Verapamil directly inhibits aldosterone synthesis by adrenal mitochondria in vitro

J.steroidBiochem. Vol.30,No. 1-6, pp. 453-456,1988

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VERAPAMIL DIRECTLY INHIBITS ALDOSTERONE SYNTHESIS BY ADRENAL MITOCHONDRIA IN VITRO N. BLANCHOUIN-EMERIC*, M. ZENA~I*,

G. DEFAYE~

and B. AUPETIT*$

*Service de Biochimie Mtdicale, FacultC de Mtdecine, Piti&SalpBtritre, 91 bd de l’hopilal, 75634 Paris Cedex 13 and tBiochimie des rkgulations cellulaires endocrines, INSERM, U244, Grenoble, BP 85X 38044, France Summary-The action of verapamil, a calcium channel blocker, on the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) was studied using duck adrenal mitochondria in the absence of regulatory factors. Results show that lo-’ M verapamil inhibits the transformation of 18-hydroxycorticosterone into aldosterone by 52.8%. Moreover, our findings show that verapamil induces

only a slight inhibition of respiratory capacity without action on respiratory control and does not displace 18-hydroxycorticosteronefrom cytochrome P,,, 1I/3which catalyses the reaction. Thus, this study does not explain the mechanism of inhibition induced by verapamil on the last step ofaldosterone synthesis but it is of interest to note, for clinical use, that this inhibition is not linked to regulatory factors of aldosterone production. Since primary hyperaldosteronisms are characterized by their independence vis-d-vis regulatory factors, administration of verapamil may be particularly interesting for treatment of primary hyperaldosteronisms.

droxy-4-pregnene-3,18,20-trione) by New England Nuclear Corporation and 18-hydroxy[ 1,2-3H]cor(1 lp, 18,2 I-trihydroxy-4-pregnene-3,20ticosterone dione) by Amersham. The other reagents were commercial products of analytical reagent grade.

INTRODUCTION

The efficacy of calcium channel blockers in various forms of hypertension has been convincingly demonstrated. The antihypertensive effects of calcium channel blockers have been attributed to their peripheral vasodilatory effect. However, clinical and experimental evidence suggests that the hypotensive action of these substances may also be due to other effects, including one on aldosterone production. It has been shown that amongst the calcium channel blockers, verapamil reduces the blood level of aldosterone in normal [l] and hypertensive subjects [2,3]. In addition, according to Fakunding et a1.[4], verapamil inhibits the in vitro production of aldosterone by isolated adrenal glomerulosa cells stimulated by angiotensin II. This inhibition preferentially affects the conversion of cholesterol into pregnenolone. To our knowledge, the effect of verapamil on the terminal portion of aldosterone synthesis in the absence of any regulatory factor has not been investigated. To carry out such an investigation, the authors studied the in vitro action of verapamil on the final step of aldosterone synthesis (conversion of 1g-hydroxycorticosterone into aldosterone) using duck adrenal mitochondria in the absence of calcium, and on reconstituted cytochrome P.+501 l/3. EXPERIMENTAL

Chemicals L-Malate, rotenone, antimycin A and verapamil were supplied by Sigma, KCN by Prolabo, ADP by Boerhinger Inc., [4J4C]aldosterone (1 lp,21-dihy-

Proceedings of the 8th International Symposium of The Journal of Steroid Biochemistry “Recent Advances in Steroid Biochemistry” (Paris, 24-27 May 1987). $To whom correspondence

should be addressed. 453

Preparation of homogenate and mitochondrial fraction Duck adrenal is the best animal gland for this study since the aldosterone biosynthesis pathway is the same as in man and the yield of transformation is sufficient to study each step in this pathway, even those with low yield [S-7]. Adrenal glands were obtained from the Musk duck (Cairina moschata) weighing 3-3.5 kg. The ducks were killed in the laboratory and the adrenals placed rapidly in an icebath (15 min). The following procedures were carried out at +4”C. To remove blood, adrenal glands were rinsed with the same buffer used for homogenization. In order to measure ATPase activity in homogenate and mitochondria, an aliquot of adrenal pool was homogenized in a buffer without phosphate: saccharose 250mM, MgCl* 5 mM, EGTA 0.5 mM, Tris lOmM, bovine serum albumin 0.075%, pH 7.00 (10 ml/g tissue). For other techniques, homogenization was carried out in a buffer containing phosphate: saccharose 450 mM, Tris 30 mM, EDTA 1 mM, bovine serum albumin 0.2%, pH 7.4 (10 ml/g tissue). The mitochondrial fraction was prepared according to the methods of Hogeboom[8] and Sauer and Mulrow[9], with a few modifications which have been previously described [lo]. The mitochondrial fraction did not undergo any physicochemical treatment which would modify membrane permeability. Mitochondrial protein concentrations were determined by the method of Lowry et al.[ 111, using bovine serum albumin as standard. The ultrastructural character-

