© 2001 by The Society for Integrative and Comparative Biology
Further Studies on Signaling Pathways for Ecdysteroidogenesis in Crustacean Y-Organs1
1 Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
2 Department of Biology, Kenyon College, Gambier, Ohio 43022
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The Y-organs of crustaceans secrete ecdysteroids (molting hormones) and are regulated (negatively) by a neurosecretory peptide, molt-inhibiting hormone (MIH). Signaling path(s) in Y-organs were explored that connect MIH receptors ultimately with suppression of receptor number for the uptake of cholesterol (ecdysteroid precursor) and of gene expression of steroidogenic enzymes. Experiments were conducted in vitro with Y-organs of crabs (Cancer antennarius, Menippe mercenaria) and crayfishes (Orconectes sp.). It was confirmed in all species that steroidogenesis occurs in the absence of external calcium (Ca++), but increases to a maximum as Ca++ is increased to 1 to 10 mM and is substantially inhibited at higher Ca++ concentrations. MIH does not require external Ca++ for inhibitory action, but inhibition is eliminated by high Ca++concentrations. Several experimental approaches failed to find evidence of phospholipase C activation, turnover of inositol triphosphate or diacylglycerol generation connected with steroidogenesis. Unbinding or chelation of intracellular Ca++ with thapsigargin or TMB-8, respectively both caused dose-dependent inhibition of ecdysteroid output. Blockade of Ca++ channels with verapamil, nifedipine or nicardipine also inhibited steroidogenesis; highest doses inhibited profoundly to below Ca++-free basal levels. Inhibition also was obtained with all doses of the Ca++ channel agonist/antagonist (–) BAY K 8644 in crabs, but in crayfishes lower doses were stimulatory. However, if the crayfish cells were depolarized, allowing greater Ca++ influx, the previously stimulatory doses of BAY K 8644 became inhibitory. Y-organ protein kinase C (PKC) is Ca++-sensitive. Activation of PKC was uniformly stimulatory in crabs, but inhibitory in crayfishes. Cytochalasin D, which disrupts the actin cytoskeleton, and which causes moderate Ca++ influx, stimulated hormone formation. These results are interpreted to indicate a regulatory role for Ca++ in ecdysteroidogenesis, involving a local, submembrane circulation of Ca++ through ion channels and Ca++ pumps and interaction with PKC in phosphorylating key proteins. An optimal local Ca++ environment fostering hormone synthesis is evident since too little or too much Ca++ is inhibitory.
Methyl farnesoate (MF) had no effect on ecdysone production in crab or crayfish Y-organs in 24-hr incubations with MF at 100 pM to 10 µM.
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INTRODUCTION |
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The ecdysteroid hormones of crustaceans directly control molting cycles; they are secreted by a pair of glands near the ventral carapace. Secretion is regulated by a defined neurosecretory peptide from the eyestalks, molt-inhibiting hormone (MIH). Both in vivo and in vitro experiments have shown that MIH regulates negatively, a feature that is unique among steroidogenic systems so far known in animals (reviews: Skinner, 1985
; Fingerman, 1987; Watson et al., 1989; Spaziani, 1990; Chang, 1993; Lachaise et al., 1993). Supporting evidence includes the finding that Y-organ activation and inhibition varies inversely with MIH mRNA levels in the eyestalks during the molting cycle (Lee et al., 1998; Watson et al., 2000).
Occupancy by MIH of its receptor in the Y-organ cell membrane (Webster, 1993) activates a signaling system that directs inhibition of cellular uptake of cholesterol (the ecdysteroid precursor), protein synthesis and the expression of steroidogenic enzymes. An objective of this laboratory is to uncover the components of the signaling system, an objective that is shared by laboratories that attempt to understand the cellular process of steroid hormone formation and its control, in arthropods and in vertebrates. Progress in this field is the subject of a recent review (Spaziani et al., 1999) in which signaling systems so far known in vertebrate steroidogenic glands (ovaries, testes, adrenal cortex), insect prothoracic glands and crustacean Y-organs are compared. A highly conserved feature is that the cells of all glands respond to their respective, regulatory tropic hormone with an increase in cyclic nucleotide. Crustaceans are unique in that the rise in cAMP (or cGMP) shuts down the cells. In the previous article (Spaziani et al., 1999) evidence was reviewed that cAMP inhibits cholesterol uptake in part by lowering the number of receptor sites for cholesterol-carrying lipoprotein, and inhibits de novo protein synthesis. A role for calcium (Ca++) also was indicated in that Ca++-calmodulin stimulates ecdysteroid production by lowering cAMP levels. Calcium also interacts with Y-organ protein kinase C, the stimulation of which fosters ecdysteroid production. Finally, it was reported that protein tyrosine kinases (PTK) are present; inhibitors of PTK activity depress Y-organ function. The present article focuses on Ca++ as second messenger and presents evidence of plasma membrane-associated Ca++ activity involving PKC and voltage-gated ion channels. Comparative data on crabs and crayfishes are included.
