- Email: [email protected]
Torsemide inhibits aldosterone secretion in vitro
PII soo24-3205(9?3)00265-3
ELSEVIER
Life sciences, Vol. 63. No. 3, Pp. PL45-50,1998 Publiskd by E?kvier Science Inc. Printed in the USA. All rights reambed am-32ospl mm t .oo
PmRhucoLoGYLEnERs Accehated Cbmnunkti
TORSEMIDE INHIBITS ALDOSTERONE SECRETION IN VITRO Theodore L. Goodfriend,’ Dennis L. Ball,’ Wolfgang Oelkers,* and Volker m? ‘William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, Wisconsin 53705 and *Freie Universitit Berlin, Universititsklinikum Benjamin Franklin, Medizinische Klinik und Poliklinik, Hindenburgdamm 30, 12200 Berlin, Germany (SubmittedJanuary29.1998, accepted March6,1998, received in final form April 27,1998) Abstract Torsemide inhibited aldosterone secretion by adrenal cells from rats, cows, and guinea pigs stimulated in vitro by potassium, angiotensin, dibutyryl cyclic AMP, ACTH, or corticosterone. Inhibitory concentrations for adrenal cells (micromolar) were comparable with those reported to inhibit ion transport in isolated renal tubules. Inhibition of aldosterone secretion could reduce kaliuresis, and that may explain why torsemide causes less kaliuresis than other diuretics. Published by Elsevier Science Inc. Key Words:
diuretics, hypokalemia,
hypertension,
torsemide
Introduction
Torsemide is a potent loop diuretic that has been reported to cause less potassium excretion than furosemide or hydrochlorothiazide (1-5). Thiazide and loop diuretics increase urinary excretion of potassium in part by stimulating the renin-angiotensin axis and the secretion of aldosterone (6). Drugs that inhibit the secretion of aldosterone or its action on the distal renal tubule minimize potassium wastage during diuretic therapy. We studied the effect of torsemide on adrenal glomerulosa cells to see if it inhibited aldosterone secretion, an action that might help to explain the relatively small kaliuresis caused by this diuretic. Aldosterone secretion is controlled by many factors in addition to angiotensin; foremost among these is extracellulax potassium (7). Potassium is thought to regulate aldosterone secretion by its effect on the membrane potential of adrenal glomerulosa cells, whose calcium channels are exquisitely sensitive to that potential. Membrane potential in the adrenal, as in other cells, is determined by the channels and pumps that regulate partition of the principal inorganic ions of extracellular and intracellular fluid. Because of the critical role that membrane potential plays in aldosterone secretion, we and others have tested reagents that affect ion pumps and channels in adrenal glomerulosa cells. Those experiments showed that ouabain, veratridine, pinacidil, monensin, nigericin, valinomycin, and 4-amino pyridine inhibit aldosterone secretion (8-10). Torsemide inhibits Na+/K+/2Cl- cotransport in the thick ascending limb of the loop of Henle, but an action on ion movements in Corresponding author: Theodore L. Goodfriend, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, Wisconsin 53705. Telephone: (608) 262-7007; FAX: (608) 262-7644; E-mail: [email protected]
PL46
Torsemide Inhibits Aldosterone Synthesis
Vol. 63, No. 3, 1998
other cells has not been reported We show here that torsemide inhibits aldosterone secretion by adrenal cells from rats, guinea pigs, and cows studied in vitro. Torsemide was more potent in this respect than furosemide. If torsemide inhibits aldosterone secretion in humans, that might help explain the clinical finding that chronic therapy with torsemide causes less potassium wastage than other diuretics. Methods Cells from the adrenal zona glomerulosa of cows, guinea pigs, and rats were prepared by collagenase-deoxyribonuclease digestion as described in previous publications (9,ll). Just before each experiment, cells were warmed to 37°C for 1 h to restore intracellular ion balance. Cell viability was assessed by trypan blue dye exclusion, and viable cells were counted in a hemocytometer. Cell viability exceeded 80% in all preparations. Contamination of glomerulosa cells by fasciculata was estimated by the difference in cell size. Fasciculata cells comprised, on average, 0.5-2.5% of the glomerulosa preparation from bovine adrenals, and S-10% of the preparation from rat and guinea pig. The presence of fasciculata in the bovine