p-Chloromercurobenzene sulfonate inhibition of active Cltransport in plasma membrane vesicles from Aplysia gut GEORGE A. GERENCSER Department of Physiology, College of Medicine, University of Florida, Gainesville, Florida 32610
GERENCSER, GEORGE A. p-Chloromercurobenzenesulfonate brane of locust rectal enterocytes that was independent inhibition of active Cl- transport in plasma membrane vesicles of both Na+ and HCOT. Additionally, Gerencser (7, 8) from Aplysia gut. Am. J. Physiol. 259 (Regulatory Integrative has demonstrated that the short-circuit current (SCC)
Comp. Physiol. 28): Rllll-Rll16, 1990.-Both a Cl-stimulated adenosinetriphosphatase(ATPase) activity and an ATPdependentCl- transport processwere found in Aplysia foregut absorptive cell plasma membranes.In an attempt to further characterize this transport process,plasmamembranevesicles from Aplysia foregut absorptive cells were prepared utilizing differential centrifugation and sucrosedensity-gradient techniques.Sulfhydryl ligand participation in ATP-dependent Cltransport was confirmed in three ways. First, 1,4-dithiothreito1 partially restored a p-chloromercurobenzene sulfonate (PCMBS)-inhibited ATP-dependent Cl- transport. Second, 1,4-dithiothreitol restored intravesicular negativity inhibited by PCMBS. Third, 1,4-dithiothreitol had no effect on either ATP-dependent Cl- transport or ATP-dependent intravesicular negativity inhibited by N-ethylmaleimide. These results are consistent with the hypothesis that surface sulfhydryl groupsparticipate in the functioning of the active electrogenic Cl- transport mechanismin Aplysia gut.
across Aplysia californica foregut is primarily a net active Cl- absorptive flux in low HCOT-containing media, whereas the remainder of the SCC is wholly or predominantly a net Na+ absorptive flux. Furthermore, Gerencser et al. (15) have shown that the SCC across A. californica foregut, bathed in a Na+-free seawater saline, is identical to a net mucosal-to-serosal Cl- flux. Because it had been demonstrated that intracellular Cl- activity in the foregut villus absorptive cells was at a lower electrochemical potential than in the extracellular medium (U), even in the absence of extracellular Na+ (9), it was hypothesized that Cl- transport across the Aplysia foregut was mediated by a primary active transport process. Furthermore, Gerencser and Lee (13) demonstrated the existence of a Cl--stimulated ATPase activity in Aplysia foregut absorptive cell plasma membranes, suggesting that this enzyme could be the mechanism mediating the active absorption of Cl- by the Aplysia gut. Lending Aplysia californica; sulfhydryl ligands;chloride absorption strength to this conclusion, Gerencser and Lee (12) and Gerencser (10) have demonstrated an ATP-dependent Cl- uptake in Aplysia foregut absorptive cell plasma GUT Cl- transport has been described by two processes membrane vesicles (FAMV). (5, 11). The first is secondarily active and is thought to The present study was therefore undertaken to further be effected through a Na+-coupled symport process with characterize the ATP-driven Cl- accumulative mechathe extra- to intracellular electrochemical potential gra- nism, especially its possible relationship to active sulfhydient for Na’ as the source of free energy driving Cldry1 ligands, in Aplysia FAMV. into the gut epithelial cells (2). Examples of this process have been demonstrated in the following gut epithelia: AND METHODS prawn (l), flounder (4), sculpin (18), marine eel (25), MATERIALS bullfrog (23), rabbit (22), and human (26). The second Materials. Seahares (A. californica) were obtained from process involves Cl--HCO; exchange and is found in the Marinus (Westchester, CA) and were maintained at 25°C gut epithelia of Amphiuma (16), rat (19), rabbit (6), and in circulating filtered seawater. Adult Aplysia (400-600 human (26). g) were used in these experiments, and in most cases There is, as yet, no rigorous, direct proof of primary only animals that had been kept in the laboratory under active Cl- transport, i.e., an adenosinetriphosphatase the above conditions for 51 wk were used. Posterior (ATPase) that translocates Cl- up its electrochemical foregut (crop) was used as in all other studies (l5), as gradient powered by the simultaneous hydrolysis of ATP. shown by Moran and Garretson (21), who demonstrated In fact, the evidence for a primary active Cl- transport that foregut, not midgut, was my experimental system. mechanism in plants (11, 15) and bacteria (15) is more N-ethylmaleimide (NEM), p-chloromercurobenzene convincing than what has been demonstrated in animal sulfonate (PCMBS), diamide, imidazole, tris( hydroxyplasma membranes (5, 11). methyl)aminomethane (Tris)-ATP, triphenylmethylHowever, there exist several gut Cl- transport studies phosphonium (TPMP+) bromide, EDTA, and phenylin which the proposed process does not coincide with methylsulfonyl fluoride (PMSF) were purchased from either the symport or countertransport models. For ex- Sigma Chemical, and 1,4-dithiothreitol (DTT) was from Research Organics. [3H]methyltriphenylphosphonium ample, Hanrahan and Phillips (17) described an electrogenie Cl- accumulative mechanism in the mucosal mem- ( [3H]TPMP+) bromide and [carboxylJ4C]inulin were ob0363-6119/90
$1.50 Copyright
0 1990 the American
Physiological
Society
Rllll
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R1112
CL- TRANSPORT
tained from New England Nuclear, and H36C1was purchased from Amersham. All other chemicals were of reagent grade purity. PCMBS was always made with equimolar EDTA to scavenge any free mercury. Preparation of membrane vesicles. The plasma membrane vesicles were prepared from Aplysia foregut by homogenization followed by differential and discontinuous sucrose density-gradient centrifugation techniques, as previously demonstrated (10, 13). The animals were decapitated, and their foreguts were removed, slit longitudinally, and rinsed off in seawater to remove any adhering residue. All subsequent steps in the procedure were carried out on ice. Briefly, the slit guts from three to four animals were transferred from seawater and rinsed with ice-cold saline (0.9% NaCl) that was buffered with 10 mM imidazole, pH 7.8. Imidazole was titrated with 1 N HCl to pH 7.8 in every bathing medium used, because both the Cl--stimulated ATPase activity (13) and the ATP-dependent Cl- transport (10) found in the plasma membrane fraction used in this study had a pH optimum of 7.8 (10, 13, 15). The mucosa was gently scraped with a glass microscope slide to remove the foregut absorptive cells. The scraped cells and adventitious tissues were weighed and dispersed in homogenizing buffer [(in mM) 250 sucrose, 10 imidazole-HCl (pH 7.8), 0.5 EDTA, and 0.1 PMSF] at 1 g tissue/20 ml buffer (1:20). The suspension was homogenized using a glassTeflon homogenizer (5 strokes at 1,000 rpm). The homogenate was filtered through four layers of gauze, and the filtrate was diluted with additional homogenization buffer to give a final ratio of 1 g tissue to 100 ml buffer. The suspension was rehomogenized using a polytron (Brinkman Instruments) equipped with a PT 20 ST probe by pulsing three times for 20 s at I-min intervals. Then the homogenate was centrifuged at 800 g for 10 min. The pellet was discarded and the supernatant centrifuged at 9,500 g for 10 min. The second pellet was removed and the supernatant recentrifuged at 35,000 g for 30 min. The resulting pellet (crude membranes) was resuspended by homogenization in 2.0 ml of a 50% sucrose solution with five strokes of a glass-Teflon homogenizer at 1,000 rpm and then placed at the bottom of a 5-ml centrifuge tube. Solutions of 40% (1.5 ml), 30% (1.0 ml), and 20% (0.5 ml) sucrose were layered over the 50% sucrose membrane suspension before a 90-min, 200,000 g centrifugation (Beckman, L5-50, SW 50.1 rotor). The membrane layer at the 40-50% interface was removed, suspended in either a choline chloride medium [(in mM) imidazole-HCl (pH 7.8), 250 sucrose, 3 MgS04, and 25 choline chloride] or a sucrose buffer [(in mM) 280 sucrose, 10 imidazole-HCl; pH 7.8) according to experimental conditions, and centrifuged at 200,000 g for 60 min to obtain a pellet that was then homogenized in a choline chloride medium (above) or a sucrose buffer (above) with a glass-Teflon homogenizer and recentrifuged at 200,000 g for 60 min. The final membrane pellet was suspended in the same respective buffer at 2 mg protein/ml. Assay of enzymes. Alkaline phosphatase (EC 3.1.3.1) and Na+-K+-ATPase were assayed as described previously (13). Cytochrome-c oxidase was measured according to the method of Cooperstein and Lazarow (3). Protein concentrations were determined using the Bio-Rad
