0013-7227/92/1303-1637$03.00/O Endocrinology Copyright Q 1992 by The Endocrine
Vol. 130, No. 3 Society
Printed
in U.S.A.
Angiotensin-II Inhibits Na+/K+ Pump in Rat Adrenal Glomerulosa Cells: Possible Contribution to Stimulation of Aldosterone Production GYGRGY HAJNGCZKY, MIKLGS P. KALAPOS,
GYijRGY CSORDAS, LASZL6 HUNYADY, TAMAS BALLA, PETER ENYEDI, AND ANDRAS
Department of Physiology and First Department School, Budapest, Hungary
of
SPAT
Biochemistry (M.P.K.), Semmelwek University Medical
ABSTRACT. The control of Na’/K’ pump activity was studied in rat adrenal glomerulosa cells. Ninety percent of K+/%Rb accumulation was blocked by ouabain, and the dose-response curve of inhibition by ouabain was monophasic (ICw, -80 PM), suggesting the role of a single type of Na’/K’ pump (cy-isoenzyme) in 8BRb accumulation by rat glomerulosa cells. The basal activity of the Na+/K+ pump was much higher in glomerulosa cells than in adrenal fasciculata cells or hepatocytes, as judged by the ouabain-sensitive uptake of WRb. In contrast to the two other cell types, increasing Na+ influx with the Na+ ionophore monensin failed to significantly affect ouabain-sensitive BBRb uptake in glomerulosa cells, suggesting that in glomerulosa cells even the resting intracellular Na’ concentration is sufficient for maximal activity of the Na’/K’ pump. Angiotensin-II (AID inhibited the ouabain-sensitive ssRb uptake by glomerulosa cells. The effect of AI1 was abolished by the
T
HE Ca2+-mobilizing agonist angiotensin-II (AII) is a major regulator of adrenal glomerulosa cells. The role of modulation of cellular Ca2’ metabolism by AI1 in its action is well established. Mobilization of Ca2+ from intracellular stores is mediated by inositol 1,4,5-trisphosphate, derived from polyphosphoinositides of the plasma membrane, through AII-induced activation of phospholipase-C (for review, see Refs. l-3). However, the mechanism of the enhancement of Ca2+ entry in AIIstimulated cells is not clear. Some reports suggest the involvement of the dihydropyridine-sensitive voltageoperated Ca2+ channels (4-8), while others show the participation of a nifedipine-insensitive Ca2+ transport mechanism (8-11). Among these latter reports, Na+/Ca2+ exchange (12) is also influenced by membrane potential and may be affected by the intracellular Na+ concentration. Considering that the Na+/K+ pump has a principal Received September 3,199l. Address all correspondence and requests for reprints to: Dr. AndrL Spat, Department of Physiology, Semmelweis University Medical School, H-1444, P.O. Box 259, Budapest 8, Hungary.
selective antagonist of the AT, type of AI1 receptors (DuP 753), while PD 123177, an AT, antagonist was ineffective. AT, receptors of glomerulosa cells coupled to phospholipase-C activation and, thus, to Ca*’ signal. The inhibitory effect of AI1 was dependent on the extracellular Ca’+ concentration, but an elevation of cytoplasmic Ca’+ by Ca*+ ionophore ionomycin failed to mimic the effect of AII. These data suggest that Ca2+ is required for but does not mediate the inhibitory effect of AI1 on the Na+/K+pump. Pharmacological activation of protein kinaseC by phorbol ester did not modify 86Rb accumulation by the cells. Ouabain induced a nifedipine-sensitive elevation in the cytoplasmic Ca*+ concentration and exerted a stimulatory effect on aldosterone production, suggesting participation of the inhibition of the Na+/K+ pump in the aldosterone stimulatory action of AII. (Endocrinology 130: 1637-X44,1992)
role in maintaining the membrane potential and the low intracellular Na+ concentration, alteration of its activity may modify cellular Ca2+ transport mechanisms. In fact, AI1 influences the activity of the Na+/K+ pump in some of its target cells (13-16). The present experiments were designed to study the properties of the Na+/K+ pump and its possible involvement in the activation of AII-stimulated rat adrenal glomerulosa cells. Materials
and Methods
Materials
AI1 (Ile6-AII) was obtained from Serva (Heidelberg, Germany); corticotropin (ACTH, Synacthen) from Ciba-Geigy (Basel, Switzerland); 12-tetradecanoyl phorbol 13-acetate (PMA), N&,ZV’,N’-tetrakis (%pyridylmethyl) ethylendiamine (TPEN), probenecid were obtained from Sigma (St. Louis, MO), ionomycin was purchased from Calbiochem (La Jolla, CA); medium 199 ((with Earle’s salts) was obtained from Gibco (Paisley, Scotland); and collagenase was purchased from Millipore-Worthington (Freehold, NJ). Nonpeptide AI1 receptor antagonists were synthesized at DuPont (Wilmington, DE.
