Regulation of intracellular calcium in dispersed fat body trophocytes of the cockroach, Periplaneta americana, by hypertrehalosemic hormone

Journal of Insect Physiology 47 (2001) 1399–1408 www.elsevier.com/locate/jinsphys

Regulation of intracellular calcium in dispersed fat body trophocytes of the cockroach, Periplaneta americana, by hypertrehalosemic hormone D. Sun, J.E. Steele

*

Department of Zoology, The University of Western Ontario, London, Ont., Canada N6A 5B7 Received 15 May 2001; accepted 31 July 2001

Abstract Incubation of trophocytes from dissaggregated fat body of Periplaneta americana with either of the hypertrehalosemic hormones, HTH-I or HTH-II, leads to an increase in the cytosolic concentration of Ca2+ from 苲80 to 苲310 nM with a rise time of approximately 110 s. The Ca2+ concentration then declines to the resting level during the ensuing 5 min. In the absence of extracellular Ca2+ the increase in [Ca2+]i due to HTH is limited to 苲100 nM. The calmodulin inhibitors calmidazolium and W-7 also limit to a similar degree the ability of HTH to increase [Ca2+]i. Phorbol 12-myristate 13-acetate, an activator of protein kinase C, was shown to block Ca2+ entry through the plasma membrane. Additional evidence to support the view that HTH enhances Ca2+ influx has been obtained by measuring the quenching of fura-2 fluorescence when Ca2+ is replaced with Mn2+.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Calcium; Calmodulin; Cockroach; Fat body; Hypertrehalosemic hormone; Periplaneta americana; Protein kinase C; Trophocyte

1. Introduction Hypertrehalosemic hormone (HTH) dependent efflux of trehalose from cockroach body has an obligatory dependence on extracellular Ca2+ (McClure and Steele, 1981), although there has been no evidence until recently to show that Ca2+ actually entered the cell. Our knowledge of how extracellular Ca2+ is mobilized for use by the fat body is modest and only now do we begin to understand how this occurs. Entry of Ca2+ into Periplaneta americana fat body, as illustrated by the uptake of 45Ca2+ in vitro, is increased by HTH (Steele and Paul, 1985). Other studies show that adipokinetic hormone (AKH) increases the entry of 45Ca2+ into intact fat body of Locusta migratoria (Van Marrewijk et al., 1993; Vroemen et al., 1995) and that the efflux of Ca2+ from the tissue increases as well. HTH-I and HTH-II have also been shown to increase 45Ca2+ influx into dispersed

* Corresponding author. Tel.: +1-519-661-3136; fax: +1-519-6612014. E-mail address: [email protected] (J.E. Steele).

trophocytes from Periplaneta americana fat body in vitro: the influx is then followed by an increase in efflux of labelled Ca2+ from the cells (Steele and Ireland, 1999). Although these studies suggest that HTH increases [Ca2+]i they are not evidence that this actually occurs. Perhaps the first direct evidence to show that [Ca2+]i is increased by hormone treatment is the qualitative study by Jahagirdar et al. (1987) showing that the intensity of fura-2 fluorescence of cultured hemocytes of Malacosoma disstria increased following treatment with HTHI and HTH-II. Dispersed fat body cells from Locusta migratoria fat body also respond to each of AKH-I, AKH-II and AKH-III with an increase in cytosolic calcium ([Ca2+]i), the concentration rising from approximately 50 to 300 nM (Goldsworthy et al., 1997). Interestingly, the duration of the response differs for each of the hormones. The key reaction in trehalose synthesis is the breakdown of glycogen catalyzed by glycogen phosphorylase (EC 2.4.1.1). Three forms of the enzyme occur in fat body (Van Marrewijk et al., 1988; Ga¨de, 1991). Phosphorylation of inactive phosphorylase b by phosphorylase kinase yields a partially active ab form, or fully