BY
THE
GUT
reagent (Bio-Rad, Richmond, CA). Assay of transport (Cl- uptake). The plasma membrane vesicles (20-30 pg of protein in 10 ~1) were incubated at 25°C in 50 ~1 of reaction mixture usually containing 10 mM imidazole-HCl (pH 7.8), 250 mM sucrose, 3 mM MgS04, and 25 mM choline chloride, as shown previously (10,13). The reaction mixture also contained trace 36C1-. Both membrane vesicles and reaction mixture were preincubated at 25°C for 5 min, and Cl- uptake was initiated by the addition of the membrane vesicles to the reaction mixture. Therefore, at this stage of the methodology, the vesicles were under isosmotic equilibrium tracer exchange (initial cis, zero trans 36Cl-) conditions. Tris-ATP (5 mM), titrated to pH 7.8 by Tris base, was routinely part of the reaction mixture in one or more of the membrane vesicle aliquots. At indicated time intervals (1.0, 2.0, 5.0, 10.0, and 60.0 min), samples were removed and immediately diluted in 2.0 ml of 0°C stop solution containing 10 mM imidazole-HCl (pH 7.8) and 250 mM sucrose or one containing 300 mM sucrose, depending upon the experimental conditions. The suspensions were rapidly filtered on a membrane filter (Millipore, 0.45 pm) and washed with 2.0 ml of the same respective buffers. The radioactivity retained on the filters was assayed in Aquasol in a liquid scintillation counter (Packard Prias). Measurement of membrane electrical potential. Transmembrane electrical potential (A$) was estimated from the distribution of the lipophilic cation TPMP+ between the extra- and intravesicular space by ultrafiltration as described above and previously shown (10). After the FAMV was equilibrated with [3H]TPMP+ for 10 min at 25°C a sample was removed and filtered but not washed. The membrane filters were presoaked in choline chloride buffer containing 10 PM nonradioactive TPMP+. The extravesicular water content of each filter was calculated from the [carboxyl-14C]inulin space. The distribution ratio of TPMP+ was estimated with a correction for the extravesicular contamination by [3H] TPMP? Nonspecific binding of TPMP+ to the vesicular membranes (10) was also corrected for by subtracting from the total A+ and was obtained by using nonionic media in the membrane preparative, reaction mixture, and ultrafiltration stages of the TPMP+ electrical potential-difference assay. When used, the reactant PCMBS (2 mM), NEM (2 mM), diamide (2 mM), or DTT (2 mM) was preincubated with the plasma membrane vesicles for 1. Distribution of marker enzymes during preparation of plasma membranes from Aplysia foregut absorptive cells TABLE
Enzyme
Na’-K’-ATPase Alkaline phosphatase Cytochrome-c oxidase
H
FAMV
0.92-1-0.38 1.06t0.43 0.72t0.20
7.28k1.85 0.86t0.30 ND
Values are means t SE from 6 different preparations. Conditions for enzyme assay were as described in MATERIALS AND METHODS. Enzyme activity is expressed as prnol. h-l mg protein-’ for Na+-K’ATPase and alkaline phosphatase and as Alog (ferrocytochrome c). min-’ . mg protein-’ for cytochrome-c oxidase. H, homogenate; FAMV, 40-50% sucrose interface; ND, not detectable. Starting gut mucosa was -5.0 g. l
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CL-
TRANSPORT
160
2 E ii
140
I-
120
I-
F a, a E 5
100
yF
60
4 I_ 0
R1113
BY THE GUT
z‘5
180
E 5
loo60-
-
80
I t
Oo
I 10
I 20
I 30
I 40
I 50
I 60
I 70
TIME (mid
2. Time course of both ATP-dependent and ATP-independent Cl- uptake in Aplysia FAMV. l , ATP present in extravesicular medium; 0, ATP absent in extravesicular medium. Intra- and extravesicular medium composition was as in Fig. 1. Values are means + SE from 2 experiments (6 animals). FIG.
40 20 0
0
04
0.8 [PCMBSI,
1.6
2.0
FIG. 1. Effect of p-chloromercurobenzene sulfonate (PCMBS) concentration on uptake of Cl- by Aplysia foregut absorptive cell plasma membrane vesicles (FAMV). Membrane vesicles were preincubated for 10 min with various concentrations of PCMBS (0.4, 0.8, 1.6, and 2.0 mM). Open bars, ATP-dependent and ATP-independent Cl- uptakes; shaded bars, ATP-independent Cl- uptakes. Intra- and extravesicular medium composition was as follows: 10 mM imidazole-HCl (pH 7.8), 250 mM sucrose, 3 mM MgS04, and 25 mM choline chloride. Values are means t SE from 6 experiments (18-24 animals).
1
2rnM PCMBS
I
mM
2. Effect of diamide or DTT on steady-state total Cl- uptake in FAMV
TABLE
Reactant
ATP
Cl- Uptake, nmol/mg protein
Control + 144.6k12.9 Control 68.Ok8.7 Diamide + 127.Ok14.1 Diamide 60.6zk4.9 DTT + 124.1k10.3 DTT 64.7k4.3 Values are means & SE from 6 different experiments (18-24 animals). Diamide (2 mM) and/or 1,4-dithiothreitol (DTT; 2 mM) was either preincubated with membrane vesicles in reaction mixture (50 ~1 containing 10 mM imidazole-HCl, 250 mM sucrose, 3 mM MgS04, and 25 mM choline chloride) at pH 7.8 for 10 min at 25°C or 5 mM ATP was added to reaction mixture to initiate incubation. Incubation for uptake of %- was measured for 2.5 min at 25°C. +, compound’s presence in reaction mixture; -, compound’s absence. Intravesicular medium matched reaction mixture (extravesicular medium) in both composition and pH.
10 min at 25°C and the time interval for inhibition of A$ in the membrane vesicles was chosen at 10 min (9). A$ was calculated from the Nernst equation (at 25°C) Arc/ = -58.8 log([TPMP+]J[TPMP’3,,t) where [TPMP+], and [TPMP+]., are the intra- and extravesicular concentrations of TPMP+, respectively. All data are reported as means + SE. Differences between means were analyzed statistically using Student’s t test with a P < 0.05 used as the statistical significant difference criterion. RESULTS
Marker enzymes. Differential
cosal homogenates
followed
centrifugation of the muby discontinuous sucrose
OoW1
20
b 40’
30
60
’
60
’
70
TIME (mid
FIG. 3. Time course of PCMBS inhibition on Cl- uptake in Aplysia FAMV. Membrane vesicles were incubated for either 5, 10, 25, 50, or 60 min with 2 mM PCMBS at 25°C. l , ATP present in extravesicular medium. 0, ATP absent in extravesicular medium. Intra- and extravesicular medium composition was as in Fig. 1. Values are means + SE from 6 experiments (18-24 animals).
160
a F al 5 E 5
loo80-
e F
60-
3 ‘G
40 20 0 0.4
0.8 INEMI.
1.6
20
mM
FIG. 4. Effect of N-ethylmaleimide (NEM) concentration on uptake of Cl- by Aplysia FAMV. Membrane vesicles were preincubated for 10 min with various concentrations of NEM (0.4, 0.8, 1.6, and 2.0 mM). Open bars, ATP-dependent and ATP-independent Cl- uptakes; shaded bars, ATP-independent Cl- uptakes. Intra- and extravesicular medium composition was as in Fig. 1. Values are means +- SE from 6 experiments (18-24 animals).
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R1114
TRANSPORT
CL-
BY THE GUT 160
160
2mM
NEM
i’ ’
140
NEM DTT
i P P1
-f - i
20
20 0
2mM 2mM
t
000 0
2
4
6
8
10
12
14
16
18
20
2
4
6
50
5. Time course of NEM inhibition on Cl- uptake in Aplysia FAMV. Membrane vesicles were incubated for either 5, 10, ‘25, 50, or 60 min with 2 mM NEM at 25°C. l , ATP present in extravesicular medium; 0, ATP absent in extravesicular medium. The intraand extravesicular medium composition was as in Fig. 1. Values are means t SE from 6 experiments (18-24 animals). FIG.