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[86Rb]RbC1 (0.5-2 Ci/g) was obtained from Izinta (Budapest, Hungary), and nitrocellulose membrane filters (pore size, 3 pm) were purchased from Schleicher and Schuell (Feldbach, Switzerland). CeU i.9olution Rat adrenal glomerulosa cells were prepared from the adrenal capsular tissues of Wistar rata (200-300 g) using collagenase and mechanical dispersion, as described previously (9). Cell yield was 2-4 x lO’/rat, and the number of fasciculata cells was less than 5% of the glomerulosa cells. Rat adrenal fasciculata cells were prepared from the decapsulated glands using the same digestion procedure. Cell yield was about 5 x lO’/rat. Isolated liver parenchymal cells were prepared from fed white CFLP mice (30 g) using collagenase, as previously described (17).
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After centrifugation and washing the cells, measurements were carried out in the presence 20 MM TPEN and 2.5 mM probenecid at 37 C. The fluorescence was monitored simultaneously at 335- and 360~nm excitation wavelenghts, and 505-nm emission wavelength, and the ratios were computed in a Deltascan fluorescence spectrophotometer (Photon Technology International, South Brunswick, NJ). Calibration of the fluorescence was carried out using the fluorescence ratios according to a previously reported method (20) after correction for the autofluorescence and the extracellular dye fluorescence. Statistics The mean f SEM of the separate experiments are given, each of which was carried out at least in duplicate. For statistical comparison, Student’s paired sample t test was applied.
Results
%Rb influx experiments In %Rb influx studies, aliquots of cell suspension (female rata) containing about l-2 x lo5 freshly isolated cells were preincubated for 15 min at 37 C in 100 ~1 of a 2:l (vol/vol) mixture of modified Krebs-Ringer HEPES-glucose solution and medium 199 (final concentrations in millimolar concentrations: Na, 145; K, 3.6; Ca, 1.2; Mg, 0.5; HEPES, 20; pH 7.4) supplemented with human serum albumin (fraction V; 2 g/ liter). Stimuli and a tracer amount of isotope (0.1-0.5 j&i) were added in 10 and 40 ~1 prewarmed medium, respectively. Incubation was terminated 5 min after addition of the isotope by vacuum filtration through nitrocellulose membrane filters. In preliminary studies %Rb accumulation by glomerulosa cells showed linear kinetics in the first 5 min of isotope uptake. The ouabain-insensitive fraction of %Rb uptake was measured on cells that were preincubated with 1 mM ouabain for 30 min. Experiments were performed at least in duplicate. In those experiments in which the effect of the Ca2+ or Na’ concentration was tested, medium 199 was omitted from the incubating medium; otherwise, the concentrations of the other ions were the same as in the medium described above. When the Na+ concentration was reduced, isosmosis was attained by Nmethyl-D-glucamine. Filter blank (100 cpm, on the average) has been submitted from the values presented. Aldosterone experiments Cells derived from male rata were preincubated for 3 h in the above medium, which was now buffered with bicarbonate (24 mM) at 37 C under a mixture of 95% 02-5% CO, (pH 7.4). Then, the cells were washed and incubated (-lo5 cells/600 ~1) for 60 min under identical conditions. The aldosterone content of the supernatant (after pelleting the cells) was measured by RIA (18). Experiments were performed in duplicate. Experiments on cytoplasmic Ca2+ The cytoplasmic Ca2+ levels were measured in fura-2-preloaded cells (isolated from female rats on the day of experiment). The applied method was based on a previously described procedure (19), with the following modifications. Cells were loaded with 0.25 pM fura- in the presence of 2.5 mM probenecid for 30 min at room temperature and then for 15 min at 37 C.
