in vivo stimulation of aldosterone biosynthesis by endothelin: loci of action and effects of doses...

J. Sterbid Biochem. Molec. Biol. Vol. 45, No. 6, pp. 555-561, 1993 0960-0760/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

I N V I V O S T I M U L A T I O N O F A L D O S T E R O N E

B I O S Y N T H E S I S B Y E N D O T H E L I N : L O C I O F A C T I O N A N D

E F F E C T S O F D O S E S A N D I N F U S I O N R A T E

ADALI PECC1,1 CELSO E. GOMEZ-SANCHEZ, 2 MARIA E. O. DE BEDNERS, l CARLOS P. LANTOS I and EDUARDO N. COZZA 1.

IDepartamento de Quimica Biol6gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and PRHOM-CONICET, Cdad. Univ. Pab. II 4 ̀0 piso, (1428) Buenos Aires, Argentina and

2Department of Internal Medicine, University of South Florida Health Science Center and James A Haley, Veterans' Hospital Tampa, FL 33612, U.S.A.

(Received 19 August 1992; accepted 8 February 1993)

Summary--Infusion of endothelin-1 (ET-1) into rats increased adrenal mitochondrial synthesis of aldosterone from deoxycorticosterone and the adrenal cytosolic content of aldosterone. The dose-response relationships for these last two effects of ET-1 were found to be biphasic with a maximum (corresponding to 80 to 200% increase) at 50 to 80 ng ET-1/kg/min, and were also dependent on the infusion rate. Plasma aldosterone levels were also increased in a similar ratio. Previous infusion of the converting enzyme inhibitor enalapril did not affect the ET-l-induced increase in steroidogenesis. Finally, pregnenolene production was also increased in incubations of mitochondria from treated rats. These results indicate that ET-I augments aldosteronogenesis by increasing the early as well as the late pathway. These effects were independent of the formation of angiotensin II. Isolated glomerulosa cells responded to ET-I increasing aldosterone production in a dose-related fashion. These results confirm a direct effect of ET-I on the adrenal gland in vivo.

INTRODUCTION

Endothelins (ET) are a group of 21 amino acid peptides with two intrachain disulfide bridges and with potent pressor/vasoconstrictor activity [1]. The porcine, canine, rat, bovine and human genes code for similar preproendothelin which have identical portions corresponding to ET [1- 5]. The human genomic D N A library has shown the existence of three distinct ET genes and the following nomenclature is suggested, ET-1 for porcine/human endothelin, ET-3 for rat ET [6] and ET-2 for a new ET differing in 2 amino acids from ET-1 [2]. ET-1 and -2 have similar powerful vasoconstrictor activities but ET-3 is significantly less potent than ET-1 [2]. Homo- logous 21 amino acid peptides have been found in the venom of the Israeli burrowing asp, Actrastaspis engaddensis, called sarafotoxins that show similar action to ETs [7, 8].

ET not only has potent vasoconstrictive/ pressor activity[l], but also affects other responses including release of atrial natriuretic peptide [9], mitogenesis in several different cell

*To whom correspondence should be addressed.

types [10-12], inhibition of renin release [13] and stimulation of aldosterone secretion [14-16].

High affinity, specific receptors have been described for ET in the adrenal gland [14, 17- 19] with a preferential localization in the zona glomerulosa[18-20]. Two populations of ET receptors exist in this zone[17]. ET-1 and -2 bind to both receptors while ET-3 and S6b bind only to one of them. The ET-1/S6b binding sites ratio is about 5 [17].

ET-1 stimulates aldosterone secretion in cultured calf[14], or freshly dispersed rabbit [21], rat [15, 16, 22, 23] and human [16] zona glomerulosa cells. ET-1 stimulation of aldosterone production is lower than that provoked by angiotensin II [14, 16, 23] depending in cultured cells on the sera used in the culture media [20, 24]. In vivo infusions of ET-1 to dogs [25, 26] and rats[15,22] showed an increase in both plasma renin activity and plasma aldosterone [25-27].