0022-1910/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 1 3 0 - 5

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active a form. The enzyme is homodimeric so that the ab form probably represents a transitional state in which only one subunit is phosphorylated. Phosphorylase kinase is calcium dependent and appears to have a requirement for calmodulin. Studies in our laboratory (Pallen and Steele, 1988) on Periplaneta americana, as well as those by Hansford and Sacktor (1970) and Yanagawa and Horie (1978), and Ashida and Wyatt (1979) on other species have shown that phosphorylase kinase is activated by low concentrations of calcium. Significantly, hormonal activation of fat body phosphorylase is strongly inhibited by the phenothiazines, chlorpromazine and trifluoperazine (Pallen and Steele, 1988; Van Marrewijk et al., 1991). These compounds have a high calcium dependent affinity for calmodulin (Levin and Weiss, 1979), which explains their potent inhibition of enzymes that require calmodulin for activity. Thus the influx of Ca2+ into fat body induced by HTH is considered to be a principal factor in the activation of phosphorylase that enables the increase in trehalose synthesis to occur. Further evidence that the hypertrehalosemic response to HTH is dependent on an increase in [Ca2+]i has been obtained using agents that release Ca2+ from the intracellular stores. Both thapsigargin and thimerosal have been used for this purpose and have been shown to activate Blaberus discoidalis fat body phosphorylase to the same level as HTH (Park and Keeley, 1996). The effect of thapsigargin is significantly less effective in stimulating trehalose efflux than is HTH although thimerosal was almost as effective as the hormone (Keeley and Hesson, 1995). The action of thapsigargin and thimerosal is postulated to occur because of an increase in [Ca2+]i although the effect of these agents on Ca2+i in the fat body has never been determined. The purpose of this study was to provide quantitative data on [Ca2+]i in the fat body trophocyte. More specifically we wished to know how [Ca2+]i is modulated by the action of the HTHs and the means by which this occurs. Hormone mediated change in the concentration of cytosolic Ca2+ is likely to be important in regulating the activity not only of glycogen phosphorylase but of numerous other reactions in the cell as well.

2. Materials and methods 2.1. Insects Adult male American cockroaches, Periplaneta americana, 4–6 weeks after the final moult, were used in the study. The cockroaches were fed a diet containing 82% rolled oats; 5% yeast extract; 10% sucrose; and 3% peanut oil. Shortly after the cockroaches moulted to become adults the males were segregated for experimental use. The insectory was operated with a 12 h light, 12 h dark

photoperiod and maintained at 28–30°C with a relative humidity of 60±10%. 2.2. Chemicals All common organic and inorganic chemicals used in these studies were of reagent grade or better and were obtained from various laboratory suppliers. Special chemicals were obtained as follows: Pluronic F-127, BAPTA AM and fura-2 AM were purchased from Molecular Probes, Eugene, OR. Phorbol 12-myristate 13acetate (PMA), bovine serum albumin (essentially fatty acid-free), collagenase A and DNAse were obtained from Boehringer Mannheim GmbH, Laval, PQ. Calmidazolium and W-7 were purchased from RBI, Natick, MA and the Bio-Rad Protein Assay kit from Bio-Rad Laboratories, Hercules, CA. The synthetic hormones HTH-I and HTH-II were obtained from Peninsula Laboratories, Belmont, CA. The HTH content was estimated by resolving a sample of the commercial peptide by HPLC and a comparing the absorption of the hormone peak at 254 nm with the remaining peaks. Solvents were supplied by Fisher Scientific Co., Toronto, Ont., VWR Canlab, Toronto, Ont., and Caledon Laboratories Ltd, Georgetown, Ont. 2.3. Physiological saline The physiological saline used in these studies contained 215 mM NaCl, 4.8 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 40 mM trehalose, 5 mM glucose and 40 mM Hepes. The pH was adjusted to 7.4 with 1 N NaOH. 2.4. Disaggregation of fat body Preparation of the trophocytes by disaggregation of the fat body followed the method described by Steele and Ireland (1994) with minor modification. Ten fat body lobes were pooled and minced on the wax surface of a dissecting dish using iridectomy scissors. The minced tissue was then transferred to a 10 ml plastic beaker containing 2.5 ml of saline, which included 1.75 mg of collagenase and 0.01% DNAse to prevent clumping of the cells. The mixture was incubated for 1.5 h at 30°C in a shaker water bath. At 15, 30, 45, 60 and 75 min during disaggregation the tissue was drawn into and expelled five times from a pipette to facilitate separation of the cells. Because lipid droplets, originating from broken cells during disaggregation, appeared to decrease the sensitivity of the fluorescence measurements they were largely removed from the trophocyte preparation. This was done by filtering the trophocytes under gravity through Sephadex G-25 with a settled bed volume of 0.8 ml which was retained in a column prepared from a syringe