Ouubain sensitivity of %Rb accumulation ghnerulosa cells
by
The transport activity of the Na+/K+ ATPase was monitored with radioactive rubidium (%Rb), which substitues for potassium in the active transport into the cell. More than 90% of 5-min @Rb uptake was abolished by the specific inhibitor of the Na+/K+ pump, ouabain (1 mM), and the concentration required for half-maximal inhibition (I&,,,) was about 80 pM (Fig. 1). Comparison of the Na+/K+ pump activities of glomerulosa cells, fasciculata cells, and hepatocytes
The ouabain-sensitive %Rb uptakes by glomerulosa cells, fasciculata cells, and hepatocytes under identical conditions were 14,815 + 1,440, 3,780 f 574, and 741 + 37 cpm/106 cells, as related to lo6 cpm added (n = 17, 3,
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FIG. 1. Effect of ouabain on %Rb accumulation by glomerulosa cells. After preincubation with or without different concentrations of ouabain for 30 min, sGRb accumulation was measured for 5 min. Results were expressed as a percentage of the control value, which was 14,825 f 1,626 cpm/106 cells, as related to lo6 cpm added (mean f SEM; n = 3).
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AI1 INHIBITS
and 3, respectively). The Na’ ionophore monensin stimulated 86Rb uptake by fasciculata cells and hepatocytes, but failed to affect it significantly in glomerulosa cells (Fig. 2). To test whether monensin failed to increase intracellular Na+ in glomerulosa cells or whether the intracellular Na+ concentration in control cells completely activated the pump, we also compared the effect of monensin on cells incubated in a medium containing 120 or 13 InM NaCl (Fig. 3, left panel). Isotope uptake by cells at the lower Na+ concentration was 45.9 + 7.2% (n = 4) of that at the higher Na+ concentration. While the effect of monensin on %Rb uptake was negligible at a Na+ concentration of 120 mM (107.2 + 14.8 of control cells; n = 5), the uptake was enhanced by 71.2 + 10.3% in a medium containing 13 mM Na+ (P < 0.01; n = 4). The intracellular sodium dependence of the Na+/K+ pump was also studied in cells that had been preincubated without K+ with varying sodium concentrations. The aim of this procedure was to block active extrusion of Na+ and, thus, equilibrating intracellular space with extracellular Na+ concentration. The sodium activation curve of hepatocytes showed a monotonic increase in the whole range of Na+ concentrations tested, while in glomerulosa cells, %Rb uptake reached a maximum value in the range of cytoplasmic Na+ concentrations and de-
Mon
AI1
FIG. 2. Comparison of the effects of monensin and AI1 on ouabainsensitive @Rb accumulation by glomendosa cells, fasciculata cells, and hepatocytes. Five-minute %Rb influx was measured, and the ouabainsensitive fraction of the total uptake is shown for glomerulosa cells (Cl), fasciculata cells (H), and hepatocytes (m). Data were normalized for the control. Control %Rb uptakes of glomerulosa cells, fasciculata cells, and hepatocytes were 20,496 + 3,875, 3,780 f 574, and 741 + 37 cpm/106 cells, as related to 10’ cpm added (n = 3). AI1 (25 nM) or monensin (Mon; 50 PM) was added with the isotope (mean + SEM; n = 3).
