Life Sciences, Vol . 25, pp . 1029-1034 Printed in the U.S .A .
Pergamon Presa
RSVERSIBLE INHIBITION OF RSNèL MICROSOMfi CELCIUM PUMP BY FUROSSMIDB Leon Moore* and Erwin J. Landon** *Department of Pharmacology, Uniformed Services University Bethesdn, Maryland 20014 **Department of Pharmacology, Vanderbilt University Nashville, Tennessee 37232 (Received in final form August 8, 1979) SUMMéRY Renal microsames have been shown to have an energydependent calcium pump activity . We now demonstrate that three diuretic compounds inhibit this calcite pump in vitro. Characterization of furosenide inhibition demonstrates that this agent is a reversible, non-competitive inhibitor of the renal microsome calFurosemide, under similar conditions does ciun pump . not inhibit Na/R-aTPase activity in the renal microsane Furosemide may be useful to define function fraction . of the microsasal calciws pump in non-muscle cells . élthough furosenide is a potent diuretic, little is known about the efIt is thought that furfects of this agent on renal transport processes . osenide acts as a diuretic by inhibiting a chloride pump in the loop of Henle (1) . However, studies of the effect of this compound on the Na/K pub Some investigators have reported that furosenide have been equivocal . inhibits the Na/R-11TPase (2-4), while others find no inhibition of this transport enzyme (5,6) . Previously, we have demonstrated a calcium pump activity in both renal These pumps may regulate cytonicrosame and plasma membrane vesicles (7) . Because of the importance of calplasmic calcium levels in kidney cells . cium ions in the regulation of membrane permeability, we have characterized the effect of furosenide on the calcium pump of renal microsanes . MATERIèLS SND METHODS Membrane Isolation . Kidneys were removed from male Sprague-Dawley rats for isolation of kidney microsome and plasma membrane vesicles as previously described (7) in 0 .25M sucrose . Calcium Uptake èssay. Calcium pump activity of both membrane fractions wns determined, as previously described (7), in a nedi~ containing : RC1 100 nM, inidazole-histidine buffer pH 6 .8 30 mM, amoniun oaalate 5 nll, sodi~ azide 5 uM, magnesium chloride 5 mM, èTP (pH adjusted to 6 .8 with inidazole) The nssay was initiated 5 mM, calcium chloride 20 Nft (0 .1 NCi ~ 6Ca/ml) . with nicrosamal protein at a final concentration of 0 .05 to 0 .1 ng/nl. (8) . Microsamnl Protein was determined by the method of Lowry et al . calcium uptake incubations were held at 37° for various time periods as indicated in the legends . èliquots of the assay were filtered on 0 .45 N
0024-3204/79/121029-06$02 .00/0
1030
Furosemide and Renal Calcium Pump
Vol . 25, No . 12, 1979
cellulose nitrate filters and 4 bCa uptake determined by liquid scintillation spectrometry . To test the effect of the diuretics, the appropriate compound was added to the calcium uptake medium before initiating the assay with membrane protein . Na/K-ATPase Assay . For Na/K-ATPase determination, microsomes were isolated in the presence of 1 mM FDTA (9) . Na/K-ATPase activity was determined as previously described (7) in a medium containing : imidazole-histidine buffer pH 6 .8 30 mM, NaCl 67 mM, RC1 3 .3 mM, magnesium chloride 5 mM, ATP (pH adjusted to 6 .8 with imidazole) 5 mM, EGTA 0 .1 mM and microsomal vesicles 0 .07 to 0 .1 mg protein/ml . Samples were incubated at 37° for 20 minutes . The reaction was terminated with ice-cold TCA . Inorganic phosphate Total released was determined by the method of Fiske and Subbarow (10) . ATPase activity was determined in the absence of ouabain . Mq-ATPase activity was determined with addition of ouabain 1 mM . Na/R-ATPase activity was calculated as the difference between total ATPase and Mq-ATPase activities . RESULTS AND DISCUSSION Three diuretic agents (amiloride, furosemide and ethacrynic acid) inhibited the calcium pump activity of both kidney microsames and plasma membranes . In measurements of calcium uptake by kidney microsames, furo semide and ethacrynic acid were potent inhibitors of calcium pump activity (Table I) . Furosemide was examined in greater detail and inhibition of Table I Inhibition of Kidney Microsome Calcium Pump by Diuretics Compound
Microsomal Calcium Pump Activity (Percentage of Control)
Aniloride O .1mM èmiloride 1mM
94 t .23 87 t 4 .1
Furosemide O .1mM Furosemide 1mM
65 t 5 .1 34 t 3 .0
Ethacrynic Acid O .1mM Ethacrynic Acid 1mM
71 t 5 .2 7 t 3 .4
