HCO3−-Dependent ATPase activity in the gills of rainbow trout Salmo gairdneri

Comp. Biochem. Physiol., 1974, Vol. 48B, pp. 581 to 589. Pergamon Press. Printed in Great Britain

HCOa--DEPENDENT ATPAsE ACTIVITY IN THE GILLS OF RAINBOW TROUT (SALMO GAIRDNERI) T H E O D O R E H. K E R S T E T T E R 1 and L E O N A R D B. K I R S C H N E R ~ 1 Department of Biology, California State University-Humboldt, Arcata, California 95521; and 2 Department of Zoology, Washington State University, Pullman, Washington 99163, U.S.A. (Received 17 August 1973) Al~tract--1. A HCOs- activated ATPase is present in both fresh water and sea water adapted rainbow trout gills. The apparent Km for HCOa- is 0.016 M. 2. Both the ATPase and active C1- uptake by intact gills are inhibited by SCN-, but by apparently independent mechanisms. 3. CI- is not required for activation of the ATPase; at [Cl-] greater than 0.010 M, enzyme activity is inhibited. 4. The intracellular location of the HCOa- ATPase is unclear, but preliminary evidence suggests it is both mitochondrial and microsomal. INTRODUCTION CHLORIDE ion absorption by the gills of teleost fishes probably occurs in exchange for bicarbonate ion (Maetz & Garcia-Romeu, 1964; Kerstetter & Kirschner, 1972); and a similar system for C1- uptake has been shown to exist in at least one species of frog (Garcia-Romeu et al., 1969). But the energetics of C1- uptake by outer boundary epithelia is poorly understood, and no known enzyme system has been shown to participate in the process. Consequently, the report of a H C O s - activated ATPase in homogenate of Necturus gill (Wiebelhaus et al., 1971) stimulated us to examine the trout gill for a similar system. One portion of this paper reports the presence of such an enzyme and describes certain of its properties. A H C O 8- activated, thiocyanate inhibited ATPase was first described in frog gastric mucosa by Kasbekar & Durbin (1965). Its characteristics and possible relationship to acid secretion in the stomach have since been intensively studied by a number of groups. Wiebelhaus et aI. (1971) localized the enzyme in the oxyntic cells of Necturus gastric mucosa; in a later paper from the same laboratory, a detailed study of its intracellular distribution was described, with evidence that it is associated with both mitochondrial and microsomal fractions of dog gastric mucosa (Sachs et al., 1972). Sachs et al. (1965) reported that HCO s- ATPase activity was present in rat liver mitochondria and hamster intestinal brush border; and recently Simon et al. (1972) have described similar activity from pancreas. Although models proposing a mechanism by which a H C O s- activated ATPase might work in ion transport have been proposed (Sachs et al., 1972; Blum et al., 1971), its participation in active transport has not been conclusively demonstrated. 581

582

THEODORE H. KERSTETTER AND LEONARD B. KIRSCHNER

One of the links between the enzyme and gastric acid secretion is its apparent presence only in the acid secreting cells of the stomach. Still another suggestive link is the action of thiocyanate ion, which inhibits the HCO 3- ATPase and also acid secretion by the gastric mucosa. In the trout gill, there is no experimental evidence that an ATPase is involved in anion transport. But since it seemed worthwhile to consider that possibility, we have included herein a report on aspects of the enzyme which might relate to ion transport. MATERIALS AND METHODS

Animals Rainbow trout (Salmo gairdneri), 150-350 g body weight, were obtained from two sources: a commercial hatchery near Soap Lake, Washington, and the experimental hatchery at California State University, Humboldt. T h e y were kept unfed in tap water at 11-13°C until used. Sea water (SW) adapted fish were prepared by first adapting to 50% SW (Instant Ocean, Aquarium Systems, Inc.), then to 100% SW for at least 1 week before use.