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clined at the highest level of Na+ tested (Fig. 3, right panel). Effect and mode of action of AII on 86Rb influx Stimulation of glomerulosa cells by the Ca’+-mobilizing AI1 (25 nM) significantly diminished the 5-min ouabain-sensitive “Rb influx (51.6 f. 3.0% of the control value; P C 0.01; n = 14). We have also observed the previously described moderate stimulatory effect of AI1 on the Na+/K+ pump in hepatocytes (121.3 + 5.1% of control; P < 0.01; n = 6) and a tendency for an increase in %Rb influx in AII-treated fasciculata cells (Fig. 2). ACTH (300 nM), a CAMP-mediated stimulus of aldosterone production, did not exert any significant effect on %Rb uptake by glomerulosa cells (97.6 f 1.6% of the control value; n = 3). The dose-response relationship for the inhibition of @jRb uptake by AI1 in glomerulosa cells is shown in Fig. 4. Half-maximal inhibition by AI1 was attained at a concentration (0.3 nM) comparable with that required for half-maximal stimulation of aldosterone production (data not shown). Two specific nonpeptide antagonists were applied to identify the subtype of AI1 receptors (AT1 or ATJ that mediates this effect of the peptide in glomerulosa cells. Neither the AT1-specific losartan potassium (previously designated DuP753) nor the ATP-specific PD123177 significantly affected the %Rb influx of unstimulated cells (data not shown). However, the inhibitory action of AI1 was prevented by losartan potassium in a concentrationdependent manner (I&, -200 nM), while PD123177 failed to modify the effect of AI1 (Fig. 5). Phospholipase-C activation and stimulation of aldosterone production are coupled to the receptor subtype AT, in rat glomerulosa cells (Hajnoczky, Gy., and A. Spat, unpublished). Therefore, we tested the possible role of known mediators of Ca’+-mobilizing stimuli in the effect of AII. The Ca*’ ionophore ionomycin (10 PM) had no effect on %Rb influx, and AI1 was able to exert its inhibitory effect in the presence of ionomycin (Fig. 6, left panel). However, in the presence of a low extracellular Ca*+ concentration, “Rb accumulation by the cells was smaller, and the inhibitory effect of AI1 disappeared (Fig. 6, rightpanel).The protein kinase-C activator PMA (50 nM; added either 10 min before the isotope or together with it) failed to modulate %Rb uptake (98.3 f 2.59% of the control value; n = 6). Effect of inhibition of Na+/K+ pump on cytoplasmic Ca*+ and aldosterone production The effect of ouabain on the cytoplasmic Ca*+ concentration of glomerulosa cells was tested. Ouabain induced a slow increase in cytoplasmic Ca*+, and its effect was sensitive to the voltage-operated Ca*+ channel blocker
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Endo. 1992 Voll30. No 3
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FIG. 3. Effect of the intracellular Na’ concentration on ouabain-sensitive -Rb accumulation by glomerulosa cells. Left panel, Cells were incubated in a medium containing 120 or 13 mM Na+ for 15 min at 37 C. Then, 5-min BBRbaccumulation was measured in the absence (0) or presence (B) of monensin (50 PM). Isotope uptake was expressed as a percentage of the control =Rb uptake (at 120 mM Na’; 15,520 f 3,254 cpm/106 cells, as related to 10’ cpm added; mean f SEM; n = 4). Right panel, Cells were preincubated in a K+-free medium containing different concentrations of sodium at 4 C. It was followed by incubation at 37 C and simultaneous addition of K+ (3.6 mM) and a tracer amount of BBRb.Five-minute isotope uptake was measured for glomerulosa cells (e n = 4) and hepatocytes (0, n = 2). The mean f SEM are shown in counts per min/lO’ cells, as related to l@ cpm added.
