Biol. Bull. 212: 161-168. (April 2007)
© 2007 Marine Biological Laboratory
Analysis of Calcium Concentration Fluctuations in Hepatopancreatic R Cells of Marsupenaeus japonicus During the Molting Cycle
Loredana Zilli*,
Roberta Schiavone,
Carlo Storelli and
Sebastiano Vilella
Laboratory of General and Comparative Physiology, Department of Biological and Environmental Sciences and Technologies, University of Lecce, Via Provinciale Lecce-Monteroni, 73100 Lecce, Italy
* To whom correspondence should be addressed. E-mail: loredana.zilli{at}unile.it
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Abstract
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In this study we examined the fluctuations of the intracellular calcium concentration in isolated hepatopancreatic R cells during the four molting stages of the prawn Marsupenaeus japonicus. In addition, we used the Fura-2-AM fluorescence technique to investigate the release of calcium from mitochondria and ATP-sensitive calcium stores (endoplasmic reticulum (ER), Golgi, and nucleus) into cytoplasm during the molting cycle. Results demonstrate that both the cytosolic free calcium concentration and the total cell calcium (free, bound to calcium-binding proteins, and stored in amorphous form) in the R cells strictly depend upon the molting cycle. Interestingly, the total cell calcium was higher (
10 mmol l–1) in postmolt than in premolt (1 mmol l–1) and intermolt (0.3 mmol l–1). The calcium released from mitochondria was higher during premolt than during postmolt and intermolt, but the amount of calcium released from ATP-sensitive calcium stores was similar during all four stages. All together, our results suggest that the mitochondria–ATP-sensitive calcium stores system does not play a key role in calcium storage during the molting cycle but that it is involved in transcellular calcium flux. We hypothesize that lysosome or membrane-clad concretion vacuoles could represent the main site of calcium storage in hepatopancreatic R cells.
Abbreviations: Cell types: B, blister-like • E, embryonic • F, fibrillar • R, resorptive
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Introduction
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The epithelial cells of the crustacean hepatopancreas play an important role in the Ca2+ balance of the whole organism during the molting cycle (Wheatly, 1996, 1997, 1999). The epithelial layer of crustacean hepatopancreas consists of four cell types: E (embryonic), R (resorptive), F (fibrillar), and B (blister-like) cells (Jacobs, 1928; Hirsch and Jacobs, 1930). At present the functions carried out by each cell type are not well understood, although different authors have commented upon the physiological roles of these cells (Gibson and Barker, 1979; Dall and Moriarty, 1983; Al-Mohanna and Nott, 1986; Paquet et al., 1993; Verri et al., 2001).
The bidirectional flux of calcium occurs mainly via a transcellular route (Roer, 1980; Wheatly, 1997; Hubbard, 2000; Ziegler, 2002) rather than a para-cellular pathway (Bronner, 1991). It has recently been suggested that of the four cell types that constitute the hepatopancreas of Homarus americanus, only the R and E types are involved in transcellular calcium flux linked to the molting cycle (Chavez-Crooker et al., 2003; Ahearn et al., 2004). Ca2+ enters the cells passively across the plasma membrane at one side of the cells and is actively extruded on the other side (Roer, 1980; Ahearn and Franco, 1993; Ahearn and Zhuang, 1996). The most critical step in transcellular epithelial Ca2+ transport, however, is the Ca2+ transport within the cells—through the cytoplasm from one side to the plasma membrane on the opposite side. Higher concentrations of Ca2+ within the cytoplasm can affect the calcium regulatory functions as well as determine cell damage and death (Berridge, 1993). To prevent a toxic rise of calcium in epithelial cells, a sequestration process involving the intracellular organelles (ER, mitochondria, and lysosomes) occurs (Simkiss, 1996). Although these intracellular structures may store and release calcium during the crustacean molting cycle, the role these organelles play in both transcellular calcium flux and storage is unclear.