2mM
DTT
Reactants
i
.f 20
0
I
I
I
I
1
2
4
6
8
10
11
I,
I
20
I
I
30
40
I
I
I
50
60
70
TIME (min) FIG. 6. Effect of 1,4dithiothreitol (DTT) on PCMBS-inhibited Cluptake in Aplysia FAMV. Membrane vesicles were first preincubated for various time periods (5 and 10 min) with 2 mM PCMBS (all in the presence of 5 mM ATP) at 25°C. Immediately after the lo-min time period, remaining samples of vesicles were preincubated with 2 mM DTT for 5, 10, and 50 min at 25°C. Intra- and extravesicular medium composition was as in Fig. 1. Values are means f: SE from 6 experiments ( 18-24 animals).
density-gradient centrifugation produced a plasma membrane fraction located at the 40-50% sucrose interface identified previously as putative basolateral membranes (13). As shown in Table 1, ouabain-sensitive Na+-K+ATPase activity (a basolateral membrane marker) was enriched approximately eight-fold in the membrane preparation compared with that found in the initial homogenate. Alkaline phosphatase activity (a brush-border membrane marker) was enriched only O.&fold in the same preparation, whereas cytochrome-c oxidase (a mitochondrial marker) was below detectable activity levels. Characteristics of inhibitors on Cl- transport. To ascertain the optimal conditions for the inhibitors PCMBS
A+, mV
ATP ATP + PCMBS
O.Ot3.6 -20.2zk1.6 -14.6t2.0
ATP ATP ATP ATP
-22.7k2.1 -14.8t1.8 -16.5t1.3 -21.5t1.6
None
0
1
30
I
I
40
50
3. Effect of reactants on ATP-dependent
L
i
’
’ 20
I
60
J
70
FIG. 7. Effect of DTT on NEM-inhibited Cl- uptake in Aplysia FAMV. Membrane vesicles were first preincubated for various time periods (5 and 10 min) with 2 mM NEM (all in presence of 5 mM ATP) at 25°C. Immediately after lo-min time period, remaining samples of vesicles were preincubated with 2 mM DTT for 5, 10, and 50 min at 25°C. Intra- and extravesicular medium composition was as in Fig. 1. Values are means of: SE from 6 experiments (18-24 animals).
TABLE
PCMBS
2mM
140
10
TIME (mid
TIME (min)
160
8
+ + + +
PCMBS + DTT NEM NEM + DTT DTT
Inhibition,
A+ %
28 27 18
Values are means t SE for 6 different experiments (18-24 animals). Reactants were preincubated with membrane vesicles in reaction mixture [50 ~1 containing 10 nM imidazole-HCl (pH 7.8), 250 mM sucrose, 3 mM MgSO,, 25 mM choline chloride, and 10 PM triphenylmethylphosphonium (TPMP+)] at concentrations of 2 mM for 10 min at 25°C. ATP (5 mM) was added to reaction mixture to initiate 15-s incubation period, which was done at 25°C. When DTT (2 mM) was added to ATP + p-chloromercurobenzene sulfonate (PCMBS) or ATP + N-ethylmaleimide (NEM) protocols, an additional lo-min incubation period was done at 25°C before samples were taken. Intravesicular medium matched the extravesicular medium in both composition and pH. TPMP+ nonspecifically bound to membrane vesicles was accounted for in final computation of transmembrane electrical potential (A$). Negative sign in A$ denotes intravesicular polarity relative to extracellular bathing medium (reaction mixture).
and NEM on ATP-stimulated uptake of Cl- in FAMV, studies on both time course and concentration dependency were conducted. As shown in Fig. 1, the concentrations of PCMBS that gave maximal inhibition of ATP-dependent Cl- uptake in FAMV was 0.8 mM (clear bars). Also shown are controls at 0.4, 0.8, 1.6, or 2.0 mM PCMBS, in the absence of ATP (shaded bars), which gave no significant difference in their Cl- uptakes from that observed for the ATP-independent Cl- uptake (Table 2). Figure 2 shows both ATP-dependent and ATP-independent Cluptake in FAMV, in the absence of any drug perturbations, as a function of time (O-70 min). As can be seen, the ATP-dependent Cl- uptake is always greater than the corresponding Cl- uptake in the absence of ATP at
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CL- TRANSPORT
BY THE GUT
Rlll5
icant effect on ATP-dependent Cl- transport, it is not unreasonable to assume that the oxidized disulfide group plays no role relative to the ATP-dependent Cl- transport mechanism because diamide is a known oxidizing agent of sulfhydryl ligands (20). Additionally, it can be assumed that sulfur moieties reduced by DTT, a specific thiol-reducing agent (24), in the absence of sulfur binding by PCMBS, play no role relative to the active Cl- transport mechanism in FAMV (Table 2). As demonstrated in the present study (Figs. 6 and 7), the addition of either PCMBS or NEM to plasma membrane vesicles of Aplysia enterocytes evoked an inhibition of ATP-dependent Cl- transport significantly different from that of control. Although PCMBS and NEM are not absolutely specific for sulfhydryl ligands and have been shown to inhibit other ligands such as carboxyl, amino, phosphoryl, and tyrosyl (24), it is strongly suggested that their inhibition was, in a major part, through sulfhydryl ligand binding, since DTT, a specific thiolreducing agent (24), partially reversed the inhibition by PCMBS (Fig. 6). Similarly, the observation that DTT restored the vesicular A$ inhibited by PCMBS (Table 3) 2. lends further support for this hypothesis. Also buttressing this argument is the fact that PCMBS binding to a Effect of DTT on PCMBS- and NEM-inhibited Cluptake. To examine the possible involvement of sulfhysulfhydryl ligand forms a mercaptide complex, which is dry1 ligands in ATP-dependent Cl- transport, DTT was an easily reversible complex in the presence of thiolreducing agents (24). Again, the reversibility of ATPutilized in concert with the inhibitors PCMBS and NEM. As shown in Fig. 6, PCMBS (2 mM) inhibition of ATPdependent Cl- transport and ATP-dependent A# by dependent Cl- uptake in FAMV at 20 min (see Figs. 1 DTT, in the presence of PCMBS (Fig. 6 and Table 3), and 2) was partially reversed at 25, 30, and even 70 min suggests sulfhydryl-ligand participation in these activiwith 2 mM DTT addition to the FAMV at the ZO- to Zlties. NEM interaction with sulfhydryl ligands is through alkylation, and these covalent bonds are not easily remin time period. However, as observed in Fig. 7, there was no restoration of ATP-dependent Cl- uptake in versed through chemical means (24). Therefore, possibly, FAMV on 2 mM DTT addition to the vesicles at the ZO- the reason why DTT had no effect on the NEM-induced of both ATP-dependent Cl- transport and to Zl-min time period after 2 mM NEM inhibition of inhibition ATP-dependent Cl- uptake, even at 70 min. ATP-dependent vesicular A$ (Fig. 7 and Table 3) was Effect of inhibitors on membrane potential measured by because DTT could not reverse the relatively strong covalent linkages between NEM and the sulfhydryl liTPMP+ distribution. To further evaluate factors involved in ATP-dependent Cl- transport, the effects of gands. Of course, this does not rule out NEM effects on other ligands, which could, directly or indirectly, inhibit several inhibitors on vesicular membrane potential Cl- transport and ATP-dependent changes (A+) induced by ATP in the presence of Cl- both ATP-dependent were studied. As shown in Table 3, the ATP-dependent A$ in FAMV. The finding that PCMBS and/or NEM inhibitions of A$ was inhibited by both 2 mM PCMBS and 2 mM NEM. DTT (2 mM) fully restored the ATP-dependent Cl- transport or A$ are discrepant will be briefly considA\C/inhibited by PCMBS; however, it did not have any ered. The most probable reason why PCMBS and NEM ATP-dependent Cl- transport and ATP-destatistically significant restorative effect on the A+ in- inhibited pendent A$ to differing degrees rests with how each of hibited by NEM. Also shown in Table 3 was the finding that 2 mM DTT had no significant effect on the ATPthese parameters was measured. ATP-dependent Cltransport is a simple, direct, radioisotopic measurement, dependent AI/ (control). whereas ATP-dependent A$ is an indirect, complex, double radioisotopic measurement in which radioisotopic DISCUSSION determinations of intravesicular TPMP+ concentrations Basic to the present study are the findings that bathed in media high in TPMP+ concentrations are affected in a large way by relatively small errors in