INTRODUCTION

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THE FRESHWATER CRAYFISH has emerged as a unique model organism for studying the transmembrane Ca²⁺ transfer processes fundamental to Ca²⁺ homeostasis in all eukaryotic organisms (28). During the crustacean molting cycle, cuticular CaCO₃ is alternately reabsorbed in premolt (negative Ca²⁺ balance) and then deposited in postmolt (positive Ca²⁺ balance) in an environment that is Ca²⁺ restricted (Ca²⁺ < 1 mM). Correspondingly, directionality (out/in) and magnitude of Ca²⁺ transfer can vary significantly during the molting cycle at the primary Ca²⁺-transporting epithelia, which are: gills (passive diffusional loss/active uptake), hepatopancreas (midgut gland analogous to liver; lumenal Ca²⁺ storage/uptake via food and drink), antennal gland (analogous to kidney; filtration/reabsorption), and the cuticular hypodermis (demineralization/mineralization).

Existing models for crustacean transepithelial Ca²⁺ transfer (28) have been derived from marine species [gill of the shore crab, Carcinus maenas (8); hepatopancreas of the lobster, Homarus americanus (2, 32, 33)]. Apical uptake of Ca²⁺ is largely passive, involving channels and electroneutral/electrogenic Ca²⁺/Na⁺ exchangers. Active transfer against an electrochemical gradient across the basolateral membrane into the hemolymph involves both a Ca²⁺ pump (Ca²⁺ ATPase) and exchanger (Ca²⁺/Na⁺). The electrochemical gradient for Na⁺ established by Na⁺-K⁺-ATPase provides energy for Ca²⁺ extrusion via the exchanger. Marine crustaceans maintain extracellular (EC) Ca²⁺ isoionically (14), produce an isosmotic urine, and absorb Ca²⁺ passively from the external seawater (SW; Ca²⁺, 10 mM) in postmolt (19).

By contrast, the intermolt freshwater (FW) crayfish hyperionically regulates EC Ca²⁺ primarily by postfiltrationally reabsorbing Ca²⁺ (30). Postmolt mineralization involves activation of branchial uptake mechanisms (13, 29) and possibly reingestion of the cast exuviate. In premolt, CaCO₃ is stored in the lumen of the cardiac stomach in the form of gastroliths that are reabsorbed rapidly in postmolt. In summary, the crayfish has evolved ionoregulatory adaptations enabling it to maintain Ca²⁺ homeostasis in FW; as such, it provides an ideal model for delineating Ca²⁺ transfer mechanisms in gills, hepatopancreas, and antennal gland.

In an earlier paper, we presented methodology for isolation of basolateral membrane vesicles (BLMV) in the inside-out orientation (IOV) from gills, hepatopancreas, and antennal gland of intermolt crayfish (31); osmotic reactivity was demonstrated, and nonspecific Ca²⁺ binding was determined. In the present paper, the vesicle preparations are used to characterize ATP-dependent Ca²⁺ uptake during intermolt. The relative role of each Ca²⁺-transporting epithelium in whole organism Ca²⁺ homeostasis during intermolt is discussed. This study will form the framework for future studies on ATP-dependent Ca²⁺ uptake in pre- and postmolt stages of the molting cycle.

ATP-dependent Ca²⁺ uptake has been partially characterized in two prior studies using marine crustacean vesicles. Flik et al. (8) examined ATP-dependent Ca²⁺ uptake into gill plasma membranes of the shore crab, C. maenas, determining K_m and maximal flux (J_max) and the effect of a Ca²⁺ ionophore. Zhuang and Ahearn (33) examined Ca²⁺ uptake into BLMV from hepatopancreas of lobster, H. americanus, describing the effects of a Ca²⁺ ionophore, EGTA, and vanadate and characterizing pH optima as well as kinetics. The ATP-dependent Ca²⁺ pump has not been characterized in any epithelium of a FW crustacean or in the antennal gland of any crustacean.