Doses and infusion rates used in previous reports[15,22,25-27] show a large diversity. We believe these parameters are important since a potent vasoconstrictor effect might reduce the adrenal blood flow and, thus, the amount

555

556 ADALI P~CCI et al.

of ET- 1 that reaches the gland and/or the ability of aldosterone to leave from the glomerulosa cells. On the other hand, when the infusion rate increases the sodium charges will, as the water flux through vascular walls also increases. These events might interfere with aldosterone production.

In the present report we confirm that ET-1 stimulates aldosterone production by rat zona glomerulosa in vivo without interfering with the renin-angiotensin system and demonstrate that such a secretagogue action of ET-1 involves the stimulation of the conversion of deoxycorticosterone into aldosterone. Since knowledge of the dose-response relationship in vivo is of importance in terms of assessing the physiological importance of the steroidogenic activity of ET-1, we also used different doses of ET-1. Finally, ET-1 was also tested at different infusion rates.

E X P E R I M E N T A L

Reagents

ET was purchased from Peninsula Labor- atories (Belmont, CA). Enalapril was a kind gift of Dr J. C. Descalzo (Merck-Sharp & Dhome, Buenos Aires, Argentina). Angiotensin I, II (AI, II) and other reagents were purchased from Sigma Chemical Company (St Louis, MO).

Glomerulosa cells.

Male CHBB-Thom rats (200 to 250 g) were killed by decapitation and adrenals removed, placed on ice-cold saline and separated into

capsules and cores. Rat adrenal glomerulosa cells were obtained from adrenal capsules by colla- genase dispersion as described by Purdy et al. [28]. Aliquots of 1.2 x 105 cells were incubated in 0.5 ml of Krebs-Ringer-bicarbonate-glucose buffer (pH 7.2) [28, 29] containing 0.2% bovine serum albumin. After incubation cells were centrifuged at 1000 rpm for 10 min and aldosterone measured in supernatants.

Infusions

CHBB-Thom rats (200 to 250g) were anesthetized using pentothal i.p. (60 mg/kg). A tracheotomy was performed in order to elimin- ate secretions. Solutions were infused through an indwelling catheter in the jugular vein. Blood samples were drawn from a catheter in the caro- tid artery. Urine was collected with a catheter placed in the urinary bladder. After completion of surgical preparations rats were allowed to stabilize for 15 min.

After this stabilizing period, saline was infused into rats for 30 min and then blood samples were withdrawn for hematocrit determination (Hc0. Urine was also collected during the last 15 min of infusion (UVt). Finally, saline (control) or different agents (ET-1, All, AI) dissolved in saline were infused for 45 min. Urine was collected for the last 15 min of this second infusion (UVz) and blood samples withdrawn for hematocrit determination (Hc2). In a separate group, enalapril was infused during the last infusion (30 min) at a rate of 0.33/~g/min and in the second infusion period (45 min) at 0.11 #g/ min.

Saline ENA

Saline ET-1 All AI ENA ENA+ET-1 ENA+AI

TIME (mln)

15 30

I t

He1 [ J

Urine collection UV1

60 75

I

1 He2

I Urine coiled|on

UV2

Fig. 1. Scheme of the infusion procedure. ENA, cnalapril; ET-I, cndothelin I; AII, angiotensin II; AI, angiotensin I; Hcl, He2, UVI and UV2, see Experimental section. For more details see the text.

Endothe l in s t imula tes a ldos te rone p roduc t ion 557

All the above mentioned experiments were performed using different infusion rates. Fig. 1 shows a scheme of the infusion procedure.

Plasma aldosterone levels

Plasma was extracted twice with 30 vol of CH2CI2, the organic solvent evaporated under N: at room temperature and the dried residue reconstituted in 0.1 M borate buffer containing 5% ethanol. Aldosterone was determined in this final solution.

Adrenals

At the end of the infusions, adrenals were removed, placed on ice-cold saline, excised of fat and connective tissues and each pair separately (except when indicated) homogenized in 20 mM Tris-HCl buffer, pH 7.2, containing 25mM sucrose, 0.5 mM MgCI2, 3 mM CaCI2 and 1 mg/ ml glucose. Mitochondria and mitochondria- supernatant fractions were obtained by two centrifugations at 10500g for 20 min. For determination of cytosolic content of aldosterone the mineralocorticoid was directly measured in mitochondria-supernatant fractions.