and guinea pig cell mixtures enabled comparison of the effect of drugs on cortisol secretion, as well as aldosterone. To test the effects of drugs, aliquots containing 200,000 cells per ml were incubated in buffered salt solutions for 2 h at 37°C in air (bovine and guinea pig) or 95% 0,/5% CO* (rat). For rat cells, the incubation medium contained two parts of tissue culture Medium 199 and one part All media contained bovine serum albumin 0.1%. balanced salt solution. Drugs were administered to cells that were stimulated by angiotensin II, ACTH, dibutyryl cyclic AMP, potassium, or the steroid substrate corticosterone. At the end of incubation, steroids were measured in the supernatants by specific radioimmunoassays for aldosterone and cortisol in experiments with bovine and guinea pig cells, and for aldosterone in experiments with rat cells. Rat adrenals lack 17-hydroxylase, so the product of their zona fasciculata is corticosterone. Data are expressed as the mean of three incubation tubes, each assayed in duplicate, plus or minus the standard error of the mean of the three tubes. Torsemide effects were tested three times in bovine, three times in rat, and once in The results depicted in Fig. 1 are from a representative guinea pig adrenal preparations. experiment. Binding of radioiodinated
angiotensin
II to cells was performed as described previously
(9).
Torsemide and two of its metabolites were the gift of Boehringer Mannheim. The chemical formulae are as follows: torsemide is l-isopropyl-3-[[4-(3-methyl-phenylamino)py~dine]-3“M3” of torsemide is 1-isopropyl-3-[[4-(3-methyl-4’-hydroxysulfonyllurea; metabolite phenylamino)pyridine]-3-sulfonyllurea; and metabolite “M5” is 1-isopropyl-3-[[4-(3’carboxyphenylamino)pyridine]-3-sulfonyllurea. Radioimmunoassay kits were purchased from Diagnostic Products Corp., Los Angeles, CA. For some assays, antialdosterone antiserum was purchased from ICN Biochemicals, Inc., Costa Mesa, CA. Angiotensin II and human ACTH (l-24) were purchased from Bachem, Inc., Torrance, CA. Results Torsemide inhibited aldosterone production by cells from bovine, rat and guinea pig adrenal glands. When the stimulus was angiotensin II at 10e7M, the average IDS, for inhibition of the increment was approximately 10-6M in bovine cells and 3 x 10mSMin rat cells (Fig. 1). In guinea
PL47
Torsemide Inhibits Aldosterone Synthesis
Vol. 63, No. 3, 1998
pig adrenals, the IDSowas 5 x 10e7M(data not shown). When potassium was the stimulus in rat or bovine cells, the efficacy of torsemide was approximately the same as when angiotensin II was added (Fig. l), but the drug was slightly less effective in inhibiting the aldosterone secretion stimulated by dibutyryl cyclic AMP or by the steroid precursor corticosterone. Basal aldosterone production of aldosterone was also inhibited by the drug (data not shown).
. I-8’ ‘t.
100
\
1 ‘*.\
K+
80
‘* 1 Corticosterone
E\ 1T I
\
60
‘I
\
\’
\
Ang II T’L. AMP \
IO-7 3x10-7
10-G 3x1o-6 10-s 3x1o-5 10-4
10-7
3x10.~
10-c 3x10+
1O-5 3x10+
1O-4
Torsemide (M)
Torsemide (M)
Fig. 1 Dose-response curves of inhibition of aldosterone production by torsemide. Adrenal glomerulosa cells from bovine (panel A) and rat (panel B) were prepared as described in the text and incubated with various stimuli and graded concentrations of torsemide for 2 h. At the end of the incubations, aldosterone in the supematant was measured by radioimmunoassay. All data are presented as a percent of the amount of aldosterone produced by each type of cell in response to the indicated stimulus on the day of the experiment. The points represent means of three incubation tubes, and the vertical bars are standard errors of the mean of those three tubes. Stimuli were added to the following final concentrations: angiotensin II (Ang II) 10w7M,ACTH 10m9M,potassium 7.2 mM, dibutyryl cyclic AMP (AMP) 1 mM, and corticosterone 3 X 10bM. The average maximum amount of aldosterone produced under stimulation by angiotensin II was 30 ng per lo6 bovine cells per h, and 34 ng per lo6 rat cells per h. Furosemide and two metabolites of torsemide were also tested. The M3 (hydroxylated) metabolite showed inhibition of aldosterone production with a potency comparable with that of torsemide, but the M5 (oxidized) metabolite was much less active. Furosemide was less potent than torsemide (Fig. 2).