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barrel fitted with a nylon mesh screen (150 µM, pore size). The Sephadex column was washed with fresh aerated saline, the fluid level lowered to the top of the bed, and the cell suspension placed on the column. The sample was then allowed to run through the column until the volume of the cell sample had been reduced to approximately 1 ml. About 3 ml of saline was added to the cells lying above the column bed and allowed to run through until approximately 1 ml of saline containing the trophocytes remained above the top of the column. This procedure was repeated four times and the trophocytes, in approximately 1 ml, were recovered from the column. The number of cells obtained was estimated with a hemocytometer. 2.5. Measurment of intracellular calcium [Ca2+]i was measured by monitoring the change in fluorescence of the Ca2+-sensitive indicator fura-2 according to the method described by Thomas and Delaville (1991). Trophocytes derived from ten fat bodies in 1 ml of saline were loaded in darkness for 1 h at 35°C with 5 µM fura-2 AM in the presence of 0.025% pluronic F-127 and 0.5% bovine serum allbumin. To avoid oxygen starvation an O2 and CO2 gas mixture (95:5, v/v) was used to flush the vial containing the cells at the beginning of loading with fura-2 AM. The vial was sealed so that the atmosphere in the vial maintained a high concentration of O2 throughout the loading period. After loading with fura-2 AM the cells were transferred to the cell harvester and washed three times with fresh saline to remove extracellular fura-2 AM and then resuspended in 2 ml of saline. In some instances calcium-free saline, as indicated in the legends to the figures, was used. For measurement of [Ca2+]i 300 µl of cell suspension was transferred to a disposable plastic cuvette containing saline to give a final valume of 2 ml. The cell suspension was gently mixed with a magnetic stirring bar (3 mm × 6.35 mm). Fura-2 fluorescence was measured using an Aminco Bowman Series 2 luminescence spectrometer with an emission wavelength of 510 nm and alternating exitation wavelengths of 340 and 380 nm. Data points were collected at 1 s intervals and the [Ca2+]i for each was calculated using a computer program incorporated into the software used to operate the AB 2 luminescence spectrometer. Fluorescence ratio values under saturating conditions were obtained by adding 50 µg/ml of digitonin to the contents of the cuvette to release cytosolic fura-2 after completing the recording of [Ca2+]i. The ratio values for Ca2+-free conditions were obtained by addition of 30 µl of 400 mM EGTA in 3 M Tris-base to chelate all calcium in the medium after the addition of the digitonin. The Kd value used for fura-2 and calcium was 224 (Grynkiewicz et al., 1985). The [Ca2+]i plots generated by the computer program

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incorporated into the AB 2 luminescence spectrometer were ‘smoothed’ by calculating the mean value for each 5 s interval. The data was then replotted using Biosoft Fig P graphics software. 2.6. Loading BAPTA AM Trophocytes derived from ten fat bodies in 1 ml of calcium-free saline were first loaded with fura-2 as described above and then loaded with 100 µM BAPTAAM in the presence of 0.025% pluronic F-127 and 0.5% bovine serum allbumin in darkness for 30 min at 35°C. After loading with BAPTA-AM the cells were transferred to the cell harvester and washed three times with fresh calcium-free saline to remove extracellular BAPTA-AM and resuspended in 2 ml of calcium-free saline. 2.7. Mn2+ quenching Mn2+ quenching of fura-2 fluorescence was performed by the addition of 1 mM MnCl2 to the saline in the cuvette and the quenching of the fluorescence measured using an excitation wavelength of 360 nm, the isosbestic wavelength, and an emission wavelength of 500 nm (Sage et al., 1989).

3. Results 3.1. HTH-induced increase in [Ca2+]i The first attempt to monitor the effect of HTH on [Ca2+]i resulted in an unexpected outcome (Fig. 1) in which the intracellular level of calcium, after responding to HTH, continued to climb for 30 min (when the measurements were stopped) and failed to return to the basal level. These observations suggested that the mechanism for removing Ca2+ from the cytosol had failed. Since removal of Ca2+ from the cytosol is energy dependent a possible source of the problem could have been a failure in the supply of ATP. To preclude this potential situation, the cells, during loading with fura-2, were incubated in capped plastic vials, which were flushed with 95% O2 and 5% CO2 at the start of incubation. Trophocytes treated in this manner retained their ability to return high [Ca2+]i to the resting level. In the unstimulated (resting) state the trophocytes have a basal intracellular Ca2+ level in the region of 80 nM. Immediately after treatment with HTH-I, [Ca2+]i increases to a maximal level in less than 2 min and then returns to the resting level during the ensuing 5 min (Fig. 2a). The maximal [Ca2+]i induced by the hormone is approximately 300 nM. Less than 8 min after initial exposure of the trophocytes to hormone the intracellular Ca2+ level has returned to the resting value. Similar