The ATP-dependent Ca²⁺ pump has been well characterized in epithelial BLMV of lower and higher vertebrates, including gills of FW eel, Anguilla rostrata (9, 5); gills of FW trout, Salmo gairdneri (20); gills of FW- and SW-adapted tilapia, Oreochromis mossambicus (26); intestine of FW tilapia, O. mossambicus, (6); gill and gut of FW tilapia (7); rat intestine (25); and rat kidney (10, 24).

The ATP-dependent Ca²⁺ uptake into BLMVs is equivalent to the basolateral Ca²⁺ pump, technically referred to as plasma membrane Ca²⁺ ATPase (PMCA), which is found in all eukaryotic cells. The cell physiology and molecular biology of PMCA have been reviewed (4). The high affinity of the pump implicates it in fine tuning of cytosolic free Ca²⁺ as well as Ca²⁺ ejection from cells. PMCA is a phosphorylated "P-type" ATPase that is regulated by calmodulin and inhibited by orthovanadate.

	MATERIALS AND METHODS

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Crayfish (Procambarus clarkii Girard) were obtained from Carolina Biological Supply and maintained in 40-liter aquariums in filtered aerated water at 21°C. BLMV were isolated from gill, hepatopancreas, and antennal gland pooled from six intermolt crayfish, as recently outlined (31). Basolateral enrichment, vesicle resealing and orientation, osmotic reactivity, and nonspecific Ca²⁺ binding are provided in the earlier study. All ATP-dependent Ca²⁺ uptake experiments were performed using fresh vesicles (within 24 h of isolation) at 37°C, the temperature at which maximal uptake rate was observed (see below).

ATP-dependent Ca²⁺ uptake. A rapid filtration procedure was used to determine ⁴⁵Ca²⁺ uptake into IOVs using cytoplasmic ATP- and Ca²⁺-binding sites (1, 2, 8, 24, 32). Vesicles suspended in isotonic sucrose buffer (31) were pelleted at 80,000 rpm in a Beckman airfuge. They were then resuspended by gentle pipetting and loaded (1 h on ice) with an intravesicular medium (IV) containing 100 mM KCl, 5 mM MgCl₂, and 25 mM HEPES-Tris at pH 7.4. The K⁺ ionophore valinomycin (50 µM) was added, which, together with equivalent concentrations of K⁺ on either side of the vesicular membrane, served to short-circuit membrane potential generated by exchanger activity. Ten minutes before the experiment, vesicles and extravesicular medium (EV, see below) were brought to experimental temperature in a water bath.

Time dependence of ATP-dependent and ATP-independent ⁴⁵Ca²⁺ uptake. Both ATP-dependent and ATP-independent ⁴⁵Ca²⁺ uptake into IOVs was assessed at incubation times ranging up to 60 min. In subsequent experiments, initial velocity conditions were measured at time 1 min.

Effects of Ca²⁺ ionophore (A-23187) and vanadate. In control experiments, ⁴⁵Ca²⁺ uptake was assessed over 20 min in either the presence or absence of ATP. In a separate experiment, the ionophore A-23187 (10 µg/ml; stock made up in DMSO) was added together with ATP either at the start of the experiment (t = 0) or 10 min after ⁴⁵Ca²⁺ loading had commenced (8). In both cases, uptake was monitored for a total of 20 min. In a separate experiment, ATP was added at the start of the experiment together with orthovanadate (0.1 mM, Na₃VO₄), an ATPase inhibitor that binds with the ATP hydrolyzing site (3).

Free [Ca²⁺] dependence: Kinetics. Free Ca²⁺ concentration ([Ca²⁺]) was varied among 0.05, 0.1, 0.2, 0.3, 0.5, 1.0, 5.0, and 10 µM (calculations made with Chelator program; see Ref. 22) in EV medium, and initial rate (1 min) of ⁴⁵Ca²⁺ uptake into BLMV was assessed. In subsequent experiments, free Ca²⁺ in EV was held at 5 µM.