0
0.01
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[antagonist] 0.1
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Angiotensin
3
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II (nM)
FIG. 4. Dose-response relationship for the inhibition of ouabain-sensitive BBRbuptake by AI1 in glomerulosa cells. Five-minute ffiRb influx was measured in the presence of different concentrations of AR. Results were expressed as a percentage of isotope uptake by unstimulated cells (10,538 f 1,387 cpm/lO cells, as related to 10’ cpm added; mean f SEM; n = 4).
nifedipine (Fig. 7). We also examined the effect of ouabain on aldosterone production by glomerulosa cells. The lowest concentration of the drug that caused a significant inhibition of the pump (10 PM; Fig. 1) was found to stimulate hormone production significantly (P < 0.05; n = 3; Fig. 8). Maximal stimulation was found at about 100 I.~M ouabain. At
100
(p;II”)
FIG. 5. Effect of specific receptor antagonists on AII-induced inhibition of ouabain-sensitive %Rb accumulation by glomerulosa cells. Fiveminute BBRbinflux was measured in the presence or absence of AI1 (25 nM), losartan potassium (AT1 receptor antagonist; l ), and PD 123177 (AT, receptor antagonist; 0). Results (mean f SEM) were expressed as a percentage of the control value (11,215 + 1,716 cpm/lO’ cells, as related to lo6 cpm added; n = 3).
this concentration, the drug induced about 60% inhibition of the Na+/K+ pump, which is comparable to the inhibitory action of 25 nM AII. Complete inhibition of the Na+/K+ pump (concentration of ouabain, 20.3 mM) caused submaximal stimulation of aldosterone production (Figs. 1 and 8). Discussion Several electrophysiological properties of adrenal glomerulosa cells resemble those of excitable cells. Highly
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FIG. 6. Effect of the Caz’ concentration on the inhibitory effect of AI1 on ouabain-sensitive =Rb accumulation by glomerulosa cells. Left panel, Effect of AI1 (25 nM) on 5min WRb influx by control and ionomycin-treated (Iono; 10 PM; 5min preincubation) glomerulosa cells. Results were expressed aa a percentage of the control value, which was 10,250 f 674 cpm/lO’ cells, aa related to lo6 cpm added. The mean f SEM are shown (n = 3). Right panel, Five-minute BBRbinflux was measured in a nominally Ca *+-free medium supplemented by the indicated concentrations of CaCh. Data were normalized for lo6 cpm/aample. The Ca’+-free medium contained Ca2+ in a concentration of about 75 PM, as checked by Ca*+-sensitive electrode. The bars of the lower part represent @Rb uptake by unstimulated cells (0) and that by AI1 (25 m&treated cells (& mean f SEM; n = 3). The ratio of isotope accumulation in the presence and absence of AI1 is shown as a function of the Ca*’ concentration in the upper part of the panel.
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FIG. 7. Effect of ouabain on the cytoplaamic Ca*+ concentration of glomeruloaa cells. Two separate mns performed on the same suspension of fura-2-preloaded cells are shown. Upper trace, Ouabain (Ou; 300 PM) and then nifedipine (Nif; 1 PM) were added. Lower trace, The same drugs were added in the opposite order. The Ca*’ concentration was computed as described in Materials and Methods. The curves are representative for four experiments.
negative membrane potential (see references in Ref. 21) and the presence and the role of voltage-operated channels in aldosterone production (see references in Ref. 19 and 22) were previously reported. The first aim of the present work was to elucidate the
;
I 10
ouabain]
I 100
1 1000
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8. Effect of ouabain on aldosterone production by glomerulosa cells. Cella were incubated in the presence of varying concentrations of ouabain for 60 min. Results are expressed aa a percentage of the control value (mean f SEM; n = 3). Mean basal aldosterone production was 0.69 f 0.11 pmol/l06 cells. h (n = 3). FIG.