PCMBS and NEM inhibited ATP-dependent Cl- transport in FAMV and that these inhibitions can be further extracellular space measurements, which are also measdefined in terms of time courses and concentration de- ured radioisotopically. Therefore, small errors in extracellular space measurements can magnify errors in intrapendencies (Figs. l-5). Thus these empirically derived optimal conditions validated any ensuing experiments vesicular concentrations ( A$). utilizing these reactants. Also fundamental to the present PCMBS is thought to interact with surface sulfhydryl study were the findings that neither diamide nor DTT groups because of its negatively charged sulfonic group, had any significant effect on ATP-dependent Cl- transwhich reduces its lipid solubility (24). In contrast, NEM is very lipid soluble and binds to both surface and intraport in FAMV (Table 2). Because diamide had no signifany given time point. As shown in Fig. 3, PCMBS (2 mM) inhibited ATP-dependent Cl- uptake in FAMV as a function of time with maximal inhibition occurring at 60 min after addition of PCMBS. Similarly, as shown in Fig. 4, the concentration of NEM that gave maximal inhibition of ATP-dependent Cl- uptake in FAMV was 1.6 mM; however, there was a further, though statistically insignificant, decrease of this flux at 2.0 mM NEM. Controls at 0.4,0.8, 1.6, or 2.0 mM NEM, in the absence of ATP, showed no significant difference in their Cl- uptakes from that observed for the ATP-independent Cl- uptake (Table 1). As seen in Fig. 5, NEM (2 mM) inhibited ATP-dependent Cl- uptake in FAMV as a function of time with maximal inhibition occurring at 10 min after addition of NEM. As shown in Table 2, both 2 mM diamide, an oxidizing agent of sulfhydryl ligands (ZO), and 2 mM DTT, a specific sulfhydryl-reducing reactant (24), had no significant effect on the ATP-dependent or ATP-independent Cl- transport in FAMV. Not shown in Table 2 was the finding that order of addition of ATP or diamide and/or DTT to FAMV had no effect on the results seen in Table
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R1116
CL-
TRANSPORT
membranous sulfhydryl ligands (24). Because PCMBS inhibited both ATP-dependent Cl- transport and ATPdependent A$ and these inhibitions were approximately equal to those induced by an equal concentration of NEM, it is highly suggestive that surface sulfhydryl ligands, not intramembranous sulfhydryl groups, are at least in part responsible, either directly or indirectly, for both ATP-dependent Cl- transport and ATP-dependent A$ in Aplysia gut. These results, coupled with previous observations (10, 13, 15), are strongly consistent with the hypothesis that the active electrogenic Cl- absorptive mechanism in Aplysia gut is an electrogenic, Cl--transporting, Cl-stimulated ATPase found in the foregut absorptive cell plasma membrane. I thank Dr. Bruce Stevens for helpful suggestions during the course of the study, acknowledge the excellent technical assistance of F. Robbins, and thank K. Fortin for assistance with the typing. This investigation was supported by the Eppley Foundation for Research. Address for reprint requests: Dept. of Physiology, Box J-274, JHMHC, Univ. of Florida, Gainesville, FL 32610. Received
3 January
1990; accepted
in final
form
17 July
1990.
REFERENCES 1. AHEARN, G. A., L. A. MAGINNISS, Y. K. SONG, AND A. TORNQUIST. Intestinal water and ion transport in fresh water malacostracan prawns (Crustacea). In: Water Relations in Membrane Transport in Plants and Animals, edited by A. M. Jungreis, T. K. Hodges, A. Kleinzeller, and S. G. Schultz. New York: Academic, 1977, p. 129142. 2. ARMSTRONG, W. McD., W. R. BIXENMAN, K. F. FREY, J. F. GARCIA-DIAZ, M. G. O’REGAN, AND J. H. OWENS. Energetics of coupled Na’ and Cl- entry into epithelial cells of bullfrog small intestine. Biochim. Biophys. Acta 551: 207-219, 1979. 3. COOPERSTEIN, S. J., AND A. LAZAROW. A microspectrophotometric method for the determination of cytochrome oxidase. J. Biol. Chem. 189: 665670,195l. 4. FIELD, M., K. J. KARNAKY, P. L. SMYTH, J. E. BOLTON, AND W. B. KINTER. Ion transport across the isolated intestinal mucosa of the winter flounder, Pseudopleuronectes americanus. I. Functional and structural properties of cellular and paracellular pathways for Na and Cl. J. Membr. Biol. 41: 265-275, 1978. 5. FRIZZELL, R. A., M. FIELD, AND S. G. SCHULTZ. Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol. 236 (Renal Fluid Electrolyte Physiol. 5): Fl-FB, 1979. 6. FRIZZELL, R. A., M. J. KOCH, AND S. G. SCHULTZ. Ion transport by rabbit colon. I. Active and passive components. J. Membr. Biol. 27: 297-316, 1976. 7. GERENCSER, G. A. Electrical characteristics of isolated Aplysia californica intestine. Comp. Biochem, Physiol. A Comp. Physiol. 61: 209-212,1978. 8. GERENCSER, G. A. Enhancement of sodium and chloride transport
BY
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
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24.
25.
26.
THE
GUT
by monosaccharides in Aplysia californica intestine. Comp. Biochem. Physiol. A Camp. Physiol. 61: 203-208, 1978. GERENCSER, G. A. Electrophysiology of chloride transport in Aplysia (mollusk) intestine. Am. J. Physiol. 244 (Regulatory Integrative Comp. Physiol. 13): R143-R149, 1983. GERENCSER, G. A. Electrogenic ATP-dependent Cl- transport by plasma membrane vesicles from Aplysia intestine. Am. J. Physiol. 254 (Regulatory Integrative Camp. Physiol. 23): R127-R133, 1988. GERENCSER, G. A., AND S. H. LEE. Cl--stimulated adenosine triphosphatase: existence, location and function. J. Exp. Biol. 106: 143-161,1983. GERENCSER, G. A., AND S. H. LEE. ATP-dependent chloride transport in plasma membrane vesicles from Aplysia gut. Biochim. Biophys. Acta 816: 415-417, 1985. GERENCSER, G. A., AND S. H. LEE. Cl--HCOT-stimulated ATPase in intestinal mucosa of Aplysia. Am. J. Physiol. 248 (Regulatory Integrative Comp. Physiol. 17): R241-R248, 1985. GERENCSER, G. A., AND J. F. WHITE. Membrane potentials and chloride activities in epithelial cells of Aplysia intestine. Am. J. Physiol. 239 (Regulatory Integrative Comp. Physiol. 8): R445-R449, 1980. GERENCSER, G. A., J. F. WHITE, D. GRADMANN, AND S. L. BONTING. Is there a Cl- pump? Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R677-R692, 1988. GUNTER-SMITH, P. J., AND J. F. WHITE. Response of Amphiuma small intestine to theophylline: effect of bicarbonate transport. Am. J. Physiol. 236 (Endocrinol. Metab. Gastrointest. Physiol. 5): E775-E783,1979. HANRAHAN, J., AND J. E. PHILLIPS. Mechanism and control of salt absorption in locust rectum. Am. J. Physiol. 244 (Regulatory Integrative Comp. Physiol. 13): R131-R142, 1983. HOUSE, C. R., AND K. GREEN. Ion and water transport in isolated intestine of the marine teleost, Cottus scorpius. J. Exp. Biol. 42: 177-189,1965. HUBEL, K. A. Effect of luminal chloride concentration on bicarbonate secretion in rat ileum. Am. J. Physiol. 217: 40-45, 1969. LAUF, P. K. Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide. J. Membr. Biol. 101: 179-188, 1988. MORAN, W. M., AND L. T. GARRETSON. Sugar-stimulated ion absorption is not different in seahare and vertebrate intestine. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R583R590,1988. NELLANS, H. N., R. A. FRIZZELL, AND S. G. SCHULTZ. Coupled sodium-chloride influx across the brush border of rabbit ileum. Am. J. Physiol. 225: 467-475, 1973. QUAY, J. F., AND W. M. ARMSTRONG. Sodium and chloride transport by isolated bullfrog small intestine. Am. J. Physiol. 217: 694702, 1969. ROTHSTEIN, A. Sulfhydryl groups in membrane structure and function. In: Current Topics in Membranes and Transport, edited by F. Bronner and A. Kleinzeller. New York: Academic, 1970, p. 135-176. SKADHAUGE, E. Coupling of transmural flows of NaCl and water in the intestine of the eel (Anguilla anguilla). J. Exp. Biol. 60: 535546,1974. TURNBERG, L. A., F. A. BISBERDORF, S. G. MORAWAKI, AND J. S. FORDTRAN. Interrelationships of chloride, bicarbonate, sodium and hydrogen transport in the human ileum. J. Clin. Invest. 49: 557567,197O.