Substrate (ATP) dependence. The ATP concentration was varied among 0, 0.01, 0.05, 0.1, 1, and 5 mM in EV medium, and initial rate (1 min) of ATP-dependent ⁴⁵Ca²⁺ uptake into BLMV was measured (23). In subsequent experiments, ATP concentration in EV medium was held at 5 mM.

Temperature dependence. The ATP-dependent ⁴⁵Ca²⁺ uptake was measured at three experimental temperatures that span the range experienced by crayfish in the natural environment: 12, 19, and 39°C.

Calmodulin dependence. Initial (1 min) ATP-dependent ⁴⁵Ca²⁺ uptake was determined in untreated vesicles (control). Uptake was then assessed after pretreatment with EGTA (15 min at 37°C; see Ref. 9). Vesicles were pelleted and then resuspended in 25 mM HEPES-Tris (7.4), 100 mM KCl, and 5 mM MgCl₂, with 5 mM EGTA. Vesicles were then pelleted and washed two times in basic assay medium (less ATP and Ca²⁺ buffer system). Uptake was then reassessed in EGTA-treated vesicles. The effect of calmodulin (0.625 µM, origin bovine testicle) was assessed in a parallel group of EGTA-treated vesicles and compared with the effect on EGTA-untreated vesicles.

Na⁺ dependence. Initial (1 min) ATP-dependent ⁴⁵Ca²⁺ uptake was assessed in EV (control, 0 mM Na) both in the presence and absence of ouabain, a known inhibitor of the Na⁺ pump. In a parallel group of vesicles, the effect of addition of 5 mM Na to the EV was assessed both in the presence and absence of ouabain. Preincubation with 1 mM ouabain occurred for 2 h on ice because the ouabain binding site of Na⁺-K⁺-ATPase is directed toward the vesicular lumen in IOVs (24).

Statistical analysis. Each experiment was repeated three to six times using vesicles prepared on different days from tissue pooled from six animals. Values are expressed as means ± SE. Statistical analysis of the data was performed using paired or unpaired Student's t-test; significance was accepted when P < 0.05.

	RESULTS

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Time dependence of ATP-dependent and ATP-independent ⁴⁵Ca²⁺ uptake. The ATP-dependent and ATP-independent uptake of ⁴⁵Ca²⁺ into IOVs of gill, hepatopancreas, and antennal gland are illustrated in Fig. 1, A-C. In the absence of ATP, ⁴⁵Ca²⁺ uptake followed a slow hyperbolic function of time. In each case, the presence of ATP significantly increased ⁴⁵Ca²⁺ accumulation compared with ATP-independent uptake. Uptake reached a plateau in each case by 15-20 min, with ATP-dependent ⁴⁵Ca²⁺ uptake exceeding ATP-independent uptake by five- to sixfold. A linear increase in ⁴⁵Ca²⁺ uptake was observed for the first 5 min of incubation in gill and the first 10-15 min of incubation in hepatopancreas and antennal gland. Maximal uptake is limited by ATP depletion in the reaction vial; accordingly, the plateau for uptake in antennal gland was higher than in gill or hepatopancreas due to lower protein content in the reaction vial. The mean initial rate of ⁴⁵Ca²⁺ uptake (first 1 min) at 37°C was calculated to be 106.4, 28.8, and 92.0 pmol · mg¹ · min¹ in gill, hepatopancreas, and antennal gland, respectively. In preliminary experiments, we confirmed that ⁴⁵Ca²⁺ uptake was promoted only by ATP and not by either ADP or the nonhydrolyzable ATP analog 5-adenylylimidodiphosphate.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of ATP (5 mM) on 45Ca2+ uptake at 37°C by short-circuited crayfish basolateral membrane vesicles (BLMV) from gill (A), hepatopancreas (B), and antennal gland (C). Vesicles were preincubated with intravesicular medium (IV) containing 100 mM KCl, 5 mM MgCl2, 25 mM HEPES-Tris at pH 7.4, and 50 µM valinomycin (K+ ionophore). Extravesicular assay medium (EV) contained (final concentration in mM except where noted otherwise) 25 HEPES-Tris at pH 7.4, 100 KCl, 0 (, control) or 5 (, experimental) Tris-ATP, 1 NaN3, 5 µg/ml oligomycin B; 0.5 EGTA, 0.5 N-(2-hydroxyethylethylenediamine)-N,N',N'-triacetic acid, 0.5 nitrilotriacetic acid, 5 MgCl2 (free Mg2+, 1.52 mM), and 0.72 CaCl2 (free Ca2+, 5 µM). pr, Protein. Values presented are means ± SE of 4 replicates.