For these reasons, in the present investigation we study the fluctuations of calcium concentration in the cytoplasm and organelles of hepatopancreatic R cells during different crustacean molting stages (early premolt, late premolt, intermolt, and postmolt).
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Materials and Methods
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Materials
Live Marsupenaeus japonicus (Bate, 1888) prawns (30 g each) were obtained from Cooperativa La Valle (Lesina, Italy) and maintained until use in recirculating seawater aquaria at 21 °C. Calcium Calibration Buffer kit, Fluo-3-AM, Fura-2-AM, thapsigargin, and pluronic F-127 were from Molecular Probes (Eugene, OR, USA). All other chemicals, reagent grade, were purchased from Merck (Darmstad, Germany), Sigma (St. Louis, MO, USA), and Fluka-Chemika (Buchs, Switzerland).
Determination of molting stage
We determined molting stages on the basis of morphological changes in the uropodes during the molting cycle, according to the method proposed by Robertson et al. (1987). Briefly (see Zilli et al., 2003, for a detailed description), in stage A, pigmented cellular matrix completely fills the setal bases. During stage B, cellular matrix retracts from the setal bases and a clear space is easily recognized in the bases. In stage C, the matrix is absent from the setal bases and the pigment seems to form an epidermal line at the bases of the setal nodes. At stage D0–D1 (early premolt), the pigment retracts from the bases of the setal nodes, leaving the old cuticle; and during D2–D3 (late premolt), the new developing setae are observed. For molting stages determination, live prawns were checked daily and the selected animals were placed on ice for experiments. We cleaned the water daily by removing the exuviae.
Preparation of isolated R-cell suspension
We prepared isolated cell suspensions according to the method detailed elsewhere (Zilli et al., 2003). For each experiment, the hepatopancreas was collected from 8–10 animals at the same molt stage, put in a petri dish, and finely minced (on ice) in citrate/EDTA buffer containing (in mmol l–1) 27 sodium citrate, 440 NaCl, 3 KCl, 5 NaH2PO4, 5.6 Na2SO4, 10 EDTA, 1 PMSF, 0.5 pepstatin A, 0.006 leupeptin; adjusted to pH 7.1 with KOH (osmotic concentration 860 mmol kg–1). Cell dissociation was accomplished by mild manual agitation with a glass rod for 20–25 min at 4 °C. The resulting crude cell suspension was filtered through a 100-µm nylon mesh to remove tissue debris. The filtrate was then transferred into 15-ml Falcon tubes and spun for 10 min at 1000 x g (2000 rpm) at 4 °C in a Beckman Allegra 6R centrifuge equipped with a GH 3.8 swinging bucket rotor (Beckman Coulter Inc., Fullerton, CA). The supernatant was poured off and the pellet resuspended in normal saline buffer: 467 mmol l–1 NaCl, 10 mmol l–1 KCl, 1 mmol l–1 NaH2PO4, 4 mmol l–1 NaHCO3, 8.4 mmol l–1 Na2SO4, 30 mmol l–1 HEPES, 10 mmol l–1 EDTA; pH 7.1 with KOH, osmotic concentration 860 mmol kg–1. After being spun again for 10 min at 1000 x g at 4 °C, the resulting pellet consisted of a stratification (from the bottom to the top) of B (dark-black), F (yellow-white), and R (pink-grey) cells. This pellet was gently resuspended to separate each stratum from the other. Each cell fraction was resuspended in normal saline, spun at 1000 x g for 10 min at 4 °C, and resuspended in a small volume of normal saline. This separation method produced enriched cell suspensions of living R cells (88% ± 4% of R cells, 4% ± 3% of B cells, 7% ± 2% of F cells, and 1% ± 1% of E cells). The viability of the cell suspensions was assessed by resuspending the cells in normal saline buffer and incubating an aliquot in a 0.6% solution of trypan blue dye. These cell suspensions were then counted, using a hemocytometer, to evaluate the proportion of living cells (without dye particles) and nonliving cells (containing dye particles). Viability greater than 90% was consistently obtained using our purification procedures.
