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Calcium Mobilization Systems During Neurogenesis

Masayuki Yamashita and Miho Sugioka

M. Yamashita and M. Sugioka are in the Dept. of Physiology, Osaka University Medical School, Yamadaoka 2-2, Suita 565, Osaka Japan.

	Abstract

 
Neuroepithelial cells have Ca²⁺ mobilization systems that are activated by acetylcholine via muscarinic receptors and by extracellular ATP via P_2U purinoceptors. The Ca²⁺ mobilization occurs during neurogenesis and diminishes in parallel with the declining of mitotic activities of the neuroepithelial cells. Capacitative Ca²⁺ influx also occurs with the Ca²⁺ mobilization.

	Introduction

Top Introduction P2U purinoceptors and muscarinic... Intracellular signal... Capacitative Ca2+ influx and... Concluding remarks References

 
Ca²⁺ mobilization plays an important role in many cellular processes including cell growth (1). Ca²⁺ signaling has a regulatory role in cell cycle control (12, 13). The activation of G protein-linked receptors and phospholipase Cß (PLCß) leads to the formation of inositol trisphosphate (IP₃). IP₃ causes the release of Ca²⁺ from IP₃-sensitive Ca²⁺ stores (1). When the intracellular Ca²⁺ store is depleted, influx of extracellular Ca²⁺ is induced by a signal from the depleted Ca²⁺ store. This type of Ca²⁺ influx is called "capacitative Ca²⁺ entry" (7).

Recently, it has been demonstrated that such Ca²⁺ mobilization and capacitative Ca²⁺ entry occur in neuroepithelial cells, or undifferentiated progenitor cells, in the early embryonic neural retina when the cells are engaged in mitotic activities (9, 11, 15). This review focuses on the pharmacological properties of the receptors that elicit embryonic Ca²⁺ mobilization, the intracellular signal transduction mechanism, and the developmental change in capacitative Ca²⁺ entry.

	P2U purinoceptors and muscarinic receptors elicit Ca2+ mobilization during neurogenesis

Top Introduction P2U purinoceptors and muscarinic... Intracellular signal... Capacitative Ca2+ influx and... Concluding remarks References

 
When adenosine 5'-triphosphate (ATP) is extracellularly applied to the neural retina dissected out of an embryonic day 3 (E3) chick, an increase in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) is evoked (Fig. 1Aa). The increase in Ca²⁺ in response to ATP is caused by the release of Ca²⁺ from intracellular Ca²⁺ stores, since the increase in Ca²⁺ occurs in a Ca²⁺-free bath solution. No Ca²⁺ response is evoked by adenosine (an agonist for P₁ purinoceptors, see Refs. 2 and 3), but uridine 5'-triphosphate (UTP) causes increases in Ca²⁺ more dramatically than does ATP. Ca²⁺ increases in response to ATP and UTP are blocked by the antagonists for P₂ purinoceptors, suramin and reactive blue 2 (see Refs. 2 and 3). From these pharmacological properties, it is concluded that P_2U purinoceptors are involved in the embryonic Ca²⁺ mobilization (11).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. A: Ca2+ fluorescence responses of neural retinas dissected from chick embryos at embryonic day 3 (E3), E6, and E13 to the application of ATP (500 µM) (a) and carbamylcholine (CCh; 100 µM) (b). Bar: period of ATP or CCh application. Neural retina was loaded with fura 2-acetoxymethyl ester (AM). Fluorescence was excited at 340 (F340) and 380 nm (F380). An increase in the ratio of the 2 fluorescence intensities (F340/F380, ordinate) indicates an increase in intracellular Ca2+ concentration ([Ca2+]i). Bathing solution contained 2.5 mM Ca2+. B: developmental profiles of Ca2+ responses of embryonic neural retina to ATP and CCh from E3 to E13. Peak amplitudes of the increases in the fluorescence ratio (F340/F380) to 500 µM ATP () and 100 µM CCh () are plotted vs. embryonic day that retina was tested. Each point indicates averaged F340/F380 recorded from 4–13 retinas. Error bars indicate SD. [Redrawn from Sugioka et al. (11) for the ATP response and from Yamashita et al. (15) for the CCh response]. , Mitotic activities of retinal cells by calculation of the whole number of cells labeled with [3H]thymidine. [Data from Prada et al. (6).]