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Animals
Crayfish were males of Orconectes immunis or O. rusticus obtained from a commercial hatchery. The animals were held in fresh-water tanks at 6°C in the dark and fed fish or shrimp feed pellets every two weeks. Animals in intermolt stages (showing firm carapaces and the absence of apolysis by microscopic examination of tails) were acclimated to 18°C. Eyestalks were then bilaterally removed (removal of molt-inhibiting hormone, MIH) and the sockets allowed to clot in air. They were held in compartmentalized steel tanks at 18°C and fed once per day. They were used three to five days after ablation, by which time the animals had entered premolt; the Y-Organs were activated (Jegla et al., 1983
) as indicated by significantly elevated levels of hemolymph ecdysteroids and the presence of apolysis.
Rock crabs, Cancer antennarius, were obtained from Marinus, Inc. (Long Beach, CA), and stone crabs, Menippe mercenaria, from Gulf Specimen Co. (Panacea, FL). They were maintained in aerated, charcoal-filtered, reconstituted sea water at 18°C, and on a 12 hr light/dark cycle. All crabs were acclimated at least four days prior to use, and were fed pieces of fish every other day. The organ donors were crabs in the intermolt stage of the molting cycle or those in premolt. Intermolt was determined by the presence of a dense carapace; crabs with very thin, compressible carapaces (indicating postmolt to very early intermolt), and those found with thin-brittle carapaces and/or leathery epidermis (stages of premolt), were not used. For premolt, animals were induced into that stage by excising the eyestalks two days prior to use. This procedure activates the Y-organs into hypersecretion of ecdysteroids (Chang et al., 1976; Watson and Spaziani, 1985a).
Materials
From Sigma Chemical Co. (St. Louis, MO): The protein kinase C (PKC) activators, phorbol 12-myristate, 13-acetate (PMA; also from Calbiochem, La Jolla, CA) and phorbol 12,13-dibutyrate (PDB); the PKC inactive phorbol, 4
-phorbol 12,13-didecanoate (PDD); the diacylglycerols, sn-1,2-dioctanoyl glycerol (DOG), 1-oleoyl-2-acetyl-glycerol (OAG) and diolein (DO); dimethyl sulfoxide (DMSO); the voltage-gated calcium channel blockers, verapamil, nefardipine and nicardipine; the intracellular calcium chelator, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, HCL (TMB-8); the calcium channel agonist, (–) S-BAY K 8644, and the inhibitor of intracellular calcium-ATPase pumps, thapsigargin. From Calbiochem (La Jolla, CA): Thapsigargin (see above); the inhibitor of cyclic nucleotide-dependent protein kinases, H-7; the calcium antagonist and inhibitor of cyclic nucleotide-and calmodulin-dependent-protein kinases, HA 1004; the protein kinase C (PKC) inhibitor, calphostin C; the phospholipase C (PLC) inhibitor, U-73122, [1-(6-((17ß-3-methoxyestra-1,3,5 (10)-trien-17-yl) amino) hexyl)-1 H-pyrrole-2,5-dione] and its inactive analog, U-73343, [1-(6-((17ß-3-methoxyestra-1,3,5 (10)-trien-17-yl) amino) hexyl)-2,5-pyrrolidine-dione] (these compounds also were purchased from Biomol, Plymouth Meeting, PA); the actin filament inhibitor, cytochalasin D.
