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Am J Physiol Regul Integr Comp Physiol 274: R725-R734, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 3, R725-R734, March 1998

Isolation, visualization, characterization, and osmotic reactivity of crayfish BLMV

Michele G. Wheatly, Jennifer R. Weil, and Phyllis B. Douglas

Department of Biological Sciences, Wright State University, Dayton, Ohio 45435

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Procedures were developed to isolate basolateral membrane vesicles (BLMV) from gill, hepatopancreas, and antennal gland of intermolt freshwater crayfish, Procambarus clarkii. Individual procedures involved a discontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopancreas), or differential centrifugation (antennal gland). BLMV were visualized, characterized (37°C), and tested for osmotic reactivity with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antennal gland). Enrichments of basolateral membranes and mitochondria in BLMV were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for hepatopancreas, and 10- and 1-fold for antennal gland. Apical contamination was negligible in BLMV. Percentages of resealing of vesicles as inside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:26:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhibited osmotic reactivity, as indicated by a linear relationship between vesicular 45Ca2+ uptake and osmolality. Nonspecific 45Ca2+ binding was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.

basolateral membrane vesicles; freshwater crayfish; gill; hepatopancreas; antennal gland

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CELLULAR MECHANISMS OF transmembrane Ca2+ transfer are of fundamental importance to eukaryotic organisms because of their involvement in intra- (IC) and extracellular (EC) Ca2+ homeostasis. These mechanisms are best exemplified in epithelia specialized for Ca2+ transport, such as gut, kidney, gills (aquatic species), and biomineralizing tissues. Crustaceans serve as an ideal model system for the study of Ca2+ transport mechanisms by virtue of their natural molting cycle. As arthropods, they periodically demineralize their calcified exoskeleton, shed, and then rapidly remineralize the new cuticle, with Ca2+ uptake rates in the range of 2-10 mmol · kg-1 · h-1 (23). The crustacean epithelia that bidirectionally transport Ca2+ are gills (passive diffusional loss and active uptake), hepatopancreas (gut, uptake via food, lumenal Ca2+ storage), antennal gland (kidney, filtration and reabsorption), and cuticular hypodermis (demineralization and mineralization).

The model for crustacean epithelial Ca2+ transport has been derived exclusively from vesicle studies in intermolt marine species, specifically, in apical vesicles of lobster Homarus americanus antennal gland (1), basolateral vesicles of Carcinus gill (6), and apical and basolateral vesicles of lobster hepatopancreas (3, 26). Briefly, Ca2+ enters the cytosol passively through apical verapamil-inhibited Ca2+ channels or via a Ca2+/nNa+ (nH+) exchanger that may be electroneutral or electrogenic (n refers to variable number of ions). Basolateral mechanisms include 1) a low-affinity, high-capacity Ca2+/Na+ exchanger responsible for Ca2+ efflux [apparent affinity (Km), 1.78 µM; maximal transport velocity (Vmax), 9.88 nmol · min-1 · mg protein-1 in crab gill, Ref. 6; Km, 310 µM; Vmax, 74 nmol · min-1 · mg protein-1 in lobster hepatopancreas; Ref. 3], 2) a high-affinity, low-capacity calmodulin-dependent Ca2+-adenosinetriphosphatase (Ca2+-ATPase) that regulates IC Ca2+ levels (Km, 0.15 µM; Vmax, 1.73 nmol · min-1 · mg protein-1; Ref. 6), and 3) a verapamil-inhibited Ca2+ channel.

Crayfish have a long evolutionary history in fresh water, a medium with reduced Ca2+ availability (<1 mM). In intermolt, their ability to hyperionically regulate EC Ca2+ is attributable primarily to their ability to produce a dilute urine (25), since active branchial Ca2+ uptake is negligible (22). Postmolt mineralization, however, involves active branchial Ca2+ uptake, described by saturation kinetics characteristic of Ca2+-ATPase (Km, 0.13 mM; saturation, 0.4 mM; Vmax, 2 mmol · g-1 · h-1;Ref. 7), as well as uptake via an Na+/Ca2+ exchanger (15). Thus freshwater crayfish potentially constitute a better crustacean model with which to study Ca2+ transport mechanisms than marine species that inhabit a Ca2+-rich environment (10 mM), regulate EC Ca2+ isoionically (8), produce an isosmotic urine, and absorb Ca2+ passively from seawater in the postmolt period (12).