Changes in purinergic and muscarinic Ca²⁺ mobilizations are closely correlated during embryonic development. These Ca²⁺ responses are intense at E3–E5 but drastically decline toward E8 and decrease further until E13 (Fig. 1B). Because the synaptic structures appear from E14 in the chick retina (10), it is unlikely that the purinergic and muscarinic Ca²⁺ mobilizations have any functional roles in synaptic transmission. A possible explanation is that the Ca²⁺ mobilization has regulatory roles in cell cycle control during neurogenesis as shown in other cell types (12, 13). It is noted that the purinergic and muscarinic Ca²⁺ mobilizations decline in parallel with the decline in mitotic activities of the cells in the neural retina (see Ref. 6) (Fig. 1B). This close relationship could indicate that the Ca²⁺ mobilization is involved in the cellular processes during the early development of the neural retina, such as the proliferation of neuroepithelial or undifferentiated progenitor cells. Alternatively, the increase in Ca²⁺ could be a function of retinal morphogenesis, since acetylcholine induces incurvation of the embryonic neural retina via muscarinic receptors and the increase in [Ca²⁺]_i (14).

The results obtained to date, however, raise a question concerning the actual occurrence of purinergic and cholinergic signals in the early embryonic retina, because synaptic structures do not appear until late in development. The actual agent that induces the Ca²⁺ mobilization and the source of the agent should be identified in the early embryonic retina.

	Intracellular signal transduction mechanisms for the purinergic and muscarinic Ca2+ mobilizations

Top Introduction P2U purinoceptors and muscarinic... Intracellular signal... Capacitative Ca2+ influx and... Concluding remarks References

 
Muscarinic receptors and P_2U purinoceptors belong to a family of G protein-linked receptors (1, 3). It can be supposed that the embryonic Ca²⁺ mobilizations in the chick retina are evoked through signal transduction pathways. The signal transduction mechanisms for the purinergic and muscarinic Ca²⁺ mobilizations have been studied (8), and the summary of the study is illustrated in Fig. 2.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Intracellular signal transduction pathways of muscarinic and purinergic Ca2+ mobilizations in the cell of early embryonic neural retina. [Ca2+]i, intracellular Ca2+ concentration; ACh, acetylcholine; ATP, adenosine 5'- triphosphate; DG, diacylglycerol; G, G protein; IP3, inositol 1,4,5-trisphosphate; mACh, muscarinic acetylcholine receptor; P2U, P2U purinoceptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PTX, pertussis toxin; PLC, phospholipase C; R, receptor; Tg, thapsigargin.

However, it is likely that both of the Ca²⁺ mobilizations are mediated by IP₃-sensitive Ca²⁺ stores, because the two Ca²⁺ responses are markedly enhanced by Li⁺, which inhibits phosphatidylinositol metabolism. Increases in Ca²⁺ in response to both ATP and CCh are abolished by thapsigargin, an inhibitor of Ca²⁺-adenosinetriphosphatase (ATPase) of IP₃-sensitive Ca²⁺ stores.

Cross-talk occurs between the purinergic and muscarinic Ca²⁺ mobilizations. When CCh is applied after successive applications of ATP in a Ca²⁺-free bath solution to deplete the Ca²⁺ store, the response to CCh is progressively less than that without the preceding ATP application. On the contrary, the ATP response is also attenuated by preceding successive applications of CCh in the Ca²⁺-free solution. These interactions may suggest that a part of the Ca²⁺ store is utilized by both the purinergic and muscarinic Ca²⁺ mobilizations. When ATP and CCh are applied simultaneously, a synergistic interaction occurs. The response to the simultaneous application of ATP and CCh is much greater than the simple summation of the individual responses. Such synergistic interactions also occur in the otocyst of the chick embryo (4). It is likely that the site of interaction is an accumulation of IP₃. The simultaneous application of ATP and CCh may produce IP₃ more effectively than the application of ATP or CCh alone.