role of the Na+/K+ pump in generation of this highly negative membrane potential. Since in rat tissues the two forms of the Na+/K+ pump [a and a(+)] have markedly different affinities for ouabain (for review, see Ref. 23), the monophasic inhibition curve and the ICso of about 80 pM are compatible with the predominant role of a-isoenzyme in 86Rb accumulation (indicator of Na+/
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K+ pump activity) by glomerulosa cells. We found that ouabain-sensitive %Rb accumulation by glomerulosa cells is several-fold higher than that by fasciculata cells and hepatocytes. A higher density of the Na+/K+ pump in the membranes of glomerulosa cells compared to that in fasciculata cells (24, 25) might explain this difference between these two cell types. However, an additional role of a higher activation of the pump by intracellular Na+ in glomerulosa cells has not yet been studied. This possibility was tested here by measuring the effect of the sodium ionophore monensin on 86Rb influx. Monensin enhances Na+ influx into several cell types, and the increased intracellular Na+ level stimulates the Na+/K+ pump (13,15, 26). In agreement with these studies, monensin induced an almost 2-fold stimulation of 86Rb accumulation by fasciculata cells and hepatocytes, but failed to affect it significantly in glomerulosa cells. However, when the intracellular Na+ content of glomerulosa cells and, thus, Na+/K+ pump activity were reduced by incubation of the cells in a low sodium medium, monensin was able to increase %Rb uptake. These data suggest that monensin enhances Na+ entry into, glomerulosa cells, but an increase in intracellular Na+ in glomerulosa cells incubated in physiological concentrations of sodium fails to enhance further the activity of the pump. An alternative experiment for assessing activation of the pump as a function of the intracellular Na+ concentration was performed on glomerulosa cells and hepatocytes that were preincubated in normal or low sodium medium with intracellular Na+ set at varying values. The maximal pump activity was reached at lower Na+ concentrations for glomerulosa cells than for hepatocytes. The sodium concentration that induced half-maximal activation (K& of the pump was about 10 mM, similar to the Ko.s of a-isoenzyme of rat adipocytes (17 mM) and far different from the K0.5 of a(+)-isoenzyme (52 mM) (27). Taken together, these data suggest that even the resting intracellular Na+ concentration in glomerulosa cells, in contrast to that in hepatocytes and fasciculata cells, is able to maintain maximal activity of the Na+/K+ pump. The higher affinity of the pump for intracellular Na+ may be a reason for this difference, but a higher intracellular Na+ concentration in glomerulosa cells (28, 29) may also contribute to this phenomenon. Thus, in addition to the high density of the pump, its greater activation by Na+ serves as an explanation for the high activity of the Na+/K+ pump in glomerulosa cells. Since the Na+/K+ pump maintains the greatest concentration gradient for K+ and Na+ between the two sides of the plasma membrane and functions in an electrogenic manner, its high activity may have an important contribution to the highly negative membrane potential of glomerulosa cells. The depolarizing effect of AI1 on glomerulosa cells was
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shown previously with electrophysiological (30, 31) and fluorescent (10) methods. The peptide may exert this effect at least in part via reducing the K+ permeability of the cells (31,32). However, AI1 decreased rather than increased the potassium content of rat glomerulosa cells (33, 34). The inhibitory action of AI1 on the Na+/K+ pump, which is a new finding of our study, may explain this apparent contradiction and may also contribute to depolarization. An intriguing point is the different effect of AI1 on its target cells. In contrast to its action in glomerulosa cells, AI1 stimulates the Na+/K+ pump of vascular smooth muscle cells (13) and hepatocytes (15). Since a supposed mechanism of AI1 action in vascular smooth muscle cells is increasing the intracellular Na+ supply (13), it is not surprising that such an action does not lead to stimulation of the Na+/K+ pump in glomerulosa cells, in which the pump appears to be completely activated by intracellular Na+ even under control conditions. In hepatocytes the previous data on the mode of action of Ca’+-mobilizing stimuli are controversial (15, 35). How is inhibition of the Na+/K+ pump associated with known messenger