Estimation of cell volume
When isolated, R cells are spherical. Thus the cell volume (mm3) of a single cell was estimated according to the formula of a sphere: volume = 4/3
r3, where r corresponds to the radius of the cell. The diameter of the R cells was estimated by coupling a calibrated ocular micrometer to a 40x objective. Fifty R cells were isolated as previously described and photographed with a digital camera (Nikon Coolpix E995) connected to the microscope (Nikon Eclipse E600). The diameter of each cell was determined by using the NIH Image program, ver. 1.61, developed at the U.S. National Institutes of Health. The mean cell volume was estimated, for each molt stage, on 50 cells obtained from three preparations (i.e., from the digestive gland of different animals). The estimated mean diameter was 23.28 ± 4.11 µm (n = 50). No differences were observed in the mean volume of cells at different molt stages.
Calibration of Ca2+ binding to Fura-2-AM
Fura-2-AM fluorescence calibration was performed using the Calcium Calibration Buffer kit. Eleven mixtures of buffer and Fura-2-AM were used to record the Fura-2 spectrum. Each mixture was prepared by adding 5 mmol l–1 of Fura-2-AM to 2 ml of one of 11 prediluted buffers, each of which contained a different ratio of K2EGTA and CaEGTA. The buffer composition was modified to accurately reproduce the solution used for the experiment. For this reason, the pH was adjusted from 7.2 to 7.1 with concentrated acid, and the ionic strength was increased by the addition of solid KCl (from 0.1 to 0.5). All fluorimetric measurements were performed at 21 °C. To reliably calculate the free Ca2+ concentration in EGTA buffers, the effect of pH on the EGTA-Kd for Ca2+ must be considered (Harrison and Bers, 1989). Corrections of Ca2+: EGTA association constants for pH, temperature, and ionic strength were performed as described previously (Harrison and Bers, 1989; Martínez-Zaguilán et al., 1991) by using the software WEBMAXCLITE ver. 1.15 (available online [accessed 14 Feb 2007] at http://www.stanford.edu/
cpatton/webmaxc/webmaxclite115.htm) (Table 1). The spectral data were plotted as the log of the [Ca2+]free (x-axis) versus the log {(F – Fmin)/(Fmax – F)} (y-axis) where Fmax represents the saturated Ca2+-dye chelate, and Fmin is the Fura-2 fluorescence ratio in the absence of Ca2+. This double log plot gives an x-intercept that is the log of the Kd expressed in moles per liter (Fig. 1). Under our experimental conditions the calculated Kd was 909 nmol l–1. This value was similar to that estimated (Kd = 850 nmol l–1) in muscular fibers and neurons of crayfish (Delaney and Tank, 1994).
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Table 1 Free [Ca2+] concentration calculated at 21 °C, in solution with pH 7.1 and ionic strength of 0.5 N in cells of Marsupenaeus japonicus
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Figure 1. Calibration curve of Fura-2 generated by the protocol described in the Materials and Methods section. As a double log plot, the Ca2+ response of the indicator is linear, with the x-intercept being equal to the log of the apparent Kd of Fura-2 AM (0.909 µmol l–1 from these data).