Preparation of enzyme Fish were anesthetized with tricaine methane sulfonate (0"1%), opened along the midline, and the gills were cleared of blood by perfusing into the ventral aorta 0"300 M sucrose, 0"020 M Tris-acetate at p H 7"7. Gill filaments were cut from the gill arches, rinsed, weighed and a 10% w/v homogenate prepared by grinding with a Teflon pestle in a solution containing 0"250 M sucrose, 0"040 M H E P E S (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 0"005 M E G T A (ethyleneglycol-bis [B-aminoethyl ether] N,N'tetra-acetic acid) adjusted to p H 7"6 with Tris base. T h e homogenate was centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was then centrifuged at 70,000 g for 1 hr at 0°C. T h e pellet was resuspended in glass distilled water, 0"001 M E G T A adjusted to p H 7"5 with Tris, and centrifuged at 70,000 g for 30 min at 0°C. T h e final pellet, representing 2-3 g of starting material from one fish, was suspended in 5 ml of the E G T A , Tris solution which had previously been made to 0"2% Triton X-100. T h e addition of Triton X-100 effectively solubilized the microsomal fraction (Wiebelhaus et al., 1971), and greatly increased the enzyme activity. Fractions prepared as above typically assayed 1-2 mg protein per ml. After removal of a 0"5 ml aliquot for protein assay, 1 vol. of 0.20 M HEPES, 0-10 M T r i s buffer, p H 7"2, was added to 9 vol. of the enzyme solution, and it was stored at 0°C with little apparent loss of activity for periods up to 7 days. When ATPase activity in high- and low-speed fractions was compared, a slightly different procedure was followed for enzyme isolation, and succinic dehydrogenase activity was simultaneously determined. T h e homogenate was centrifuged at 600 g for 15 min, the supernatant was then centrifuged at 10,000 g for 10 min, the resulting pellet resuspended in homogenizing solution, and again centrifuged at 10,000 g for 10 min. T h e pellet (10 ~ g min fraction) was then suspended in 1 m M E G T A , 0"2% Triton X-100 neutralized with Tris. T h e supernatant from the 105 x g rain fraction was centrifuged, washed and suspended as described above for preparation of the microsomal (4-2 x 106 g rain) fraction. This procedure gave fractions which had both ATPase and S D H activity, the latter serving as a mitochondrial marker.

Enzyme assay T h e reaction mixture for assays of the H C O 3 - ATPase was buffered at p H 8"30 with 0-03 M T r i s - H E P E S or Tris acetate; the choice of buffer did not affect the activity. Bicarbonate was added as K H C O a to a final concentration of 0"03 M except in concentration dependence work. After the addition of enzyme (0.1 or 0'2 ml), the reaction was started by

HCOs--DEPENDENT

ATPase

ACTIVITY IN THE GILLS OF RAINBOW TROUT

583

adding 0"1 ml of Mg-Tris-ATP made up to give a final concentration of 0"005 M Mg 2+ and 0"0035 M ATP in the reaction tubes. The final volume was 1-0 ml. For the determination of Mg ATPase activity, the KHCO8 was omitted. The use of Tris-HEPES or Tris-acetate and Mg-Tris-ATP made possible reaction mixtures which were free of measurable inorganic anions, except when added for experimental purposes. The Mg-Tris-ATP was made by passing di-Na ATP through Dowex-50, hydrogen form, ion exchange resin, adding an appropriate amount of magnesium carbonate, and adjusting the final pH to 8"3 with 'Iris. Early on in our work, we discovered that the HCOs-activated ATPase activity could be markedly increased by preincubating the enzyme solution for 15-30 min at 37°C. This also decreased the Mg ATPase activity. Therefore, preincubation of the enzyme solution for 15 min was routinely carried out before each assay. The reaction was run at 25°C for 30 rain and then stopped by the addition of 1"0 nil of cold trichloroacetic acid (15%). Reaction tubes were centrifuged at 30,000 g for 10 min and 1-0 ml of the supernatant was removed for the measurement of inorganic phosphate. This was done by the method of Fiske & SubbaRow (1925). The reaction is not linear with time, therefore enzyme activity is expressed as Fmoles of inorganic phosphate released in 30 min per mg of protein. Succinic dehydrogenase was assayed by the method of King (1967); activity is expressed in/,moles of O~/min per mg of protein. Protein was assayed according to Lowry et al. (1951), with bovine serum albumin (Calbiochem) serving as a standard. Ion influx measurements through gills of intact fish were measured by methods previously described (Kerstetter & Kirschner, 1972). RESULTS