mechanisms activated by AII? The inhibition seems to be coupled to the AT1 type of angiotensin receptors through which the Ca2+ signal and aldosterone response are also induced. However, some of our data are against a role of Ca2+ or protein kinase-C (known mediators of Ca2+-mobilizing ligands) in inhibition of the pump. While some previous studies (15, 26) suggested that increased cytoplasmic Ca2+ is associated with inhibition of the Na+/K+ pump in different tissues, the inhibitory effect of AI1 in glomerulosa cells cannot be explained with such a mechanism, as elevation of cytoplasmic Ca2+ by the Ca2+ ionophore ionomycin did not modify the activity of the pump, and AI1 was able to exert its effect also in ionomycin-pretreated cells. However, a permissive role of Ca2’ is likely in the effect of AII, since the action of AI1 disappeared in a low calcium medium. Agonist-induced modulation of Na+/K+ pump activity seems to be mediated by protein kinase-C in some ceil types (15, 36). However, activation of protein kinase-C by phorbol ester in glomerulosa cells did not affect the activity of the Na+/K+ pump. Elucidation of whether the effect of AI1 on the Na+/K+ pump is related to phospholipase-C activation or is mediated by an unknown mediator coupled to AT, receptors or in another way requires further experiments. Whether AI1 exerts its inhibitory effect directly on the Na+/K+ pump or modifies the ionic enviroment of the pump has not been elucidated. It was demonstrated that AI1 has no significant effect on ouabain-sensitive or potassium-stimulated ATPase activity in adrenal capsular membranes (24,37,38). However, the signal transduction pathway from the AI1 receptor to the pump may
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have been lacking in membrane preparations. The contribution of the inhibition of Na+/K+ pump to elevation of cytoplasmic Ca*+ by AI1 is also suggested by our data. The ensuing depolarization may enhance Ca*+ entry via dihydropyridine-sensitive voltage-operated Ca*+ channels. Such a component is likely to participate in the ouabain-induced cytoplasmic Ca*+ signal, since it was almost completely abolished by nifedipine. In addition to this mechanism, ouabain-induced elevation of the intracellular Na+ concentration may modify the ion transport via Na+/Ca+ exchanger of glomerulosa cells (12,39), resulting in a net increase in Ca*+ entry into the cells. Considering that nifedipine abolished the effect of ouabain on the cytoplasmic Ca*+ concentration, Na+/ Ca*+ exchange that is dihydropyridine insensitive (39) may have a minor role in generation of the cytoplasmic Ca*+ signal induced by inhibition of the Na+/K+ pump. The participation of inhibition of the Na+/K+ pump in the biological response to AI1 is supported by the effect of ouabain on aldosterone production. In this respect previous data were controversial (see references in Ref. 40). In two studies the stimulatory effect of 10 PM ouabain on aldosterone production by rat glomerulosa cells was observed (41, 42), and it was sensitive to Ca*+ channel blockers (42). In our experiments there was a close correlation between the effects of ouabain on the Na+/K+ pump and stimulation of aldosterone production up to 100 pM of the drug. Thus, partial inhibition of the pump that is also induced by AI1 enhances aldosterone production. In summary, the inhibitory effect of AI1 on the Na+/ K+ pump may elicit depolarization, which, in turn, enhances Ca*+ entry. This mechanism may serve as an additional transduction pathway, leading to the aldosterone response of glomerulosa cells. Acknowledgments Nonpeptide angiotensin receptor antagonists were kindly provided by Andrew T. Chiu. The excellent technical assistance of Miss Erika Kovacs, Mrs. Zsuzsa Legeza, and Mrs. Agnes RibC are greatly appreciated. The aldosterone antibody was a gift from the NIAMD (Bethesda, MD).
References Catt KJ, Carson MC, Hausdorff CM, Leach-Harper CM, Baukal AJ, Guillemette G, Balla T, Aguilera G 1987 Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells. J Steroid Biochem 27:915-927 Spat A 1988 Stimulus-secretion coupling in angiotensin-stimulated aldosterone secretion. J Steroid Biochem 29443-453 Barrett PQ, Bollag WB, Isales CM, McCarthy RT, Rasmussen H 1989 Role of calcium in angiotensin 11mediated aldosterone secretion. Endocr Rev l&496-518 Kojima I, Kojima K, Rasmussen H 1985 Characteristics of angiotensin II:, K’- and ACTH-induced calcium influx in adrenal glomerulosa cells. J Biol Chem 260:9171-9176
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