PL-48
Torsemide Inhibits Aidosterone Synthesis
Vol. 63, No. 3, 1998
Cortisol production by bovine and guinea pig adrenal cells was much less sensitive to torsemide than aldosterone production; the ID,,, was lo-20 times greater for cortisol than for aldosterone in the same incubation tubes (data not shown). Torsemide had no effect on the saturable binding adrenal glomerulosa cells (data not shown).
of radio-iodinated
angiotensin
II to bovine
Furosemide
100
0
tl
11
I11111
10-7 3x1o-7
I
II
I11111
10-a 3x1o-6
I
II
I,,,,
1O-5 3x1o-5
Drug Concentration
J
10-4
(M)
Fig. 2 Effects of torsemide, two metabolites of torsemide, and furosemide on rat adrenal cells stimulated by angiotensin II (10m7M). The metabolites were tested in three different experiments, one of which is presented in the figure. Discussion Our experiments show that torsemide, a diuretic that inhibits the Na+/K+/2Cl- cotransporter in the ascending limb of the loop of Henle, inhibits aldosterone production by adrenal cells from rat, The drug was active at micromolar concentrations in the adrenal guinea pig, and cow. glomerulosa where aldosterone is produced, and at lo- to 20-fold higher concentrations in the zona Torsemide inhibited aldosterone production fasciculata where glucocorticoids are produced. stimulated by each of the classical secretagogues - angiotensin II, potassium, and ACTH. Inhibition was also seen, albeit at higher drug concentrations, when steroid production was
Vol. 63, No. 3, 1998
TorsemideInhibitsAldost~one Synthesis
PL-49
increased by addition of the aldosterone precursor corticosterone, a test of the “late pathway” of aldosteronogenesis. First, our results suggest that inhibition of Our observations have several implications. aldosterone could occur in humans taking torsemide for treatment of edema, heart failure, or hypertension. Indeed, the plasma concentrations of torsemide achieved in humans are similar to those that were used in our experiments (12). However, the high proportion of the drug bound to plasma proteins makes it difficult to compare the two situations using total concentration alone. A more direct comparison is offered by experiments on rabbit renal tubules in virro where the EDso for torsemide’s effect on sodium current was between 10m7and 10dM, similar to the EDSo we found for inhibition of aldosterone production by bovine and guinea pig adrenal cells (13). The diuretic and aldosterone effects show similar relative susceptibilities to torsemide and two of its metabolites. If aldosterone secretion were reduced by torsemide in humans, there would be less tendency for potassium excretion in response to the drug. Less potassium loss has been reported in patients taking torsemide compared with furosemide (2,5). That is only indirect evidence for lower aldosterone secretion. Although some direct measurements suggest lower aldosterone levels in patients on torsemide compared with furosemide, it is difficult to conclude that the drug inhibited the zona glomerulosa unless accurate measurements are made of the many stimuli and inhibitors that can influence that cell (14). Furthermore, potassium loss with diuretic therapy is largely caused by increased levels of sodium at the distal tubule, an effect largely independent of aldosterone. There are two other reasonable explanations for reduced kaliuresis during treatment with torsemide compared with furosemide: torsemide may be a weaker stimulus to the renin-angiotensinaldosterone axis because its effects have a slower rate of onset (15), and/or torsemide may antagonize the effects of aldosterone at the distal tubule (15,16). The sensitivity of the adrenal glomerulosa to torsemide suggests that the drug has effects in cells outside of the kidney. This susceptibility may not be widespread. Cells of the zonal glomerulosa are exquisitely sensitive to their ionic milieu, especially to the concentration of potassium in cytosol and extracellular fluid (7). Aldosterone production is also sensitive to ionophores and inhibitors of monovalent cation transport (8-10). The relative insensitivity of the adjacent zona fasciculata to ions, ionophores, and torsemide shows how unusual the zona glomerulosa is. One other nonrenal action of torsemide has been reported - inhibition of thromboxane-induced constriction of canine coronary artery in vitro (17). It is not known whether the coronary effect is like the diuretic effect in that it results from an action of torsemide on ion transport in the cell Wall.