Effect of Ca²⁺ ionophore A-23187 and vanadate. The effects of A-23187 and vanadate on ⁴⁵Ca²⁺ uptake into BLMV of gill, hepatopancreas, and antennal gland are illustrated in Fig. 2, A-C. The ATP-dependent uptake followed the same time course as outlined above, with uptake rates reaching a plateau after 10-15 min at levels that were elevated severalfold over ATP-independent rates. Incubation of vesicles from the start of the experiment with ATP and either the Ca²⁺ ionophore A-23187 or vanadate produced ⁴⁵Ca²⁺ uptake rates that were virtually identical to those measured in the absence of ATP. When A-23187 was added after 10 min to Ca²⁺-loaded vesicles, 60-70% of loaded ⁴⁵Ca²⁺ was lost within 10 min.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of Ca2+ ionophore A-23187 and vanadate on time-dependent 45Ca2+ uptake at 37°C by short-circuited crayfish BLMV from gill (A), hepatopancreas (B), and antennal gland (C). Vesicles were preincubated with IV and EV as outlined in legend to Fig. 1. In individual experiments, the EV medium also contained 5 mM Tris-ATP from time 0 () and 5 mM Tris-ATP from time 0 with addition of A-23187 (10 µg/ml) at time 10 min (arrow, ). , 3 treatments (controls) that produced uptake data that were not significantly different: 0 mM Tris-ATP, 5 mM Tris ATP and A-23187 (10 µg/ml), and 5 mM Tris-ATP and vanadate (0.1 mM). Values presented are means ± SE of 4 replicates.

Free [Ca²⁺] dependence: Kinetics. Initial (1 min) ATP-dependent ⁴⁵Ca²⁺ uptake rates into BLMV of gill, hepatopancreas, and antennal gland were determined as a function of EV free Ca²⁺ (Fig. 3, A-C; left), and an Eadie-Hofstee transformation of the data was undertaken to determine enzyme kinetics (Fig. 3, A-C; right). ATP-dependent ⁴⁵Ca²⁺ uptake was a hyperbolic function of EV free Ca²⁺, suggesting the presence of a carrier system that saturates at free Ca²⁺ concentrations below 1 µM. The Eadie-Hofstee transformation plots ⁴⁵Ca²⁺ uptake flux rate (J) against ⁴⁵Ca²⁺ uptake flux rate/[free Ca²⁺] (or J/[S], where [S] is substrate concentration), providing an intercept of J_max on the y-axis as J/[S] tends to 0. The slope of the line is equal to K_m (apparent dissociation constant or substrate concentration at which half-maximal flux rate occurs). The Eadie-Hofstee plot is considered more accurate and generally superior to a Lineweaver-Burk transformation because it does not compress data points at high substrate concentrations. Linear regression on Eadie-Hofstee transformed data produced the following kinetics: gill, K_m = 0.28 µM, J_max = 108.84 pmol · mg¹ · min¹;hepatopancreas, K_m = 0.27 µM, J_max = 20.26 pmol · mg¹ · min¹; and antennal gland, K_m = 0.11 µM, J_max = 122.66 pmol · mg¹ · min¹. Values for J_max in gill and antennal gland were comparable and in both cases were fivefold higher than in hepatopancreas. The K_m for Ca²⁺ was lowest in antennal gland; K_m was higher and equivalent in both gill and hepatopancreas.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Free Ca2+ dependence of ATP-dependent 45Ca2+ uptake into crayfish BLMV from gill (A; ), hepatopancreas (B; ), and antennal gland (C; ) at 37°C. Rates are mean values of 3 experiments. Vesicles were preincubated with IV and EV as outlined in Fig. 1 legend, except that EV contained CaCl2 ranging from 0.15 to 0.83 (free Ca2+, 0.05-10 µM). Left: kinetic plot of initial flux rate (1 min) of ATP-dependent 45Ca2+ uptake (corrected for ATP-independent 45Ca2+ uptake) plotted as a function of EV free Ca2+ (substrate concentration). Right: mean uptake data at left linearized in an Eadie-Hofstee plot, where 45Ca2+ uptake rate in pmol · mg1 · min1 is plotted against 45Ca2+ uptake rate/free Ca2+ concentration ([Ca2+]) in pmol · mg1 · min1 · µM1. Linear regression produced the following kinetics: gill, Km = 0.28 µM, Jmax = 108.84 pmol · mg1 · min1 (r2 = 0.665, P < 0.05); hepatopancreas, Km = 0.27 µM, Jmax = 20.26 pmol · mg1 · min1 (r2 = 0.862, P < 0.05); antennal gland, Km = 0.11 µM, Jmax = 122.66 pmol · mg1 · min1 (r2 = 0.905, P < 0.05).