Microsomal calcium uptake activity was determined as described in the text over a 30 minute incubation period . Values reported are the mean t S_E .M . of the determination in three to six microsome preparations . the micrsomal calcium pump by furosemide as reversible as demonstrated in Table 2 . In this experiment, microsomes were isolated and preincubated for 15 minutes at 37° t furosemide 1 m1i . The preincubation was terminated with a 10-fold dilution into ice-cold 0 .25M sucrose . Preincubated microsomal material was collected by centrifugation at 123,0009 for 30 minutes . Both pellets (control and furosemide pretreated) were resuspended and assayed for calcium pump activity in the absence (left column) or presence (right column) of furosemide . Data in the table indicate that furosemide did not inhibit the microsome calcium pump after a single wash of furosemide pretreated microsomes . The calcium pump activity of both preparations (control and furosemide pretreated) was inhibited if furosemide was added to the assay to the mediwn (Table II) .
Vol. 25, No . 12, 1979
Furosemide and Renal Calciua Pump
1031
Table II
Preincubation Condition
Washed Nicrosomes
Washed Kicrosaaes èssayed in the Pre sence of Furosenide (laN)
nmoles calcite/aq protein/30ain Control Furoseaide 1aN
14 .7 * 1 .17
6 .58 t 0 .91
14 .7 t 3 .00
6 .42 t 0 .78
Nicrosomal calcium uptake activity wns determined as described in the test over a 30 minute incubation period . Values reported are the mean t S .S .N . of the determination of three to sin aicrosame preparations . The kinetics of furoseaide inhibition of the nicrosoral calcüa pure was exaained . In Figure ls, ricrosare calcium poop activity was determined over range of calcium concentrations (5 to 50 N!I) in the presence of two fuorseaide concentrations . èt either concentration aasiaal pump velocity was decreased, but the apprent k of the syster for calcüm rained constant at 16 .7 Nlt . In Figure 1B, ~ump activity was determined over a range of Ng-àTP concentrations (0 .625 to 5 nli) . again, naainal pure velocity was decreased at either furoseaide concentration, but the k of the systw for Ng-èTP was not significantly changed . These data leadmus to conclude the furosemide is a reversible, non-competitive inhibitor of the kidney aicrosone calcite pump . Furoseaide inhibition of the calcium pump was not Baited to kidney mfcrosooes . The compound also inhibited the renal plasma membrane calciur pure (figure 2) . The plasma membrane calcium pump was sosewhat lean sensiThe plasma membrane calcina pump was inhibited 51 t tive to furosemide . 5 .1$ by 1nN furoseaide, while the microsame puep was inhibited 70 t 5 .4$. Ethacryaic acid also was a note potent inhibitor of the microsaoe puap than of the plasma nenbrene pump (data not shown) . There ere other differences of biochemical characteristics between the aicrosame end plasma membrane calcite poops (7) . àlthou~ aicrosames are a heterogeaoua population of membrane vescicles, the calcüa pump activity found in the microaaae fraction is associated with merbraaes of the endoplasmic reticuhm (7) . To determine if inhibition of the aicrosase calci~a poop was specific we esaained the effect of this compound on Na/R-aTPase activity in kidney microsames . Under siailar conditions, the Na/R-èTPase was not inhibited by furoseaide (1 rN) . Na/R-STPase activity of kidney microsames, under the assay conditions described above, was 2 .13 t 0 .31 Emoles PO s/mg protein/20 mantes . When 1 mM furoseaide was added to the assay media, Na/R-aTPase activity was 2 .5410 .46 moles P04/mg protein/20 rinutes in five psired e:Qeriments . Likewise, Hg-àTPase activity was not affected by this concentration of furosenide . These data suggest that under siailar conditions furosemide inhibits the calcium, but not the Na/R-purp found is membranes of the renal microsaae fraction . These data also show that furosemide inhibits not only an anion pump (1) but also inhibits a cation pump in renal membranes . The physiological importance of inhibition of renal calciuo poops by furose-
Furoeemide and Renal Calcium Pump
1032
Vol . 25, No . 12, 1979
aide is not known . It is possible that inhibition of this pub by furosemide may play soa+e role in furosemide produced diuresis . èlternately, in view of the importance of calcium in volume regulation (11-13) and the effect of furosemide on ouabain insensitive sodium transport (14-15), inhibition of the calcium pump by furosemide may suggest a role for the pump in volume regulation .