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Fig. 1. Unaerated saline prevents the return of [Ca2+]i to the resting level in HTH-II activated trophocytes. Trophocytes were loaded with fura-2 as described in the Section 2 with the exception that the saline used to load and incubate the cells was not oxygenated. Trophocytes loaded with fura-2 in 200 µl of saline (苲1×105 cells) were added to 1.8 ml of saline in the spectrofluorimeter cuvette and the [Ca2+]i determined as described in the Section 2. About 10 µl of HTH-II was added at 100 s to give a final concentration of 100 pmol/ml and the [Ca2+]i recorded continuously for 1000 s. The plot of [Ca2+]i shown is representative of four experiments.

results were obtained when trophocytes were treated with HTH-II (Fig. 2b). Although chelation of extracelluar Ca2+ prevents activation of phosphorylase (McClure and Steele, 1981) this effect is most likely manifested by a decrease in [Ca2+]i since removal of all extracellular Ca2+ by EGTA probably leads to a loss of Ca2+ from the cell because of the steep outwardly directed concentration gradient. To test this possibility the effect that chelation of extracellular Ca2+ might have on [Ca2+]i was determined. The data show that the intracellular concentration of free Ca2+ is lowered by more than 50% to approximately 30 nM within 1 min after addition of EGTA to the extracellular medium (Fig. 3). Significantly, the increase in concentration of cytosolic free Ca2+ following addition of HTH to the medium does not exceed 100 nM so that the final concentration of Ca2+ in the cell is approximately 130 nM. These results suggest that the maximum increase in the cytosolic level of Ca2+ in response to HTH is largely dependent on an influx of extracellular Ca2+. Because calmodulin has been implicated in the stimulation of trehalose synthesis by HTH, it was of interest to know whether antagonists of calmodulin could block the increase in [Ca2+]i induced by HTH. Fura-2 loaded trophocytes were pretreated with 10 µM calmidazolium, an antagonist of calmodulin, for 5 min followed by the addition of HTH-II. The results show that calmidazolium does not affect the resting level of intracellular Ca2+ whereas the increase in [Ca2+]i that normally follows the

Fig. 2. An experiment showing that HTH-I and HTH-II increase [Ca2+]i in dispersed trophocytes. Trophocytes loaded with fura-2 in 200 µl of saline (苲1×105 cells) were added to 1.8 ml of saline in the spectrofluorimeter cuvette. (a) After recording the resting level of [Ca2+]i for 200 s HTH-I (final concentration 100 pmol/ml) was added and recording of the [Ca2+]i continued to 900 s. The [Ca2+]i plot is representative of four experiments. (b) The experimental details are identical to those described for (a) with the exception that HTH-II was used. The plot showing [Ca2+]i is representative of four experiments.

addition of HTH-II is significantly diminished by the inhibitor (Fig. 4a). Similar results were obtained using the napthalenesulfonamide derivative W-7 (0.1 mM), another antagonist of calmodulin (Fig. 4b). These data suggest that calmodulin participates in the intracellular Ca2+ signalling process evoked by the hormones. 3.2. Regulation of capacitative Ca2+ entry The results show that the full HTH-induced increase in [Ca2+]i depends on an influx of extracellular calcium into the trophocyte. A related problem is the mechanism that regulates the entry of Ca2+ in response to HTH. Generally, InsP3, a product of the reaction catalyzed by phos-

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Fig. 3. Inhibition of the HTH-II induced [Ca2+]i increase in trophocytes by chelation of extracellular Ca2+ with EGTA. Trophocytes in 200 µl of saline and loaded with fura-2 were added to 1.6 ml of saline in the spectrofluorimeter cuvette and the [Ca2+]i recorded. About 200 µl of EGTA yielding a final concentration of 2 mM was added to the saline at 100 s followed by the addition of HTH-II, final concentration 100 pmol/ml, at 150 s. [Ca2+]i was recorded up to 500 s. The experiment shows that addition of EGTA brings [Ca2+]i down from 苲75 to 苲30 nM. The subsequent addition of HTH-II is able to raise the concentration of Ca2+ by only 苲100 nM to a maximum level of 苲130 nM.