To study transepithelial Ca2+ transport mechanisms in freshwater crayfish, it is necessary to develop appropriate isolation procedures for both apical and basolateral membrane vesicles (BLMV). The purpose of this study was to develop individual isolation procedures for BLMV from the gill, hepatopancreas, and antennal gland of the freshwater crayfish, Procambarus clarkii. BLMV have been visualized and characterized. Equilibrium uptake of 45Ca2+ into an osmotically reactive space was demonstrated providing a foundation for continued functional studies on Ca2+ uptake mechanisms into inside-out vesicles (IOV). To date, BLMV have not been isolated from any crustacean antennal gland. Isolation procedures for BLMV are established for gill and hepatopancreas in marine crustaceans. They have been isolated from lobster H. americanus hepatopancreas (2) to study proton-stimulated Cl-/HCO3 antiport. Gill vesicles have been isolated from the crab Callinectes sapidus (10, 11, 20) to study, respectively, HCO3-dependent adenosinetriphosphatase (ATPase), Na+-independent HCO3/Cl- exchange, and ATP-dependent Na+ transport and from the crab Carcinus maenas (6) to study ATP- and Na+- dependent Ca2+ transport. Initial attempts to replicate published isolation procedures for hepatopancreas and gill were unsuccessful confirming that methodology is not necessarily transferable among distantly related crustacean species. Other studies have similarly concluded that there are interspecific differences in vesicle isolation procedures among marine crabs. Although plasma membrane vesicles have been isolated from Carcinus that exhibit Ca2+ uptake (6), other preparations (9, 17 20) had sufficient IOVs to demonstrate energized Na+ transport but insufficient IOVs for measurement of Ca2+ transport.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Crayfish (P. clarkii) (Girard) were obtained from Carolina Biological Supply and maintained in 40 liters aquaria in filtered aerated water at 21°C (22). Individual protocols were developed for preparation of BLMV from gill, hepatopancreas, and antennal gland of intermolt crayfish. Vesicles were kept at 4°C until use and were used within 3 days of preparation. Pelleted BLMV were visualized and resuspended in buffer for characterization.

Preparation of Gill BLMV

The isolation procedure for crayfish gill BLMV (Fig. 1) borrowed some elements from procedures published on marine crabs (6, 10, 20). Crayfish were killed by decerebration. Gills dissected from 6-10 medium crayfish (combined tissue mass, 16.22 g) were homogenized using a glass-on-glass Dounce homogenizer in 15-20 ml of ice-cold isotonic sucrose buffer (24) containing (in mM) 250 sucrose, 6 EDTA, and 20 imidazole, at pH 6.8, with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). A "crude" homogenate sample (500 µl) was removed for characterization. The homogenate was centrifuged (30 min at 480 g), and the pellet (P1) was discarded. The supernatant (SN1) was passed through cheesecloth and then centrifuged to pellet the membranes (30 min at 100,000 g). This pellet (P2) was resuspended in 4-5 ml isotonic buffer and layered on top of a discontinuous sucrose gradient made from 40, 45, 50, 55, 60, and 65% sucrose. The gradient was centrifuged (4 h at 100,000 g). In initial preparations, the two visible bands, B1 and B2, were removed, and the remaining gradient was divided into two fractions, as indicated in Fig. 1. Each fraction was slowly reequilibrated with isotonic buffer (4-8 h) and then pelleted (30 min at 100,000 g). The BLMV pellet (P3 from top band, B1) was resuspended in 1 ml isotonic buffer, passed 15 times through a 23-gauge needle, and incubated at room temperature for 30 min to reseal vesicles.


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  		Fig. 1.   Flow diagram for preparation of crayfish gill basolateral membrane vesicles (BLMV). Isotonic buffer was 250 mM sucrose, 6 mM EDTA, and 20 mM imidazole, pH 6.8 with HEPES; wt/v, wt/vol.