Substrate (ATP) dependence. Initial (1 min) ATP-dependent ⁴⁵Ca²⁺ uptake rates into BLMV of gill, hepatopancreas, and antennal gland were determined as a function of EV ATP concentration (Fig. 4, A-C, left), and an Eadie-Hofstee transformation of the data was performed to determine kinetics (Fig. 4, A-C, right). The K_m for ATP was comparable in gill and hepatopancreas (0.006 and 0.012 mM, respectively) but was higher in antennal gland (0.039 mM). There was excellent correspondence between J_max determined for ATP dependence and that reported above for free [Ca²⁺] dependence (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Substrate (ATP) dependence of ATP-dependent 45Ca2+ uptake into crayfish BLMV from gill (A; ), hepatopancreas (B; ), and antennal gland (C; ) at 37°C. Vesicles were preincubated with IV and EV as outlined in Fig. 1 legend, except that EV contained ATP concentration ranging from 0 to 5 mM. Left: kinetic plot of initial flux rate (1 min) of ATP-dependent 45Ca2+ uptake (corrected for ATP-independent 45Ca2+ uptake) plotted as a function of EV ATP concentration ([ATP]). Right: mean uptake data at left linearized using an Eadie-Hofstee plot (45Ca2+ uptake rate in pmol · mg1 · min1 vs. 45Ca2+ uptake rate/[ATP] in pmol · mg1 · min1 · mM1). Linear regression produced the following kinetics: gill, Km = 0.006 mM, Jmax = 105.45 pmol · mg1 · min1 (r2 = 0.972, P < 0.05); hepatopancreas, Km = 0.0123 mM, Jmax = 20.41 pmol · mg1 · min1 (r2 = 0.995, P < 0.05); antennal gland, Km = 0.039 mM, Jmax = 135.37 pmol · mg1 · min1 (r2 = 0.996, P < 0.05).

Temperature dependence. ATP-dependent ⁴⁵Ca²⁺ uptake rate was temperature dependent between 12 and 39°C, exhibiting a mean temperature coefficient (Q₁₀) of 2.06 ± 0.38 (n = 3) in gill, 1.43 ± 0.12 (n = 4) in hepatopancreas, and 1.64 ± 0.05 (n = 3) in antennal gland.