Incubation of mitochondria

Mitochondria corresponding to one adrenal (approx. 0.8mg of mitochondrial protein) were incubated in duplicate at 37°C for 15 min in 0.65 ml of Krebs-Ringer-bicarbonate-glucose buffer [29, 30] containing 0.5 mM NADP ÷, 7 mM sodium malate in the absence or presence of 30#M deoxycorticosterone (DOC). The incubation was started by addition of malate and NADP ÷. After incubation, the tubes were placed in an ice-water bath for 15 min, centrifuged at 4500 rpm for 30 min at 4°C and aldosterone measured in the supernatant.

Miscellaneous

Aldosterone was measured by direct RIA using a monoclonal antibody [31]. Protein was measured by the method of Bradford [32].

Calculations

Data are expressed as mean -+ SEM of the number of separate experiments indicated in Results. Statistical analysis was performed by Student t-test or ANOVA as indicated in Results. Differences were considered significant at P < 0.05.

Hematocrit (Hc) and urine volume (UV) are presented as the value after treatment (second infusion, see Fig. 1) over the value after the first

infusion, i.e. Hc2/Hc I and UV2/UVI. These ratios were calculated dividing every single H c 2

(or UV2) value by the mean of HCl (or UV0. Abosolute values for Hc are also shown in the Results section.

Cytosolic content of aldosterone, mitochondrial synthesis of aldosterone after infusions and aldosterone production by isolated glomerulosa cells are presented as the treated/control (T/C) ratios. These T/C ratios were calculated by dividing treated values by the mean of control values. Absolute values are also shown in Results section.

R E S U L T S

Hc and UV

The Hc of control rats after the first infusion (HCl) were 47.9--+1.9 (n= 13); 46.7-+0.7* (n = 33) and 42.9 -+ 1.1"* (n = 17) at infusion rates of 50, 125 and 180/~l/min, respectively (*P < 0.05, **P < 0.01 with respect to 50 #1/ min; ANOVA). As expected, Hc decreased as the infusion rate increased.

ET-1 increased the Hc in a dose-dependent manner (Table 1). The maximal effects of ET-I on Hc were 13, 27 and 36% using infusion rates of 50, 125 and 180#1/min, respectively (Table 1). Thus, the effect of ET-1 on Hc was more evident as the infusion rate increased.

UV was dramatically decreased by ET-1. In effect, the UVJUV 1 ratio (see Fig. 1) varies from 3.1 -+ 0.8 for control rats to 0.4-+ 0.1 for rats treated with 80 ng ET-1/kg/min at an infusion rate of 125/zl/min.

Aldosterone production by isolated glomerulosa cells

Isolated adrenal glomerulosa cells incubated with different doses of ET-1 responded with a

Table 1. Effect of ET-I on Hc at different infusion rates

Infusion rates ET-1 (#l/rain) doses

(ng/kg/min) 50 125 180

0 0.96 + 0.04 (4) 0.90 + 0.04 (6) 0.81 + 0.13 (3) 20 0.97 + 0.04 (3) 0.95 +_ 0.06 (3) ND 50 0.98 + 0.03 (2) 1.02 _+ 0.08 (3) 1.23 + 0.19 (2) 80 1.10 _+ 0.20 (2) 1.12 _+ 0.06* (5) 1.26 -+ 0.13* (2)

110 1.13+0.11(2) 1.27+0.13.(3) 1.36+_0.10"*(2)

Rats were infused according to Fig. 1. T/C ratios were calculated as indicated in the Experimental section. Data are mean + SEM of the number of separated experiments indicated between round brackets. *P < 0.05, **P < 0.02 with respect to no ET-I (ANOVA). The Hc of control rats after the first infusion (He0 were 47.9_+ 1.9 (n = 13); 46.7+0.7 (n =33) and 42.9-+ 1.17 (n = 17) at infusion of 50, 125 and 180 #l/min, respectively ( tP < 0.05 with respect to 50 g l/rain; ANOVA). ND, Not determined.