Preparation of Hepatopancreas BLMV

The hepatopancreas isolation procedure (Fig. 2) was modified from the method for lobsters (2). Hepatopancreas was removed from 6-10 medium-size decerebrated crayfish (combined tissue mass, 15.5 g) and placed in 15-20 ml of ice-cold homogenizing buffer. In the case of hepatopancreas, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) was added to the homogenizing buffer to inhibit digestive proteases. Tissue was homogenized, and a crude sample (500 µl) was removed for characterization. The homogenate was centrifuged (30 min at 480 g). The surface lipid layer and the pellet (P1) were removed and discarded. The supernatant (SN1) was filtered through cheesecloth and then centrifuged (30 min at 100,000 g). The lipid layer was again removed. A double pellet was formed consisting of a clear chocolate-colored upper region (P2b) and a lighter cloudy lower region (P2a). The supernatant (SN2) was removed along with the upper pellet while the lower pellet was carefully discarded. It was then layered on top of 2 ml of 65% sucrose and centrifuged (60 min to 100,000 g) to separate the mitochondria from the BLMV. After this spin, a light layer (band 1) was visible on top of the sucrose cushion. The band was slowly reequilibrated in isotonic buffer without PMSF on ice (4-8 h). It was then centrifuged (30 min at 100,000 g) to pellet the BLMV (P3), which were brought up in 1 ml of buffer, passed 15 times through a 23-gauge needle, and incubated at room temperature for 30 min to reseal vesicles.


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  		Fig. 2.   Flow diagram for preparation of crayfish hepatopancreas BLMV.

Preparation of Antennal Gland BLMV

Antennal glands dissected from 6-10 medium-size decerebrated crayfish (combined tissue mass, 1.1 g) were homogenized in 5-10 ml of isotonic sucrose buffer (Fig. 3). A "crude" homogenate sample (500 µl) was removed for characterization. The homogenate was centrifuged (30 min at 480 g). The pellet (P1) was discarded while the supernatant (SN1) was retained and centrifuged to pellet (P2) membranes (30 min at 100,000 g). The resulting pellet consisted of two distinct regions: a darker upper pellet (P2b) and a lighter lower pellet (P2a). The supernatant (SN2) and lower pellet (P2a) were carefully separated and discarded. The upper pellet (P2b) was resuspended in isotonic buffer and repelleted as in the previous step (30 min at 100,000 g). Again, a double pellet was formed, and the upper pellet (P3b) was retained while the supernatant and lower pellet (P3a) were discarded. This upper pellet (P3b) was resuspended in 1 ml isotonic buffer, passed 15 times through a 23-gauge needle, and incubated at room temperature for 30 min to reseal vesicles.


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  		Fig. 3.   Flow diagram for preparation of crayfish antennal gland BLMV.

Visualization of BLMV

Pelleted BLMV were fixed (2% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.4), washed (0.1 M sodium cacodylate), postfixed in 2% osmium tetroxide, dehydrated, and embedded in epoxy resin. Thin sections were stained in uranyl acetate and lead citrate and examined in a Zeiss 900 transmission electron microscope at 50 kV. The mean vesicle diameter was determined by measuring 100 vesicles from low-magnification electron micrographs (×4,300-6,800). Student's t-test was used to determine significance between means using P < 0.05 as the confidence limit.

Characterization of BLMV

Protein assay. The protein content was determined using the Bio-Rad microassay procedure (Bio-Rad protein assay kit I with bovine serum albumin standard ranging from 0.001 to 0.03 mg/ml).

Membrane polarity. Polarity was identified biochemically by determining specific activities (at 37°C) of domain-specific marker enzymes: basolateral (Na+-K+-ATPase) (19), apical (alkaline phosphatase) (2), and mitochondrial (cytochrome-c oxidase) (21).

Na+-K+-ATPase. The activity of Na+-K+-ATPase was assessed by the appearance of Pi, due to the splitting of ATP substrate by the ATPase (13). The reaction was run in a solution containing (in mM) 100 NaCl, 12.5 KCl, 5 MgCl2, 0.1 EDTA, 5 NaN3, 20 imidazole (pH 7.5 with HEPES), and 3 Na2ATP, with or without 1 mg/ml ouabain. At intervals (0-25 min) following the addition of vesicles (0.25 mg/ml), aliquots were removed and immediately placed into ice-cold 8.6% trichloroacetic acid (stop solution). Color was developed in these tubes with the addition of 4% FeSO4/1% ammonium molybdate (20 min). Pi released was determined colorimetrically as the increase in absorbance at 700 nm (5). Na+-K+-ATPase activity was calculated as the difference in rate of Pi release in the presence and absence of ouabain.

Alkaline phosphatase. Alkaline phosphatase activity was determined using Sigma assay kit no. 104. The reaction is based on the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol (measured at 400-420 nm) and phosphate.

Cytochrome-c oxidase. Cytochrome-c oxidase activity was measured as electron transfer rate (18). Phosphate buffer (50 mM KH2PO4, pH 7.5), 18 mM sodium ascorbate, 40 µM cytochrome-c (Sigma), and N,N,N',N'-tetramethyl-p-phenylenediamine were combined, and an autooxidation rate was determined. Sample was added, and enzyme activity was calculated from the difference in the oxidation rates. The oxidation rate was measured with a Clark O2 electrode (Yellow Springs Instruments, Yellow Springs, OH) contained in a thermostatted cell.