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Spectrofluorimetric measurement of cytoplasmic Ca2+ concentration
After washing the cells three times by pelleting (100 x g for 5 min), the cells (4 x 106) were incubated with 5 mmol l–1 Fura 2-AM and 0.2% pluronic F-127 for 45 min at 21 °C with continuous shaking (100 cycles min–1). After the loading period, the cells were washed twice with a saline solution for hepatopancreatic cells (see above), incubated again for 10 min at room temperature to facilitate hydrolysis of the esterified probe, and washed once again. The cells were resuspended in 2 ml of the same buffer containing 0.1% BSA; 20 µl of this cell suspension was added to a 2-ml fluorescence cuvette kept at 21 °C and stirred throughout the experiment. The fluorescence intensity was measured with a JASCO FP 750 fluorimeter (Jasco Corporation, Japan). The excitation wavelengths were 340 and 380 nm, and emission was measured at 510 nm. The maximal fluorescence value was determined at the end of each experiment by adding 10 µl of 2 mol l–1 CaCl2 and 20 µl of 10% SDS; the minimal fluorescence value was determined by adding 20 µl of 0.5 mol l–1 EGTA solution, pH 9.0. The cytoplasmic Ca2+ concentration at time t was calculated by using the software of the fluorimeter with the calculated dissociation constant (see above) for the Fura 2-Ca2+ complex, according to the Grynkiewicz equation (Grynkiewicz et al., 1985):
where F denotes the time course of the fluorescence at 510 nm after dual excitation at 340/380 nm, and F380 is the fluorescence at 510 nm after excitation at 380 nm.
At the end of the experiment, Fmax was determined by adding 10 µl of 2 mol l–1 CaCl2 and 20 µl of 10% SDS, and Fmin was determined by adding 20 µl of 0.5 mol l–1 of EGTA.
Measurement of ATPase activity
The R cell fraction was homogenized in cold (0–4 °C) saline buffer (see above), using a glass-Teflon homogenizer. The homogenate was kept at 4 °C until ATPase activity was assayed. Pi was determined according to Fiske and Subbarow (1925). A 100-µl sample of cell homogenate (containing 600 µg of protein) was added to 900 µl of assay buffer containing (in mmol l–1) 1 MgCl2, 100 KCl, 5 NaN3, 0.125 CaCl2, 0.11 EGTA, 40 maleic acid (pH 6.8 with Tris) and maintained at 4 °C for 10 min. The samples were transferred into a water bath at 21 °C, and 50 µl of ATP (100 mmol l–1) was added after 5 min. After 30 min the activity of the ATPases was blocked by inactivation in boiling water for 2 min. The samples were centrifuged at 13,000 x g rpm for 5 min, the resulting supernatant was added to 350 µl of distilled water, and 200 µl of 20% HClO4 and 200 µl of 5% ammonium molybdate were added to each one. After 1 min, 200 µl of Fiske's reagent (sodium metabisulfite 110 g l–1, L-amino-2 naphtalenesulfonic acid 2 g l–1, sodium sulfite 24 g l–1) was added and the absorbance read, after 30 s, at 700 nm (spectrophotometer DU 530 Beckman). The specific activity of the ATPases was calculated as nanomoles of Pi released per milligram of protein in 1 min. The value of absorbance of each sample was corrected for a "blank" obtained by boiling the samples after addition of ATP and before incubation for 30 min at 21 °C.
Protein concentration was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.
Atomic absorption determination of total cell calcium
R cell suspensions prepared from animals at different molting stages, as previously described, were diluted to a final concentration of 107 cell ml–1. The total calcium concentration was determined by atomic absorption spectrophotometry (Varian AA-220) according to De Flora et al. (1985). Results obtained were expressed as micrograms of calcium per sample, and these values were used to calculate the micromoles of calcium per sample. The mmolar concentration of calcium was calculated taking into account the number of cells per sample and the total cell volume per sample.
Statistics
Individual experiments are presented throughout this article. For enzymatic activity and spectrofluorimetric measurement, statistical significance (between different experimental conditions, within a single experiment) was determined by two-way analysis of variance followed by a Tukey test of pairwise multiple comparisons. Data points were expressed as means ± standard deviation (SD). In all cases, differences were accepted at P < 0.05.