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GERENCSER, GEORGE A. p-Chloromercurobenzenesulfonate brane of locust rectal enterocytes that was independent inhibition of active Cl- transport in plasma membrane vesicles of both Na+ and HCOT. Additionally, Gerencser (7, 8) from Aplysia gut. Am. J. Physiol. 259 (Regulatory Integrative has demonstrated that the short-circuit current (SCC)
Comp. Physiol. 28): Rllll-Rll16, 1990.-Both a Cl-stimulated adenosinetriphosphatase(ATPase) activity and an ATPdependentCl- transport processwere found in Aplysia foregut absorptive cell plasma membranes.In an attempt to further characterize this transport process,plasmamembranevesicles from Aplysia foregut absorptive cells were prepared utilizing differential centrifugation and sucrosedensity-gradient techniques.Sulfhydryl ligand participation in ATP-dependent Cltransport was confirmed in three ways. First, 1,4-dithiothreito1 partially restored a p-chloromercurobenzene sulfonate (PCMBS)-inhibited ATP-dependent Cl- transport. Second, 1,4-dithiothreitol restored intravesicular negativity inhibited by PCMBS. Third, 1,4-dithiothreitol had no effect on either ATP-dependent Cl- transport or ATP-dependent intravesicular negativity inhibited by N-ethylmaleimide. These results are consistent with the hypothesis that surface sulfhydryl groupsparticipate in the functioning of the active electrogenic Cl- transport mechanismin Aplysia gut.
across Aplysia californica foregut is primarily a net active Cl- absorptive flux in low HCOT-containing media, whereas the remainder of the SCC is wholly or predominantly a net Na+ absorptive flux. Furthermore, Gerencser et al. (15) have shown that the SCC across A. californica foregut, bathed in a Na+-free seawater saline, is identical to a net mucosal-to-serosal Cl- flux. Because it had been demonstrated that intracellular Cl- activity in the foregut villus absorptive cells was at a lower electrochemical potential than in the extracellular medium (U), even in the absence of extracellular Na+ (9), it was hypothesized that Cl- transport across the Aplysia foregut was mediated by a primary active transport process. Furthermore, Gerencser and Lee (13) demonstrated the existence of a Cl--stimulated ATPase activity in Aplysia foregut absorptive cell plasma membranes, suggesting that this enzyme could be the mechanism mediating the active absorption of Cl- by the Aplysia gut. Lending Aplysia californica; sulfhydryl ligands;chloride absorption strength to this conclusion, Gerencser and Lee (12) and Gerencser (10) have demonstrated an ATP-dependent Cl- uptake in Aplysia foregut absorptive cell plasma GUT Cl- transport has been described by two processes membrane vesicles (FAMV). (5, 11). The first is secondarily active and is thought to The present study was therefore undertaken to further be effected through a Na+-coupled symport process with characterize the ATP-driven Cl- accumulative mechathe extra- to intracellular electrochemical potential gra- nism, especially its possible relationship to active sulfhydient for Na’ as the source of free energy driving Cldry1 ligands, in Aplysia FAMV. into the gut epithelial cells (2). Examples of this process have been demonstrated in the following gut epithelia: AND METHODS prawn (l), flounder (4), sculpin (18), marine eel (25), MATERIALS bullfrog (23), rabbit (22), and human (26). The second Materials. Seahares (A. californica) were obtained from process involves Cl--HCO; exchange and is found in the Marinus (Westchester, CA) and were maintained at 25°C gut epithelia of Amphiuma (16), rat (19), rabbit (6), and in circulating filtered seawater. Adult Aplysia (400-600 human (26). g) were used in these experiments, and in most cases There is, as yet, no rigorous, direct proof of primary only animals that had been kept in the laboratory under active Cl- transport, i.e., an adenosinetriphosphatase the above conditions for 51 wk were used. Posterior (ATPase) that translocates Cl- up its electrochemical foregut (crop) was used as in all other studies (l5), as gradient powered by the simultaneous hydrolysis of ATP. shown by Moran and Garretson (21), who demonstrated In fact, the evidence for a primary active Cl- transport that foregut, not midgut, was my experimental system. mechanism in plants (11, 15) and bacteria (15) is more N-ethylmaleimide (NEM), p-chloromercurobenzene convincing than what has been demonstrated in animal sulfonate (PCMBS), diamide, imidazole, tris( hydroxyplasma membranes (5, 11). methyl)aminomethane (Tris)-ATP, triphenylmethylHowever, there exist several gut Cl- transport studies phosphonium (TPMP+) bromide, EDTA, and phenylin which the proposed process does not coincide with methylsulfonyl fluoride (PMSF) were purchased from either the symport or countertransport models. For ex- Sigma Chemical, and 1,4-dithiothreitol (DTT) was from Research Organics. [3H]methyltriphenylphosphonium ample, Hanrahan and Phillips (17) described an electrogenie Cl- accumulative mechanism in the mucosal mem- ( [3H]TPMP+) bromide and [carboxylJ4C]inulin were ob0363-6119/90
$1.50 Copyright
0 1990 the American
Physiological
Society
Rllll
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R1112
CL- TRANSPORT
tained from New England Nuclear, and H36C1was purchased from Amersham. All other chemicals were of reagent grade purity. PCMBS was always made with equimolar EDTA to scavenge any free mercury. Preparation of membrane vesicles. The plasma membrane vesicles were prepared from Aplysia foregut by homogenization followed by differential and discontinuous sucrose density-gradient centrifugation techniques, as previously demonstrated (10, 13). The animals were decapitated, and their foreguts were removed, slit longitudinally, and rinsed off in seawater to remove any adhering residue. All subsequent steps in the procedure were carried out on ice. Briefly, the slit guts from three to four animals were transferred from seawater and rinsed with ice-cold saline (0.9% NaCl) that was buffered with 10 mM imidazole, pH 7.8. Imidazole was titrated with 1 N HCl to pH 7.8 in every bathing medium used, because both the Cl--stimulated ATPase activity (13) and the ATP-dependent Cl- transport (10) found in the plasma membrane fraction used in this study had a pH optimum of 7.8 (10, 13, 15). The mucosa was gently scraped with a glass microscope slide to remove the foregut absorptive cells. The scraped cells and adventitious tissues were weighed and dispersed in homogenizing buffer [(in mM) 250 sucrose, 10 imidazole-HCl (pH 7.8), 0.5 EDTA, and 0.1 PMSF] at 1 g tissue/20 ml buffer (1:20). The suspension was homogenized using a glassTeflon homogenizer (5 strokes at 1,000 rpm). The homogenate was filtered through four layers of gauze, and the filtrate was diluted with additional homogenization buffer to give a final ratio of 1 g tissue to 100 ml buffer. The suspension was rehomogenized using a polytron (Brinkman Instruments) equipped with a PT 20 ST probe by pulsing three times for 20 s at I-min intervals. Then the homogenate was centrifuged at 800 g for 10 min. The pellet was discarded and the supernatant centrifuged at 9,500 g for 10 min. The second pellet was removed and the supernatant recentrifuged at 35,000 g for 30 min. The resulting pellet (crude membranes) was resuspended by homogenization in 2.0 ml of a 50% sucrose solution with five strokes of a glass-Teflon homogenizer at 1,000 rpm and then placed at the bottom of a 5-ml centrifuge tube. Solutions of 40% (1.5 ml), 30% (1.0 ml), and 20% (0.5 ml) sucrose were layered over the 50% sucrose membrane suspension before a 90-min, 200,000 g centrifugation (Beckman, L5-50, SW 50.1 rotor). The membrane layer at the 40-50% interface was removed, suspended in either a choline chloride medium [(in mM) imidazole-HCl (pH 7.8), 250 sucrose, 3 MgS04, and 25 choline chloride] or a sucrose buffer [(in mM) 280 sucrose, 10 imidazole-HCl; pH 7.8) according to experimental conditions, and centrifuged at 200,000 g for 60 min to obtain a pellet that was then homogenized in a choline chloride medium (above) or a sucrose buffer (above) with a glass-Teflon homogenizer and recentrifuged at 200,000 g for 60 min. The final membrane pellet was suspended in the same respective buffer at 2 mg protein/ml. Assay of enzymes. Alkaline phosphatase (EC 3.1.3.1) and Na+-K+-ATPase were assayed as described previously (13). Cytochrome-c oxidase was measured according to the method of Cooperstein and Lazarow (3). Protein concentrations were determined using the Bio-Rad