	Capacitative Ca2+ influx and its developmental change

Top Introduction P2U purinoceptors and muscarinic... Intracellular signal... Capacitative Ca2+ influx and... Concluding remarks References

 
Depletion of intracellular Ca²⁺ stores induces the influx of extracellular Ca²⁺ or capacitative Ca²⁺ influx (7). The capacitative Ca²⁺ influx occurs in nonexcitable cells or the cells of nonnervous systems, such as platelets, lymphocytes, macrophages, neutrophils, parotid acinar cells, epithelial cells, and endothelial cells (7). Nonexcitable cells lack voltage-operated Ca²⁺ channels but have developed the Ca²⁺ entry mechanism, which is coupled with the depletion of intracellular Ca²⁺ stores to activate the Ca²⁺-release-activated Ca²⁺ channel (CRAC) (5). Thus it is granted that the capacitative Ca²⁺ influx is a general property of these nonneural cells, but it is not known whether the capacitative Ca²⁺ influx occurs in neural cells. Evidence for the occurrence of the capacitative Ca²⁺ influx in the cells of neural origin has been provided only in neuroblastoma and pheochromocytoma cell lines (see introduction of Ref. 9). Our recent study (9) revealed that the capacitative Ca²⁺ influx really occurs in the cells of the neural retina, but only at the early stages of development.

Figure 3A shows the rise in [Ca²⁺]_i due to the capacitative Ca²⁺ influx. After the depletion of intracellular Ca²⁺ stores by application of thapsigargin in a Ca²⁺-free bath solution, reintroduction of extracellular Ca²⁺ induces a rapid rise in [Ca²⁺]_i (Fig. 3A). This Ca²⁺ increase is suppressed by Zn²⁺ and Ni²⁺ (Fig. 2). Such Ca²⁺ increases are clearly observed at E3 and E5 but are not seen at E13 (see Fig. 3A). The developmental changes in the thapsigargin-induced Ca²⁺ increase were studied from E3 to E13 (9). The thapsigargin-induced Ca²⁺ increase is greatest at E3, declines rapidly toward E6, and then decreases gradually until E13, when the Ca²⁺ increase almost disappears (Fig. 3B). This developmental profile is very close to or somewhat precedes the decline of purinergic and muscarinic Ca²⁺ mobilizations and of the mitotic activity of the retinal cells (cf. Fig. 1B).

View larger version (13K): [in this window] [in a new window]   FIGURE 3. A: Ca2+ fluorescence responses of 3 neural retinas dissected from chick embryos at E3, E5, and E13 to the bath application of a Ca2+-free medium containing thapsigargin (Tg, 250 nM). Tg-containing Ca2+-free solution was applied for 5 min (indicated by bar), and then the normal bath solution (containing 2.5 mM Ca2+) was reintroduced. B: developmental profile of Tg-induced Ca2+ rise. Rapid rise in F340/F380 when the bath solutions were changed from Ca2+-free solution containing Tg (250 nM) to normal bath solution was measured from bottom to top and is represented as F340/F380. F340/F380 is plotted vs. embryonic day. Each point indicates the mean F340/F380 recorded from 4–12 retinas. Error bars indicate SD. [Reproduced from Sakaki et al. (9). Copyright © 1997 John Wiley & Sons.]

	Concluding remarks

Top Introduction P2U purinoceptors and muscarinic... Intracellular signal... Capacitative Ca2+ influx and... Concluding remarks References

 
Neuroepithelial cells, or undifferentiated progenitor cells in the early embryonic neural retina, have a Ca²⁺ mobilization system that includes phosphatidylinositol metabolism, IP₃-sensitive Ca²⁺ stores, PLCß, and G protein-linked receptors such as P_2U purinoceptors and muscarinic receptors. Capacitative Ca²⁺ influx also occurs in these cells with the Ca²⁺ mobilization. It is noteworthy that the Ca²⁺ mobilization and the capacitative Ca²⁺ influx are physiological characteristics of the early embryonic cells in the nervous system. The change in Ca²⁺ signaling during development may underlie neuronal differentiation. The differentiation of neuroepithelial cells or undifferentiated progenitor cells into neurons is accompanied by the elimination of the physiological properties that are common to nonneural cells.

	References

Top Introduction P2U purinoceptors and muscarinic... Intracellular signal... Capacitative Ca2+ influx and... Concluding remarks References

 

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