N. BLANCHOUIN-EMERIC etal.

454

istics of the mitochondria were studied by electron microscopy using a method previously described [ 121. The purity of the mitochondrial preparation was determined using enzymatic markers: succinate cytochrome C reductase, cytochrome C oxidase, malate dehydrogenase, ATPase, glucose-6-phosphate dehydrogenase and 2 1-hydroxylase, using methods previously reported [ 131. The respiratory characteristics of the mitochondrial fraction and the action of verapamil on these characteristics was determined polarographically with a Clark oxygen electrode at 40°C in 2 ml of “respiratory buffer” (saccharose 250mM, MgClz 5 mM, EGTA 0.5 mM, KHzPO, 10 mM, Tris 10 mM, bovine serum albumin 0.075%, pH 7.00).

ble 1). The oxidative phosphorylation chain functioned normally (Table 2). Rotenone did not inhibit respiration in the presence of succinate as it did in the presence of r.-malate. Respiratory control was lower than that in other organs [ 17, 181. As shown in Table 3, inhibition of respiratory intensity by verapamil (0.01 mM) was only 11.5O/o(PtO.OOl), and no statistically significant inhibition of respiratory-control was observed, consistent with results reported by other authors [ 191.

Table 1. Enzymatic activities of the mitochondrial fraction and homogenate Enzymes

Conditions of incubation The general conditions of incubation were the following: 1 mg of mitochondrial protein in 2 ml of “respiratory buffer” was incubated aerobically in a Dubnoff metabolic shaker at 40°C for 30 min in the presence of 136 pmol of 18-hydroxy[ 1,2-3H]corticosterone with sp. act. 46 Ci/mmol. Before starting the reaction by 15 mM of L-malate, 0.0 1 mM of veranamil was added in 25 ul of ethanol. The assays without verapamil were carried out in the presence of the same volume of ethanol. The reaction was stopped by addition of chloroform and samples were then frozen at - 18°C until extraction.

Cytochrome c oxidase Succinate cytochrome c reductase Malate dehydrogenase ATPase 2 1-Hydroxylase Glucose 6 phosphate dehvdroaenase

Mitochondria

Homogenate

1185k25.13

39Ok 8.67

89k6.18

24 + 3.45

401& 13.87 46lkl3.40 0.01 kO.005

123 2 8.42 13Ok7.25 29k2.48

0

29k2.38

The specific activity of 21-hydroxylase (a microsomal enzyme catalysing the transformation of progesterone into deoxycorticosterone) is expressed as pmol/mg protein per min. The other specific activities are expressed as nmol/mg protein per min.

Aldosterone purification and identification An internal standard [4-r4C]aldosterone _ _

was ineluded to determine the yield. The aldosterone was purified and identified by paper chromatography as previously described [ 141. The tritiated aldosterone formed was calculated from the dpm (liquid scintillation counter, Tricarb 2000). The results were expressed as pmole of aldosterone formed per mg of mitochondrial protein per 30 min.

Table 2. Respiratory characteristics of adrenal mitochondria Inhibitors

Succinate

0 Rotenone Antimycin A KCN

56.03 + 2.83 54.11* 2.94 0 0

t_-Malate

0 Rotenone Antimycin A KCN

43.94 5 1.47 3.14+ 1.62 0 0

Spectrophotometric study Cytochrome P45,1 18 was prepared according to Katagiri et al.[ 151 with minor modifications [ 161. Cytochrome Pdsol 18 was diluted in phosphate buffer (50 mM, pH 7.4) containing dithiothreitol (0.1 mM), EDTA (0.1 mM) and Tween 20 (0.3%). Signal-producing ligand binding was determined at the appropriate wavelengths (1390-420 nm). Spectra were recorded with a Uvicon Kontron spectrophotometer.