phatidylinositol phospholipase C, increases [Ca2+]i by mobilizing the ion from intracelluar Ca2+ stores. Mobilization of this Ca2+ activates the plasma membrane Ca2+ influx pathway through Ca2+ release-activated channels and is termed capacitative Ca2+ entry or Ca2+ releaseactivated Ca2+ entry (iCRAC) (Putney and Bird, 1993; Berridge, 1995). To test whether this capacitative Ca2+ entry also exists in the trophocytes, the fura-2 loaded cells were incubated in Ca2+-free medium for 100 s followed by the addition of 1 mM CaCl2. The addition of 1 mM CaCl2 induced a rapid increase in [Ca2+]i because of Ca2+ influx from the extracellular fluid (Fig. 5a). Treatment of the cells with HTH-II (100 pmol/ml) in Ca2+-free medium produced a transient increase in the concentration of Ca2+i followed by a decrease to the resting level (Fig. 5b). This reflects the emptying of the intracellular Ca2+ stores. The subsequent addition of 1 mM CaCl2 to the medium dramatically enhances influx of Ca2+ (Fig. 5b), thus supporting the idea of HTHinduced capacitative Ca2+ entry. Because protein kinase C (PKC) has been implicated in HTH mediated stimulation of trehalose synthesis (unpublished data), it was of interest to know whether it may be associated with capacitative Ca2+ entry. Trophocytes bathed in Ca2+-free saline maintain a lower than normal, but measurable level of Ca2+ in the cytosol. Addition of Ca2+ to the saline is then followed by a sharp rise in the concentration of cytosolic Ca2+ as shown in Fig. 5a. If, however, PKC is activated by the inclusion of 0.1 µM PMA in the Ca2+-free saline the subsequent

Fig. 4. Calmodulin inhibitors decrease Ca2+ influx into trophocytes due to stimulation with HTH-II. Trophocytes (苲1×105 cells) loaded with fura-2 in 200 µl of Ca2+-free saline (containing 1 mM EGTA) were added to 1.8 ml of the same saline in the spectrofluorimeter cuvette. (a) About 5 µl of calmidazolium, final concentration 10 µM, was added to the saline 200 s after the addition of the trophocytes. HTH-II was added at 500 s to give a final concentration of 100 pmol/ml. The [Ca2+]i was recorded continuously up to 1000 s. (b) The protocol was identical to that described in (a) except that the inhibitor used was W-7 (0.1 mM). Note that the maximum [Ca2+]i achieved in the presence of HTH-II in both instances is only 苲150 nM. The [Ca2+]i plots shown are representative of four experiments.

elevation of [Ca2+]i which normally follows the addition of 1 mM CaCl2 to the medium is significantly decreased (Fig. 6). This implies that PMA exhibits an inhibitory effect on Ca2+ influx through the plasma membrane. To determine whether PMA might also inhibit capacitative Ca2+ entry induced by HTH, the fura-2 loaded trophocytes were incubated with 0.1 µM PMA in Ca2+free saline prior to stimulation of the cells with HTHII. The data (Fig. 7) show that PMA does not significantly affect the expected discharge of Ca2+ from the intracellular stores following treatment of the trophocytes with HTH in Ca2+-free saline. In contrast, the sub-

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Fig. 6. Phorbol myristate acetate (PMA) inhibits basal Ca2+ influx in trophocytes. Trophocytes (苲1×105 cells) loaded with fura-2 in 200 µl of Ca2+-free saline (containing 1 mM EGTA) were added to 1.8 ml of the same saline in the spectrofluorimeter cuvette and [Ca2+]i recorded. PMA, 0.1 µM final concentration, was added to the saline at 100 s followed by the addition of calcium chloride at 400 s to yield 2 mM. [Ca2+]i was recorded continuously up to 600 s. The data show that PMA inhibits the Ca2+ stimulated Ca2+ influx. Compare this figure with Fig. 5a. The plot showing [Ca2+]i is representative of four experiments.

Fig. 5. Evidence for iCRAC in trophocytes. Trophocytes (苲1×105 cells) in 200 µl of Ca2+-free saline (containing 1 mM EGTA) were loaded with fura-2. The sample was added to 1.8 ml of the same saline in the spectrofluorimeter cuvette and the [Ca2+]i recorded. (a) Calcium chloride (2 mM, final concentration) was added after 100 s and the [Ca2+]i recorded to 300 s. (b) HTH-II, final concentration 100 pmol/ml, was added to the Ca2+-free saline after 150 s incubation. This was followed by the addition of calcium chloride at 350 s to yield a final concentration of 2 mM. [Ca2+]i was recorded to 700 s. The [Ca2+]i record is representative of four experiments. 2+

sequent influx of extracellular Ca after addition of CaCl2 to the saline is significantly diminished by the presence of PMA (Fig. 7; cf. Fig. 5b). If the PMA is applied after the addition of HTH-II a similar inhibitory effect of PMA on capacitative Ca2+ entry occurs (Fig. 8; cf. Fig. 5b). These results suggest that PKC has a direct inhibitory effect on HTH-induced capacitative Ca2+ entry. To provide confirming evidence that PKC inhibits influx of extracellular Ca2+ advantage has been taken of the fact that Mn2+ has certain characteristics in common with Ca2+. Mn 2+ enters cells through the same channels