Resealing, leakiness, and orientation of BLMV. Vesicle resealing, leakiness, and orientation [IOVs vs. right-side-out vesicles (ROVs)] were determined by assessing percent inhibition of Na+-K+-ATPase by ouabain in the absence or presence of the detergent saponin (0.2 mg/ml; Ref. 2). Total ATPase activity was measured under four conditions: 1) absence of ouabain and saponin, 2) presence of ouabain and absence of saponin, 3) absence of ouabain and presence of saponin, and 4) presence of ouabain and saponin. The relative proportions of leaky vesicles or membrane sheets (LS), IOVs, and ROVs were calculated using standard equations (2).

Osmotic Reactivity of BLMV

The effect of transmembrane osmotic gradients on 60-min equilibrium uptake of 45Ca2+ was used to assess vesicle closure and to determine whether uptake of 45Ca2+ reflected binding to the membrane or transport into an osmotically reactive space (4, 14). Because the intravesicular (IV) space decreases as osmolarity increases, reduced uptake at increased osmolarity implicates transport into an osmotically reactive space and extrapolation to infinite medium osmolarity (zero IV space) can be used to estimate nonspecific membrane binding.

Vesicles were loaded with 250 mM sucrose, 6 mM EDTA, and 20 mM imidazole at pH 6.8, with HEPES. They were then incubated in extravesicular (EV) medium containing 125 mM NaCl, 6 mM EDTA, 20 mM imidazole at pH 6.8, with HEPES, 0.5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.5 mM N-(2-hydroxyethylethylenediamine)-N,N',N'-triacetic acid (HEEDTA), 0.5 mM nitrilotriacetic acid (NTA), and 0.72 mM CaCl2 that contained varying concentrations of sucrose (0, 250, 500, 2,000 mM) and 2 µCi of 45Ca2+ (purchased as CaCl2; DuPont, sp act 26.3 mCi/mg). The free Ca2+ concentration (5 µM) was calculated using the program Chelator (16). The first and second protonations of the ligands in the calcium buffers (ATP, EGTA, HEEDTA, and NTA) were taken into account, and the stability constants were adjusted in accordance with the pH, temperature, and ionic strength of the medium. The osmolarity of each solution was measured on a Wescor 5500 vapor pressure osmometer. The reaction was set up in a total volume of 165 µl with 20-µl vesicles (0.1-1.0 mg total protein) at the optimum temperature of 37°C. After 60-min incubation, a 50-µl subsample was removed and added to fluor to determine the specific activity. A second 50-µl subsample was filtered (Schleicher & Schuell, ME 24; pore diameter, 0.2 µm presoaked). Retained radioactivity was determined by placing the filter in 5 ml Ecoscint fluor and counting on a scintillation counter. Uptake of 45Ca2+ was calculated as nanomoles per milligram of protein, using specific activity of 45Ca2+ in the incubation medium. Nonspecific surface binding was extrapolated to infinite osmolarity, using a plot of 45Ca2+ uptake vs. the reciprocal of osmolarity.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation Procedures for BLMV From Gill, Hepatopancreas, and Antennal Gland

Crayfish gill BLMV were successfully isolated using the method described above. Characterization of fractions in the purification procedure (Table 1) revealed that BLMV and mitochondria were closely cosedimented on a discontinuous sucrose gradient. Membranes recovered from pellet P3 (sedimented from top band, B1) showed the greatest enrichment of Na+-K+-ATPase with the lowest mitochondrial contamination. Basolateral enrichment, however, occurred to a lesser extent throughout the entire gradient (B2, F3, and F4). The mitochondria, as evidenced by cytochrome-c oxidase activity, were minimally enriched in B1 but were greatly enriched in the band B2 and were found in reasonably high concentrations at higher sucrose percentages in the gradient fractions F3 and F4. Alkaline phosphatase activity was only detectable in the crude homogenate: there was negligible contamination in any subsequent fraction.