Thermal activation of HCOa- ATPase Exposing the enzyme solution to 37°C in the absence of substrate increased the subsequent bicarbonate activation and also decreased the baseline ( M g z+) activity. Results of one experiment are shown in T a b l e 1. Although H C O 3- activity TABLE I ~ E F F E C T S OF EXPOSING ENZYME SUSPENSION TO

37°C FOR SELECTED TIMES

Specific activity* Time of exposure (rain)

Mg 2+ ATPase

H C O s - ATPase

No exposure 15 30 60

1 "93 1"55 1"39 1"24

1"90 3.27 3.27 3"05

* #moles Pt x (30 min)-a x (rag protein) -1. Results of one experiment are given. reached a peak by the end of 15 min exposure, the baseline activity continued to decline throughout the 60 min period. Fifteen rain was routinely used as the preincubation time in all remaining experiments.

Time course T h e time course of the H C O s - activated A T P a s e was not linear (Fig. 1). A S0-min incubation was selected as sufficient to give consistently good activity, but

584

THEODORE H. KERSTETTER AND LEONARD B. KIRSCHNER

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Fro. 1. T i m e course of the H C O 3 - A T P A S E . T h e 5 min value is the mean of two experiments; the remaining values are means of five experiments + S.E.M.

clearly the values for Pi released in 30 min cannot validly be extrapolated in either direction. In the four experiments relating to the time course of the FW enzyme, the total ATP broken down ranged from 17 to 29 per cent of the initial amount. This is far too little to account for the rate change shown in Fig. 1 on the basis of

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FIG. 2a. Bicarbonate concentration dependence of the H C O a - ATPase. Vertical bars are S.E.M. from four experiments on FW enzyme. Mean values from two SW enzyme experiments are also included, b. Reciprocal plot of the FW enzyme concentration dependence values.

,,

HCO3--DEPENDENT

ATPase ACTIVITY IN

simple first-order kinetics. systematically investigated.

THE GILLS OF RAINBOW TROUT

585

The ATP dependence of the enzyme was not

Bicarbonate dependence Bicarbonate was added to reaction tubes as KHCOs since control experiments indicated that potassium had no detectable effect on the activity of either the Mg ATPase or the HCO 3- ATPase. Figure 2 shows the HCO 3- dependence as the fraction of the activity at 0.05 M KHCO3. The apparent Km for HCO3-, calculated from a reciprocal plot (Fig. 2), is 16.6 raM. The data used for the plots derive from assays of FW gill, but the HCOa- ATPase activity in two SW gill preparations showed a similar relationship (Fig. 2). The effect of NaHCO a was identical to that of KHCOa in the one experiment in which the substitution was made. In none of the HCOa- dependence experiments were K + and Na + present at the same time in the reaction tubes, thus ruling out the possibility that any of the observed activity was due to the (Na++K+)ATPase. As a further check, the HCO 3- activity was measured in the presence of 10 -3 M ouabain. Specific activity was 3.19, compared to a control value of 2.97.

Effects of other anions The effect of chloride, with and without HCO3- , was tested with chloride concentrations ranging from 10 -a to 10 -1 M. The C1- was added as tetramethylammonium chloride in two experiments and as KCI in two experiments. Both SW and FW preparations were tested. In the absence of HCOa- , chloride had no detectable effect; when HCO 3- was also present (0.030 M), C1- inhibited at concentrations of 10 -2 M and above. Table 2 presents the results from four fish. TABLE 2--INHIBITION OF

HCOs- A T P A s E

BY CHLORIDE

Final CI- concentration (m-moles/L) Adaptation medium FW SW FW SW

CI- added as TMAC* TMAC* KC1 KCI

10'0

20"0

30.0

50-0

100-0

0"0 12"6 7"5 10"6

--11"1 15"0

17"2 18"0 ---

--26"2 32"8

-71 "2 53"8 59-2

* Tetramethylammonium chloride. Results of four separate experiments are shown, each with 30 mM KHCO8 in reaction tubes. Results are expressed as per cent inhibition. Both sulfate and acetate can substitute for HCOs- to a slight extent. When 0.030 M KHCO 3 was replaced by 0.015 M K2SO 4 in two experiments, the increase in activity over the Mg 2+ baseline was 15-2 and 13.8 per cent of the previously measured HCO~- activity. Potassium acetate, 0"030 M, gave 9.6 per cent of the activity of equimolar KHCOa in one experiment, but gave no additional activity when added to reaction tubes containing 0.030 M KHCOa, nor was there any inhibition of H C O s - activation.