558 ADALIPEccI et al.

Table 2. Effect of ET-I on aldosterone synthesis by isolated glomerulosa cells

ET-I (nM) Aldosterone: T/C ratio

0 1.00 5:0.07 0.0l 1.30 -+ 0.18" 0.1 1.66 -+ 0.20* 1 1.84 5: 0.21"*

Data are mean -+ SEM (n = 4) T/C ratios calculated as indicated under Exper- imental. Control incubation (0 nM ET-1) produced 1.21 -+ 0.21 ng aldosterone/tube. *P < 0.05; **P < 0.02, with respect to no ET-I.

stimulation of aldosterone release in a dose- dependent manner (Table 2).

Plasma aldosterone levels

When the infusion rate was 180 pl/min, 80 ng ET-1/kg/min increased plasma aldosterone levels from 34.4 + 2.8 ng/dl (control) to 58.2 + 4.3 ng/dl (P < 0.05) and the T/C ratio was 1.7. Similarly, at an infusion rate of 125 pl/min, 110 ng ET-1/kg/min increased plasma aldosterone levels from 31.1 +0.7 to 42.5_ 1.8 ng/dl (P < 0.05), the T/C ratio was 1.4. Thus, the increase in plasma aldosterone was higher than the increase in Hc (see Table 1 and below).

Cytosolic content of aldosterone

The adrenal cytosolic content of aldosterone was significantly increased by ET-1 (Table 3). The dose-response relationship showed to be biphasic with a maximum at about 50 to 80 ng ET/kg/min. Maximal stimulations at different rates were 120% at 50 #l/min (corresponding to 37.4 _ 2.6, control vs 78.0 + 7.1 ng aldosterone/ adrenal, treated); 100% at 125#l/min (corresponding to 29.1 + 2.0, control vs 56.5 + 5.0 ng aldosterone/adrenal, treated) and 140% at 180 #l/min (corresponding to 25.0_ 4.1, control vs 35.4 + 2.5 ng aldosterone/adrenal, treated).

Table 3. Effect of ET-I on cytosolic content of aldosterone

Infusion rates ET- 1 ( # I/rain) doses

(ng/kg/min) 50 125 180

0 1.00 + 0.06 (8) 1.00 -+_ 0.04 (13) 1.00 -+ 0.06 (8) 20 1.06 5:0.09 (4) 0.90 -+ 0.11 (6) ND 50 1.13 + 0.07 (4) 1.26 -+ 0.20 (4) 2.43 5:0.22 (8)* 80 2 .20+0.13(4)* 1.91 +0.14"**(7) 2.435:0.29**(7)

110 1.31 5: 0.05* (4) 1.43 5: 0.19" (6) 1.86 5: 0.06* (4)

Data are mean _+ SEM of T/C ratios calculated as indicated under Experimental. The number of separated experiments is indicated between round brackets. *P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA). Control rats, 37.4 -+ 2.6; 29.1 + 2.0 and 25.0 -+ 4.1 ng aldosterone/adrenal at 50, 125 and 180/~l/min. ND, not determined.

Table 4. Effect of ET- 1 on mitochondrial synthesis of aldosterone

Infusion rates ET-1 (#l/rain) doses

(ng/kg/min) 50 125 180

0 1.00 + 0.07 (8) 1.00 + 0.05 (9) 1.00 -+ 0.04 (6) 20 1.78 _+ 0.35 (6) 0.98 + 0.05 (6) ND 50 1.61 + 0.16" (4) 2.53 5: 0.26* (4) 2.98 + 0.23** (8) 80 1.80+0.24(4) 1.51 +0.10"*(6) 1.71 -+0.16"*(8)

110 1.48 -+ 0.24 (4) 1.59 -+ 0.16"* (6) 1.14 -+ 0.03 (4)

Effect of ET on mitochondrial synthesis of aldosterone from deoxycorticosterone under saturating conditions. Data are mean + SEM of T/C ratios calculated as indicated under Experimental. The number of separated experiments is indicated between round brackets. Control rats were 0.45 -+ 0.03, 0.42 + 0.06 and 0.50 + 0.11 pg aldosterone/gg prot at 50, 125 and 180 gl/min. ND, not determined. *P < 0.05, **P < 0.01 (ANOVA).