_c
80
E 0 50 Z w O 40 a a 30 E w 20 O c 10
Figure 1 Kinetics of Mq-8TP dependent calcium uptake by rat renal Reaction conditions are microsomes in the presence of furosenide . described in the teat and furosemide concentrations are indicated in the figure . Binding of calcium in the absence of èTP was subtracted The data are from total calcitm~ uptake in the presence of Mq-èTP . Increasing eapressed in a single reciprocal form (ßadie-Hofstee) . calcium (è) or Hq-èTP (B) does not appear to decrease furose~ide inhibition and the calculated concentrations of either substrate necessary for half mammal uptake are not altered by the presence of furosemide . In è each point represents the mean of the detersination in four preparations, while each point in B represents the mean of the determination of three preparations .
Vol. 25, No . 12, 1979
Ftirosemide and Renal Calcium Pump
1033
15
Z W H ~ 10
a a E w c
0
Figure 2 . !ig-aTP stimilated calcium uptake by rat renal plasma oembranes in the presence of furosemide . Calcite uptake was measured as described in the teat and the addition of furose mide is indicated in the figure . The binding of calcite to the membrane fraction in the absence of èTP was subtracted from the total calcium uptake in the presence of Kg-STP . ßach point represents the mean of nooles calcite per mg protein t S .ß .li . for sia meiabrane preparations . è Supported in part by grants from USPHS (HL 14681 and RR 05424) . R8FSR8NCSS 1. 2. 3. 4. 5.
N . BURG, L . STOd~t, J . CéRDINèL, 8ND N . GRBSN, èm . J . Physiol . _225, 119-124 (1973) . J . B . Fi00R, èND H . 8 . WILLIèMSON, Proc . Soc . ßap . Biol . Hed . _l20, 358-360 (1965) . U . SCHlIIDT, èl~ U . C . DIIBèCH, Nephron _7, 447-458 (1970) . D . R . FSRGUSON, éND B . R . TWITB, Naunyn-Schmiedenberq's arch . Pharmacol . 285, 111-116 (1975) . H . SBSL, J . BHRICH, N . G . D8 SèNTO, él~ U . DOBRRSN, Pflegers arch . _335, 224-234 (1972) .
1034
Furoaemide and Renal Calcium Pump
6.
H . W . RADTRB,
7.
L . lIOORS, D . F . FITZPATRICK, T . S . CHßri, èND B . J . LANDON , Biochim . Biophys . Acta _345, 405-418 (1974) . 0 . H . LOWREY, N . J . ROSSNBROUGH, A . L . FARR, AND R . J . RANDALL, J . Biol . Chem . _193, 473-490 (1951) . S . J . LANDON, and J . L . NORRIS, Biochim . Biophys . Acta . 71, 266-276 (1963) . C . H . FISRB, AND Y . SUBBAROW, J . Biol . Chem . _66, 375-400 (1925) . G . RORIVB, AND A . RLSINZBLLER, Biochim . Biophys . Acte _274, 226-239 (1972) . P . K . RANGACHARI, S . 8 . DANIEL, èND D . M . PATTON, Biothun . Biophys . Acta _323, 297-307 (1973) . J . C . PARRSR, H . J . GITBL!lAN, P . S . GLOSSON, AND D . L . LfiONARD, J . Gen . Physiol _65, 84-96 (1975) . J . R . BACHS, J . Gen . Physiol . _57, 259-282 (1971) . J . S . WILSY, AND R . A . COOPER, J . Clin . Invest . _53, 745-755 (1974) .
8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
G.
RUNRICH, B . RINNS-SUFFRAN, AND R .