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Results
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Calcium concentration in R cells during molting cycle
Table 2 reports the values of the resting [Ca2+]i in R cells during different molting stages of the prawn Marsupenaeus japonicus, as determined by the Fura-2-AM fluorescence technique (see Materials and Methods section). The resting [Ca2+]i was about 0.20 µmol l–1 during both post- and intermolt phases but was significantly (P < 0.05) higher ( 0.40 µmol l–1) during early and late premolt. To evaluate the total cell calcium (free, bound to calcium-binding proteins, and stored in amorphous form) in R cells during the molting cycle, we used the atomic absorption method. Results shown in Table 2 demonstrate that the total calcium concentration in R cells was lower in intermolt, increased significantly during premolt (early and late), and was highest in postmolt.
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Table 2 Measurement of calcium concentration in R cells during different molting stages of Marsupenaeus japonicus
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Calcium released from intracellular stores in R cells during molting cycle
To evaluate the possible quantitative fluctuations of calcium that was stored in the mitochondria of R cells during different phases of the molting cycle, we performed experiments using the Fura-2 technique. After obtaining a baseline trace (resting [Ca2+]i), we added to the cuvette buffer a mixture of CCCP (carbonyl cyanide 3-chlorophenylhydrazone) and ruthenium red. CCCP dissipates the electrochemical proton gradient at the mitochondrial level and causes release of pooled Ca2+ (Herrington et al., 1996); ruthenium red inhibits mitochondrial calcium uptake (Klein and Ahearn, 1999). As Figure 2 shows, the addition of 2 mmol l–1 CCCP plus 2 mmol l–1 ruthenium red produced a rapid rise in cytosolic calcium concentration that achieved a maximum within 2–3 min and slowly returned to control levels within 5–7 min. During premolt the mitochondria calcium depletion doubled the cytoplasmic calcium concentration, whereas during intermolt and postmolt phases the cytosolic calcium increase was lower (Fig 2). These results indicate that the amount of calcium that can be released from mitochondria depends upon the molting phase.
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Figure 2. Effect of 2 µmol l–1 carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and 2 µmol l–1 ruthenium red on [Ca2+]i in R cells during intermolt, early premolt, late premolt, and postmolt. Arrow indicates time point at which CCCP and ruthenium red were added. Results shown are representative experiments.
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To investigate the role of endoplasmic reticulum as a calcium store in R cells during the molting cycle, we used thapsigargin, the specific inhibitor of the family of SERCA pumps (Thastrup et al., 1990; Lytton et al., 1991). Under our experimental conditions, thapsigargin (0.1, 1, and 10 mmol l–1) did not elicit calcium release from intracellular stores. Furthermore, no mobilization of thapsigargin-sensitive pools of calcium was noted when cells were incubated with thapsigargin for 10 min before analysis (data not shown). However, results reported in Table 3 demonstrate that R cells have thapsigargin-sensitive SERCA pumps that are detectable only in early premolt and not in other molting stages.
Since thapsigargin failed to determine calcium release from endoplasmic stores, we tested different calcium agonists: ATP (Schwiebert and Zsembery, 2003), carbachol (Turner et al., 2003), and isoproterenol (Dehaye et al., 1993). All the agonists were tested at three concentrations (1, 10, and 100 mmol l–1). Among these, only 100 mmol l–1 ATP caused an increase in [Ca2+]i of R cells during all the molting stages: an initial spike was followed by a progressive decrease lasting more than 8 min (Fig. 3). It has been demonstrated (Grapengiesser et al., 2005; Stamatakis and Mantzaris, 2006) that ATP acts as an extracellular messenger, determining IP3 production that in turn opens channels through which Ca2+ ions are secreted in the cytosol.
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Figure 3. Effect of 100 µmoll–1 ATP on [Ca2+]i in R cells during intermolt, early premolt, late premolt, and postmolt. Arrow indicates time point at which ATP was added. Results shown are representative experiments.
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Finally, our results (Figs. 4 and Table 2) demonstrate the additive effect of CCCP and ATP.