                              
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  		Table 1.   Characterization of fractions in the purification of crayfish gill basolateral membrane vesicles

Crayfish hepatopancreas BLMV were also successfully isolated using the method described above. Characterization of the fractions in the purification procedure (Table 2) revealed that BLMV were cleanly isolated in pellet P3, which originated from upper pellet P2b. Mitochondrial contamination of this BLMV fraction was minimal based on deenrichment of cytochrome-c oxidase. Mitochondria were effectively separated in the lower pellet P2a, which exhibited greatest enrichment of cytochrome-c oxidase. As in the gill preparation, apical membranes were only detectable in the original homogenate.

                              
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  		Table 2.   Characterization of fractions in purification of crayfish hepatopancreas basolateral membrane vesicles

This is the first published method for isolation of BLMV in the antennal gland of any crustacean. Characterization of the fractions in the purification procedure (Table 3) reveals that BLMV were best enriched in upper pellet P3b, which originated from upper pellet P2b. This fraction had the same degree of mitochondrial contamination as the corresponding gill fraction. Mitochondria were most enriched in the lower pellet P2a but not to the same extent as observed in corresponding gill fractions. The antennal gland was the only tissue where apical contamination could be detected in the supernatant following the first 480-g spin.

                              
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  		Table 3.   Characterization of fractions in purification of crayfish antennal gland basolateral membrane vesicles

Visualization of BLMV

Gill, hepatopancreas, and antennal gland BLMV were visualized in the transmission electron micrograph (Fig. 4). In each preparation, the transmission electron microscope revealed large numbers of resealed vesicles along with some membrane sheets. The lipid bilayer was visible under higher magnification. There was minimal visual contamination with mitochondria. The mean diameters of BLMV were, respectively, 159 ± 9.42 (gill), 363 ± 14.6 (hepatopancreas), and 226 ± 13.6 nm (antennal gland). Maximum diameters observed in each preparation were 396 (gill), 836 (hepatopancreas), and 540 (antennal gland) nm. The mean diameters of both hepatopancreas and antennal gland vesicles were both significantly greater than gill.


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  		Fig. 4.   Electron micrograph of crayfish BLMV from gill (A; bar, 0.6 µm), hepatopancreas (B; bar, 1.7 µm), and antennal gland (C; bar, 1.1 µm). D: high-magnification electron micrograph of antennal gland BLMV (bar, 0.6 µm), demonstrating lipid bilayer structure (arrowheads).

Characterization of BLMV

Characterization of crayfish gill BLMV (Table 4) revealed that total protein recovery was 2.4%. Basolateral membranes, as indicated by Na+-K+-ATPase activity, were enriched 18-fold in the BLMV fraction with enzyme recovery of 16%. Mitochondria, as indicated by cytochrome-c oxidase activity, were marginally enriched in the BLMV fraction (1.7-fold). Apical membranes, as indicated by alkaline phosphatase activity, were undetectable in the BLMV fraction. Saponin unmasked significant ATPase that was ouabain inhibitable (Table 5). These data indicated that 56% of the membrane preparation was LS, 17% was IOV, and 27% was ROV.

                              
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  		Table 4.   Purification and recovery of marker enzymes in crayfish gill basolateral membrane vesicles

                              
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  		Table 5.   Effects of ouabain and saponin on ATPase activity and orientation of crayfish gill basolateral membrane vesicles

Characterization of crayfish hepatopancreas BLMV (Table 6) revealed that total protein recovery was 9.8%. Basolateral membranes, as indicated by Na+-K+-ATPase activity, were enriched ninefold in the BLMV fraction with enzyme recovery of 32%. Cytochrome-c oxidase activity was deenriched (0.4-fold) in the BLMV fraction, indicating minimal mitochondrial contamination. Alkaline phosphatase activity was undetectable, indicating minimal apical contamination. Saponin unmasked significant ATPase that was ouabain inhibitable (Table 7). These data indicated that 60% of the membrane preparation was LS, 14% was IOV, and 26% was ROV.

                              
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  		Table 6.   Purification and recovery of marker enzymes in crayfish hepatopancreas basolateral membrane vesicles

                              
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  		Table 7.   Effects of ouabain and saponin on ATPase activity and orientation of crayfish hepatopancreas basolateral membrane vesicles

Characterization of crayfish antennal gland BLMV (Table 8) revealed that protein recovery was 8.3%. The Na+-K+-ATPase activity indicated a 10-fold enrichment of basolateral membranes, whereas mitochondria, as indicated by cytochrome-c oxidase levels, were maintained. Alkaline phosphatase activity was again minimal, indicating negligible apical contamination. As for the other preparation, saponin unmasked significant ATPase activity that was ouabain inhibitable (Table 9). Calculations revealed that 40% of the membrane preparation was LS, 21% was resealed in the IOV orientation, and 39% was resealed as ROVs.