Vol . 25, No . 12, 1979
J . ULLRICH, Kidney
Pergamon Presa
RSVERSIBLE INHIBITION OF RSNèL MICROSOMfi CELCIUM PUMP BY FUROSSMIDB Leon Moore* and Erwin J. Landon** *Department of Pharmacology, Uniformed Services University Bethesdn, Maryland 20014 **Department of Pharmacology, Vanderbilt University Nashville, Tennessee 37232 (Received in final form August 8, 1979) SUMMéRY Renal microsames have been shown to have an energydependent calcium pump activity . We now demonstrate that three diuretic compounds inhibit this calcite pump in vitro. Characterization of furosenide inhibition demonstrates that this agent is a reversible, non-competitive inhibitor of the renal microsome calFurosemide, under similar conditions does ciun pump . not inhibit Na/R-aTPase activity in the renal microsane Furosemide may be useful to define function fraction . of the microsasal calciws pump in non-muscle cells . élthough furosenide is a potent diuretic, little is known about the efIt is thought that furfects of this agent on renal transport processes . osenide acts as a diuretic by inhibiting a chloride pump in the loop of Henle (1) . However, studies of the effect of this compound on the Na/K pub Some investigators have reported that furosenide have been equivocal . inhibits the Na/R-11TPase (2-4), while others find no inhibition of this transport enzyme (5,6) . Previously, we have demonstrated a calcium pump activity in both renal These pumps may regulate cytonicrosame and plasma membrane vesicles (7) . Because of the importance of calplasmic calcium levels in kidney cells . cium ions in the regulation of membrane permeability, we have characterized the effect of furosenide on the calcium pump of renal microsanes . MATERIèLS SND METHODS Membrane Isolation . Kidneys were removed from male Sprague-Dawley rats for isolation of kidney microsome and plasma membrane vesicles as previously described (7) in 0 .25M sucrose . Calcium Uptake èssay. Calcium pump activity of both membrane fractions wns determined, as previously described (7), in a nedi~ containing : RC1 100 nM, inidazole-histidine buffer pH 6 .8 30 mM, amoniun oaalate 5 nll, sodi~ azide 5 uM, magnesium chloride 5 mM, èTP (pH adjusted to 6 .8 with inidazole) The nssay was initiated 5 mM, calcium chloride 20 Nft (0 .1 NCi ~ 6Ca/ml) . with nicrosamal protein at a final concentration of 0 .05 to 0 .1 ng/nl. (8) . Microsamnl Protein was determined by the method of Lowry et al . calcium uptake incubations were held at 37° for various time periods as indicated in the legends . èliquots of the assay were filtered on 0 .45 N
0024-3204/79/121029-06$02 .00/0
1030
Furosemide and Renal Calcium Pump
Vol . 25, No . 12, 1979
cellulose nitrate filters and 4 bCa uptake determined by liquid scintillation spectrometry . To test the effect of the diuretics, the appropriate compound was added to the calcium uptake medium before initiating the assay with membrane protein . Na/K-ATPase Assay . For Na/K-ATPase determination, microsomes were isolated in the presence of 1 mM FDTA (9) . Na/K-ATPase activity was determined as previously described (7) in a medium containing : imidazole-histidine buffer pH 6 .8 30 mM, NaCl 67 mM, RC1 3 .3 mM, magnesium chloride 5 mM, ATP (pH adjusted to 6 .8 with imidazole) 5 mM, EGTA 0 .1 mM and microsomal vesicles 0 .07 to 0 .1 mg protein/ml . Samples were incubated at 37° for 20 minutes . The reaction was terminated with ice-cold TCA . Inorganic phosphate Total released was determined by the method of Fiske and Subbarow (10) . ATPase activity was determined in the absence of ouabain . Mq-ATPase activity was determined with addition of ouabain 1 mM . Na/R-ATPase activity was calculated as the difference between total ATPase and Mq-ATPase activities . RESULTS AND DISCUSSION Three diuretic agents (amiloride, furosemide and ethacrynic acid) inhibited the calcium pump activity of both kidney microsames and plasma membranes . In measurements of calcium uptake by kidney microsames, furo semide and ethacrynic acid were potent inhibitors of calcium pump activity (Table I) . Furosemide was examined in greater detail and inhibition of Table I Inhibition of Kidney Microsome Calcium Pump by Diuretics Compound
Microsomal Calcium Pump Activity (Percentage of Control)
Aniloride O .1mM èmiloride 1mM
94 t .23 87 t 4 .1
Furosemide O .1mM Furosemide 1mM
65 t 5 .1 34 t 3 .0
Ethacrynic Acid O .1mM Ethacrynic Acid 1mM
71 t 5 .2 7 t 3 .4