586

THEODORE H . KERSTETTER AND LEONARD B. KIRSCHNER

S W vs F W activity Transition from fresh water to sea water puts markedly different demands on ion transport systems in teleost gills. Thus one way of checking possible involvement of an enzyme in the process of ion transport is to test the activity in both SW and F W adapted forms of a euryhaline species like the rainbow trout. Since both the vector and kinetics of ion movement change in the process of SW adaptation, it is not unreasonable to suppose that the activity of a transport enzyme will also change. The H C O a - ATPase activity in three FW and three SW preparations was compared: at 0.030 M H C O 3- the F W enzyme averaged 3.26 + 0.46 (S.E.), and the SW enzyme was 4.03 + 0.89. The difference is not significant.

.Effects of thiocyanate Thiocyanate, added as NaSCN, was tested at selected concentrations between 10 -s and 10 -~ M. In these experiments, ouabain (5 x 10 -4 M) was also present to prevent any (Na + + K+)-ATPase activity. Inhibition of bicarbonate activity was evident at 10 -4 M SCN-, and was essentially complete at 5 x 10 -3 M. Figure 3 illustrates S C N - effects on both SW and FW preparations. The effect of thiocyanate on chloride transport by the gill was tested on four intact fish. When added to water bathing the gill, complete inhibition of chloride

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FIG. 3. T h e effect of thiocyanate on the H C O 3 - ATPase. Each point represents a value from one experiment. T h e 100 per cent level is the activity of a S C N - - f r e e reaction tube with 30 m i K H C O , . Thiocyanate was added as N a S C N , and 5 x 10 -4 M ouabain was present to prevent any ( N a + + K ÷ ) - A T P a s e activity.

HCO3--DEPENDENT

ATPase ACTIVITY IN THE GILLS OF RAINBOW TROUT

587

influx from a 1.0 m M NaC1 solution was noted at a S C N - concentration of 10 -3 M. At 10 -3 M S C N - , the per cent inhibition was 66.7__. 9.3. Replacement of the bathing medium by a SCN--free solution restored C1- influx to normal. Sodium influx, measured at the same time by dual isotope labeling, was unaffected. In an additional series of experiments, NaSCN was given via intracardiac injection at a dosage of 1 ml of 0.2 M NaSCN/100 g body weight. Of the six fish so treated, there was a significant increase in CI- influx in one, a significant decrease in one and no change in the remaining four. Plasma analysis of two fish indicated a S C N concentration of about 5 x 10 -3 M 1 hr after the injection. Thiocyanate analysis was by photometry after complexing with iron. Thiocyanate inhibition of C1uptake by goldfish (Carassius) was recently reported by Epstein et al. (1973). Their results were qualitatively similar, i.e. S C N - inhibited from the outside at 0.15 raM, but plasma S C N - of about 7 m M had no effect.

Intracellular distribution A preliminary attempt to determine the intracellular location of the enzyme in the trout gill was made by assaying activity in two cell fractions and comparing the resulting activity ratio with the ratio of succinic dehydrogenase in the same two fractions. Centrifugal times and forces are described in the previous section. Table 3 summarizes the results of these experiments. The ratios of S D H and TABLE3--SPECIFIC ACTIVITIESOF HCOs- ATPAsE, Mg 2+ ATPAsE, AND SDH tN THE 106 g MIN AND 4"2 X 106 g MIN FRACTIONS Enzyme

Specific activity _+S.E. (106 g min fraction)

SDH Mg ~+ ATPase HCO~- ATPase

0"0065 + 0.0006 3.04 + 0.28 4.55 + 0"96

Specific activity _ S.E. Activity* (4"2 x 106 g rain) ratio _ S.E. 0"0026 _+0.0002 1'45 _+0.24 2"74 +_0"45