Inhibition of aldosterone production from its precursor corticosterone indicates that torsemide acts on the late pathway of aldosterone biosynthesis, most of which takes place inside mitochondria. Most stimuli and many inhibitors of aldosterone biosynthesis act at a step in the early pathway, the transport of cholesterol across the mitochondrial membrane to the side chain cleavage enzyme that forms pregnenolone (7). Inhibition of later steps suggests that drugs like torsemide, which affect ion transport by the outer cell membrane, can impact events inside the mitochondrion. The same indirect mitochondrial effect is apparently displayed by ionophores for monovalent cations; they are inactive against isolated mitochondria, but inhibit the late pathway in intact cells (9).
PL-50
Torsemide Inhibits Aldosterone Synthesis
Vol. 63, No. 3, 1998
Torsemide’s effects on adrenal cells were shared by one of its metabolites, “M3,” the hydroxylated derivative that is almost as potent a diuretic as the parent (18). “M5,” the carboxy derivative that is virtually devoid of diuretic potency, was less potent than M3 in the adrenal. Insofar as those three compounds can be instructive, the diuretic and adrenal inhibitory activities of torsemide appear to be related. Furosemide, which is chemically distinct from torsemide but roughly equal to it in diuretic potency, did not show as much activity against aldosterone production. Our results suggest that candidate diuretics, or drugs that might be used with diuretics, should be tested for their effects on aldosterone secretion, since inhibition of aldosterone secretion could ameliorate the potassium wastage that frequently complicates diuretic therapy, and the cardiovascular fibrosis that may be stimulated by aldosterone in some disease states. Acknowledgements We acknowledge the expert technical assistance of A. Redmann. Torsemide and its metabolites were the gift of Boehringer Mannheim GmbH, facilitated by Drs. T. Bolke and L. Piesche. The experiments were supported by grants from the U.S. Department of Veterans Affairs, and from Boehringer Mannheim GmbH. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
R. LAMBE, 0. KENNEDY, M. KENNY, and A. DARRAGH, Eur. J. Clin. Pharmacol. 31 (suppl) 9-14 (1986). M. LESNE, Arzneim. Forsch./Drug Res. 38 160-163 (1988). I. ACHHAMMER and P. METZ, Drugs 41 (suppl. 3) 80-91 (1991). A.J. REYES, J. Cardiovasc. Pharmacol. 22 (suppl. 3) Sll-S23 (1993). C.J. DUNN, A. FITTON, and R.N. BROGDEN, Drugs 49 121-142 (1995). G. GIEBISCH, Klin. Wochenshr. 63 877-885 (1985). S.J. QUINN and G.H. WILLIAMS, Am. Rev. Physiol. 50 409-426 (1988). M.E. ELLIOTT, N.E. HADJOKAS, and T.L. GOODFRIEND, Endocrinology 118 1469-1475 (1986). N.E. HADJOKAS, T.L. GOODFRIEND, M.E. ELLIOTT, and S.-F. WEN, J. Cardiovasc. Pharmacol. 15 291-301 (1990). N. HADJOKAS and T. GOODFRIEND, Pharmacology 43 141-150 (1991). A. REDMANN, K. MOBIUS, H.H. HILLER, W. OELKERS, and V. BOHR, Eur. J. Endocrinol . 133 499-506 ( 1995). A. HERCHUELZ, F. DEGER, J. DOUCHAMPS, H. DUCARNE, and 1. BROEKHUYSEN, Arzneim.-Forsch./Drug Res. 38 180-183 (1988). M. WITTNER, A. DI STEFANO, P. WANGEMANN, and R. GREGER, Drugs 41 (suppl. 3) 1-13 (1991). A.J. REYES, Drugs 41 (suppl. 3) 35-59 (1991). G. GIEBISCH and G. KLEIN-ROBBENHAAR, J. Cardiovasc. Pharmacol. 22 (suppl. 3) Sl-SlO (1993). T. UCHIDA, K. YAMANAGA, M. NISHIKAWA, Y. OHTAKI, H. KIDO, and M. WATANABE, Eur. J. Pharmacol. 205 145-150 (1991). T. UCHIDA, H. KIDO, K. YAMANAGA, M. OKITA, and M. WATANABE, Prostaglandins Leukot. Essent. Fatty Acids 45 121-124 (1992). H. KNAUF, H. SPAHN, and E. MUTSCHLER, Drugs 41 (suppl. 3) 23-34 (1991).