Statistical analysis Statistical t-test.

analysis

was performed

0, I uutake 1 ~~ (nmol/mg protein per min)

Substrate

:oncentrations of the reagents (mM) are the following Lmalate and succinate 15, ADP 3, rotenone 25 x IO-‘, antimycin A 9 x 10e5, KCN 10-r. Each value represents the mean + SD of five different experiments. Respiratory control ratio for duck adrenal mitochondria are 1.2 both for succinate and t-malate, in the presence of 3 mM ADP.

using Student’s

RESULTS The purity of the mitochondrial fraction was satisfactory. The mitochondria-associated enzymes were all enriched in the mitochondrial fraction, whereas the microsomal marker 2 1-hydroxylase was considerably reduced and the cytosolic enzyme glucose-6-phosphate dehydrogenase was absent (Ta-

In the absence of regulatory factors, verapamil (0.0 1 mM) inhibited the conversion of 1I-hydroxycorticosterone into aldosterone by 52.8% (Table 4). Verapamil did not bind to reconstituted cytochrome P4,01 18 which catalyses the transformation of 18hydroxycorticosterone into aldosterone. The change in optical density caused by the binding of metopirone to cytochrome P,,, (O.D. 0.420-0.3 10) was not observed with verapamil (0.01 mM).

Aldosterone synthesis Table 3. Effect of verapamil on respiratory characteristics of adrenal mitochondria Verapamil (M)

Or uptake (nmoVmg protein per min)

Succinate

0 10-5

62.82k2.16 56.82 * 2.33

L-Malate

0 10-5

47.31+ 1.56 40.94+ 1.31

Substrate

Concentrations of the reagents (mM) are the following: Lmalate and succinate 15. The assays without verapamil are carried out in the presence of the corresponding volume of ethanol. Each value represents the mean c SD of five different experiments. Respiratory control ratios for duck adrenal mitochondria are 1.15,for both succinate and L-malate in the presence pf ADP 3 mM, and are not affected by verapamil. Table 4. Verapamil inhibits the transformation hydroxycorticosterone to aldosterone

of 18-

Verapamil Aldosterone formed % Inhibition (pmol/mg protein per 30 min) of the reaction (M) 0 10-s

4.62 + 0.09 2.18+0.11

52.8

Each value represents the mean k SD of four experiments. The reaction was carried out in a final volume of 2 ml of buffer (pH 7), 1 mg of protein, 136 pmol of 18hydroxy[ 1,2-3H]corticosterone at 40°C for 30 min. For each assay, the reaction was started by adding 15 mM Lmalate. DISCUSSION AND CONCLUSION This study showed that verapamil, at the concentration usually used in vitro, inhibited (52.8%) the conversion of 18-hydroxycorticosterone into aldosterone. Previous studies showed that this reaction is a cytochrome P.,sol l/3 hydroxylation [20] and is linked to energy metabolism [2 11. Our findings showed that verapamil induced a slight inhibition of respiratory capacity without action on respiratory control, and did not displace 18-hydroxycorticosterone from the cytochrome P4sol l/I binding site (verapamil did not bind to reconstituted cytochrome P,sol 18). This inhibition of aldosterone biosynthesis from 18-hydroxycorticosterone by verapamil cannot be explained by an inhibition of electron transfer nor by an inhibition at the cytochrome P4s0 level. We are continuing our research in order to find out how

verapamil inhibits the last step of aldosterone biosynthesis: is it an inhibitor of mitochondrial enzymatic activities or an artificial electron transport between NADH and cytochrome c as is ruthenium red [22]? Although this study does not explain the mechanism of inhibition induced by verapamil on the last step of aldosterone synthesis, it is of interest to note, for clinical use, that this inhibition is not linked to regulatory factors of aldosterone production. As a matter of fact, this inhibition takes place without calcium (including endogenous calcium released in the medium and chelated by EGTA) and in the

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absence of regulatory factors of aldosterone production (ACTH, Angiotensin II, potassium). Since primary hyperaldosteronisms are characterized by their independence vis-his regulatory factors, administration of verapamil may be particularly interesting for the treatment of primary hyperaldosteronisms. Acknowledgement-This work was supported in part by research grants from INSERM (contract no. 854004).