Fig. 7. An experiment showing that PMA does not affect the release of Ca2+ from the intracellular stores in trophocytes challenged with HTH-II. Trophocytes (苲1×105 cells) loaded with fura-2 in 200 µl of Ca2+-free saline (containing 1 mM EGTA) were added to 1.8 ml of the same saline in the spectrofluorimeter cuvette and the [Ca2+]i recorded. PMA, final concentration 0.1 µM, was added to the Ca2+-free saline after incubation for 100 s. At 400 s, HTH-II,100 pmol/ml final concentration, was added followed by the addition at 700 s of CaCl2 to give a final concentration of 2 mM. This figure should be compared with Fig. 5b. The data show that PMA does not block the release of Ca2+ by HTH-II from intracellular stores but does inhibit the Ca2+ stimulated Ca2+ influx evoked by HTH-II. The plot of [Ca2+]i is representative of four experiments.

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Fig. 8. PMA inhibits HTH-II stimulated Ca2+ influx into trophocytes by. Trophocytes (苲1×105 cells) loaded with fura-2 in 200 µl of Ca2+free saline (containing 1 mM EGTA) were added to 1.8 ml of the same saline in the spectrofluorimeter cuvette and the [Ca2+]i recorded. HTHII,final concentration 100 pmol/ml, was added to the Ca2+-free saline after incubation for 100 s and was followed by the addition of PMA, 0.1 µM final concentration at 400 s and CaCl2 (2 mM, final concentration) at 700 s. This figure should be compared with Fig. 5b. The results show that the effect of PMA occurs subsequent to the action of HTH-II on the release of Ca2+ from the internal stores. The data shown are representative of four experiments.

as does as Ca2+, but irreversibly quenches fura-2 fluorescence (Cobbold and Rink, 1987; Hallam et al., 1989). The rate of fluorescence decay can therefore be used to trace the entry of the cation. As shown in Fig. 9a, the rate of fluorescence quenching which resulted from the influx of Mn2+ was accelerated following addition of HTH-II (100 pmol/ml). HTH-II, however, did not change the rate of Mn2+ quenching when the cells were preincubated with 0.1 µM PMA for 5 min (Fig. 9b), thus confirming the inhibitory effect of PMA on capacitative cation influx. An important aspect of the mechanism responsible for regulating Ca2+ entry is a requirement for Ca2+. This Ca2+ may act directly on the membrane to activate the putative plasma membrane ion channels. If this interpretation is correct, chelation of the stored Ca2+ immediately after its release should prevent the subsequent entry of extracellular Ca2+. To test this hypothesis BAPTA-AM (100 µM) was used to trap intracellular Ca2+. The chelator was incorporated into the trophocytes after they had been loaded with fura-2. Since direct [Ca2+]i measurement could not be performed on cells loaded with BAPTA, because the Ca2+ chelator would compete with the fura-2, the rate of divalent cation entry was monitored by determining the quenching of fura-2 fluorescence as a result of the entry of Mn2+. As illustrated in Fig. 9c, no subsequent increase in Mn2+ entry occurred in BAPTA-loaded trophocytes upon addition of HTHII (100 pmol/ml) compared with the control cells. This

suggests that there is a direct requirement for Ca2+ to activate capacitative Ca2+ entry. 4. Discussion This study shows that the increased [Ca2+]i in trophocytes in response to HTH occurs primarily because of an