ELSEVIER
Life sciences, Vol. 63. No. 3, Pp. PL45-50,1998 Publiskd by E?kvier Science Inc. Printed in the USA. All rights reambed am-32ospl mm t .oo
PmRhucoLoGYLEnERs Accehated Cbmnunkti
TORSEMIDE INHIBITS ALDOSTERONE SECRETION IN VITRO Theodore L. Goodfriend,’ Dennis L. Ball,’ Wolfgang Oelkers,* and Volker m? ‘William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, Wisconsin 53705 and *Freie Universitit Berlin, Universititsklinikum Benjamin Franklin, Medizinische Klinik und Poliklinik, Hindenburgdamm 30, 12200 Berlin, Germany (SubmittedJanuary29.1998, accepted March6,1998, received in final form April 27,1998) Abstract Torsemide inhibited aldosterone secretion by adrenal cells from rats, cows, and guinea pigs stimulated in vitro by potassium, angiotensin, dibutyryl cyclic AMP, ACTH, or corticosterone. Inhibitory concentrations for adrenal cells (micromolar) were comparable with those reported to inhibit ion transport in isolated renal tubules. Inhibition of aldosterone secretion could reduce kaliuresis, and that may explain why torsemide causes less kaliuresis than other diuretics. Published by Elsevier Science Inc. Key Words:
diuretics, hypokalemia,
hypertension,
torsemide
Introduction
Torsemide is a potent loop diuretic that has been reported to cause less potassium excretion than furosemide or hydrochlorothiazide (1-5). Thiazide and loop diuretics increase urinary excretion of potassium in part by stimulating the renin-angiotensin axis and the secretion of aldosterone (6). Drugs that inhibit the secretion of aldosterone or its action on the distal renal tubule minimize potassium wastage during diuretic therapy. We studied the effect of torsemide on adrenal glomerulosa cells to see if it inhibited aldosterone secretion, an action that might help to explain the relatively small kaliuresis caused by this diuretic. Aldosterone secretion is controlled by many factors in addition to angiotensin; foremost among these is extracellulax potassium (7). Potassium is thought to regulate aldosterone secretion by its effect on the membrane potential of adrenal glomerulosa cells, whose calcium channels are exquisitely sensitive to that potential. Membrane potential in the adrenal, as in other cells, is determined by the channels and pumps that regulate partition of the principal inorganic ions of extracellular and intracellular fluid. Because of the critical role that membrane potential plays in aldosterone secretion, we and others have tested reagents that affect ion pumps and channels in adrenal glomerulosa cells. Those experiments showed that ouabain, veratridine, pinacidil, monensin, nigericin, valinomycin, and 4-amino pyridine inhibit aldosterone secretion (8-10). Torsemide inhibits Na+/K+/2Cl- cotransport in the thick ascending limb of the loop of Henle, but an action on ion movements in Corresponding author: Theodore L. Goodfriend, William S. Middleton Memorial Veterans Hospital, 2500 Overlook Terrace, Madison, Wisconsin 53705. Telephone: (608) 262-7007; FAX: (608) 262-7644; E-mail: [email protected]
PL46
Torsemide Inhibits Aldosterone Synthesis
Vol. 63, No. 3, 1998