Mitochondrial synthesis of aldosterone

Preliminary experiments showed that 30/~ M deoxycorticosterone is a saturating concentration for aldosterone production (data not shown). Mitochondrial synthesis of aldosterone from deoxycorticosterone was increased after infusion of ET-1 (Table 4). The dose-response relationship for this effect of ET-1 also appears to be biphasic. The maximal responses were seen at 50 to 80ng/kg/min and corresponded to stimulations of 80 (0.45+0.06, control vs 0.84___0.06pg aldosterone//~g prot, treated); 150% at 125#1/ min (0.42 +__0.03, control vs 1.07 ___ 0.11 pg aldosterone/pg prot, treated) and 200% at 180 #l/min (0.50+0.11, control vs 1.56+0.12pg aldosterone/pg prot, treated). The effect of ET-I at 50#l/min was almost independent of the dose used (Table 4), suggesting that with lower infusion rates, ET reaches maximal responses more easily. Thus, the dose-response relationships for cytosolic content and mitochondrial synthesis of aldosterone are very similar.

Pregnenolene was measured simultaneously in incubations of mitochondria from control and treated rats (ET-1 dose 50ng/kg/min, infusion rate 125 #l/min, conditions for maximal effect of ET-1 on the conversion of deoxycorticosterone to aldosterone, see Table 4). Pregnenolone increased from 7.15 to 20.40 pg pregnenolone/#g protein (185%).

Effect of enalapril

In order to ascertain whether or not the ET- induced increases in cytosolic aldosterone as well as in mitochondrial synthesis of aldosterone might be attributed to AII through an ET-mediated increase in plasma renin activity, infusions were also carried out in the presence of the angiotensin converting enzyme (ACE) inhibitor

Endo the l in s t imula tes a ldos te rone p roduc t ion 559

Table 5. Effect of enalapril

Cystosolic aldosterone content Treatment (ng/adrenal)

Control 30.1 _+ 0. I Enalapril 25.3 _+ 5.6 AI 39.3 + 2.9 Enalapril + AI 19.7 + 4.4 All 51.6 + 0.5 ET-I 56.3 + 4.6 Enalapril + ET-I 65.8 +_ 7.4

Rats were infused according to different treat- ments following the protocol shown in Fig. I. A1 was infused at 300 ng/kg/min, All at 40ng/kg/min, and ET-I at 80ng/kg/min. After infusions, adrenals were removed, homogenized, centrifuged (see Experimental) and aldosterone measured in supernatants. Infusion rate was 125 #l/rain. Data (mean __. range) are from 1 of 2 different experiments performed in triplicate.

enalapril. To evaluate the inhibition of ACE activity by enalapril, AI was infused (see Fig. 1) in the absence or presence of enalapril.

AI provoked a sizable increase in cytosolic content of aldosterone which was prevented and even diminished by enalapril. However, enalapril failed to abolish the increase in aldosterone provoked by ET (Table 5).

D I S C U S S I O N

As expected, infusion of ET- 1 increased Hc in a dose-dependent manner. The increase in Hc caused by ET-1 is not fully accounted for by fluid losses suggesting that the hemo concentration might be due to vascular leakage [26, 33, 34]. The increase in Hc is not associated with a pro- portional increase in plasma proteins (data not shown) indicating that protein extravasation occurs with ET-1 infusion. Albumin leaks occur in the heart, skeletal muscle, and intestine, but not in liver, lung, testis or brain [34].

The herein reported decrease in UV after infusion of ET is in agreement with previous reports in other species[25,26]. This effect of ET-1 was proposed to occur because of a decrease in kidney blood flow provoked by ET-1 mediated vasoconstriction [25] (see below).

When our cells were incubated in the presence of ET-1, aldosterone production was increased. These results confirm a direct in vitro action of the peptide on aldosteronogenesis [15, 16, 21-23]. On the other hand, infusion of ET-1 also increased plasma aldosterone levels which is in agreement with previous reports [25-27]. This increase was higher than the ET-1 mediated increase in Hc.