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Fig. 9. Quenching of fura-2 flurescence in trophocytes by Mn2+. (a) Trophocytes (苲1×105 cells) loaded with fura-2 in 200 µl of Ca2+-free saline (containing 1 mM EGTA) were added to 1.8 ml of the same saline in the spectrofluorimeter cuvette and the fluorescence measured at 500 nm with excitation at 360 nm. Manganous chloride (1 mM) was added at 60 s followed by HTH (100 pmol/ml final concentration) at 150 s and the fluorescence recorded. (b) Trophocytes loaded with fura2 in the Ca2+-free saline (containing 1 mM EGTA) were pre-incubated with 0.1 µM PMA for 5 min before beginning recording of the fluorescence. Manganous chloride was added at 60 s followed by HTH, 100 pmol/ml, at 150 s. PMA decreases quenching of the fluorescence due to Mn2+ and is evidence that Mn2+ entry into the cell has been reduced. (c) An experiment showing that BAPTA inhibits HTH-II induced Mn2+ quenching. After loading the trophocytes with fura-2 the cells in Ca2+-free saline were loaded with 100 µM BAPTA-AM for 30 min. The extracellular BAPTA-AM was then removed and the cells in 200 µl of Ca2+-free saline were transferred to the cuvette. The fluorescence was recorded at 500 nm with excitation at 360 nm. Manganous chloride was added at 60 s to give a final concentration of 2 mM and HTH-II (final concentration 100 pmol/ml) at 150 s. The data are evidence that HTH-II promotes entry of Mn2+ into the cell.

influx of extracellular Ca2+. Treatment with either HTH-I or HTH-II increases [Ca2+]i to its maximal level approximately 150 s after addition of the hormone, the duration of the Ca2+ pulses being similar for both hormones. The elevated level of cytosolic calcium is maintained only momentarily and then decays to the resting level over a period of approximately 5 min. The only other insect for which there is comparable data is the locust, Locusta migratoria (Goldsworthy et al., 1997). In that species AKH I, II and III increase [Ca2+]i in dispersed fat body cells to approximately the same extent as does HTHI and HTH-II in the cockroach. The locust hormones, however, differ in that they increase Ca2+ levels faster than do HTH-I or HTH-II in cockroach trophocytes. Furthermore, the duration of the elevated Ca2+ in Periplaneta americana trophocytes is shorter than that in Locusta migratoria. In considering the magnitude of the Ca2+ pulse and the time taken to return to the resting level in vitro it is notable that a low oxygen level in the incubation saline appears to block the return of [Ca2+]i to the resting level, the consequence being that [Ca2+]i climbs to an unphysiologically high level. Since return of the cytosolic Ca2+ to the resting level is probably dependent on a Ca2+-ATPase in the endoplasmic reticulum and plasma membrane, and is subject to regulation by PKC (Lagast et al., 1984), the need for ATP to drive the system is obvious. A convenient mammalian model for comparison with the cockroach trophocyte is the rat hepatocyte. Activation of these cells by vasopressin causes glycogenolysis resulting in the release of glucose from the tissue. Vasopressin increases [Ca2+]i which becomes maximal within 5–10 s (Thomas et al., 1985) in contrast to the 150 s in the trophocyte. The hepatocyte also differs in that the increased [Ca2+] is maintained for 400 s or more (Rooney et al., 1989). The sustained elevation of [Ca2+]i in hepatocytes reflects a series of Ca2+ oscil-

lations of equal magnitude which at high agonist concentration become fused. Whether this also occurs in the trophocyte is not known. The increase in trophocyte [Ca2+]i induced by HTH requires extracelluar Ca2+. This is evident from the fact that chelation of extracellular Ca2+ with EGTA decreases [Ca2+]i by two-thirds during the subsequent 100 s. The increment in [Ca2+]i following addition of HTH-II in the presence of EGTA is approximately 100 nM and only one-half as great as that which occurs when extracellular Ca2+ is available. The concentration rises to only 120 nM which is well below the expected level of 280–300 nM that normally arises after treatment with HTH. Implicit in this view is the idea that Ca2+ enters the cell from the extracellular fluid. The stimulatory effect of HTH on Ca2+ flux across the trophocyte plasma membrane has been described by Steele and Ireland (1999). They show that the influx of Ca2+ nearly doubled during the first 30 s following a challenge with HTH-I or HTH-II. Furthermore, the rapid return of cytosolic [Ca2+] to the resting level is consistent with the finding that 45Ca2+ taken up under the influence of HTH is removed a few minutes later (Steele and Ireland, 1999). A detectable increase in [Ca2+]i occurs as early as 20 s after addition of HTH. This supports the view that elevated InsP3 levels precede the Ca2+ increase since InsP3 increases by more than 100% as early as 15 s after application of HTH (unpublished data). There is consensus that InsP3 acts to deplete intracellular Ca2+ stores and this is responsible for the signal that initiates the entry of extracellular Ca2+ into the cell. Entry of Ca2+ under these circumstances is described as capacitative Ca2+ entry (Putney and Bird, 1993; Berridge, 1995) or calcium release-activated calcium current (iCRAC). It is likely that InsP3, in the course of elevating cytosolic Ca2+, depletes the Ca2+ store in the ER. There is, however, less certainty about events leading to the influx of Ca2+ across the plasma membrane. In mast cells and T lymphocytes depletion of intracellular Ca2+ stores activates a calcium current that brings Ca2+ into the cell (Hoth and Penner, 1992; Zweifach and Lewis, 1993). An alternative suggestion is that depleted stores generate a calcium influx factor (CIF) which then diffuses to the store-operated channels (SOC) to stimulate Ca2+ entry across the plasma membrane (Ranchiamampita and Tsien, 1993; Parekh et al., 1993; Davies and Hallett, 1995). No direct evidence for such a factor has yet been obtained. A more attractive proposal is that the membrane of the intracellular Ca2+ stores and the plasma membrane are coupled through the cytoplasmic head of the InsP3 receptor. As the store empties, the InsP3 receptor undergoes a conformational change which is transmitted to the SOC in the plasma membrane to enable influx of external calcium (Irvine, 1990; Berridge 1990, 1995; Hardie and Minke, 1993). This study supports the idea that depletion of internal