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-32P]ATP (3,000 Ci/mmol) was purchased from Amersham Corp. (Arlington Heights, IL); [3H]ecdysone (60 Ci/mmol) was from New England Nuclear (Bedford, MA) and ecdysteroid standards were from Research Plus (Bayonne, NJ). Ecdysone antiserum (antibody H-21-B, Horn et al., 1976) was a gift from Dr. W.E. Bollenbacher (Dept. of Biology, University of North Carolina, Chapel Hill, NC). Methyl farnesoate (MF) was a gift from Dr. Hans Laufer (Dept. of Molecular and Cellular Biology, University of Connecticut, Storrs, CT).
Molt-inhibiting hormone (MIH) was prepared as an eyestalk extract as previously described (Mattson and Spaziani, 1985a).
Y-organ incubations and experimental treatments
Crayfish
Activated Y-organs were collected in cold Van Harreveld's crayfish Ringer solution (Van Harreveld, 1936); the standard medium contained 10 mM calcium. They were then preincubated, one Y-organ per well, for 30 min in a 96-well culture plate, each well containing 100 µl of Van Harreveld's solution and experimental drug. Following preincubation, the glands were placed in 100 µl of fresh experimental medium and incubated for 24 hr in the dark in a humidified chamber. Verapamil, thapsigargin, PMA, DOG, TMB-8, calphostin C and MF were diluted to appropriate concentration with the crayfish saline containing 1.0% DMSO; in some experiments, verapamil and PMA were in the medium with 0.01% ethanol. The drugs H-7 and HA 1004 were dissolved in dH2O. All control incubations contained the corresponding solvent.
Crabs
Activated Y-organs were collected in cold Pantin's crustacean saline (Pantin, 1934), cut in half or quarters, depending upon experiment; the pieces were randomly assigned to control or experimental wells in 24-well culture plates. A well contained a single quarter (for ecdysteroid RIA in the medium) or a half (for measurement, by high-performance liquid chromatography [HPLC] of specific ecdysteroid secretion into the medium). The pieces were incubated for the indicated periods in Pantin's saline, adjusted to 1 mM calcium, and supplemented 10% with crab serum and antibiotics (for details see Watson and Spaziani, 1985a) or in calcium-free medium that included EGTA (0.1 mM). All incubations were in 0.5 ml of medium, at room temperature, with slow rotary shaking in a humidified atmosphere of high oxygen tension: 95% air/O2 (50:50 by volume) and 5% CO2.
The experimental agents were dissolved in 95% ethanol and added to incubations such that the alcohol concentration did not exceed 2%. Ethanol alone at this dilution did not affect steroidogenesis or tissue cAMP levels. (It should be noted that suppliers of several of the drugs recommend DMSO among solvents for dissolving and applying these agents. We found that DMSO alone profoundly inhibited steroidogenesis in Y-organs of Menippe; DMSO at 2% in the medium inhibited Y-organs to 12% of control levels, at 6% concentration inhibited to 5% of controls. However, DMSO did not inhibit crafish Y-organs.)
Ecdysteroid measurement
Radioimmunoassay (RIA)
Measurements were of total ecdysteroids secreted by Y-organs into incubation media. For crab studies the method used was described by Mattson and Spaziani (1985a); for crayfish, the method of Borst and O'Connor (1972) was employed. Since Y-organs in vitro secrete more than one ecdysteroid (Watson and Spaziani, 1985b; Spaziani et al., 1989), assayed levels are expressed as ecdysone equivalents. Ecdysteroid values were normalized as amount per Y-organ or as a percent of control.
High-performance liquid chromatography (HPLC)
In most experiments with crabs, HPLC was used to measure specifically the major ecdysteroid secretion of crustacean Y-organs, 3-dehydroecdysone (3DE). Following incubations, pooled media samples were passed through Sep Pak C-18 chromatographic cartridges (Waters; Milford, MA) to isolate ecdysteroids (Watson and Spaziani, 1982). The Sep Pak eluant volume was reduced to dryness with rotary evaporation under vacuum and dissolved in 35% methanol for HPLC analysis. 3DE was isolated with a Nova-Pak (Waters) C-18 reversed-phase HPLC column, using a 35–70% methanol gradient (Rudolph and Spaziani, 1992), and measured by integration of peaks.