Microsomal calcium uptake activity was determined as described in the text over a 30 minute incubation period . Values reported are the mean t S_E .M . of the determination in three to six microsome preparations . the micrsomal calcium pump by furosemide as reversible as demonstrated in Table 2 . In this experiment, microsomes were isolated and preincubated for 15 minutes at 37° t furosemide 1 m1i . The preincubation was terminated with a 10-fold dilution into ice-cold 0 .25M sucrose . Preincubated microsomal material was collected by centrifugation at 123,0009 for 30 minutes . Both pellets (control and furosemide pretreated) were resuspended and assayed for calcium pump activity in the absence (left column) or presence (right column) of furosemide . Data in the table indicate that furosemide did not inhibit the microsome calcium pump after a single wash of furosemide pretreated microsomes . The calcium pump activity of both preparations (control and furosemide pretreated) was inhibited if furosemide was added to the assay to the mediwn (Table II) .
Vol. 25, No . 12, 1979
Furosemide and Renal Calciua Pump
1031
Table II
Preincubation Condition
Washed Nicrosomes
Washed Kicrosaaes èssayed in the Pre sence of Furosenide (laN)
nmoles calcite/aq protein/30ain Control Furoseaide 1aN
14 .7 * 1 .17
6 .58 t 0 .91
14 .7 t 3 .00
6 .42 t 0 .78
Nicrosomal calcium uptake activity wns determined as described in the test over a 30 minute incubation period . Values reported are the mean t S .S .N . of the determination of three to sin aicrosame preparations . The kinetics of furoseaide inhibition of the nicrosoral calcüa pure was exaained . In Figure ls, ricrosare calcium poop activity was determined over range of calcium concentrations (5 to 50 N!I) in the presence of two fuorseaide concentrations . èt either concentration aasiaal pump velocity was decreased, but the apprent k of the syster for calcüm rained constant at 16 .7 Nlt . In Figure 1B, ~ump activity was determined over a range of Ng-àTP concentrations (0 .625 to 5 nli) . again, naainal pure velocity was decreased at either furoseaide concentration, but the k of the systw for Ng-èTP was not significantly changed . These data leadmus to conclude the furosemide is a reversible, non-competitive inhibitor of the kidney aicrosone calcite pump . Furoseaide inhibition of the calcium pump was not Baited to kidney mfcrosooes . The compound also inhibited the renal plasma membrane calciur pure (figure 2) . The plasma membrane calcium pump was sosewhat lean sensiThe plasma membrane calcina pump was inhibited 51 t tive to furosemide . 5 .1$ by 1nN furoseaide, while the microsame puep was inhibited 70 t 5 .4$. Ethacryaic acid also was a note potent inhibitor of the microsaoe puap than of the plasma nenbrene pump (data not shown) . There ere other differences of biochemical characteristics between the aicrosame end plasma membrane calcite poops (7) . àlthou~ aicrosames are a heterogeaoua population of membrane vescicles, the calcüa pump activity found in the microaaae fraction is associated with merbraaes of the endoplasmic reticuhm (7) . To determine if inhibition of the aicrosase calci~a poop was specific we esaained the effect of this compound on Na/R-aTPase activity in kidney microsames . Under siailar conditions, the Na/R-èTPase was not inhibited by furoseaide (1 rN) . Na/R-STPase activity of kidney microsames, under the assay conditions described above, was 2 .13 t 0 .31 Emoles PO s/mg protein/20 mantes . When 1 mM furoseaide was added to the assay media, Na/R-aTPase activity was 2 .5410 .46 moles P04/mg protein/20 rinutes in five psired e:Qeriments . Likewise, Hg-àTPase activity was not affected by this concentration of furosenide . These data suggest that under siailar conditions furosemide inhibits the calcium, but not the Na/R-purp found is membranes of the renal microsaae fraction . These data also show that furosemide inhibits not only an anion pump (1) but also inhibits a cation pump in renal membranes . The physiological importance of inhibition of renal calciuo poops by furose-
Furoeemide and Renal Calcium Pump
1032
Vol . 25, No . 12, 1979
aide is not known . It is possible that inhibition of this pub by furosemide may play soa+e role in furosemide produced diuresis . èlternately, in view of the importance of calcium in volume regulation (11-13) and the effect of furosemide on ouabain insensitive sodium transport (14-15), inhibition of the calcium pump by furosemide may suggest a role for the pump in volume regulation .