REFERENCES 1. Guthrie G. P., McAllister R. G. and Kotchen T. A.: Effects of verapamil upon the pressor and steroidogenic actions of angiotensin and ACTH. Clin. Res. 30 (1982) 734A. 2. Spivack C., Ocken S. and Frishman W. H.: Clinical use in the treatment of systemic hypertension. Drugs 25 (1983) 154-177. 3. Guerrero J. R., Martin S. S.: Full spectrum calcium channel blocking agent; an overview. Med. Rex Rev. 4 (1984) 87-109. 4. Fakunding J. L. and Catt K. J.: Dependence of aldosterone stimulation in adrenal glomerulosa cells on calcium uptake: effects of lanthanum and verapamil. Endocrinology 107 (1980) 1345-1353. 5. Sandor T. and Lanthier A.: Etude comparee de la biosynthtse de l’aldosterone et d’autres corticosteroides chez diverses especes de verttbrts. Union Med. Can. 97 (1967) 1208-1211. 6. Sandor T. and Lanthier A.: Studies on the sequential hydroxylation of progesterone to corticosteroids by domestic duck (Anas platyrinchos). Endocrinology 86 (1970) 552-559. 7. Aupetit B.: Etude de la reaction de transformation de la 18-hydroxycorticosterone en aldosterone dans la surrenale de canard (Anas platyrinchos). These de Doctorat d’Etat, Universite Pierre et Marie Curie, Paris VI (1978). 8. Hogeboom G. H.: In Methods in Enzymology. Academic Press, New York, Vol. 1 (1955) pp. 16-19. 9. Sauer L. A. and Mulrow P. J.: Steroid hydroxylations in rat adrenal mitochondria. Archs Biochem. Biophys. 134 (1969) 486-496. 10. Aupetit B., Aubry-Marais F. and Legrand J. C.: Comportement compare de la 18-hydroxycorticosterone de synthtse et de la 18-hydroxycorticosttrone “endogene” dans la synthtse de l’aldosterone. Biochimie 59 (1977) 311-321. 11. Lowry 0. H., Rosenbrough N. J., Farr A. L. and Randall R. L.: Protein measurement with the Folin phenol reaaent. J. biol. Chem. 193 (1952) 265-275. 12. Aupetit B., Emeric N., To&y R.,‘Racadot O., Racadot J., Vonarx V. and Legrand J. C.: Stimulation of oxygen consumption at the cytochrome a, level inhibits aldosterone biosynthesis from 18hydroxycorticosterone. Biochim. biophys. Acta 884 (1986) 270-275. 13. Aupetit B., Accarie C., Emeric N., Vonarx V. and Legrand J. C.: The final step of aldosterone biosynthesis requires reducing power: it is not a dehydrogenation. Biochim. biophvs. Acta 752 (1983) 73-78. 14. Aupetit B., Antreassian J. and Legrand J. C.: Caractbre enzymatique et localisation subcellulaire de la transformation de la 18-hydroxycorticosterone en aldosterone. Biochimie 59 (1977) 705-712. 15. Katagiri M., Takemori S., Itagati E. and Suhara K.: Purification of adrenal cytochrome PdsO (cholesterol desmolase and steroid 118 and 18 hydroxylase). In Methods in Enzymology (Edited by S. Fleisher and L.

456

16.

17.

18.

19.

N. BJ..ANCHOLJIN-EMERIC et al. Packer). Academic Press, New York, Vol. II, part C (1978) pp. 124-132. Lombard0 A., Laine M., Defaye G., Monnier N., Guidicelli C. and Chambaz E. M.: Molecular organization (topography) of cytochrome P4s01 lp in mitochondrial membrane and phospholipid vesicles are studied by trypsinolysis. Biochim. biophys. Acta 863 (1986) 71-81. Cammer W. and Estabrook R. W.: Respiratory activity of adrenal cortex mitochondria during steroid hydroxylation. Archs Biochem. Biophys. 122 (1967) 721-734. Wakabayashi T., Kurono C., Asano M., Kimura H. and Ozana I.: Steroidogenesis in the zona glomerulosa of the adrenal cortex. II. Distribution of cytochrome PdsO in the zona glomerulosa of the bovine adrenal cortex. Bioenergetics 8 (1976) 55-71. Vaghy P. L., Matlib M. A., Szekeres L. and Schwartz A.:

Protective effects of verapamil and diltiazem against inorganic phosphate induced impairment of oxidative phosphorylation of isolated heart mitochondria. Biothem. Pharmac. 30 (198 1) 2603-26 IO. 20. Yanagibashi K., Haniu M., Shively J. E., Shen W. H. and Hall P.: The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 1 Ip, ll-hydroxylation, and aldehyde synthesis. J. biol. Chem. 261 (1986) 3556-3562. 2 1. Aupetit B., Toury R. and Legrand J. C.: Relation between energy metabolism and conversion of 18hydroxycorticosterone to aldosterone in adrenals. Biochimie 62 (1980) 823-827. 22. Schwerzmann K., Gazzotti P. and Carafoli E.: Ruthenium red as a carrier of electrons between external NADH and cytochrome C in rat liver mitochondria. Biochem. biophys. Res. Commun. 69 (1976) 8 12-8 15.