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Figure 4. Additive effect of 100 µmol l–1 ATP and 2 µmol l–1 carbonyl cyanide 3-chlorophenylhydrazone (CCCP) plus 2 µmol l–1 ruthenium red on [Ca2+]i in R cells during intermolt, early premolt, late premolt, and postmolt. Arrow indicates time point at which the substances were added. Results shown are representative experiments.
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Discussion
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It is generally accepted that the hepatopancreatic cells play a considerable role in massive calcium movements between the exoskeleton and the hepatopancreatic and gastrolith storage sites during premolt and postmolt stages of the molting cycle in crustaceans (Wheatly, 1996, 1997, 1999). Recently Chavez-Crooker et al. (2003) demonstrated that in Homarus americanus both R and E cells show a significant increase in calcium content during premolt, whereas F and B cells do not. Our data demonstrate that the cytosolic free calcium concentration in R cells isolated from the hepatopancreas of Marsupenaeus japonicus also depends strictly upon the molting cycle. Resting [Ca2+]i was lower during both postmolt and intermolt than in premolt phase. Resting [Ca2+]i values (reported in Table 2) were of the same order of magnitude as those reported for cells in freshwater and saltwater crustaceans (Wheatly, 1999) and more generally for eukaryotic cells (Simkiss, 1996). On the other hand, it is known that absolute values for cytosolic ionized calcium obtained by Fura-2 measurements should be regarded with some caution, even if changes in ionized calcium are reliable.
In the R cells of Marsupenaeus japonicus digestive gland, the total cell calcium content (free, bound to calcium-binding proteins, and stored in amorphous form) also depends upon the molting cycle (Table 2). The total cell calcium content was lower in intermolt, increased during premolt (early and late), and was highest in postmolt. Our data could be explained by considering the process of calcium homeostasis in the hepatopancreas during the molting cycle. During the premolt phase, calcium solubilized from the old exoskeleton enters the hemolymph and, following its electrochemical gradient, flows into hepatopancreatic cells through channels or reversal antiporters in the basolateral membrane (Wheatly, 1999; Zanotto and Wheatly, 2003). Some calcium is sequestered in mitochondria, ER, or as CaPO4/CaSO4 granules in membrane-bound vacuoles. The rest is transported out of the apical membrane to form gastroliths. This explains the greater content of calcium in R cells during premolt compared to intermolt. The presence of higher total cell calcium during postmolt compared to premolt could have at least two explanations.
- The amount of calcium that flows into R cells is higher during postmolt, since the calcium coming from reingestion of exuviae is added to the calcium released from gastroliths and intracellular stores (Zanotto and Wheatly, 2003). It is possible that during this stage, when the total cell calcium concentration was 8–10 times higher than in the premolt stage (Table 2), the binding proteins are up-regulated to keep the total concentration of free calcium low. This hypothesis was sustained by the demonstration that different calcium transporters are up-regulated during postmolt (Wheatly et al., 2002; Ziegler, 2002).
- We have previously demonstrated that the number of R cells is affected by the molting cycle (Zilli et al., 2003). During the premolt phase, R cells represent 65%–75% of the total hepatopancreatic cell population. This number decreases to about 40% in postmolt. The higher calcium concentration that we measured during postmolt could therefore be due to the lower number of R cells and the simultaneous massive calcium flux through these cells.
Within the hepatopancreatic cells, possible mechanisms of calcium sequestration include attachment to calcium-binding proteins (calmodulin, calreticulin); formation of CaPO4/CaSO4 granules; and storage in many intracellular organelles such as mitochondria, ER, lysosome, nucleus, Golgi, and endosomes (Wheatly, 1999; Zanotto and Wheatly, 2003; Ahearn et al., 2004). A possible role of crustacean epithelial ER and mitochondria in transcellular calcium transport and storage has already been demonstrated (Chen and Lehninger, 1973; Becker et al., 1974; Chen et al., 1974; Klein and Ahearn, 1999; Hagedorn and Ziegler, 2002; Chavez-Crooker et al., 2003). Our results (Figs. 2 and Table 2) show that the amount of calcium released by mitochondria is very low during inter- and postmolt, and rises during premolt when calcium massively enters cells from the hemolymph. One possible explanation of these results is that the mitochondria take part in the calcium homeostasis and calcium storage in R cells during premolt. These data were also in agreement with previous studies that demonstrated a differential mitochondrial calcium transport and storage throughout the molting cycle (Ueno, 1980; Rogers and Wheatly, 1997; Klein and Ahearn, 1999).
