof & Rizo, 1996; Thomas & Elferink, 1998; Mikoshiba et al. 1999) for the exocytosis of LVs, but they suggest an additional mechanism for the Ca2+-dependent exocytosis of SVs.
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We have systematically examined the ion selectivities of exocytosis by directly increasing the cytosolic concentrations of various divalent cations with the use of caged-Ca2+ compounds. Our results indicate that the two components of exocytosis in PC12 cells exhibit distinct ion selectivities. They thus provide a new line of evidence for the hypothesis that the two components of exocytosis are mediated by two distinct types of secretory vesicle, SVs and LVs, in PC12 cells (Kasai, 1999). Our data are not inconsistent with a model that assumes transitions of state within a single population of vesicles in adrenal chromaffin cells (Gillis et al. 1996; Smith et al. 1998; Voets, 2000), given that, unlike in PC12 cells (Ninomiya et al. 1997), the major component of exocytosis is attributed to LVs in chromaffin cells (Haller et al. 1998; Kasai, 1999).
The slow component of exocytosis in PC12 cells, representing LV exocytosis, was induced by all divalent metal ions investigated, with half-maximal rates apparent at cytosolic concentrations of 18 pm for Cd2+, 500 nm for Mn2+, 900 nm for Co2+, 8 μm for Ca2+, 180 μm for Sr2+ and 280 μm for Ba2+. The extent of synaptotagmin- phospholipid binding is half-maximal at Ca2+, Sr2+ and Ba2+ concentrations of 5.4, 177 and 254 μm, respectively (Li et al. 1995), values that are similar to those that give rise to half-maximal rates for the slow component of exocytosis in PC12 cells. In addition, exocytosis of LVs in endocrine cells monitored either biochemically (Berggren, 1981; Douglas et al. 1983; Brown et al. 1990), by capacitance measurement (Seward et al. 1996; Nucifora & Fox, 1998) or by amperometry (von Ruden et al. 1993; Borges et al. 1997) has been shown to be supported by Ba2+ in place of Ca2+ in the external solution. Thus, the ion selectivity of the slow component of exocytosis quantified in the present study supports a major role for synaptotagmin-phospholipid in the triggering of exocytosis of LVs.
The fast component of exocytosis in PC12 cells, representing synchronous SV exocytosis, appeared more selective for divalent cations than did the slow component. The rate of the fast component was half-maximal at 26 pm Cd2+, 620 nm Mn2+, 24 μm Ca2+ and 320 μm Sr2+, and this component was little activated by Ba2+ (< 1 mm) or Co2+. Moreover, the fast component of exocytosis was competitively inhibited by high concentrations of Na+ but not by K+, Li+ or Cs+. This pattern of ion selectivity can be explained by the ionic radii of these cations, given that the divalent cations (Mn2+, Cd2+ and Sr2+) and monovalent cation (Na+) with radii most similar to that of Ca2+ triggered and inhibited, respectively, the fast component of exocytosis (Fig. 7). A similar divalent ion selectivity is exhibited by EF-hand proteins such as calmodulin (Chao et al. 1984). The binding of Ca2+ to the EF-hand proteins α-lactalbumin (Eberhard & Erne, 1991) and parvalbumin (Permyakov et al. 1983; Eberhard & Erne, 1994) is also inhibited by Na+. Previous studies have shown that synchronous synaptic transmission is maintained when external Ca2+ is replaced by Sr2+, but is supported to only a small extent, or not at all, by external Ba2+ (Dodge et al. 1969; Alvarez-Leefmans et al. 1978; Augustine & Eckert, 1984; Medina et al. 1994; Ohno-Shosaku et al. 1994). Thus, the ion selectivity of both synchronous synaptic transmission and the fast component of exocytosis in PC12 cells is more stringent than that of exocytosis of LVs.
Figure 7. Ionic radii and ion selectivities of exocytosis in PC12 cells
The exocytosis of LVs was induced by all divalent cations studied, whereas synchronous SV exocytosis was induced by the divalent cations with ionic radii most similar to that of Ca2+ and was blocked selectively by Na+. 1 Å= 0.1 nm.
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There is some uncertainty as to whether the fast component of the capacitance increase faithfully reflects the exocytosis of SVs. First, this component may also reflect changes in other electrical properties of the plasma membrane caused by a sudden increase in [Ca2+]i. However, the complete absence of the fast component of the capacitance increase in PC12 cells exposed to Ba2+ (< 1 mm) or Co2+ jumps or to Ca2+ jumps (< 58 μm) in the presence of 155 mm Na+ is consistent with the conclusion that fast synchronous SV exocytosis is not induced by Ba2+ (< 1 mm) or Co2+ and is blocked by Na+. Second, the fast component of the capacitance increase may be curtailed by concurrent endocytosis even at the peak of the increase that is apparent within 0.2 s. However, endocytosis occurs only in the presence of exocytosis and the ion selectivities of each component of the capacitance increase in PC12 cells were identical when measured at the minimal effective concentrations or the median effective concentrations (Fig. 4E and F).
The ion selectivity of synchronous SV exocytosis in PC12 cells appears inconsistent with that of the synaptotagmin-phospholipid interaction. The more stringent ion selectivity and higher [Ca2+]i requirement of synchronous SV exocytosis suggests a larger coordination number and smaller negative charge for the Ca2+ binding sites that underlie synchronous SV exocytosis. Such Ca2+ binding sites may be provided by (1) the synaptotagmin- phospholipid interaction in a distinct conformational state (Davis et al. 1999), (2) the interaction of synaptotagmin with syntaxin (Li et al. 1995) or SNAP25 (Gerona et al. 2000), or (3) another Ca2+ binding protein with a high ion selectivity, such as an EF-hand protein (Peters & Mayer, 1998; Quetglas et al. 2000). Testing the actions of Ba2+ and Na+ in the experiments cited above (Li et al. 1995; Peters & Mayer, 1998; Davis et al. 1999; Quetglas et al. 2000; Gerona et al. 2000) may help to clarify the molecular events that underlie synchronous SV exocytosis.