2"57 _+0.22 2"37 _+0.32 1"74 + 0.30

N 7 7 7

* Ratio of activity in 106 g min fraction to activity in 4-2 x 106 g rain fraction. The figure given is the mean of ratios calculated for the individual experiments. ATPase activity in # moles Pl x (30rain) -1 x (rag protein) -x, SDH activity in /~moles 02 x rain -~ x (rag protein) -1. H C O 3- ATPase distribution in the two fractions are significantly different at the 0.05 per cent level, with the data indicating that H C O 3 - activity in the 4-2 × 106 g min fraction is higher than would be expected if the ATPase was exclusively mitochondrial. Mg 2+ ATPase distribution is not significantly different from that of SDH. DISCUSSION The evidence that chloride is absorbed by an exchange mechanism in outer boundary epithelia is compelling. This was first pointed out by Krogh (1937, 1938) for frog skin, teleost (Carassius) gill and crustacean gill. It was verified in crayfish gill by Shaw (1960). The two most probable candidates for the counter

588

THEODORE H. KERSTETTER AND LEONARD B. KIRSCHNER

ion are HCO3- and OH-. Garcia-Romeu et al. (1969) showed that HCO3accumulated in the medium bathing intact Chilean frogs under conditions whereby only chloride uptake was occurring and sodium uptake was blocked. (But it should be pointed out that OH-, by combining with CO 2 also excreted by the skin, would appear as HCOz-. ) Chloride uptake by teleost gill has been stimulated by injections of NaHCO3 in both goldfish (Maetz & Garcia-Romeu, 1964) and rainbow trout (Kerstetter & Kirschner, 1972). Evidence that an ATP-dependent process is involved in epithelial C1- transport is scantier; however, Kristensen (1972) inhibited net CI- influxes in short-circuited, isolated frog skins with both KCN and dinitrophenol. In an earlier paper, we pointed out that the chloride uptake system in the trout gill shows saturation kinetics, with an apparent K m of 0.25 raM. Also the mucosal membrane of the gill epithelium poses an electrical barrier to inward CI- movement (Kerstetter & Kirschner, 1972). There is presumably a marked concentration gradient against CI- movement there too, since net uptake can occur from solutions with less than 0.2 mmoles/1, of chloride. From this, we postulated an energyrequiring step at the outer membrane. Although it is tempting to offer the HCO 3ATPase as the energetic step in the gill chloride transport system, the data do not warrant such an interpretation. Thiocyanate inhibition of both the bicarbonate ATPase and the chloride transport system was initially suggestive. But the two observations may be unrelated: S C N - inhibits transport probably by interaction at a CI- site on the outer membrane, yet the HCO 3- ATPase has no apparent CIsite. Whether or not S C N - inhibition of the ATPase in vivo would also inhibit transport is an unresolved question, since we have no way of knowing if the HCOz- ATPase is accessible to the inhibitor under our experimental conditions. On balance we prefer to draw no conclusions from the dual effects of SCN-. Our other observations on the HCO z- ATPase neither support nor deny the hypothesis of a transport function. The similarity of the enzyme in both SW and FW adapted trout is in contrast to the (Na + + K +) ATPase (Pfeiler & Kirschner, 1972), but the lack of a parallel by itself means nothing. Moreover, the lack of stimulation by C1- need not argue against possible involvement in C1- movement, and even the inhibition by CI- could be fitted to a model: unpublished observations by T H K indicate the transport system may be inhibited at external [CI-] higher than 5 raM. The intraeellular distribution of the HCO 3- ATPase remains in doubt. Based on activity ratios of SDH and the ATPase in the two cell fractions assayed, we can tentatively conclude that the HCO 3- ATPase is in both mitoehondrial and microsomal fractions. Sachs et al. (1972) showed this type of distribution in dog gastric mucosa, with the highest specific activity associated with membrane fractions. The trout gill enzyme, however, seems to be more concentrated in the mitochondrial fraction. But intraeellular localization of enzymes in teleost gills is complicated by difficulties in tissue disruption. The presence of cartilaginous columns in the gill filaments makes rotating pestle homogenization difficult and probably leads to extensive organelle destruction. This may well cause heavy cross-contamination

HCOs--DEPENDENT

ATPase

ACTIVITY IN THE GILLS OF RAINBOW TROUT

589

a m o n g fractions. T h e resolution of the question m u s t await the outcome of density gradient fracdonation, planned for the near future.