BY
THE
GUT
reagent (Bio-Rad, Richmond, CA). Assay of transport (Cl- uptake). The plasma membrane vesicles (20-30 pg of protein in 10 ~1) were incubated at 25°C in 50 ~1 of reaction mixture usually containing 10 mM imidazole-HCl (pH 7.8), 250 mM sucrose, 3 mM MgS04, and 25 mM choline chloride, as shown previously (10,13). The reaction mixture also contained trace 36C1-. Both membrane vesicles and reaction mixture were preincubated at 25°C for 5 min, and Cl- uptake was initiated by the addition of the membrane vesicles to the reaction mixture. Therefore, at this stage of the methodology, the vesicles were under isosmotic equilibrium tracer exchange (initial cis, zero trans 36Cl-) conditions. Tris-ATP (5 mM), titrated to pH 7.8 by Tris base, was routinely part of the reaction mixture in one or more of the membrane vesicle aliquots. At indicated time intervals (1.0, 2.0, 5.0, 10.0, and 60.0 min), samples were removed and immediately diluted in 2.0 ml of 0°C stop solution containing 10 mM imidazole-HCl (pH 7.8) and 250 mM sucrose or one containing 300 mM sucrose, depending upon the experimental conditions. The suspensions were rapidly filtered on a membrane filter (Millipore, 0.45 pm) and washed with 2.0 ml of the same respective buffers. The radioactivity retained on the filters was assayed in Aquasol in a liquid scintillation counter (Packard Prias). Measurement of membrane electrical potential. Transmembrane electrical potential (A$) was estimated from the distribution of the lipophilic cation TPMP+ between the extra- and intravesicular space by ultrafiltration as described above and previously shown (10). After the FAMV was equilibrated with [3H]TPMP+ for 10 min at 25°C a sample was removed and filtered but not washed. The membrane filters were presoaked in choline chloride buffer containing 10 PM nonradioactive TPMP+. The extravesicular water content of each filter was calculated from the [carboxyl-14C]inulin space. The distribution ratio of TPMP+ was estimated with a correction for the extravesicular contamination by [3H] TPMP? Nonspecific binding of TPMP+ to the vesicular membranes (10) was also corrected for by subtracting from the total A+ and was obtained by using nonionic media in the membrane preparative, reaction mixture, and ultrafiltration stages of the TPMP+ electrical potential-difference assay. When used, the reactant PCMBS (2 mM), NEM (2 mM), diamide (2 mM), or DTT (2 mM) was preincubated with the plasma membrane vesicles for 1. Distribution of marker enzymes during preparation of plasma membranes from Aplysia foregut absorptive cells TABLE
Enzyme
Na’-K’-ATPase Alkaline phosphatase Cytochrome-c oxidase
H
FAMV
0.92-1-0.38 1.06t0.43 0.72t0.20
7.28k1.85 0.86t0.30 ND
Values are means t SE from 6 different preparations. Conditions for enzyme assay were as described in MATERIALS AND METHODS. Enzyme activity is expressed as prnol. h-l mg protein-’ for Na+-K’ATPase and alkaline phosphatase and as Alog (ferrocytochrome c). min-’ . mg protein-’ for cytochrome-c oxidase. H, homogenate; FAMV, 40-50% sucrose interface; ND, not detectable. Starting gut mucosa was -5.0 g. l
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CL-
TRANSPORT
160
2 E ii
140
I-
120
I-
F a, a E 5
100
yF
60
4 I_ 0
R1113
BY THE GUT
z‘5
180
E 5
loo60-
-
80
I t
Oo
I 10
I 20
I 30
I 40
I 50
I 60
I 70
TIME (mid
2. Time course of both ATP-dependent and ATP-independent Cl- uptake in Aplysia FAMV. l , ATP present in extravesicular medium; 0, ATP absent in extravesicular medium. Intra- and extravesicular medium composition was as in Fig. 1. Values are means + SE from 2 experiments (6 animals). FIG.
40 20 0
0
04
0.8 [PCMBSI,
1.6
2.0
FIG. 1. Effect of p-chloromercurobenzene sulfonate (PCMBS) concentration on uptake of Cl- by Aplysia foregut absorptive cell plasma membrane vesicles (FAMV). Membrane vesicles were preincubated for 10 min with various concentrations of PCMBS (0.4, 0.8, 1.6, and 2.0 mM). Open bars, ATP-dependent and ATP-independent Cl- uptakes; shaded bars, ATP-independent Cl- uptakes. Intra- and extravesicular medium composition was as follows: 10 mM imidazole-HCl (pH 7.8), 250 mM sucrose, 3 mM MgS04, and 25 mM choline chloride. Values are means t SE from 6 experiments (18-24 animals).
1
2rnM PCMBS
I
mM
2. Effect of diamide or DTT on steady-state total Cl- uptake in FAMV
TABLE
Reactant
ATP
Cl- Uptake, nmol/mg protein
Control + 144.6k12.9 Control 68.Ok8.7 Diamide + 127.Ok14.1 Diamide 60.6zk4.9 DTT + 124.1k10.3 DTT 64.7k4.3 Values are means & SE from 6 different experiments (18-24 animals). Diamide (2 mM) and/or 1,4-dithiothreitol (DTT; 2 mM) was either preincubated with membrane vesicles in reaction mixture (50 ~1 containing 10 mM imidazole-HCl, 250 mM sucrose, 3 mM MgS04, and 25 mM choline chloride) at pH 7.8 for 10 min at 25°C or 5 mM ATP was added to reaction mixture to initiate incubation. Incubation for uptake of %- was measured for 2.5 min at 25°C. +, compound’s presence in reaction mixture; -, compound’s absence. Intravesicular medium matched reaction mixture (extravesicular medium) in both composition and pH.
10 min at 25°C and the time interval for inhibition of A$ in the membrane vesicles was chosen at 10 min (9). A$ was calculated from the Nernst equation (at 25°C) Arc/ = -58.8 log([TPMP+]J[TPMP’3,,t) where [TPMP+], and [TPMP+]., are the intra- and extravesicular concentrations of TPMP+, respectively. All data are reported as means + SE. Differences between means were analyzed statistically using Student’s t test with a P < 0.05 used as the statistical significant difference criterion. RESULTS
Marker enzymes. Differential
cosal homogenates
followed
centrifugation of the muby discontinuous sucrose
OoW1
20
b 40’
30
60
’
60
’
70
TIME (mid
FIG. 3. Time course of PCMBS inhibition on Cl- uptake in Aplysia FAMV. Membrane vesicles were incubated for either 5, 10, 25, 50, or 60 min with 2 mM PCMBS at 25°C. l , ATP present in extravesicular medium. 0, ATP absent in extravesicular medium. Intra- and extravesicular medium composition was as in Fig. 1. Values are means + SE from 6 experiments (18-24 animals).
160
a F al 5 E 5
loo80-
e F
60-
3 ‘G
40 20 0 0.4
0.8 INEMI.
1.6
20
mM
FIG. 4. Effect of N-ethylmaleimide (NEM) concentration on uptake of Cl- by Aplysia FAMV. Membrane vesicles were preincubated for 10 min with various concentrations of NEM (0.4, 0.8, 1.6, and 2.0 mM). Open bars, ATP-dependent and ATP-independent Cl- uptakes; shaded bars, ATP-independent Cl- uptakes. Intra- and extravesicular medium composition was as in Fig. 1. Values are means +- SE from 6 experiments (18-24 animals).
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R1114
TRANSPORT
CL-
BY THE GUT 160
160
2mM
NEM
i’ ’
140
NEM DTT
i P P1
-f - i
20
20 0
2mM 2mM
t
000 0
2
4
6
8
10
12
14
16
18
20
2
4
6
50
5. Time course of NEM inhibition on Cl- uptake in Aplysia FAMV. Membrane vesicles were incubated for either 5, 10, ‘25, 50, or 60 min with 2 mM NEM at 25°C. l , ATP present in extravesicular medium; 0, ATP absent in extravesicular medium. The intraand extravesicular medium composition was as in Fig. 1. Values are means t SE from 6 experiments (18-24 animals). FIG.