The effect of ET- 1 on aldosterone production could also be visualized by measuring the

increase of the adrenal cytosolic content of the mineralocorticoid after infusion of the peptide. Thus, cytosolic content of aldosterone was a useful and much easier tool to evaluate the effects of ET-1 on aldosterone production. In effect, (a) the percentages of increase for plasma aldosterone levels and cytosolic content of aldosterone were of the same order (see Results and Table 3); and (b) the very good correlation between dose-response relationships for cytosolic content of aldosterone (Table 3) and activation of enzymatic conversion of deoxycorticosterone into aldosterone (Table 4) suggests that the measured increase in cytosolic content of aldosterone after infusion with ET-1 is a conse- quence of increased mitochondrial synthesis of the mineralocorticoid.

ACTH has been documented to increase both activity of aldosteronogenic enzymes and aldosterone secretion [35-37]. In effect ACTH- treated animals present increased plasma aldosterone levels while the cytosolic content of aldosterone was almost unaffected [37]. Accord- ing to our data, ET-1 only stimulates the production, but not the secretion, of aldosterone.

An alternative hypothesis might attribute the ET-1 mediated increase in cytosolic content of aldosterone to plasma concentration and/or the accumulation of aldosterone in the adrenal gland due to vasoconstriction-induced reduction in adrenal blood flow. However, this is unlikely because of (a) dose-response relationships are different for Hc and cytosolic content of aldosterone; (b) ET-1 mediated augmentation of plasma aldosterone and cytosolic content of aldosterone are of the same order; (c) dose- response relationships for mitochondrial synthesis and cytosolic content of aldosterone are very similar; (d) higher doses of ET- 1 decreased rather than further increased the cytosolic content of aldosterone; and (e) increases in cytosolic content of aldosterone were always higher than in Hc.

Miller et al. [25] have proposed that the aldosteronogenic effect of ET-1 might not be due to a direct action on the adrenal but through increases in plasma renin activity. However, we have found that coadministration of enalapril, a very well known converting enzyme inhibitor, at a dose proved to inhibit AI-induced increases in aldosterone production, did not affect the increase of aldosterone provoked by infusion of ET-I. Thus it appears likely that in vivo stimulation of aldosterone production by ET-1 is independent of the renin-angiotensin system. Our results are in agreement with the report of

560 ADALI P~CCl et al.

Cao and Banks [27] according to which captopril did not affect stimulation of aldosterone provoked by ET-1.

In order to evaluate the loci of action of ET-1 on aldosterone stimulation, mitochondria from control and treated rats were incubated in the presence of saturating concentrations of deoxycorticosterone. Our results show that ET-1 activates the late mitochondrial step of aldosterone formation, at least in part by increasing the apparent Vmax. Our results also show that mitochondrial production of pregnenolone is increased after infusion with ET-1, suggesting that this peptide activates the early mitochondrial step of aldosterone formation as well. We have previously reported that ET-1 potentiates the early and late pathways of aldosterone production stimulated by ACTH and All [38]. These results taken together suggest that ET-1 pro- vokes both activation by itself and potentiation of the already stimulated late and early pathways of aldosterone production.

The dose-response relationship for the ET-1 mediated augmentation in cytosolic content of aldosterone and mitochondrial synthesis of aldosterone reached a peak and exhibited less stimulation at higher doses. The explanation is unclear but it is unlikely that ET-l-mediated vasoconstriction of the adrenal arteries decreases adrenal blood flow and the access of stimulants to the adrenal.

Adrenal blood flow has been shown to play a role in ACTH and ET-1 actions on aldosterone stimulation [16]. ET-l-mediated reduction of blood flow in other organs is supported by the herein reported decrease in UV.

These ET-l-induced effects on aldosterone production were shown to be dependent on ET doses and on infusion rates. In effect, dose- response experiments showed significant differences at different infusion rates. Surprisingly, at 50 pl/min the ET-mediated increase in mitochondrial aldosterone synthesis was almost independent of the ET dose. These results clearly indicate the importance of the selection of the dose and the infusion rate in designing an experiment or in comparing the results from different reports. To the best of our knowledge this is the first report in which the use of different doses of ET-1 and infusion rates has been shown and its critical importance demonstrated.

Acknowledgements--These studies were supported by grants from CONICET and Fundacion Antorchas, Argentina, to Drs Cozza and Lantos and the National Institutes of Health to Dr Gomez-Sanchez (HL 27737).

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in vivo stimulation of aldosterone biosynthesis by endothelin: loci of action and effects of doses...

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