Acknowledgements~This work was supported by National Science Foundation Grant No. GB-35537 and by National Institutes of Health Research Grant No. GM-04254. REFERENCES BLUM A. L., SHAH G . , ST. Pmmm T., HELANDERH., SUNG C. P., WIEBELHAUSV. D. & SACHS G. (1971) Properties of soluble ATPase of gastric mucosa--II. Effect of HCOs-. Biochim. biophys. Acta 249, 101-113. FISK~ C. & SUBBAROWY. (1925) The colorimettic determination of phosphorus, ft. biol. Chem. 66, 375--400. G~a~CIA-Ro~u F., SAt.IBt~U'~A. & P E z z ~ I - H ~ ' ~ D E Z S. (1969) The nature of the in vivo sodium and chloride uptake mechanisms through the epithelium of the Chilean frog, Calyptocephallela gayi (Dum. et Bibr., 1841). Exchanges of hydrogen against sodium and of bicarbonate against chloride, aY.gen. Physiol. 53, 816--835. K~BEK~ D. K. & DUPXIN R. P. (1965) An adenosine triphosphatase from frog gastric mucosa. Biochim. biophys. Acta 105, 472--482. KERSTETTEnT. H. & KIRSCH~mRL. B. (1972) Active chloride transport by the gills of rainbow trout (Salmo gairdneri), a7. exp. Biol. 56, 263-272. KING T. E. (1967) Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. In Methods in Enzymology (Edited by ESTABROOKR. W. & PULLMANM. E.), Vol. X, pp. 322-331. Academic Press, New York. KRISTENSENP. (1972) Chloride transport across isolated frog skin. Acta physiol, scand. 84, 338-346. KROOH A. (1937) Osmotic regulation in fresh water fishes by active absorption of chloride ions. Z. vergl. Physiol. 24, 656--666. LowRy O. H., ROSEBROUCHN. J., FAm~A. L. & RA_~DALt.R. J. (1951) Protein measurement with the Folin phenol reagent, if. biol. Chem. 193, 265-275. MAETZ J. & GARCIA-RoivmUF. (1964) The mechanism of sodium and chloride uptake by the gills of a fresh water fish, Carassius auratus--II. Evidence for NH4+/Na and HCOs-/ CI- exchanges, aq. gen. Physiol. 47, 1209-1227. MOTAIS R. (1970) Effect of actinomyein D on the branchial N a - K dependent ATPase activity in relation to sodium balanee of the eel. Comp. Biochem. Physiol. 34, 497-501. P~ILER E. & KIaSCH~R L. B. (1972) Studies on gill ATPase of rainbow trout (Nalmo gairdneri). Biochim. biophys. ~Icta 282, 301-320. SACHS G., MITCH W. E. & HmSCHOWITZB. I. (1965) Frog gastric mucosal ATPase. Proc. Soc. exp. Biol. Med. 119, 1023-1027. SACHSG., SHAHG., STRYCHA., CLINEG. & HmSCHOWITZB. I. (1972) Properties of ATPase of gastric mucosa--III. Distribution of HCOa--stimulated ATPase in gastric mucosa. Biochim. biophys. Acta 266, 625-638. SHAWJ. (1960) The absorption of chloride ions by the crayfish, Astacus pallipes Lereboullet. aT. exp. Biol. 37, 557-572. SIMON B., KINNE R. & SACHSG. (1972) The presence of a HCO3--ATPase in panereatic tissue. Biochim. biophys. Acta 282, 293-300. WIEBELHAUSV. D., SUNC C. P., HEL~DER H. F., SHAH G., BLUMA. L. & SACHSG. (1971) Solubilization of anion ATPase from Necturus oxyntic cells. Biochim. biophys, dcta 241, 49-56.

Key Word Index--ATPase; HCOs--ATPase; gills; rainbow trout; CI- transport; thiocyanate.