2mM
DTT
Reactants
i
.f 20
0
I
I
I
I
1
2
4
6
8
10
11
I,
I
20
I
I
30
40
I
I
I
50
60
70
TIME (min) FIG. 6. Effect of 1,4dithiothreitol (DTT) on PCMBS-inhibited Cluptake in Aplysia FAMV. Membrane vesicles were first preincubated for various time periods (5 and 10 min) with 2 mM PCMBS (all in the presence of 5 mM ATP) at 25°C. Immediately after the lo-min time period, remaining samples of vesicles were preincubated with 2 mM DTT for 5, 10, and 50 min at 25°C. Intra- and extravesicular medium composition was as in Fig. 1. Values are means f: SE from 6 experiments ( 18-24 animals).
density-gradient centrifugation produced a plasma membrane fraction located at the 40-50% sucrose interface identified previously as putative basolateral membranes (13). As shown in Table 1, ouabain-sensitive Na+-K+ATPase activity (a basolateral membrane marker) was enriched approximately eight-fold in the membrane preparation compared with that found in the initial homogenate. Alkaline phosphatase activity (a brush-border membrane marker) was enriched only O.&fold in the same preparation, whereas cytochrome-c oxidase (a mitochondrial marker) was below detectable activity levels. Characteristics of inhibitors on Cl- transport. To ascertain the optimal conditions for the inhibitors PCMBS
A+, mV
ATP ATP + PCMBS
O.Ot3.6 -20.2zk1.6 -14.6t2.0
ATP ATP ATP ATP
-22.7k2.1 -14.8t1.8 -16.5t1.3 -21.5t1.6
None
0
1
30
I
I
40
50
3. Effect of reactants on ATP-dependent
L
i
’
’ 20
I
60
J
70
FIG. 7. Effect of DTT on NEM-inhibited Cl- uptake in Aplysia FAMV. Membrane vesicles were first preincubated for various time periods (5 and 10 min) with 2 mM NEM (all in presence of 5 mM ATP) at 25°C. Immediately after lo-min time period, remaining samples of vesicles were preincubated with 2 mM DTT for 5, 10, and 50 min at 25°C. Intra- and extravesicular medium composition was as in Fig. 1. Values are means of: SE from 6 experiments (18-24 animals).
TABLE
PCMBS
2mM
140
10
TIME (mid
TIME (min)
160
8
+ + + +
PCMBS + DTT NEM NEM + DTT DTT
Inhibition,
A+ %
28 27 18
Values are means t SE for 6 different experiments (18-24 animals). Reactants were preincubated with membrane vesicles in reaction mixture [50 ~1 containing 10 nM imidazole-HCl (pH 7.8), 250 mM sucrose, 3 mM MgSO,, 25 mM choline chloride, and 10 PM triphenylmethylphosphonium (TPMP+)] at concentrations of 2 mM for 10 min at 25°C. ATP (5 mM) was added to reaction mixture to initiate 15-s incubation period, which was done at 25°C. When DTT (2 mM) was added to ATP + p-chloromercurobenzene sulfonate (PCMBS) or ATP + N-ethylmaleimide (NEM) protocols, an additional lo-min incubation period was done at 25°C before samples were taken. Intravesicular medium matched the extravesicular medium in both composition and pH. TPMP+ nonspecifically bound to membrane vesicles was accounted for in final computation of transmembrane electrical potential (A$). Negative sign in A$ denotes intravesicular polarity relative to extracellular bathing medium (reaction mixture).
and NEM on ATP-stimulated uptake of Cl- in FAMV, studies on both time course and concentration dependency were conducted. As shown in Fig. 1, the concentrations of PCMBS that gave maximal inhibition of ATP-dependent Cl- uptake in FAMV was 0.8 mM (clear bars). Also shown are controls at 0.4, 0.8, 1.6, or 2.0 mM PCMBS, in the absence of ATP (shaded bars), which gave no significant difference in their Cl- uptakes from that observed for the ATP-independent Cl- uptake (Table 2). Figure 2 shows both ATP-dependent and ATP-independent Cluptake in FAMV, in the absence of any drug perturbations, as a function of time (O-70 min). As can be seen, the ATP-dependent Cl- uptake is always greater than the corresponding Cl- uptake in the absence of ATP at
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CL- TRANSPORT
BY THE GUT
Rlll5
icant effect on ATP-dependent Cl- transport, it is not unreasonable to assume that the oxidized disulfide group plays no role relative to the ATP-dependent Cl- transport mechanism because diamide is a known oxidizing agent of sulfhydryl ligands (20). Additionally, it can be assumed that sulfur moieties reduced by DTT, a specific thiol-reducing agent (24), in the absence of sulfur binding by PCMBS, play no role relative to the active Cl- transport mechanism in FAMV (Table 2). As demonstrated in the present study (Figs. 6 and 7), the addition of either PCMBS or NEM to plasma membrane vesicles of Aplysia enterocytes evoked an inhibition of ATP-dependent Cl- transport significantly different from that of control. Although PCMBS and NEM are not absolutely specific for sulfhydryl ligands and have been shown to inhibit other ligands such as carboxyl, amino, phosphoryl, and tyrosyl (24), it is strongly suggested that their inhibition was, in a major part, through sulfhydryl ligand binding, since DTT, a specific thiolreducing agent (24), partially reversed the inhibition by PCMBS (Fig. 6). Similarly, the observation that DTT restored the vesicular A$ inhibited by PCMBS (Table 3) 2. lends further support for this hypothesis. Also buttressing this argument is the fact that PCMBS binding to a Effect of DTT on PCMBS- and NEM-inhibited Cluptake. To examine the possible involvement of sulfhysulfhydryl ligand forms a mercaptide complex, which is dry1 ligands in ATP-dependent Cl- transport, DTT was an easily reversible complex in the presence of thiolreducing agents (24). Again, the reversibility of ATPutilized in concert with the inhibitors PCMBS and NEM. As shown in Fig. 6, PCMBS (2 mM) inhibition of ATPdependent Cl- transport and ATP-dependent A# by dependent Cl- uptake in FAMV at 20 min (see Figs. 1 DTT, in the presence of PCMBS (Fig. 6 and Table 3), and 2) was partially reversed at 25, 30, and even 70 min suggests sulfhydryl-ligand participation in these activiwith 2 mM DTT addition to the FAMV at the ZO- to Zlties. NEM interaction with sulfhydryl ligands is through alkylation, and these covalent bonds are not easily remin time period. However, as observed in Fig. 7, there was no restoration of ATP-dependent Cl- uptake in versed through chemical means (24). Therefore, possibly, FAMV on 2 mM DTT addition to the vesicles at the ZO- the reason why DTT had no effect on the NEM-induced of both ATP-dependent Cl- transport and to Zl-min time period after 2 mM NEM inhibition of inhibition ATP-dependent Cl- uptake, even at 70 min. ATP-dependent vesicular A$ (Fig. 7 and Table 3) was Effect of inhibitors on membrane potential measured by because DTT could not reverse the relatively strong covalent linkages between NEM and the sulfhydryl liTPMP+ distribution. To further evaluate factors involved in ATP-dependent Cl- transport, the effects of gands. Of course, this does not rule out NEM effects on other ligands, which could, directly or indirectly, inhibit several inhibitors on vesicular membrane potential Cl- transport and ATP-dependent changes (A+) induced by ATP in the presence of Cl- both ATP-dependent were studied. As shown in Table 3, the ATP-dependent A$ in FAMV. The finding that PCMBS and/or NEM inhibitions of A$ was inhibited by both 2 mM PCMBS and 2 mM NEM. DTT (2 mM) fully restored the ATP-dependent Cl- transport or A$ are discrepant will be briefly considA\C/inhibited by PCMBS; however, it did not have any ered. The most probable reason why PCMBS and NEM ATP-dependent Cl- transport and ATP-destatistically significant restorative effect on the A+ in- inhibited pendent A$ to differing degrees rests with how each of hibited by NEM. Also shown in Table 3 was the finding that 2 mM DTT had no significant effect on the ATPthese parameters was measured. ATP-dependent Cltransport is a simple, direct, radioisotopic measurement, dependent AI/ (control). whereas ATP-dependent A$ is an indirect, complex, double radioisotopic measurement in which radioisotopic DISCUSSION determinations of intravesicular TPMP+ concentrations Basic to the present study are the findings that bathed in media high in TPMP+ concentrations are affected in a large way by relatively small errors in PCMBS