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Ca2+ stores initiates influx of extracellular Ca2+ through SOCs in the plasma membrane according to the scheme described above. In trophocytes, as in most stimulated cells, the increase in [Ca2+]i induced by HTH is of short duration. The rapid return of [Ca2+]i to the basal level suggests that not only mobilization, but the return of Ca2+ to the resting level is tightly controlled. Thus an important objective of the study has been to determine how the rising level of [Ca2+]i is regulated and eventually returned to the resting state. The data suggest that PKC and calmodulin are part of this regulatory mechanism. HTH-induced capacitative Ca2+ entry was significantly decreased by the PKC activator PMA. This action of PMA on capacitive Ca2+ entry is not dependent on the Ca2+ pulse generated by HTH since it also occurs after the Ca2+ pulse generated by HTH had dissipated. Furthermore, the effect of PMA is not dependent on HTH since PMA decreased Ca2+ influx into trophocytes following addition of Ca2+ to the previously Ca2+-free medium. These results are evidence that PKC is not an activator of capacitative Ca2+ entry but instead is an antagonist that down regulates Ca2+ entry. Our data agree with those of Mene` et al. (1996) who showed that capacitative Ca2+ influx in human mesangial cells is inhibited by PMA, presumably because of activation of PKC. A similar effect has been shown for thyroid cells (Tornquist, 1993) and human neutrophils (McCarthy et al., 1989). In Drosophila, PKC also inhibits capacitative Ca2+ entry due to photoreceptor activation (Hardie et al., 1993). Thus there is evidence from several sources in support of the idea that PKC inhibits capacitative Ca2+ entry to the trophocyte. The demonstration that the divalent ion chelator BAPTA decreases quenching of the fura-2 fluorescent signal when Mn2+ replaces Ca2+ in the extracellular medium confirms this view. Mn2+ is a selective tracer for Ca2+ entry because it enters the cell through the same route as Ca2+ but cannot be pumped out of the cell (Hallam et al., 1989). Once inside the cell the trapped Mn2+ quenches fura-2 fluorescence generated by the bound Ca2+ (Cobbold and Rink, 1987; Hallam et al., 1989). These results illustrate the importance of a rise in [Ca2+]i so that the inwardly directed capacitative Ca2+ entry can be activated. The finding that PMA blocked HTH stimulated Mn2+ influx in the trophocytes is similar to results obtained for human mesangial cells (Mene` et al., 1996), and shows that PKC inhibits capacitative Ca2+ influx. The inhibitory effect of PKC on capacitative Ca2+ influx implies that protein phosphorylation is involved although there is no data to suggest how this might be accomplished, at least in insects. The calmodulin inhibitors, calmidazolium or W-7, diminished the ability of HTH-II to increase [Ca2+]i by imposing an upper limit of only 苲150 nM. This is approximately one-half the normal response and represents the release of Ca2+ from the internal stores alone. The calmodulin inhibitors appear to block capacitative

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Ca2+ influx into trophocytes completely, but have no effect on the basal level of [Ca2+]i. The same inhibitors caused significant inhibition of capacitative Ca2+ influx in human mesangial cells but only partially blocked Ca2+ influx induced by angiotensin II (Mene` et al., 1996). These observations suggest that the Ca2+-calmodulin complex may be an integral part of the capacitative Ca2+ influx pathway in the trophocyte.

Acknowledgements This study was supported by a Research Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) to J.E.S.

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