                              
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  		Table 8.   Purification and recovery of marker enzymes in crayfish antennal gland basolateral membrane vesicles

                              
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  		Table 9.   Effects of ouabain and saponin on ATPase activity and orientation of crayfish antennal gland basolateral membrane vesicles

Osmotic Reactivity of BLMV

For each BLMV preparation, there was a linear relationship between vesicular 45Ca2+ uptake at equilibrium and the reciprocal of the incubation medium osmolarity (Fig. 5), suggesting that a population of vesicles were completely resealed and exhibited osmotic reactivity. Each linear relationship was extrapolated to infinite osmolarity to estimate nonspecific membrane binding. The binding component was estimated as a percentage of equilibrium 45Ca2+ uptake in the NaCl medium lacking sucrose (1/osmolarity = 3.36 osmol-1), that is, under control osmotic conditions. Membrane binding was highest in hepatopancreas BLMV (39.4%), intermediate in the antennal gland BLMV (31.1%), and least in the gill BLMV (20.2%).


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  		Fig. 5.   Effect of a transmembrane osmotic gradient on equilibrium uptake (60 min at 37°C) of 45Ca2+ by crayfish BLMV from gill (A), hepatopancreas (B), and antennal gland (C). Vesicles were loaded with (in mM) 250 sucrose, 6 EDTA, and 20 imidazole, at pH 6.8 with HEPES. They were then incubated in a medium containing (in mM) 125 NaCl, 6 EDTA, 20 imidazole, at pH 6.8 with HEPES, 0.5 EGTA, 0.5 N-(2-hydroxyethylenediamine)-N,N',N'-triacetic acid, and 0.5 nitriloacetic acid, with CaCl2 added to achieve a free Ca2+ of 5 µM that contained varying concentrations of sucrose (0, 250, 500, and 2,000 mM) and 2 µCi of 45Ca2+. Osmolarity was measured using a Wescor 5500 vapor pressure osmometer. Points are means ± SE of measurements on 3 different vesicle preparations. Lines were fitted through the data using linear regression. A: 45Ca2+ uptake = 17.36 + 19.77 (osmolarity-1); r2 = 0.98. B: 45Ca2+ uptake = 22.26 + 9.93 (osmolarity-1); r2 = 0.95. C: 45Ca2+ uptake = 76.02 + 54.66 (osmolarity-1); r2 = 0.96.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isolation Procedures for BLMV

The present crayfish gill BLMV isolation procedure can be compared with published procedures for marine crabs (6, 10, 20). In the present study, all gill sets were combined to prepare vesicles, having shown earlier (24) that ion transport enzymes are uniformly distributed on crayfish gills. In marine species, vesicles are typically prepared from the posterior gills, which are the primary site of ion transport (6, 10, 11, 20). In those studies, prior to vesicle isolation, euryhaline crabs are typically acclimated to reduced-salinity water to stimulate ion uptake. Two studies (10, 20) similarly utilized a sucrose buffer for the initial homogenization. Early attempts to homogenize crayfish gills in an NaCl buffer, as recommended in another study (6), were unsuccessful. Two of the published crab protocols (6, 10) separated mitochondria and plasma membranes by differential centrifugation (10,000 and 50-100,000 g, respectively), eliminating the need to run a sucrose gradient and thereby greatly simplifying the isolation procedure. In crayfish, a discontinuous sucrose gradient was necessary to adequately separate the mitochondria and plasma membranes, as recommended in a third protocol (20). In that study, 0.1% deoxycholate was added to the homogenizing buffer to separate mitochondria (37% sucrose) and plasma membranes (25% sucrose) on a linear sucrose gradient. Whereas mitochondrial contamination was lower in that preparation, basolateral membranes were only one-fourth as enriched. Both studies (Ref. 20 and this study) emphasize the technical difficulty of obtaining mitochondria-free basolateral membranes from crustacean gill tissue.

Published gill vesicle preparations have also differed in their use of protease inhibitors (such as PMSF) and a disulfhydryl reducing agent [such as dithiothreitol (DTT)]. In the present study, use of PMSF significantly inhibited Na+-K+-ATPase activity, and so it was omitted from the final procedure. DTT has been included in the homogenization buffer in some gill protocols (6, 10), typically to prevent nonspecific protein aggregation of sulfhydryl groups by maintaining the reducing atmosphere present in the cytoplasm. Plasma membrane proteins are exposed to an oxidizing atmosphere on the EC surface and a reducing atmosphere on the IC surface, and so the utility of DTT in a BLMV preparation remains questionable.