and NEM inhibited ATP-dependent Cl- transport in FAMV and that these inhibitions can be further extracellular space measurements, which are also measdefined in terms of time courses and concentration de- ured radioisotopically. Therefore, small errors in extracellular space measurements can magnify errors in intrapendencies (Figs. l-5). Thus these empirically derived optimal conditions validated any ensuing experiments vesicular concentrations ( A$). utilizing these reactants. Also fundamental to the present PCMBS is thought to interact with surface sulfhydryl study were the findings that neither diamide nor DTT groups because of its negatively charged sulfonic group, had any significant effect on ATP-dependent Cl- transwhich reduces its lipid solubility (24). In contrast, NEM is very lipid soluble and binds to both surface and intraport in FAMV (Table 2). Because diamide had no signifany given time point. As shown in Fig. 3, PCMBS (2 mM) inhibited ATP-dependent Cl- uptake in FAMV as a function of time with maximal inhibition occurring at 60 min after addition of PCMBS. Similarly, as shown in Fig. 4, the concentration of NEM that gave maximal inhibition of ATP-dependent Cl- uptake in FAMV was 1.6 mM; however, there was a further, though statistically insignificant, decrease of this flux at 2.0 mM NEM. Controls at 0.4,0.8, 1.6, or 2.0 mM NEM, in the absence of ATP, showed no significant difference in their Cl- uptakes from that observed for the ATP-independent Cl- uptake (Table 1). As seen in Fig. 5, NEM (2 mM) inhibited ATP-dependent Cl- uptake in FAMV as a function of time with maximal inhibition occurring at 10 min after addition of NEM. As shown in Table 2, both 2 mM diamide, an oxidizing agent of sulfhydryl ligands (ZO), and 2 mM DTT, a specific sulfhydryl-reducing reactant (24), had no significant effect on the ATP-dependent or ATP-independent Cl- transport in FAMV. Not shown in Table 2 was the finding that order of addition of ATP or diamide and/or DTT to FAMV had no effect on the results seen in Table
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R1116
CL-
TRANSPORT
membranous sulfhydryl ligands (24). Because PCMBS inhibited both ATP-dependent Cl- transport and ATPdependent A$ and these inhibitions were approximately equal to those induced by an equal concentration of NEM, it is highly suggestive that surface sulfhydryl ligands, not intramembranous sulfhydryl groups, are at least in part responsible, either directly or indirectly, for both ATP-dependent Cl- transport and ATP-dependent A$ in Aplysia gut. These results, coupled with previous observations (10, 13, 15), are strongly consistent with the hypothesis that the active electrogenic Cl- absorptive mechanism in Aplysia gut is an electrogenic, Cl--transporting, Cl-stimulated ATPase found in the foregut absorptive cell plasma membrane. I thank Dr. Bruce Stevens for helpful suggestions during the course of the study, acknowledge the excellent technical assistance of F. Robbins, and thank K. Fortin for assistance with the typing. This investigation was supported by the Eppley Foundation for Research. Address for reprint requests: Dept. of Physiology, Box J-274, JHMHC, Univ. of Florida, Gainesville, FL 32610. Received
3 January
1990; accepted
in final
form
17 July
1990.
REFERENCES 1. AHEARN, G. A., L. A. MAGINNISS, Y. K. SONG, AND A. TORNQUIST. Intestinal water and ion transport in fresh water malacostracan prawns (Crustacea). In: Water Relations in Membrane Transport in Plants and Animals, edited by A. M. Jungreis, T. K. Hodges, A. Kleinzeller, and S. G. Schultz. New York: Academic, 1977, p. 129142. 2. ARMSTRONG, W. McD., W. R. BIXENMAN, K. F. FREY, J. F. GARCIA-DIAZ, M. G. O’REGAN, AND J. H. OWENS. Energetics of coupled Na’ and Cl- entry into epithelial cells of bullfrog small intestine. Biochim. Biophys. Acta 551: 207-219, 1979. 3. COOPERSTEIN, S. J., AND A. LAZAROW. A microspectrophotometric method for the determination of cytochrome oxidase. J. Biol. Chem. 189: 665670,195l. 4. FIELD, M., K. J. KARNAKY, P. L. SMYTH, J. E. BOLTON, AND W. B. KINTER. Ion transport across the isolated intestinal mucosa of the winter flounder, Pseudopleuronectes americanus. I. Functional and structural properties of cellular and paracellular pathways for Na and Cl. J. Membr. Biol. 41: 265-275, 1978. 5. FRIZZELL, R. A., M. FIELD, AND S. G. SCHULTZ. Sodium-coupled chloride transport by epithelial tissues. Am. J. Physiol. 236 (Renal Fluid Electrolyte Physiol. 5): Fl-FB, 1979. 6. FRIZZELL, R. A., M. J. KOCH, AND S. G. SCHULTZ. Ion transport by rabbit colon. I. Active and passive components. J. Membr. Biol. 27: 297-316, 1976. 7. GERENCSER, G. A. Electrical characteristics of isolated Aplysia californica intestine. Comp. Biochem, Physiol. A Comp. Physiol. 61: 209-212,1978. 8. GERENCSER, G. A. Enhancement of sodium and chloride transport
BY
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26.
THE
GUT
by monosaccharides in Aplysia californica intestine. Comp. Biochem. Physiol. A Camp. Physiol. 61: 203-208, 1978. GERENCSER, G. A. Electrophysiology of chloride transport in Aplysia (mollusk) intestine. Am. J. Physiol. 244 (Regulatory Integrative Comp. Physiol. 13): R143-R149, 1983. GERENCSER, G. A. Electrogenic ATP-dependent Cl- transport by plasma membrane vesicles from Aplysia intestine. Am. J. Physiol. 254 (Regulatory Integrative Camp. Physiol. 23): R127-R133, 1988. GERENCSER, G. A., AND S. H. LEE. Cl--stimulated adenosine triphosphatase: existence, location and function. J. Exp. Biol. 106: 143-161,1983. GERENCSER, G. A., AND S. H. LEE. ATP-dependent chloride transport in plasma membrane vesicles from Aplysia gut. Biochim. Biophys. Acta 816: 415-417, 1985. GERENCSER, G. A., AND S. H. LEE. Cl--HCOT-stimulated ATPase in intestinal mucosa of Aplysia. Am. J. Physiol. 248 (Regulatory Integrative Comp. Physiol. 17): R241-R248, 1985. GERENCSER, G. A., AND J. F. WHITE. Membrane potentials and chloride activities in epithelial cells of Aplysia intestine. Am. J. Physiol. 239 (Regulatory Integrative Comp. Physiol. 8): R445-R449, 1980. GERENCSER, G. A., J. F. WHITE, D. GRADMANN, AND S. L. BONTING. Is there a Cl- pump? Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R677-R692, 1988. GUNTER-SMITH, P. J., AND J. F. WHITE. Response of Amphiuma small intestine to theophylline: effect of bicarbonate transport. Am. J. Physiol. 236 (Endocrinol. Metab. Gastrointest. Physiol. 5): E775-E783,1979. HANRAHAN, J., AND J. E. PHILLIPS. Mechanism and control of salt absorption in locust rectum. Am. J. Physiol. 244 (Regulatory Integrative Comp. Physiol. 13): R131-R142, 1983. HOUSE, C. R., AND K. GREEN. Ion and water transport in isolated intestine of the marine teleost, Cottus scorpius. J. Exp. Biol. 42: 177-189,1965. HUBEL, K. A. Effect of luminal chloride concentration on bicarbonate secretion in rat ileum. Am. J. Physiol. 217: 40-45, 1969. LAUF, P. K. Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide. J. Membr. Biol. 101: 179-188, 1988. MORAN, W. M., AND L. T. GARRETSON. Sugar-stimulated ion absorption is not different in seahare and vertebrate intestine. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R583R590,1988. NELLANS, H. N., R. A. FRIZZELL, AND S. G. SCHULTZ. Coupled sodium-chloride influx across the brush border of rabbit ileum. Am. J. Physiol. 225: 467-475, 1973. QUAY, J. F., AND W. M. ARMSTRONG. Sodium and chloride transport by isolated bullfrog small intestine. Am. J. Physiol. 217: 694702, 1969. ROTHSTEIN, A. Sulfhydryl groups in membrane structure and function. In: Current Topics in Membranes and Transport, edited by F. Bronner and A. Kleinzeller. New York: Academic, 1970, p. 135-176. SKADHAUGE, E. Coupling of transmural flows of NaCl and water in the intestine of the eel (Anguilla anguilla). J. Exp. Biol. 60: 535546,1974. TURNBERG, L. A., F. A. BISBERDORF, S. G. MORAWAKI, AND J. S. FORDTRAN. Interrelationships of chloride, bicarbonate, sodium and hydrogen transport in the human ileum. J. Clin. Invest. 49: 557567,197O.
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