other cells has not been reported We show here that torsemide inhibits aldosterone secretion by adrenal cells from rats, guinea pigs, and cows studied in vitro. Torsemide was more potent in this respect than furosemide. If torsemide inhibits aldosterone secretion in humans, that might help explain the clinical finding that chronic therapy with torsemide causes less potassium wastage than other diuretics. Methods Cells from the adrenal zona glomerulosa of cows, guinea pigs, and rats were prepared by collagenase-deoxyribonuclease digestion as described in previous publications (9,ll). Just before each experiment, cells were warmed to 37°C for 1 h to restore intracellular ion balance. Cell viability was assessed by trypan blue dye exclusion, and viable cells were counted in a hemocytometer. Cell viability exceeded 80% in all preparations. Contamination of glomerulosa cells by fasciculata was estimated by the difference in cell size. Fasciculata cells comprised, on average, 0.5-2.5% of the glomerulosa preparation from bovine adrenals, and S-10% of the preparation from rat and guinea pig. The presence of fasciculata in the bovine and guinea pig cell mixtures enabled comparison of the effect of drugs on cortisol secretion, as well as aldosterone. To test the effects of drugs, aliquots containing 200,000 cells per ml were incubated in buffered salt solutions for 2 h at 37°C in air (bovine and guinea pig) or 95% 0,/5% CO* (rat). For rat cells, the incubation medium contained two parts of tissue culture Medium 199 and one part All media contained bovine serum albumin 0.1%. balanced salt solution. Drugs were administered to cells that were stimulated by angiotensin II, ACTH, dibutyryl cyclic AMP, potassium, or the steroid substrate corticosterone. At the end of incubation, steroids were measured in the supernatants by specific radioimmunoassays for aldosterone and cortisol in experiments with bovine and guinea pig cells, and for aldosterone in experiments with rat cells. Rat adrenals lack 17-hydroxylase, so the product of their zona fasciculata is corticosterone. Data are expressed as the mean of three incubation tubes, each assayed in duplicate, plus or minus the standard error of the mean of the three tubes. Torsemide effects were tested three times in bovine, three times in rat, and once in The results depicted in Fig. 1 are from a representative guinea pig adrenal preparations. experiment. Binding of radioiodinated
angiotensin
II to cells was performed as described previously
(9).
Torsemide and two of its metabolites were the gift of Boehringer Mannheim. The chemical formulae are as follows: torsemide is l-isopropyl-3-[[4-(3-methyl-phenylamino)py~dine]-3“M3” of torsemide is 1-isopropyl-3-[[4-(3-methyl-4’-hydroxysulfonyllurea; metabolite phenylamino)pyridine]-3-sulfonyllurea; and metabolite “M5” is 1-isopropyl-3-[[4-(3’carboxyphenylamino)pyridine]-3-sulfonyllurea. Radioimmunoassay kits were purchased from Diagnostic Products Corp., Los Angeles, CA. For some assays, antialdosterone antiserum was purchased from ICN Biochemicals, Inc., Costa Mesa, CA. Angiotensin II and human ACTH (l-24) were purchased from Bachem, Inc., Torrance, CA. Results Torsemide inhibited aldosterone production by cells from bovine, rat and guinea pig adrenal glands. When the stimulus was angiotensin II at 10e7M, the average IDS, for inhibition of the increment was approximately 10-6M in bovine cells and 3 x 10mSMin rat cells (Fig. 1). In guinea
PL47
Torsemide Inhibits Aldosterone Synthesis
Vol. 63, No. 3, 1998
pig adrenals, the IDSowas 5 x 10e7M(data not shown). When potassium was the stimulus in rat or bovine cells, the efficacy of torsemide was approximately the same as when angiotensin II was added (Fig. l), but the drug was slightly less effective in inhibiting the aldosterone secretion stimulated by dibutyryl cyclic AMP or by the steroid precursor corticosterone. Basal aldosterone production of aldosterone was also inhibited by the drug (data not shown).