Calmodulin dependence. Pretreatment of hepatopancreas and antennal gland BLMV with EGTA significantly decreased ATP-dependent ⁴⁵Ca²⁺ uptake (Table 1) by 50 and 90%, respectively. In EGTA-treated hepatopancreas and antennal gland vesicles, ⁴⁵Ca²⁺ uptake was restorable to levels observed in untreated vesicles by addition of calmodulin. Calmodulin addition did not affect ATP-dependent ⁴⁵Ca²⁺ uptake into untreated hepatopancreas or antennal gland vesicles. Gill vesicles exhibited a completely different response from that observed in hepatopancreas and antennal gland. EGTA treatment of vesicles did not significantly reduce ATP-dependent ⁴⁵Ca²⁺ uptake. However, at the concentration used in the present experiment (0.625 µM), calmodulin addition significantly reduced ⁴⁵Ca²⁺ uptake into EGTA-treated vesicles as well as EGTA-untreated vesicles (P = 0.051).

Na⁺ dependence. Addition of 5 mM Na to the EV resulted in a significant (58%) increase in ATP-dependent ⁴⁵Ca²⁺ uptake in both hepatopancreas and antennal gland BLMV (Table 2). Pretreatment of membranes with ouabain had no effect on ATP-dependent ⁴⁵Ca²⁺ uptake in Na⁺-free EV buffer. Pretreatment of hepatopancreas BLMV with ouabain produced ATP-dependent ⁴⁵Ca²⁺ uptake rates in 5 mM Na buffer that were significantly below levels in 5 mM buffer untreated with ouabain, indicating that the Na⁺-dependent increase was ouabain inhibitable. These levels were also significantly below the levels observed in Na-free buffer, suggesting that ouabain had an effect over and above inhibition of the Na⁺-dependent increase in ATP-dependent ⁴⁵Ca²⁺ uptake. Similarly, in antennal gland BLMV, the Na⁺-dependent increase in ATP-dependent ⁴⁵Ca²⁺ uptake was virtually inhibited by pretreatment with ouabain (P = 0.063). In gill vesicles, neither addition of Na⁺ to the EV buffer nor pretreatment with ouabain had any significant effect on ATP-dependent ⁴⁵Ca²⁺ uptake. Mean ⁴⁵Ca²⁺ uptake increased 16% after addition of 5 mM Na to the EV buffer. However, high variance among ⁴⁵Ca²⁺ uptake rates in these vesicles meant that the trend was not significant.

	DISCUSSION

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The present study is the first to characterize ATP-dependent ⁴⁵Ca²⁺ uptake into antennal gland BLMV of any intermolt crustacean and the first to demonstrate uptake into gill and hepatopancreas BLMV in the intermolt FW crayfish. ATP-dependent ⁴⁵Ca²⁺ uptake was shown to be homogeneous, exhibit half-maximal saturation at IC [Ca²⁺], be prevented by the ionophore A-23187 and vanadate, and be temperature sensitive and calmodulin and Na⁺ dependent. As such, the ATP-dependent ⁴⁵Ca²⁺ uptake process in Ca²⁺-transporting epithelia of FW crayfish demonstrates similarities with the Ca²⁺ transport system described for BLMV of other crustaceans (8, 33), fish (9, 20), and mammals (10, 24).

Time dependence of ATP-dependent and ATP-independent ⁴⁵Ca²⁺ uptake. In the two published crustacean studies (8, 33), ⁴⁵Ca²⁺ uptake reached a plateau more rapidly (2-5 min) than in the present study, consistent with ATP depletion resulting from increased uptake rates in marine species. Linear ⁴⁵Ca²⁺ uptake was reported over the initial 1-3 min of incubation under similar reaction conditions in studies in crustaceans (8, 33), fish (9, 20), and mammals (24, 10). In the literature, addition of ATP stimulates ⁴⁵Ca²⁺ uptake above ATP-independent uptake anywhere from twofold [lobster hepatopancreas (33)] to fivefold [crab gill (8, present study)] and up to 20-fold in vertebrates (9, 10, 20, 24).