Inositol triphosphate (IP3) and diacylglycerol (DAG) measurements
Y-organs from de-eyestalked and intact crabs were compared. For IP3 determinations, Y-organs were collected and directly homogenized in ice-cold 20% perchloric acid. After centrifugation, IP3 was determined in the supernatant, employing the D-myo-inositol 1,4,5-triphosphate [3H] immunoassay kit and protocol (Biotrak TRK 1000) from Amersham Life Sciences (Arlington Heights, IL). DAG was determined by the procedures of Preiss et al. (1986). In brief, glands were extracted with chloroform/methanol. An aliquot was taken to determine total lipid phosphorus; DAG in the remainder was purified by thin-layer chromatography (TLC). The purified DAG was analyzed for total mass by measuring its conversion to [32P]phosphatidic acid in an incubation that included diacylglycerol kinase and [32P]ATP. Labeled phosphatidic acid was isolated by TLC, identified by autoradiography and measured by scintillation counting.
Cell permeabilization and added IP3
Y-organ halves or quarters from de-eyestalked and intact crabs were permeabilized by incubating 2 min in Pantin's saline containing 0.1 mg/ml saponin (Geras-Raaka and Gershengorn, 1987). The tissues were then washed and transferred to standard serum-supplemented medium containing 10 µM 1,5,6-IP3 or 4,5,6-IP3 and incubated for 6 hr to determine any effect of added IP3 on ecdysteroid output. Three kinds of controls were tested: those preincubated with saponin, but without IP3 in the subsequent incubation; those preincubated without saponin, but with subsequent IP3; those incubated in both intervals with neither saponin nor IP3. Permeabilization was monitored by observing trypan blue penetration. The two IP3 variants were isolated and purified by the methods of Hull et al. (1999).
Statistics
Results were assessed statistically by one-way analysis of variance and Student's t-test for post hoc tests.
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Effects of external calcium concentration on ecdysteroidogenesis
The principal basis for interpretation of experiments (further below), that examine the effects of perturbing cellular calcium (Ca++) concentration on Y-organ steroidogenesis, are measurements of ecdysteroid output as affected by changing extracellular Ca++ concentration in vitro (Fig. 1). In the absence of external Ca, a substantial basal secretion occurs in Y-organs of crabs (Cancer antennarius) and crayfishes (Orconectes rusticus and O. immunis) (Fig. 1). The secretion by crayfish glands is shown to be lower than in crabs as the glands are much smaller and the data are expressed as amount secreted per Y-organ. With increasing extracellular Ca++, secretion increases to a maximum at 1 mM Ca++ in crabs and 10 mM in crayfishes (see also results of Mattson and Spaziani, 1986
). Higher Ca++ consistently depresses secretion. Accordingly, we routinely adjust Pantin's crustacean saline to 1 mM Ca++ for incubations of crab glands. Both Pantin's (1934) and Van Harreveld's (1936) crustacean formulations contain Ca++ at 10 mM based on an average found for total hemolymph calcium at intermolt (v. Greenaway, 1985). However, it should be noted that only about 50% of total hemolymph calcium is in ionic (unbound) form and therefore physiologically effective. Addition of molt-inhibiting hormone (MIH) lowers the basal secretion significantly in the absence of Ca++ (Fig. 1). Thus, the inhibiting activity of MIH does not require Ca++. Raising external Ca++ above 1 mM eliminates MIH inhibition (see also Mattson and Spaziani, 1986). This suggests that excess Ca++ interferes with the binding of MIH to its receptor.
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Does a phospholipase C/IP3/diacylglycerol signaling system operate in regulating Y-organ ecdysteroidogenesis?
Due to uniformly negative results, the data on the following experiments are not shown. The phospholipase C (PLC) inhibitor U-73122 (Yule and Williams, 1992
) did not significantly affect ecdysteroid production in vitro over the dosage range, 10–4 to 10–9 M, in crab and crayfish glands. Direct extraction of inositol triphosphates (IP3) from crab Y-organs showed no differences in content between glands from intact and de-eyestalked crabs. The same was found for diacylglycerol (DAG) content. Y-organs, from both intact and de-eyestalked crabs, were permeabilized with saponin and tested for effects of adding purified IP3 isomers. In six-hr incubations, neither 1,5,6-IP3 nor 4,5,6-IP3 (Hull et al., 1999), 10 µM, effected any significant change in ecdysteroid output beyond saponin-treated controls. In a control experiment, saponin alone (0.1 mg/ml) reduced output 30% below non-saponin controls. We conclude that a PLC-linked receptor system of signaling is not involved in regulating Y-organ ecdysteroid production.