_c
80
E 0 50 Z w O 40 a a 30 E w 20 O c 10
Figure 1 Kinetics of Mq-8TP dependent calcium uptake by rat renal Reaction conditions are microsomes in the presence of furosenide . described in the teat and furosemide concentrations are indicated in the figure . Binding of calcium in the absence of èTP was subtracted The data are from total calcitm~ uptake in the presence of Mq-èTP . Increasing eapressed in a single reciprocal form (ßadie-Hofstee) . calcium (è) or Hq-èTP (B) does not appear to decrease furose~ide inhibition and the calculated concentrations of either substrate necessary for half mammal uptake are not altered by the presence of furosemide . In è each point represents the mean of the detersination in four preparations, while each point in B represents the mean of the determination of three preparations .
Vol. 25, No . 12, 1979
Ftirosemide and Renal Calcium Pump
1033
15
Z W H ~ 10
a a E w c
0
Figure 2 . !ig-aTP stimilated calcium uptake by rat renal plasma oembranes in the presence of furosemide . Calcite uptake was measured as described in the teat and the addition of furose mide is indicated in the figure . The binding of calcite to the membrane fraction in the absence of èTP was subtracted from the total calcium uptake in the presence of Kg-STP . ßach point represents the mean of nooles calcite per mg protein t S .ß .li . for sia meiabrane preparations . è Supported in part by grants from USPHS (HL 14681 and RR 05424) . R8FSR8NCSS 1. 2. 3. 4. 5.
N . BURG, L . STOd~t, J . CéRDINèL, 8ND N . GRBSN, èm . J . Physiol . _225, 119-124 (1973) . J . B . Fi00R, èND H . 8 . WILLIèMSON, Proc . Soc . ßap . Biol . Hed . _l20, 358-360 (1965) . U . SCHlIIDT, èl~ U . C . DIIBèCH, Nephron _7, 447-458 (1970) . D . R . FSRGUSON, éND B . R . TWITB, Naunyn-Schmiedenberq's arch . Pharmacol . 285, 111-116 (1975) . H . SBSL, J . BHRICH, N . G . D8 SèNTO, él~ U . DOBRRSN, Pflegers arch . _335, 224-234 (1972) .
1034
Furoaemide and Renal Calcium Pump
6.
H . W . RADTRB,
7.
L . lIOORS, D . F . FITZPATRICK, T . S . CHßri, èND B . J . LANDON , Biochim . Biophys . Acta _345, 405-418 (1974) . 0 . H . LOWREY, N . J . ROSSNBROUGH, A . L . FARR, AND R . J . RANDALL, J . Biol . Chem . _193, 473-490 (1951) . S . J . LANDON, and J . L . NORRIS, Biochim . Biophys . Acta . 71, 266-276 (1963) . C . H . FISRB, AND Y . SUBBAROW, J . Biol . Chem . _66, 375-400 (1925) . G . RORIVB, AND A . RLSINZBLLER, Biochim . Biophys . Acte _274, 226-239 (1972) . P . K . RANGACHARI, S . 8 . DANIEL, èND D . M . PATTON, Biothun . Biophys . Acta _323, 297-307 (1973) . J . C . PARRSR, H . J . GITBL!lAN, P . S . GLOSSON, AND D . L . LfiONARD, J . Gen . Physiol _65, 84-96 (1975) . J . R . BACHS, J . Gen . Physiol . _57, 259-282 (1971) . J . S . WILSY, AND R . A . COOPER, J . Clin . Invest . _53, 745-755 (1974) .
8. 9. 10 . 11 . 12 . 13 . 14 . 15 .
G.
RUNRICH, B . RINNS-SUFFRAN, AND R .
Vol . 25, No . 12, 1979
J . ULLRICH, Kidney