. I-8’ ‘t.
100
\
1 ‘*.\
K+
80
‘* 1 Corticosterone
E\ 1T I
\
60
‘I
\
\’
\
Ang II T’L. AMP \
IO-7 3x10-7
10-G 3x1o-6 10-s 3x1o-5 10-4
10-7
3x10.~
10-c 3x10+
1O-5 3x10+
1O-4
Torsemide (M)
Torsemide (M)
Fig. 1 Dose-response curves of inhibition of aldosterone production by torsemide. Adrenal glomerulosa cells from bovine (panel A) and rat (panel B) were prepared as described in the text and incubated with various stimuli and graded concentrations of torsemide for 2 h. At the end of the incubations, aldosterone in the supematant was measured by radioimmunoassay. All data are presented as a percent of the amount of aldosterone produced by each type of cell in response to the indicated stimulus on the day of the experiment. The points represent means of three incubation tubes, and the vertical bars are standard errors of the mean of those three tubes. Stimuli were added to the following final concentrations: angiotensin II (Ang II) 10w7M,ACTH 10m9M,potassium 7.2 mM, dibutyryl cyclic AMP (AMP) 1 mM, and corticosterone 3 X 10bM. The average maximum amount of aldosterone produced under stimulation by angiotensin II was 30 ng per lo6 bovine cells per h, and 34 ng per lo6 rat cells per h. Furosemide and two metabolites of torsemide were also tested. The M3 (hydroxylated) metabolite showed inhibition of aldosterone production with a potency comparable with that of torsemide, but the M5 (oxidized) metabolite was much less active. Furosemide was less potent than torsemide (Fig. 2).
PL-48
Torsemide Inhibits Aidosterone Synthesis
Vol. 63, No. 3, 1998
Cortisol production by bovine and guinea pig adrenal cells was much less sensitive to torsemide than aldosterone production; the ID,,, was lo-20 times greater for cortisol than for aldosterone in the same incubation tubes (data not shown). Torsemide had no effect on the saturable binding adrenal glomerulosa cells (data not shown).
of radio-iodinated
angiotensin
II to bovine
Furosemide
100
0
tl
11
I11111
10-7 3x1o-7
I
II
I11111
10-a 3x1o-6
I
II
I,,,,
1O-5 3x1o-5
Drug Concentration
J
10-4
(M)
Fig. 2 Effects of torsemide, two metabolites of torsemide, and furosemide on rat adrenal cells stimulated by angiotensin II (10m7M). The metabolites were tested in three different experiments, one of which is presented in the figure. Discussion Our experiments show that torsemide, a diuretic that inhibits the Na+/K+/2Cl- cotransporter in the ascending limb of the loop of Henle, inhibits aldosterone production by adrenal cells from rat, The drug was active at micromolar concentrations in the adrenal guinea pig, and cow. glomerulosa where aldosterone is produced, and at lo- to 20-fold higher concentrations in the zona Torsemide inhibited aldosterone production fasciculata where glucocorticoids are produced. stimulated by each of the classical secretagogues - angiotensin II, potassium, and ACTH. Inhibition was also seen, albeit at higher drug concentrations, when steroid production was
Vol. 63, No. 3, 1998
TorsemideInhibitsAldost~one Synthesis
PL-49
increased by addition of the aldosterone precursor corticosterone, a test of the “late pathway” of aldosteronogenesis. First, our results suggest that inhibition of Our observations have several implications. aldosterone could occur in humans taking torsemide for treatment of edema, heart failure, or hypertension. Indeed, the plasma concentrations of torsemide achieved in humans are similar to those that were used in our experiments (12). However, the high proportion of the drug bound to plasma proteins makes it difficult to compare the two situations using total concentration alone. A more direct comparison is offered by experiments on rabbit renal tubules in virro where the EDso for torsemide’s effect on sodium current was between 10m7and 10dM, similar to the EDSo we found for inhibition of aldosterone production by bovine and guinea pig adrenal cells (13). The diuretic and aldosterone effects show similar relative susceptibilities to torsemide and two of its metabolites. If aldosterone secretion were reduced by torsemide in humans, there would be less tendency for potassium