Release and binding of intracellular calcium
The probability was explored that increases of intracellular calcium may have a regulatory role, if not from release by IP3 then perhaps from greater influx of external Ca++ (see Fig. 1). Thapsigargin was applied to release intracellular calcium (Fig. 2). The drug inhibits Calcium-ATPase linked to pumping Ca++ into repositories, principally the smooth endoplasmic reticulum (Thastrup et al., 1990). Somewhat surprisingly, thapsigargin caused significant dose-dependent inhibition of ecdysteroid production in Orconectes; inhibition also was observed in the crab, Menippe although it was not cleanly dose-dependent (Fig. 2). Ecdysteroid output was measured in incubations with crayfish Y-organs and the intracellular Ca++ chelator, TMB-8 (Fig. 3). A dose dependent depression of output was observed, both in the presence and absence of external Ca++. However, another intracellular Ca++ chelator, BAPT-AM had no effect in the same species and dosage range (data not shown). Thus, inhibition occurred both upon increasing Ca++ from within and of binding intracellular Ca++. In view of results in Figure 1, these outcomes could have resulted from too much intracellular Ca++ in the first instance and not enough in the second.
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Effects of calcium channel agonists and antagonists
Blockade of L-type voltage-gated Ca++ channels with nifedipine and verapamil led to dose-dependent inhibition of ecdysteroid production in two species of crayfish (Fig. 4). Similar results were obtained with nifedipine and nicardipine in crab Y-organs. In the crab, nifedipine also was an effective steroid inhibitor in the absence of external Ca++ (Fig. 5). It is noted that inhibition at the higher doses of these drugs (100 µM to 1 mM) was profound in all species, lowering ecdysteroid output well below the basal levels seen without external Ca++ in Figure 1. In crayfishes, flunarizine (10 µM to 1 mM), a specific inhibitor of n-type Ca++channels, had no effect (data not shown). Interestingly in crayfishes, Y-organ cells are inhibited below 50% if they are depolarized with saline containing 50 mM KCl. If verapamil is added, in doses that formerly had no effect or were inhibiting (0.1 to 10 µM), verapamil now becomes distinctly stimulatory (data not shown). These data suggest that depolarization alone causes depressing levels of Ca++ to enter cells, but in combination with the Ca++ channel blocker, permits just enough Ca++ influx to stimulate ecdysteroid production. Generally, the signaling system for steroidogenesis appears to require a membrane-local Ca++ circulation that is in delicate balance between influx and efflux.
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The negative enantiomer of the Ca++ channel effector, BAY K 8644 is a channel agonist, where as the mixed enantiomers are antagonists in many systems (Ferrante et al., 1989
). However, in both intact and eyestalkless crabs, M. mercenaria (–) BAY K 8644 caused a dose-responsive depression of ecdysteroid production, with profound inhibition at 50 µM (Fig. 6). At the latter dose, the drug also was effectively inhibitory in the absence of external Ca++ (Fig. 6). The same compound in crayfish Y-organs, however, showed a bimodal response with dose (Fig. 7). Concentrations up to 1 µM significantly stimulated ecdysteroid production, whereas higher doses to 1 mM were inhibitory. However, when the cells were depolarized with 50 mM KCl, the doses of BAY K 8644 that had been stimulatory were now inhibitory (Fig. 8). These results are consistent with our interpretation that small amounts of Ca++ influx are stimulatory, whereas augmenting Ca++ entry (in this case by depolarization) depresses steroidogenesis.