The crayfish hepatopancreas isolation procedure was quite similar to the one published for lobster (2), except for some minor details. The protease inhibitor PMSF was added to the homogenizing buffer to counteract activity of digestive enzymes. As for gill, homogenization was most effectively performed in an isosmotic sucrose buffer, although a hypotonic ionic buffer (25 mM NaCl) had been employed with good results in lobster (2). In that study, membranes were separated from mitochondria on a sorbitol gradient (65% sorbitol overlain with 25% sorbitol) with BLMV visible at the 65-25% interface. The antennal gland isolation procedure developed in this study was similar to that used for hepatopancreas, except that BLMV were pelleted via differential centrifugation. A sucrose gradient may have further deenriched mitochondrial contamination but at the expense of protein recovery.

Visualization of BLMV

Visualization in the transmission electron microscope confirmed the existence of resealed vesicles with minimal mitochondrial contamination. The mean diameter of crayfish gill BLMV was on the lower end of the range of diameters (100-500 nm) observed in crab gill vesicles (20). The mean diameter of crayfish hepatopancreas BLMV was in the center of the range (100-700 nm) observed in the lobster BLMV (2). Crayfish antennal gland BLMV were of intermediate diameter. The mean diameter measured by transmission electron microscope may underestimate the true vesicle diameter, since a section through a population of vesicles will capture some at greatest diameter and other vesicles at the periphery. Furthermore, in preparation for electron microscopy, vesicles were fixed, embedded, and sectioned in the pelleted state, and BLMV diameter under these conditions may not necessarily approximate to the diameter when suspended in EV medium. One of the major difficulties encountered in the use of membrane vesicles is their size heterogeneity (19), since smaller vesicles typically will reach equilibrium sooner than larger vesicles.

Characterization of BLMV

Characterization data for crayfish gill BLMV were compared with published studies on marine crab BLMV (6, 10, 11, 20), following correction for experimental temperature. As expected, less protein was recovered in procedures that included a sucrose gradient (Ref. 6 and present study). Activity of Na+-K+-ATPase was two orders of magnitude higher in both crab gill homogenate and BLMV (6, 11) associated with increased branchial ion fluxes (24). Overall, the crayfish preparation had a higher basolateral purification factor and lower apical contamination than published crab protocols, whereas mitochondrial contamination was comparable. Vesicle resealing and orientation were comparable in crayfish and Carcinus (6). Higher resealing was reported in Callinectes vesicles (11); however, only 2% of the vesicles were in the preferred IOV orientation.

Characterization data for crayfish hepatopancreas BLMV were compared with an existing protocol on lobster (H. americanus) hepatopancreas BLMV (2), following correction for experimental temperature. In the crayfish procedure, use of a sucrose cushion improved protein recovery, compared with lobster BLMV prepared on a gradient. Activities of both Na+-K+-ATPase and cytochrome-c oxidase were greater in lobster than in crayfish. Both preparations had comparable basolateral membrane enrichment and mitochondrial deenrichment, although crayfish BLMV exhibited less apical contamination and higher enzyme recoveries for both Na+-K+-ATPase and cytochrome-c oxidase. Both preparations exhibited comparable resealing, although crayfish BLMV contained more than twice as many IOVs.

Because BLMV have not previously been isolated from any crustacean antennal gland, characterization and orientation data for crayfish antennal gland BLMV are compared with the gill and hepatopancreas BLMV prepared in the present study. The Na+-K+-ATPase activity was threefold higher in the antennal gland than in gill and hepatopancreas. Past studies have indicated that the crayfish antennal gland plays a more important role in ion reclamation than the gill (23); renal reabsorption rates (25) exceed branchial unidirectional influx rates for most electrolytes (22), and specific activities of transport marker enzymes are typically higher in antennal gland than in gills. Recovery of Na+-K+-ATPase was lowest in the gill and antennal gland and was virtually double in the hepatopancreas. Purification factor for Na+-K+-ATPase was comparable in the hepatopancreas and antennal gland but was twofold higher in the gill. Cytochrome-c oxidase activity was lowest in the gill homogenate and highest in the hepatopancreas, possibly associated with metabolic activity of the digestive gland. The hepatopancreas preparation was most effective in deenriching mitochondrial contamination. Total ATPase activity was higher in gill and antennal gland but significantly lower in hepatopancreas, reflecting again reduced involvement of this epithelium in ion transport. Percentage of resealing and percentage IOVs and ROVs were comparable in gill and hepatopancreas preparations. Antennal gland exhibited better resealing with higher proportions of IOVs and ROVs; however, the ratio of IOVs to ROVs was 2:3 in all three preparations.