excretion in response to the drug. Less potassium loss has been reported in patients taking torsemide compared with furosemide (2,5). That is only indirect evidence for lower aldosterone secretion. Although some direct measurements suggest lower aldosterone levels in patients on torsemide compared with furosemide, it is difficult to conclude that the drug inhibited the zona glomerulosa unless accurate measurements are made of the many stimuli and inhibitors that can influence that cell (14). Furthermore, potassium loss with diuretic therapy is largely caused by increased levels of sodium at the distal tubule, an effect largely independent of aldosterone. There are two other reasonable explanations for reduced kaliuresis during treatment with torsemide compared with furosemide: torsemide may be a weaker stimulus to the renin-angiotensinaldosterone axis because its effects have a slower rate of onset (15), and/or torsemide may antagonize the effects of aldosterone at the distal tubule (15,16). The sensitivity of the adrenal glomerulosa to torsemide suggests that the drug has effects in cells outside of the kidney. This susceptibility may not be widespread. Cells of the zonal glomerulosa are exquisitely sensitive to their ionic milieu, especially to the concentration of potassium in cytosol and extracellular fluid (7). Aldosterone production is also sensitive to ionophores and inhibitors of monovalent cation transport (8-10). The relative insensitivity of the adjacent zona fasciculata to ions, ionophores, and torsemide shows how unusual the zona glomerulosa is. One other nonrenal action of torsemide has been reported - inhibition of thromboxane-induced constriction of canine coronary artery in vitro (17). It is not known whether the coronary effect is like the diuretic effect in that it results from an action of torsemide on ion transport in the cell Wall.
Inhibition of aldosterone production from its precursor corticosterone indicates that torsemide acts on the late pathway of aldosterone biosynthesis, most of which takes place inside mitochondria. Most stimuli and many inhibitors of aldosterone biosynthesis act at a step in the early pathway, the transport of cholesterol across the mitochondrial membrane to the side chain cleavage enzyme that forms pregnenolone (7). Inhibition of later steps suggests that drugs like torsemide, which affect ion transport by the outer cell membrane, can impact events inside the mitochondrion. The same indirect mitochondrial effect is apparently displayed by ionophores for monovalent cations; they are inactive against isolated mitochondria, but inhibit the late pathway in intact cells (9).
PL-50
Torsemide Inhibits Aldosterone Synthesis
Vol. 63, No. 3, 1998
Torsemide’s effects on adrenal cells were shared by one of its metabolites, “M3,” the hydroxylated derivative that is almost as potent a diuretic as the parent (18). “M5,” the carboxy derivative that is virtually devoid of diuretic potency, was less potent than M3 in the adrenal. Insofar as those three compounds can be instructive, the diuretic and adrenal inhibitory activities of torsemide appear to be related. Furosemide, which is chemically distinct from torsemide but roughly equal to it in diuretic potency, did not show as much activity against aldosterone production. Our results suggest that candidate diuretics, or drugs that might be used with diuretics, should be tested for their effects on aldosterone secretion, since inhibition of aldosterone secretion could ameliorate the potassium wastage that frequently complicates diuretic therapy, and the cardiovascular fibrosis that may be stimulated by aldosterone in some disease states. Acknowledgements We acknowledge the expert technical assistance of A. Redmann. Torsemide and its metabolites were the gift of Boehringer Mannheim GmbH, facilitated by Drs. T. Bolke and L. Piesche. The experiments were supported by grants from the U.S. Department of Veterans Affairs, and from Boehringer Mannheim GmbH. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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