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The role of protein kinase C (PKC)
Previous work demonstrated that Y-organ membranes of the crab, Cancer antennarius contain PKC activity that was directly shown to be activated by phorbol esters. Moreover, the phorbols stimulated ecdysteroid production, and did so without affecting levels of cAMP, nor did they affect the rise in cAMP due to MIH (Mattson and Spaziani, 1987
). Data on the effects of phorbols and of diacylglycerols (DG) are summarized in Table. 1. The phorbol, PMA increased ecdysteroidogenesis dose-dependently in crabs. The phorbol, PDB at 100 µM also was stimulatory, whereas the PKC-inactive phorbol, PDD was mildly inhibitory. Consistent with these results, the synthetic DG, OAG stimulated ecdysteroid output, but the DG, diolein (DO) that is inactive on PKC had no significant effect on the Y-organs (Table 1). However, the natural DG, DOG was inactive in crabs.
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In contrast with these results in crabs, a range of PMA doses was distinctly inhibitory in the crayfish, O. immunis. Similarly, in O. virilis the DGs, DOG and OAG were inhibitory (Table 1). These actions having to do with PKC were the only effects that were consistently different between crabs and crayfishes in our comparative studies. Otherwise, the results were mixed in crayfishes. The PKC inhibitors H-7 and calphostin were without effect over a considerable dosage range, as was DOG, in O. immunis and O. rusticus, respectively. Moreover, the phorbol PMA that was demonstrably inhibitory in O. immunis, was without effect on Y-organs of O. rusticus over the same range (Table 1).
Miscellaneous effectors
Cytochalasin D disrupts actin filaments in cells. The effects of this agent were observed in the present studies because disrupting the cytoskeleton in vertebrate steroidogenic glands has been reported generally to inhibit the glands. In M. mercenaria, however, a dose-dependent stimulation was seen (Fig. 9). The stimulation may be attributed in part to the observation that cytochalasin D mildly increases cellular calcium (Aszalos et al., 1994).
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Methyl farnesoate (MF) has been reported to stimulate Y-organs of the crab, C. magister, at 1 and 10 µM, over 24 to 72 hr of incubation (Tamone and Chang, 1993
). In our hands, MF had no effect over 24 hr in O. immunis Y-organs in the range 100 pM to 1 µM, nor in the glands of M. menippe at 10 µM (data not shown; see also unpublished negative findings for Carcinus maenas and Cancer pagarus reported by Smith et al., 2000).
General
These data together with other information recently reported and reviewed on Y-organ signaling (Spaziani et al., 1999) indicate that ecdysteroidogenesis in crustaceans is cAMP (or cGMP)-driven and does not involve PLC nor IP3. The cellular level of cyclic nucleotide is inversely related to degree of steroid output; the sole established hormonal controller of the Y-organs (MIH) regulates negatively by raising cyclic nucleotide concentrations (Mattson and Spaziani, 1985b).
The evidence is convincing that PKC is present and linked to steroid output, but is independent of the cyclic nucleotide signaling system. In connection with steroidogenesis, the natural activator of Y-organ PKC is not established, but in crabs at least the evidence strongly suggests that it is Ca++ rather than an endogenous DG. For example, PKC activity could not be stimulated by phorbols in the absence of Ca++ (Mattson and Spaziani, 1987). Lanthanum, which blocks Ca++ channels, inhibited ecdysteroid formation and this effect could not be overcome with treatment with the phorbol, PMA. Similarly, trifluoperizine (TFP, which blocks Ca++-calmodulin and secondarily PKC, Mattson and Spaziani, 1986) profoundly inhibited ecdysteroid production; simultaneous treatment with PMA did not overcome the effect of TFP alone (Mattson and Spaziani, 1987). While PKC is activated by some synthetic diacylglycerols, there is little evidence that an endogenous DG acts as a signal in this system. Thus in the present studies we could not find changes in Y-organ DG content after de-eyestalking. The natural DG, DOG was effective in only one of three species tested. (Table 1).