Effect of Ca²⁺ ionophore A-23187 and vanadate. In crayfish, the addition of the Ca²⁺ ionophore A-23187 produced ⁴⁵Ca²⁺ efflux from preloaded vesicles (Fig. 2), proving that ATP-dependent ⁴⁵Ca²⁺ uptake occurred against a concentration gradient. It also confirmed that uptake was into the IV space and not from superficial binding as outlined in an earlier study (31). However, in crayfish vesicles, only 60-70% of preloaded ⁴⁵Ca²⁺ was lost within 10 min of addition of A-23187, the other 30% remaining bound inside the vesicles. Other studies have similarly shown that ionophore-stimulated Ca²⁺ efflux is incomplete [60-80%; lobster hepatopancreas (33), rat kidney cortex (24), eel gill (9), trout gill (20)], whereas other studies have demonstrated complete unloading [crab gill (8), rat hepatic (16), rat duodenum (10)] often occurring rapidly (within 1 min). In the present study, ATP-dependent ⁴⁵Ca²⁺ uptake into crayfish vesicles was completely inhibited when A-23187 was added at the start of the experiment, as demonstrated in FW trout vesicles (20). In other vesicle populations [rat enterocyte (10), rat kidney cortex (24), eel gill (9)], only 70-90% of uptake was prevented.

Free [Ca²⁺] dependence: Kinetics. A comparison of J_max among different crayfish epithelia can provide an indication of density of Ca²⁺ pumps and their relative importance in Ca²⁺ influx. In the present study, J_max was higher in crayfish gill and antennal gland than in hepatopancreas, possibly suggesting that in intermolt crayfish the branchial/renal epithelia may potentially play a greater role in overall Ca²⁺ homeostasis than the digestive epithelium (28). This assumes that Ca²⁺-transporting proteins contribute equally to total protein in each tissue, because uptake rates are expressed per milligram total protein. Prior epithelial and whole tissue studies in our lab have confirmed that Ca²⁺ influx at the kidney (reabsorption of Ca²⁺ from urine) is higher than branchial unidirectional Ca²⁺ influx in the intermolt stage of the molting cycle. In aquatic organisms, J_max is typically higher in marine species (8, 26, 33) than in FW species (6, 9, 20, 26, present study) associated with increased whole organism ion fluxes. Also, branchial uptake is more important in aquatic species than uptake at the gut (6, present study). Evolution into terrestrial environments and loss of contact with water increase reliance on gastric epithelia for Ca²⁺ influx from food, with the renal reabsorption of Ca²⁺ in urinary filtrate also contributing to Ca²⁺ homeostasis (10, 16, 24).

Substrate (ATP) dependence. Substrate (ATP) dependence of ATP-dependent ⁴⁵Ca²⁺ uptake in crayfish vesicles (Fig. 4) revealed a K_m close to values determined for Na⁺-K⁺-ATPase in crab gill [0.03 (23)]. Both were two orders of magnitude lower than K_m for ATP for HCO₃-dependent ATPase (4.4 mM) in gills of crabs acclimated to either high- or low-salinity water (17).

Temperature dependence. Temperature dependence of ATP-dependent Ca²⁺ uptake is of physiological significance to a poikilothermic organism such as the crayfish. Q₁₀ values determined for ATP-dependent ⁴⁵Ca²⁺ uptake in crayfish were consistent with thermal coefficients reported for Ca²⁺ pumps in rat kidney cortex BLMV [1.54 (24)] and with pancreatic islet BLMV [1.49 (15)]. For blue crab gills, the HCO₃-dependent ATPase had a Q₁₀ of 1.46 in crabs acclimated to full-strength SW and 1.71 in crabs acclimated to low-salinity water [temperature range was 13-37°C (17)].