Osmotic Reactivity of BLMV

There was significant nonspecific 45Ca2+ binding to BLMV. Because the vesicle preparations contained a high proportion of membrane sheets, as well as leaky vesicles and sealed vesicles of either orientation (ROV and IOV), binding represents the combined association of labeled ion to both inside and outside surfaces. High nonspecific 45Ca2+ binding has also been reported in lobster hepatopancreas brush-border membrane vesicles (26), whereas significant but lower nonspecific 22Na+ binding has been reported in crab gill BLMV (20). Meanwhile, nonspecific Cl- binding to crab gill and lobster hepatopancreas BLMV appears to be relatively low (<10%; Refs. 2, 11). Thus nonspecific binding is a serious consideration for crustacean vesicles, more so for Ca2+ than Na+ or Cl-. Membranes may bind Ca2+ via two mechanisms. First, many membrane or membrane-associated proteins routinely bind Ca2+ or use it as a cofactor. Second, divalent ions are more attracted to the negative charge maintained on membrane phospholipid than monovalent cations. Nonspecific binding in excess of 10% of total uptake is considered significant and must be taken into consideration during future 45Ca2+ uptake experiments. The present study has shown that >60% of 45Ca2+ uptake into BLMV is into an osmotically reactive interior space. Studies continue to functionally characterize the primary Ca2+ uptake mechanisms (Ca2+-ATPase, Ca2+/Na+ exchanger).

Perspectives

Vesicles have been routinely employed for transport studies in mammals and other vertebrates. Because vertebrates share a common evolutionary origin, established techniques are easily transferable among species. By contrast, in aquatic invertebrates and, more specifically, in crustaceans, very few vesicle studies have been conducted primarily focusing on gill tissue of marine species. Extant crustaceans exemplify a variety of osmotic histories, ranging from marine to freshwater origins and via either origin onto land. Evolution into freshwater is associated with marked changes in osmoregulation (from isosmotic to hyperosmotic), with gills and antennal gland assuming increased importance in hyperionic regulation. The hepatopancreas, meanwhile, as a digestive organ, is less important in ion transport. Not surprisingly, different isolation procedures are required for BLMV isolation in marine vs. freshwater species. To more completely understand the evolving role of transporting epithelia in intermolt crustaceans, it will be necessary to isolate and characterize BLMV from terrestrial species. Loss of contact with water is associated with decreased involvement of the gills in ion exchange and increased reliance on the hepatopancreas, as food becomes the primary source of electrolyte influx. Meanwhile, the antennal gland shifts its emphasis from ion regulation to water conservation.

Isolated BLMV will be used to functionally characterize Ca2+ uptake mechanisms at the gills, antennal gland, and hepatopancreas in intermolt crayfish. A longer-term goal is to compare the relative role of the primary ion transporting epithelia in Ca2+ transport during premolt and postmolt for comparison with intermolt. Because crayfish are seldom synchronized in their natural molting cycle, it will be necessary to scale down the isolation procedure and enhance sensitivity of the characterization assays, so that BLMV can be successfully isolated from individual crayfish. Once basolateral processes have been characterized, it will be interesting to assess the evolving function of apical Ca2+ transfer mechanisms.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Greg Ahearn (Univ. of Hawaii), Dr. Gert Flik (Univ. of Nijmegen), and Dr. David Towle (Lake Forest College) for technical advice over the past two years. We also thank Dr. Robert Putnam (Dept. of Physiology and Biophysics, Wright State Univ.) and Dr. Adrian Corbett (Dept. of Biochemistry and Molecular Biology, Wright State Univ.) for many useful discussions in designing the vesicle preparation and for access to equipment. Dr. Lawrence J. Prochaska (Dept. of Biochemistry and Molecular Biology) assisted with the cytochrome-c oxidase determinations, and Mr. James Rogers prepared the electron micrographs.

    FOOTNOTES

This investigation was supported by National Science Foundation Grants DCB-8916412, IBN-9307290, and 9603723 (M. G. Wheatly).

Address reprint requests to M. G. Wheatly.

Received 18 March 1997; accepted in final form 7 November 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Regul Integr Compar Physiol 274(3):R725-R734
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