Thus it appears that PKC is importantly sensitive to Ca++ and that Ca++ via PKC is involved in regulating ecdysteroid output. Moreover, the control by Ca++ is delicately balanced in that Ca++ seems to be basically stimulatory or supportive of greater output, but too much or not enough of the ion is inhibitory. Thus Ca++ was stimulatory up to an external Ca++ concentration of 1 or 10 mM (species-dependent), but higher amounts in all cases were distinctly inhibitory. Intracellular Ca++ releasers or chelators were inhibitory (Figs. 2, 3), but a Ca++ ionophore was stimulatory in the absence of external Ca++ (Mattson and Spaziani, 1986). Voltage-gated Ca++ channel blockers were profoundly inhibitory at higher doses, but the Ca++ channel agonist/antagonist Bay K 8644 could enhance ecdysteroid output at lower doses and depress at higher. Also relevant is the observation that Y-organ inhibition by MIH is accompanied by increased Ca++ efflux (Mattson and Spaziani, 1986).
Based upon all the observations, it is our hypothesis that some form of juxtamembrane cycling of Ca++ occurs in Y-organs as part of a signaling system (Alkan and Rasmussin, 1988). Ca++ influx through voltage-gated Ca++ channels is coupled with Ca++ pumps that remove cellular Ca++. The extruded Ca++ accumulating near the channels re-enters, etc. The Ca++ as messenger occurs when an increase in local cycling results in a transient net increase in cytosolic Ca++ near the plasma membrane. This activates membrane-associated PKC which increases the rate at which PKC phosphorylates other proteins, thereby mediating a sustained cellular response. The response in crabs is increased ecdysteroid production. In crayfishes activation of PKC is inhibitory. In crayfishes it is probable that the relevant proteins are inactivated when phosphorylated by PKC. Part of the mechanism in crabs at least is that Ca++ combines with calmodulin, activating cyclic nucleotide phosphodiesterase, resulting in a decrease in cAMP (Mattson and Spaziani, 1986). The net effect is sustained ecdysteroid hormone production by at least two mechanisms activated by Ca++.
Depression of hormone production in crustacean species is usually triggered by MIH via cAMP (Mattson and Spaziani, 1985c; Böcking and Sedlmeier, 1994) (or cGMP; Sedlmeier and Fenrich, 1993; Von Gliscynski and Sedlmeier, 1993). The cyclic nucleotide would (a) activate kinases that deactivate enzymes through phosphorylation, and (b) increase Ca++ efflux which would decrease the rate of cAMP degradation (Mattson and Spaziani, 1986). This scenario would lead to a sustained response in the negative direction. However, large Ca++ influxes, or release from intracellular sites, such as employed experimentally in the present studies, could have the same effect. The Ca++ pumps would be overwhelmed, leading to a large increase in intracellular Ca++ concentration and inhibition of steroidogenesis as we have observed experimentally. The mechanism remains to be uncovered. A similar condition would exist close to the molt, at which time total hemolymph calcium rises dramatically as an animal reabsorbs carapace minerals (Greenaway, 1985). The rise in calcium coincides with, and may be the cause of, a sharp drop in ecdysteroid titer, ultimately to the basal levels of intermolt.
Clearly, the system is complex. A requisite next step in attempting to refine understanding of the mechanisms that control hormone production in Y-organs should be to deploy methods that visualize the amounts and cellular distributions of Ca++ and of PKC, as a function of adding MIH and of manipulating extracellular Ca++ concentration. Studies that directly measure cellular Ca++ have been initiated in insect prothoracic glands, as influenced by the controlling peptide, prothoracicotropic hormone (PTTH). The results thus far (Birkenbeil, 1996, 2000) are generally consistent with the hypothesis outlined above: The action of PTTH on ecdysteroid production depends intrinsically upon the Ca++ that is transported across the plasma membrane rather than that released from intracellular stores.
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ACKNOWLEDGMENTS |
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The authors wish to acknowledge with thanks the services provided by Dr. Mark Yorek and the University of Iowa Diabetes and Endocrinology Research Center, NIH grant DK25295, for analyses of inositol triphosphate and diacylglycerol.
This work was supported by successive grants IBN 9221790 and 9603547 (to E.S.) from the National Science Foundation. Upon his imminent retirement, one of us (E.S.) wishes to express his appreciation to the NSF staffs for their consistently dedicated, fair and conscientious service in a difficult job.
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FOOTNOTES |
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1 From the Symposium Recent Progress in Crustacean Endocrinology: A Symposium in Honor of Milton Fingerman presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
2 To whom correspondence should be addressed. E-mail: eugene-spaziani{at}uiowa.edu
3 Address: Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
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References |
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