Under our experimental conditions, the amount of ATP-stimulated calcium was similar during all four stages. ATP acts as an extracellular messenger; in fact, it binds receptors in the membrane of the cell, thus activating phospholipase C, which catalyses IP3 production from PIP2. IP3 binds synergistically with Ca2+ in receptors on the surface of the intracellular calcium stores and opens channels through which Ca2+ ions are secreted in the cytosol (Grapengiesser et al., 2005; Stamatakis and Mantzaris, 2006). The ER is the organelle in which IP3 receptors are most abundantly expressed (Bush et al., 1994; Pozzan et al., 1994). However, IP3-evoked Ca2+ mobilization has also been reported to occur from the nuclear envelope (Stehno-Bittel et al., 1995; Mak and Foskett, 1997), the Golgi apparatus (Pinton et al., 1998), and plasma membranes (Dellis et al., 2006). Under our experimental conditions, the observed [Ca2+]i increase following ATP administration cannot be due to the influx of calcium throughout plasma membrane channels, since calcium was not present in the extracellular medium. This means that the increase of calcium is attributable to ion release from intracellular stores (ER, nucleus, and Golgi). Within these, the major calcium-storing, buffering, and signaling organelle is the ER (Rizzuto et al., 2004). However, our data do not allow us to rule out a possible contribution of Golgi and nucleus.
It must be underlined that under our experimental conditions, thapsigargin inhibited the SERCA activity expressed by R cells (Table 3) but failed to determine calcium efflux from the ER. The failure of thapsigargin to empty the ER is probably due to low calcium permeability. Similar findings were reported by Pozzan et al. (1994). The presence of a SERCA activity measurable only during early premolt (Table 3) suggests that calcium is actively accumulated within the ER during this stage, after calcium re-absorption from the hemolymph. A molt-cycle-dependent SERCA activity was already demonstrated in Porcellio scaber (Hagedorn and Ziegler, 2002).
Figure 4 and Table 2 also show that the total calcium released by the mitochondria/ER/Golgi/nucleus system led to an increase in cytosolic calcium concentration of about 0.20–0.46 mmol l–1 in postmolt and intermolt, and 0.56–0.94 mmol l–1 during premolt. These values represent 0.1% of the total cell calcium content during intermolt, about 0.05%–0.06% during early and late premolt, and only 0.003% in postmolt (Table 2).
Our results suggest that these organelles do not play a key role in calcium storage during the molting cycle, and that they are involved in transcellular calcium flux. We hypothesize that lysosome or membrane-clad concretion vacuoles could represent the main site of calcium storage in hepatopancreatic R cells.
Our hypothesis is sustained by many previous studies (Becker et al., 1974; Johnson, 1980; Al-Mohanna and Nott, 1985, 1987; Simkiss, 1986; Chavez-Crooker et al., 2003) reporting the presence in the absorptive R cells of preferential calcium storage sites that are different from mitochondria and ER.
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Acknowledgments
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We are indebted to Prof. Francesco Paolo Fanizzi, director of Consorzio Interuniversitario di Chimica dei Metalli nei Sistemi Biologici (CIRCMSB) which performed the atomic absorption determination of total cell calcium. The authors wish to thank Mrs. Nicolette S. James for her language assistance.
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Footnotes
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Received 30 June 2006; accepted 6 February 2007.
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Abstract
Introduction