Calmodulin dependence. High-affinity PMCA is typically calmodulin dependent. The fact that calmodulin addition did not significantly increase ATP-dependent ⁴⁵Ca²⁺ uptake into untreated hepatopancreas or antennal gland vesicles tends to suggest that endogenous calmodulin was not lost during vesicle isolation. The present study has confirmed that ATP-dependent ⁴⁵Ca²⁺ uptake into hepatopancreas and antennal gland BLMV is calmodulin dependent. In vesicles from these two tissues, EGTA treatment, which removes endogenous calmodulin by chelating Ca²⁺, had the effect of significantly reducing ATP-dependent ⁴⁵Ca²⁺ uptake. The remaining uptake that was calmodulin independent was higher in hepatopancreas (50%) but was virtually negligible (10%) in antennal gland. Calmodulin repletion restored ATP-dependent ⁴⁵Ca²⁺ uptake in EGTA-treated vesicles to rates that were not significantly different from those measured in untreated vesicles. Similarly, in other vesicle populations, 50% of Ca²⁺ uptake has been shown to be calmodulin dependent [eel gill (9), trout gill (20), rat kidney cortex (24), and lobster hepatopancreas (33)]. In rat intestinal BLMV, only 25% of uptake was calmodulin dependent (25). Although calmodulin is believed to affect primarily J_max, an affect on affinity was reported in rat duodenum (10) and both were affected in intestinal BLMV and rat renal BLMV, where the readdition of exogenous calmodulin shifted the affinity for Ca²⁺ from 1.2 to 0.24 µM Ca²⁺ and increased the J_max by 30% (12).

Na⁺ dependence. In crayfish hepatopancreas and antennal gland, an increase in ATP-dependent ⁴⁵Ca²⁺ uptake was observed with addition of 5 mM Na. This increase was fully inhibitable by preincubation with ouabain in hepatopancreas (and largely inhibitable in antennal gland), suggesting that basolateral Na⁺-K⁺-ATPase generated an Na⁺ gradient favorable for additional Ca²⁺ accumulation via a basolateral Ca²⁺/Na⁺ exchanger. In these two vesicle populations, the increase in ⁴⁵Ca²⁺ uptake suggests that the Ca²⁺/Na⁺ exchanger capacity may approach 60% of the J_max of the ATP-dependent Ca²⁺ pump.

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Comparison of in vitro and in vivo ATP-dependent Ca²⁺ uptake capacity in crayfish epithelia. The capacity for ATP-dependent ⁴⁵Ca²⁺ uptake in each tissue of an individual crayfish was calculated from in vitro vesicle studies and compared with in vivo ⁴⁵Ca²⁺ uptake rates in Table 3. In vitro ATP-dependent ⁴⁵Ca²⁺ uptake was calculated (26) as maximal initial rate of ATP-dependent ⁴⁵Ca²⁺ uptake (J_max) × total protein in the homogenate adjusted to 100% IOVs and 100% recovery of basolateral membranes (calculated from percentage recovery of Na⁺-K⁺-ATPase specific activity). This value was divided by six because tissues from six crayfish had been pooled for vesicle isolation. The total capacity for ATP-dependent ⁴⁵Ca²⁺ uptake was calculated as 27.3 µmol/h for gill, 3.1 µmol/h for hepatopancreas, and 3.7 µmol/h for antennal gland. Again on the assumption that each tissue has a similar density of Ca²⁺ pumps per milligram protein, the gills of intermolt crayfish have a capacity for ATP-dependent ⁴⁵Ca²⁺ uptake that is an order of magnitude greater than either the hepatopancreas or the antennal gland. This relationship confirms the relative importance of the three exchange epithelia in overall Ca²⁺ uptake.

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Perspectives

A logical progression of this work is to integrate cellular physiology with molecular characterization of the Ca²⁺ pump. Due to its fragility, the hypodermal epithelium has largely evaded most in vitro approaches, and yet its role in Ca²⁺ transfer during the molting cycle is paramount. This epithelium will continue to challenge comparative physiologists desiring to understand its role in Ca²⁺ homeostasis.